Photoreceptor rescue cell (PRC) compositions and methods of treating ocular conditions

Abstract

The present invention provides populations of photoreceptor rescue cells (PRCs) having a unique profile of markers and resulting from in vitro differentiation of early progenitor cells, including pluripotent stem cells and embryonic stem cells (ESCs). Also provided are methods of generating populations of photoreceptor-rescuing cells and methods of using them to treat ocular conditions.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 373,298, filed August 23, 2022, and U.S. Provisional Patent Application No. 63 / 432,948, filed December 15, 2022, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] Several retinal diseases or conditions can lead to vision loss or even blindness. These include rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration (such as age-related macular degeneration (wet or dry)), geographic atrophy secondary to AMD, leber congenital amaurosis, and Staggart's disease. Some retinal diseases or conditions result from the loss of cells in the nuclear layer, primarily the outer nuclear layer, including photoreceptor cells. Replacing degenerated photoreceptors with new cells offers a potential way to slow or prevent cell degeneration and vision loss.

[0004] Potential replacement sources for photoreceptor cells include stem cells. Early studies have introduced heterologous populations of mouse cells, mouse stem cells, or retinal progenitor cells as potential cellular sources for replacing lost photoreceptors. These early studies described the transplantation of photoreceptor progenitor cells from the retina of mice on day 1 after birth (Maclaren et al., Nature 444(9):203-207, 2006), the in vitro generation of retinal progenitor cells from mouse embryonic stem cells (Ikeda et al., Proc. Natl. Acad. Sci. 102(32):11331-11336, 2005), the generation of retinal progenitor cells from the retina of mice on day 1 after birth (Klassen et al., Invest. Ophthal. Vis. Sci. 45(11):4167-4175, 2004), the transplantation of bone marrow mesenchymal stem cells in a rat model of retinal degeneration (Inoue et al., Exp. Eye Res. 8(2):234-241, 2007), and the generation of retinal progenitor cells from HI human embryonic stem cell lines, including ganglion cells, amacrine neurons, and photoreceptors expressing S-opsin or rhodopsin, and bipolar cells. Photoreceptor progenitor cells (PRCs) and horizontal cells (Lamba et al., Proc. Natl. Acad. Sci. 10(34):12769-12774, 2006) and induced pluripotent stem cells (iPSCs) from human fibroblasts to generate retinal progenitor cells (Lamba et al., PLoS ONE 5(l):e8763.doi:10.1371 / journal.pone.0008763). None of these methods generated homologous populations of photoreceptor progenitor cells or photoreceptor cells for transplantation. None of these methods generated populations of photoreceptor progenitor cells or photoreceptor cells that exhibit in vivo rod or cone cell function (e.g., detectable by conferring improved visual acuity). Differentiating stem cells into photoreceptor rescue cell populations could provide an alternative to currently available therapeutic approaches. Summary of the Invention

[0005] The present invention provides a photoreceptor rescue cell (PRC) composition comprising a variety of heterologous photoreceptor rescue cells with unique portfolios, and also provides a method of using them in the treatment of eye conditions.

[0006] Therefore, in one aspect, the present invention provides a photoreceptor rescue cell composition comprising a plurality of heterologous photoreceptor rescue cells, wherein the plurality of heterologous photoreceptor rescue cells cumulatively express at least two of the markers selected from FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

[0007] In one embodiment, the photoreceptor cell rescue composition further comprises a medium (culture medium) suitable for maintaining cell viability.

[0008] In one embodiment, the cells are generated through in vitro differentiation of pluripotent cells.

[0009] In one embodiment, the pluripotent cell is an embryonic cell (ESC) or an induced pluripotent stem cell (iPSC).

[0010] In one embodiment, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition are photoreceptor-rescued cells.

[0011] In one embodiment, the multiple heterologous cells cumulatively express FOXG1 and MAP2.

[0012] In one embodiment, (i) about 50% to about 100%, about 50% to about 90%, about 55% to about 85%, or about 55% to about 73% of the cells in the composition express FOXG1; and / or (ii) at least about 50%, 55%, 57%, 60%, 65%, 70%, 71%, 75%, 78%, 79%, 80%, 81%, 85%, 87%, 90%, 92%, 95%, 97%, or 100% of the cells in the composition express FOXG1; and / or (iii) the cells in the composition express about 55 transcripts per million (T). (iv) FOXG1 transcripts of about 55 TPM to about 200 TPM, about 60 TPM to about 170 TPM, about 140 TPM to about 165 TPM, or about 149 TPM to about 170 TPM; and / or (iv) cells of the composition expressing FOXG1 transcripts of at least 55 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, or 200 TPM.

[0013] In one embodiment, (i) about 75% to about 100%, about 75% to about 98%, about 75% to about 95%, or about 77% to about 93% of the cells in the composition express MAP2; and / or (ii) at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition express MAP2; and / or (iii) the cells in the composition express about 250 transcripts per million (TPM) to about 700 TPM, about 290 TPM to about 650 TPM, about 450 TPM to about 625 TPM or about 490 TPM to about 615 TPM; and / or (iv) the cells of the composition express at least 250 TPM, 300 TPM, 350 TPM, 400 TPM, 450 TPM, 475 TPM, 490 TPM, 510 TPM, 525 TPM, 550 TPM, 575 TPM, 600 TPM, 610 TPM, 625 TPM, 650 TPM, 675 TPM or 700 TPM of MAP2 transcripts.

[0014] In one embodiment, the plurality of heterologous cells cumulatively express at least one other biomarker, the other biomarker being selected from STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In one embodiment, the plurality of heterologous cells cumulatively express at least 3, 4, 5, 6, or 7 of the biomarkers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

[0015] In one embodiment, (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, or about 75% to about 80% of the cells in the composition express STMN2; and / or (ii) at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or more of the cells in the composition express STMN2; and / or (ii) at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or more of the cells in the composition express STMN2. 100% of the cells express STMN2; and / or (iii) the cells of the composition express about 150 transcripts / million (TPM) to about 600 TPM, about 190 TPM to about 560 TPM, about 400 TPM to about 600 TPM, or about 450 TPM to about 560 TPM of STMN2 transcripts; and / or (iv) the cells of the composition express at least 150 TPM, 185 TPM, 200 TPM, 250 TPM, 300 TPM, 350 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 525 TPM, 550 TPM, 575 TPM, or 600 TPM of STMN2 transcripts.

[0016] In one embodiment, (i) about 65% to about 95%, about 65% to about 85%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 75% to about 95%, about 75% to about 90%, or about 75% to about 89% of the cells in the composition express DCX; and / or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of the cells in the composition express DCX; and / or (iii) the cells in the composition express about 200 transaminases. The composition contains DCX transcripts at a rate of approximately 200 TPM, 250 TPM, 600 TPM, 750 TPM, or 850 TPM per million (TPM); and / or (iv) the cells of the composition express at least 200 TPM, 250 TPM, 350 TPM, 400 TPM, 450 TPM, 500 TPM, 550 TPM, 600 TPM, 650 TPM, 700 TPM, 750 TPM, 800 TPM, 850 TPM, or 900 TPM.

[0017] In one embodiment, (i) about 65% to about 98%, about 65% to about 95%, about 70% to about 98%, about 70% to about 95%, about 70% to about 90%, about 75% to about 98%, about 75% to about 90%, or about 80% to about 95% of the cells in the composition express LINC00461; and / or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 98% of the cells in the composition express LINC00461; and / or (iii) the... The cells of the composition express about 50 transcripts per million (TPM) to about 100 TPM, about 50 TPM to about 95 TPM, about 85 TPM to about 95 TPM, or about 87 TPM to about 93 TPM; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 87 TPM, 89 TPM, 90 TPM, 92 TPM, 95 TPM, or 100 TPM of the LINC00461 transcript.

[0018] In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 1% to about 8%, about 1% to about 7%, about 1% to about 5%, or about 2% to about 4% of the cells in the composition express NEUROD2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, or 25% of the cells in the composition express NEUROD2. D2; and / or (iii) the cells of the composition express about 0 transcripts per million (TPM) to about 10 TPM, about 0.01 TPM to about 9 TPM, about 0.2 TPM to about 2 TPM, or about 0.4 TPM to about 1.2 TPM of NEUROD2 transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.4 TPM, 0.6 TPM, 0.8 TPM, 1.0 TPM, 1.2 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM, or 10 TPM of NEUROD2 transcripts.

[0019] In one embodiment, (i) about 35% to about 70%, about 35% to about 68%, about 35% to about 67%, about 35% to about 66%, about 35% to about 65%, about 40% to about 70%, about 40% to about 68%, about 40% to about 67%, about 40% to about 66%, about 40% to about 65%, about 42% to about 70%, about 42% to about 68%, about 42% to about 67%, about 42% to about 66%, or about 42% to about 65% of the cells in the composition express GAD1; and / or (ii) at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, or 68% of the cells in the composition. (iii) 69% or 70% of the cells express GAD1; and / or (iv) the cells of the composition express about 10 transcripts per million (TPM) to about 50 TPM, about 12 TPM to about 45 TPM, about 12 TPM to about 40 TPM, or about 25 TPM to about 42 TPM of GAD1 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 12 TPM, 16 TPM, 18 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 47 TPM, or 50 TPM of GAD1 transcripts.

[0020] In one embodiment, (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 89%, about 60% to about 88%, about 60% to about 87%, about 60% to about 86%, about 65% to about 95%, about 65% to about 90%, about 65% to about 89%, about 65% to about 88%, about 65% to about 87%, about 65% to about 86%, about 69% to about 90%, about 69% to about 89%, about 69% to about 88%, about 69% to about 87%, or about 69% to about 86% of the cells in the composition express NFIA; and / or (ii) at least about 50%, 55%, 60%, 65%, 67%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, or 95% of the cells express NFIA; and / or (iii) the cells of the composition express about 30 transcripts per million (TPM) to about 120 TPM, about 33 TPM to about 117 TPM, about 60 TPM to about 85 TPM, or about 65 TPM to about 80 TPM of NFIA transcripts; and / or (iv) the cells of the composition express at least 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, or 120 TPM of NFIA transcripts.

[0021] In one embodiment, the heterologous cells cumulatively express each of the biomarkers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In another embodiment, the heterologous cells individually express at least one of the biomarkers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, or NFIA.

[0022] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprises one or more cell types selected from inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and alternative neurons. In one embodiment, the plurality of heterologous photoreceptor rescue cells comprises each of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and alternative neurons.

[0023] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise inhibitory neurons expressing one or more markers selected from DLX5, TUBB3, SCGN, ERBB4, and CALB2. In another embodiment, the plurality of heterologous photoreceptor rescue cells comprise a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0024] In one embodiment, (i) about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 52% to about 69%, about 52% to about 75%, about 54% to about 69%, about 54% to about 68%, or about 54% to about 66% of the cells in the composition express DLX5; and / or (ii) at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 72%, 74%, 76%, 78%, or 80% of the cells in the composition express DLX5. ; and / or (iii) the cells of the composition express about 30 transcripts / million (TPM) to about 150 TPM, about 50 TPM to about 140 TPM, about 80 TPM to about 138 TPM, or about 130 TPM to about 140 TPM; and / or (iv) the cells of the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 135 TPM, 140 TPM, or 150 TPM of DLX5 transcripts.

[0025] In one embodiment, (i) about 60% to about 95%, about 70% to about 95%, about 72% to about 95%, about 75% to about 95%, about 76% to about 94%, about 77% to about 93%, about 78% to about 93%, about 70% to about 90%, about 72% to about 89%, about 73% to about 88%, about 74% to about 87%, about 75% to about 87%, about 76% to about 86%, about 77% to about 86%, or about 79% to about 86% of the cells in the composition express TUBB3; and / or (ii) at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93% of the cells in the composition express TUBB3; and / or (ii) at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93% of the cells in the composition express TUBB3; % or 95% of the cells express TUBB3; and / or (iii) the cells of the composition express about 150 transcripts / million (TPM) to about 500 TPM, about 160 TPM to about 450 TPM, about 300 TPM to about 450 TPM, about 350 TPM to about 430 TPM, or about 375 TPM to about 430 TPM of TUBB3 transcripts; and / or (iv) the cells of the composition express at least 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 430 TPM, 450 TPM, 475 TPM, or 500 TPM of TUBB3 transcripts.

[0026] In one embodiment, (i) about 45% to about 70%, about 45% to about 65%, about 50% to about 70%, about 50% to about 65%, about 50% to about 64%, about 50% to about 63%, about 50% to about 62%, about 50% to about 61%, about 46% to about 52%, about 47% to about 51%, about 48% to about 51%, or about 48% to about 50% of the cells in the composition express SCGN; and / or (ii) at least about 45%, 47%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 65%, 67%, or 70% of the cells in the composition express SCGN; and / or (i) ii) The cells of the composition express about 50 transcripts / million (TPM) to about 200 TPM, about 70 TPM to about 180 TPM, about 75 TPM to about 175 TPM, or about 120 TPM to about 175 TPM; and / or (iv) The cells of the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 171 TPM, 173 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, or 200 TPM of SCGN transcripts.

[0027] In one embodiment, (i) about 60% to about 85%, about 60% to about 80%, about 60% to about 79%, about 60% to about 78%, about 60% to about 77%, about 60% to about 76%, about 63% to about 75%, about 63% to about 70%, about 63% to about 79%, about 63% to about 78%, about 63% to about 77%, about 63% to about 75%, about 63% to about 73%, about 63% to about 72%, about 63% to about 71%, about 65% to about 72%, or about 66% to about 71% of the cells in the composition express ERBB4; and / or (ii) at least about 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71% of the cells in the composition express ERBB4. % , 72% , 75% , 80% or 85% of the cells express ERBB4; and / or (iii) the cells of the composition express about 15 transcripts / million (TPM) to about 120 TPM, about 17 TPM to about 100 TPM, about 18 TPM to about 95 TPM, about 70 TPM to about 95 TPM or about 73 TPM to about 94 TPM of ERBB4 transcripts; and / or (iv) the cells of the composition express at least 15 TPM, 30 TPM, 50 TPM, 70 TPM, 73 TPM, 75 TPM, 80 TPM, 82 TPM, 85 TPM, 88 TPM, 90 TPM, 91 TPM, 93 TPM, 95 TPM, 100 TPM, 110 TPM or 120 TPM of ERBB4 transcripts.

[0028] In one embodiment, (i) about 35% to about 75%, about 40% to about 70%, about 40% to about 65%, about 41% to about 75%, about 41% to about 70%, about 41% to about 65%, about 41% to about 64%, about 41% to about 63%, about 41% to about 62%, about 35% to about 55%, about 40% to about 52%, or about 41% to about 52% of the cells in the composition express CALB2; and / or (ii) at least about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 45%, 47%, 50%, 51%, 52%, 55%, 60%, 65%, 70%, or 75% of the cells in the composition express CALB2; and / or (i) ii) The cells of the composition express about 30 transcripts per million (TPM) to about 220 TPM, about 50 TPM to about 200 TPM, about 70 TPM to about 200 TPM, or about 75 TPM to about 199 TPM; and / or (iv) The cells of the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 195 TPM, 196 TPM, 220 TPM, 210 TPM, or 220 TPM of CALB2 transcripts.

[0029] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise excitatory neurons expressing one or more markers selected from NEUROD2, NEUROD6, SLA, NELL2, and SATB2. In another embodiment, the plurality of heterologous photoreceptor rescue cells comprise excitatory neurons that cumulatively express each of the plurality of markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

[0030] In one embodiment, (i) about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 24%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 24%, about 0.8% to about 30%, about 0.8% to about 25%, about 0.8% to about 24%, about 1% to about 5%, about 1% to about 4.5%, about 2% to about 5%, about 2% to about 4.5%, or about 2% to about 4% of the cells in the composition express NEUROD6; and / or (ii) at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 2%, 4%, 5%, 7%, 10%, 12% of the cells in the composition. 15%, 17%, 20%, 22%, 23%, 24%, 26%, 28%, or 30% of the cells express NEUROD6; and / or (iii) the cells of the composition express about 1 transcript per million (TPM) to about 210 TPM, about 1 TPM to about 205 TPM, about 1 TPM to about 25 TPM, or about 10 TPM to about 20 TPM of NEUROD6 transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 5 TPM, 10 TPM, 12 TPM, 15 TPM, 19 TPM, 50 TPM, 100 TPM, 150 TPM, 175 TPM, 200 TPM, 203 TPM, or 210 TPM of NEUROD6 transcripts.

[0031] In one embodiment, (i) about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express SLA; and / or (ii) at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 18%, or 20% of the [unspecified ingredient] in the composition. Cells express SLA; and / or (iii) the cells of the composition express about 0.1 transcripts per million (TPM) to about 60 TPM, about 0.1 TPM to about 50 TPM, about 1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 3 TPM to about 6 TPM of SLA transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 10 TPM, 20 TPM, 30 TPM, 40 TPM, 50 TPM, or 60 TPM of SLA transcripts.

[0032] In one embodiment, (i) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 30%, or about 20% to about 28% of the cells in the composition express NELL2; and / or (ii) at least about 10%, 12%, 15%, 17%, 20%, 21%, 24%, 25%, 27%, 30%, 32%, 35%, 40%, or 45% of the cells in the composition express NELL2; and / or (iii) the cells in the composition... The NELL2 transcripts are expressed at a rate of about 1 transcript per million (TPM) to about 150 TPM, about 4 TPM to about 130 TPM, about 4 TPM to about 35 TPM, about 20 TPM to about 30 TPM, or about 25 TPM to about 28 TPM; and / or (iv) the cells of the composition express at least 1 TPM, 5 TPM, 15 TPM, 20 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 120 TPM, or 150 TPM.

[0033] In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 12%, about 1% to about 11%, about 2% to about 20%, about 2% to about 15%, about 2% to about 12%, about 2% to about 11%, about 3% to about 20%, about 3% to about 15%, about 3% to about 12%, about 3% to about 11%, about 2% to about 6%, about 2% to about 5%, or about 3% to about 4% of the cells in the composition express SATB2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17% of the cells in the composition express SATB2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17% of the cells in the composition express SATB2; % or 20% of the cells express SATB2; and / or (iii) the cells of the composition express about 0.1 transcripts / million (TPM) to about 30 TPM, about 0.5 TPM to about 20 TPM, about 1 TPM to about 5 TPM, or about 2 TPM to about 3 TPM of SATB2 transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 25 TPM, or 30 TPM of SATB2 transcripts.

[0034] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise progenitor cells expressing one or more markers selected from VIM, MKI67, CLU, and GLI3. In another embodiment, the plurality of heterologous photoreceptor rescue cells comprise progenitor cells that cumulatively express each of the plurality of markers VIM, MKI67, CLU, and GLI3.

[0035] In one embodiment, (i) about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 40% to about 75%, about 40% to about 70%, about 40% to about 69%, about 40% to about 60%, or about 42% to about 47% of the cells in the composition express VIM; and / or (ii) at least about 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 69%, 72%, 75%, or 80% of the cells in the composition express VI. M; and / or (iii) the cells of the composition express about 250 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 865 TPM, about 200 TPM to about 350 TPM, or about 250 TPM to about 340 TPM; and / or (iv) the cells of the composition express at least 250 TPM, 260 TPM, 270 TPM, 300 TPM, 320 TPM, 350 TPM, 370 TPM, 400 TPM, 500 TPM, 600 TPM, 700 TPM, 800 TPM, or 900 TPM of VIM transcripts.

[0036] In one embodiment, (i) about 5% to about 20%, about 5% to about 15%, about 5% to about 12%, about 6% to about 15%, about 6% to about 12%, about 7% to about 15%, about 7% to about 12%, or about 6% to about 8% of the cells in the composition express MKI67; and / or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the cells in the composition express MKI67; and / or (iii) the cells in the composition express... The MKI67 transcripts are expressed at approximately 5 TPM to approximately 40 TPM, approximately 10 TPM to approximately 35 TPM, approximately 15 TPM to approximately 25 TPM, or approximately 18 TPM to approximately 22 TPM; and / or (iv) the cells of the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 33 TPM, 35 TPM, 37 TPM, or 40 TPM.

[0037] In one embodiment, (i) about 10% to about 60%, about 15% to about 55%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 40%, about 20% to about 35%, or about 25% to about 32% of the cells in the composition express CLU; and / or (ii) at least about 10%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 55%, or 60% of the cells in the composition express CLU; and / or (iii) the cells in the composition express about 30 transcripts per million (TPM) to about 400 TPM, about 40 TPM to about 150 TPM, about 60 TPM to about 150 TPM, or about 60 TPM to about 105 TPM of CLU transcripts; and / or (iv) the cells of the composition express at least 30 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 365 TPM, 375 TPM, or 400 TPM of CLU transcripts.

[0038] In one embodiment, (i) about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 12% to about 50%, about 12% to about 35%, about 12% to about 29%, about 15% to about 29%, about 15% to about 29%, or about 15% to about 17% of the cells in the composition express GLI3; and / or (ii) at least about 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GLI3. LI3; and / or (iii) the cells of the composition express about 5 transcripts / million (TPM) to about 60 TPM, about 10 TPM to about 45 TPM, about 15 TPM to about 30 TPM, or about 20 TPM to about 25 TPM of GLI3 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, or 60 TPM of GLI3 transcripts.

[0039] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise astrocytes expressing one or more markers selected from GFAP, LUCAT1, MIR99AHG, and FBXL7. In another embodiment, the plurality of heterologous photoreceptor rescue cells comprise astrocytes cumulatively expressing each of the plurality of markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0040] In one embodiment, (i) about 1% to about 50%, about 1% to about 20%, about 1% to about 15%, about 1% to about 13%, about 1% to about 10%, about 1% to about 7%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express GFAP; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GFAP; and / or (iii) the cells in the composition express about 0.1 The composition is expressed in cells containing approximately 0.1 TPM to 150 TPM, approximately 0.1 TPM to 125 TPM, approximately 1 TPM to 20 TPM, or approximately 3 TPM to 15 TPM of GFAP transcripts; and / or (iv) the composition expresses at least 0.1 TPM, 0.2 TPM, 0.5 TPM, 1 TPM, 5 TPM, 7 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 30 TPM, 40 TPM, 50 TPM, 80 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 140 TPM, or 150 TPM of GFAP transcripts.

[0041] In one embodiment, (i) about 5% to about 20%, about 5% to about 17%, about 5% to about 15%, about 5% to about 13%, about 7% to about 20%, about 7% to about 17%, about 7% to about 15%, about 7% to about 13%, about 5% to about 12%, or about 7% to about 10% of the cells in the composition express LUCAT1; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17%, or 20% of the cells in the composition express LUCAT1.

[0042] In one embodiment, (i) about 50% to about 100%, about 50% to about 90%, about 50% to about 88%, about 60% to about 100%, about 60% to about 90%, about 60% to about 88%, about 70% to about 90%, about 70% to about 88%, or about 75% to about 82% of the cells in the composition express MIR99AHG; and / or (ii) at least about 50%, 60%, 65%, 70%, 72%, 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 96%, 98%, or 100% of the cells in the composition express MIR99AHG; and / or (iii) the composition The composition expresses about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 30 TPM, about 6 TPM to about 25 TPM, or about 10 TPM to about 15 TPM in cells; and / or (iv) the composition expresses at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM in cells.

[0043] In one embodiment, (i) about 20% to about 70%, about 20% to about 60%, about 25% to about 70%, about 25% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 40%, about 32% to about 39%, or about 34% to about 39% of the cells in the composition express FBXL7; and / or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 60%, 65%, or 70% of the cells in the composition express FBXL7; and / or (iii) the cells in the composition express about 5 transgenes. The FBXL7 transcripts are expressed at least 5 TPM to 40 TPM, about 5 TPM to 30 TPM, about 7 TPM to 25 TPM, about 10 TPM to 15 TPM, or about 11 TPM to 14 TPM; and / or (iv) the cells of the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM, or 40 TPM.

[0044] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise substitute neurons expressing one or more markers selected from MEIS2, PBX3, GRIA2, and CACNA1C. In another embodiment, the plurality of heterologous photoreceptor rescue cells comprise substitute neurons that cumulatively express each of the plurality of markers MEIS2, PBX3, GRIA2, and CACNA1C.

[0045] In one embodiment, (i) about 30% to about 80%, about 30% to about 90%, about 40% to about 90%, about 45% to about 85%, about 45% to about 80%, about 49% to about 79%, or about 50% to about 78% of the cells in the composition express MEIS2; and / or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 81%, or 82% of the cells in the composition express MEIS2; and / or (iii) the cells in the composition express about 5 transcripts per million (TPM) to about MEIS2 transcripts of 200 TPM, about 10 TPM to about 180 TPM, about 50 TPM to about 180 TPM, or about 60 TPM to about 173 TPM; and / or (iv) cells of the composition expressing at least 5 TPM, 10 TPM, 15 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 135 TPM, 136 TPM, 138 TPM, 140 TPM, 145 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, or 200 TPM.

[0046] In one embodiment, (i) about 30% to about 90%, about 35% to about 85%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, or about 49% to about 75% of the cells in the composition express PBX3; and / or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 80%, 85%, or 90% of the cells in the composition express PBX3; and / or (iii) the cell surface of the composition... The composition comprises PBX3 transcripts ranging from about 5 TPM to about 100 TPM, about 5 TPM to about 90 TPM, about 25 TPM to about 90 TPM, or about 29 TPM to about 88 TPM; and / or (iv) cells expressing at least 5 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, or 100 TPM.

[0047] In one embodiment, (i) about 20% to about 60%, about 20% to about 50%, about 20% to about 50%, about 20% to about 48%, about 22% to about 55%, about 22% to about 50%, about 22% to about 47%, or about 23% to about 47% of the cells in the composition express GRIA2; and / or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 46%, 47%, 50%, 52%, 54%, 56%, 58%, or 60% of the cells in the composition express GRIA2; and / or (iii) the cells in the composition express about 2 Transcripts per million (TPM) to about 40 TPM, about 2 TPM to about 30 TPM, about 4 TPM to about 35 TPM, or about 10 TPM to about 30 TPM; and / or (iv) cells of the composition expressing at least 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 17 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 28 TPM, 29 TPM, 30 TPM, 35 TPM, or 40 TPM.

[0048] In one embodiment, (i) about 20% to about 70%, about 30% to about 60%, about 35% to about 70%, about 35% to about 60%, about 33% to about 60%, about 35% to about 60%, or about 39% to about 60% of the cells in the composition express CACNA1C; and / or (ii) at least about 20%, 25%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 56%, 57%, 58%, 59% of the cells in the composition express CACNA1C. 60%, 65%, or 70% of the cells express CACNA1C; and / or (iii) the cells of the composition express about 1 transcript per million (TPM) to about 15 TPM, about 1 TPM to about 10 TPM, about 1 TPM to about 7 TPM, or about 3 TPM to about 7 TPM of CACNA1C transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 10 TPM, 12 TPM, or 15 TPM of CACNA1C transcripts.

[0049] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise: (i) inhibitory neurons expressing one or more markers selected from DLX5, TUBB3, SCGN, ERBB4, and CALB2; (ii) excitatory neurons expressing one or more markers selected from NEUROD2, NEUROD6, SLA, NELL2, and SATB2; (iii) progenitor cells expressing one or more markers selected from VIM, MKI67, CLU, and GLI3; (iv) astrocytes expressing one or more markers selected from GFAP, LUCAT1, MIR99AHG, and FBXL7; and (v) surrogate neurons expressing one or more markers selected from MEIS2, PBX3, GRIA2, and CACNA1C.

[0050] In one embodiment, the plurality of heterologous photoreceptor rescue cells comprise: (i) inhibitory neurons of each of the plurality of cumulative expression markers DLX5, TUBB3, SCGN, ERBB4, and CALB2; (ii) excitatory neurons of each of the plurality of cumulative expression markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2; (iii) progenitor cells of each of the plurality of cumulative expression markers VIM, MKI67, CLU, and GLI3; (iv) astrocytes of each of the plurality of cumulative expression markers GFAP, LUCAT1, MIR99AHG, and FBXL7; and (v) replacement neurons of each of the plurality of cumulative expression markers MEIS2, PBX3, GRIA2, and CACNA1C.

[0051] In one embodiment, the composition comprises: (i) about 25% to about 55%, about 25% to about 50%, about 30% to about 55%, about 30% to about 50%, about 35% to about 55%, about 35% to about 50%, or about 38% to about 49% inhibitory neurons; and / or (ii) about 0% to about 15%, about 0% to about 12%, about 0% to about 10%, about 0% to about 8%, or about 0.5% to about 9% excitatory neurons; and / or (iii) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 17% to about 45%, about 17% to about 40%, about 17% to about 35%, or about 20% to 35% progenitor cells; and / or (iv) about 0% to 6%, about 0% to 5%, about 0% to 4%, about 0% to 3%, about 0% to 2%, about 0.5% to 6%, about 0.5% to 5%, about 0.5% to 4%, about 0.5% to 4%, about 0.5% to 3%, about 0.5% to 2%, or about 0.5% to 1.5% astrocytes; and / or (v) about 10% to 50%, about 10% to 45%, about 10% to 40%, about 12% to 50%, about 12% to 45%, about 12% to 40%, about 15% to 50%, about 15% to 45%, about 15% to 40%, or about 17% to 37% mixed neurons.

[0052] In one embodiment, the cells in the composition further express one or more eyefield progenitor markers, rod / cone photoreceptor markers, and / or neuronal markers. In one embodiment, the eyefield progenitor markers are selected from PAX6, LHX2, SIX3, NES, and SOX2. In one embodiment, the plurality of heterologous cells cumulatively express at least one, two, three, or four of the eyefield progenitor markers PAX6, LHX2, SIX3, NES, or SOX2. In one embodiment, the plurality of heterologous cells cumulatively express at least each of the eyefield progenitor markers PAX6, LHX2, SIX3, NES, and SOX2. In one embodiment, the plurality of heterologous cells cumulatively express SOX2. In one embodiment, the composition substantially does not contain cells expressing the eyefield progenitor markers RAX, SIX6, and / or TBX3.

[0053] In one embodiment, (i) about 25% to about 60%, about 25% to about 55%, about 25% to about 52%, about 25% to about 45%, about 30% to about 60%, about 30% to about 55%, about 30% to about 45%, or about 30% to about 42% of the cells in the composition express PAX6; and / or (ii) at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the cells in the composition express PAX6; and / or (iii) the cells in the composition express about 20 transcripts per million (TPM) to about 125 TPM, about 30 TPM to about 110 TPM, or about 35 TPM. The PAX6 transcripts are expressed at TPM to about 100 TPM, about 70 TPM to about 80 TPM, or about 73 TPM to about 78 TPM; and / or (iv) the cells of the composition express at least 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 78 TPM, 80 TPM, 82 TPM, 85 TPM, 87 TPM, 90 TPM, 92 TPM, 95 TPM, 97 TPM, 100 TPM, 105 TPM, 110 TPM, 115 TPM, 120 TPM, or 125 TPM.

[0054] In one embodiment, (i) about 3% to about 35%, about 5% to about 35%, about 5% to about 35%, about 6% to about 35%, about 7% to about 35%, about 3% to about 30%, about 5% to about 30%, about 6% to about 30%, about 7% to about 30%, about 3% to about 25%, about 5% to about 25%, about 6% to about 25%, or about 7% to about 9% of the cells in the composition express LHX2; and / or (ii) at least about 3%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 32%, or 35% of the cells in the composition express LHX2; and / or (i) ii) The cells of the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 36 TPM, about 5 TPM to about 10 TPM, or about 6 TPM to about 8 TPM of LHX2 transcripts; and / or (iv) The cells of the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 36 TPM, 38 TPM, or 40 TPM of LHX2 transcripts.

[0055] In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or 2% to about 10% of the cells in the composition express SIX3; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, or 25% of the cells in the composition express SIX3; and / or (iii) the cells of the composition express about 1 transcript per million (TPM) to about 50 TPM, about 2 TPM to about 30 TPM, about 1 TPM to about 25 TPM, or about 5 TPM to about 20 TPM of the SIX3 transcript; and / or (iv) the cells of the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, or 50 TPM of the SIX3 transcript.

[0056] In one embodiment, (i) about 15% to about 40%, about 15% to about 35%, about 15% to about 34%, about 15% to about 33%, about 15% to about 32%, about 15% to about 31%, about 18% to about 40%, about 18% to about 35%, about 18% to about 34%, about 18% to about 33%, about 18% to about 32%, or about 18% to about 31% of the cells in the composition express NES; and / or (ii) at least about 15%, 17%, 20%, 25%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, or 40% of the cells in the composition express NES; and / or (iii) the composition The cell culture of the composition expresses NES transcripts at about 5 transcripts per million (TPM) to about 35 TPM, about 5 TPM to about 28 TPM, about 7 TPM to about 15 TPM, about 10 TPM to about 15 TPM, or about 11 TPM to about 13 TPM; and / or (iv) the cell culture of the composition expresses NES transcripts at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 22 TPM, 24 TPM, 25 TPM, 26 TPM, 27 TPM, 30 TPM, or 35 TPM.

[0057] In one embodiment, (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 75% of the cells in the composition express SOX2; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or 90% of the cells in the composition express SOX2; and / or (iii) the cells in the composition express about 50% SOX2. The composition is expressed in cells of the composition at a rate of approximately 50 TPM, 60 TPM, 70 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, 200 TPM, 210 TPM, 220 TPM, 230 TPM, 240 TPM, or 250 TPM.

[0058] In one embodiment, the rod / cone photoreceptor markers are selected from ASCL1, RORB, NR2E3, and NRL. In one embodiment, the various heterologous cells cumulatively express each of the rod / cone photoreceptor markers ASCL1, RORB, NR2E3, and NRL. In one embodiment, the composition is substantially free of cells expressing the rod / cone photoreceptor markers CRX, RHO, OPN1SW, PDE6B, RCRRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0059] In one embodiment, (i) about 10% to about 60%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 22% to about 45%, about 22% to about 43%, about 25% to about 420%, or about 28% to about 30% of the cells in the composition express ASCL1; and / or (ii) at least about 10%, 15%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 41%, 42%, 45%, 50%, 55%, or 60% of the cells in the composition express ASCL1; and / or (iii) the composition The cells of the composition express about 50 transcripts per million (TPM) to about 150 TPM, about 60 TPM to about 140 TPM, about 65 TPM to about 130 TPM, or about 95 TPM to about 130 TPM; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 125 TPM, 130 TPM, 140 TPM, or 150 TPM of ASCL1 transcripts.

[0060] In one embodiment, (i) about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 10% to about 25%, or about 11% to about 22% of the cells in the composition express RORB; and / or (ii) at least about 5%, 7%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express RORB; And / or (iii) the cells of the composition express about 0.1 transcripts / million (TPM) to about 20 TPM, about 0.1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 1 TPM to about 6 TPM; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 18 TPM, or 20 TPM of RORRB transcripts.

[0061] In one embodiment, (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or about 2% to about 5% of the cells in the composition express NR2E3; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express N R2E3; and / or (iii) the cells of the composition express about 0.1 transcripts / million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 3 TPM or about 0.1 TPM to about 1 TPM of NR2E3 transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 2 TPM, 5 TPM, 7 TPM, 9 TPM or 10 TPM of NR2E3 transcripts.

[0062] In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, or about 2% to about 8% of the cells in the composition express NRL; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express NRL; and / or (iii) the cells in the composition express NRL. Cells expressing NRL transcripts at a rate of about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 7 TPM, or about 0.1 TPM to about 2 TPM; and / or (iv) cells of the composition expressing at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM, or 10 TPM.

[0063] In one embodiment, the neuronal markers are selected from TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. In one embodiment, the various heterologous cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0064] In one embodiment, (i) about 50% to about 100%, about 60% to about 100%, about 60% to about 95%, about 70% to about 100%, about 70% to about 95%, about 80% to about 100%, about 80% to about 95%, or about 80% to about 90% of the cells in the composition express NFIB; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 100% of the cells in the composition express NFIB; and / or (iii) the composition Cells expressing approximately 150 transcripts per million (TPM) to approximately 650 TPM, approximately 180 TPM to approximately 610 TPM, approximately 400 TPM to approximately 500 TPM, or approximately 400 TPM to approximately 480 TPM; and / or (iv) cells of the composition expressing at least 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 550 TPM, 600 TPM, 625 TPM, or 650 TPM.

[0065] In one embodiment, (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, or about 2% to about 6% of the cells in the composition express OTX2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20% of the cells in the composition express OTX2; and / or (iii) the cells in the composition express about 5 transcripts / million ( (iv) OTX2 transcripts of about 50 TPM, about 8 TPM to about 40 TPM, about 8 TPM to about 25 TPM, or about 12 TPM to about 15 TPM; and / or (iv) cells of the composition expressing at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 26 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, or 50 TPM.

[0066] In one embodiment, (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 78%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 78%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 78% of the cells in the composition express ELAVL3; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or 90% of the cells in the composition express ELAVL3; and / or (iii) the cells in the composition express about 10 transcripts per million (TPM). (iv) ELAVL3 transcripts of about 120 TPM, about 15 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 60 TPM to about 90 TPM, or about 70 TPM to about 80 TPM; and / or (iv) cells of the composition expressing at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, or 120 TPM.

[0067] In one embodiment, (i) about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 78%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 78%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, or about 50% to about 65% of the cells in the composition express ELAVL4; and / or ( ii) at least about 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 75%, 80%, 85%, or 90% of the cells in the composition express ELAVL4; and / or (iii) the cells in the composition express about 1 ELAVL4 transcripts at 0 transcripts / million (TPM) to about 120 TPM, about 15 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 50 TPM to about 90 TPM, about 70 TPM to about 90 TPM, or about 79 TPM to about 85 TPM; and / or (iv) cells expressing the composition at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, or 35 TPM. ELAVL4 transcripts at TPM, 40TPM, 45TPM, 50TPM, 55TPM, 60TPM, 65TPM, 70TPM, 71TPM, 72TPM, 73TPM, 74TPM, 75TPM, 76TPM, 77TPM, 78TPM, 80TPM, 82TPM, 84TPM, 86TPM, 90TPM, 100TPM, 110TPM, or 120TPM.

