Photoreceptor rescue cell (PRC) compositions and methods for treating ocular disorders

Abstract

The present invention provides populations of photoreceptor rescue cells (PRCs) with unique marker profiles and generated by in vitro differentiation from early precursors, including pluripotent stem cells and embryonic stem cells (ESCs). Methods for generating the populations of photoreceptor rescue cells and their use for the treatment of ocular disorders are also provided.

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 373,298, filed August 23, 2022, and U.S. Provisional Application No. 63 / 432,948, filed December 15, 2022, the entire contents of each of which are hereby incorporated by reference into this specification. [Background technology]

[0002] background

[0002] There are several retinal diseases or disorders that can lead to vision loss or even blindness. Among them are rod or cone dystrophies, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration (such as age-related macular degeneration (wet or dry), geographic atrophy secondary to AMD), Leber's congenital amaurosis, and Stargardt's disease. Some retinal diseases or disorders are the result of cell loss in the granular layer, primarily the outer nuclear layer, which contains photoreceptor cells. Replacement of degenerated photoreceptors with new cells offers a potential method for slowing or halting cell degeneration and vision loss.

[0003]

[0003] Potential replacement sources of photoreceptor cells include stem cells. Early studies incorporated the use of heterogeneous populations of mouse cells, mouse stem cells, or retinal progenitor cells as potential sources of cells for replacement of lost photoreceptors. These early studies included transplantation of photoreceptor progenitor cells from postnatal day 1 mouse retinas (Maclaren et al., Nature 444(9):203-207, 2006), in vitro generation of retinal progenitor cells from mouse embryonic stem cells (Ikeda et al., Proc. Natl. Acad. Sci. 102(32):11331-11336, 2005), generation of retinal progenitor cells from postnatal day 1 mouse retinas (Klassen et al., Invest. Ophthal. Vis. Sci. 45(11):4167-4175, 2004), and implantation of bone marrow mesenchymal stem cells in the RCS rat model of retinal degeneration (Inoue et al., Exp. Eye Res. 8(2):234-241, 2007), generation of retinal progenitor cells, including ganglion cells, amacrine cells, and photoreceptors, bipolar cells, and horizontal cells from the H1 human embryonic stem cell line, with 0.01% of the total cells expressing S-opsin or rhodopsin (Lamba et al., Proc. Natl. Acad. Sci. 10(34):12769-12774, 2006), and derivation of induced pluripotent stem cells (iPSCs) from human fibroblasts to generate retinal progenitor cells (Lamba et al., PLoS ONE 5(1):e8763. doi:10.1371 / journal.pone.0008763). None of these approaches generated homogeneous populations of photoreceptor progenitor cells or photoreceptor cells for implantation. None of these approaches generated populations of photoreceptor progenitor or photoreceptor cells that exhibited rod or cone function in vivo (e.g., detectable by conferring improved vision). Differentiation of stem cells into populations of photoreceptor rescue cells may offer an alternative to currently available therapeutic approaches. Summary of the Invention

[0004] overview The present invention provides photoreceptor rescue cell (PRC) compositions comprising a plurality of heterogeneous photoreceptor rescue cells with unique marker portfolios, and methods for their use in the treatment of ocular disorders.

[0005]

[0005] Accordingly, in one aspect, the present invention provides a photoreceptor rescue cell composition comprising a plurality of heterogeneous photoreceptor rescue cells, wherein the plurality of heterogeneous 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.

[0006] In one embodiment, the photoreceptor rescue cell composition further comprises a suitable medium for maintaining cell viability.

[0007] In one embodiment, the cells are produced by in vitro differentiation of pluripotent cells.

[0008]

[0008] In one embodiment, the pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0009] 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 rescue cells.

[0010]

[0010] In one embodiment, the plurality of heterogeneous cells cumulatively expresses FOXG1 and MAP2.

[0011] 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) about 55 transcripts per million (TPM) to about 2 00 TPM, about 60 TPM to about 170 TPM, about 140 TPM to about 165 TPM, or about 149 TPM to about 170 TPM of FOXG1 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0012] 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) about 250 transcripts per million (TPM) to about 700 TPM, about 290 MAP2 transcripts of from about 450 TPM to about 625 TPM, or from about 490 TPM to about 615 TPM are expressed by the cells of the composition; and / or (iv) MAP2 transcripts of 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 are expressed by the cells of the composition.

[0013] In one embodiment, the plurality of heterogeneous cells cumulatively express at least one additional marker selected from the group consisting of STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In one embodiment, the plurality of heterogeneous cells cumulatively express at least three, four, five, six, or seven of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

[0014]

[0014] 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% 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) 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 of STMN2 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0015] 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) a transcript count per million (Tm) of about 200 is obtained. (iv) 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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0016] 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) about 50%, Transcripts per million (TPM) between about 100 TPM, between about 50 TPM and about 95 TPM, between about 85 TPM and about 95 TPM, or between about 87 TPM and about 93 TPM of LINC00461 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0017] 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; and / or or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0018] 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%, 68%, 69%, or 70% of the cells in the composition express GAD1. , express GAD1; and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0019] 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% and / or (ii) at least about 50%, 55%, 60%, 65%, 67%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180 7%, 88%, 89%, 90%, 92%, or 95% express NFIA; and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0020] In one embodiment, the plurality of heterogeneous cells cumulatively express each of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. In one embodiment, the heterogeneous cells each individually express at least one of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, or NFIA.

[0021] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises one or more cell types selected from the group consisting of inhibitory neurons, excitatory neurons, precursors, astrocytes, and replacement neurons. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises each of inhibitory neurons, excitatory neurons, precursors, astrocytes, and replacement neurons.

[0022] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises inhibitory neurons that express one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4, and CALB2. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of inhibitory neurons that cumulatively express each of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0023] 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) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0024] 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 78% to about 9 ... %, 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) 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 of TUBB3 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0025] 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 (iii) 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 of SCGN transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0026]

[0026] 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%, 72%, 75%, 80% of the cells in the composition express ERBB4. or 85% express ERBB4; and / or (iii) about 15, about 120, about 17, about 100, about 18, about 95, about 70, or about 95 TPM of ERBB4 transcripts are expressed by the cells of the composition; and / or (iv) at least 15, 30, 50, 70, 73, 75, 80, 82, 85, 88, 90, 91, 93, 95, 100, 110, or 120 TPM of ERBB4 transcripts are expressed by the cells of the composition.

[0027] 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 (iii) about 30, transcripts per million (TPM) between about 220 TPM, between about 50 TPM and about 200 TPM, between about 70 TPM and about 200 TPM, or between about 75 TPM and about 199 TPM of CALB2 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0028] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises excitatory neurons that express one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2, and SATB2. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of excitatory neurons that cumulatively express each of the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

[0029] 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%, 15%, 17%, 20%, 22%, 24%, 26%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 2%, 23%, 24%, 26%, 28%, or 30% express NEUROD6; and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0030] 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 cells in the composition express SLA; and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0031] 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) about 1 per million Transcript numbers (TPM) of from about 150 TPM, from about 4 TPM to about 130 TPM, from about 4 TPM to about 35 TPM, from about 20 TPM to about 30 TPM, or from about 25 TPM to about 28 TPM of NELL2 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0032] 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%, or 20% of the cells in the composition express SATB2. and / or (iii) 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 of SATB2 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0033] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises progenitors that express one or more markers selected from the group consisting of VIM, MKI67, CLU, and GLI3. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises progenitors that cumulatively express each of the markers VIM, MKI67, CLU, and GLI3.