[0068] In one embodiment, (i) about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 73%, about 51% to about 73%, or about 52% to about 66% of the cells in the composition express SLC1A2; and / or (ii) at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 75%, 80%, 85%, or 90% of the cells in the composition express SLC1A2; and / or (ii) at least about 40%, 45%, 50%, 51%, 52%, 53%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 75%, 80%, 85%, or 90% of the cells in the composition express SLC1A2. % of cells express SLC1A2; and / or (iii) cells of the composition express about 10 transcripts / million (TPM) to about 120 TPM, about 10 TPM to about 90 TPM, about 20 TPM to about 90 TPM, about 40 TPM to about 70 TPM, about 50 TPM to about 70 TPM, or about 58 TPM to about 63 TPM of SLC1A2 transcripts; and / or (iv) cells of the composition express at least 10 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, or 120 TPM of SLC1A2 transcripts.

[0069] In one embodiment, (i) about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 11% to about 19%, or about 10% to about 12% of the cells in the composition express SLC1A3; and / or (ii) at least about 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 25%, 30%, 35%, or 40% of the cells in the composition express SLC1A3; and / or (iii) the cells in the composition express about 50 transcripts per million (TPM) to about 200 T. SLC1A3 transcripts of about 70 TPM to about 190 TPM, about 60 TPM to about 100 TPM, about 60 TPM to about 80 TPM, or about 72 TPM to about 79 TPM; and / or (iv) cells of the composition expressing at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 79 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 175 TPM, 180 TPM, or 200 TPM.

[0070] In one embodiment, (i) about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 3% to about 4% of the cells in the composition express HCN1; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the cells in the composition express HCN1; and / or (iii) the cells in the composition express about 0.1 transcripts per million (TPM). HCN1 transcripts at approximately 10 TPM, approximately 0.1 TPM to approximately 5 TPM, approximately 0.1 TPM to approximately 1 TPM, or approximately 0.2 TPM to approximately 0.6 TPM; and / or (iv) cells of the composition expressing at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1.0 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM, or 10 TPM.

[0071] In one embodiment, (i) about 5% to about 30%, about 5% to about 25%, about 1% to about 30%, about 1% to about 25%, about 5% to about 25%, about 10% to about 25%, or about 10% to about 15% of the cells in the composition express HES5; and / or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or 30% of the cells in the composition express HES5; and / or (iii) the composition The composition expresses HES5 transcripts at approximately 10 transcripts per million (TPM) to approximately 60 TPM, approximately 5 TPM to approximately 50 TPM, approximately 12 TPM to approximately 46 TPM, or approximately 15 TPM to approximately 39 TPM in cells; and / or (iv) the composition expresses HES5 transcripts at least 10 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, or 60 TPM in cells.

[0072] In one embodiment, the plurality of heterologous cells cumulatively express: (i) one or more of the ocular progenitor cell markers PAX6, LHX2, SIX3, NES, and SOX2; (ii) one or more of the rod / cone photoreceptor cell markers ASCL1, RORB, NR2E3, and NRL; and (iii) one or more of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0073] In one embodiment, the plurality of heterologous cells cumulatively express: (i) each of the ocular progenitor cell markers PAX6, LHX2, SIX3, NES, and SOX2; (ii) each of the rod / cone photoreceptor cell markers ASCL1, RORB, NR2E3, and NRL; and (iii) each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0074] In one embodiment, the composition is substantially free of at least one cell type selected from pluripotent stem cells, retinal ganglion cells, photoreceptor cells, and amacrine cells. In another embodiment, the composition is substantially free of pluripotent stem cells, retinal ganglion cells, photoreceptor cells, and amacrine cells.

[0075] In one embodiment, the composition is substantially free of retinal progenitor cells expressing VSX2 and / or POU5F1. In one embodiment, the composition has less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of cells expressing SSEA4, optionally determined by flow cytometry, or the composition is free of cells expressing SSEA4, optionally determined by flow cytometry.

[0076] In one embodiment, the cells in the composition have phagocytic activity, optionally the ability to phagocytose isolated photoreceptor outer segment, dye conjugate, or both.

[0077] In one embodiment, the cells in the composition secrete one or more neuroprotective factors. In one embodiment, the neuroprotective factor is selected from CNTF, MIF, S100B, GFAP, TAU, NCAM1, and TNC.

[0078] In one embodiment, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are alive. In one embodiment, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are alive. In one embodiment, at least 50% of the cells in the composition are alive. In one embodiment, at least 55% of the cells in the composition are alive. In one embodiment, at least 60% of the cells in the composition are live. In one embodiment, at least 65% of the cells in the composition are live. In one embodiment, at least 68% of the cells in the composition are live.

[0079] In one embodiment, the composition is produced according to a method comprising: 1) culturing pluripotent stem cells in rescue induction medium (RIM) and Noggin; 2) culturing cells from step 1 in neural differentiation medium (NDM) and Noggin; and 3) expanding cells from step 2 in the absence of Noggin in NDM, the step comprising a) culturing cells in Noggin-free NDM under low adherence or non-adherence conditions, and b) culturing cells in Noggin-free NDM under adherence conditions.

[0080] In one embodiment, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0081] In one embodiment, step 3 is performed at least once, at least twice, at least three times, at least four times, at least five times, or at least six times. In one embodiment, cells in the composition are harvested after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In one embodiment, cells in the composition are harvested after the fifth repetition of step 3.

[0082] In one embodiment, the cells harvested from the composition are cryopreserved. In one embodiment, the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. In one embodiment, the composition is cryopreserved after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In one embodiment, step 3 is repeated five times, and the composition is cryopreserved after the fifth repetition of step 3.

[0083] In one embodiment, after cryopreservation and thawing, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells are viable. In one embodiment, after cryopreservation and thawing, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells are viable. In one embodiment, after cryopreservation and thawing, at least about 50% of the cells in the composition are viable. In one embodiment, after cryopreservation and thawing, at least about 55% of the cells in the composition are viable. In one embodiment, after cryopreservation and thawing, at least about 60% of the cells in the composition are viable. In one embodiment, after cryopreservation and thawing, at least about 65% of the cells in the composition are viable. In one embodiment, after cryopreservation and thawing, at least 68% of the cells in the composition are viable.

[0084] In one embodiment, the composition comprises cell spheroids. In one embodiment, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are cell spheroids.

[0085] In one embodiment, during steps 1, 2, and / or 3, cells of the composition are cultured in a cell culture container selected from culture dishes, culture flasks, and culture chambers. In one embodiment, the cell culture container has a diameter of approximately 50 cm². 2 Approximately 800cm 2 Approximately 60cm 2 Approximately 800cm 2 Approximately 100cm 2 Approximately 800cm 2 Approximately 150cm 2 Approximately 800cm 2 Approximately 175cm 2 Approximately 800cm 2 Approximately 200cm2 Approximately 800cm 2 Approximately 250cm 2 Approximately 800cm 2 Approximately 300cm 2 Approximately 800cm 2 Approximately 400cm 2 Approximately 800cm 2 Approximately 500cm 2 Approximately 800cm 2 Approximately 600cm 2 Approximately 800cm 2 Approximately 700cm 2 Approximately 800cm 2 Approximately 30cm 2 Approximately 100cm 2 Approximately 50cm 2 Approximately 100cm 2 Approximately 100cm 2 Approximately 300cm 2 Approximately 150cm 2 Approximately 250cm 2 or about 150cm 2 Approximately 200cm 2 Cell growth area. In one embodiment, the cell culture container has a surface area of ​​at least about 60 cm². 2 Approximately 175cm 2 or approximately 636cm 2 Cell growth area.

[0086] In one embodiment, the culture chamber is a stackable rectangular chamber. In one embodiment, step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5, or 1 to 2 culture chambers. In one embodiment, step 3 has at least 1, 2, 5, 10, or 40 culture chambers.

[0087] In one embodiment, the cell culture vessel is coated for use in step 3a for low-adherence or non-adherent cell culture, and / or step 3b for adherent cell culture. In one embodiment, the cells are enzymatically dissociated from the plate into a cell suspension. In one embodiment, the enzymatic dissociation utilizes an enzyme selected from thermolysin, liberase, accutase, and combinations thereof. In one embodiment, the enzyme used for cell dissociation is accutase. In one embodiment, cell dissociation from the plate does not involve manual scraping.

[0088] Therefore, in another aspect, the present invention provides a pharmaceutical preparation comprising a photoreceptor-rescue cell composition according to various embodiments of the above aspects described herein or any other aspect of the invention, and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutically acceptable excipient is suitable for ocular delivery.

[0089] Therefore, in another aspect, the present invention provides formulations comprising: a) a composition according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention, or a pharmaceutical preparation according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention; and b) about 4-10% (v / v) cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose and buffer.

[0090] In one embodiment, the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol. In one embodiment, the cryoprotectant is DMSO. In one embodiment, the formulation comprises about 4-6% (v / v) cryoprotectant. In one embodiment, the formulation comprises about 0.08-0.10% (w / v) glucose. In one embodiment, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer. In one embodiment, the formulation comprises about 0.6% (w / v) glucose. In one embodiment, the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

[0091] In one embodiment, the albumin is human albumin. In another embodiment, the albumin is recombinant human albumin.

[0092] In one embodiment, the buffer is a buffered saline solution. In one embodiment, the buffer is phosphate-buffered saline (PBS). In one embodiment, the buffered saline solution contains Ca. 2+ and Mg 2+ In one embodiment, the buffered brine does not contain Ca. 2+ and Mg 2+ In one embodiment, the preparation is cryopreserved.

[0093] Therefore, in another aspect, the present invention provides a formulation comprising: a) a composition according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention, or a pharmaceutical preparation according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention; and b) a solution comprising (1) a buffer that maintains the solution at a physiological pH; and (2) at least 2 mM or at least 0.05% (w / v) glucose; and (3) an osmotic agent that maintains the solution at a physiological osmolarity.

[0094] In one embodiment, the solution contains at least 5 mM or at least 0.1% (w / v) glucose; or at least 7.5 mM or at least 0.14% (w / v) glucose; or at least 10 mM or at least 0.2% (w / v) glucose; or at least 15 mM or at least 0.25% (w / v) glucose; or at least 20 mM or at least 0.4% (w / v) glucose; or at least 25 mM or at least 0.5% (w / v) glucose.

[0095] In one embodiment, the solution further comprises (4) a divalent cation source, optionally wherein the divalent cation source comprises a calcium and / or magnesium source, and / or (5) an acetate buffer and / or a citrate buffer.

[0096] Therefore, in another aspect, the present invention provides a formulation comprising: a) a composition according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention, or a pharmaceutical preparation according to various embodiments of the foregoing aspects described herein or any other aspect of the present invention; and b) a solution comprising (1) a buffer that maintains the solution at a physiological pH, wherein the buffer is not a bicarbonate buffer; and (2) glucose; and (3) an osmotic agent that maintains the solution at a physiological osmolar concentration; and (4) a divalent cation source, optionally wherein the divalent cation source comprises a calcium source and / or a magnesium source, and / or wherein the buffer comprises an acetate buffer and / or a citrate buffer.

[0097] In one embodiment, the calcium source comprises a pharmaceutically acceptable calcium salt, and / or the magnesium source comprises a pharmaceutically acceptable magnesium salt. In one embodiment, the pharmaceutically acceptable calcium salt and / or pharmaceutically acceptable magnesium salt is selected from calcium and / or magnesium salts formed with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid, and sulfuric acid. In one embodiment, the calcium source comprises calcium chloride, optionally wherein the calcium source comprises calcium chloride dihydrate. In one embodiment, the magnesium source comprises magnesium chloride, optionally wherein the magnesium source comprises magnesium chloride hexahydrate. In one embodiment, the citrate buffer is provided as sodium citrate. In one embodiment, the glucose is D-glucose (dextrose).

[0098] In one embodiment, the permeabilizer is a salt, optionally a sodium salt, and more preferably sodium chloride. In one embodiment, the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose.

[0099] In one embodiment, the pH of the solution is 6.8-7.8, or 7.4-7.5, or about 7.5.

[0100] In one embodiment, the solution is isotonic or hypertonic.

[0101] In one embodiment, the solution exhibits an osmotic molar concentration of about 270-345 mOsm / l or about 315 mOsm / l.

[0102] In one embodiment, the concentration of the calcium source is (a) 0.25-0.75 mM, or 0.4-0.65 mM, or 0.5-0.6 mM or about 0.6 mM; or (b) 0.5-0.9 mM, or 0.6-0.8 mM or about 0.7 mM.

[0103] In one embodiment, the concentration of the magnesium source is 0.05-5 mM, or 0.1-0.3 mM, or about 0.3 mM. In one embodiment, the concentration of glucose is 5-50 mM, or 10-25 mM, or 10-20 mM, or about 16 mM. In one embodiment, the concentration of the permeabilizer is about 100-200 mM, or about 125-175 mM, or about 150 mM. In one embodiment, the concentration of the citrate or acetate is 0.1-5 mM, or 0.5-2 mM, or about 1 mM.

[0104] In one embodiment, the solution further comprises a potassium salt, optionally said potassium salt being potassium chloride, and further optionally said KCl concentration being 0.2-5 mM, or 1-2.5 mM, or about 2 mM.

[0105] In one embodiment, the solution comprises (a) about 0.7 mM CaCl2 (calcium chloride), about 0.3 mM MgCl2 (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, and about 145 mM NaCl; or (b) about 0.5-0.9 mM CaCl2 (calcium chloride), about 0.2-0.4 mM MgCl2 (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, and about 116-174 mM NaCl; or (c) about 0.85% NaCl, about 0.01% CaCl2 dihydrate (calcium chloride dihydrate), about 0.006% MgCl2 hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate, and about 0.29% dextrose; or (d) about 0.68-1.02%. NaCl, approximately 0.008-0.012% CaCl2 dihydrate (calcium chloride dihydrate), approximately 0.0048-0.0072% MgCl2 hexahydrate (magnesium chloride hexahydrate), approximately 0.028-0.042% sodium citrate dihydrate, and approximately 0.23-0.35% dextrose.

[0106] In one embodiment, the solution further comprises (a) about 2 mM KCl and / or (b) a viscoelastic polymer, optionally wherein the polymer is hyaluronic acid or a salt or solvate thereof, and further optionally wherein the polymer is sodium hyaluronate.

[0107] In one embodiment, the polymer is present at a concentration that effectively reduces the exposure of cells to shear stress in solution, optionally wherein the concentration of the polymer is 0.005-5% w / v or about 0.05% w / v.

[0108] In one embodiment, the solution comprises (a) about 0.7 mM CaCl2 (calcium chloride), about 0.3 mM MgCl2 (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, and about 0.05% hyaluronic acid, or (b) about 0.5-0.8 mM CaCl2 (calcium chloride), about 0.2-0.4 mM MgCl2 (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid.

[0109] In one embodiment, the solution does not contain (a) a carbonate buffer, and / or (b) glutathione or glutathione disulfide (oxidized glutathione, GSSG), and / or (c) a zwitterionic organic buffer.

[0110] In one embodiment, the solution may (a) be stored at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable solute precipitation and / or measurable loss of the ability of the solution to support the survival and viability of the cells stored in the solution, and / or (b) be stored at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable solute precipitation and / or measurable loss of the ability of the solution to support the survival and viability of the cells stored in the solution.

[0111] In one embodiment, the solution (a) is suitable for administration to a subject, suitable for administration to the subject's eye, and / or suitable for transplanting cells to the subject's eye, and / or (b) is essentially (substantially) pyrogen-free, and / or (c) is sterile, and / or (d) is used for rinsing, cell remodeling, cell storage, cell delivery, and / or administration to a subject.

[0112] Therefore, in another aspect, the present invention provides a method for producing compositions according to various embodiments of the above aspects described herein or any other aspect of the invention, the method comprising: 1) culturing pluripotent stem cells in rescue-induction medium (RIM) and Noggin; 2) culturing cells from step 1 in neural differentiation medium (NDM) and Noggin; 3) expanding cells from step 2 in Noggin-free NDM, the step comprising a) culturing cells in Noggin-free NDM under low-adherence or non-adherence conditions, and b) culturing cells in Noggin-free NDM under non-adherence conditions, thereby differentiating pluripotent stem cells into photoreceptor rescue cells.

[0113] Therefore, in another aspect, the present invention provides a method for producing a photoreceptor rescue cell composition comprising multiple heterologous cells, the method comprising: 1) culturing pluripotent stem cells in rescue induction medium (RIM) and Noggin; 2) culturing the cells in neural differentiation medium (NDM) and Noggin; 3) expanding the cells in Noggin-free NDM, the step comprising a) culturing the cells in Noggin-free NDM under low adherence or non-adherence conditions, and b) culturing the cells in Noggin-free NDM under adherence conditions, thereby differentiating the pluripotent stem cells into multiple cell types of the photoreceptor rescue cell composition.

[0114] In one embodiment, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0115] In one embodiment, step 3 is performed at least once, at least twice, at least three times, at least four times, at least five times, or at least six times. In one embodiment, the cells in the composition are harvested after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In one embodiment, the composition is harvested after the fifth repetition of step 3.

[0116] In one embodiment, the cells harvested from the composition are cryopreserved.

[0117] In one embodiment, the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3. In one embodiment, the composition is cryopreserved after the third repetition of step 3, after the fourth repetition of step 3, or after the fifth repetition of step 3. In one embodiment, step 3 is repeated five times, and the composition is cryopreserved after the fifth repetition of step 3.

[0118] In one embodiment, after cryopreservation and thawing, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are alive. In one embodiment, after cryopreservation and thawing, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable. In one embodiment, after cryopreservation and thawing, less than about 50% of the cells in the composition are viable. In one embodiment, less than about 55% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, less than about 60% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, less than about 65% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, less than about 68% of the cells in the composition are viable after cryopreservation and thawing.

[0119] In one embodiment, the composition comprises cell spheroids. In one embodiment, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are cell spheroids.

[0120] In one embodiment, during steps 1, 2 and / or 3, cells of the composition are cultured in a cell culture container selected from culture dishes, culture flasks and culture chambers.

[0121] In one embodiment, the cell culture container has a diameter of approximately 50 cm. 2 Approximately 800cm 2 Approximately 60cm 2 Approximately 800cm 2 Approximately 100cm 2 Approximately 800cm 2 Approximately 150cm 2 Approximately 800cm 2 Approximately 175cm 2 Approximately 800cm 2 Approximately 200cm 2 Approximately 800cm 2 Approximately 250cm 2 Approximately 800cm 2 Approximately 300cm 2 Approximately 800cm 2 Approximately 400cm 2 Approximately 800cm 2 Approximately 500cm 2 Approximately 800cm 2 Approximately 600cm 2 Approximately 800cm 2 Approximately 700cm 2 Approximately 800cm 2 Approximately 30cm 2 Approximately 100cm 2 Approximately 50cm 2 Approximately 100cm 2 Approximately 100cm 2 Approximately 300cm 2 Approximately 150cm 2 Approximately 250cm 2 or about 150cm 2 Approximately 200cm 2 Cell growth area. In one embodiment, the cell culture container has a surface area of ​​at least about 60 cm². 2Approximately 175cm 2 or approximately 636cm 2 Cell growth area.

[0122] In one embodiment, the culture chamber is a stackable rectangular chamber.

[0123] In one embodiment, step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5, or 1 to 2 culture chambers. In another embodiment, step 3 has at least 1, 2, 5, 10, or 40 culture chambers.

[0124] In one embodiment, the cell culture container is coated for use in step 3a for low-adherence or non-adherence cell culture, and / or in step 3b for adherence cell culture.

[0125] In one embodiment, the cells are enzymatically dissociated from the plate into a cell suspension. In one embodiment, the enzyme used for cell dissociation is a thermophilic protease, liberase, and / or accutase. In one embodiment, the enzyme used for cell dissociation is accutase. In one embodiment, cell dissociation from the plate does not involve manual scraping.

[0126] Therefore, in another aspect, the present invention provides photoreceptor-rescue cell compositions produced by various embodiments of the methods described above or any other aspect of the present invention.

[0127] Therefore, in another aspect, the present invention provides a method for treating an eye disease or condition in a subject, the method comprising administering to the subject a photoreceptor cell-saving composition according to various embodiments of the present invention described herein, or a pharmaceutical preparation according to various embodiments of the present invention described herein, or an formulation according to various embodiments of the present invention described herein.

[0128] Therefore, in another aspect, the present invention provides a method for increasing the secretion of neuroprotective factors in the eye of a subject suffering from an eye disease or condition, the method comprising administering to the subject a photoreceptor cell-saving composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0129] In one embodiment, the method increases the secretion of neuroprotective factors by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the secretion of neuroprotective factors before administration.

[0130] Therefore, in another aspect, the present invention provides a method for improving visual acuity in subjects suffering from retinal diseases or conditions, the method comprising administering to the subject a photoreceptor-rescue cell composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0131] In one implementation, the method improves visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application visual acuity.

[0132] In one implementation, visual acuity is measured by optomotor response (OMR) and / or electroretinogram (ERG).

[0133] In one implementation, the spatial frequency threshold of the treated eye, as measured by OMR, is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the spatial frequency threshold before application.

[0134] In one implementation, the scotopic b-wave amplitude of the treated eye, as measured by ERG, is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the pre-application scotopic b-wave amplitude.

[0135] Therefore, in another aspect, the present invention provides a method for preventing or mitigating the loss of photoreceptor cells in a subject suffering from a retinal disease or condition, the method comprising administering to the subject a photoreceptor cell-saving composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0136] In one embodiment, the prevention of photoreceptor cell loss is measured by CNFT expression. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to CNFT expression in the eye before application.

[0137] Therefore, in another aspect, the present invention provides a method for improving phagocytic activity in the eye of a subject suffering from a retinal disease or condition, the method comprising administering to the subject a photoreceptor-rescue cell composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0138] In one embodiment, the method increases phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the activity prior to application.

[0139] Therefore, in another aspect, the present invention provides a method for inhibiting the activation of microglia in the eye of a subject suffering from a retinal disease or condition, the method comprising administering to the subject a photoreceptor cell-saving composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0140] In one embodiment, the inhibition of microglial activation is measured by the expression of CNFT and / or MIF. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application CNFT expression. In one embodiment, the method increases MIF expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application MIF expression.

[0141] Therefore, in another aspect, the present invention provides a method for reducing oxidative stress in the eyes of a subject suffering from retinal disease or condition, the method comprising administering to the subject a photoreceptor cell-saving composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0142] In one embodiment, the reduction in oxidative stress is measured by CNFT expression. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT expression before administration.

[0143] Therefore, in another aspect, the present invention provides a method for increasing the expression of an anti-apoptotic factor in the eye of a subject suffering from a retinal disease or condition, the method comprising administering to the subject a photoreceptor cell rescue composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0144] In one embodiment, the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before administration.

[0145] In one embodiment, the anti-apoptotic factor is S100B.

[0146] Therefore, in another aspect, the present invention provides a method for preventing degeneration of the outer nuclear layer (ONL) of the eye in a subject suffering from retinal disease or condition, the method comprising administering to the subject a photoreceptor-rescue cell composition according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical preparation according to various embodiments of the present invention described above or any other aspect thereof, or a formulation according to various embodiments of the present invention described above or any other aspect thereof.

[0147] In one embodiment, the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, moderate dry age-related macular degeneration (AMD), leber congenital amaurosis, or Stargardt disease. In one embodiment, the ocular disease is macular degeneration or retinitis pigmentosa. In one embodiment, the disease is a retinal degenerative disease.

[0148] In one embodiment, the disease is associated with the loss of photoreceptor cells.

[0149] In one embodiment, the disease is associated with the loss of photoreceptor cells in the outer nuclear layer of the retina. In one embodiment, the photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered to the subretinal space, suprachoroidal space, via a depot to the eye, or via systemic delivery to other parts of the subject.

[0150] In one embodiment, the cell preparation or formulation is administered by injection or transplantation (implantation).

[0151] In one implementation, the injection is administered intraocularly.

[0152] In one embodiment, an intraocular administration of an aqueous solution, optionally an isotonic solution, and / or a saline solution is administered into the subretinal space to form a pre-bleb, and the aqueous solution is removed before the photoreceptor rescue cell composition is administered into the same subretinal space as the aqueous solution.

[0153] In one embodiment, the cell preparation or formulation is applied at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after the onset of symptoms.

[0154] In one implementation, (i) the subject has intermediate or near-terminal disease; (ii) the subject's best corrected visual acuity (BCVA) is in the range of 20 / 50 to 20 / 200; (iii) the subject's BCVA is less than 20 / 200, but light perception is maintained; or (iv) the subject is diagnosed with retinitis pigmentosa by genotyping.

[0155] In one embodiment, the method further includes administering one or more anti-inflammatory agents to the subject.

[0156] In one embodiment, one or more anti-inflammatory agents are applied simultaneously. In another embodiment, one or more anti-inflammatory agents are applied separately.

[0157] In one embodiment, the one or more anti-inflammatory agents are applied prior to the cell preparation or formulation, or the cell preparation or formulation is applied prior to the one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents are applied both before and after the application of the cell preparation or formulation. In one embodiment, the cell preparation or formulation is applied without the one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents include dexamethasone and / or cyclosporine.

[0158] Therefore, in another aspect, the present invention provides a population of extracellular vesicles (EVs) secreted by a photoreceptor rescue cell composition from various embodiments of the invention described herein or any other aspect thereof.

[0159] In one embodiment, the EVs secreted by the photoreceptor-rescued cells are expressed from at least one of the following proteins: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, DLX5, TUBB3, SCGN, ERBB4, CALB2, NEUROD2, NEUROD6, SLA, NELL2, SATB2, VIM, MKI67, CLU, GLI3, GFAP, LUCAT1, MIR99AHG, FBXL7, MEIS2, PBX3, GRIA2, CACNA1C, PAX6, LHX2, SIX3, NES, SOX2, ASCL1, RORB, NR2E3, NRL, TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5, AGT, ACBLN2, CDH7, DNAH11, EGR1, FAM216B, FO S, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, Nestin, TRβ2, MASH1, RORβ, MAP2, ELAVL3, NFIA, DCX, LHX2, SLC1A2, ELAVL4, PAX6, EMX2, ASC L1, DLL1, NFIB, ENOXI, TUBB3, MAP2, DCLK1 / 2, DCX, KALRN, LINC00461, C1orf61, NCAM1, SETBP1, PAK3, AKAP6, RTN1, CRMP1, FOXG1, TRIM2, BACH2, Recoverin, Opsin, Rhodopsin, rod and cone cGMP phosphodiesterase.

[0160] Therefore, in another aspect, the present invention provides pharmaceutical compositions comprising an EV population and a pharmaceutically acceptable carrier according to various embodiments of the above or any other aspects of the invention described herein.

[0161] Therefore, in another aspect, the present invention provides formulations comprising: a) a group of EVs according to various embodiments of the present invention described herein or any other aspect thereof, or a pharmaceutical composition according to various embodiments of the present invention described herein or any other aspect thereof; and b) about 4-10% (v / v) of a cryoprotectant, about 2-3% (w / v) of albumin, about 0-1.5% (w / v) of glucose and a buffer.

[0162] In one embodiment, the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol. In one embodiment, the cryoprotectant is DMSO. In one embodiment, the formulation comprises about 4-6% (v / v) cryoprotectant. In one embodiment, the formulation comprises about 0.08-0.10% (w / v) glucose. In one embodiment, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer. In one embodiment, the formulation comprises about 0.6% (w / v) glucose. In one embodiment, the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

[0163] In one embodiment, the albumin is human albumin. In another embodiment, the albumin is recombinant human albumin.

[0164] In one embodiment, the buffer is a buffered saline solution. In one embodiment, the buffer is phosphate-buffered saline (PBS). In one embodiment, the buffered saline solution contains Ca. 2+ and Mg 2+ In one embodiment, the buffered brine does not contain Ca. 2+ and Mg 2+ .

[0165] In one embodiment, the preparation is cryopreserved.

[0166] Therefore, in another aspect, the present invention provides a method for treating an eye disease in a subject, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0167] Therefore, in another aspect, the present invention provides a method for increasing the secretion of neuroprotective factors in the eye of a subject, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0168] In one embodiment, the method increases the secretion of neuroprotective factors by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the secretion of neuroprotective factors before administration.

[0169] Therefore, in another aspect, the present invention provides a method for improving visual acuity in subjects suffering from retinal diseases or conditions, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0170] In one embodiment, the method improves visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application visual acuity. In one embodiment, visual acuity is measured by optomotor response (OMR) and / or electroretinography (ERG). In one embodiment, the spatial frequency threshold of the treated eye, as measured by OMR, is improved by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application spatial frequency threshold. In one implementation, the scotopic b-wave amplitude measured by ERG in the treated eye is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the pre-application scotopic b-wave amplitude.

[0171] Therefore, in another aspect, the present invention provides a method for preventing or slowing the loss of photoreceptor cells in a subject suffering from a retinal disease or condition, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0172] In one embodiment, the prevention or mitigation of photoreceptor cell loss is measured by CNFT expression. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to CNFT expression in the eye before application.

[0173] Therefore, in another aspect, the present invention provides a method for improving phagocytic activity in the eye of a subject suffering from a retinal disease or condition, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0174] In one embodiment, the method increases phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the activity prior to application.

[0175] Therefore, in another aspect, the present invention provides a method for inhibiting microglial activation in the eye of a subject suffering from retinal disease or condition, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0176] In one embodiment, the inhibition of microglial activation is measured by the expression of CNFT and / or MIF. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application CNFT expression. In one embodiment, the method increases MIF expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to pre-application MIF expression.

[0177] Therefore, in another aspect, the present invention provides a method for reducing oxidative stress in the eyes of a subject suffering from retinal disease or condition, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0178] In one embodiment, the reduction in oxidative stress is measured by CNFT expression. In one embodiment, the method increases CNFT expression by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to CNFT expression before administration.

[0179] Therefore, in another aspect, the present invention provides a method for increasing the expression of anti-apoptotic factors in the eyes of subjects suffering from retinal diseases or conditions, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0180] In one embodiment, the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of the anti-apoptotic factor before application. In one embodiment, the anti-apoptotic factor is S100B.

[0181] Therefore, in another aspect, the present invention provides a method for preventing degeneration of the outer nuclear layer (ONL) of the eye in a subject suffering from retinal disease or condition, the method comprising administering to the subject an EV population according to various embodiments of the present invention described above or any other aspect thereof, a pharmaceutical composition comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof, or a formulation comprising an EV population according to various embodiments of the present invention described above or any other aspect thereof.

[0182] In one embodiment, the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, moderate dry age-related macular degeneration (AMD), leber congenital amaurosis, or Staggart's disease. In one embodiment, the ocular disease is macular degeneration or retinitis pigmentosa. In one embodiment, the disease is a retinal degenerative disease. In one embodiment, the disease is associated with the loss of photoreceptor cells. In one embodiment, the disease is associated with the loss of photoreceptor cells in the outer nuclear layer of the retina.

[0183] In one embodiment, the EV population, a pharmaceutical composition comprising the EV population, or a formulation comprising the EV population is administered to the subretinal or suprachoroidal space of the subject.

[0184] In one embodiment, the EV population, a pharmaceutical composition containing the EV population, or a formulation containing the EV population is administered by injection or transplantation.

[0185] In one embodiment, the injection is administered intraocularly.

[0186] In one embodiment, an intraocular administration of an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space is performed to form a pre-bleb, and the aqueous solution is removed before the photoreceptor rescue cell composition is applied to the same subretinal space as the aqueous solution.

[0187] In one embodiment, the EV population, a pharmaceutical composition comprising the EV population, or a formulation comprising the EV population is administered at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after the onset of symptoms.

[0188] In one embodiment, the method further includes administering one or more anti-inflammatory agents to the subject.

[0189] In one embodiment, the one or more anti-inflammatory agents and the EV population, a pharmaceutical composition containing the EV population, or a formulation containing the EV population are administered simultaneously.

[0190] In one embodiment, the one or more anti-inflammatory agents and the EV population, a pharmaceutical composition containing the EV population, or a formulation containing the EV population are administered separately.

[0191] In one embodiment, 1) the one or more anti-inflammatory agents are administered before the EV group, a pharmaceutical composition containing the EV group, or a formulation containing the EV group, or 2) the EV group, a pharmaceutical composition containing the EV group, or a formulation containing the EV group are administered before the one or more anti-inflammatory agents.

[0192] In one embodiment, the one or more anti-inflammatory agents are applied before and after the application of the EV group, a pharmaceutical composition containing the EV group, or a formulation containing the EV group.

[0193] In one embodiment, the EV population, a pharmaceutical composition comprising the EV population, or a formulation comprising the EV population is administered without the presence of one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. Attached Figure Description

[0194] Figure 1A Cell clustering analysis of ESC / PRE / PRC is shown. ESC: Undifferentiated, research grade J1-ESC and Kd-ESC. RPE: J1-RPE at P3+4 months. PRC: J1-PRC (P4), Kd-PRC (P4), GB2R-PRC-2019 (P4), GB2R-PRC-PR1 (P4), GB2R-PRC-CN2 (P4), GB2R-PRC-CN3 (P4).

[0195] Figure 1B The qPCR validation of neuronal genes, including NANOG, NEUROD2, GAD1, DCX, MAP2, and NFIA, was demonstrated.

[0196] Figure 1C Single-cell analysis of PRC-P4 (GB2R-PRC-2019(P4), GB2R-PRC-PR1(P4), GB2R-PRC-CN2(P4), and GB2R-PRC-CN3(P4)) is shown. Cells were classified into inhibitory neurons, excitatory neurons, mixed neurons, progenitor cells, and astrocytes.

[0197] Figure 1D The qPCR characterization assays show the upregulation of markers FOXG1, MAP2, STMN2, DCX, and LINC00461 in GB2R-PRC, GB2R-ESC, RPE, and HMC cells. GB2R-FCP: GB2R-final cell products, including GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019.

[0198] Figure 1E It shows, as Figure 1C The heatmap shows the expression of ocular progenitor cell markers, rod / cone photoreceptor cell markers, and neuronal markers in inhibitory neurons, excitatory neurons, surrogate neurons, progenitor cells, and astrocytes.

[0199] Figure 1F-1T The table shows the expression of cellular markers from the PRC compositions listed in Table 10 at days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3), and 107 (D107 / P4). PRC markers FOXG1, MAP2, STMN2, and DCX (… Figure 1F ); PRC markers EINC00461, NEUROD2, GAD1 and NFIA ( Figure 1G Inhibitory neuronal markers DEX5, TUBB3, and SCGN ( Figure 1H ); inhibitory neuronal markers ERBB4 and CALB2 ( Figure 1I ); excitatory neuronal markers NEUROD2, NEUROD6 and SLA ( Figure 1J ); excitatory neuronal markers NELL2 and SATB2 ( Figure 1K ); progenitor cell markers VIM, MKI67, CLU and GLI3 ( Figure 1L ); astrocyte markers GFAP, MIR99AHG and FBXL7 ( Figure 1M ); Alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C ( Figure 1N ); ocular progenitor cell markers PAX6, LHX2, and SIX3 ( Figure 10 ); Ocular progenitor cell markers NES and SOX2 ( Figure 1P ); Rod / cone photoreceptor markers ASCL1, RORB, NR2E3 and NRL ( Figure 1Q Neuronal markers TUBB3, NFIA, and NFIB ( Figure 1R Neuronal markers OTX2, ELAVL3, and ELAVL4 Figure 1S ); and neuronal markers SLC1A2, SLC1A3, HCN1, and HES5 ( Figure 1T ).

[0200] Figure 2A Bright-field image of PRC-P4 cells is shown.

[0201] Figure 2B A scorecard analysis of PRC differentiation, which observes markers of self-renewal, ectoderm, mesoderm, and endoderm, is presented.

[0202] Figure 2C Immunocytochemical (ICC) staining in PRC-P4 cells reveals the expression of PAX6 / OTX2 in PRC-NPC cells (neural progenitor cells), as well as the expression of STMN2, CALB2, SCGN, and DCX in PRC-P4 cells.

[0203] Figure 2D The expression of markers NEUROD2, FOXG1, and HMGA1 in PRC-P4 cells was shown, indicating successful differentiation.

[0204] Figure 2E Flow cytometry analysis of GB2R-CN3-P4 cells based on FOXG1 and MAP2 expression is shown. Percentage of cells expressing purity markers: mean % (GB2R-PRC-CN3 FCP, N=3). FOXG1: 95.6 (94.2–96.7); MAP2: 88.4 (84.7–91.4); and SSEA4: 0.44 (0.15–0.88).

[0205] Figure 2F Differentially expressed genes between PRC-P3 and PRC-P4, identified by RNA-seq analysis, are shown. The mean log10 ratio of these genes is either greater than 3 or less than -3.

[0206] Figure 3AThe PRC stimulation using TBHP (oxidative stress) and the Luminex assay for detecting secreted factors from the PRC after stimulation are shown. PRC: GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer and GB2R-PRC-2019.

[0207] Figure 3B The levels of secreted neuroprotective factors detected in the culture medium when PRC cells were not under oxidative stress are shown. PRC stimulation with TBHP (oxidative stress) and Luminex assays were used to detect secreted factors from stimulated PRC cells. CNTF, MIF, and S100B in the culture medium were quantified when unstimulated (without oxidative stress). PRCs: GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019.

[0208] Figure 3C Confocal images are shown that confirm subretinal implantation of PRCs inhibits microglial infiltration into ONL (P60) in RCS rats. Stained retinal sections were imaged using confocal z-stack analysis on a Leica SP8.