[0034] 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 VIM; and / or (iii) 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 of VIM transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0035] 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) about 5 transcripts per million. Number of products (TPM) of from about 40 TPM, from about 10 TPM to about 35 TPM, from about 15 TPM to about 25 TPM, or from about 18 TPM to about 22 TPM of MKI67 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0036] 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) about 30 transcripts per million (TPM) to about 400 TPM, about 4 0 TPM to about 150 TPM, about 60 TPM to about 150 TPM, or about 60 TPM to about 105 TPM of CLU transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0037] 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; and / or or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0038] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG, and FBXL7. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of astrocytes cumulatively expressing each of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0039] 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) about 0.1 transcripts per million. (iv) from about 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 are expressed by the cells of the composition.

[0040]

[0040] 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.

[0041] 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) a) 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) at least about 5, 100%, ... Transcripts per million (TPM) between about 40 TPM, between about 5 TPM and about 30 TPM, between about 6 TPM and about 25 TPM, or between about 10 TPM and about 15 TPM of MIR99AHG transcript are expressed by the cells of the composition; and / or (iv) 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 MIR99AHG transcript are expressed by the cells of the composition.

[0042] 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) a transcript count per million of about 5 ( FBXL7 transcripts of 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 are expressed by the cells of the composition; and / or (iv) FBXL7 transcripts of 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 are expressed by the cells of the composition.

[0043] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises replacement neurons that express one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2, and CACNA1C. In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises a plurality of replacement neurons that cumulatively express each of the markers MEIS2, PBX3, GRIA2, and CACNA1C.

[0044] 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) 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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0045] 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) about 5 translocations per million. Transcript numbers (TPM) of PBX3 transcripts ranging from 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0046] 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) a transcript count per million of about 2. (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 transcript is expressed by the cells of the composition; and / or (iv) 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 of GRIA2 transcript is expressed by the cells of the composition.

[0047] 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%, 60%, 65%, or 70% of the cells in the composition express CACNA1C. 0% express CACNA1C; and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0048]

[0048] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells include (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 the group consisting of NEUROD2, NEUROD6, SLA, NELL2, and SATB2; (iii) precursors 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.

[0049]

[0049] In one embodiment, the plurality of heterogeneous photoreceptor rescue cells comprises: (i) a plurality of inhibitory neurons cumulatively expressing each of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2; (ii) a plurality of excitatory neurons cumulatively expressing each of the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2; (iii) a plurality of precursors cumulatively expressing each of the markers VIM, MKI67, CLU, and GLI3; (iv) a plurality of astrocytes cumulatively expressing each of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7; and (v) a plurality of replacement neurons cumulatively expressing each of the markers MEIS2, PBX3, GRIA2, and CACNA1C.

[0050] 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 18% to about 20%. or about 20% to about 35% of the precursor; 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 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.

[0051] In one embodiment, the cells in the composition further express one or more eye field progenitor markers, rod / cone photoreceptor markers, and / or neuronal markers. In one embodiment, the eye field progenitor markers are selected from the group consisting of PAX6, LHX2, SIX3, NES, and SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express at least one, two, three, or four of the eye field progenitor markers PAX6, LHX2, SIX3, NES, or SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express at least each of the eye field progenitor markers PAX6, LHX2, SIX3, NES, and SOX2. In one embodiment, the plurality of heterogeneous cells cumulatively express SOX2. In one embodiment, the composition is substantially free of cells expressing the eye field progenitor markers RAX, SIX6, and / or TBX3.

[0052] 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) a transcripts per million (TPM) of about 20 to about 125 TPM, about 30 TPM to about 110 TPM, about 35 TPM to about 1 00 TPM, about 70 TPM to about 80 TPM, or about 73 TPM to about 78 TPM of PAX6 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0053] 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 (iii) about 5, about 5 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0054] 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 (i) ii) 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 are expressed by the cells of the composition; and / or (iv) 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 SIX3 transcripts are expressed by the cells of the composition.

[0055] 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 an 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 an NES; and / or (iii) about 5, 100% or more of the cells in the composition express an NES. Transcripts per million (TPM) between about 35 TPM, between about 5 TPM and about 28 TPM, between about 7 TPM and about 15 TPM, between about 10 TPM and about 15 TPM, or between about 11 TPM and about 13 TPM of NES transcripts are expressed by the cells of the composition; and / or (iv) 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 of NES transcripts are expressed by the cells of the composition.

[0056] 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) a transcript count per million of about 50. (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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0057] In one embodiment, the rod / cone photoreceptor marker is selected from the group consisting of ASCL1, RORB, NR2E3, and NRL. In one embodiment, the plurality of heterogeneous 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, RCVRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0058] 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) about 50, 100 Transcripts per million (TPM) between about 150 TPM, between about 60 TPM and about 140 TPM, between about 65 TPM and about 130 TPM, or between about 95 TPM and about 130 TPM of ASCL1 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0059] 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) 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 of RORB transcripts are expressed by the cells of the composition; and / or (iv) 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 transcripts are expressed by the cells of the composition.

[0060] 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 NR2E3; and and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0061] 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) about 0.1 to about 1 million (iv) 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 are expressed by the cells of the composition.

[0062] In one embodiment, the neuronal marker is selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. In one embodiment, the plurality of heterogeneous cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0063] 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% of the cells in the composition express NFIB. %, 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% express NFIB; and / or (iii) about 150, 100 and / or (iv) 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 are expressed by the cells of the composition.

[0064] 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) 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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0065] 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 are ELAVL 3; 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) a transcripts per million (TPM) of between about 10 and about 120. 10 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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0066] 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) a) 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) a transcript level of about 10 per million and / or (iv) 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, 75 TPM, 80 TPM, 85 TPM, 90 TPM, 95 TPM, 100 TPM, 120 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 45 TPM, 50 TPM, 55 TPM, 60 TPM, 70 TPM, 75 TPM, 80 TPM, 90 TPM, 100 TPM, 120 TPM, 15 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 100 TPM, 150 TPM, 100 TPM, 150 TPM, 100 TPM, 150 TPM, 100 TPM, 150 TPM, 150 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 4 ...50 TPM, 150 TPM, 20 TPM, 25 TPM, 30 TPM, 35 TPM, 40 TPM, 150 TPM, 0 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 the ELAVL4 transcript is expressed by the cells of the composition.

[0067] 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 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 are SLC1A2. and / or (iii) 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 are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0068] 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) about 5% to about 40%, about 5% to about 35%, about 5 ... 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 sample express SLC1A3; and / or (iii) a transcript count per million (TPM) of between about 50 and about 200 TPM, between about 70 and about 80 TPM, or between about 80 and about 90 TPM. 100 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 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0069] 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) about 0.1 transcripts per million (TPM) to about 10 TPM. M, 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 of HCN1 transcript is expressed by the cells of the composition; and / or (iv) 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 transcript is expressed by the cells of the composition.

[0070] 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) about 10%, Transcripts per million (TPM) of from about 60 TPM, from about 5 TPM to about 50 TPM, from about 12 TPM to about 46 TPM, or from about 15 TPM to about 39 TPM of HES5 transcripts are expressed by the cells of the composition; and / or (iv) 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 are expressed by the cells of the composition.

[0071]

[0071] In one embodiment, the plurality of heterogeneous cells cumulatively express (i) one or more of the eye field progenitor markers PAX6, LHX2, SIX3, NES, and SOX2; (ii) one or more of the rod / cone photoreceptor 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.

[0072]

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

[0073] In one embodiment, the composition is substantially free of at least one cell type selected from the group consisting of pluripotent stem cells, retinal ganglion cells, photoreceptors, and amacrine cells. In one embodiment, the composition is substantially free of pluripotent stem cells, retinal ganglion cells, photoreceptors, and amacrine cells.

[0074] In one embodiment, the composition is substantially free of retinal progenitors that express 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% cells that express SSEA4, optionally as determined by flow cytometry, or the composition is free of cells that express SSEA4, optionally as determined by flow cytometry.

[0074]

[0075] In one embodiment, the cells in the composition have phagocytic activity, optionally the ability to phagocytose isolated photoreceptor outer segments, dye conjugates, or both.

[0075]

[0076] In one embodiment, the cells in the composition secrete one or more neuroprotective factors, hi one embodiment, the neuroprotective factors are selected from the group consisting of CNTF, MIF, S100B, GFAP, TAU, NCAM1, and TNC.

[0076]

[0077] 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 viable. 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 viable. In one embodiment, at least 50% of the cells in the composition are viable. In one embodiment, at least 55% of the cells in the composition are viable. In one embodiment, at least 60% of the cells in the composition are viable. In one embodiment, at least 65% of the cells in the composition are viable. In one embodiment, at least 68% of the cells in the composition are viable.

[0077]

[0078] In one embodiment, the composition is produced by a method comprising the steps of: 1) culturing pluripotent stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells from step 1 in neural differentiation medium (NDM) and noggin; and 3) expanding the cells from step 2 in NDM in the absence of noggin, comprising: a) culturing the cells under low-adhesion or non-adhesion conditions in NDM without noggin; and b) culturing the cells under adherence conditions in NDM without noggin.

[0078]

[0079] In one embodiment, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0079]

[0080] 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 cells in the composition are harvested after the fifth repetition of step 3.

[0080]

[0081] In one embodiment, the cells in the collected composition are cryopreserved. In one embodiment, the composition is cryopreserved between the first and second iterations of step 3, between the second and third iterations of step 3, between the third and fourth iterations of step 3, or between the fourth and fifth iterations of step 3. In one embodiment, the composition is cryopreserved after the third iteration of step 3, after the fourth iteration of step 3, or after the fifth iteration of step 3. In one embodiment, step 3 is repeated five times, and the composition is cryopreserved after the fifth iteration of step 3.

[0081]

[0082] 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 are viable after cryopreservation and thawing. 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% are viable after cryopreservation and thawing. In one embodiment, at least about 50% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, at least about 55% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, at least about 60% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, at least about 65% of the cells in the composition are viable after cryopreservation and thawing. In one embodiment, at least 68% of the cells in the composition are viable after cryopreservation and thawing.

[0082]

[0083] In one embodiment, the composition comprises cell spheres, in which 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 spheres.

[0083]

[0084] In one embodiment, during steps 1, 2, and / or 3, the cells of the composition are cultured in a cell culture vessel selected from the group consisting of a culture dish, a culture flask, and a culture chamber. In one embodiment, the cell culture vessel is about 50 cm 2 ~about 800cm 2 , about 60cm 2~about 800cm 2 , about 100cm 2 ~about 800cm 2 , about 150cm 2 ~about 800cm 2 , about 175cm 2 ~about 800cm 2 , about 200cm 2 ~about 800cm 2 , about 250cm 2 ~about 800cm 2 , about 300cm 2 ~about 800cm 2 , about 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 ~about 100cm 2 , about 50cm 2 ~about 100cm 2 , about 100cm 2 ~about 300cm 2 , about 150cm 2 ~about 250cm 2 , or approximately 150 cm 2 ~about 200cm 2 In one embodiment, the cell culture vessel has a cell growth area of ​​at least about 60 cm 2 , about 175cm 2 , or approximately 636 cm 2 has a cell growth area of ​​.

[0084]

[0085] In one embodiment, the culture chambers are stackable rectangular chambers. 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.

[0085]

[0086] In one embodiment, the cell culture vessel is coated for low-adherent or non-adherent cell culture for step 3a and / or adherent cell culture for step 3b. 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 the group consisting of thermolysin, liberase, accutase, and combinations thereof. In one embodiment, the enzyme used to dissociate the cells is accutase. In one embodiment, the dissociation of the cells from the plate does not involve manual scraping.

[0086]

[0087] Thus, in another aspect, the invention provides pharmaceutical preparations comprising the photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein and a pharmaceutically acceptable excipient, hi one embodiment, the pharmaceutically acceptable excipient is suitable for ocular delivery.

[0087]

[0088] Accordingly, in another aspect, the present invention provides a) a composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein; and b) a formulation comprising about 4-10% (v / v) cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer.

[0088]

[0089] 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.

[0089]

[0090] In one embodiment, the albumin is human albumin. In one embodiment, the albumin is recombinant human albumin.

[0090]

[0091] In one embodiment, the buffer is buffered saline. In one embodiment, the buffer is phosphate buffered saline (PBS). In one embodiment, the buffered saline contains Ca2+ and Mg2+. In one embodiment, the buffered saline does not contain Ca2+ and Mg2+. In one embodiment, the formulation is stored frozen.

[0091]

[0092] Accordingly, in another aspect, the invention provides a) a composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein; and b) a formulation comprising a solution comprising: (1) a buffer that maintains the solution at physiological pH; and (2) at least 2 mM or at least 0.05% (w / v) glucose; and (3) an osmotically active agent that maintains the solution at physiological osmolality.

[0092]

[0093] In one embodiment, 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.

[0093]

[0094] In one embodiment, the solution further comprises (4) a source of divalent cations, optionally comprising a calcium source and / or a magnesium source, and / or (5) an acetate buffer and / or a citrate buffer.

[0094]

[0095] Accordingly, in another aspect, the invention provides a) a composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein; and b) a formulation comprising a solution comprising: (1) a buffer that maintains a solution at physiological pH, where the buffer is not a bicarbonate buffer; and (2) glucose; and (3) an osmotically active agent that maintains a solution at physiological osmolality; 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 the buffer comprises an acetate buffer and / or a citrate buffer.