[0209] Figure 3D The study showed stimulation of the microglial cell line SIM-A9 with LPS and inhibition with L-carnitine.

[0210] Figure 3E The phagocytosis assay of GB2R-PRC-PR1 incubated with pHrodo Escherichia coli (E. coli) bioparticles is shown.

[0211] Figure 3F This study demonstrates the internalization of extracellular rod segment (ROS) debris via PRC in the retina of RCS (Royal College of Surgeons) rats. RHO+ debris was observed in the GFP+PRC implant (left panel), and EM in the right panel shows ROS detachment from the cytoplasm of the PRC. GFAP+PRC was co-stained with HuNu (data not shown).

[0212] Figure 4A A schematic diagram showing the time course of RCS rat dosage administration and in vivo experiments is presented.

[0213] Figure 4BOptomodynamic response (OMR) and electroretinogram (ERG) analyses were presented in RCS rats injected with GB2R-PRC-2019PRC at P25 with 100,000 cells / eye. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT), comparing eyes injected with the test product to those not injected and those injected with the GS2 mediator. Cell viability: 78.4% (manual).

[0214] Figure 4C Representative images are shown, demonstrating that PRC implantation significantly reduced outer nuclear layer (ONL) degradation 3 months post-implantation. Confocal images show that PRC implantation remained highly correlated with ONL. Stained retinal sections were imaged using a Leica MDi8 epifluorescence microscope.

[0215] Figure 4D Quantification of outer nuclear layer (ONL) thickness was shown, which demonstrated that PRC implantation was significantly associated with retention of P35, P60, and P120. Cell viability: P35 57.1% and 54%; P60 57.1% (Cellometer); P120 >70% (manual). n = 148 ROI / 7 eyes.

[0216] Figure 4E Area plots of GB2R-PRC-2019(P4) implanted PRCs vs. ONL photoreceptor cells in a P120 RCS rat model of retinal degeneration are shown. Statistical analysis was performed using Pearson correlation coefficient. Cell viability: >70% (manual), n = 4 eyes (12 slices / eye).

[0217] Figure 5A Immunohistochemical analysis of glial fibrillary acidic protein (rabbit anti-GFAP; ABCAM) in P120 RCS rats is shown. GFAP is expressed in Miller glial cells (MG) and optic nerve fiber astrocytes (white arrows) of the host rat retina, as well as in PRCs implanted in the subretinal space. MG hypertrophy with increased GFAP expression in MG and astrocytes, and the expanded distribution of GFAP expression throughout the radial MG cell body, are characteristic of disease-associated reactive gliosis and are visible at the distal implantation site (yellow arrows, white arrows). At the subretinal PRC implantation site, GFAP upregulation and increased distribution, MG hypertrophy, and reduced or absent outer nuclear layer (ONL) degeneration are observed. Cell viability: >70% (manual).

[0218] Figure 5BTUNEL quantification (TUNEL-labeled mixture, Sigma catalog number #11767291910, TUNEL enzyme Sigma catalog number #11767305001) performed at P35 is shown, during which peak apoptotic activity was observed in the RCS retina. Implanted PRCs in the subretinal space were identified by Ku80+ staining (Abeam, ab80592) of adjacent sections (see below). Very few TUNEL+ nuclei (if present) were observed in the PRC implantation region, indicating little or no apoptotic activity at the implantation site. However, the non-implantation region contained significant TUNEL+ nuclei in the ONL, indicating extensive photoreceptor death at P35. Quantification of TUNEL+ nuclei in the ONL showed significantly reduced apoptotic activity at the implantation site compared to the non-implantation region. TUNEL quantification was performed on retinal sections penetrating the PRC implant, and Ku80 staining was used to locate the implanted PRC and DAPI to identify the ONL. Three to four regions of interest (ROIs) were imaged in total, both centrally and peripherally in each retina. TUNEL quantification was performed blinded using only the DAPI channel. Subsequently, ROIs were identified as “implanted” or “non-implanted” based on the presence of subretinal Ku80+PRCs. Cell viability: 3.5% / 52.3% (Cellometer).

[0219] Figure 5C Extracellular vesicles (EVs) isolated from PRC maintained an OMR response in RCS rats until P90. EVs were isolated from PRC conditioned medium and subretinally injected into RCS rats at a dose of 9.42 × 10^8 EVs / eye. Other RCS rats received subretinal injections of PRC (100,000 cells / eye) or the medium. Injections were performed at age P25. OMR analysis was performed at P60, 90, and 120 and compared with uninjected (NI) animals of the same age.

[0220] Figure 5D This demonstrates how a single subretinal injection of PRC-EV maintains ONL thickness up to P90. Quantification of retinal ONL thickness in PRC-EV-injected RCS rats relative to uninjected eyes. Retinal cryosections were stained with DAPI to visualize ONL. The stained retinal sections were imaged using a Leica DMi8 epifluorescence microscope. Figure 4CThe ONL thickness was determined in the central and peripheral regions by line graph measurements across the ONL. P90: ONL retention is correlated with OMR retention. ONL thickness in Long-evans rats (sighted): ~55 μm (Weber et al., 1996). The P-prefix indicates age at birth.

[0221] Figure 5E This demonstrates a single PRC-EV injection. ONL thickness was maintained up to P90.

[0222] Figure 5F The image shows ONL cells without PRC-EV injection. Small plaques of ONL cells are preserved, with an average of 1-3 rows of nuclei.

[0223] Figure 5G The ONL (on-cell lymphocytes) after PRC-EV injection is shown. Compared with uninjected ONL, the retained ONL has a longer extension, with an average of 3-6 rows of nuclei.

[0224] Figure 5H The image shows the ONL thickness after PRC-EV injection at P120. A single PRC-EV injection cannot maintain the ONL thickness at P120.

[0225] Figure 5I The optomotor response following PRC-EV injection is shown. Visual function was evaluated by recording the optomotor responses of PRC-EV-injected rats at P60, P90, and P120.

[0226] Figure 5J Analysis of ONL in PRC-EV-treated rats showed that a single injection of PRC-EV maintained the morphology of ONL up to P90, but not up to P120.

[0227] Figure 5K The image shows the ONL after subretinal cell transplantation in PRC. Subretinal cell transplantation in PRC maintained the ONL thickness up to P120.

[0228] Figure 6A Toluidine blue staining is shown in the PRC implantation and non-implantation areas. ONL retention is clearly observed, while the non-implantation area shows severely degenerated ONL. ONL is shown as a thick, dark band (long arrow). Note: ONL retention is absent on the non-injection side (short arrow).

[0229] Figure 6B Transmission electron microscopy (TEM) analysis of implanted and unimplanted ONL sites is shown. The implanted ONL site showed significant preservation compared to the site without PRC implantation.

[0230] Figure 6C TEM ultrastructural analysis is shown. TEM reveals that in the presence of subretinal PRC, the overall morphology of photoreceptor cells and finer structures, such as the connecting cilia of the inner segments of rod cells, is preserved. Finally, PRC implantation minimizes the debris area in the subretinal space. In the absence of subretinal PRC, outer segment debris of rod cells occupies most of the subretinal space due to the incomplete removal of detached outer segments. GB2R-PRC-2019 was injected at P25 postnatally. The black arrows point to the preserved connecting cilia of the inner and outer segments. Cell viability: 57.1% (Cellometer).

[0231] Figure 7A A schematic diagram of PRC administration using the rd10 mouse model is shown. At 14 days postnatal time (P14), 100k cells were transplanted into the subretinal space of the right eye, while GS2 mediator was injected into the left eye as a control. OMR was performed at P21, P28, P35, P42, and P49. Cell viability: 68.5% (manual).

[0232] Figure 7B The figures shown on P21 and P28 illustrate the presence of tracking in both eyes. On P35, P42, and P49, the control eye was blinded, while the eye injected with cells still showed tracking ability.

[0233] Figure 7C A graph showing a morphological analysis of the preservation of the extranuclear layer (ONL) of photoreceptors in P28 rd10 mice is presented. The location of the subretinal implantation in the PRC retina, compared to adjacent unimplanted central and peripheral retina, can be identified as the ONL area (mm²). 2 Quantitative ONL retention. Cell viability: 73.3% (manual).

[0234] Figure 7D A graph showing the cone cell length analysis in P28 rd10 mice is displayed. The length of arrestin-labeled cone photoreceptors was significantly increased at the PRC subretinal implantation site compared to adjacent areas of the central and peripheral retina without subretinal implantation. Cell viability: 73.3% (manual).

[0235] Figure 7E Confocal imaging was shown, revealing CtBP2 / RIBEYE-labeled rod cell synapses at the implantation site and at non-implanted central and peripheral retinal sites.

[0236] Figure 7FA map showing the quantitative analysis of rod cell synapses in P28 rd10 mice is presented. The number of RIBEYE-positive / arrestin-negative rod cell synapses was significantly increased at the PRC implantation site compared to the unimplanted central and peripheral retina, indicating PRC-related maintenance of rod photoreceptors in the rd10 mouse retina. Cell viability: 73.3% (manual).

[0237] Figure 8A The OMR at P35 is shown, with multiple PRC batches showing survival rates below 70% (sub-70%). PRC survival rates of 66.05%, 68.5%, and 68.8% show similar retention effects. Eyes injected with PRC showed better OMR performance than control eyes.

[0238] Figure 8B Anatomical preservation of PRC cells was demonstrated. Anatomical preservation was observed in multiple PRC batches, with survival rates below 70%. Histological analysis confirmed the OMR data. At P35, injected PRC with a survival rate of 66.05% maintained cone cell anatomy, as evaluated by ONL thickness, axon length, and morphology. At P50, injected PRC with a survival rate of 68.5% maintained ONL thickness and density of the RIBEYE+ synapse. At P70, injected PRC with a survival rate of 68.8% still maintained ONL thickness and density of the RIBEYE+ synapse.

[0239] Figure 9A ONL retention in RD10 mice was shown. At 14 days postnatal time (P14), 100k cells were transplanted into the subretinal space of the right eye, while GS2 mediator was injected into the left eye as a control. At P50, eyes were collected for IHC staining. DAPI staining revealed only one row of photoreceptor cells in the unimplanted retina, while the implanted retina contained multiple rows. Quantitative analysis of data from 3 sections per animal from 5 animals showed that the thickness and area of ​​ONL in the implanted retina were significantly greater than in the unimplanted retina (6 μm vs 18 μm; 5000 μm² vs 10000 μm²). Cell viability: 68.5% (manual).

[0240] Figure 9BThe image shows ICC staining of cone cell repressor protein (Millipore AB15282 1:500) used to detect cone cell morphology (left panel). In the unimplanted retina, the repressor protein is expressed only in the cell bodies of cones, with degenerative changes in the outer segment and axon. In the implanted retina, cone cells are maintained by preserving normal morphology, and the repressor protein is expressed in the outer segment, cell body, axon, and pedicle. Quantitative data show that cone cells in the implanted retina have significantly longer axons than those in the unimplanted retina (8 μm vs 4 μm). (Right panel) Ribeye (also known as ct-BP2, BD transduction Laboratories 612044, 1:500) is a marker of presynaptic structures located at the axonal ends of photoreceptor cells. It shows the synaptic connections between photoreceptor cells and horizontal cells. Quantitative data show that Ribeye spot expression is higher in the implanted retina than in the unimplanted retina (40 vs 20 / 100 μm retina). Cell viability: 68.5% (manual).

[0241] Figure 9C A schematic diagram of the administration of PRC cells to hemizygous rats is shown. A summary of the dosage administration and time course of in vivo experiments in P23H rats is also provided.

[0242] Figure 9D A plot showing the preservation of OMR in P23H hemizygous rats of GB2R-PRC-P4-FDA PRC injected with 100,000 cells / eye at P25 is presented. The plot shows statistically significant preservation of the OMR response compared to uninjected controls at P60, P90, and P120, and compared to controls injected with the medium at P90. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons test (MCT). Cell viability: 71.7% (manual).

[0243] Figure 10A A schematic diagram of the HuNu+ cell quantification process is shown. 100,000 GB2R PRC or GS2 mediators were injected subretinously into RCS rats aged P23 to P25. Animals were sacrificed at P120 for retinal tissue cryosection and histological analysis. Every 10th retinal section penetrating the implant was selected for quantification, where HuNu+ PRC was quantified. Cell counts were calculated between each section using interpolation, and the total implantation was calculated. Total PRC implantation was calculated from n=4.

[0244] Figure 10BThe number of PRCs implanted in four transplant animals at P120 or 3 months post-transplantation is quantified. Quantification of total HuNu+PRCs in the subretinal implants showed an average of 80,000 transplanted cells per eye, which translates to an overall implantation rate of 80% compared to the initial injection of 100,000 cells per injection.

[0245] Figure 10C The differences in PRC implantation in retinal degeneration models with varying disease onset and post-treatment severity are shown. Specific batches and doses of PRC used in this analysis are shown in the top box of the figure. Bar chart of relative subretinal implantation: Comparative implantation of GB2R-PRC was confirmed in RCS rat and P23H hemizygous rat models of retinal degeneration, with maximum implantation covering 54% and 41% of the subretinal space, respectively. rd10 mouse and P23H homozygous rat models of retinal degeneration showed significantly less implantation, with 19% (rd10 mice) and 2% (P23H homozygous rats) of the subretinal space showing PRC implantation at maximum length. Relative retinal length implantation coverage plot: Relative calculations provide coverage relative to different sizes of mouse and rat eyes, while the maximum length plot shows the absolute length of implantation. rd10 mice and P23H rats showed reduced implantation in both relative and absolute terms compared to RCS and P23H hemizygous rats.

[0246] Figure 11A A graph showing the downregulation of proliferation in subretinal PRCs up to P120. HuNu (Millipore, MAB1281) and Ki67 (Abeam ab15580) double-positive cells in the subretinal implant were quantified in retinal cross-sections from animals at P35, P60 (not shown), and P120. A total of three animals were used at each time point. Confocal images show Ki67 and HuNu double-positive cells in the subretinal space at P35. However, no proliferating (Ki67+) PRCs were observed by P120. Quantification shows a significant downregulation of Ki67 and HuNu double-positive PRCs by P120 or 3 months post-transplantation (bar chart in the lower left figure). Cell viability: P35: 43.5% and 52.3%; P60: 43.5% and 52.3% (Cellometer); and P120: >70% (manual).

[0247] Figure 11BConfocal images of the implanted PRCs are shown. The absence of Oct4 (Abeam ab27985) expression in the implanted HuNu+ PRCs indicates a lack of pluripotency at all time points in the post-transplant study (P35, P60, and P120). A total of three animals were used at each time point. Cultured GMP1 iPSCs were used as a positive control for Oct4 expression, with subcellular compartment localization determined by WGA-647 (Thermo Scientific W32466) and DAPI staining (small images in the lower middle figure).

[0248] Figure 12A A schematic diagram of the procedure for preparing PRC cells is shown. There are approximately 200M cells in P4-PRC(DS), and the scale-up to 500-600M cells is currently scalable in P4-PRC(DS). Preferred methodological changes in the procedure include: i) changing the culture dishes (to T-flasks), and ii) replacing 2D with 3D lifting, and changing from mechanical dissociation to enzymatic dissociation (expected to be implemented in GLPTox studies and first-in-human studies). RIM : Rescue induction medium; NDM : Neural differentiation culture medium; ICC Immunocytochemistry; IFA Immunofluorescence assay.

[0249] Figure 12B A schematic diagram of the procedure for preparing PRC cells is shown, which includes a cryopreservation step between P3 and P4. Approximately 200M cells are present in P4-PRC(DS), and the scale-up is currently possible to 500-600M cells based on P4-PRC(DS). Methodological changes implemented during development include: i) changing the culture dishes (to T-flasks), ii) replacing 2D with 3D lifting, and changing from mechanical dissociation to enzymatic dissociation (expected to be implemented in GLP Tox studies and first-in-human studies), and iii) implementing the P3-sphere Cell Stock intermediate. RIM : Rescue induction medium; NDM : Neural differentiation culture medium; ICC Immunocytochemistry; IFA Immunofluorescence assay.

[0250] Figure 12C A schematic diagram of the PRC production method is shown.

[0251] Figure 13 A schematic diagram showing the production, remodeling, and injection of PRC cells is displayed. CZ Crystal Zenith; CRF Rate-controlled refrigeration unit.

[0252] Figure 14A The study demonstrates the cell viability of photoreceptor rescued cells (PRCs) prepared in formulations containing 2.5% rHA, DPBS containing Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO or 10% DMSO) after thawing, as well as in commercially available formulations containing 10% DMSO. Cell viability of PRC cells formulated in the CS10 cell cryopreservation formulation (for comparison) after thawing.

[0253] Figure 14B The percentage of P4 PRC cells that survived cryopreservation under different conditions is shown. Values ​​are shown as mean ± SD.

[0254] Figure 14C Displayed in the selected Figure 14B Percentage of P4PRC cells survived cryopreservation under different conditions. Values ​​are shown as mean ± SD.

[0255] Figure 15A A comparison of OMR responses in animals subretinally injected with 100,000 or 200,000 GB2R PRC cells is shown. Twelve spatial frequencies (SPs) were implemented, and OMR responses (tracking intensity) were recorded in animals subretinally injected with 100k (left panel) or 200k (middle panel) PRC cells. A response of 1.2 on the Y-axis is the threshold for the presence of OMR (indicated by the green dashed line). Any value below 1.2 indicates that the eye could not track. At P35, P42, and P50, the control eye was blind, while the eye injected with 200k PRC cells consistently showed a larger and wider amplitude curve than the 100k group. A rightward shift of the SP threshold indicates better visual acuity in the 200k eye. Survival rate: 80.6% / 76.64% (manual, 2 vials).

[0256] Figure 15B A schematic timeline of injections and analyses in rd10 mice is shown.

[0257] Figure 15CICC analysis confirmed that ONL thickness was greater in the 200k group than in the 100k group. Quantification of ONL thickness at implantation sites versus non-implantation sites was performed in animals receiving 100k and 200k cells. This data is consistent with the hypothesis that higher doses lead to greater potency. ONL thickness at P70: In the 200k group, the OMR curve consistently showed a larger amplitude or wider band compared to the 100k group, while in the 200k group, ONL thickness remained larger at P70 compared to the 100k group. This data is consistent with the hypothesis that greater retinal coverage (through higher doses) leads to greater potency.

[0258] Figure 16A OMR recorded in rd10 mice injected with a 300k cryopreserved PRC formulation (cryo-PRC) is shown (identical to previous rd10 mice, injected subretinally at P14). Cryo-PRC was functionally effective at P35 and P42. However, at P49, OMR was absent in both the mediator and cryo-PRC eyes. Cryo: cryoprotective formulation with PRC or mediator. Cell viability: 69.56% (manual). N = 8.

[0259] Figure 16B OMR analysis of RCS rats injected with 100,000 cells / eye of GB2R-PRC (CN2 batch); GB2R-PRC (P3INT DS); or GB2R-PRC (freezing: cryoprotectant) at P25 is presented. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT) to compare eyes that were not injected with and those injected with GS2 mediator.

[0260] Figure 16C ERG analysis is shown for RCS rats injected with 100,000 cells / eye of GB2R-PRC (CN2 batch); GB2R-PRC (P3INT DS); or GB2R-PRC (freezing: cryoprotectant) at P25. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT) to compare eyes that were not injected with and those injected with GS2 mediator.

[0261] Figure 17A and 17B OCT showing an eye treated with GS2 buffer and supplemented IMT. Figure 17A This includes GS2+ / freezing buffer (1:4) without IMT (left column) and GS2+ / freezing buffer (1:4) with Dex (right column). Figure 17BThe left column shows GS2+ / freezing buffer (1:4) with Dex / CsA and BSS without IMT (right column).

[0262] Figures 18A-18C The ERG reactions of group 1 (GS2+ / freezing buffer (1:4), without IMT); group 2 (GS2+ / freezing buffer (1:4), with DEX); group 3 (GS2+ / freezing buffer (1:4), with Dex / CsA); and group 4 (BSS without IMT) are shown. Figure 18A The α-wave response was shown. Figure 18B It shows the scotopic b-wave response. Figure 18C The photopic b-wave response was displayed.

[0263] Figure 19 The retinal morphology of group 1 (GS2+ / cryobuffer (1:4), without IMT); group 2 (GS2+ / cryobuffer (1:4), with DEX); group 3 (GS2+ / cryobuffer (1:4), with Dex / CsA); and group 4 (BSS without IMT) are shown.

[0264] Figure 20 OCT images of group 1 (GS2+ / cryobuffer (1:4), without IMT); group 2 (GS2+ / cryobuffer (1:4), with DEX); group 3 (GS2+ / cryobuffer (1:4), with Dex / CsA); and group 4 (BSS without IMT) are shown.

[0265] Figure 21 The ONL thicknesses of Group 1 (GS2+ / freezing buffer (1:4), without IMT); Group 2 (GS2+ / freezing buffer (1:4), with DEX); Group 3 (GS2+ / freezing buffer (1:4), with Dex / CsA); and Group 4 (BSS without IMT) are shown.

[0266] Figure 22 The study demonstrates intraretinal migration following PRC injection of cells that were frozen in an intermediate step using P3 and further differentiated into P4 after thawing.

[0267] Figure 23 The OMR readings (readings) at post-injection time points P35, P42, and P49 are shown, comparing P4(d) and P4(i).

[0268] Figure 24Immunohistochemistry of the inner plexiform layer (IPL) was shown, which revealed intraretinal migration between P4(d) and P4(i) injections.

[0269] Figure 25 Immunohistochemistry of the IPL was shown, which demonstrated enhanced migration to the IPL after injection of P4(i)PRC.

[0270] Figure 26 The OMR readings for group 1, where high doses of P4(d) or P4(i) were administered, are shown.

[0271] Figure 27 The OMR readings for group 1, where high doses of P4(d) or P4(i) were administered, are shown.

[0272] Figure 28 Showing with (using as) Figure 12A-12B Compared to the CMC control prepared in the culture dish shown, the one injected with... Or the OMR readings of the eye of a PRC prepared from a PDL-pre-coated bottle.

[0273] Figure 29 Retinal OCT images showing D7 and D38 (days post-injection) are compared. Preparation of PRC using PDL pre-coated bottles.

[0274] Figures 30A-30B Immunohistochemical staining of CAR / HuNu was shown. Figure 30A and Figure 30B This immunohistochemical staining method compared the use of CMC control with... Alternatively, PRC prepared from PDL pre-coated bottles. Figure 30A and Figure 30B All show usage The prepared PRC has a larger implant and better cone cell retention.

[0275] Figure 31 Immunohistochemical staining of GFAP and HuNu was shown, comparing the results with CMC control. Alternatively, PRC can be prepared using PDL pre-coated bottles.

[0276] Figure 32 Immunohistochemical staining of IBA1 and HuNu is shown, which compares the use of IBA1 and HuNu with the CMC control. Alternatively, PRC can be prepared using PDL pre-coated bottles.

[0277] Figure 33Immunohistochemical staining of Ki67 and HuNu is shown, comparing the results with CMC controls. Alternatively, PRC prepared from PDL pre-coated bottles.

[0278] Figure 34 Immunohistochemical staining of OCT4 and HuNu is shown, comparing the results with CMC controls. Alternatively, PRC can be prepared using PDL pre-coated bottles.

[0279] Figure 35 This diagram illustrates the experimental design for delayed PRC injection in RCS rats. Injections were administered at P25, P45, or P60 after disease onset. OMR and ERG readings were collected from P60 to P150.

[0280] Figure 36A and 36B The OMR (Operational Morphological Ratio) was displayed at P60, P90, P120, and P150 after each injection time point. Figure 36A ) and ERG ( Figure 36B )reading.

[0281] Figure 37 Immunohistochemical staining of STEM21 was shown in RCS rats after delayed injection of PRC at P45.

[0282] Figure 38A and 38B The quantitative data of subretinal PRC implantation at injection time points P25, P45, and P60 are presented. Figure 38A The maximum implant length is displayed, and Figure 38B The maximum implant / retinal length ratio is shown.

[0283] Figure 39A and 39B The quantification of ONL retention at injection time points P25, P45, and P60 is shown. Figure 39A The maximum holdable ONL length is shown, and Figure 39B The maximum ONL / retinal length ratio is shown. Gb2R-GMP-MCBPRC-P4 RCS injected at P25 (P150); Gb2R-GMP-MCBPRC-P4 RCS injected at P45 (P150); Gb2R-GMP-MCBPRC-P4 RCS injected at P60 (P150).

[0284] Figure 40A-40D In the eyes of those injected with PRC, the optomotor response was maintained. At birth, P28 ( Figure 40A P35 Figure 40B P42 Figure 40C) and P49 ( Figure 40D OMR response was measured. Statistically significant differences were observed in group 1 OD relative to the control eye (mediator or uninjected), while only a trend relative to the control eye was observed in group 2 OD. No statistically significant differences were observed between the cell injection groups. *p<0.05, **p<0.01.

[0285] Figure 41A-41F It showed that group D0 (OD) ≥ 70% Figure 41A ); D0 group 2 (OD) ≤ 60% ( Figure 41B ); D9 group 1 (OD) ≥ 70% ( Figure 41C ); D9 group 2 (OD) ≤ 60% ( Figure 41D Group D35 1 (OD) ≥ 70% Figure 41E ); and D35 group 2 (OD) ≤ 60% ( Figure 41F Retinal OCT images.

[0286] Figure 42A and 42B Figure 42A Positive HNA staining was observed in the cells injected into the eyes of groups 1 and 2. Red = HNA, blue = DAPI. Scale bar is 50 μm. Figure 42B The results showed that group 1 (OD) > 70% and group 2 (OD) < 60%.

[0287] Figures 43A-43F Examples of subretinal PRC implants associated with elongated cone cell morphology were found in the treated eyes of groups 1 and 2. Injection of ≥70% live PRC ( Figure 43A ≤60% live PRC ( Figure 43B ) or medium ( Figure 43C Example image of cone cell morphology in the eye. Green = CAR, red = HNA. Scale bar is 50 μm. Figure 43D Quantification of cone cell length is shown. One-way ANOVA (p<0.05). Error bars are SEM. CAR staining for all eyes, see [link to CAR staining chart]. Figure 43E (Group 1) and Figure 43F (Group 2)

[0288] Figure 44 The subretinal PRC implant was shown to be associated with increased ONL thickness at the implantation site. One-way ANOVA (p = 0.21). Error bars are represented by SEM.

[0289] Figure 45 Miller glial cell immunoreactivity with GFAP was significant in all studies. Example GFAP staining in the study group's eyes showed strong vertical banding characteristics of GFAP morphology in the diseased retina. Green = GFAP, Red = HNA. Scale bar: 50 μm.

[0290] Figures 46A-46J The image shows an OCT image at D28, which reveals surgical-related damage at the injection site. The OCT image shows the eye compared to the uninjected (naive) group 4. Figure 46J Compared to group 1, group 1 Figure 46A , Figure 46B and Figure 46C Group 2 Figure 46D , Figure 46E and Figure 46F ) and group 3 ( Figure 46G , Figure 46H and Figure 46I Surgical damage and accumulation of subretinal material in the injected eyes. The presence of subretinal material in the eyes of group 1 indicates that the subretinal masses (masses) observed by OCT in the cells-injected eyes (groups 2 and 3) can contain a large number of host cells. In both eyes of individual female mice from group 1, various medium-sized areas of retinal atrophy (red dashed lines) were observed at the injection center.

[0291] Figures 47A-47C The ERG records (D29-32) showed no statistically significant differences between the study groups. Figure 47A The a-wave amplitude measurement via two-factor ANOVA is shown; Figure 47B The scotopic b-wave amplitude measurement via two-factor ANOVA is shown; and Figure 47C The amplitude measurement of the photopic b-wave is shown by one-way ANOVA.

[0292] Figures 48A-48F HNA immunostaining and retinal laminar structures at the injection center are shown. In group 1 ( Figure 48A and Figure 48B Group 2 Figure 48C and Figure 48D ) and group 3 ( Figure 48E and Figure 48F No nuclear HNA staining was detected in groups 2 and 3 (individual HNA channels, HNA and DAPI overlay, bright field). However, DAPI-containing staining was found in groups 2 and 3. + Subretinal implant-like structures of cells and autofluorescent debris (*)( Figure 48C , Figure 48E and Figure 48F Further bright autofluorescent fragments (#) were observed in groups 1-3, which were located subretinal or embedded in the outer retina. Figure 48B and Figure 48D Consistent with surgical damage, at the injection center, the retinal layers in all groups were locally disrupted. Figures 48A-48F(HNA and DAPI superposition). In many cases, the brightest autofluorescent structure appears yellow / pigmented on bright-field imaging. Figure 48B (Examples in the text). Red = HNA, Blue = DAPI. Scale bar is 100μm.

[0293] Figures 49A-49H The study showed that Miller glial cells exhibited GFAP immunoreactivity concentration (enrichment) at the injection site in the injected eye, consistent with injection-related damage. (AH) Miller glial cell reactivity was assessed using GFAP immunostaining: Group 1 ( Figure 49A and Figure 49B Group 2 Figure 49C and Figure 49D Group 3 Figure 49E and Figure 49F ) and group 4 ( Figure 49G and Figure 49H (Separate GFAP channel; GFAP and DAPI overlay). Figure 49C , 49E and 49F and Figure 49A In contrast to Miller glial cell hypertrophy, GFAP also marks subretinal cell clusters with a thin (fine) curled morphology. Green = GFAP, blue = DAPI. Scale bar is 100 μm.

[0294] Figures 50A-50D Showing the difference between the uninjected eyes in group 4 ( Figure 50D Compared to group 1, group 1 Figure 50A Group 2 Figure 50B Group 3 Figure 50C Elevated Iba1 immunoreactivity at the injection site in the eye was observed, consistent with inflammation following injection-related lesions. #Autofluorescent fragments. Green = Iba1, Blue = DAPI. Scale bar: 100 μm. Detailed Implementation

[0295] This invention relates to photoreceptor rescue cell compositions comprising a variety of heterologous photoreceptor rescue cells with unique biomarker profiles. The heterologous photoreceptor rescue cell (PRC) compositions of this invention cumulatively express the biomarkers: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. The photoreceptor rescue cell compositions of this invention comprise inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and hybrid neurons. PRCs can be phenotype-defined, for example, by the expression of intracellular or extracellular biomarkers. Photoreceptor rescue cells may also be characterized by the expression or absence of ocular progenitor cell biomarkers, neural biomarkers, and / or rod / cone photoreceptor biomarkers.

[0296] These photoreceptor rescue cell compositions can be generated from early progenitor cells (including pluripotent stem cells, such as embryonic stem cells (ESCs), cells undergoing transdifferentiation or partial reprogramming into a progenitor state, and induced pluripotent stem cells (iPSCs)) through in vitro differentiation. This invention provides compositions of photoreceptor rescue cells from original sources that are not yet available or unavailable, and thus possess a unique spectrum of non-natural biomarkers.

[0297] The photoreceptor rescue cell composition can be used in a variety of in vivo and in vitro methods. For example, the photoreceptor rescue cells can be used to treat retinal conditions, including but not limited to macular degeneration (including age-related macular degeneration (AMD), such as dry and wet AMD, retinitis pigmentosa, and geographic atrophy secondary to AMD). The photoreceptor rescue cells can be used in vitro for screening assays to identify hypothetical therapeutic or preventative treatment candidates.

[0298] Functionally, PRC has demonstrated the ability to treat ocular diseases and improve visual acuity in subjects with retinal diseases or conditions, for example, by increasing the secretion of neuroprotective factors, by preventing or slowing the loss of photoreceptor cells, by increasing phagocytic activity (e.g., the ability to phagocytose the extracellular segments of photoreceptor cells), by inhibiting microglia activation (e.g., by increasing the expression of CNFT and / or MIF), by reducing oxidative stress (e.g., by increasing the expression of CNFT), by increasing the expression of antiapoptotic factors, and / or by preventing outer nuclear layer degeneration (denaturation).

[0299] definition

[0300] As defined herein, the singular form is provided for illustrative purposes, but the singular form may also be applied to the plural form of the phrase. The following definitions are intended to supplement the conventional definitions of terms that will be understood by one of ordinary skill in the art.

[0301] As used herein, the term "photoreceptor rescue cell composition" refers to a composition comprising a heterologous combination of photoreceptor rescue cells (PRCs). As used herein, the photoreceptor rescue cell composition comprises heterologous cells, including but not limited to inhibitory neurons, excitatory neurons, hybrid neurons, progenitor cells, and astrocytes. The cells in the photoreceptor rescue cell composition cumulatively express at least 2, 3, 4, 5, 6, or 7 of the biomarkers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and / or NFIA. In one embodiment, the cells in the composition cumulatively express at least FOXG1 and MAP2. In another embodiment, the cells in the composition cumulatively express at least each of the biomarkers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

[0302] As used herein, the excitatory neurons in the photoreceptor rescue cell composition of the present invention refer to cells expressing one or more of the markers NEUROD2, NEUROD6, SLA, NELL2, and / or SATB2.

[0303] As used herein, the inhibitory neurons in the photoreceptor rescue cell composition of the present invention refer to cells expressing one or more of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0304] As used herein, alternative neurons refer to cells expressing one or more of the biomarkers MEIS2, PBX3, GRIA2, and CACNA1C in a photoreceptor rescue cell composition.

[0305] As used herein, when referring to a specific cell type contained in the photoreceptor rescue cell composition of the present invention, progenitor cells refer to cells expressing one or more of the markers VIM, MKI67, CLU, and GLI3.

[0306] As used herein, the photoreceptor rescue cell composition of the present invention contains one or more of the astrocyte expression markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0307] The photoreceptor rescue cell composition may also contain photoreceptor rescue cells that express ocular progenitor cell markers, rod / cone photoreceptor cell markers, and / or neuronal markers.

[0308] Exemplary ocular progenitor cell markers expressed by cells in the photoreceptor rescue cell compositions of the present invention include PAX6, LHX2, SIX3, NES, or SOX2. Some ocular progenitor cell markers, such as PAX6, LHX2, SOX2, and NES, are widely expressed in a variety of neuronal progenitor cells and are also highly expressed in the PRCs of the compositions of the present invention. Accordingly, in one embodiment, the compositions of the present invention comprise heterologous photoreceptor rescue cells cumulatively expressing at least PAX6, LHX2, SOX2, NES, and optionally, SIX3. However, certain genes that are ocular progenitor cell-specific markers in neuronal progenitor cells, such as RAX, SIX6, and TBX3, are rarely or not expressed in the PRCs of the compositions of the present invention. Therefore, in one embodiment, the compositions of the present invention substantially do not contain photoreceptor rescue cells expressing RAX, SIX6, or TBX3, or ordinary cells (cell totality). In one embodiment, the compositions of the present invention substantially do not contain photoreceptor rescue cells expressing RAX and / or TBX3, or ordinary cells.

[0309] Exemplary rod / cone photoreceptor markers expressed by cells in the photoreceptor rescue cell compositions of the present invention include Mash1 / ASCL1 and RORB. Some rod / cone photoreceptor markers, such as Mash1 / ASCL1 and RORB, are widely expressed in neuroectoderm and neuroectoderm-derived neural progenitor cells, and are highly expressed in the PRCs of the compositions of the present invention. Accordingly, in one embodiment, the compositions of the present invention comprise heterologous photoreceptor rescue cells cumulatively expressing at least Mash1 / ASCL1 and RORB. However, certain genes that are specific rod / cone photoreceptor markers in neuroectoderm and neuroectoderm-derived neural progenitor cells, such as NRL and NR2E3, are rarely or not expressed in the PRCs of the compositions of the present invention. Therefore, in one embodiment, the compositions of the present invention substantially do not contain photoreceptor rescue cells, or normal cells, expressing CRX, RHO, OPN1SW, PDE6B, RCRRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0310] Exemplary neuronal markers expressed by cells in the photoreceptor rescue cell compositions of the present invention include TUBB3, NFIA, DCX, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. Some neuronal markers, such as TUBB3, NFIA, DCX, and NFIB, are robustly expressed in the PRCs of the compositions of the present invention. Accordingly, in one embodiment, the compositions of the present invention comprise heterologous photoreceptor rescue cells that cumulatively express TUBB3, NFIA, DCX, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. However, certain genes that are specific neuronal markers in neuronal progenitor cells, such as OTX2, are expressed at low levels in the PRCs of the compositions of the present invention. Therefore, in one embodiment, the compositions of the present invention substantially do not contain photoreceptor rescue cells expressing OTX2, or ordinary cells.

[0311] In other embodiments, the compositions of the present invention are substantially free of photoreceptor rescue cells, or normal cells, that express the pluripotent markers SSEA4 and / or OCT4.

[0312] “Retinal cells” refers to the nerve cells of the eye, which are layered into three nuclear layers consisting of photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, Miller glial cells, and ganglion cells. Retinal cells include neuroretinal cells (also referred to herein as photoreceptor cells), retinal pigment epithelial (RPE) cells, iris epithelial cells, and their precursors. As used herein, “retinal pigment epithelial (RPE) cells” refers to the cells of the outermost layer of the retina. RPE cells function to support retinal photoreceptor cells and are responsible for the metabolic digestion of discarded neuroretinal outer segments. As used herein, “neuroreretinal cells” refers to the layer of photoreceptor cells (i.e., rods and cones) located beneath the RPE cell layer in the retina. Neuroretinal (NR) cells are modified light-sensitive neurons. As used herein, the term “cumulative” and its grammatical variations refer to marker expression in a heterologous cell population in a composition. Specifically, cumulative expression refers to a composition in which at least one cell expresses one of its markers, resulting in a totality of cells in the composition expressing all listed genes. For example, the description "cumulative expression of FOXG1 and MAP2 in multiple heterologous cells" could refer to a composition in which at least one cell expresses FOXG1 and at least one cell expresses MAP2, or a composition in which at least one single cell expresses FOXG1 and MAP2, thereby causing multiple cells in the composition to cumulatively express both FOXG1 and MAP2. As a further example, the description "cumulative expression of FOXG1, MAP2, STMN2, and DCX in multiple heterologous cells" could refer to, but is not limited to, a composition in which at least one cell expresses FOXG1, at least one cell expresses MAP2, at least one cell expresses STMN2, and at least one cell expresses DCX. Alternatively, this phrase is intended to cover a composition in which at least one cell expresses FOXG1 and MAP2 and at least one other cell expresses STMN2 and DCX. As a further example, this phrase is intended to cover a composition in which at least one cell expresses FOXG1 and STMN2, at least one cell expresses DCX, and at least one cell expresses MAP2.