[0095]

[0096] 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 are selected from the group of calcium salts and / or magnesium salts formed with an acid selected from the group including 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 including calcium chloride dihydrate. In one embodiment, the magnesium source comprises magnesium chloride, optionally including magnesium chloride hexahydrate. In one embodiment, the citrate buffer is provided as sodium citrate. In one embodiment, the glucose is D-glucose (dextrose).

[0096]

[0097] In one embodiment, the osmotically active agent is a salt, optionally the osmotically active agent is a sodium salt, and further optionally the osmotically active agent is sodium chloride, hi one embodiment, the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose.

[0097]

[0098] In one embodiment, the pH of the solution is 6.8 to 7.8, or 7.4 to 7.5, or about 7.5.

[0098]

[0099] In one embodiment, the solution is isotonic or hypertonic.

[0099]

[0100] In one embodiment, the solution exhibits an osmolality of about 270-345 mOsm / l or about 315 mOsm / l.

[0100]

[0101] In one embodiment, the concentration of the calcium source is (a) 0.25 to 0.75 mM, or 0.4 to 0.65 mM, or 0.5 to 0.6 mM, or about 0.6 mM; or (b) 0.5 to 0.9 mM, or 0.6 to 0.8 mM, or about 0.7 mM.

[0101]

[0102] 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 osmotically active agent is about 100-200 mM, or about 125-175 mM, or about 150 mM. In one embodiment, the concentration of citrate or acetate is 0.1-5 mM, or 0.5-2 mM, or about 1 mM.

[0102]

[0103] In one embodiment, the solution further comprises a potassium salt, optionally the potassium salt is potassium chloride, and further optionally the concentration of KCl is 0.2-5 mM, or 1-2.5 mM, or about 2 mM.

[0103]

[0104] In one embodiment, the solution contains (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% Ca (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;

[0104]

[0105] 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.

[0105]

[0106] In one embodiment, the polymer is present at a concentration effective to reduce exposure of cells in solution to shear stress, optionally the concentration of the polymer is 0.005-5% w / v or about 0.05% w / v.

[0106]

[0107] 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.

[0107]

[0108] In one embodiment, the solution does not contain (a) a carbonate buffer, and / or (b) glutathione or glutathione disulfide (GSSG), and / or (c) a zwitterionic organic buffer.

[0108]

[0109] In one embodiment, the solution (a) can 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 one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and / or measurable loss of the solution's ability to support the survival and viability of cells stored in the solution, and / or (b) can 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 one week, at least two weeks, at least three weeks, or at least one month without measurable precipitation of solutes and / or measurable loss of the solution's ability to support the survival and viability of cells stored in the solution.

[0109]

[0110] In one embodiment, the solution is (a) 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) essentially pyrogen-free, and / or (c) sterile, and / or (d) for irrigation, cell reconstitution, cell storage, cell transport, and / or administration to a subject.

[0110]

[0111] Thus, in another aspect, the present invention provides methods for producing compositions of various embodiments of the above aspect or any other aspect of the invention detailed herein, comprising the steps of: 1) culturing pluripotent stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells from step 1 in neural differentiation medium (NDM) and noggin; and 3) expanding the cells from step 2 in NDM without noggin, comprising: a) culturing the cells under low-adherent or non-adherent conditions in NDM without noggin; and b) culturing the cells under non-adherent conditions in NDM without noggin, thereby differentiating the pluripotent stem cells into photoreceptor rescue cells.

[0111]

[0112] Thus, in another aspect, the present invention provides a method for producing a photoreceptor rescue cell composition comprising a plurality of heterogeneous cells, the method comprising the steps of: 1) culturing pluripotent stem cells in rescue induction medium (RIM) and noggin; 2) culturing the cells in neural differentiation medium (NDM) and noggin; and 3) expanding the cells in NDM without noggin, comprising: a) culturing the cells under low-adhesion or non-adhesion conditions in NDM without noggin; and b) culturing the cells under adhesion conditions in NDM without noggin, thereby differentiating the pluripotent stem cells into a plurality of cells of the photoreceptor rescue cell composition.

[0112]

[0113] In one embodiment, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0113]

[0114] 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 iteration of step 3, after the fourth iteration of step 3, or after the fifth iteration of step 3. In one embodiment, the composition is harvested after the fifth iteration of step 3.

[0114]

[0115] In one embodiment, the cells in the harvested composition are cryopreserved.

[0115]

[0116] In one embodiment, the composition is cryopreserved between the first and second iterations of step 3, between the second and third iterations of step 3, between the third and fourth iterations of step 3, or between the fourth and fifth iterations of step 3. In one embodiment, the composition is cryopreserved after the third iteration of step 3, after the fourth iteration of step 3, or after the fifth iteration of step 3. In one embodiment, step 3 is repeated five times, and the composition is cryopreserved after the fifth iteration of step 3.

[0116]

[0117] 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 viable after cryopreservation and thawing. 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 viable after cryopreservation and thawing. In one embodiment, less than about 50% of the cells in the composition are viable after cryopreservation and thawing. 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.

[0117]

[0118] In one embodiment, the composition comprises cell spheres, in which 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 spheres.

[0118]

[0119] In one embodiment, during steps 1, 2 and / or 3, the cells of the composition are cultured in a cell culture vessel selected from the group consisting of a culture dish, a culture flask, and a culture chamber.

[0119]

[0120] In one embodiment, the cell culture vessel is about 50 cm2 ~about 800cm 2 , about 60cm 2 ~about 800cm 2 , about 100cm 2 ~about 800cm 2 , about 150cm 2 ~about 800cm 2 , about 175cm 2 ~about 800cm 2 , about 200cm 2 ~about 800cm 2 , about 250cm 2 ~about 800cm 2 , about 300cm 2 ~about 800cm 2 , about 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 ~about 100cm 2 , about 50cm 2 ~about 100cm 2 , about 100cm 2 ~about 300cm 2 , about 150cm 2 ~about 250cm 2 , or approximately 150 cm 2 ~about 200cm 2 In one embodiment, the cell culture vessel has a cell growth area of ​​at least about 60 cm 2 , about 175cm 2 , or approximately 636 cm 2 has a cell growth area of ​​.

[0120]

[0121] In one embodiment, the culture chamber is a stackable rectangular chamber.

[0121]

[0122] 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.

[0122]

[0123] In one embodiment, the cell culture vessel is coated for low-adherent or non-adherent cell culture for step 3a and / or for adherent cell culture for step 3b.

[0123]

[0124] In one embodiment, the cells are enzymatically dissociated from the plate into a cell suspension. In one embodiment, the enzyme used to dissociate the cells is thermolysin, liberase, and / or accutase. In one embodiment, the enzyme used to dissociate the cells is accutase. In one embodiment, dissociating the cells from the plate does not involve manual scraping.

[0124]

[0125] Thus, in another aspect, the present invention provides photoreceptor rescue cell compositions produced by the methods of various embodiments of the above aspects or any other aspects of the invention detailed herein.

[0125]

[0126] Thus, in another aspect, the present invention provides a method of treating an ocular disease or disorder in a subject, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0126]

[0127] Thus, in another aspect, the present invention provides a method for increasing secretion of neuroprotective factors in the eye of a subject having an ocular disease or disorder, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0127]

[0128] In one embodiment, the method increases 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 secretion of the neuroprotective factor before administration.

[0128]

[0129] Thus, in another aspect, the present invention provides a method of improving vision in a subject having a retinal disease or disorder, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0129]

[0130] In one embodiment, 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.

[0130]

[0131] In one embodiment, visual acuity is measured by optokinetic response (OMR) and / or electroretinogram (ERG).

[0131]

[0132] In one embodiment, the treated eye has an increased spatial frequency threshold as measured by OMR of 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.

[0132]

[0133] In one embodiment, the treated eye has an increased dark-adapted b-wave amplitude as measured by ERG of 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 dark-adapted b-wave amplitude before administration.

[0133]

[0134] Thus, in another aspect, the invention provides a method of preventing or slowing the loss of photoreceptor cells in a subject having a retinal disease or disorder, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0134]

[0135] 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 administration.

[0135]

[0136] Thus, in another aspect, the present invention provides a method of increasing phagocytic activity in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0136]

[0137] 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 phagocytic activity before administration.

[0137]

[0138] Thus, in another aspect, the invention provides 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 a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0138]

[0139] In one embodiment, the inhibition of microglial activation is measured by the expression of CNFT and / or MIF.In one embodiment, 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 with the expression of CNFT before administration.In one embodiment, 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 with the expression of MIF before administration.

[0139]

[0140] Thus, in another aspect, the present invention provides 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 a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0140]

[0141] In one embodiment, the reduction in oxidative stress is measured by the expression of CNFT. In one embodiment, 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.

[0141]

[0142] Thus, in another aspect, the present invention provides a method for increasing 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 a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0142]

[0143] In one embodiment, the method increases 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.

[0143]

[0144] In one embodiment, the anti-apoptotic factor is S100B.

[0144]

[0145] Thus, in another aspect, the invention provides a method of preventing degeneration of the outer nuclear layer (ONL) in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject a photoreceptor rescue cell composition of various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical preparation of various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation of various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0145]

[0146] In one embodiment, the disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber's congenital amaurosis, or Stargardt's disease. In one embodiment, the eye disease is macular degeneration or retinitis pigmentosa. In one embodiment, the disease is a retinal degenerative disease.

[0146]

[0147] In one embodiment, the disease is associated with loss of photoreceptor cells.

[0147]

[0148] In one embodiment, the disease is associated with loss of photoreceptor cells in the outer nuclear layer of the retina, hi one embodiment, the photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered to the subretinal space, to the suprachoroidal space, by depot in the eye, or by systemic delivery to another part of the subject's body.

[0148]

[0149] In one embodiment, the cell preparation or formulation is administered by injection or implantation.

[0149]

[0150] In one embodiment, the injection is administered intraocularly.

[0150]

[0151] In one embodiment, intraocular administration comprises injecting an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space, thereby forming a pre-bleb, and removing the aqueous solution prior to administration of the photoreceptor rescue cell composition into the same subretinal space as the aqueous solution.

[0151]

[0152] In one embodiment, the cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years of the onset of symptoms.

[0152]

[0153] In one embodiment, (i) the subject has intermediate or near-end stage disease; (ii) the subject has best corrected visual acuity (BCVA) in the range of 20 / 50 to 20 / 200; (iii) the subject has BCVA worse than 20 / 200 but maintains light sensitivity; or (iv) is diagnosed with retinitis pigmentosa by genotyping.

[0153]

[0154] In one embodiment, the method further comprises administering one or more anti-inflammatory agents to the subject.

[0154]

[0155] In one embodiment, the one or more anti-inflammatory agents are administered simultaneously. In one embodiment, the one or more anti-inflammatory agents are administered separately.

[0155]

[0156] In one embodiment, one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before the one or more anti-inflammatory agents. In one embodiment, one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation. In one embodiment, the administration of the cell preparation or formulation is without the administration of one or more anti-inflammatory agents. In one embodiment, the one or more anti-inflammatory agents include dexamethasone and / or cyclosporine.

[0156]

[0157] Thus, in another aspect, the present invention provides a population of extracellular vesicles (EVs) secreted from the photoreceptor rescue cell compositions of various embodiments of the above aspects or any other aspects of the invention detailed herein.

[0157]

[0158] In one embodiment, the EVs secreted from photoreceptor rescue cells are selected from the group consisting of 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, F BXL7, 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, L GI2, LOC221946, LRRC4C, MAP3k19, OLFM3, PRND, PTGER3, RELN, TCERGIL, TSHR, UNC13C, TRb2, PDE6B, CNGb1, Tuj1, CHX10, nestin, TR beta 2, MASH1, ROR beta, MAP2, ELAVL3, NFIA, DCX, LHX2, SLC1A2, ELAVL4, PAX6, EMX2, ASCL1 , DLL1, NFIB, ENOX1, 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.

[0158]

[0159] Thus, in another aspect, the present invention provides a pharmaceutical composition comprising a population of EVs of various embodiments of the above aspects or any other aspects of the invention detailed herein and a pharmaceutically acceptable carrier.

[0159]

[0160] Accordingly, in another aspect, the present invention provides a) a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a pharmaceutical composition according to various embodiments of the above aspect or any other aspect of the invention detailed herein; and b) a formulation comprising about 4-10% (v / v) cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer.

[0160]

[0161] 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.

[0161]

[0162] In one embodiment, the albumin is human albumin. In one embodiment, the albumin is recombinant human albumin.

[0162]

[0163] In one embodiment, the buffer is buffered saline. In one embodiment, the buffer is phosphate buffered saline (PBS). In one embodiment, the buffered saline contains Ca2+ and Mg2+. In one embodiment, the buffered saline does not contain Ca2+ and Mg2+.

[0163]

[0164] In one embodiment, the formulation is stored frozen.

[0164]

[0165] Accordingly, in another aspect, the present invention provides a method of treating an eye disease in a subject, the method comprising administering to the subject a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0165]

[0166] Accordingly, in another aspect, the present invention provides a method of increasing secretion of a neuroprotective factor in the eye of a subject, the method comprising administering to the subject a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0166]

[0167] In one embodiment, the method increases 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 secretion of the neuroprotective factor before administration.

[0167]

[0168] Accordingly, in another aspect, the present invention provides a method of improving vision in a subject having a retinal disease or disorder, the method comprising administering to the subject a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0168]

[0169] In one embodiment, 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 the visual acuity before administration. In one embodiment, visual acuity is measured by optokinetic response (OMR) and / or electroretinogram (ERG). In one embodiment, the treated eye has an increased spatial frequency threshold, as measured by OMR, of 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. In one embodiment, the treated eye has an increased dark-adapted b-wave amplitude as measured by ERG of 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 dark-adapted b-wave amplitude before administration.