[0313] The term "human neural stem cell" or "hNSC" is used herein to refer to cells that self-renew and are generated through neurogenesis throughout adult life. These pluripotent adult stem cells produce the major phenotypes of the nervous system, differentiating into neurons, astrocytes, and oligodendrocytes.

[0314] The term “neural progenitor cell” or “NPC” is used herein to refer to progenitor cells of the central nervous system (CNS) that produce a variety (if not all) of the glial cell and neuronal cell types present in the CNS.

[0315] The term “multiple” is used in this document to refer to a state of multiples, i.e., at least two, such as cell types, for example, multiple heterologous photoreceptor rescue cells.

[0316] The terms “substantially free” or “essentially free” are used herein to mean that more than about 95%, 96%, 97%, 98%, 99%, or 100% of the cells are free. For example, the phrase “the composition thereof is substantially free of cells expressing progenitor cell markers RAX, SIX6, and / or TBX3” means that at least 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition do not express any of the aforementioned markers.

[0317] The compositions of the present invention are characterized in that at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the cells in the composition are PRC. In some embodiments, the methods described herein can produce the compositions of the present invention, characterized in that about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, or 55% of the cells in the composition. About 100%, about 55% to 95%, about 55% to 90%, about 55% to 85%, about 55% to 80%, about 55% to 75%, about 55% to 70%, about 55% to 65%, about 55% to 60%, 60% to 100%, about 60% to 95%, about 60% to 90%, about 60% to 85%, about 60% to 80%, about 60% to 75%, about 60% to About 70%, about 60% to about 65%, 65% to about 100%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, 75% to about 1 00%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, 85% to about 100%, about 85% to about 95%, about 85% to about 90%, 90% to about 100%, about 90% to about 95%, or about 95% to about 100% of cells are PRC.

[0318] The compositions of the present invention are characterized in that at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% of the cells in the composition are alive. In some embodiments, the methods described herein can produce the compositions of the present invention, characterized in that at least 50% to about 85%, at least 50% to about 80%, at least 50% to about 75%, at least 50% to about 70%, at least 50% to about 65%, at least 50% to about 60%, at least 50% to about 55%, at least 55% to about 85%, at least 55% to about 80%, at least 55% to about 75%, at least 55% to about 70%, at least 55% to about 65%. Approximately 55% to 60%, approximately 60% to 85%, approximately 60% to 80%, approximately 60% to 75%, approximately 60% to 70%, approximately 60% to 65%, approximately 65% ​​to 85%, approximately 65% ​​to 80%, approximately 65% ​​to 75%, approximately 65% ​​to 70%, approximately 70% to 85%, approximately 70% to 80%, approximately 70% to 75%, approximately 75% to 85%, approximately 75% to 80%, or approximately 80% to 85% of the cells are alive.

[0319] As used herein, the phrase “basically pure photoreceptor rescue cell composition” refers to a heterologous photoreceptor rescue cell composition (e.g., a composition containing cells) comprising a significant percentage of cells sharing the same expression when it relates to a specific biomarker profile, such as at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. All or most PRC cells possess a substantially equal capacity to contact their surrounding medium, and therefore to the factors within that medium, leading to those progenitor cells differentiating at similar frequencies and to similar degrees. This similar differentiation timeline of the PRC cell population indicates that these cells are synchronized. In some cases, PRCs may also be cell cycle synchronized. This synchronicity results in the emergence of homologous or nearly homologous cell subpopulations when correlated with a specific biomarker expression profile.

[0320] For example, purity can refer to the percentage of photoreceptor cells in the composition that exhibit a specific expression profile. For instance, for the expression of FOXG1 and MAP2, a substantially pure photoreceptor rescue cell composition can refer to a composition of heterologous photoreceptor rescue cells in which at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, about at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the photoreceptor rescue cells express FOXG1 and MAP2. In some embodiments, the heterologous photoreceptor rescue cell composition is at least 50% pure, at least 55% pure, at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, about at least 80% pure, at least 85% pure, at least 90% pure, or at least 95% pure. In some embodiments, the cells are approximately 50% to 95% pure, approximately 55% to 95% pure, approximately 60% to 95% pure, approximately 70% to 95% pure, approximately 75% to 95% pure, approximately 80% to 95% pure, approximately 85% to 95% pure, approximately 90% to 95% pure, approximately 50% to 90% pure, approximately 55% to 90% pure, or approximately 60% pure. Pure to about 90% pure, about 65% pure to about 90% pure, about 70% pure to about 90% pure, about 75% pure to about 90% pure, about 80% pure to about 90% pure, about 85% pure to about 90% pure, about 50% pure to about 85% pure, about 55% pure to about 85% pure, about 60% pure to about 85% pure, about 65% pure to about 85% pure, about 70% pure to about 85% pure, about 75% pure to about 85% pure Approximately 80% pure to approximately 85% pure, approximately 50% pure to approximately 80% pure, approximately 55% pure to approximately 80% pure, approximately 60% pure to approximately 80% pure, approximately 65% ​​pure to approximately 80% pure, approximately 70% pure to approximately 80% pure, approximately 75% pure to approximately 80% pure, approximately 50% pure to approximately 75% pure, approximately 55% pure to approximately 75% pure, approximately 60% pure to approximately 75% pure, approximately 65% ​​pure to approximately 75% pure, approximately 70% pure About 75% pure, about 50% pure to about 70% pure, about 55% pure to about 70% pure, about 60% pure to about 70% pure, about 65% pure to about 70% pure, about 70% pure to about 75% pure, about 50% pure to about 65% pure, about 55% pure to about 65% pure, about 60% pure to about 65% pure, about 50% pure to about 60% pure, about 55% pure to about 60% pure, or about 50% pure to about 55% pure.

[0321] As used herein, most cells represent at least 50%, and according to embodiments, may include at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of cells.

[0322] The purity achievable using the method of this invention is particularly important when these cell populations will be used in vivo for therapeutic or preventative purposes. The ability to obtain highly pure cell populations avoids additional operations, such as enrichment or selection steps, which could result in unnecessary cell loss. This is especially important when the cell population may be small or the cell number may be limited.

[0323] For example, purity levels can be quantified by determining the proportion of cells expressing one or more biomarkers, such as those of PRC (including those identified in this application), relative to the total number of cells in the preparation, for example, by detecting cells that express or do not express said one or more biomarkers. Optionally, the expression of biomarkers indicating non-PRC cells can also be detected, thereby aiding in the detection and / or quantification of said cells. Exemplary methods that can be used to detect and / or quantify biomarker expression include, without limitation, flow cytometry, fluorescence-activated cell sorting (FACS), immunohistochemistry, in situ hybridization, scRNA-seq, immunofluorescence, single-cell proteomics (e.g., by EC-MS), possibly single-cell metabolomics, lipidomics, scPCR, and other suitable methods known in the art. Optionally, the purity of the composition can be determined by the percentage of live cells present in the composition.

[0324] "Embryoid bodies" refer to clumps or clusters of pluripotent cells (e.g., iPSCs or ESCs) that can be formed by culturing pluripotent cells under non-adherent conditions, such as on a low-adherent matrix or in a "hanging drop." In these cultures, pluripotent cells can form clumps or clusters of cells that are called embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 February; 6(2):88-95, the entire contents of which are incorporated herein by reference. Typically, embryoid bodies initially form as solid clumps or clusters of pluripotent cells, and over time, some embryoid bodies begin to include fluid-filled cavities; the former are referred to in the literature as "simple" EBs, while the latter are referred to as "cystic" embryoid bodies.

[0325] This disclosure provides compositions of heterologous PRCs, such as (but not limited to) pluripotent stem cells (e.g., ESCs and iPSCs), based on the ability of the disclosed methods to directly differentiate progenitor cells. As used herein, directed differentiation aims to differentiate a population of progenitor cells into or towards a desired lineage due to factors or other stimuli provided to these progenitor cells, thereby avoiding differentiation into other undesirable lineages and thus avoiding potential lineage contamination. In some embodiments, the methods provided herein drive, for example, the differentiation of pluripotent stem cells into PRCs without generating embryoid bodies (EBs). As described below, EBs are three-dimensional clusters of cells (including progenitor cells) that can form during the differentiation of pluripotent stem cells (including, but not limited to, embryonic stem (ES) cells and iPSCs) and typically contain mesoderm, ectoderm, and endoderm lineages. The three-dimensional nature of EBs can produce environments different from those occurring in the non-EB-based methods described herein, including different cell-cell interactions and different cell-cell signal transduction. Furthermore, cells within the EB may not all receive similar doses of exogenously added agents, such as differentiation factors present in the surrounding medium, and this can lead to a variety of differentiation events and decisions during EB development.

[0326] Conversely, the PRC cell culture method of the present invention does not require and preferably avoids EB formation. Instead, these methods culture cells under conditions that provide equal contact between the cells and the surrounding medium (including factors in the medium). In some embodiments, for example, under adherent conditions, PRCs can grow as a monolayer or near-monolayer adhered to the culture surface. In some embodiments, the culture methods disclosed herein provide for culturing PRCs under non-adherent or low-adherence conditions (e.g., suspension).

[0327] As used in the art, the term “embryonic stem cell” (ES cell or ESC) is used herein. This term includes cells derived from the inner cell mass of a human blastocyst or morula, including those cells that have been continuously passaged as a cell line. ES cells can be derived from the fertilization of an egg and sperm, and from methods using DNA, nuclear transfer, parthenogenesis, or by generating ES cells that are homozygous in the HLA region. ES cells are also derived from cells of conidia, blastomeres, or blastocyst-stage mammalian embryos produced by sperm-egg fusion, nuclear transfer, parthenogenesis, androgenesis, or chromatin reprogramming and subsequent integration of reprogrammed chromatin into the plasma membrane to generate cells. Regardless of their origin or the specific method used to generate them, embryonic stem cells can be identified based on the following: (i) their ability to differentiate into cells with all three germ layers, (ii) expression of at least OCT 4 and alkaline phosphatase, and (iii) their ability to generate teratomas when transplanted into immunodeficient animals. Embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human ES cells (“ESC” or “hES cells”), such as MA01, MA09, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Other exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that can be used is the MA09 cell. The isolation and preparation of MA09 cells were previously described in Klimanskaya et al. (2006), “Human Embryonic Stem Celllines Derived from Single Blastomeres.” Nature 444:481-485. The human ES cells used in exemplary embodiments of the present invention can be obtained and maintained according to GMP standards.

[0328] The term “ES cells” does not mean, and should not be meant, to mean that the cells are produced by destroying the embryo. Instead, several methods are available and can be used to produce ES cells without destroying the embryo, such as a human embryo. For example, ES cells can be produced from a single blastomer from an embryo in a manner similar to blastomer extraction used for preimplantation genetic diagnosis (PGD). Examples of these cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. An exemplary human embryonic stem cell line that can be used is the MA09 cell. The isolation and preparation of MA09 cells were previously described in Klimanskaya et al. (2006), “Human Embryonic Stem Cell lines Derived from Single Blastomeres.” Nature 444:481-485. See also Chung et al. 2008, Cell Stem Cell, 2:113. All of these lines are produced without destroying the embryo. As used herein, without regard to methods of obtaining (derived) pluripotent stem cells, the term "pluripotent stem cell" includes, but is not limited to, tissue-derived stem cells, embryonic stem cells, embryonic-derived stem cells, induced pluripotent stem cells, and stimulation-triggered pluripotency acquisition (STAP) cells. Without regard to methods used to generate these cells, the term also includes pluripotent stem cells possessing the functional and phenotypic characteristics of the aforementioned cells. Pluripotent stem cells are functionally defined as stem cells possessing the following characteristics: (a) the ability to induce teratomas when transplanted into immunodeficient (SCID) mice; (b) the ability to differentiate into cell types possessing all three germ layers (e.g., the ability to differentiate into ectodermal, mesodermal, and endoderm cell types); (c) the expression of one or more markers of embryonic stem cells (e.g., expression of OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REXI, etc.); and (d) the ability to self-renew. The term "pluripotent" refers to the ability of cells to form all organismal or somatic cell (i.e., embryonic) lineages. In some embodiments, pluripotent stem cells express one or more markers selected from the following: OCT4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst-stage embryos, and embryonic stem cells derived from one or more blastomeres of cleavage-stage or morula-stage embryos (optionally without destroying the rest of the embryo). Other exemplary pluripotent stem cells include induced pluripotent stem cells (iPSCs) generated by reprogramming somatic cells through the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells.As used herein, without regard to the method of obtaining pluripotent stem cells, the terms "pluripotent stem cell," "PS cell," or "PSC" include embryonic stem cells, induced pluripotent stem cells, and embryonic-derived pluripotent stem cells. For example, embryonic stem cells and induced pluripotent stem cells are a class of pluripotent stem cells capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum ranging from incomplete or partially pluripotent cells that cannot produce a complete organism to more primitive, more potent cells (e.g., embryonic stem cells) capable of producing a complete organism. Exemplary pluripotent stem cells can be produced using methods known in the art, for example. Exemplary pluripotent stem cells include, but are not limited to, embryonic stem cells derived from the inner cell mass of blastocyst embryos, embryonic stem cells derived from one or more blastomeres of cleavage-stage or morula-stage embryos (optionally without destroying the rest of the embryo), induced pluripotent stem cells generated by reprogramming somatic cells to a pluripotent state, and pluripotent cells generated from embryonic embryo (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF). These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis.

[0329] In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, combinations of OCT 4 (sometimes referred to as OCT 3 / 4), SOX2, c-Myc, and KLF4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, combinations of OCT 4, SOX2, Nanog, and Lin28. In some embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, other reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. Induced pluripotent stem cells are functionally defined and include cells reprogrammed using any of a variety of methods (integrative vectors, non-integrative vectors, chemical methods, etc.). Pluripotent stem cells can be genetically modified or otherwise modified to enhance lifespan, potency, homing, prevent or reduce alloimmune responses, or deliver desired factors to cells differentiated from these pluripotent cells (e.g., photoreceptor rescue cells, photoreceptor progenitor cells, rod cells, cone cells, and other cell types described herein, such as in the examples). In one embodiment, pluripotent stem cells can be genetically engineered or otherwise modified, for example, to enhance lifespan, potency, homing, prevent or reduce immune responses, or to deliver desired factors to cells derived from these pluripotent cells (e.g., photoreceptor rescue cells or cells present in a photoreceptor rescue cell composition). For example, pluripotent stem cells and the resulting differentiated cells can be engineered or otherwise modified to lack or have reduced expression of class I β2 microglobulin genes, including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, TAP1, TAP2, Tapasin, CTIIA, RFX5, TRAC, or TRAB genes. As described in WO2012145384 and WO2013158292 (the entire contents of which are incorporated herein by reference), in some embodiments, the cells, such as pluripotent stem cells and the resulting differentiated cells, such as photoreceptor rescue cells or cells present in a photoreceptor rescue cell composition, contain genetically engineered disruption of the β-2 microglobulin (B2M) gene.In some embodiments, the cell further comprises a polynucleotide encoding a class I single-stranded fusion human leukocyte antigen (HLA) protein, said protein comprising at least a portion of a B2M protein covalently linked directly or via a linker sequence to at least a portion of the HLA-1α chain. In some embodiments, the HLA-1α chain is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the cell comprises genetically engineered disruption of a class II HLA-related gene. In some embodiments, the class II HLA-related gene is selected from proteins containing factor X-associated ankylosing protein (RFXANK), factor 5 (RFX5), factor X-associated protein (RFXAP), class II transactivator (CIITA), HLA-DPA (α chain), HLA-DPB (β chain), HLA-DQA, HLA-DQB, HLA-DRA, HLA-DRB, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB. In some embodiments, the cell contains one or more polynucleotides encoding a single-stranded fusion class II HLA protein or a class II HLA protein. In some embodiments, the cell further contains one or more factors selected from the following: CD200, CD24, PD-L1, HLA-AQ or H2-M3, Cd47, FASLG or Fas1, Ccl21 or Ccl21b, Mfge8, serine protease inhibitor (Serpin) B9 or Spi6 or DUX4.

[0330] Induced pluripotent stem cells (iPS cells or iPSCs) can be generated through protein transduction of reprogramming factors in somatic cells. In some embodiments, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram them. In other embodiments, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram them. In still other embodiments, at least four reprogramming proteins are transduced into somatic cells to successfully reprogram them.

[0331] Pluripotent stem cells can be derived from any species. Embryonic stem cells have been successfully derived from, for example, mice, various non-human primates, and humans, and embryonic stem cell-like cells have been generated from a variety of other species. Therefore, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells (embryo-derived stem cells) from any species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), felines (domestic cats and wild felines such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In some embodiments, the species are endangered species. In some embodiments, the species are currently extinct species.

[0332] Similarly, iPS cells can be derived from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, neonatal, and adult tissues. Therefore, iPS cells can be readily generated using donor cells from any species. Thus, iPS cells can be generated from any species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), felines (domestic cats and wild felines such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In some embodiments, the species are endangered species. In some embodiments, the species are currently extinct species.

[0333] Induced pluripotent stem cells can be generated using any somatic cell at virtually any developmental stage as a starting point. For example, the cells can be derived from an embryo, placenta, fetus, newborn, juvenile, or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as skin fibroblasts obtained from skin samples or biopsies, synovial cells from synovial tissue, prepuce cells, buccal cells, or lung fibroblasts. In some embodiments, the somatic cells are placental cells. While skin and buccal cells provide readily available and readily accessible suitable cell sources, virtually any cell can be used. In some embodiments, the somatic cells are not fibroblasts. In some embodiments, the somatic cells are derived from the placenta.

[0334] Induced pluripotent stem cells can be generated by expressing or inducing the expression of one or more reprogramming factors in somatic cells. The somatic cells can be fibroblasts, such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblast somatic cells. Somatic cells can be reprogrammed by inducing the expression of at least 1, 2, 3, 4, or 5 reprogramming factors (e.g., via viral transduction, integration or non-integration vectors) and / or by contacting at least 1, 2, 3, 4, or 5 reprogramming factors (e.g., using protein transduction domains, electroporation, microinjection, cationic amphiphilic molecules, fusion with a lipid bilayer, detergent permeation, etc.). The reprogramming factors can be selected from OCT 3 / 4, SOX2, NANOG, LIN28, C-MYC, and KLF4. Expression of the reprogramming factors can be induced by contacting somatic cells with at least one agent that induces the expression of a reprogramming factor, such as a small organic molecule agent.

[0335] Other exemplary pluripotent stem cells include induced pluripotent stem cells (iPSCs) resulting from somatic cell reprogramming through the expression of combinations of expression factors (“reprogramming factors”) or inducing factors. iPSCs can be obtained from cell banks. The preparation of iPSCs can be a starting step in the production of differentiated cells. iPSCs can be specifically generated using materials from a specific patient or matched donor with the goal of generating tissue-matched photoreceptor rescue cells. iPSCs can be generated from cells that are substantially non-immunogenic in the intended recipient, for example, from autologous cells or cells that are tissue-compatible with the intended recipient.

[0336] Somatic cell reprogramming can also be achieved using combinatorial approaches, where reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and their expression is induced (e.g., using small organic molecules). For example, reprogramming factors can be expressed in somatic cells by infection with viral vectors, such as retroviral or lentiviral vectors. Alternatively, non-integrating vectors, such as episomal plasmids, can be used to express reprogramming factors in somatic cells. See, for example, Yu et al., Science. 2009 May 8; 324(5928):797-801, the entire contents of which are incorporated herein by reference. When using non-integrating vectors to express reprogramming factors, the factors can be expressed in cells by electroporation, transfection, or transformation of somatic cells with the vector. For example, in mouse cells, the expression of four factors (OCT3 / 4, SOX2, C-MYC, and KLF4) using an integrated viral vector is sufficient to reprogram somatic cells. In human cells, the expression of four factors (OCT3 / 4, SOX2, NANOG, and LIN28) using an integrated viral vector is sufficient to reprogram somatic cells.

[0337] Once the reprogramming factor is expressed in the cells, the cells can be cultured. Over time, cells exhibiting ES characteristics appear in the culture dish. Cells can be selected and subcultured based on, for example, ES morphology or on the expression of selectable or detectable markers. Cells can be cultured to produce cell cultures that resemble ES cells.

[0338] To confirm the pluripotency of iPS cells, the cells can be tested in one or more pluripotency assays. For example, the expression of ES cell markers can be tested; the ability of cells to generate teratomas can be evaluated when transplanted into SCID mice; and the ability of cells to differentiate into cell types with all three germ layers can be evaluated. Once pluripotent iPSCs are obtained, they can be used to generate cell types disclosed herein, such as photoreceptor rescue cells.

[0339] Stimulus-triggered pluripotency acquisition (STAP) cells are pluripotent stem cells generated by reprogramming somatic cells with sublethal stimuli (such as low pH exposure). Reprogramming does not require nuclear transfer to somatic cells or genetic manipulation of somatic cells. See Obokata et al., Nature, 505:676-680, 2014.

[0340] As used herein, the term "stem cell" refers to a master cell, a specialized cell capable of proliferating indefinitely to form tissues and organs. Stem cells are pluripotent or multipotent cells. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor cell ("transitional cell"), and then proliferate into mature, fully formed cells of the tissue.

[0341] As used herein, the term "adult stem cell" refers to stem cells that are at a later stage of development than the embryonic stage, isolated from tissues or organs (e.g., bone marrow stem cells, umbilical cord blood stem cells, and adipose stem cells) of an animal (e.g., a human). In one aspect, the stem cells of the present invention can be isolated at the postnatal stage. These cells can preferably be isolated from mammals, such as humans. Adult stem cells differ from embryonic stem cells, which are defined by their origin, namely the inner cell mass of the blastocyst. The adult stem cells according to the present invention can be isolated from any non-embryonic tissue and will include neonates, infants, adolescents, and adult patients. Typically, the stem cells of the present invention will be isolated from non-neonatal mammals, and more preferably from non-neonatal humans. These adult stem cells are characterized in that, in their undifferentiated state, they express telomerase, and they do not exhibit gap junction intercellular communication (GJIC) and do not possess a transformative phenotype.

[0342] As used in this article, the “sign” of a disease broadly refers to any abnormality that can be found during examination of a patient that indicates the disease; the objective manifestation of the disease, as opposed to a symptom, which is the subjective manifestation of the disease.

[0343] As used in this article, the “symptoms” of a disease broadly refer to any pathological phenomena or deviations from normal in structure, function, or sensation that a patient experiences and that indicate the disease.

[0344] As used herein, “therapeutic,” “therapeutic,” and “treatment” broadly refer to treating a disease, preventing or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease or causing the remission of a disease or its clinical symptoms. Therapy encompasses prevention, averting, treating, curing, remedying, reducing, alleviating a disease, its signs and / or symptoms, and / or providing relief from the disease, its signs and / or symptoms. Therapy encompasses the reduction of signs and / or symptoms in patients with persistent signs and / or symptoms of the disease. Therapy also encompasses “prevention” and “avoidance.” Prevention includes preventing the occurrence of disease following treatment of a patient or reducing the incidence or severity of disease in a patient. For the purposes of therapy, the term “reduction” broadly refers to a significant reduction in clinical signs and / or symptoms. Therapy includes treating recurrent or recurring signs and / or symptoms. Therapy encompasses (but is not limited to) eliminating the occurrence of signs and / or symptoms at any time, as well as reducing and eliminating existing signs and / or symptoms. Therapy includes treating chronic diseases (“maintenance”) and acute diseases. For example, treatment includes treating or preventing the recurrence or relapse of symptoms and / or symptom recurrence.

[0345] Conditions to be treated according to the present invention, and therefore treated using one or more of the preparations provided herein, include, but are not limited to, macular degeneration, including age-related macular degeneration, which may be early or late. Other conditions to be treated include, but are not limited to, retinitis pigmentosa, retinal dysplasia, retinal degeneration, diabetic retinopathy, age-related macular degeneration (e.g., wet or dry), geographic atrophy secondary to AMD, congenital retinal dystrophy, rod dystrophy, cone dystrophy, cone-rod dystrophy, leber congenital amaurosis, Staggart's disease, retinal detachment, glaucoma, optic neuropathy, and trauma affecting the eye.

[0346] Cell markers:

[0347] Photoreceptor rescue cell compositions comprising multiple heterologous photoreceptor rescue cells exhibit a unique spectrum of biomarkers that distinguish them from naturally occurring cells. Specifically, the compositions of the present invention are characterized by the expression of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the compositions of the present invention are characterized by the cumulative expression of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention are characterized by the expression of FOXG1 and / or MAP2. In some embodiments, the multiple photoreceptor rescue cells in the compositions of the present invention are characterized by the cumulative expression of FOXG1 and MAP2.

[0348] In some embodiments, the cells in the photoreceptor-rescue cell compositions of the present invention comprise inhibitory neurons and are therefore characterized by the expression of DLX5, TUBB3, SCGN, ERBB4, CALB2, or combinations thereof. In some embodiments, the multiple photoreceptor-rescue cells in the compositions of the present invention are characterized by the cumulative expression of DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0349] In some embodiments, the cells in the photoreceptor-rescue cell compositions of the present invention comprise excitatory neurons and are therefore characterized by the expression of NEUROD2, NEUROD6, SLA, NELL2, SATB2, or combinations thereof. In some embodiments, the multiple photoreceptor-rescue cells in the compositions of the present invention are characterized by the cumulative expression of NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

[0350] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include progenitor cells and are therefore characterized by the expression of VIM, MKI67, CLU, GLI3, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of VIM, MKI67, CLU, and GLI3.

[0351] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention include astrocytes and are therefore characterized by the expression of GFAP, LUCAT1, MIR99AHG, FBXL7, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0352] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise substitute neurons and are therefore characterized by the expression of MEIS2, PBX3, GRIA2, CACNA1C, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the compositions of the present invention are characterized by the cumulative expression of MEIS2, PBX3, GRIA2, and CACNA1C.

[0353] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are characterized by the expression of ocular progenitor cell markers selected from the following: PAX6, LHX2, SIX3, NES, SOX2, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of PAX6, LHX2, SIX3, NES, and SOX2.

[0354] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing ocular progenitor cell markers RAX, SIX6, and / or TBX3. In some embodiments, the various photoreceptor rescue cells in the composition of the present invention are characterized by little or no cumulative expression of RAX, SIX6, and TBX3.

[0355] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are characterized by the expression of rod / cone photoreceptor markers selected from the following: ASCL1, RORB, NR2E3, NRL, or combinations thereof. In some embodiments, the multiple photoreceptor rescue cells in the composition of the present invention are characterized by the cumulative expression of ASCL1, RORB, NR2E3, and NRL.

[0356] In some embodiments, the cells in the photoreceptor-rescue cell composition of the present invention are characterized by the expression of neuronal markers selected from the following: TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5, or combinations thereof. In some embodiments, the multiple photoreceptor-rescue cells in the composition of the present invention are characterized by the cumulative expression of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0357] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing CRX, RHO, OPN1SW, PDE6B, RCRRN, ARR3, CNGB1, GNAT1, GNAT2, or combinations thereof. In some embodiments, the various photoreceptor rescue cells in the composition of the present invention are characterized by little or no cumulative expression of CRX, RHO, OPN1SW, PDE6B, RCRRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0358] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing VSX2, POU5F1, or a combination thereof. In some embodiments, the various photoreceptor rescue cells in the composition of the present invention are characterized by little or no cumulative expression of VSX2 and POU5F1.

[0359] In some embodiments, the cells in the photoreceptor rescue cell composition of the present invention are substantially free of cells expressing OCT4, SSEA4, or a combination thereof. In some embodiments, the various photoreceptor rescue cells in the composition of the present invention are characterized by little or no cumulative expression of OCT4 and SSEA4.

[0360] In some embodiments, the cells in the photoreceptor-rescue cell composition of the present invention are characterized by AGT, ACBLN2, CDH7, DNAH11, EGR1, FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, nestin, TRβ2, MASH1, RORβ, MAP2, ELAVL3, NFIA, and DC. The expression of X, LHX2, SLC1A2, ELAVL4, PAX6, EMX2, ASCL1, DLL1, NFIB, ENOXI, TUBB3, MAP2, DCLK1 / 2, DCX, KALRN, LINC00461, C1orf61, NCAM1, SETBP1, PAK3, AKAP6, RTN1, CRMP1, FOXG1, TRIM2, BACH2, recovery protein, opsin, rhodopsin, and cGMP phosphodiesterase in rod and cone cells. These markers can be evaluated at the protein and / or mRNA levels (see Fischer). AJ, Reh TA, Dev Neurosci. 2001; 23(4-5):268-76; Baumer et al., Development. 2003 July; 130(13):2903-15; Swaroop et al., Nat Rev Neurosci. 2010 August; 11(8):563-76; Agathocleous and Harris, Annu. Rev. Cell Dev. Biol. 2009. 25:45-69. (The full contents of each of the above references are included in this article).

[0361] In some embodiments, cells in the photoreceptor rescue cell composition of the present invention are identified by evaluating the expression of cell markers compared to their expression in pluripotent stem cells, such as iPSCs or embryonic-derived PSCs. In some embodiments, cells in the photoreceptor rescue cell composition of the present invention are identified by decreased expression of FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4c, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, and SSEA4 compared to pluripotent stem cells, such as iPSCs or embryonic-derived PSCs. In some embodiments, cells in the photoreceptor rescue cell composition of the present invention are identified by increased expression of AGT, ACBLN2, DCH7, DNA11, and EGR1 compared to ESCs or iPSCs. These markers are typically human, for example, unless otherwise specified in the context. Cellular markers can be identified using conventional immunocytochemistry, conventional PCR, transcriptomic analysis including RNAseq, real-time quantitative PCR, flow cytometry, FACS, scRNAseq, whole RNAseq, single-cell or whole qRT-PCR, immunocytochemistry, immunofluorescence, single-cell or whole proteomics (e.g., via LC-MS), possibly metabolomics, lipidomics, and other suitable methods known in the art.

[0362] As used herein, the term "scRNAseq" refers to single-cell RNA sequencing. In some implementations, scRNAseq provides data that clusters individual cells of a cell population based on the expression of gene markers. In other implementations, scRNAseq provides data that identifies the percentage of individual cells expressing (one or more) gene markers in a population, such as a PRC cell population. The scRNAseq localization sequence data is filtered for quality control metrics, dimensionality reduction is performed for visualization, and clustering is performed using the Louvain algorithm with a shared nearest neighbor approach. Differential expression is then applied to the clusters, and the most differentially expressed genes are compared between the clusters and some publicly available data sources. Multiple human developmental biology reviews, along with datasets of the human retina (www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE142526), ​​human hippocampus (www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE119212), human midbrain (www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE76381), and human cortex (www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE132672), were used to assign the best guessed cell characteristics (identities) to clusters based on differentially expressed genes and expression patterns during development and in the review datasets.

[0363] As used herein, the term "holistic RNAseq" refers to RNA sequencing analysis of a cell population. Holistic RNAseq provides transcripts per million (TPM), which is the amount of RNA molecules from the gene of interest per 1,000,000 RNA molecules in an RNA-seq sample.

[0364] Cell culture medium:

[0365] In embodiments of the invention, cells are stored, proliferated, or differentiated in various cell culture media. A rescue-inducing medium (RIM) is used for the differentiation of stem cells into early-stage neuronal progenitor cells. The RIM may contain D-glucose, N2 supplement (e.g., 0.1-5%), B27 supplement (e.g., 0.005-0.2%), a MEM non-essential amino acid solution, and optionally insulin and / or Noggin, and may be in DMEM / F12 (Invitrogen) or a similar basal medium. For example, the RIM may include at least insulin. Additionally, insulin concentrations may be altered or increased, which may promote cell survival and / or the acquisition of differentiated cells. For example, insulin concentrations may be altered within a range, and survival and / or differentiation may be monitored to identify insulin concentrations that improve one or both of these properties. The addition of Noggin is believed to be non-essential, but increased expression of neuronal progenitor-related transcription factors has been observed.

[0366] Tables 1-4 provide the components of DMEM / F12, neural basal medium, N2 serum supplement, and B27 serum supplement. It should be understood that this invention considers the use of these specific culture media and supplements, or culture media or supplements containing, substantially consisting of, or composed of these components.

[0367] Table 1

[0368]

[0369]

[0370]

[0371] Table 2

[0372]

[0373]

[0374]

[0375] Table 3

[0376]

[0377] Table 4

[0378]

[0379] The method described in this article can use human factors, such as human Noggin and human insulin.

[0380] Noggin is a secreted bone morphogenetic protein (BMP) inhibitor, reported to bind with high affinity to BMP2, BMP4, and BMP7 to block TGFβ family activity. SB431542 is a small molecule reported to inhibit TGFβ / activin / Nodal by blocking phosphorylation of ACTRIB, TGFβR1, and ACTRIC receptors. SB431542 is thought to destabilize activin- and Nanog-mediated pluripotent networks and inhibit BMP-induced trophoblast, mesoderm, and endoderm cell fate by blocking endogenous activin and BMP signaling. Agents with one or more of the above-described activities are expected to replace or amplify (enhance) the functions of one or both of Noggin and SB431542, for example, as they are used in the context of the disclosed methods. For example, the applicant envisions replacing or amplifying the protein Noggin and / or the small molecule SB4312542 with one or more inhibitors affecting any one or all of the following three target regions: 1) preventing the binding of ligands (e.g., bone morphogenetic proteins (BMPs), such as BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14) to receptors, such as bone morphogenetic protein receptors; 2) blocking receptor activation (e.g., dorsomorphin); and 3) inhibiting intracellular proteins / transcription factors of SMAD. Exemplary potentially suitable factors include the naturally secreted BMP inhibitors chordin (which blocks BMP4) and follistatin (which blocks activin), and their analogues or mimics. Other exemplary factors that can mimic the effects of Noggin include the use of dominant or negative receptors that multivalently chelate BMP2, BMP4, and / or BMP7, or blocking antibodies. In addition, regarding the blocking of receptor phosphorylation, dorsomorphin (or compound C) has been reported to have similar effects on stem cells. Soluble inhibitors such as SIS3 (6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline, a specific inhibitor of Smad3, SIS3), overexpression of one or more of the inhibitors of SMAD (e.g., SMAD6, SMAD7, SMAD10), or RNAi of one of the receptors SMAD (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8 / 9) can also be used to inhibit SMAD proteins.Another combination of factors expected to be suitable for generating neural progenitor cells includes a mixture of leukemia suppressor factor (LIF), a GSK3 inhibitor (CHIR99021), compound E (a γ-secretase inhibitor XXI), and SB431542, a TGFβ inhibitor previously shown to be effective for generating neural crest stem cells (Li et al., Proc Natl Acad Sci USA. 2011 May 17; 108(20):8299-304). Other exemplary factors may include derivatives of SB431542, such as molecules comprising one or more added or different substituents, similar functional groups, etc., and having similar inhibitory effects on one or more SMAD proteins. Suitable factors or combinations of factors can be identified, for example, by exposing pluripotent cells to the factors and monitoring early neuronal progenitor phenotypes, such as the adoption of characteristic gene expression (including the expression of markers described herein, the expression of reporter genes coupled to early neuronal progenitor promoters, etc.) or the ability to form cell types disclosed herein, such as early neuronal progenitors, late neuronal progenitors, retinal neuronal progenitors, photoreceptor progenitors, rod progenitors, cones, rods and / or photoreceptor rescue cells.

[0381] Preferably, cells are treated or cultured in rescue induction medium before being cultured with neural differentiation medium (NDM). NDM can be used to promote further maturation of early neural progenitor cells. In one embodiment, neural differentiation medium is used to promote the differentiation and development of early neuronal progenitor cells. Neural differentiation medium may contain D-glucose, penicillin, streptomycin, GlutaMAX, etc. TM The medium may contain N2 supplements, B27 supplements, MEM non-essential amino acid solution, and optionally Noggin. The neural differentiation medium can also be used for the differentiation and maturation of early neuronal progenitor cells with transcriptional characteristics of excitatory or inhibitory neurons, and neurons or "photoreceptor rescue cells," but without Noggin. In some embodiments, Noggin is not required once PSCs and PSC subsets are no longer present in the mature cell product.

[0382] The composition of the neural differentiation medium is as follows: N2: 1% (1 ml N2 per 100 ml), B27: 2% (2 ml B27 per 100 ml) and Noggin: 50 ng / ml.

[0383] Noggin is no longer needed after all cells have become early neuronal progenitor cells.

[0384] Stem cells, embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSs):

[0385] The ESCs, adult stem cells, or iPS cells used in this article can proliferate in feeder-free cell systems, such as Matrigel. TM (A soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) or another matrix for proliferation. Alternatively, the pluripotent cells can be cultured on a matrix selected from laminins (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332 / laminin-3A32, laminin-3B32, laminin-311 / laminin-3A11, laminin-321 / laminin-3A21, laminin-411, laminin-421, laminin-511 (e.g., iMatrix)). TM Laminin-511, laminin-521, laminin-213, laminin-432, laminin-522, laminin-532, and / or laminin fragments, fibronectin, hylocinin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel TM (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The matrix may contain Matrigel. TM (A soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), comprising or substantially comprising thereof. In some embodiments, the stem cells do not form germ-like structures during culture, which is an improvement over the prior art. In one embodiment, stem cells, such as ESCs or iPSCs, differentiate into photoreceptor rescue cells in the presence of Noggin.