[0169]

[0170] Accordingly, in another aspect, the present invention provides 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 a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0170]

[0171] In one embodiment, the prevention or slowing 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 administration.

[0171]

[0172] Accordingly, in another aspect, the present invention provides a method of increasing phagocytic activity in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0172]

[0173] 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 phagocytic activity before administration.

[0173]

[0174] Accordingly, in another aspect, the present invention provides 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 a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0174]

[0175] In one embodiment, the inhibition of microglial activation is measured by the expression of CNFT and / or MIF.In one embodiment, 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 with the expression of CNFT before administration.In one embodiment, 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 with the expression of MIF before administration.

[0175]

[0176] Accordingly, in another aspect, the present invention provides 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 a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0176]

[0177] In one embodiment, the reduction in oxidative stress is measured by the expression of CNFT. In one embodiment, 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.

[0177]

[0178] Accordingly, in another aspect, the present invention provides a method of increasing 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 a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0178]

[0179] In one embodiment, the method increases 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. In one embodiment, the anti-apoptotic factor is S100B.

[0179]

[0180] Accordingly, in another aspect, the present invention provides a method of preventing degeneration of the outer nuclear layer (ONL) in the eye of a subject having a retinal disease or disorder, the method comprising administering to the subject a population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, a pharmaceutical composition comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein, or a formulation comprising the population of EVs according to various embodiments of the above aspect or any other aspect of the invention detailed herein.

[0180]

[0181] In one embodiment, the disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber's congenital amaurosis, or Stargardt'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 loss of photoreceptor cells. In one embodiment, the disease is associated with loss of photoreceptor cells in the outer nuclear layer of the retina.

[0181]

[0182] In one embodiment, the population of EVs, a pharmaceutical composition comprising the population of EVs, or a formulation comprising the population of EVs is administered to the subretinal space or suprachoroidal space of a subject.

[0182]

[0183] In one embodiment, the population of EVs, a pharmaceutical composition comprising the population of EVs, or a formulation comprising the population of EVs is administered by injection or implantation.

[0183]

[0184] In one embodiment, the injection is administered intraocularly.

[0184]

[0185] In one embodiment, intraocular administration comprises injecting an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space, thereby forming a pre-bleb, and removing the aqueous solution prior to administration of the photoreceptor rescue cell composition into the same subretinal space as the aqueous solution.

[0185]

[0186] In one embodiment, the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs 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 the onset of symptoms.

[0186]

[0187] In one embodiment, the method further comprises administering one or more anti-inflammatory agents to the subject.

[0187]

[0188] In one embodiment, the one or more anti-inflammatory agents and the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs are administered simultaneously.

[0188]

[0189] In one embodiment, the one or more anti-inflammatory agents and the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs are administered separately.

[0189]

[0190] In one embodiment, 1) the one or more anti-inflammatory agents are administered before the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs, or 2) the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs is administered before the one or more anti-inflammatory agents.

[0190]

[0191] In one embodiment, the one or more anti-inflammatory agents are administered before and after administration of the population of EVs, a pharmaceutical composition comprising the population of EVs, or a formulation comprising the population of EVs.

[0191]

[0192] In one embodiment, the population of EVs, pharmaceutical composition comprising the population of EVs, or formulation comprising the population of EVs is administered without one or more anti-inflammatory agents, hi one embodiment, the one or more anti-inflammatory agents comprise dexamethasone and / or cyclosporine. [Brief explanation of the drawings]

[0192] [Figure 1A]

[0193] Figure 1A shows cell cluster analysis of ESC / PRE / PRC. 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). [Figure 1B]

[0194] FIG. 1B is a graph showing qPCR validation of neuronal genes, including NANOG, NEUROD2, GAD1, DCX, MAP2, and NFIA. [Figure 1C]

[0195] Figure 1C shows single-cell analysis of PRC-P4 (GB2R-PRC-2019(P4), GB2R-PRC-PR1(P4), GB2R-PRC-CN2(P4), and GB2R-PRC-CN3(P4)). Cells were classified as inhibitory neurons, excitatory neurons, mixed neurons, precursors, and astrocytes. [Figure 1D]

[0196] Figure 1D is a graph showing a qPCR identity assay comparing 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 include GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019. [Figure 1E]

[0197] Figure 1E shows a heat map of the expression of eye field progenitor markers, rod / cone photoreceptor markers, and neuronal markers in the inhibitory neurons, excitatory neurons, replacement neurons, precursors, and astrocytes shown in Figure 1C. [Figure 1F]

[0198] Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1G]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1H]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1I]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1J]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1K]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1L]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1M]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1N]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1O]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1P]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1Q]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1R]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1S]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 1T]Figures 1F-1T are graphs showing the expression of cell markers on days 2 (D2), 12 (D12), 19 (D19), 37 (D37 / P0), 55 (D55 / P1), 72 (D72 / P2), 90 (D90 / P3) and 107 (D107 / P4) from the PRC compositions listed in Table 10. PRC markers FOXG1, MAP2, STMN2, and DCX (Figure 1F); PRC markers LINC00461, NEUROD2, GAD1, and NFIA (Figure 1G); inhibitory neuron markers DLX5, TUBB3, and SCGN (Figure 1H); inhibitory neuron markers ERBB4 and CALB2 (Figure 1I); excitatory neuron markers NEUROD2, NEUROD6, and SLA (Figure 1J); excitatory neuron markers NELL2 and SATB2 (Figure 1K); progenitor markers VIM, MKI67, CLU, and GLI3 (Figure 1L); and astrocyte markers GFAP, MIR99AHG. , and FBXL7 (Figure 1M); alternative neuronal markers MEIS2, PBX3, GRIA2, and CACNA1C (Figure 1N); eye field progenitor markers PAX6, LHX2, and SIX3 (Figure 1O); eye field progenitor 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). [Figure 2A]

[0199] FIG. 2A is a bright field image of PRC-P4 cells. [Figure 2B]

[0200] FIG. 2B shows a scorecard analysis of PRC differentiation identifying markers of self-renewal, ectoderm, mesoderm, and endoderm. [Figure 2C]

[0201] Figure 2C is an image showing immunocytochemistry (ICC) staining in PRC-P4 cells showing the expression of PAX6 / OTX2 in PRC-NPC cells (neural progenitor cells) and the expression of STMN2, CALB2, SCGN, and DCX in PRC-P4 cells. [Figure 2D]

[0202] FIG. 2D is a graph showing the expression of the markers NEUROD2, FOXG1, and HMGA1 in PRC-P4 cells, indicating successful differentiation. [Figure 2E]

[0203] Figure 2E is a graph showing flow cytometry of GB2R-CN3-P4 cells for purity based on expression of FOXG1 and MAP2. 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). [Figure 2F]