[0386] stem cells

[0387] In a developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells play a role in the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerative organs such as blood, skin, or intestinal tissue.

[0388] Pluripotent stem cells, such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can proliferate in vitro for extended periods while retaining the potential to differentiate into all cell types in the body, including photoreceptor rescue cells. Therefore, these cells could potentially provide an unlimited supply of patient-specific functional photoreceptor rescue cells for drug development and transplantation therapies. In vitro differentiation of pluripotent stem cells into photoreceptor rescue cells may involve the addition of different growth factors at different differentiation stages and may require approximately 10–30 days of differentiation (see, for example, Figure 12). The unlimited proliferative capacity of pluripotent stem cells offers an advantage over somatic cells as a starting cell population for generating photoreceptor rescue cells.

[0389] Pluripotent stem cells, such as embryonic stem (ES) cells or iPS cells, can be the starting material for the disclosed methods. In any embodiment herein, the pluripotent stem cells can be human pluripotent stem cells (hPSCs). Pluripotent stem cells (PSCs) can be cultured in any manner known in the art, such as with or without feeder cells. Additionally, PSCs generated using any method can be used as starting material for generating photoreceptor rescue cells. For example, hES cells can be derived from blastocyst-stage embryos, which are the product of in vitro fertilization of oocytes and sperm. Alternatively, hES cells can be derived from one or more blastomeres removed from early cleavage-stage embryos, optionally without damaging the rest of the embryo. In other embodiments, hES cells can be generated using nuclear transfer. In other embodiments, iPSCs can be used. Previously cryopreserved PSCs can be used as starting material. In another embodiment, PSCs that have never been cryopreserved can be used.

[0390] In one aspect of the invention, PSCs are plated onto an extracellular matrix under feeder or no feeder conditions. In one embodiment, PSCs can be cultured on an extracellular matrix including, but not limited to, laminin, fibronectin, hydrin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the extracellular matrix is ​​a laminin with or without e-cadherin. In some embodiments, the laminin may be selected from the group consisting of laminin 521, laminin 511, or iMatrix 511. In some embodiments, the feeder cells are human feeder cells, such as human skin fibroblasts (HDF). In other embodiments, the feeder cells are mouse embryonic fibroblasts (MEF).

[0391] In some embodiments, the culture medium used when culturing PSCs can be selected from any culture medium suitable for culturing PSCs. In some embodiments, any culture medium capable of supporting PSC culture can be used. For example, those skilled in the art can choose between commercially available or proprietary culture media.

[0392] The culture medium supporting pluripotency can be any such medium known in the art. In some embodiments, the culture medium supporting pluripotency is Nutristomum. TM In some implementations, the culture medium supporting pluripotency is TeSR. TM In some implementations, the culture medium supporting pluripotency is StemFit. TM In other embodiments, the culture medium supporting pluripotency is Knockout. TM DMEM (Gibco) can supplement Knockout TM Serum substitutes (Gibco), LIF, bFGF, or any other factor. Each of these exemplary culture media is known in the art and is commercially available. In other embodiments, the culture medium supporting pluripotency may be supplemented with a ROCK inhibitor, bFGF, or any other factor. In one embodiment, bFGF may be supplemented at a low concentration (e.g., 4 ng / mL). In another embodiment, bFGF may be supplemented at a higher concentration (e.g., 100 ng / mL), which can induce PSC differentiation.

[0393] The concentration of PSC used in the production method of this invention is not specifically limited. For example, when using a 10cm culture dish, 1×10⁻⁶ PSC can be used. 4 -1×10 8 5 × 10 cells per dish, preferably 5 × 10 4 -5×10 6 1 × 10 cells per dish, more preferably 1 × 10 5 -1×10 7 One cell per dish. For example, when using When using containers, use 1×10 4 -1×10 8 5 × 10 cells per container, preferably 5 × 10 4 -5×10 6 1.5 × 10 cells per container 6 -2.2×10 6 10 cells per container, more preferably 1×10 5 -1×10 7 Cells per container. For example, when using flasks (e.g., PDL pre-coated T175 flasks), use 1 × 10⁻⁶ cells per container. 4-1×10 8 5 × 10 cells per bottle, preferably 5 × 10 4 -5×10 6 4.3 × 10 cells per flask 5 -6.2×10 6 10 cells per vial, more preferably 1×10 5 -1×10 7 One cell per bottle.

[0394] In some implementations, 3×10 is used 3 and 6×10 3 cells / cm 2 The target inoculation density is used to amplify hESCs. In some embodiments, a 6-well plate (3 × 10⁻⁶) is preferably used. 4 -6×10 4 1 cell per container, for example, 2 × 10 4 -7×10 4 (cells per container) or in T75 flasks (2 × 10⁶ cells per container) 5 -5×10 5 1 cell per container, for example, 1×10 5 and 6×10 5 (Each cell per container) to amplify hESCs. In some implementations, Containers or T175 vials are used for amplifying hESC.

[0395] In some implementations, hESCs are seeded to initiate the differentiation process into PRCs. In some implementations, the target seeding density is 3,000 cells / cm². 2 The target density is approximately 1.5 × 10⁻⁶. 6 -2.2×10 6 Each cell Container, and 4.3 × 10 5 -6.2×10 6 175 flasks per T cells.

[0396] In some implementations, the concentration is approximately 1,000-100,000 cells / cm². 2 PSCs are plated at a cell density of approximately 5,000-100,000 cells / cm². 2 Approximately 5,000-50,000 cells / cm³ 2 Or approximately 5,000-15,000 cells / cm 2 The cell density of PSCs was plated. In other embodiments, approximately 10,000 cells / cm² was used. 2 Density PSC (Polyester Slab)

[0397] In some implementations, the culture medium supporting pluripotency is replaced with a differentiation medium, such as StemFit. TM Alternatively, other similar culture media can be used to differentiate cells into photoreceptors to rescue cells. In some embodiments, the culture medium replacement from a pluripotency-supporting medium to a differentiation medium can be performed at different time points during PSC cell culture and can also depend on the initial plating density of the PSCs. In some embodiments, the culture medium replacement can be performed after the PSCs have been cultured in pluripotency medium for 2–14 days. In some embodiments, the culture medium replacement can be performed on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0398] In some embodiments, the stem cells useful for the methods described herein include, but are not limited to, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, chondrocyte progenitor cells, epidermal stem cells, gastrointestinal stem cells, neural stem cells, liver stem cells, adipose-derived mesenchymal stem cells, pancreatic progenitor cells, hair follicle stem cells, endothelial progenitor cells, and smooth muscle progenitor cells.

[0399] In some embodiments, stem cells useful for the methods described herein are isolated from the umbilical cord, placenta, amniotic fluid, chorion villi, blastocyst, bone marrow, adipose tissue, brain, peripheral blood, gastrointestinal tract, umbilical cord blood, blood vessels, skeletal muscle, skin, liver, and menstrual blood.

[0400] Detailed procedures for isolating human stem cells from multiple sources are described in Current Protocols in Stem Cell Biology (2007), the entire contents of which are incorporated herein by reference. Methods for isolating and culturing stem cells from multiple sources are also described in U.S. Patent Nos. 5,486,359, 6,991,897, 7,015,037, 7,422,736, 7,410,798, 7,410,773, and 7,399,632; the entire contents of each of these patents are incorporated herein by reference.

[0401] somatic cells

[0402] In some aspects of the invention, a transdifferentiation method may also be provided, which involves directly converting one somatic cell type into another somatic cell type, for example, obtaining photoreceptor rescue cells from other somatic cells.

[0403] However, the supply of human cells can be limited, especially somatic cells from living donors. To provide an unlimited supply of initiating cells for photoreceptor rescue cell differentiation, somatic cells can be immortalized by introducing immortalizing genes or proteins, such as hTERT and / or other oncogenes. Cell immortalization can be reversible (e.g., using removable expression cassettes) or inducible (e.g., using inducible promoters).

[0404] In some aspects of the invention, the somatic cells may be primary cells (non-immortalized cells), such as those freshly isolated from animals, or may be derived from cell lines (immortalized cells). The cells may be maintained in cell culture after isolation from the subject. In some embodiments, the cells are passaged once or more (e.g., 2-5, 5-10, 10-20, 20-50, 50-100 times or more) before being used in the methods of the invention. In some embodiments, the cells will be passaged no more than 1, 2, 5, 10, 20, or 50 times before being used in the methods of the invention.

[0405] The somatic cells used or described herein can be natural somatic cells or engineered somatic cells, i.e., genetically modified somatic cells. The somatic cells of this invention are typically mammalian cells, such as human cells, primate cells, or mouse cells. They can be obtained by well-known methods and can be obtained from any organ or tissue containing living somatic cells, such as blood, bone marrow, skin, lungs, pancreas, liver, stomach, intestines, heart, reproductive organs, bladder, kidneys, urethra, and other urinary organs.

[0406] The mammalian somatic cells useful in this invention include, but are not limited to, Sertoli cells, endothelial cells, granular epithelial cells, neurons, pancreatic islet cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, cardiomyocytes, and other muscle cells.

[0407] The methods described herein can be used to program one or more somatic cells, such as colonies or populations of somatic cells, into photoreceptor-rescue cells. In some embodiments, the cell populations of the present invention are substantially homogeneous, wherein at least 90% of the cells exhibit the phenotype or characteristic of interest. In some embodiments, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, or more of the cells exhibit the phenotype or characteristic of interest. In some embodiments of the present invention, the somatic cells are capable of division, i.e., the somatic cells are not post-mitotic.

[0408] Somatic cells can differentiate partially or completely. As described in this article, both partially differentiated and fully differentiated somatic cells can differentiate to produce photoreceptors to rescue cells.

[0409] Photoreceptor-rescued cells (PRC):

[0410] PRCs can differentiate from pluripotent stem cells (e.g., ESCs or iPSCs in the absence of Noggin and in a neural differentiation medium). PRCs are FOXG1(+) and MAP2(+), as determined by flow cytometry. In one embodiment, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the PRCs are FOXG1(+) and MAP2(+). PRCs can also be STMN2(+), DCX(+), LINC00461(+), NEUROD2(+), GAD1(+), and / or NFIA(+). PRCs can also be SSEA4(-) and / or OCT4(-), as determined by flow cytometry. Cells can be grown as spheres or neurospheres (e.g., on low-adherent plates or optionally in hanging drop cultures, in low-gravity environments, in Aggrewell, or under other suitable culture conditions).

[0411] Figure 12A and 12B The diagram illustrates an alternative PRC production method. In some embodiments, hESCs or pluripotent cells are expanded prior to PRC differentiation. Culture chambers (e.g., culture dishes, culture flasks, such as T75 culture flasks coated with iMatrix), or culture containers (e.g., TC-coated containers) can be used. The expansion of hESCs or pluripotent cells is performed. In some embodiments, the expansion of hESCs or pluripotent cells includes thawing hESCs or pluripotent cells on day -10, counting and seeding them in culture vessels coated with iMatrix (lamin-511, Matrixome), and using StemFit culture supplemented with 100 ng / mL bFGF (Peprotech) and 10 μM ROCK inhibitor (Y-27632; Fujifilm / Wako). TM The culture medium (Ajinomoto) was used to culture the cells under feeder-free conditions. After changing the medium (StemFit + bFGF, without ROCK inhibitor) daily for four days, hESCs or pluripotent cells were harvested using cell dissociation buffer (Gibco) and reseeded under the same culture conditions.

[0412] After culturing for another 4 days (day -2), harvest hESCs as described above, obtain the survival rate percentage and live cell count, and record the results at 3000 cells / cm³. 2 hESC inoculation is used in culture chambers (e.g., petri dishes, culture flasks, such as T75 culture flasks coated with iMatrix, or culture containers, such as those coated with TC). PRC differentiation was performed in StemFit+bFGF medium supplemented with ROCK inhibitor (Y-27632). After inoculation on day -2, the cultures were fed with the following: (1) day -1 - StemFit+bFGF (without ROCK inhibitor); (2) day 0 to day 3 (daily) - rescue induction medium (RIM: DMEM / F12+B27+N2+non-essential amino acids (all from Gibco)+glucose (Sigma)+insulin (Akron Biotech)+Noggin (Gibco)); and (3) day 4 to day 19 (every 2-3 days) - neural differentiation medium plus Noggin (NDM+: neural basal medium+B27+N2+non-essential amino acids+glucose+glutamax (Gibco)+Noggin).

[0413] On day 19, the culture was "lifted" to suspension culture (2D to 3D) by incubation with a combination of liberase and thermophilic protease (Roche Custom Labs) and then inoculated into culture chambers (e.g., petri dishes, culture flasks, such as ultra-low adherence T75 flasks, or culture containers, such as ultra-low adherence). On the 3D culture, maintain the culture for 3-4 days using NDM minus Noggin (NDM-) to allow for the formation of neurospheres (“spheres”). Then, inoculate the spheres in suspension (3D to 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to start generation 0 (P0d0) and culture for 14 days (up to P0d14) using NDM- (feed every 2-3 days).

[0414] The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, with 3D cultured for 3-4 days and 2D cultured for 14 days) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using acutase (Innovative Cell Technologies) and cultured for 24 hours in ultra-low adherence T75 flasks. The P3-spheres were then cryopreserved by resuspending them in Cryptotor CS10 (Stemcell Technologies) and freezing to -80°C, followed by storage in the vapor phase of liquid nitrogen. The cryopreserved P3-spheres are referred to as Cell Stock (CS). In some embodiments, the cells in the composition are expanded by growth under culture, adherent conditions, and then low adherent conditions. In some embodiments, the culture is repeated at least once, at least twice, at least three times, at least four times, at least five times, or at least six times under adherent conditions and then under low adherent conditions. In some embodiments, after a third repeat of culture under adherent conditions and then under low-adherent conditions, a fourth repeat of culture under adherent conditions and then under low-adherent conditions, or a fifth repeat of culture under adherent conditions and then under low-adherent conditions, cells in the composition are harvested.

[0415] In some embodiments, cells are cultured under adherent conditions for the fifth replicate, and then harvested from the composition after culturing under low adherent conditions. In some embodiments, the harvested cells from the composition are cryopreserved.

[0416] In some embodiments, the culture is grown in a petri dish, flask, or container from suspension culture to adherent culture (2D to 3D). In some embodiments, the culture container is from Corning. Container. In some embodiments, it is treated with a low-adhesion or adhesion coating. In one embodiment, cells are cultured under low adhesion conditions and coated with an ultra-low adhesion coating. In one embodiment, cells are cultured under adherent conditions and coated with a TC-treated coating.

[0417] Examples of methods for “lifting” cells into suspension include, but are not limited to, the methods shown in Table 13.

[0418] Table 13

[0419]

[0420] To produce the composition of the present invention, CS vials were thawed in a 37°C water bath, resuspended in NDM-, and transferred to ultra-low adherence T75 flasks. The vials were cultured in 3D suspension for 2-3 days, then re-coated into T75 flasks coated with poly-D-lysine / fibronectin / laminoid-521 and cultured in NDM- under 2D conditions for 14 days to complete passage. At P4d14, cells were harvested using acutase, resuspended in NDM-, milled, and filtered through a 40 μm cell filter to obtain a single-cell suspension constituting the PRC composition of the present invention, which can then be formulated with a cryoprotectant.

[0421] The PRC production protocol can be modified to allow for scale-up. For example, the bottles used can be changed from T75 bottles to T225 bottles or cell stacks (e.g., ).

[0422] Additionally, the PRC production process may include any one or more intermediate cryopreservation steps between P0 and P1, between P1 and P2, between P2 and P3, and / or between P3 and P4. In some embodiments, the PRC production process may exclude intermediate cryopreservation steps.

[0423] The intermediate cryopreservation step may include 5% DMSO instead of 10% DMSO. In some embodiments, the intermediate cryopreservation step may include about 1% DMSO, about 2% DMSO, about 3% DMSO, about 4% DMSO, about 5% DMSO, about 6% DMSO, about 7% DMSO, about 8% DMSO, about 9% DMSO, about 10% DMSO, about 11% DMSO, about 12% DMSO, about 14% DMSO, or about 15% DMSO.

[0424] Intermediate cryopreservation steps may include cryoprotectants as described herein. The PRC manufacturing process may include thermophilic proteases and liberases from D19, along with accutase, followed by overnight culture with a Rock inhibitor. The resulting cells or cell clusters can be seeded into Aggrewells. TMThis allows for the formation of uniformly sized spheroids. On day 19, the cultures were "lifted" to suspension culture (2D to 3D) by incubation with accutase (Innovative Cell Technologies) and inoculated on ultra-low adherent T75 flasks using NDM-subtracted Noggin (NDM-) medium supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako). After 24 hours, the growth medium was replaced with NDM- without Y-27632, and the 3D cultures were maintained for another 2–3 days using NDM- to allow for the formation of neurospheres ("spheres"). The spheres in suspension were then inoculated (3D to 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to begin passage 0 (P0d0) and cultured for 14 days under 2D conditions using NDM- (feeding every 2–3 days) (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, 3D culture for 3–4 days, 2D culture for 14 days) until P3d14 was reached after 90 days of differentiation. The P3d14 cultures were harvested using acutase (Innovative Cell Technologies) and cultured for 24 hours in ultra-low adhesion T75 flasks. The P3-spheres were then cryopreserved by resuspending in Cryptotor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to the vapor phase of liquid nitrogen for storage. Cryopreserved P3 spheres are called cell banks (CS).

[0425] In some implementations, the PRC production protocol uses Aggrewells. According to this method, on day 19, the culture is “lifted” to suspension culture (2D to 3D) by incubation with accutase (Innovative Cell Technologies), and then seeded onto Aggrewell plates (Stemcell Technologies) using NDM-subtracted Noggin (NDM-) medium supplemented with Y-27632 (a ROCK inhibitor, Fujifilm Wako) to allow the formation of neurospheres (“spheres”). After 24 hours, cells are harvested from the Aggrewell plates and transferred to ultra-low adherence T75 flasks, where the 3D culture is maintained for another 2–3 days using NDM- without Y-27632. The spheres in suspension were then inoculated (3D to 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virus-inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to begin passage 0 (P0d0) and cultured for 14 days under 2D conditions using NDM- (feeding every 2–3 days) (until P0d14). The 2D to 3D to 2D transition was repeated three times (through P1, P2, and P3, 3D culture for 3–4 days, 2D culture for 14 days) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using acutase (Innovative Cell Technologies) and cultured for 24 hours in ultra-low adhesion T75 flasks. The P3-spheres were then cryopreserved by resuspending in Cryptotor CS10 (Stemcell Technologies) and freezing to -80°C, and then transferred to the vapor phase of liquid nitrogen for storage. Cryopreserved P3 spheres are called cell banks (CS). PRC compositions can be pretreated with sucrose, for example, on D14 and P4, before being formulated with cryoprotectants.

[0426] Finally, the cryopreservation step for the final PRC formulation may include poloxamer 188 (a nonionic block linear copolymer) and / or sucrose.

[0427] Extracellular vesicles (EVs)

[0428] The present invention also provides for the rescue of extracellular vesicles secreted by cells from photoreceptors and their use in methods of treating eye diseases in subjects.

[0429] Rescuing extracellular vesicles secreted by cells from photoreceptors

[0430] The present invention also provides extracellular vesicles that are isolated, derived, secreted, or released from cells, for example, from photoreceptors of the present invention.

[0431] As used herein, the term “extracellular vesicle” or “EV” refers to lipid-bound vesicles secreted by cells into the intercellular space (extracellular space). The three main subtypes of EVs are microvesicles (MVs), exosomes (exosomes), and apoptotic bodies, distinguished by their biogenesis, release pathway, size, abundance, and function (Zaborowski MP, et al. Bioscience. 2015; 65:783-797). Typically, extracellular vesicles range in diameter from 20 nm to 5000 nm and can contain a variety of macromolecular payloads displayed on the outer surface of the extracellular vesicle and / or transmembrane within their internal space (i.e., lumen). These payloads can include nucleic acids, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), mRNAs, DNA fragments; proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. Extracellular vesicles include, for example and without limitation, apoptotic bodies, cell fragments, vesicles derived from / secreted from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicularized organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can originate from / secrete living or dead organisms, transplanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0432] As used herein, the term "exogenous body" refers to a small vesicle derived from a cell, which contains an internal space (i.e., a cavity) and is formed by the cell through direct budding of the plasma membrane or through the fusion of late endosomes with the plasma membrane. (J. Extracell. Vesicles. 2015; 4:27066). Specifically, exosomes are involved in protein sorting, recycling, storage, transport, and release. Exosomes typically range in diameter from 20 to 300 nm. They are secreted by all cell types and have been found in plasma, urine, semen, saliva, bronchial fluid, cerebrospinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid.

[0433] Exosomes have been found to participate in cell-cell communication, cell maintenance, and tumor development. Furthermore, they have been found to stimulate immune responses by acting as antigen-presenting vesicles (Bobrie A. et al., Traffic. 2077; 12:1659-1668). In the nervous system, exosomes have been found to contribute to myelin formation, axonal growth, and neuronal survival, thus playing a role in tissue repair and regeneration (Faure J. et al., Mol. Cell. Neurosci. 2006; 31:642-648). Exosomes in the central nervous system (CNS) have also been found to contain pathogenic proteins such as β-amyloid peptides, superoxide dismutase, and α-synuclein, which can aid in disease development (Fevrier B. et al., Proc. Natl. Acad. Sci. USA. 2004; 101:9683-9688). Exosomes have also been shown to be carriers of disease biomarkers. The use of exogenous bodies as carriers of biomarkers is ideal because these vesicles are present in bodily fluids such as blood and urine, which allows for minimally invasive to non-invasive “liquid biopsy”-type methods to diagnose and even monitor a patient’s response to treatment.

[0434] In addition to their natural role in cell-cell interactions, exogenous bodies can load various payloads, such as drugs and exogenous nucleic acids or proteins, and deliver these payloads to different cells. The payloads can be coupled to, embedded within, encapsulated within, or otherwise carried by extracellular vesicles, or any combination thereof. Therefore, as used herein, references to the "presence" of a payload in an extracellular vesicle or its lumen should be understood to include any of the aforementioned methods of carrying the payload.

[0435] The loading material can be endogenous, exogenous, or a combination thereof. Examples of loading materials that can be conjugated, embedded, encapsulated, or otherwise carried by extracellular vesicles as described herein include, without limitation, nucleic acid molecules (e.g., DNA, cDNA, antisense oligonucleotides, mRNA, repressive RNA (e.g., antisense RNA, miRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), and agomiR), antagomiR, primary miRNA (pri-miRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and microbial RNA), peptides (e.g., enzymes, antibodies), lipids, hormones, vitamins, minerals, small molecules, and drugs, or any combination thereof. Importantly, exosomes are natural carriers of miRNAs and other non-coding RNAs, and direct membrane fusion with the target cell allows for direct delivery of contents into the cytosol. This makes exosomes an excellent delivery system for small molecules (Lai RC, et al. Biotechnol. Adv. 2013; 31:543-551).

[0436] Microvesicles are extracellular organisms (EVs) formed directly outward from the cytoplasmic membrane via budding or pinching. Microvesicle size typically ranges from 100 nm to 1000 nm in diameter. The pathway of microvesicle formation is not fully understood; however, it is thought to require cytoskeletal components such as actin and microtubules, as well as molecular motors (kinesins and myosins) and fusion mechanisms (SNARE and tethering factors) (Cai H. et al., Dev. Cell. 2007; 12:671-682). The number of microvesicles produced depends on the physiological state and microenvironment of the donor cell (Zaborowski MP et al., Bioscience. 2015; 65:783-797). Similarly, it has been previously shown that the number of microvesicles consumed depends on the physiological state and microenvironment of the recipient cell. Like extracellular organisms (EVs), microvesicles also participate in cell-cell communication between local and distant cells. The ability of these EVs to alter recipient cells has been well demonstrated (Harding CN. et al. J. Cell Biol. 2013; 200:367-371; White IJ et al., EMBOJ. 2006; 25:1-12). EVs are unique in that they can package active loads (proteins, nucleic acids, and lipids) and deliver those loads to another cell, either nearby or distant, thereby altering the function of the recipient cell.

[0437] Apoptotic bodies are released from dying cells into the intercellular space. They have been reported to range in size from 50 nm to 5000 nm in diameter, with most bodies tending to be larger on the larger side (Borges F. et al., Braz. J. Med. Biol. Res. 2013; 46:824-830). These bodies are formed due to the separation of the cell membrane from the cytoskeleton caused by increased hydrostatic pressure after cell contraction (Wickman G. et al., Cell Death Differ. 2012; 19:735-742). The composition of apoptotic bodies is the opposite of that of exosomes and microvesicles. Unlike exosomes and microvesicles, apoptotic bodies contain intact organelles, chromatin, and a small amount of glycosylated proteins (Borges F. et al., Braz. J. Med. Biol. Res. 2013; 46:824-830; Thery C. et al., J. Immunol. 2001; 166:7309-7318).

[0438] Methods for isolating extracellular vesicles

[0439] The EVs of the present invention can be isolated, secreted, obtained, or isolated from culture media or other source materials (e.g., photoreceptor rescued cells of the present invention) using conventional methods known in the art (see, for example, Taylor et al., Serum / Plasma Proteomics, Chapter 15, “Extracellular vesicle Isolation for Proteomic Analyses and RNA Profiling,” Springer Science, 2011; and Tauro et al., Methods 56(2012) 293-304, and the references cited therein) and described in the Examples section below. The most commonly used methods include multiple centrifugation and ultracentrifugation steps.

[0440] The physical properties of EVs can be used for EV separation, purification, or enrichment, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieve separation, etc.), density (e.g., conventional or gradient centrifugation), and Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or in addition, separation can be based on one or more biological properties and includes methods that can use surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, immunomagnetic capture of EVs using magnetic beads coated with antibodies against proteins exposed on the EV membrane, etc.).

[0441] Methods based on the use of size-exclusion polymers (such as PEG) have recently been described by several different groups (US Patent Application No. 20130273544, US Patent Application No. 20130337440). Two such products are ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). These polymers work by binding water molecules and forcing less soluble components (such as extracellular vesicles and proteins) to precipitate from solution, allowing them to be collected by short, low-speed centrifugation.

[0442] In some implementations, separation, purification, and enrichment can be performed in a conventional and non-selective manner, typically involving continuous centrifugation. Alternatively, separation, purification, and enrichment can be performed in a more specific and selective manner, e.g., using cell-specific surface markers. For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, or for bead-bound ligands used for magnetic separation.

[0443] In some implementations, tangential flow filtration can be used to separate or purify EVs.

[0444] In some embodiments, size exclusion chromatography can be used to separate or purify EVs. Size exclusion chromatography techniques are known in the art. In some embodiments, density gradient centrifugation can be used to separate EVs. In some embodiments, EV separation can include ion chromatography, such as anion exchange, cation exchange, or mixed-mode chromatography. In some embodiments, EV separation can include desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some embodiments, EV separation can include a combination of methods, including but not limited to differential centrifugation, size-based membrane filtration, concentration, and / or rate zonal centrifugation. In some embodiments, EV separation can include one or more centrifugation steps. Centrifugation can be performed at about 50,000 to 150,000 × g. Centrifugation can be performed at about 50,000 × g, 75,000 × g, 100,000 × g, 125,000 × g, or 150,000 × g. In another embodiment, EVs are separated from non-membrane particles using their relatively low buoyancy density (Raposo et al., 1996; Escola et al., 1998; van Niel et al., 2003; Wubbolts et al., 2003). Kits for this separation are commercially available from, for example, Qiagen, InVitrogen, and SBI. Methods for loading therapeutic agents into EVs are known in the art and include lipid transfection, electroporation, and any standard transfection method.

[0445] Applications and uses

[0446] Screening assay

[0447] This invention provides a method for screening various reagents that regulate retinal progenitor cell differentiation. It can also be used to discover therapeutics that support and / or rescue mature photoreceptor cells generated from retinal progenitor cells in culture. For the purposes of this invention, "reagent" is intended to include, but is not limited to, biological or chemical compounds such as simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (e.g., antisense polynucleotides), or ribozymes. A wide range of compounds can be synthesized, such as polymers like polypeptides and polynucleotides, as well as synthetic organic compounds based on a variety of core structures, and these are also included in the term "reagent." Furthermore, a variety of natural sources can provide compounds for screening, such as plant or animal extracts. Although not always explicitly stated, it should be understood that the reagent is used alone or in combination with another reagent having the same or different biological activity as the reagent identified by screening according to this invention.

[0448] To practice the screening method in vitro, isolated cell populations can be obtained as described herein. When the reagent is a composition other than DNA or RNA (such as the small molecules described above), it can be added directly to the cells or to the culture medium for addition. As will be apparent to those skilled in the art, an "effective" amount must be added, which can be determined empirically. When the reagent is a polynucleotide, it can be added directly using a gene gun or electroporation. Alternatively, it can be inserted into the cells using a gene delivery medium or other methods described above. Positive and negative controls can be assayed to confirm the expected activity of the drug or other reagent.

[0449] Biocompatible carriers for photoreceptor cell rescue

[0450] Biocompatible carriers for cells can be biodegradable polyester membrane carriers for rescuing cells from photoreceptors. The biodegradable polyester can be any biodegradable polyester suitable for use as a substrate or scaffold. The polyester should be able to form a thin film, preferably a microtextured film, and should be biodegradable if used for tissue or cell transplantation. Suitable biodegradable polyesters for use in this invention include polylactic acid (PLA), polylactide, polyhydroxyalkanoates, homopolymers and copolymers such as polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyhydroxybutyrate-co-hydroxyhexanoate (PHBHx), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO), and polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), polycaprolactone (PCL), polyamide esters (PEA), aliphatic copolyesters such as polybutylene succinate (PBS) and polybutylene succinate / polybutylene adipate (PBSA), and aromatic copolyesters. High and low molecular weight polyesters, substituted and unsubstituted polyesters, block, branched or random, as well as polyester mixtures and blends can be used. Preferably, the biodegradable polyester is polycaprolactone (PCL).

[0451] In some embodiments, the biocompatible carrier is a poly(p-xylene) polymer, such as parylene N, parylene D, parylene-C, parylene AF-4, parylene SF, parylene HT, parylene VT-4, and parylene CF, with parylene-C being the most preferred.

[0452] Polymer supports can typically be formed into thin films using known techniques. The film thickness is advantageously from about 1 micrometer to about 50 micrometers, and preferably about 5 micrometers. The film surface can be smooth, or it can be partially or completely microtextured. Suitable surface textures include, for example, microgrooves or micropillars. The film can be cut and shaped to form a shape suitable for transplantation.

[0453] Photoreceptor-rescued cells can be directly plated onto a membrane to form a biocompatible scaffold. Alternatively, the polymer membrane can be coated with suitable coating materials such as poly-D-lysine, poly-L-lysine, fibronectin, and laminins (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332 / laminin-3A32, laminin-3B32, laminin-311 / laminin-3A11, laminin-321 / laminin-3A21, laminin-411, laminin-421, laminin-511 (e.g., iMatrix)). TM Laminin-511, laminin-521, laminin-213, laminin-432, laminin-522, laminin-532 and / or laminin fragments, collagen I, collagen IV, hyalin and Matrigel TM Cells can be plated to any desired density, but a monolayer of PRC cells (PRC monolayer) is preferred.

[0454] Alternatively, photoreceptor rescue cells (PRCs) can be administered together with other cell types, including other retinal cell types such as, but not limited to, retinal ganglion cells, retinal ganglion progenitor cells, retinal pigment epithelium (RPE) cells, or RPE progenitor cells. Photoreceptor rescue cells (PRCs) can be administered with one or any combination of these different cell types, such as corneal endothelial cells. As described above, the cells can be administered on a matrix or scaffold or membrane, or they can be administered as cell clumps, or they can be administered as dissociated cell suspensions. In some embodiments, the cells can be administered on top of a monolayer of RPE cells, and may or may not be located on the matrix or substrate. The cells to be administered can be entirely derived from in vitro differentiation of hES cells or iPS cells, or in some cases, they may be derived from or obtained from other sources. Some cells may be derived from in vitro differentiation of hES cells or iPS cells, and other cells may be derived from or obtained from other sources. At least, the photoreceptor rescue cells are derived from in vitro differentiation of pluripotent stem cells, such as hES cells or iPS cells. Any of these multiple cell combinations can be used in combination with other therapeutic agents, such as those described in this article.

[0455] Photoreceptor-rescued cells can be administered via devices such as syringes, Oxulumis-illuminated suprachoroidal microcatheters (Oxular), POD3 Gold (Oxular), Oxuspheres (Oxular), SCS microinjectors (Clearside Biomedical), and Orbit SDS (Gyroscope), via grafts, via cell-seedling substrates or transplantable membranes, alone or in combination with another cell type (e.g., retinal pigment epithelial cells).

[0456] Therapeutic uses

[0457] The present invention also provides a method for improving the activity / function of photoreceptor cells in patients requiring such treatment, the method comprising administering to the patient a pharmaceutical preparation comprising a variety of heterologous photoreceptor rescue cells, derived cells thereof, or combinations thereof of the present invention. As described herein, the pharmaceutical preparation may be a cell suspension or cells forming a transplantable tissue in vitro or on a substrate. In many cases, the cells are administered to the subretinal or suprachoroidal space of a diseased or degenerated retina. However, because the various heterologous photoreceptor rescue cells of the present invention also have neuroprotective effects, the cells may be administered locally, but extraretinically (e.g., in the vitreous), on the choroid, or delivered to other parts of the body via a depot or systemically. For example, the photoreceptor rescue cells of the present invention may be administered via a patch or other transplantable device, wherein the cell population secretes neuroprotective factors. In one embodiment, such a device may be reloaded with photoreceptor cells for repeated administration to improve the durability of the therapeutic effect.

[0458] The pharmaceutical preparations of this invention can be used for a wide range of diseases and conditions that lead to the deterioration of the visual system, including retinal degeneration-related diseases. These diseases and conditions can be caused by aging, so that there appears to be no lesion or disease that can be identified as a substantial source of deterioration. Those skilled in the art will understand the established methods for diagnosing such disease states and / or examining the known signs of such lesions. Furthermore, the literature is rich with information on age-related decline or deterioration of the visual system in animals. The term "retinal degeneration-related disease" is intended to refer to any disease caused by congenital or postnatal retinal degeneration or abnormality. Examples of retinal degeneration-related diseases include retinal developmental abnormalities, retinal degeneration, age-related macular degeneration (wet or dry), geographic atrophy secondary to AMD, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, leber congenital amaurosis, Staggart's disease, retinal detachment, glaucoma, optic neuropathy, and trauma.

[0459] In addition to this, or as an alternative, deterioration of visual system components (such as the neurosensory retina) can be caused by injury, such as trauma to the visual system itself (e.g., the eye), to the head or brain, or more generally to the body. Some of these injuries are known to be age-related, meaning their likelihood or frequency increases with age. Examples of such injuries include retinal tears, macular holes, epiretinal membranes, and retinal detachments, each of which can occur in animals of any age, but are more likely to occur or occur more frequently in aging animals (including other healthy aging animals).

[0460] Deterioration of the visual system or its components can also be caused by disease. These diseases include a variety of age-related disorders that affect the visual system. These diseases are more likely and / or more frequent in older animals than in younger animals. Examples of diseases that can affect the visual system (including, for example, the neurosensory retinal layer) and cause its deterioration include various forms of retinitis, optic neuritis, macular degeneration (wet or dry), geographic atrophy secondary to AMD, proliferative or non-proliferative diabetic retinopathy, diabetic macular edema, progressive retinal atrophy, progressive retinal degeneration, sudden acquired retinal degeneration, immune-mediated retinopathy, retinal dysplasia, chorioretinitis, retinal ischemia, retinal hemorrhage (preretinal, intraretinal, and / or subretinal), hypertensive retinopathy, retinitis, retinal edema, retinoblastoma, or retinitis pigmentosa.

[0461] Some of the diseases listed above are often specific to certain animals, such as companion animals like dogs and / or cats. A general list is provided for some diseases, noting that there may be multiple types of retinitis or retinal hemorrhage; therefore, some diseases are not caused by a single specific pathogen (causative agent), but rather are descriptions of the type or outcome of the disease. Multiple diseases can cause deterioration or worsening of one or more components of the visual system, and these diseases can have primary, secondary, or even more distant effects on the animal's visual system.

[0462] Advantageously, the pharmaceutical preparations of the present invention can be used to compensate for the lack or reduction of photoreceptor cell function. As shown in the examples, the cells of the present invention, including photoreceptor rescue cells, can be used as a cell replacement therapy in subjects who have completely or partially lost photoreceptor cell (photoreceptor) function. The visual acuity of these subjects (if human) can be characterized as 20 / 60 or worse, including 20 / 80 or worse, or 20 / 100 or worse, or 20 / 120 or worse, or 20 / 140 or worse, or 20 / 160 or worse, or 20 / 180 or worse, or 20 / 200 or worse. Therefore, this disclosure is contemplated for the treatment of subjects with a certain level of visual acuity as well as subjects without discernible visual acuity.

[0463] The characteristic of the photoreceptor rescue cells of this disclosure lies in their ability to reconstruct a certain level of visual acuity in animal models, such as mouse models. In some cases, suitable animal models may be those exhibiting visual deficits manifested as optomotor responses that are 10% or lower, 20% or lower, 30% or lower, 40% or lower, or 50% or lower compared to wild-type responses. Optomotor responses can be measured using the assays described in the examples. Upon transplantation of the photoreceptor rescue cells of this invention, such optomotor responses will increase, preferably by a statistically significant amount, as shown in the examples.