[0204] Figure 2F is a graph showing genes that are differentially expressed between PRC-P3 and PRC-P4 as determined via RNA-seq analysis. These genes have a log10 ratio of mean expression that is greater than 3 or less than -3. [Figure 3A]

[0205] Figure 3A is a graph showing stimulation of PRCs using TBHP (oxidative stress) and Luminex assays to detect secreted factors from stimulated PRCs: GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019. [Figure 3B]

[0206] Figure 3B is a graph showing the levels of secreted neuroprotective factors detected in the medium of PRC cells without oxidative stress. Stimulation of PRCs using TBHP (oxidative stress) and Luminex assays to detect secreted factors from stimulated PRCs. CNTF, MIF, and S100B were quantified in the medium of unstimulated (without oxidative stress) PRCs: GB2R-PRC-CN3, GB2R-PRC-CN2, Pioneer, and GB2R-PRC-2019. [Figure 3C]

[0207] Figure 3C shows confocal images demonstrating that subretinal engraftment of PRCs suppresses microglial infiltration into the ONL (P60) in RCS rats. Stained retinal sections were imaged using confocal z-stack analysis on a Leica SP8. [Figure 3D]

[0208] FIG. 3D is a graph showing stimulation of the microglial line SIM-A9 using LPS and inhibition using L-carnitine. [Figure 3E]

[0209] FIG. 3E shows a phagocytosis assay using GB2R-PRC-PR1 incubated with pHrodo E. coli bioparticles. [Figure 3F]

[0210] Figure 3F is a photograph showing the internalization of rod outer segment (ROS) debris in the RCS rat retina by PRCs. RHO debris was observed in GFP PRC explants (left panel), and EM in the right panel shows ROS flowing into the cytoplasm of PRCs. GFAP PRCs co-stain with HuNu (data not shown). [Figure 4A]

[0211] Figure 4A is a schematic representation of the dosing and time course of the RCS rat in vivo experiment. [Figure 4B]

[0212] Figure 4B shows graphs showing optokinetic response (OMR) and electroretinogram (ERG) analyses of RCS rats injected with 100,000 cells / eye of GB2R-PRC-2019PRC at P25. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT) comparing uninjected eyes with the test article compared to GS2 vehicle-injected eyes. Cell viability: 78.4% (manual). [Figure 4C]

[0213] Figure 4C shows representative images demonstrating that PRC engraftment significantly attenuates outer nuclear layer (ONL) degeneration up to 3 months after transplantation. Confocal images show that PRC engraftment is highly correlated with ONL preservation. Stained retinal sections were imaged using a Leica MDi8 epifluorescence microscope. [Figure 4D]

[0214] Figure 4D is a graph showing quantification of outer nuclear layer (ONL) thickness, demonstrating that PRC engraftment correlates with significant preservation at P35, P60, and P120. Cell viability: P35: 57.1%, 54%; P60: 57.1% (cell counter); P120: >70% (manual). n = 148 ROIs / 7 eyes. [Figure 4E]

[0215] Figure 4E shows an area plot of PRCs engrafted with GB2R-PRC-2019 (P4) versus photoreceptor ONL in a P120 RCS rat model of retinal degeneration. Statistical analysis was performed using Pearson's correlation coefficient. Cell viability: >70% (manual). n = 4 eyes (12 sections / eye). [Figure 5A]

[0216] Figure 5A shows an immunohistochemical analysis of glial fibrillary acidic protein (rabbit anti-GFAP; ABCAM) in a P120 RCS rat. GFAP is expressed in both Müller glia (MG) and optic nerve fiber astrocytes (white arrows) of the host rat retina, as well as in PRCs engrafted into the subretinal space. Increased GFAP expression in MG and astrocytes, as well as MG hypertrophy with expanded distribution of GFAP expression throughout the radial MG cell bodies, are characteristic of disease-associated reactive gliosis and can be confirmed distal to the transplantation site (yellow arrowheads, white arrowheads). GFAP upregulation and expanded distribution, MG hypertrophy, and outer nuclear layer (ONL) degeneration are attenuated or absent at the subretinal PRC transplantation site. Cell viability: >70% (manual). [Figure 5B]

[0217] Figure 5B shows TUNEL quantification (TUNEL Label Mix, Sigma catalog no. 11767291910, TUNEL Enzyme, Sigma catalog no. 11767305001) performed at P35, during which peak apoptotic activity was observed in RCS retinas. PRCs engrafted into the subretinal space were identified by Ku80+ staining (Abcam, ab80592) of adjacent sections (see below). In areas engrafted with PRCs, very few TUNEL+ nuclei were observed, indicating little to no apoptotic activity at the transplant site. However, non-engrafted areas contained significant TUNEL+ nuclei in the ONL, indicating widespread photoreceptor death at P35. Quantification of TUNEL+ nuclei in the ONL demonstrates a significant attenuation of apoptotic activity in the transplant site versus non-transplant areas. Retinal sections through the PRC graft were selected for TUNEL quantification and stained with Ku80 to identify the location of engrafted PRCs and DAPI to identify the ONL. A total of three to four regions of interest (ROIs) were imaged in the central and peripheral regions per retina. TUNEL quantification was performed in a blinded fashion using only the DAPI channel. ROIs were then identified subretinal as "graft" or "no graft" based on the presence of Ku80+ PRCs. Cell viability: 3.5% / 52.3% (cell counter). [Figure 5C]

[0218] Figure 5C is a graph showing that extracellular vesicles (EVs) isolated from PRC preserve OMR responses in RCS rats up to P90. EVs were isolated from PRC-conditioned medium and injected subretinal into RCS rats at a dose of 9.42 x 10^8 EVs / eye. Other RCS rats received subretinal injections of PRC (100,000 cells / eye) or vehicle. Injections were performed at P25. OMR analysis was performed at P60, 90, and 20 and compared with age-matched non-injected (NI) animals. [Figure 5D]

[0219] Figure 5D is a graph showing that a single subretinal injection of PRC-EV preserves ONL thickness until P90. Quantification of retinal ONL thickness from RCS rats injected with PRC-EV versus uninjected eyes. Retinal cryosections were stained with DAPI to visualize the ONL. Stained retinal sections were imaged using a Leica DMi8 epifluorescence scope. ONL thickness was determined by measuring the line graph through the ONL in the central and peripheral regions as described in Figure 4C. P90: ONL preservation correlates with OMR preservation. ONL thickness in Long-evans rats (sighted): approximately 55 μm (Weber et al., 1996). The prefix P indicates postnatal age. [Figure 5E]

[0220] Figure 5E shows a single PRC-EV injection. ONL thickness was preserved until P90. [Figure 5F]

[0221] Figure 5F shows an image of the ONL following injection without PRC-EVs. Small patches of preserved ONL with an average of 1-3 rows of nuclei. [Figure 5G]

[0222] Figure 5G shows an image of the ONL after PRC-EV injection. Compared to uninjected cells, the ONL was preserved in a longer stretch with an average of 3 to 6 rows of nuclei. [Figure 5H]