[0464] Therefore, this disclosure contemplates the application of compositions comprising the various heterologous photoreceptors described herein to rescue cells in order to completely or partially prevent disease progression, or to improve the vision of the receptor, or some combination thereof. The degree to which any mechanism contributes to the improvement of the outcome will depend on the extent of retinal degeneration in the receptor.

[0465] Preferably, the photoreceptor-rescue cell composition of the present invention is administered to a subject who still has functional photoreceptors to be retained / protected at the time of administration. Accordingly, the target patient group is subjects with intermediate-stage disease. In one specific embodiment, the subject has no substantial or near-complete loss of photoreceptors.

[0466] Examples of retinal dysfunction that can be treated by the retinal stem cell population and photoreceptor salvage cell compositions and methods of the present invention include, but are not limited to: partial or complete photoreceptor degeneration (such as occurring in, for example, retinitis pigmentosa, cone dystrophy, cone-rod and / or rod-cone dystrophy and macular degeneration); retinal detachment and retinal trauma; photopathologies caused by laser or sunlight; macular holes; macular edema; night blindness and color blindness; ischemic retinopathy caused by diabetes or vascular occlusion; retinopathy of prematurity; infectious conditions such as, for example, CMV retinitis and toxoplasmosis; inflammatory conditions such as uveitis; tumors such as retinoblastoma and ocular melanoma; and replacement of internal retinal neurons affected in ocular neuropathy, including glaucoma, traumatic optic neuropathy and radiation optic neuropathy and retinopathy.

[0467] In one aspect, photoreceptor rescue cells can treat or alleviate symptoms of retinitis pigmentosa in patients requiring treatment. In another aspect, said cells can treat or alleviate symptoms of macular degeneration in patients requiring such treatment, such as age-related macular degeneration (wet or dry), Stagger's disease, myopic macular degeneration, etc. For all these treatments, the cells can be autologous or allogeneic to the patient. In yet another aspect, the cells of the present invention can be administered in combination with other treatments.

[0468] Retinitis pigmentosa (RP) is a heterogeneous group of inherited eye diseases characterized by progressive vision loss due to the gradual degeneration of photoreceptors. It is estimated that 100,000 people in the United States have RP. This group of conditions is classified into several subgroups based on the most commonly observed clinical features in these patients. RP is characterized by night blindness and decreased peripheral vision, narrowing of retinal vessels, and the migration of pigment from ruptured retinal pigment epithelial cells to the retina, forming clumps of various sizes, usually located near retinal vessels.

[0469] Typically, patients first notice difficulty seeing at night due to the loss of rod photoreceptor cells; then, the remaining cone photoreceptor cells become the backbone of visual function. However, over years or even decades, the cones also degenerate, leading to progressive vision loss. In most patients with recurrent visual field (RP), the visual field defect begins at the equatorial region of the retina, within a fixed range of 30° to 50°. The defect area gradually expands, leaving peripheral visual islands and a constricted central visual field (called tubular visual field). When the visual field constricts to 200 degrees or less and / or central visual acuity is 20 / 200 or worse, the patient becomes legally blind.

[0470] Inheritance patterns indicate that RP can be transmitted in X-linked (XLRP), autosomal dominant (ADRP), or recessive (ARRP) patterns. Of the three inheritance types of RP, ADRP is the mildest. These patients typically maintain good central vision into their 60s. Conversely, patients with the XLRP type typically become legally blind between the ages of 30 and 40. However, the severity of symptoms and age of onset vary considerably among RP patients with the same genotype. This variation is evident even within the same family, assuming all affected members share the same genetic mutation. Several RP-induced mutations have been described. Among the genes currently identified, several encode photoreceptor-specific proteins, some of which are involved in light transduction in rod cells, such as rhodopsin, the cGMP phosphodiesterase subunit, and cGMP-gated Ca2+. 2+ The pathway. Multiple mutations have been found in each cloned gene. For example, in the case of the rhodopsin gene, 90 different mutations have been identified in ADRP patients.

[0471] Regardless of specific mutations, the most critical vision loss in patients with RP is due to the gradual degeneration of cone cells. In many cases, the proteins affected by RP mutations are not even expressed in cone cells; a prime example is rhodopsin—the rod-specific visual pigment. Therefore, the loss of cone cells can be an indirect consequence of rod-specific mutations. The ability to replace damaged photoreceptors offers a potential avenue for treatment of this disease.

[0472] In one specific implementation, for example, a subject is diagnosed with RP through genotyping. Specifically, prior to treatment, a subject is diagnosed with RP based on the identification of mutations affecting the RPE gene or photoreceptors. In another specific implementation, the patient has visual impairment, but not to the extent of complete blindness or non-light perception (NLP). Preferably, the best-corrected visual acuity (BCVA) of the subject suitable for treatment ranges from 20 / 50 (visual impairment) to 20 / 200 (legal blindness, but not NLP). In other implementations, the visual acuity of the subject suitable for treatment is below 20 / 200, but light perception is maintained.

[0473] Age-related macular degeneration (AMD) causes progressive loss of central vision and is the most common cause of vision loss in people over 55 years of age. The underlying pathology is the degeneration of photoreceptors. Multiple studies have identified genetic factors, cardiovascular disease, environmental factors (such as smoking and light exposure), and nutritional factors as contributing factors to the development of AMD. RPE degeneration is accompanied by varying degrees of loss of perfusion in the overlying photoreceptors and the underlying choroid. When the RPE atrophies and leads to secondary loss of the overlying photoreceptors it supplies, visual acuity loss or visual field defects occur. The ability to replace the RPE and / or photoreceptors provides a means of treating AMD that has already occurred.

[0474] AMD is diagnosed by fundus examination, and early / intermediate / late AMD is determined based on the anatomical features of the disease stage observed on fundus images. Intermediate AMD is defined as the presence of at least one large drusen (>125 μm) and / or pigmentary abnormality. AMD pigmentary abnormality is defined as hyperpigmentation or hypopigmentation within two optic disc diameters at the center of the macula in an eye with a choroidal drusen ≥63 μm in diameter and no known retinal disease entity or other cause of this abnormality. (See García-Layana A, Cabrera-López F, García-Arumí J, Arias-Barquet L, Ruiz-Moreno JM. Early and intermediate age-related macular degeneration: update and clinical review. Clin Interv Aging. 2017 Oct 3; 12:1579-1587). This definition covers a wide range of clinical manifestations of intermediate AMD, including a single large choroidal wart (>125 μm) or multiple large choroidal warts and numerous pigmentary abnormalities. These are two forms of advanced AMD, provided they do not meet the criteria for geographic atrophy or neovascular AMD.

[0475] Age-related macular degeneration (AMD) is broadly classified into two types. In exudative neovascular or “wet” AMD, which accounts for 10% of all cases, abnormal vascular growth occurs beneath the macula. The formation of a subretinal network of choroidal neovascularization is often associated with intraretinal hemorrhage, subretinal fluid, detachment of the pigment epithelium, and hyperpigmentation. Eventually, this complex contracts, leaving a prominent, raised scar at the posterior pole. These vessels leak fluid and blood into the retina, thus damaging the photoreceptors. Wet AMD tends to progress rapidly and can cause severe damage; central vision can be lost rapidly within just a few months.

[0476] The remaining 90% of AMD cases are atrophic macular degeneration (the dry form), in which pigmentary disturbances are present in the macular region, but without raised macular scars, hemorrhages, or exudates. In these patients, retinal pigment epithelial cells (RPE) gradually disappear, leading to localized areas of atrophy. Because the loss of photoreceptors follows closely after the disappearance of RPE, the affected retinal areas have very low or no visual function. Vision loss in dry AMD occurs gradually over many years. These patients often retain some central vision, although this loss can be severe enough to impair performance on tasks requiring the ability to see details.

[0477] When there is appropriate age and clinical findings accompanied by loss of visual acuity, visual field, or other visual function, this condition is usually classified as AMD. Sometimes, if the patient has characteristic choroidal conjunctivitis and a relevant family history, the steps preceding the onset of vision loss are classified as AMD.

[0478] Sometimes, macular degeneration occurs at a much younger age. Many of these cases are caused by gene mutations. There are several forms of hereditary macular degeneration, each with its own clinical presentation and genetic cause. The most common form of juvenile macular degeneration is called Stagger's disease, an autosomal recessive genetic disorder. Patients are usually diagnosed before the age of 20. Although the progression of vision loss is variable, most of these patients become legally blind before the age of 50. The mutation that causes Stagger's disease has been identified in the ABCR gene, which encodes a protein that transports retinoids across the photoreceptor membrane.

[0479] The photoreceptor-rescue cell composition of the present invention has found use in the treatment of degenerative diseases. The cells are administered in a manner that allows them to implant or migrate to the intended retinal site (such as in the outer nuclear layer) and to rebuild or regenerate areas of functional deficiency.

[0480] The examples demonstrate the ability of the photoreceptor rescue cells disclosed herein to regenerate visual acuity in a mouse model of blindness due to photoreceptor degeneration. Visual acuity in such mouse models can be evaluated using optokinetic responses (or optokinetic nystagmus responses).

[0481] In another aspect, this disclosure provides a method of drug delivery comprising administering to the patient a photoreceptor-rescue cell composition described herein or generated by any of the methods described herein, wherein the photoreceptor-rescue cell composition delivers the drug. A wide range of drugs can be used. Engineered photoreceptor rescue cell compositions can be prepared, thereby comprising one or more compounds selected from the following: drugs acting on synaptic and nerve effector junctions; drugs acting on the central nervous system; drugs modulating inflammatory responses, such as anti-inflammatory agents, including nonsteroidal anti-inflammatory agents; drugs affecting renal and / or cardiovascular function; drugs affecting gastrointestinal function; antibiotics; antiviral agents, antitumor agents, and anticancer agents; immunomodulators; anesthetics, steroidal drugs, antigens, vaccines, antibodies, decongestants, antihypertensive agents, sedatives, contraceptives, progestins, anticholinergic drugs, analgesics, antidepressants, antipsychotics, β-adrenergic blockers, diuretics, cardiovascular activators, vasoactive agents, nutrients; drugs acting on blood and / or hematopoietic organs; hormones; hormone antagonists; agents affecting calcification and bone turnover; vitamins; gene therapy agents; or other agents, such as targeting agents, etc.

[0482] For example, photoreceptor rescue cell compositions can be prepared such that they comprise one or more compounds selected from the following: drugs acting on synaptic and nerve effector junction sites (e.g., acetylcholine, methacholine, pilocarpine, atropine, scopolamine, physostigmine, succinylcholine, adrenaline, noradrenaline, dopamine, dobutamine, isoproterenol, albuterol, propranolol, serum...). Drugs acting on the central nervous system (e.g., clonazepam, diazepam, lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine, amitriptyline, fluoxetine, paroxetine, valproic acid, carbamazepine, bromocriptine, morphine, fentanyl, naltrexone, naloxone); drugs regulating inflammatory responses (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, cromoglycate). Drugs affecting renal and / or cardiovascular function (e.g., furosemide, thiazide, amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine, verapamil, digoxin, isorhizol nitrate, dobutamine, lidocaine, quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, mevalonate); drugs affecting gastrointestinal function (e.g., omeprazole, sucralfate); antibiotics (e.g., tetracycline, clindamycin, amphotericin B). B) Quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, acyclovir, zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin, streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid, fluconazole, amantadine, interferon;Anticancer drugs (e.g., cyclophosphamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, decarbazine); immunomodulators (e.g., interleukins, interferon, GM-CSF, TNFα, TNFβ, cyclosporine, FK506, azathioprine, steroids); drugs acting on blood and / or hematopoietic organs (e.g., interleukins, G-CSF, GM-CSF, erythropoietin, vitamins, iron, copper, vitamin B12, folic acid, heparin, warfarin, coumarin); hormones (e.g., growth hormone (GH), prolactin, luteinizing hormone). Hormones, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgens, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodothyronine; hormone antagonists; agents affecting calcification and bone turnover (e.g., calcium, phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride), vitamins (e.g., riboflavin, niacin, pyridoxine, pantothenic acid, biotin, choline, inositol, carnitine, vitamin C, vitamin A, vitamin E, vitamin K), gene therapy agents (e.g., viral vectors, liposomes carrying nucleic acids, DNA-protein conjugates, antisense agents); or other agents, such as targeting agents. The photoreceptor-rescue cell compositions of the present invention can be engineered to include one or more therapeutic agents, which are released or secreted by these cells in a passive manner (diffused out of the cells over time) or in an active manner (through intentional cell rupture or lysis). hESCs and / or hiPSCs can be genetically modified and used to generate photoreceptor rescue cells (PRCs) expressing agents desired for treating diseases. In one aspect, hESCs, hiPSCs, and / or multi-lymphocyte progenitor cells (MLPs) can be genetically modified to express antitumor agents. Photoreceptor rescue cells (PRCs) generated from such genetically modified hESCs and hiPSCs, adult stem cells, and multi-lymphocyte progenitor cells (MLPs) can be used to deliver such antitumor agents to tumors to treat cancerous diseases, including, for example, retinoblastoma.

[0483] In some embodiments, photoreceptor rescue cells of the present invention are generated by increasing the expression of one or more transcription factors selected from the following: PAX6, FOXG1, HMGA1 / 2, OTX2, ASCL1, POU3F2, NR2F1 / 2, NR6A1, MEIS2, NEUROD6, HES5, ATF4 / 5, and RXRG. In one embodiment, photoreceptor rescue cells are generated by increasing the expression of one or more transcription factors selected from the following in pluripotent stem cells, early neuronal progenitor cells, or late neuronal progenitor cells: PAX6, FOXG1, HMGA1 / 2, OTX2, ASCL1, POU3F2, NR2F1 / 2, NR6A1, MEIS2, NEUROD6, HES5, ATF4 / 5, and RXRG.

[0484] In some implementations, photoreceptor rescue cells (PRCs) have been engineered to include one or more therapeutic agents, such as small molecule drugs, aptamers or other nucleic acid reagents, or recombinant proteins.

[0485] Genetically engineered photoreceptor rescue cells can also be used to target gene products to degenerative sites. These gene products can include survival-promoting factors that rescue naturally degenerated neurons, factors that can act autocrinely to promote the survival of implanted cells and their differentiation into site-specific neurons, or deliver neurotransmitters to allow for functional recovery. Ex vivo gene therapies, such as recombinant engineered pluripotent stem cells, progenitor cells, early neural progenitor cells, late neural progenitor cells, or photoreceptor rescue cells, can be effectively used as a neuroprotective strategy to prevent retinal cell loss in RP (retinitis pigmentosa), AMD, and glaucoma, as well as diseases that cause retinal detachment, by delivering growth factors and neurotrophic proteins such as FGF2, NGF, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) (which have been shown to significantly slow cell death in models of retinal degeneration). Therapies using PRCs engineered to synthesize growth factors or combinations of growth factors can not only ensure the sustained delivery of neuroprotective agents but also reconstruct damaged retinas.

[0486] The photoreceptor-rescue cells of this invention can be administered in combination with one or more other therapeutic agents. As used herein, the phrases “combined administration” and “co-administered” refer to any form of administration of a combination of two or more different therapeutic entities, such that a second agent is administered while the previously administered therapeutic agent (e.g., cells) remains effective in vivo (e.g., both therapeutic agents are effective simultaneously in the patient, which may include synergistic effects between the two agents). For example, different therapeutic agents can be administered in the same formulation, wherein the cells comply with the co-formulation, or in separate formulations, either simultaneously or sequentially. Thus, individuals receiving this treatment can benefit from the combined effects of transplanted cells and one or more different therapeutic agents.

[0487] One or more angiogenesis inhibitors may be administered in combination (i.e., in combination) with cell preparations, preferably in a therapeutically effective amount for the prevention or treatment of eye diseases, such as angiogenesis-related eye diseases. Exemplary eye diseases include macular degeneration (e.g., wet AMD or dry AMD), diabetic retinopathy, and choroidal neovascularization. Exemplary angiogenesis inhibitors include VEGF antagonists, such as inhibitors of VEGF and / or VEGF receptors (VEGFR, e.g., VEGFR1 (FLT1, FLT), VEGFR2 (KDR, FLK1, VEGFR, CD309), VEGFR3 (FLT4, PCL)), such as peptides, peptide mimics, small molecules, chemicals, or nucleic acids, e.g., pegaptanib sodium, aflibercept, bevasiranib, rapamycin, AGN-745, vitalanib, pazopanib, NT-502, NT-503, or PLG101, CPD791 (a bis-Fab' polyethylene glycol (PEG) conjugate that inhibits VEGFR-2), anti-VEGF antibodies, or functional fragments thereof (e.g., bevacizumab). Or ranibizumab ) or anti-VEGF receptor antibodies (such as IMC-1121(B) (a monoclonal antibody against VEGFR-2) or IMC-18F1 (an antibody against the extracellular binding domain of VEGFR-1)). Other exemplary VEGF activity inhibitors include fragments or domains of the VEGFR receptor, an example of which is VEGF-Trap (aflibercept), a fusion protein of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 with the FC fragment of IgG1. Another exemplary VEGFR inhibitor is AZD-2171 (sildenafil), which inhibits VEGF receptors 1 and 2. Other exemplary VEGF antagonists include tyrosine kinase inhibitors (TKIs), including TKIs that have been reported to inhibit VEGFR-1 and / or VEGFR-2, such as sorafenib (Nexavar), SU5416 (Simasanib), SU11248 / sunitinib (Sutent), and vandetanib (ZD6474). Other exemplary VEGF antagonists include Ly317615 (inzatore), which is believed to target downstream kinases (protein kinase C) involved in VEGFR signaling. Other exemplary angiogenesis inhibitors include α5β1 integrin activity inhibitors, including anti-α5β1 integrin antibodies or functional fragments thereof (such as volociximab), peptides, peptide mimics, small molecules, chemicals, or nucleic acids such as 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidine-3-yloxy}-acetamido)-2-(alkyl-amino)-propionic acid, (S)-2-[(2,4,6-trimethylphenyl)sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]carbonylaminopropionic acid, EMD478761, or RC*D(thioP)C*(Arg-Cys-Asp-thioproline-Cys; asterisk indicates cyclization via disulfide bond through cysteine ​​residues).Other exemplary angiogenesis inhibitors include 2-methoxyestradiol, αVβ3 inhibitors, angiopoietin 2, angiogenesis-inhibiting steroids and heparin, angiostatin, angiostatin-related molecules, anti-α5β1 integrin antibody, anti-cathepsin S antibody, antithrombin III fragment, bevacizumab, calreticulin, angiostatin, carboxytamitonazole, cartilage-derived angiogenesis inhibitors, CDAI, CM101, CXCL10, endostatin, IFN-α, IFN-β, IFN-γ, IL-12, IL-18, IL-4, linolamine, mammary filament inhibitor (maspin), matrix metalloproteinase inhibitors, Meth-1, Meth-2, osteopontin, pegaptanib, platelet factor-4, prolactin, and proliferin-related protein. Protein), prothrombin (Kringle domain-2), ranibizumab, restin, soluble NRP-1, soluble VEGFR-1, SPARC, SU5416, suramin, tecogallan, tetrathiomolybdate, thalidomide, lenalidomide, thrombospondin, TIMP, TNP-470, TSP-1, TSP-2, angiogenesis inhibitors, VEGFR antagonists, VEGI, volociximab (also known as M200), fibronectin fragments such as anastellin (see Yi and Ruoslahti, Proc Natl Acad Sci USA. 2001 Jan. 16; 98(2):620-4) or any combination thereof. The angiogenesis inhibitor is preferably in an amount sufficient to prevent or eliminate proliferative (neovascular) eye diseases, such as choroidal neovascular membrane (CNV) and other retinal diseases associated with wet AMD. Other exemplary angiogenesis inhibitors include lenvatinib (E7080), motixaib (AMG 706), pazopanib (Votrient), and IL-6 antagonists, such as anti-IL-6 antibodies. Other exemplary angiogenesis inhibitors include any of the above-described fragments, mimics, chimeras, fusions, analogs, and / or domains. Other exemplary angiogenesis inhibitors include any combinations of the above. In an exemplary embodiment, the photoreceptor rescue cell composition comprises an anti-VEGF antibody, such as bevacizumab, such as about 0.1 mg to about 6.0 mg per ocular injection, for example, about 1.25 mg to about 2.5 mg of bevacizumab. In other exemplary embodiments, the photoreceptor rescue cell composition comprises one or more VEGF activity inhibitors and one or more α5β1 integrin activity inhibitors.

[0488] One or more anti-inflammatory agents can be administered in combination with photoreceptor-rescue cell compositions. Exemplary anti-inflammatory agents include: glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), aspirin, ibuprofen, naproxen, cyclooxygenase (COX) inhibitors, aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, fluocinolone acetonide (e.g., fluocinolone acetonide). ), glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids and triamcinolone.

[0489] One or more antioxidants, antioxidant cofactors, and / or other factors that help enhance antioxidant activity can be administered in combination with photoreceptor cell rescue compositions, examples of which may include OT-551 (Othera), vitamin C, vitamin E, beta-carotene, zinc (e.g., zinc oxide), and / or copper (e.g., copper oxide).

[0490] One or more lutein for yellow spots (such as lutein and / or zeaxanthin) can be applied in combination with a photoreceptor-rescue cell composition.

[0491] One or more long-chain ω-3 fatty acids, such as docosahexaenoic acid (DHA) and / or eicosapentaenoic acid (EPA), can be administered in combination with photoreceptor-rescue cell compositions.

[0492] One or more amyloid inhibitors, such as fenretinide, Arc-1905, Copaxone (glatiramer acetate, Teva), RN6G (PF-4382923, Pfizer) (a humanized monoclonal antibody against Aβ40 and Aβ42), and GSK933776 (GlaxoSmithKline) (an anti-amyloid antibody), can be administered in combination with photoreceptor-rescue cell compositions.

[0493] One or more ciliary neurotrophic factor (CNTF) agonists (e.g., CNTF that can be delivered in an intraocular device, such as NT-501 (Neurotech)) can be administered in combination with a photoreceptor rescue cell composition.

[0494] One or more inhibitors of RPE65, such as ACU-4429 (Aculea, Inc.), can be administered in combination with photoreceptor cell rescue compositions.

[0495] One or more factors targeting A2E and / or lipofuscin accumulation, such as fenivel-Aamine and ACU-4429, can be administered in combination with photoreceptor-rescue cell compositions.

[0496] One or more downregulatory factors or inhibitors of photoreceptor function and / or metabolism, such as fenivel-Amin and ACU-4429, can be administered in combination with photoreceptor rescue cell compositions.

[0497] One or more α2-adrenergic receptor agonists, such as brimonidine tartrate, can be administered in combination with photoreceptor rescue cell compositions.

[0498] One or more selective serotonin 1A agonists, such as tandospirone (AL-8309B), can be administered in combination with photoreceptor-rescue cell compositions.

[0499] In combination with photoreceptor-rescue cell compositions, one or more factors can be administered targeting C-5, the membrane attack complex (C5b-9), and / or any other choroidal wart component, examples of which include inhibitors of complement factor D, C-3, C-3a, C5, and C5a, and / or agonists of factor H, such as ARC 1905 (Ophthotec) (an anti-C5 aptamer that selectively inhibits C5), POT-4 (Potentia) (a campstatin derivative that inhibits C3), complement factor H, eculizumab (Soliris, Alexion) (a humanized IgG antibody that inhibits C5), and / or FCFD4514S (Genentech, San Francisco) (a monoclonal antibody against complement factor D).

[0500] One or more immunosuppressants, such as sirolimus (rapamycin), can be administered in combination with photoreceptor-rescue cell compositions.

[0501] One or more agents for the prevention or treatment of lipofuscin accumulation, such as piracetam, centrophenoxine, acetyl-L-carnitine, ginkgo or its extracts or preparations and / or DMAE (dimethylethanolamine), may be administered in combination with photoreceptor-rescue cell compositions.

[0502] When one or more agents (such as angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors that help enhance antioxidant activity, macular lutein, long-chain ω-3 fatty acids, amyloid inhibitors, CNTF agonists, RPE65 inhibitors, factors targeting A2E and / or lipofuscin accumulation, downregulatory factors or inhibitors of photoreceptor function and / or metabolism, α2-adrenergic receptor agonists, selective serotonin 1A agonists, C-5-targeting factors, membrane attack complex (C5b-9) and / or any other choroidal wart component, immunosuppressants, agents for the prevention or treatment of lipofuscin accumulation, etc.) are administered in combination with a photoreceptor rescue cell composition, the agents may be administered simultaneously with, before, and / or after the photoreceptor rescue cell composition. For example, the agents may be administered to the patient's eye during a procedure to introduce the photoreceptor rescue cell composition into the patient's eye. Administration of the agents may begin before and / or continue after the cells are administered to the patient's eye. For example, the agent may be in solution, suspension, as a sustained-release form, and / or in a continuous delivery system (e.g., Allergan Novadur). TM It is provided in a delivery system, NT-501 or another intraocular device or continuous release system.

[0503] In some embodiments, the cells may be engineered to include recombinant expression constructs that, when expressed in vivo by the cells, produce a recombinant form of the reagents described herein. In the case of antibodies, this includes double-stranded monoclonal antibodies and their epitope-binding fragments, such as Fab, Fab', and F(ab')2, Fd, Fv, single-stranded Fv (scFv), disulfide-linked Fv (sdFv), and fragments containing VL or VH domains, as well as fibronectin scaffolds and other antibody CDR mimics. Engineered cells may include expression constructs encoding recombinant peptides and proteins, and constructs that, upon transcription, form transcripts that produce RNA interference agents (such as siRNA, hairpin RNA, etc.), aptamers, decoys (which bind to transcription factors and repress the expression of native genes), antisense molecules, etc. Recombinant genes can be operatively linked to transcriptional regulatory elements, such as promoters and / or enhancers, which are active in transplanted cells (e.g., constitutively active elements or photosensitive active elements) or can be regulated by small molecules.

[0504] Exemplary recombinant agents to be expressed by transplanted cells include anti-angiogenic agents, such as those that reduce the occurrence of choroidal neovascularization (wet AMD). These include agents that inhibit VEGF-mediated ocular angiogenesis, such as anti-VEGF antibodies and VEGF receptor traps. These proteins include antibodies and antibody analogs (such as single-chain antibodies, monomers, antigen-binding sites, etc.), such as ranibizumab, and VEGF traps, such as aflibercept, which is a soluble protein including a ligand-binding domain from the VEGF receptor that binds to either VEGF or the VEGF receptor and blocks receptor activation.

[0505] Activation of the alternative complement pathway is associated with disease development in some patients, particularly in the case of dry AMD. Another class of exemplary recombinants expressed by transplanted cells includes complement inhibitors, such as inhibitors of complement factor D, factor C5, and / or factor C3. For illustrative purposes only, these can be RNA reagents or recombinant antibodies.

[0506] The choroidal wart deposits in dry AMD resemble amyloid deposits. Therefore, transplanted cells can be engineered to express anti-β-amyloid agents. These include recombinant antibodies, β-secretase inhibitors, etc.

[0507] Transplanted cells can also be engineered to express one or more anti-inflammatory agents, such as antagonists / inhibitors of pro-inflammatory cytokines like IL-1, IL-2, IL-3, and TNF-α, or anti-inflammatory cytokines like IL-37. Antagonists / inhibitors of pro-inflammatory cytokines include recombinant antibodies, receptor traps, aptamers, etc. In one embodiment, transplanted cells can be engineered to express recombinant lipocortin, a potent anti-inflammatory protein.

[0508] In the method of this invention, cells to be transplanted are transferred to a recipient in any physiologically available excipient, said excipient comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For the general principles of the pharmaceutical formulation, the reader refers to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, eds. G. Morstyn & W. Sheridan, Cambridge University Press, 1996. The selection of any accompanying elements of the cell excipient and composition will be adjusted according to the route of administration and device. Cells can be introduced by injection, catheter, etc. Cells can be frozen and stored for extended periods at liquid nitrogen temperatures and can be used by thawing. If frozen, cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0509] The pharmaceutical preparations of the present invention are optionally packaged in suitable containers having written instructions for use for the desired purpose. These preparations may contain a mixture of retinal differentiation and / or nutritional factors in a form suitable for combination with PRCs. Such compositions may also contain suitable buffers and / or excipients suitable for transfer to animals. Such compositions may further contain cells to be implanted.

[0510] Pharmaceutical preparations

[0511] PRCs can be formulated with pharmaceutically acceptable carriers. For example, PRCs can be administered alone or as a component of a pharmaceutical formulation. The subject compound can be formulated for administration in any convenient manner for medical use. Suitable pharmaceutical preparations may comprise PRCs together with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions, or emulsions, or sterile powders that can be immediately reconstituted into sterile injectable solutions or dispersions before use, said solutions may contain antioxidants, buffers, antibacterial agents, solutes or suspending agents, or thickeners. Exemplary pharmaceutical preparations comprise PRCs together with BSS (Equilibrium salt solution, each mL of the solution contains 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg disodium hydrogen phosphate, 2.1 mg sodium bicarbonate, 0.92 mg dextrose, 0.184 mg glutathione disulfide (oxidized glutathione), hydrochloric acid and / or sodium hydroxide (to adjust the pH to approximately 7.4)).

[0512] When administered, the pharmaceutical preparation used in this disclosure may be in a pyrogen-free, physiologically usable form.

[0513] The PRC-containing preparations used in the methods described herein can be implanted in suspensions, gels, colloids, slurries, or mixtures. Furthermore, the preparations can be desirably encapsulated or injected into the vitreous humor in a viscous form for delivery to retinal or choroidal lesion sites. Additionally, upon injection, cryopreserved PRC can be resuspended in a commercially available balanced salt solution to achieve the desired permeability and concentration for subretinal injection. The preparations can be applied to the pericentral macular region that is not yet fully diseased, which can promote the adhesion and / or survival of the applied cells.

[0514] As described herein, PRCs can be frozen (cryopreserved). Once thawed, the viability of these cells can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% of the cells harvested after thawing are alive, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100% of the initial frozen cell number harvested after thawing). In some cases, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, and at least about 85% of the cells in the composition are viable before and after cryopreservation and thawing. In some cases, after thawing, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% of the cells in the composition are viable. In some cases, before and after cryopreservation and thawing, the composition contains approximately 50% to approximately 85%, approximately 50% to approximately 80%, approximately 50% to approximately 75%, approximately 50% to approximately 70%, approximately 50% to approximately 65%, approximately 50% to approximately 60%, approximately 50% to approximately 55%, approximately 55% to approximately 85%, approximately 55% to approximately 80%, approximately 55% to approximately 75%, approximately 55% to approximately 70%, approximately 55% to approximately 65%, and approximately 55% to approximately 60%. About 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 85%, about 70% to about 75%, about 75% to about 85%, about 75% to about 80%, or about 80% to about 85% of the cells are viable. In some cases, after cryopreservation and thawing, at least about 50% of the cells in the composition are viable. In some cases, after cryopreservation and thawing, at least about 55% of the cells in the composition are viable. In some cases, after cryopreservation and thawing, at least about 60% of the cells in the composition are viable. In some cases, after cryopreservation and thawing, at least about 65% of the cells in the composition are viable. In some cases, at least about 70% of the cells in the composition are viable after cryopreservation and thawing. In some cases, at least about 75% of the cells in the composition are viable after cryopreservation and thawing. In some cases, the cell viability is about 80% before and after thawing. In some cases, at least 90% or at least 95% or about 95% of the frozen cells are recovered.Cells can be frozen as single cells or as aggregates. For example, they can be frozen as neurosphere cells.

[0515] The PRC of this disclosure can be delivered in a pharmaceutically acceptable ophthalmic formulation via intraocular injection. When the formulation is administered via intravitreal injection, for example, the solution can be concentrated to deliver a minimal volume. Based on the factors described herein, the concentration used for injection can be any effective and non-toxic amount. It can be at least about 10 4 Pharmaceutical preparations for treating patients with PRC are formulated at a dose of approximately 10 cells / mL. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 Or 10 10 PRC preparations are formulated at doses of 1 PRC cells / mL for the treatment of patients. For example, photoreceptor rescue cells are formulated in pharmaceutically acceptable carriers or excipients.

[0516] The pharmaceutical preparations of PRCs described herein may contain at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, or at least about 9,000 PRCs. The PRC pharmaceutical preparations may contain at least about 1 × 10⁻⁶ PRCs. 4 , at least about 2×10 4 At least approximately 3×10 4 , at least about 4×10 4 At least approximately 5×10 4 , at least about 6×10 4 At least approximately 7×10 4 , at least about 8×10 4 At least approximately 9×10 4 , at least about 1×10 5 , at least about 2×10 5 At least approximately 3×10 5 , at least about 4×10 5 At least approximately 5×10 5 , at least about 6×10 5 At least approximately 7×10 5 , at least about 8×10 5 At least approximately 9×10 5 , at least about 1×10 6 , at least about 2×10 6 At least approximately 3×10 6 , at least about 4×10 6At least approximately 5×10 6 , at least about 6×10 6 At least approximately 7×10 6 , at least about 8×10 6 At least approximately 9×10 6 , at least about 1×10 7 , at least about 2×10 7 At least approximately 3×10 7 , at least about 4×10 7 At least approximately 5×10 7 , at least about 6×10 7 At least approximately 7×10 7 , at least about 8×10 7 At least approximately 9×10 7 , at least about 1×10 8 , at least about 2×10 8 At least approximately 3×10 8 , at least about 4×10 8 At least approximately 5×10 8 , at least about 6×10 8 At least approximately 7×10 8 , at least about 8×10 8 At least approximately 9×10 8 , at least about 1×10 9 , at least about 2×10 9 At least approximately 3×10 9 , at least about 4×10 9 At least approximately 5×10 9 , at least about 6×10 9 At least approximately 7×10 9 , at least about 8×10 9 At least approximately 9×10 9 , at least about 1×10 10 , at least about 2×10 10 At least approximately 3×10 10 , at least about 4×10 10 At least approximately 5×10 10 , at least about 6×10 10 At least approximately 7×10 10 , at least about 8×10 10 Or at least about 9×10 10 One PRC. A PRC pharmaceutical preparation may contain at least approximately 1 × 10⁻⁶. 2 To approximately 1×10 3 , about 1×10 2 To approximately 1×10 4 , about 1×10 4 To approximately 1×10 5 Or approximately 1×103 To approximately 1×10 6 PRC pharmaceutical preparations may contain at least about 10,000, at least about 20,000, at least about 25,000, at least about 50,000, at least about 75,000, at least about 100,000, at least about 125,000, at least about 150,000, at least about 175,000, at least about 180,000, at least about 185,000, at least about 190,000, or at least about 200,000 PRCs. For example, a PRC pharmaceutical preparation may contain at least about 20,000 to about 200,000 PRCs in a volume of at least about 50 to about 200 μL. Furthermore, the PRC drug preparation may contain approximately 50,000 PRCs in a volume of approximately 150 μL, approximately 200,000 or more PRCs in a volume of approximately 150 μL, or approximately 180,000 PRCs in a volume of at least approximately 150 μL.

[0517] In the aforementioned pharmaceutical preparations and compositions, the number or concentration of PRCs can be determined by counting live cells and excluding non-live cells. For example, non-live PRCs can be detected by the inability to exclude a vital dye (such as trypan blue) or by using functional assays (such as adhesion to culture medium, phagocytic ability, etc.). Alternatively, the number or concentration of PRCs can be determined by counting cells expressing one or more PRC markers and / or excluding cells expressing one or more markers indicating cell types other than PRCs.

[0518] PRCs can be formulated for delivery in pharmaceutically acceptable ophthalmic mediators, thereby maintaining contact between the preparation and the ocular surface for a sufficient period of time to allow cells to penetrate diseased areas of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous fluid, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal cavity, extracorneal cavity, pars plana, surgically induced avascular areas, or macula.

[0519] PRCs can be contained in sheets of cells. For example, cell sheets containing PRC can be prepared by culturing PRC, such as a thermosensitive polymer, like thermosensitive poly(N-isopropylacrylamide) (PNIPAAm), on a substrate from which intact cell sheets can be released. Cells adhere and proliferate on the substrate at the culture temperature, and then, once the temperature changes, the surface characteristics change, resulting in the release of the cell culture sheets (e.g., by cooling to below the minimum critical dissolution temperature (LCST)). (See da Silva et al., Trends Biotechnol. 2007 December; 25(12):577-83; Hsiue et al., Transplantation. 2006 Feb. 15; 81(3):473-6; Ide, T. et al. (2006); Biomaterials 27, 607-614; Sumide, T. et al. (2005), FASEB J. 20, 392-394; Nishida, K. et al. (2004), Transplantation. 77, 379-385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187-1196, each of which is incorporated herein by reference in its entirety. Cell sheets can adhere to a substrate suitable for transplantation, such as a substrate that can dissolve in vivo when said sheet is transplanted into a host organism, for example, by culturing cells on a substrate suitable for transplantation, or by releasing cells from another substrate (such as a thermosensitive polymer) to a substrate suitable for transplantation. Potentially suitable exemplary substrates for transplantation include... It may contain gelatin (see Hsiue et al., as above). Alternative substrates suitable for transplantation include fibrin-based matrices, etc. The cell sheets can be used in the production of agents for the prevention or treatment of retinal degenerative diseases. PRC sheets can be formulated for introduction into the eyes of subjects in need. For example, the cell sheets can be introduced into the eyes in need via subfoveal membranectomy with PRC sheet transplantation, or the cell sheets can be used to produce agents for transplantation after subfoveal membranectomy.

[0520] The volume of the preparatio...

Claims

1. A photoreceptor rescue cell composition comprising a plurality of heterologous photoreceptor rescue cells, wherein the plurality of heterologous photoreceptor rescue cells cumulatively express at least two of the markers selected from the group consisting of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA.

2. The photoreceptor-salvage cell composition according to claim 1, further comprising a medium suitable for maintaining cell viability. 3 . The photoreceptor-salvaged cell composition according to claim 1 , wherein the cells are produced by in vitro differentiation of pluripotent cells. The photoreceptor-salvaged cell composition according to claim 3 , wherein the pluripotent cells are embryonic cells (ESCs) or induced pluripotent stem cells (iPSCs).

5. The photoreceptor-salvage cell composition of any of the preceding claims, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the composition are photoreceptor-salvage cells. 6 . The photoreceptor-salvage cell composition of any one of the preceding claims, wherein the plurality of heterologous cells cumulatively express FOXG1 and MAP2.

7. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 50% to about 100%, about 50% to about 90%, about 55% to about 85%, or about 55% to about 73% of the cells in the composition express FOXG1; and / or (ii) at least about 50%, 55%, 57%, 60%, 65%, 70%, 71%, 75%, 78%, 79%, 80%, 81%, 85%, 87%, 90%, 92%, 95%, 97% or 100% of the cells in the composition express FOXG1, and / or (iii) cells of the composition express FOXG1 transcripts from about 55 transcripts per million (TPM) to about 200 TPM, from about 60 TPM to about 170 TPM, from about 140 TPM to about 165 TPM, or from about 149 TPM to about 170 TPM; and / or (iv) the cells of the composition express at least 55 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM or 200 TPM of FOXG1 transcripts.

8. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 75% to about 100%, about 75% to about 98%, about 75% to about 95%, about 77% to about 93% of the cells in the composition express MAP2; and / or (ii) at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the composition express MAP2; and / or (iii) cells of the composition express about 250 transcripts per million (TPM) to about 700 TPM, about 290 TPM to about 650 TPM, about 450 TPM to about 625 TPM, or about 490 TPM to about 615 TPM of MAP2 transcripts; and / or (iv) the cells of the composition express at least 250 TPM, 300 TPM, 350 TPM, 400 TPM, 450 TPM, 475 TPM, 490 TPM, 510 TPM, 525 TPM, 550 TPM, 575 TPM, 600 TPM, 610 TPM, 625 TPM, 650 TPM, 675 TPM or 700 TPM of MAP2 transcript. 9 . The photoreceptor-rescue cell composition of claim 6 , wherein the plurality of heterologous cells cumulatively express at least one other marker selected from STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

10. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein the plurality of heterologous cells cumulatively express at least 3, 4, 5, 6 or 7 of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 and NFIA.

11. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, or about 75% to about 80% of the cells in the composition express STMN2; and / or (ii) at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 100% of the cells in the composition express STMN2; and / or (iii) cells of the composition express STMN2 transcripts at about 150 transcripts per million (TPM) to about 600 TPM, about 190 TPM to about 560 TPM, about 400 TPM to about 600 TPM, or about 450 TPM to about 560 TPM; and / or (iv) the cells of the composition express at least 150 TPM, 185 TPM, 200 TPM, 250 TPM, 300 TPM, 350 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 525 TPM, 550 TPM, 575 TPM or 600 TPM of STMN2 transcripts.

12. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 65% to about 95%, about 65% to about 85%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 75% to about 95%, about 75% to about 90%, or about 75% to about 89% of the cells in the composition express DCX; and / or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95% of the cells in the composition express DCX; and / or (iii) the cells of the composition express about 200 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 900 TPM, about 600 TPM to about 900 TPM, or about 750 TPM to about 850 TPM of DCX transcripts; and / or (iv) the cells of the composition express at least 200 TPM, 250 TPM, 350 TPM, 400 TPM, 450 TPM, 500 TPM, 550 TPM, 600 TPM, 650 TPM, 700 TPM, 750 TPM, 800 TPM, 850 TPM or 900 TPM of DCX transcripts.

13. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 65% to about 98%, about 65% to about 95%, about 70% to about 98%, about 70% to about 95%, about 70% to about 90%, about 75% to about 98%, about 75% to about 90%, or about 80% to about 95% of the cells in the composition express LINC00461; and / or (ii) at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or 98% of the cells in the composition express LINC00461; and / or (iii) cells of the composition express about 50 transcripts per million (TPM) to about 100 TPM, about 50 TPM to about 95 TPM, about 85 TPM to about 95 TPM, or about 87 TPM to about 93 TPM of LINC00461 transcripts; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 87 TPM, 89 TPM, 90 TPM, 92 TPM, 95 TPM or 100 TPM of LINC00461 transcripts.

14. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 1% to about 8%, about 1% to about 7%, about 1% to about 5%, or about 2% to about 4% of the cells in the composition express NEUROD2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% or 25% of the cells in the composition express NEUROD2; and / or (iii) cells of the composition express about 0 transcripts per million (TPM) to about 10 TPM, about 0.01 TPM to about 9 TPM, about 0.2 TPM to about 2 TPM, or about 0.4 TPM to about 1.2 TPM of NEUROD2 transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.4 TPM, 0.6 TPM, 0.8 TPM, 1.0 TPM, 1.2 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of NEUROD2 transcripts.

15. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 35% to about 70%, about 35% to about 68%, about 35% to about 67%, about 35% to about 66%, about 35% to about 65%, about 40% to about 70%, about 40% to about 68%, about 40% to about 67%, about 40% to about 66%, about 40% to about 65%, about 42% to about 70%, about 42% to about 68%, about 42% to about 67%, about 42% to about 66%, or about 42% to about 65% of the cells in the composition express GAD1; and / or (ii) at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69% or 70% of the cells in the composition express GAD1; and / or (iii) cells of the composition express about 10 transcripts per million (TPM) to about 50 TPM, about 12 TPM to about 45 TPM, about 12 TPM to about 40 TPM, or about 25 TPM to about 42 TPM of GAD1 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 12 TPM, 16 TPM, 18 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 47 TPM or 50 TPM of GAD1 transcripts.

16. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 60% to about 95%, about 60% to about 90%, about 60% to about 89%, about 60% to about 88%, about 60% to about 87%, about 60% to about 86%, about 65% to about 95%, about 65% to about 90%, about 65% to about 89%, about 65% to about 88%, about 65% to about 87%, about 65% to about 86%, about 69% to about 90%, about 69% to about 89%, about 69% to about 88%, about 69% to about 87%, or about 69% to about 86% of the cells in the composition express NFIA; and / or (ii) at least about 50%, 55%, 60%, 65%, 67%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 92% or 95% of the cells in the composition express NFIA; and / or (iii) the cells of the composition express about 30 transcripts per million (TPM) to about 120 TPM, about 33 TPM to about 117 TPM, about 60 TPM to about 85 TPM, or about 65 TPM to about 80 TPM of NFIA transcripts; and / or (iv) the cells of the composition express at least 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM or 120 TPM of NFIA transcripts.

17. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein the plurality of heterologous cells cumulatively express each of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

18. The photoreceptor-salvage cell composition according to any one of the preceding claims, wherein the heterologous cells express at least one of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1 or NFIA, respectively.

19. The photoreceptor-salvage cell composition of any one of claims 1-18, wherein the plurality of heterologous photoreceptor-salvage cells comprises one or more cell types selected from inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and replacement neurons.

20. The photoreceptor rescue cell composition of claim 19, wherein the plurality of heterologous photoreceptor rescue cells comprises each of inhibitory neurons, excitatory neurons, progenitor cells, astrocytes, and replacement neurons.

21. The photoreceptor rescue cell composition of claim 19 or claim 20, wherein the plurality of heterologous photoreceptor rescue cells comprises inhibitory neurons expressing one or more markers selected from DLX5, TUBB3, SCGN, ERBB4 and CALB2.

22. The photoreceptor rescue cell composition according to any one of claims 19 to 21, wherein the plurality of heterologous photoreceptor rescue cells comprises a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2.

23. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 52% to about 69%, about 52% to about 75%, about 54% to about 69%, about 54% to about 68%, or about 54% to about 66% of the cells in the composition express DLX5; and / or (ii) at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 72%, 74%, 76%, 78% or 80% of the cells in the composition express DLX5; and / or (iii) cells of the composition express about 30 transcripts per million (TPM) to about 150 TPM, about 50 TPM to about 140 TPM, about 80 TPM to about 138 TPM, or about 130 TPM to about 140 TPM of DLX5 transcripts; and / or (iv) the cells of the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 135 TPM, 140 TPM or 150 TPM of DLX5 transcripts.

24. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 60% to about 95%, about 70% to about 95%, about 72% to about 95%, about 75% to about 95%, about 76% to about 94%, about 77% to about 93%, about 78% to about 93%, about 70% to about 90%, about 72% to about 89%, about 73% to about 88%, about 74% to about 87%, about 75% to about 87%, about 76% to about 86%, about 77% to about 86%, or about 79% to about 86% of the cells in the composition express TUBB3; and / or (ii) at least about 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93% or 95% of the cells in the composition express TUBB3; and / or (iii) cells of the composition express TUBB3 transcripts at about 150 transcripts per million (TPM) to about 500 TPM, about 160 TPM to about 450 TPM, about 300 TPM to about 450 TPM, about 350 TPM to about 430 TPM, or about 375 TPM to about 430 TPM; and / or (iv) the cells of the composition express at least 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 430 TPM, 450 TPM, 475 TPM or 500 TPM of TUBB3 transcripts.

25. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 45% to about 70%, about 45% to about 65%, about 50% to about 70%, about 50% to about 65%, about 50% to about 64%, about 50% to about 63%, about 50% to about 62%, about 50% to about 61%, about 46% to about 52%, about 47% to about 51%, about 48% to about 51%, or about 48% to about 50% of the cells in the composition express SCGN; and / or (ii) at least about 45%, 47%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 65%, 67%, or 70% of the cells in the composition express SCGN; and / or (iii) cells of the composition express SCGN transcripts at about 50 transcripts per million (TPM) to about 200 TPM, about 70 TPM to about 180 TPM, about 75 TPM to about 175 TPM, or about 120 TPM to about 175 TPM; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 171 TPM, 173 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM or 200 TPM of SCGN transcripts.

26. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 60% to about 85%, about 60% to about 80%, about 60% to about 79%, about 60% to about 78%, about 60% to about 77%, about 60% to about 76%, about 63% to about 75%, about 63% to about 70%, about 63% to about 79%, about 63% to about 78%, about 63% to about 77%, about 63% to about 75%, about 63% to about 73%, about 63% to about 72%, about 63% to about 71%, about 65% to about 72%, or about 66% to about 71% of the cells in the composition express ERBB4; and / or (ii) at least about 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 80% or 85% of the cells in the composition express ERBB4; and / or (iii) cells of the composition express ERBB4 transcripts at about 15 transcripts per million (TPM) to about 120 TPM, about 17 TPM to about 100 TPM, about 18 TPM to about 95 TPM, about 70 TPM to about 95 TPM, or about 73 TPM to about 94 TPM; and / or (iv) the cells of the composition express at least 15 TPM, 30 TPM, 50 TPM, 70 TPM, 73 TPM, 75 TPM, 80 TPM, 82 TPM, 85 TPM, 88 TPM, 90 TPM, 91 TPM, 93 TPM, 95 TPM, 100 TPM, 110 TPM or 120 TPM of ERBB4 transcripts.

27. The photoreceptor-salvaged cell composition according to any one of the preceding claims, wherein (i) about 35% to about 75%, about 40% to about 70%, about 40% to about 65%, about 41% to about 75%, about 41% to about 70%, about 41% to about 65%, about 41% to about 64%, about 41% to about 63%, about 41% to about 62%, about 35% to about 55%, about 40% to about 52%, or about 41% to about 52% of the cells in the composition express CALB2; and / or (ii) at least about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 45%, 47%, 50%, 51%, 52%, 55%, 60%, 65%, 70% or 75% of the cells in the composition express CALB2; and / or (iii) cells of the composition express CALB2 transcripts at about 30 transcripts per million (TPM) to about 220 TPM, about 50 TPM to about 200 TPM, about 70 TPM to about 200 TPM, or about 75 TPM to about 199 TPM; and / or (iv) the cells of the composition express at least 30 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 195 TPM, 196 TPM, 220 TPM, 210 TPM or 220 TPM of CALB2 transcripts.

28. The photoreceptor rescue cell composition of any one of claims 19-27, wherein the plurality of heterogeneous photoreceptor rescue cells comprises excitatory neurons expressing one or more markers selected from NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

29. The photoreceptor rescue cell composition according to any one of claims 19 to 28, wherein the plurality of heterologous photoreceptor rescue cells comprises a plurality of excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

30. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 24%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 24%, about 0.8% to about 30%, about 0.8% to about 25%, about 0.8% to about 24%, about 1% to about 5%, about 1% to about 4.5%, about 2% to about 5%, about 2% to about 4.5%, or about 2% to about 4% of the cells in the composition express NEUROD6; and / or (ii) at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 2%, 4%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 23%, 24%, 26%, 28% or 30% of the cells in the composition express NEUROD6; and / or (iii) cells of the composition express about 1 transcripts per million (TPM) to about 210 TPM, about 1 TPM to about 205 TPM, about 1 TPM to about 25 TPM, or about 10 TPM to about 20 TPM of NEUROD6 transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 5 TPM, 10 TPM, 12 TPM, 15 TPM, 19 TPM, 50 TPM, 100 TPM, 150 TPM, 175 TPM, 200 TPM, 203 TPM or 210 TPM of NEUROD6 transcripts.

31. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 0.5% to about 20%, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express SLA; and / or (ii) at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 18%, or 20% of the cells in the composition express SLA; and / or (iii) cells of the composition express about 0.1 transcripts per million (TPM) to about 60 TPM, about 0.1 TPM to about 50 TPM, about 1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 3 TPM to about 6 TPM of SLA transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 10 TPM, 20 TPM, 30 TPM, 40 TPM, 50 TPM or 60 TPM of SLA transcripts.

32. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 30%, or about 20% to about 28% of the cells in the composition express NELL2; and / or (ii) at least about 10%, 12%, 15%, 17%, 20%, 21%, 24%, 25%, 27%, 30%, 32%, 35%, 40% or 45% of the cells in the composition express NELL2; and / or (iii) cells of the composition express about 1 transcripts per million (TPM) to about 150 TPM, about 4 TPM to about 130 TPM, about 4 TPM to about 35 TPM, about 20 TPM to about 30 TPM, or about 25 TPM to about 28 TPM of NELL2 transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 5 TPM, 15 TPM, 20 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 120 TPM or 150 TPM of NELL2 transcripts.

33. The photoreceptor-salvaged cell composition of any one of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 12%, about 1% to about 11%, about 2% to about 20%, about 2% to about 15%, about 2% to about 12%, about 2% to about 11%, about 3% to about 20%, about 3% to about 15%, about 3% to about 12%, about 3% to about 11%, about 2% to about 6%, about 2% to about 5%, or about 3% to about 4% of the cells in the composition express SATB2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17% or 20% of the cells in the composition express SATB2; and / or (iii) cells of the composition express SATB2 transcripts at about 0.1 transcripts per million (TPM) to about 30 TPM, about 0.5 TPM to about 20 TPM, about 1 TPM to about 5 TPM, or about 2 TPM to about 3 TPM; and / or (iv) the cells of the composition express at least 0.1 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 25 TPM or 30 TPM of SATB2 transcripts.

34. The photoreceptor rescue cell composition of any one of claims 19-33, wherein the plurality of heterologous photoreceptor rescue cells comprises progenitor cells expressing one or more markers selected from VIM, MKI67, CLU and GLI3.

35. The photoreceptor rescue cell composition of any one of claims 19-34, wherein the plurality of allogeneic photoreceptor rescue cells comprises a plurality of progenitor cells that cumulatively express each of the markers VIM, MKI67, CLU, and GLI3.

36. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 40% to about 75%, about 40% to about 70%, about 40% to about 69%, about 40% to about 60%, or about 42% to about 47% of the cells in the composition express VIM; and / or (ii) at least about 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 69%, 72%, 75%, or 80% of the cells in the composition express VIM; and / or (iii) cells of the composition express VIM transcripts at about 250 transcripts per million (TPM) to about 900 TPM, about 250 TPM to about 865 TPM, about 200 TPM to about 350 TPM, or about 250 TPM to about 340 TPM; and / or (iv) the cells of the composition express at least 250 TPM, 260 TPM, 270 TPM, 300 TPM, 320 TPM, 350 TPM, 370 TPM, 400 TPM, 500 TPM, 600 TPM, 700 TPM, 800 TPM or 900 TPM of VIM transcripts.

37. The photoreceptor-salvaged cell composition of any one of the preceding claims, wherein (i) about 5% to about 20%, about 5% to about 15%, about 5% to about 12%, about 6% to about 15%, about 6% to about 12%, about 7% to about 15%, about 7% to about 12%, or about 6% to about 8% of the cells in the composition express MKI67; and / or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the cells in the composition express MKI67; and / or (iii) the cells of the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 10 TPM to about 35 TPM, about 15 TPM to about 25 TPM, or about 18 TPM to about 22 TPM of MKI67 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 17 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 33 TPM, 35 TPM, 37 TPM or 40 TPM of MKI67 transcripts.

38. The photoreceptor-salvaged cell composition of any one of the preceding claims, wherein (i) about 10% to about 60%, about 15% to about 55%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 40%, about 20% to about 35%, or about 25% to about 32% of the cells in the composition express CLU; and / or (ii) at least about 10%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 55%, or 60% of the cells in the composition express CLU; and / or (iii) cells of the composition express CLU transcripts from about 30 transcripts per million (TPM) to about 400 TPM, from about 40 TPM to about 150 TPM, from about 60 TPM to about 150 TPM, or from about 60 TPM to about 105 TPM; and / or (iv) the cells of the composition express at least 30 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 125 TPM, 150 TPM, 175 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 365 TPM, 375 TPM or 400 TPM of CLU transcripts.

39. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 12% to about 50%, about 12% to about 35%, about 12% to about 29%, about 15% to about 29%, about 15% to about 29%, or about 15% to about 17% of the cells in the composition express GLI3; and / or (ii) at least about 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GLI3; and / or (iii) the cells of the composition express about 5 transcripts per million (TPM) to about 60 TPM, about 10 TPM to about 45 TPM, about 15 TPM to about 30 TPM, or about 20 TPM to about 25 TPM of GLI3 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 10 TPM, 12 TPM, 15 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 25 TPM, 30 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM or 60 TPM of GLI3 transcripts.

40. The photoreceptor rescue cell composition of any one of claims 19-39, wherein the plurality of heterologous photoreceptor rescue cells comprises astrocytes expressing one or more markers selected from GFAP, LUCAT1, MIR99AHG, and FBXL7.

41. The photoreceptor rescue cell composition of any one of claims 19-40, wherein the plurality of heterologous photoreceptor rescue cells comprises a plurality of astrocytes that cumulatively express each of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

42. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 50%, about 1% to about 20%, about 1% to about 15%, about 1% to about 13%, about 1% to about 10%, about 1% to about 7%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3% of the cells in the composition express GFAP; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express GFAP; and / or (iii) cells of the composition express GFAP transcripts from about 0.1 transcripts per million (TPM) to about 150 TPM, from about 0.1 TPM to about 125 TPM, from about 1 TPM to about 20 TPM, or from about 3 TPM to about 15 TPM; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.5 TPM, 1 TPM, 5 TPM, 7 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 30 TPM, 40 TPM, 50 TPM, 80 TPM, 100 TPM, 110 TPM, 115 TPM, 120 TPM, 130 TPM, 140 TPM or 150 TPM of GFAP transcripts.

43. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 5% to about 20%, about 5% to about 17%, about 5% to about 15%, about 5% to about 13%, about 7% to about 20%, about 7% to about 17%, about 7% to about 15%, about 7% to about 13%, about 5% to about 12%, or about 7% to about 10% of the cells in the composition express LUCAT1; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17% or 20% of the cells in the composition express LUCAT1.

44. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 50% to about 100%, about 50% to about 90%, about 50% to about 88%, about 60% to about 100%, about 60% to about 90%, about 60% to about 88%, about 70% to about 90%, about 70% to about 88%, or about 75% to about 82% of the cells in the composition express MIR99AHG; and / or (ii) at least about 50%, 60%, 65%, 70%, 72%, 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 96%, 98% or 100% of the cells in the composition express MIR99AHG; and / or (iii) cells of the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 30 TPM, about 6 TPM to about 25 TPM, or about 10 TPM to about 15 TPM of MIR99AHG transcripts; and / or (iv) the cells of the composition express at least 5TPM, 6TPM, 7TPM, 8TPM, 9TPM, 10TPM, 11TPM, 12TPM, 13TPM, 14TPM, 15TPM, 16TPM, 17TPM, 18TPM, 19TPM, 20TPM, 21TPM, 22TPM, 23TPM, 24TPM, 28TPM, 30TPM, 34TPM, 38TPM or 40TPM of MIR99AHG transcripts.

45. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 20% to about 70%, about 20% to about 60%, about 25% to about 70%, about 25% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 40%, about 32% to about 39%, or about 34% to about 39% of the cells in the composition express FBXL7; and / or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 60%, 65%, or 70% of the cells in the composition express FBXL7; and / or (iii) the cells of the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 30 TPM, about 7 TPM to about 25 TPM, about 10 TPM to about 15 TPM, or about 11 TPM to about 14 TPM of FBXL7 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 28 TPM, 30 TPM, 34 TPM, 38 TPM or 40 TPM of FBXL7 transcripts.

46. ​​The photoreceptor rescue cell composition of any one of claims 19-45, wherein the plurality of heterologous photoreceptor rescue cells comprises replacement neurons expressing one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2, and CACNA1C.

47. The photoreceptor rescue cell composition of any one of claims 19-46, wherein the plurality of heterologous photoreceptor rescue cells comprises a plurality of replacement neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2, and CACNA1C.

48. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 30% to about 80%, about 30% to about 90%, about 40% to about 90%, about 45% to about 85%, about 45% to about 80%, about 49% to about 79%, or about 50% to about 78% of the cells in the composition express MEIS2; and / or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 81% or 82% of the cells in the composition express MEIS2; and / or (iii) cells of the composition express about 5 transcripts per million (TPM) to about 200 TPM, about 10 TPM to about 180 TPM, about 50 TPM to about 180 TPM, or about 60 TPM to about 173 TPM of MEIS2 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 10 TPM, 15 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 135 TPM, 136 TPM, 138 TPM, 140 TPM, 145 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM or 200 TPM of MEIS2 transcripts.

49. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 30% to about 90%, about 35% to about 85%, about 40% to about 85%, about 40% to about 80%, about 45% to about 75%, or about 49% to about 75% of the cells in the composition express PBX3; and / or (ii) at least about 30%, 35%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 80%, 85% or 90% of the cells in the composition express PBX3; and / or (iii) cells of the composition express about 5 transcripts per million (TPM) to about 100 TPM, about 5 TPM to about 90 TPM, about 25 TPM to about 90 TPM, or about 29 TPM to about 88 TPM of PBX3 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM or 100 TPM of PBX3 transcripts.

50. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 20% to about 60%, about 20% to about 50%, about 20% to about 50%, about 20% to about 48%, about 22% to about 55%, about 22% to about 50%, about 22% to about 47%, or about 23% to about 47% of the cells in the composition express GRIA2; and / or (ii) at least about 20%, 22%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 46%, 47%, 50%, 52%, 54%, 56%, 58% or 60% of the cells in the composition express GRIA2; and / or (iii) cells of the composition express about 2 transcripts per million (TPM) to about 40 TPM, about 2 TPM to about 30 TPM, about 4 TPM to about 35 TPM, or about 10 TPM to about 30 TPM of GRIA2 transcripts; and / or (iv) the cells of the composition express at least 2TPM, 3TPM, 4TPM, 5TPM, 6TPM, 7TPM, 8TPM, 9TPM, 10TPM, 11TPM, 12TPM, 15TPM, 17TPM, 20TPM, 21TPM, 22TPM, 23TPM, 24TPM, 25TPM, 26TPM, 27TPM, 28TPM, 29TPM, 30TPM, 35TPM or 40TPM of GRIA2 transcripts.

51. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 20% to about 70%, about 30% to about 60%, about 35% to about 70%, about 35% to about 60%, about 33% to about 60%, about 35% to about 60%, or about 39% to about 60% of the cells in the composition express CACNA1C; and / or (ii) at least about 20%, 25%, 30%, 32%, 34%, 35%, 37%, 38%, 39%, 40%, 42%, 45%, 47%, 50%, 52%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70% of the cells in the composition express CACNA1C; and / or (iii) cells of the composition express about 1 transcripts per million (TPM) to about 15 TPM, about 1 TPM to about 10 TPM, about 1 TPM to about 7 TPM, or about 3 TPM to about 7 TPM of CACNA1C transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 10 TPM, 12 TPM or 15 TPM of CACNA1C transcripts.

52. The photoreceptor-rescue cell composition of any one of claims 1-51, wherein the plurality of heterologous photoreceptor-rescue cells comprises: (i) inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4 and CALB2; (ii) excitatory neurons expressing one or more markers selected from NEUROD2, NEUROD6, SLA, NELL2 and SATB2; (iii) progenitor cells expressing one or more markers selected from the group consisting of VIM, MKI67, CLU and GLI3; (iv) astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG and FBXL7; and (v) replacement neurons expressing one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2 and CACNA1C.

53. The photoreceptor-rescue cell composition of any one of claims 1-52, wherein the plurality of heterologous photoreceptor-rescue cells comprises: (i) multiple inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2; (ii) multiple excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2; (iii) accumulation of multiple progenitor cells expressing each of the markers VIM, MKI67, CLU, and GLI3; (iv) multiple astrocytes accumulating each of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7; and (v) Multiple replacement neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2, and CACNA1C.

54. The photoreceptor-salvage cell composition of any one of claims 1-53, wherein the composition comprises: (i) about 25% to about 55%, about 25% to about 50%, about 30% to about 55%, about 30% to about 50%, about 35% to about 55%, about 35% to about 50%, or about 38% to about 49% inhibitory neurons; and / or (ii) about 0% to about 15%, about 0% to about 12%, about 0% to about 10%, about 0% to about 8%, or about 0.5% to about 9% excitatory neurons; and / or (iii) about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 17% to about 45%, about 17% to about 40%, about 17% to about 35%, or about 20% to about 35% progenitor cells; and / or (iv) about 0% to about 6%, about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, about 0% to about 2%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, or about 0.5% to about 1.5% astrocytes; and / or (v) about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, or about 17% to about 37% mixed neurons.

55. The photoreceptor-salvage cell composition of any one of claims 1-54, wherein the cells in the composition further express one or more eye field progenitor cell markers, rod / cone photoreceptor cell markers, and / or neuronal markers.

56. The photoreceptor-salvaged cell composition of claim 55, wherein the eye field progenitor cell marker is selected from PAX6, LHX2, SIX3, NES and SOX2.

57. The photoreceptor-salvage cell composition of claim 55 or claim 56, wherein the plurality of heterologous cells cumulatively express at least 1, 2, 3 or 4 of the eye field progenitor cell markers PAX6, LHX2, SIX3, NES or SOX2.

58. The photoreceptor-salvage cell composition of any one of claims 55-57, wherein the plurality of heterologous cells cumulatively express at least each of the eye field progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2.

59. The photoreceptor-salvaged cell composition of any one of claims 55-58, wherein the plurality of heterologous cells cumulatively express SOX2.

60. The photoreceptor-salvaged cell composition of any one of claims 55-59, wherein the composition is substantially free of cells expressing the eye field progenitor cell markers RAX, SIX6 and / or TBX3.

61. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 25% to about 60%, about 25% to about 55%, about 25% to about 52%, about 25% to about 45%, about 30% to about 60%, about 30% to about 55%, about 30% to about 45%, or about 30% to about 42% of the cells in the composition express PAX6; and / or (ii) at least about 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the cells in the composition express PAX6; and / or (iii) cells of the composition express about 20 transcripts per million (TPM) to about 125 TPM, about 30 TPM to about 110 TPM, about 35 TPM to about 100 TPM, about 70 TPM to about 80 TPM, or about 73 TPM to about 78 TPM of PAX6 transcripts; and / or (iv) the cells of the composition express at least 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 50 TPM, 60 TPM, 70 TPM, 75 TPM, 78 TPM, 80 TPM, 82 TPM, 85 TPM, 87 TPM, 90 TPM, 92 TPM, 95 TPM, 97 TPM, 100 TPM, 105 TPM, 110 TPM, 115 TPM, 120 TPM or 125 TPM of PAX6 transcripts.

62. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 3% to about 35%, about 5% to about 35%, about 5% to about 35%, about 6% to about 35%, about 7% to about 35%, about 3% to about 30%, about 5% to about 30%, about 6% to about 30%, about 7% to about 30%, about 3% to about 25%, about 5% to about 25%, about 6% to about 25%, or about 7% to about 9% of the cells in the composition express LHX2; and / or (ii) at least about 3%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 32% or 35% of the cells in the composition express LHX2; and / or (iii) the cells of the composition express about 5 transcripts per million (TPM) to about 40 TPM, about 5 TPM to about 36 TPM, about 5 TPM to about 10 TPM, or about 6 TPM to about 8 TPM of LHX2 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 13 TPM, 14 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 36 TPM, 38 TPM or 40 TPM of LHX2 transcripts.

63. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or 2% to about 10% of the cells in the composition express SIX3; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% or 25% of the cells in the composition express SIX3; and / or (iii) cells of the composition express about 1 transcripts per million (TPM) to about 50 TPM, about 2 TPM to about 30 TPM, about 1 TPM to about 25 TPM, or about 5 TPM to about 20 TPM of SIX3 transcripts; and / or (iv) the cells of the composition express at least 1 TPM, 2TPM, 3TPM, 4TPM, 5TPM, 6TPM, 7TPM, 8TPM, 9TPM, 10TPM, 12TPM, 15TPM, 17TPM, 18TPM, 19TPM, 20TPM, 25TPM, 30TPM, 35TPM, 40TPM, 45TPM or 50TPM of SIX3 transcripts.

64. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 15% to about 40%, about 15% to about 35%, about 15% to about 34%, about 15% to about 33%, about 15% to about 32%, about 15% to about 31%, about 18% to about 40%, about 18% to about 35%, about 18% to about 34%, about 18% to about 33%, about 18% to about 32%, or about 18% to about 31% of the cells in the composition express NES; and / or (ii) at least about 15%, 17%, 20%, 25%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or 40% of the cells in the composition express the NES; and / or (iii) cells of the composition express about 5 transcripts per million (TPM) to about 35 TPM, about 5 TPM to about 28 TPM, about 7 TPM to about 15 TPM, about 10 TPM to about 15 TPM, or about 11 TPM to about 13 TPM of NES transcripts; and / or (iv) the cells of the composition express at least 5TPM, 6TPM, 7TPM, 8TPM, 9TPM, 10TPM, 11TPM, 12TPM, 13TPM, 14TPM, 15TPM, 17TPM, 19TPM, 20TPM, 22TPM, 24TPM, 25TPM, 26TPM, 27TPM, 30TPM or 35TPM of NES transcripts.

65. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 75% of the cells in the composition express SOX2; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or 90% of the cells in the composition express SOX2; and / or (iii) cells of the composition express about 50 transcripts per million (TPM) to about 250 TPM, about 90 TPM to about 200 TPM, about 125 TPM to about 190 TPM, or about 155 TPM to about 175 TPM of CACNA1C transcripts; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 70 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 180 TPM, 190 TPM, 200 TPM, 210 TPM, 220 TPM, 230 TPM, 240 TPM or 250 TPM of CACNA1C transcripts.

66. The photoreceptor-salvaged cell composition of any one of claims 55-65, wherein the rod / cone photoreceptor cell marker is selected from ASCL1, RORB, NR2E3 and NRL.

67. The photoreceptor-salvage cell composition of claim 66, wherein the plurality of heterologous cells cumulatively express each of the rod / cone photoreceptor cell markers ASCL1, RORB, NR2E3, and NRL.

68. The photoreceptor-salvage cell composition of any one of claims 55-67, wherein the composition is substantially free of cells expressing the rod / cone photoreceptor cell markers CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, and GNAT2.

69. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 10% to about 60%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 22% to about 45%, about 22% to about 43%, about 25% to about 420%, or about 28% to about 30% of the cells in the composition express ASCL1; and / or (ii) at least about 10%, 15%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 41%, 42%, 45%, 50%, 55%, or 60% of the cells in the composition express ASCL1; and / or (iii) the cells of the composition express about 50 transcripts per million (TPM) to about 150 TPM, about 60 TPM to about 140 TPM, about 65 TPM to about 130 TPM, or about 95 TPM to about 130 TPM of ASCL1 transcripts; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM, 120 TPM, 125 TPM, 130 TPM, 140 TPM or 150 TPM of ASCL1 transcripts.

70. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 10% to about 25%, or about 11% to about 22% of the cells in the composition express RORB; and / or (ii) at least about 5%, 7%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the composition express RORB; and / or (iii) cells of the composition express RORB transcripts at about 0.1 transcripts per million (TPM) to about 20 TPM, about 0.1 TPM to about 10 TPM, about 2 TPM to about 8 TPM, or about 1 TPM to about 6 TPM; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 1 TPM, 2 TPM, 3 TPM, 4 TPM, 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 12 TPM, 14 TPM, 16 TPM, 18 TPM or 20 TPM of RORB transcript.

71. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 25%, about 1% to about 20%, 1% to about 18%, 1% to about 16%, about 1% to about 14%, about 1% to about 12%, 1% to about 10%, about 2% to about 25%, about 2% to about 20%, 2% to about 18%, 2% to about 16%, about 2% to about 14%, about 2% to about 12%, or about 2% to about 5% of the cells in the composition express NR2E3; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express NR2E3; and / or (iii) cells of the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 3 TPM, or about 0.1 TPM to about 1 TPM of NR2E3 transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 2 TPM, 5 TPM, 7 TPM, 9 TPM or 10 TPM of NR2E3 transcripts.

72. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, or about 2% to about 8% of the cells in the composition express NRL; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express NRL; and / or (iii) cells of the composition express about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 9 TPM, about 0.1 TPM to about 7 TPM, or about 0.1 TPM to about 2 TPM of NRL transcripts; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1 TPM, 1.5 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of NRL transcripts.

73. The photoreceptor-salvaged cell composition of any one of claims 55-72, wherein the neuronal marker is selected from TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

74. The photoreceptor-salvage cell composition of claim 73, wherein the plurality of heterologous cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

75. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 50% to about 100%, about 60% to about 100%, about 60% to about 95%, about 70% to about 100%, about 70% to about 95%, about 80% to about 100%, about 80% to about 95%, or about 80% to about 90% of the cells in the composition express NFIB; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 100% of the cells in the composition express NFIB; and / or (iii) cells of the composition express about 150 transcripts per million (TPM) to about 650 TPM, about 180 TPM to about 610 TPM, about 400 TPM to about 500 TPM, or about 400 TPM to about 480 TPM of NFIB transcripts; and / or (iv) the cells of the composition express at least 150 TPM, 175 TPM, 180 TPM, 185 TPM, 190 TPM, 200 TPM, 225 TPM, 250 TPM, 275 TPM, 300 TPM, 325 TPM, 350 TPM, 375 TPM, 400 TPM, 425 TPM, 450 TPM, 475 TPM, 500 TPM, 550 TPM, 600 TPM, 625 TPM or 650 TPM of NFIB transcripts.

76. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 2% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, or about 2% to about 6% of the cells in the composition express OTX2; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% or 20% of the cells in the composition express OTX2; and / or (iii) cells of the composition express about 5 transcripts per million (TPM) to about 50 TPM, about 8 TPM to about 40 TPM, about 8 TPM to about 25 TPM, or about 12 TPM to about 15 TPM of OTX2 transcripts; and / or (iv) the cells of the composition express at least 5 TPM, 6 TPM, 7 TPM, 8 TPM, 9 TPM, 10 TPM, 11 TPM, 12 TPM, 15 TPM, 16 TPM, 17 TPM, 18 TPM, 19 TPM, 20 TPM, 21 TPM, 22 TPM, 23 TPM, 24 TPM, 26 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM or 50 TPM of OTX2 transcripts.

77. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 78%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 78%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, or about 60% to about 78% of the cells in the composition express ELAVL3; and / or (ii) at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or 90% of the cells in the composition express ELAVL3; and / or (iii) cells of the composition express about 10 transcripts per million (TPM) to about 120 TPM, about 15 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 60 TPM to about 90 TPM, or about 70 TPM to about 80 TPM of ELAVL3 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 110 TPM or 120 TPM of ELAVL3 transcripts.

78. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 78%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 78%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, or about 50% to about 65% of the cells in the composition express ELAVL4; and / or (ii) at least about 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 75%, 80%, 85% or 90% of the cells in the composition express ELAVL4; and / or (iii) cells of the composition express about 10 transcripts per million (TPM) to about 120 TPM, about 15 TPM to about 100 TPM, about 20 TPM to about 90 TPM, about 50 TPM to about 90 TPM, about 70 TPM to about 90 TPM, or about 79 TPM to about 85 TPM of ELAVL4 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 80 TPM, 82 TPM, 84 TPM, 86 TPM, 90 TPM, 100 TPM, 110 TPM or 120 TPM of ELAVL4 transcripts.

79. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 73%, about 51% to about 73%, or about 52% to about 66% of the cells in the composition express SLC1A2; and / or (ii) at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 75%, 80%, 85%, or 90% of the cells in the composition express SLC1A2; and / or (iii) the cells of the composition express about 10 transcripts per million (TPM) to about 120 TPM, about 10 TPM to about 90 TPM, about 20 TPM to about 90 TPM, about 40 TPM to about 70 TPM, about 50 TPM to about 70 TPM, or about 58 TPM to about 63 TPM of SLC1A2 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 60 TPM, 65 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM or 120 TPM of SLC1A2 transcripts.

80. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 11% to about 19%, or about 10% to about 12% of the cells in the composition express SLC1A3; and / or (ii) at least about 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 25%, 30%, 35%, or 40% of the cells in the composition express SLC1A3; and / or (iii) cells of the composition express about 50 transcripts per million (TPM) to about 200 TPM, about 70 TPM to about 190 TPM, about 60 TPM to about 100 TPM, about 60 TPM to about 80 TPM, or about 72 TPM to about 79 TPM of SLC1A3 transcripts; and / or (iv) the cells of the composition express at least 50 TPM, 60 TPM, 65 TPM, 70 TPM, 71 TPM, 72 TPM, 73 TPM, 74 TPM, 75 TPM, 76 TPM, 77 TPM, 78 TPM, 79 TPM, 80 TPM, 90 TPM, 100 TPM, 110 TPM, 120 TPM, 130 TPM, 140 TPM, 150 TPM, 160 TPM, 170 TPM, 175 TPM, 180 TPM or 200 TPM of SLC1A3 transcripts.

81. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 3% to about 4% of the cells in the composition express HCN1; and / or (ii) at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the cells in the composition express HCN 1; and / or (iii) cells of the composition express HCN1 transcripts at about 0.1 transcripts per million (TPM) to about 10 TPM, about 0.1 TPM to about 5 TPM, about 0.1 TPM to about 1 TPM, or about 0.2 TPM to about 0.6 TPM; and / or (iv) the cells of the composition express at least 0.1 TPM, 0.2 TPM, 0.3 TPM, 0.4 TPM, 0.5 TPM, 0.6 TPM, 0.7 TPM, 0.8 TPM, 0.9 TPM, 1.0 TPM, 2 TPM, 4 TPM, 6 TPM, 8 TPM or 10 TPM of HCN1 transcripts.

82. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein (i) about 5% to about 30%, about 5% to about 25%, about 1% to about 30%, about 1% to about 25%, about 5% to about 25%, about 10% to about 25%, or about 10% to about 15% of the cells in the composition express HES5; and / or (ii) at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or 30% of the cells in the composition express HES5; and / or (iii) the cells of the composition express about 10 transcripts per million (TPM) to about 60 TPM, about 5 TPM to about 50 TPM, about 12 TPM to about 46 TPM, or about 15 TPM to about 39 TPM of HES5 transcripts; and / or (iv) the cells of the composition express at least 10 TPM, 15 TPM, 17 TPM, 20 TPM, 22 TPM, 25 TPM, 27 TPM, 30 TPM, 32 TPM, 35 TPM, 37 TPM, 40 TPM, 42 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM of HES5 transcripts.

83. The photoreceptor-salvage cell composition of any one of claims 1-82, wherein the plurality of heterologous cells cumulatively express: (i) one or more of the eye domain progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) one or more of the rod / cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) one or more of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5.

84. The photoreceptor-salvage cell composition of any one of claims 1-83, wherein the plurality of heterologous cells cumulatively express: (i) each of the eye field progenitor cell markers PAX6, LHX2, SIX3, NES and SOX2; (ii) each of the rod / cone photoreceptor cell markers ASCL1, RORB, NR2E3 and NRL; and (iii) each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1 and HES5.

85. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein the composition is substantially free of at least one cell type selected from the group consisting of pluripotent stem cells, retinal ganglion cells, photoreceptor cells, and amacrine cells.

86. The photoreceptor-salvage cell composition of any of the preceding claims, wherein the composition is substantially free of pluripotent stem cells, retinal ganglion cells, photoreceptor cells, and amacrine cells.

87. The photoreceptor-salvage cell composition of any of the preceding claims, wherein the composition is substantially free of retinal progenitor cells expressing VSX2 and / or POU5F1.

88. The photoreceptor-salvage cell composition of any of the preceding claims, wherein the composition has less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5% of cells expressing SSEA4, optionally as determined by flow cytometry, or wherein the composition is free of cells expressing SSEA4, optionally as determined by flow cytometry.

89. The photoreceptor-salvage cell composition of any one of the preceding claims, wherein the cells in the composition have phagocytic activity, optionally the ability to phagocytose isolated photoreceptor outer segments, dye conjugates, or both.

90. The photoreceptor-salvage cell composition of any preceding claim, wherein cells in the composition secrete one or more neuroprotective factors.

91. The photoreceptor-salvage cell composition of claim 90, wherein the neuroprotective factor is selected from the group consisting of CNTF, MIF, S100B, GFAP, TAU, NCAM1 and TNC.

92. The photoreceptor-salvage cell composition of any of the preceding claims, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, or at least 80% of the cells in the composition are viable.

93. The photoreceptor-salvage cell composition of any of the preceding claims, wherein about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable.

94. The photoreceptor-salvage cell composition of any preceding claim, wherein at least 50% of the cells in the composition are viable.

95. The photoreceptor-salvage cell composition of any preceding claim, wherein at least 55% of the cells in the composition are viable.

96. The photoreceptor-salvage cell composition of any preceding claim, wherein at least 60% of the cells in the composition are viable.

97. The photoreceptor-salvage cell composition of any preceding claim, wherein at least 65% of the cells in the composition are viable.

98. The photoreceptor-salvage cell composition of any preceding claim, wherein at least 68% of the cells in the composition are viable.

99. The photoreceptor-salvaged cell composition of any one of claims 1-98, wherein the composition is produced according to a method comprising the steps of: 1) Cultivate pluripotent stem cells in rescue induction medium (RIM) and Noggin; 2) Cultivate the cells from step 1 in neural differentiation medium (NDM) and Noggin; 3) expanding the cells from step 2 in NDM in the absence of Noggin, the steps comprising a) culturing the cells under low-adherence or non-adherence conditions in NDM without Noggin, and b) culturing the cells under adherence conditions in NDM without Noggin.

100. The photoreceptor-salvage cell composition of claim 99, wherein the pluripotent stem cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

101. The photoreceptor rescue cell composition of claim 99 or 100, wherein step 3 is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times.

102. The photoreceptor-salvage cell composition of any one of claims 99-101, wherein the cells in the composition are harvested after a third repetition of step 3, after a fourth repetition of step 3, or after a fifth repetition of step 3.

103. The photoreceptor-salvage cell composition of claim 102, wherein after the fifth repetition of step 3, the cells in the composition are harvested.

104. The photoreceptor-salvage cell composition of claim 102 or 103, wherein the harvested cells in the composition are cryopreserved.

105. The photoreceptor-salvage cell composition of any one of claims 99-104, wherein the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3.

106. The photoreceptor-salvage cell composition of any one of claims 99-104, wherein the composition is cryopreserved after a third repetition of step 3, after a fourth repetition of step 3, or after a fifth repetition of step 3.

107. The photoreceptor rescue cell composition of claim 106, wherein step 3 is repeated five times, and wherein after the fifth repetition of step 3, the composition is cryopreserved.

108. The photoreceptor-salvage cell composition of any one of claims 99-107, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75% or at least 80% of the cells are viable after cryopreservation and thawing.

109. The photoreceptor salvage cell composition of any one of claims 99-108, wherein about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% are viable after cryopreservation and thawing.

110. The photoreceptor-salvage cell composition of any one of claims 99-109, wherein at least about 50% of the cells in the composition are viable after cryopreservation and thawing.

111. The photoreceptor-salvage cell composition of any one of claims 99-110, wherein at least about 55% of the cells in the composition are viable after cryopreservation and thawing.

112. The photoreceptor-salvage cell composition of any one of claims 99-111, wherein at least about 60% of the cells in the composition are viable after cryopreservation and thawing.

113. The photoreceptor-salvage cell composition of any one of claims 99-112, wherein at least about 65% of the cells in the composition are viable after cryopreservation and thawing.

114. The photoreceptor-salvage cell composition of any one of claims 99-113, wherein at least 68% of the cells in the composition are viable after cryopreservation and thawing.

115. The photoreceptor-salvage cell composition of any one of claims 99-114, wherein the composition comprises a spheroid.

116. The photoreceptor-salvage cell composition of claim 115, wherein about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids.

117. The photoreceptor-salvage cell composition according to any one of claims 99-116, wherein during steps 1, 2 and / or 3, the cells of the composition are cultured in a cell culture container selected from the group consisting of a culture dish, a culture flask and a culture chamber.

118. The photoreceptor-salvage cell composition of claim 117, wherein the cell culture container has a size of about 50 cm 2 About 800cm 2 、Approx. 60cm 2 About 800cm 2 , about 100cm 2 About 800cm 2 、Approx. 150cm 2 About 800cm 2 、Approx. 175cm 2 About 800cm 2 , about 200cm 2 About 800cm 2 、About 250cm 2 About 800cm 2 、About 300cm 2 About 800cm 2 、Approx. 400cm 2 About 800cm 2 、About 500cm 2 About 800cm 2 、About 600cm 2 About 800cm 2 、About 700cm 2 About 800cm 2 、About 30cm 2 To about 100cm 2 、Approx. 50cm 2 To about 100cm 2 , about 100cm 2 About 300cm 2 、Approx. 150cm 2 To about 250cm 2 or about 150cm 2 To about 200cm 2 Cell growth area.

119. The photoreceptor-salvage cell composition of claim 117 or 118, wherein the cell culture container has a size of at least about 60 cm 2 、Approx. 175cm 2 or about 636cm 2 Cell growth area.

120. The photoreceptor-salvage cell composition of any one of claims 117-119, wherein the culture chamber is a stackable rectangular chamber.

121. The photoreceptor rescue cell composition of any one of claims 117-120, wherein step 3 has 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5 or 1 to 2 culture chambers.

122. The photoreceptor rescue cell composition of claim 121, wherein step 3 has at least 1, 2, 5, 10 or 40 culture chambers.

123. The photoreceptor-salvage cell composition according to any one of claims 117-122, wherein the cell culture container is coated for low-adherent or non-adherent cell culture in step 3a, and / or adherent cell culture in step 3b.

124. The photoreceptor-salvage cell composition of any one of claims 99-123, wherein the cells are enzymatically dissociated from a plate into a cell suspension.

125. The photoreceptor rescue cell composition of claim 124, wherein the enzymatic dissociation utilizes an enzyme selected from the group consisting of thermolysin, liberase, accutase, and combinations thereof.

126. The photoreceptor-salvage cell composition of claim 125, wherein the enzyme used to dissociate the cells is accutase.

127. The photoreceptor-salvage cell composition of any one of claims 99-126, wherein dissociation of the cells from the plate does not involve manual scraping.

128. A pharmaceutical preparation comprising the photoreceptor-salvage cell composition of any one of claims 1-127 and a pharmaceutically acceptable excipient.

129. The pharmaceutical preparation of claim 128, wherein the pharmaceutically acceptable excipient is suitable for ocular delivery.

130. A formulation comprising: a) a composition according to any one of claims 1 to 127, or a pharmaceutical preparation according to claim 128 or claim 129; and b) about 4-10% (v / v) cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer.

131. The formulation of claim 130, wherein the cryoprotectant is selected from the group consisting of DMSO, glycerol, and ethylene glycol.

132. The formulation of claim 130 or 131, wherein the cryoprotectant is DMSO.

133. The formulation of any one of claims 130-132, wherein the formulation comprises about 4-6% (v / v) cryoprotectant.

134. The formulation of any one of claims 130-133, wherein the formulation comprises about 0.08-0.10% (w / v) glucose.

135. The formulation of any one of claims 130-133, wherein the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer.

136. The formulation of any one of claims 130-134, wherein the formulation comprises about 0.6% (w / v) glucose.

137. The formulation of any one of claims 130-134 or 136, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

138. The formulation of any one of claims 130-137, wherein the albumin is human albumin.

139. The formulation of any one of claims 130-138, wherein the albumin is recombinant human albumin.

140. The formulation of any one of claims 130-139, wherein the buffer is buffered saline.

141. The formulation of any one of claims 130-140, wherein the buffer is phosphate buffered saline (PBS).

142. The formulation of claim 140 or 141, wherein the buffered saline comprises Ca 2+ and Mg 2+ .

143. The formulation of claim 140 or 141, wherein the buffered saline does not contain Ca 2+ and Mg 2+ .

144. The formulation of any one of claims 130-143, wherein the formulation is a cryopreserved formulation.

145. A formulation comprising: a) a composition according to any one of claims 1 to 127, or a pharmaceutical preparation according to claim 128 or claim 129; and b) a solution comprising (1) a buffer that maintains the solution at a physiological pH; and (2) at least 2 mM or at least 0.05% (w / v) glucose; and (3) An osmotic agent that maintains the solution at a physiological osmotic molarity.

146. The formulation of claim 145, wherein the solution comprises at least 5 mM or at least 0.1% (w / v) glucose; or at least 7.5 mM or at least 0.14% (w / v) glucose; or at least 10 mM or at least 0.2% (w / v) glucose; or at least 15 mM or at least 0.25% (w / v) glucose; or at least 20 mM or at least 0.4% (w / v) glucose; or at least 25 mM or at least 0.5% (w / v) glucose.

147. The formulation of claim 145 or 146, wherein the solution further comprises (4) a source of divalent cations, optionally wherein the source of divalent cations comprises a source of calcium and / or magnesium, and / or (5) Acetate buffer and / or citrate buffer.

148. A formulation comprising: a) a composition according to any one of claims 1 to 127, or a pharmaceutical preparation according to claim 128 or claim 129; and b) a solution comprising (1) a buffer that maintains the solution at a physiological pH, wherein the buffer is not a bicarbonate buffer; and (2) Glucose; and (3) an osmotic agent that maintains the solution at a physiological osmolarity; and (4) a source of divalent cations, Optionally wherein the source of divalent cations comprises a calcium source and / or a magnesium source, and / or wherein the buffer comprises an acetate buffer and / or a citrate buffer.

149. The formulation of claim 147 or 148, wherein the calcium source comprises a pharmaceutically acceptable calcium salt, and / or the magnesium source comprises a pharmaceutically acceptable magnesium salt.

150. The formulation of claim 149, wherein the pharmaceutically acceptable calcium salt and / or pharmaceutically acceptable magnesium salt is selected from the group consisting of calcium salts and / or magnesium salts formed with an acid selected from the group consisting of acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid.

151. The formulation of any one of claims 147-150, wherein the calcium source comprises calcium chloride, optionally wherein the calcium source comprises calcium chloride dihydrate.

152. The formulation of any one of claims 147-151, wherein the magnesium source comprises magnesium chloride, optionally wherein the magnesium source comprises magnesium chloride hexahydrate.

153. The formulation of any one of claims 147-152, wherein the citrate buffer is provided as sodium citrate.

154. The formulation of any one of claims 145-153, wherein the glucose is D-glucose (dextrose).

155. The formulation of any one of claims 145-154, wherein the osmotically active agent is a salt, optionally wherein the osmotically active agent is a sodium salt, further optionally wherein the osmotically active agent is sodium chloride.

156. The formulation of any one of claims 145-155, wherein the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and dextrose.

157. The formulation of any one of claims 145-156, wherein the pH of the solution is 6.8-7.8, or 7.4-7.5, or about 7.

5.

158. The formulation of any one of claims 145-157, wherein the solution is an isotonic or hypertonic solution.

159. The formulation of any one of claims 145-157, wherein the solution exhibits an osmolarity of about 270-345 mOsm / l or about 315 mOsm / l.

160. The formulation of any one of claims 145-159, wherein the concentration of the calcium source is (a) 0.25-0.75 mM, or 0.4-0.65 mM, or 0.5-0.6 mM or about 0.6 mM; or (b) 0.5-0.9 mM, or 0.6-0.8 mM or about 0.7 mM.

161. The formulation of any one of claims 145-160, wherein the concentration of the magnesium source is 0.05-5 mM, or 0.1-0.3 mM or about 0.3 mM.

162. The formulation of any one of claims 145-161, wherein the concentration of glucose is 5-50 mM, or 10-25 mM, or 10-20 mM or about 16 mM.

163. The formulation of any one of claims 145-162, wherein the concentration of the osmotically active agent is about 100-200 mM, or about 125-175 mM, or about 150 mM.

164. The formulation of any one of claims 145-163, wherein the concentration of citrate or acetate is 0.1-5 mM, or 0.5-2 mM, or about 1 mM.

165. The formulation of any one of claims 145-164, wherein the solution further comprises a potassium salt, optionally wherein the potassium salt is potassium chloride, further optionally wherein the concentration of KCl is 0.2-5 mM, or 1-2.5 mM or about 2 mM.

166. The formulation of any one of claims 145-165, wherein the solution comprises (a) about 0.7 mM CaCl2 (calcium chloride), about 0.3 mM MgCl2 (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, and about 145 mM NaCl, or (b) about 0.5-0.9 mM CaCl2 (calcium chloride), about 0.2-.4 mM MgCl2 (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, and about 116-174 mM NaCl, or (c) about 0.85% NaCl, about 0.01% CaCl2 dihydrate (calcium chloride dihydrate), about 0.006% MgCl2 hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate, and about 0.29% dextrose, or (d) about 0.68-1.02% NaCl, about 0.008-0.012% CaCl2 dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl2 hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate, and about 0.23-0.35% dextrose.

167. The formulation of any one of claims 145-166, wherein the solution further comprises (a) about 2 mM KCl, and / or (b) a viscoelastic polymer, optionally wherein the polymer is hyaluronic acid or a salt or solvate thereof, further optionally wherein the polymer is sodium hyaluronate.

168. The formulation of claim 167, wherein the polymer is present at a concentration effective to reduce exposure of cells to shear stress in the solution, optionally wherein the polymer is at a concentration of 0.005-5% w / v or about 0.05% w / v.

169. The formulation of any one of claims 145-168, wherein the solution comprises (a) about 0.7 mM CaCl2 (calcium chloride), about 0.3 mM MgCl2 (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl and about 0.05% hyaluronic acid, or (b) about 0.5-0.8 mM CaCl2 (calcium chloride), about 0.2-.4 mM MgCl2 (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid.

170. The formulation of any one of claims 145-169, wherein the solution does not comprise (a) carbonate buffer, and / or (b) glutathione or glutathione disulfide (GSSG), and / or (c) Zwitterionic organic buffers.

171. The formulation of any one of claims 145-170, wherein the solution is capable of (a) storage at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution, and / or (b) stored at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution.

172. The formulation of any one of claims 145-171, wherein the solution (a) is suitable for administration to a subject, suitable for administration to the eye of a subject, and / or suitable for transplanting cells into the eye of a subject, and / or (b) is substantially non-pyrogenic, and / or (c) is sterile, and / or (d) for flushing, cell reconstitution, cell storage, cell delivery, and / or administration to a subject.

173. A method of producing a composition according to any one of claims 1-127, comprising 1) Cultivate pluripotent stem cells in rescue induction medium (RIM) and Noggin; 2) Cultivate the cells from step 1 in neural differentiation medium (NDM) and Noggin; 3) expanding the cells from step 2 in Noggin-free NDM, the steps comprising a) culturing the cells under low-adherence or non-adherence conditions in Noggin-free NDM, and b) culturing the cells under non-adherence conditions in Noggin-free NDM, thereby differentiating the pluripotent stem cells into photoreceptor rescue cells.

174. A method for producing a photoreceptor-salvage cell composition comprising a plurality of heterologous cells, the method comprising: 1) Cultivate pluripotent stem cells in rescue induction medium (RIM) and Noggin; 2) culturing the cells in neural differentiation medium (NDM) and Noggin; 3) expanding the cells in Noggin-free NDM, the step comprising a) culturing the cells under low-adherence or non-adherence conditions in Noggin-free NDM, and b) culturing the cells under adherence conditions in Noggin-free NDM, thereby differentiating the pluripotent stem cells into a plurality of cells of the photoreceptor rescue cell composition.

175. The method of claim 173 or claim 174, wherein the pluripotent stem cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

176. The method of any one of claims 173-175, wherein step 3 is performed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times.

177. The method of any one of claims 173-176, wherein the cells in the composition are harvested after a third repetition of step 3, after a fourth repetition of step 3, or after a fifth repetition of step 3.

178. The method of claim 177, wherein after the fifth repetition of step 3, the composition is harvested.

179. The method of claim 177 or 178, wherein the cells in the harvested composition are cryopreserved.

180. The method of any one of claims 173-179, wherein the composition is cryopreserved between the first and second repetitions of step 3, between the second and third repetitions of step 3, between the third and fourth repetitions of step 3, or between the fourth and fifth repetitions of step 3.

181. The method of any one of claims 173-180, wherein the composition is cryopreserved after a third repetition of step 3, after a fourth repetition of step 3, or after a fifth repetition of step 3.

182. The method of claim 180, wherein step 3 is repeated five times, and wherein after the fifth repetition of step 3, the composition is cryopreserved.

183. The method of any one of claims 173-182, wherein after cryopreservation and thawing, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75% or at least 80% of the cells in the composition are viable.

184. The method of any one of claims 173-183, wherein after cryopreservation and thawing, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 50% to about 60%, about 55% to about 60%, or about 50% to about 55% of the cells in the composition are viable.

185. The method of any one of claims 173-184, wherein after cryopreservation and thawing, less than about 50% of the cells in the composition are viable.

186. The method of any one of claims 173-185, wherein after cryopreservation and thawing, less than about 55% of the cells in the composition are viable.

187. The method of any one of claims 173-186, wherein after cryopreservation and thawing, less than about 60% of the cells in the composition are viable.

188. The method of any one of claims 173-187, wherein after cryopreservation and thawing, less than about 65% of the cells in the composition are viable.

189. The method of any one of claims 173-188, wherein after cryopreservation and thawing, less than about 68% of the cells in the composition are viable.

190. The method of any one of claims 173-189, wherein the composition comprises a spheroid.

191. The method of claim 190, wherein about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 70% to about 90% of the cells are spheroids.

192. The method of any one of claims 173-191, wherein during step 1, step 2 and / or step 3, the cells of the composition are cultured in a cell culture container selected from the group consisting of a culture dish, a culture flask and a culture chamber.

193. The method of claim 192, wherein the cell culture container has a diameter of about 50 cm 2 About 800cm 2 、Approx. 60cm 2 About 800cm 2 , about 100cm 2 About 800cm 2 、Approx. 150cm 2 About 800cm 2 、Approx. 175cm 2 About 800cm 2 , about 200cm 2 About 800cm 2 、About 250cm 2 About 800cm 2 、About 300cm 2 About 800cm 2 、Approx. 400cm 2 About 800cm 2 、About 500cm 2 About 800cm 2 、About 600cm 2 About 800cm 2 、About 700cm 2 About 800cm 2 、About 30cm 2 To about 100cm 2 、Approx. 50cm 2 To about 100cm 2 , about 100cm 2 About 300cm 2 、Approx. 150cm 2 To about 250cm 2 or about 150cm 2 To about 200cm 2 Cell growth area.

194. The method of claim 192 or 193, wherein the cell culture container has a diameter of at least about 60 cm 2 、Approx. 175cm 2 or about 636cm 2 Cell growth area.

195. The method of claim 192 or 193, wherein the culture chamber is a stackable rectangular chamber.

196. The method of claim 195, wherein step 3 comprises 1 to 40, 2 to 40, 5 to 40, 10 to 40, 20 to 40, 1 to 20, 2 to 20, 5 to 20, 10 to 20, 1 to 10, 2 to 10, 5 to 10, 1 to 5 or 1 to 2 culture chambers.

197. The method of claim 196, wherein step 3 comprises at least 1, 2, 5, 10 or 40 culture chambers.

198. The method of any one of claims 192-197, wherein the cell culture vessel is coated for low adherent or non-adherent cell culture in step 3a, and / or adherent cell culture in step 3b.

199. The method of any one of claims 173-198, wherein the cells are enzymatically dissociated from a plate into a cell suspension.

200. The method of claim 199, wherein the enzyme used to dissociate the cells is thermolysin, liberase and / or accutase.

201. The method of claim 200, wherein the enzyme used to dissociate the cells is accutase.

202. The method of any one of claims 173-201, wherein dissociation of the cells from the plate does not involve manual scraping.

203. A photoreceptor-salvaged cell composition produced by the method of any one of claims 173-202.

204. A method of treating an ocular disease or condition in a subject, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

205. A method of increasing the secretion of a neuroprotective factor in the eye of a subject suffering from an ocular disease or condition, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

206. The method of claim 205, wherein the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the secretion of the neuroprotective factor before administration.

207. A method of improving visual acuity in a subject having a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

208. The method of claim 207, wherein the method improves visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity prior to administration.

209. The method of claim 207 or 208, wherein visual acuity is measured by optomotor response (OMR) and / or electroretinogram (ERG).

210. The method of claim 209, wherein the spatial frequency threshold of the treated eye as measured by OMR is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the spatial frequency threshold before administration.

211. The method of claim 209 or 210, wherein the scotopic b-wave amplitude of the treated eye as measured by ERG is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the scotopic b-wave amplitude before administration.

212. A method of preventing or slowing photoreceptor cell loss in a subject having a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

213. The method of claim 212, wherein prevention of photoreceptor cell loss is measured by expression of CNFT.

214. The method of claim 213, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% compared to the expression of CNFT in the eye prior to administration.

215. A method of increasing phagocytic activity in the eye of a subject suffering from a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

216. The method of claim 215, wherein the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the phagocytic activity before administration.

217. A method of inhibiting microglial activation in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

218. The method of claim 217, wherein inhibition of microglial activation is measured by expression of CNFT and / or MIF.

219. The method of claim 218, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of CNFT before administration.

220. The method of claim 217 or claim 218, wherein the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration.

221. A method of reducing oxidative stress in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

222. The method of claim 221, wherein the reduction in oxidative stress is measured by expression of CNFT.

223. The method of claim 222, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of CNFT before administration.

224. A method of increasing the expression of an anti-apoptotic factor in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

225. The method of claim 224, wherein the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of the anti-apoptotic factor before administration.

226. The method of claim 224 or 225, wherein the anti-apoptotic factor is S100B.

227. A method of preventing outer nuclear layer (ONL) degeneration in an eye of a subject suffering from a retinal disease or disorder, the method comprising administering to the subject the photoreceptor-salvage cell composition of any one of claims 1-127 or 203, the pharmaceutical preparation of any one of claims 128 or 129, or the formulation of any one of claims 130-172.

228. The method of any one of claims 204-227, wherein the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, moderate dry form of age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt's disease.

229. The method of claim 228, wherein the eye disease is macular degeneration or retinitis pigmentosa.

230. The method of any one of claims 204-229, wherein the disease is a retinal degenerative disease.

231. The method of any one of claims 204-230, wherein the disease is associated with a loss of photoreceptor cells.

232. The method of claim 231, wherein the disease is associated with a loss of photoreceptor cells in the outer nuclear layer of the retina.

233. The method of any one of claims 204-232, wherein the photoreceptor-salvage cell composition, formulation, or pharmaceutical preparation is administered to the subretinal space, suprachoroidal space, to the eye via a reservoir, or to other parts of the subject's body via systemic delivery.

234. The method of any one of claims 204-233, wherein the cell preparation or formulation is administered by injection or implantation.

235. The method of claim 234, wherein the injection is administered intraocularly.

236. The method of claim 234 or 235, wherein intraocular administration comprises injecting an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space, thereby forming an anterior vesicle, and removing the aqueous solution before administering the photoreceptor salvage cell composition to the same subretinal space as the aqueous solution.

237. The method of any one of claims 204-237, wherein the cell preparation or formulation is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset.

238. The method of any one of claims 204-237, wherein (i) the subject suffers from an intermediate or near-terminal disease; (ii) the subject's best corrected visual acuity (BCVA) is in the range of 20 / 50 to 20 / 200; (iii) the subject has a BCVA of less than 20 / 200 but maintains light perception; or (iv) diagnosed with retinitis pigmentosa by genotyping.

239. The method of any one of claims 204-238, further wherein one or more anti-inflammatory agents are administered to the subject.

240. The method of claim 239, wherein the one or more anti-inflammatory agents are administered simultaneously.

241. The method of claim 239, wherein the one or more anti-inflammatory agents are administered separately.

242. The method of claim 241, wherein the one or more anti-inflammatory agents are administered prior to the cell preparation or formulation, or the cell preparation or formulation is administered prior to the one or more anti-inflammatory agents.

243. The method of any one of claims 239, 241, and 242, wherein the one or more anti-inflammatory agents are administered before and after administration of the cell preparation or formulation.

244. The method of any one of claims 204-238, wherein the cell preparation or formulation is administered in the absence of one or more anti-inflammatory agents.

245. The method of any one of claims 243, wherein the one or more anti-inflammatory agents comprise dexamethasone and / or cyclosporine.

246. A population of extracellular vesicles (EVs) secreted from the photoreceptor-salvage cell composition of any one of claims 1-127 and 203.

247. The EV population of claim 246, wherein the EVs secreted from the photoreceptor rescue cells express at least one of the following proteins: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, DLX5, TUBB3, SCGN, ERBB4, CALB2, NEUROD2, NEUROD6, SLA, NELL2, SATB2, VIM, MKI67, CL U, GLI3, GFAP, LUCAT1, MIR99AHG, FBXL7, MEIS2, PBX3, GRIA2, CACNA1C, PAX6, LHX2, SIX3, NES, SOX2, ASCL1, RORB, NR2E3, NRL, TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5, AGT, ACBLN2, CDH7 , DNAH11, EGR1, FAM216B, FOS, KCNC2, LGI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, nestin, TRβ2, MASH1, RORβ, MAP2, ELAVL3, NFIA, DCX, LHX2 , SLC1A2, ELAVL4, PAX6, EMX2, ASCL1, DLL1, NFIB, ENOXI, TUBB3, MAP2, DCLK1 / 2, DCX, KALRN, LINC00461, C1orf61, NCAM1, SETBP1, PAK3, AKAP6, RTN1, CRMP1, FOXG1, TRIM2, BACH2, recoverin, opsin, rhodopsin, rod and cone cGMP phosphodiesterase.

248. A pharmaceutical composition comprising the EV population of claim 246 or claim 247 and a pharmaceutically acceptable carrier.

249. A formulation comprising: a) the EV population of claim 192 or 193, or the pharmaceutical composition of claim 194; and b) about 4-10% (v / v) cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer.

250. The formulation of claim 249, wherein the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol.

251. The formulation of claim 249 or 250, wherein the cryoprotectant is DMSO.

252. The formulation of any one of claims 248-251, wherein the formulation comprises about 4-6% (v / v) cryoprotectant.

253. The formulation of any one of claims 248-252, wherein the formulation comprises about 0.08-0.10% (w / v) glucose.

254. The formulation of any one of claims 248-253, wherein the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer.

255. The formulation of any one of claims 248-252, wherein the formulation comprises about 0.6% (w / v) glucose.

256. The formulation of any one of claims 248-252 or 255, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

257. The formulation of any one of claims 248-256, wherein the albumin is human albumin.

258. The formulation of any one of claims 248-257, wherein the albumin is recombinant human albumin.

259. The formulation of any one of claims 248-258, wherein the buffer is buffered saline.

260. The formulation of any one of claims 248-259, wherein the buffer is phosphate buffered saline (PBS).

261. The formulation of claim 259 or 260, wherein the buffered saline comprises Ca 2+ and Mg 2+ .

262. The formulation of claim 259 or 260, wherein the buffered saline does not contain Ca 2+ and Mg 2+ .

263. The formulation of any one of claims 249-262, wherein the formulation is a cryopreserved formulation.

264. A method for treating an ocular disease in a subject, comprising administering to the subject the EV population described in claim 246 or 247, the pharmaceutical composition comprising the EV population described in claim 248, or the preparation comprising the EV population described in any one of claims 249-263.

265. A method for increasing the secretion of neuroprotective factors in the eyes of a subject, comprising administering to the subject the EV population described in claim 246 or 247, the pharmaceutical composition comprising the EV population described in claim 248, or the preparation comprising the EV population described in any one of claims 249-263.

266. The method of claim 265, wherein the method increases the secretion of the neuroprotective factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the secretion of the neuroprotective factor before administration.

267. A method for improving visual acuity in a subject suffering from a retinal disease or condition, comprising administering to the subject the EV population of claim 246 or 247, the pharmaceutical composition comprising the EV population of claim 248, or the preparation comprising the EV population of any one of claims 249-263.

268. The method of claim 267, wherein the method increases visual acuity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to visual acuity before administration.

269. The method of claim 267 or 268, wherein visual acuity is measured by optomotor response (OMR) and / or electroretinogram (ERG).

270. The method of claim 269, wherein the spatial frequency threshold of the treated eye as measured by OMR is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the spatial frequency threshold before administration.

271. The method of claim 269 or 270, wherein the scotopic b-wave amplitude of the treated eye as measured by ERG is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the scotopic b-wave amplitude before administration.

272. A method for preventing or slowing the loss of photoreceptor cells in a subject suffering from a retinal disease or condition, comprising administering to the subject the EV population of claim 246 or 247, the pharmaceutical composition comprising the EV population of claim 248, or the preparation comprising the EV population of any one of claims 249-263.

273. The method of claim 272, wherein the prevention or reduction of photoreceptor cell loss is measured by expression of CNFT.

274. The method of claim 273, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% compared to the expression of CNFT in the eye prior to administration.

275. A method for increasing phagocytic activity in the eye of a subject suffering from a retinal disease or condition, comprising administering to the subject the EV population of claim 246 or 247, the pharmaceutical composition comprising the EV population of claim 248, or the preparation comprising the EV population of any one of claims 249-263.

276. The method of claim 275, wherein the method increases the phagocytic activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the phagocytic activity before administration.

277. A method for inhibiting ocular microglial activation in a subject suffering from a retinal disease or condition, comprising administering to the subject an EV population as described in claim 246 or 247, a pharmaceutical composition comprising the EV population as described in claim 248, or a preparation comprising the EV population as described in any one of claims 249-263.

278. The method of claim 277, wherein inhibition of microglial activation is measured by expression of CNFT and / or MIF.

279. The method of claim 278, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of CNFT before administration.

280. The method of claim 277 or claim 278, wherein the method increases the expression of MIF by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the expression of MIF before administration.

281. A method for reducing oxidative stress in the eye of a subject suffering from a retinal disease or condition, comprising administering to the subject an EV population as described in claim 246 or 247, a pharmaceutical composition comprising the EV population as described in claim 248, or a preparation comprising the EV population as described in any one of claims 249-263.

282. The method of claim 281, wherein the reduction in oxidative stress is measured by expression of CNFT.

283. The method of claim 282, wherein the method increases the expression of CNFT by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of CNFT before administration.

284. A method for increasing the expression of anti-apoptotic factors in the eye of a subject suffering from a retinal disease or condition, comprising administering to the subject the EV population of claim 246 or 247, the pharmaceutical composition comprising the EV population of claim 248, or the preparation comprising the EV population of any one of claims 249-263.

285. The method of claim 284, wherein the method increases the expression of the anti-apoptotic factor by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to the expression of the anti-apoptotic factor before administration.

286. The method of claim 284 or 285, wherein the anti-apoptotic factor is S100B.

287. A method for preventing degeneration of the outer nuclear layer (ONL) of the eye of a subject suffering from a retinal disease or condition, comprising administering to the subject the EV population of claim 246 or 247, the pharmaceutical composition comprising the EV population of claim 248, or the preparation comprising the EV population of any one of claims 249-263.

288. The method of any one of claims 264-287, wherein the disease or condition is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, moderate dry form of age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt's disease.

289. The method of claim 288, wherein the eye disease is macular degeneration or retinitis pigmentosa.

290. The method of any one of claims 264-289, wherein the disease is a retinal degenerative disease.

291. The method of any one of claims 264-290, wherein the disease is associated with a loss of photoreceptor cells.

292. The method of claim 291, wherein the disease is associated with a loss of photoreceptor cells in the outer nuclear layer of the retina.

293. The method of any one of claims 264-292, wherein the EV population, a pharmaceutical composition comprising the EV population, or a preparation comprising the EV population is administered to the subretinal space or suprachoroidal space of the subject.

294. The method of any one of claims 264-293, wherein the EV population, the pharmaceutical composition comprising the EV population, or the preparation comprising the EV population is administered by injection or implantation.

295. The method of claim 294, wherein the injection is administered intraocularly.

296. The method of claim 294 or 295, wherein intraocular administration comprises injecting an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space, thereby forming an anterior vesicle, and removing the aqueous solution before administering the photoreceptor salvage cell composition into the same subretinal space as the aqueous solution.

297. The method of any one of claims 287-296, wherein the EV population, the pharmaceutical composition comprising the EV population, or the preparation comprising the EV population is administered within at least 1 week, at least 1 month, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years of symptom onset.

298. The method of any one of claims 287-297, further wherein one or more anti-inflammatory agents are administered to the subject.

299. The method of claim 298, wherein the one or more anti-inflammatory agents and the EV population, a pharmaceutical composition comprising the EV population, or a preparation comprising the EV population are administered simultaneously.

300. The method of claim 298, wherein the one or more anti-inflammatory agents and the EV population, a pharmaceutical composition comprising the EV population, or a preparation comprising the EV population are administered separately.

301. The method according to claim 300, wherein 1) administering the one or more anti-inflammatory agents prior to the EV population, the pharmaceutical composition comprising the EV population, or the formulation comprising the EV population, or 2) administering the EV population, a pharmaceutical composition comprising the EV population, or a preparation comprising the EV population prior to the one or more anti-inflammatory agents.

302. The method of any one of claims 298, 300, and 301, wherein the one or more anti-inflammatory agents are administered before and after administration of the EV population, a pharmaceutical composition comprising the EV population, or a preparation comprising the EV population.

303. The method of any one of claims 287-297, wherein the EV population, the pharmaceutical composition comprising the EV population, or the preparation comprising the EV population is administered in the absence of one or more anti-inflammatory agents.

304. The method of claim 303, wherein the one or more anti-inflammatory agents comprise dexamethasone and / or cyclosporine.

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