[0223] Figure 5H shows the ONL after PRC-EV injection at P120. A single PRC-EV injection does not preserve ONL thickness at P120. [Figure 5I]

[0224] Figure 5I is a graph showing the optokinetic response after PRC-EV injection. Visual function was assessed by recording the optokinetic response in rats injected with PRC-EV at P60, P90, and P120. [Figure 5J]

[0225] FIG. 5J is a graph showing that ONL analysis of PRC-EV-treated rats shows that a single injection of PRC-EV confers morphological preservation of the ONL up to P90 (but not P120). [Figure 5K]

[0226] Figure 5K is a graph showing ONL after subretinal cell transplantation of PRC. Subretinal cell transplantation of PRC preserves ONL thickness up to P120. [Figure 6A]

[0227] Figure 6A shows images of toluidine blue staining of PRC-engrafted and non-engrafted areas. ONL preservation is clearly observed, while the non-engrafted area shows severely degenerated ONL. The ONL is visualized by a thick, dark band (long arrow). Note: There is no ONL preservation in the non-injected side (short arrow). [Figure 6B]

[0228] Figure 6B is an image showing transmission electron microscopy (TEM) analysis of engrafted and non-engrafted ONL sites. There was significant preservation of ONL in engrafted areas compared to ONL sites without PRC engraftment. [Figure 6C]

[0229] Figure 6C shows an image showing TEM ultrastructural analysis. TEM demonstrates overall photoreceptor morphological preservation, as well as fine structures such as the contacting cilia of rod inner segments in the presence of subretinal PRCs. Finally, PRC engraftment minimizes the debris zone in the subretinal space. In the absence of subretinal PRCs, rod outer segment debris settles in much of the subretinal space because it does not have clearance after outer segment shedding. GB2R-PRC-2019 was injected at postnatal day P25. The black arrowhead points to the preserved contacting cilia of the inner and outer segments. Cell viability: 57.1% (Cell Counter). [Figure 7A]

[0230] Figure 7A is a schematic diagram for the administration of PRCs using the rd10 mouse model. On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye, and GS2 vehicle was injected into the left eye as a control. OMR was performed on P21, P28, P35, P42, and P49. Cell viability: 68.5% (manual). [Figure 7B]

[0231] Figure 7B is a graph showing that both eyes show the presence of tracking at P21 and P28. At P35, P42, and P49, the control eye is blind and the cell-injected eye still shows the ability to track. [Figure 7C]

[0232] Figure 7C is a graph showing morphometric analysis of photoreceptor outer nuclear layer (ONL) preservation in P28 rd10 mice. ONL preservation, quantified as ONL area (mm), can be identified at the location of PRC subretinal engraftment compared with adjacent untransplanted central and peripheral retina. Cell viability: 73.3% (manual). [Figure 7D]

[0233] Figure 7C is a graph showing cone length analysis of P28 rd10 mice. Cone arrestin-labeled cone photoreceptor length is significantly increased at the location of PRC subretinal engraftment compared to adjacent areas of the central and peripheral retina where no subretinal engraftment is present. Cell viability: 73.3% (manual). [Figure 7E]

[0234] FIG. 7E is a confocal image showing rod synapses labeled with CtBP2 / RIBEYE at the site of engraftment as well as non-engrafted central and peripheral retinal locations. [Figure 7F]

[0235] Figure 7C shows a graph quantifying rod synapses in rd10 mice at P28. The number of ribeye-positive / cone-arrestin-negative rod synapses was significantly increased at the site of PRC engraftment compared to the untransplanted central and peripheral retina, indicating PRCs associated with rod photoreceptor preservation in the rd10 mouse retina. Cell viability: 73.3% (manual). [Figure 8A]

[0236] Figure 8A is a graph showing OMR at P35, where viability was 70% or less from multiple PRC lots. PRCs with viability of 66.05%, 68.5%, and 68.8% demonstrated similar preservation efficacy. PRC-injected eyes performed better than control eyes. [Figure 8B]

[0237] Figure 8B shows the anatomical preservation of PRC cells. Anatomical preservation was observed across multiple PRC lots with viabilities of 70% or less. Histological analysis confirmed the OMR data. At P35, injected PRCs with 66.05% viability preserved pyramidal anatomical structure as assessed by ONL thickness, axon length, and morphology. At P50, injected PRCs with 68.5% viability preserved ONL thickness and density of ribeye+ synapses. At P70, injected PRCs with 68.8% viability also preserved ONL thickness and density of ribeye+ synapses. [Figure 9A]

[0238] Figure 9A shows ONL preservation in RD10 mice. On postnatal day 14 (P14), 100k cells were transplanted into the subretinal space of the right eye, and GS2 vehicle was injected into the left eye as a control. At P50, eyes were harvested for IHC staining. DAPI staining showed that only one row of photoreceptors remained in the non-grafted retina, while numerous rows of photoreceptors were observed in the grafted retina. Quantification of data from five animals, three sections from each animal, showed that the thickness and area of ​​the ONL in the grafted retina significantly exceeded those in the non-grafted retina (6µm vs. 18µm; 5000µm vs. 10000µm). Cell viability: 68.5% (manual). [Figure 9B]

[0239] Figure 9B (left panel) shows an ICC image stained for cone arrestin (Millipore AB15282 1:500), which was used to detect cone morphology. In non-grafted retinas, arrestin was expressed only in the cone cell bodies, while the outer segments and axons were degenerated. In grafted retinas, cones were preserved by maintaining normal morphology, and arrestin was expressed in the outer segments, cell bodies, axons, and stalks. Quantification of the data shows that cones had significantly longer axons in grafted retinas than in non-grafted retinas (8 μm vs. 4 μm). (right panel) Ribeye (also known as ct-BP2, BD Transduction Laboratories 612044, 1:500) is a marker for presynaptic structures located at the axon terminals of photoreceptors. This indicates synaptic connections between photoreceptors and horizontal cells. Quantification of the data shows that the expression of Ribeye puncta was higher in the grafted retinas than in the non-grafted retinas (40 vs. 20 / 100um retinas). Cell viability: 68.5% (manual). [Figure 9C]

[0240] Figure 9C is a schematic diagram for administration of PRC cells to hemizygous rats. Summary of the dosing and time course of the in vivo experiment in P23H rats. [Figure 9D]

[0241] Figure 9D is a graph of OMR preservation in P23H hemizygous rats injected with 100,000 cells / eye of GB2R-PRC-P4-FDA PRC at P25, demonstrating statistically significant preservation of the OMR response compared to uninjected controls at P60, P90, and P120, and compared to vehicle-injected controls at P90. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test (MCT). Cell viability: 71.7% (manual). [Figure 10A]

[0242] Figure 10A is a schematic diagram of the quantification process for HuNu+ cells. RCS rats aged P23–P25 were subretinal injected with 100,000 GB2R PRCs or GS2 vehicle. Animals were sacrificed on P120 for cryosectioning and histological analysis of retinal tissue. Every 10th retinal section through the graft was selected for quantification to quantify HuNu+ PRCs. Cell numbers were interpolated between each section to generate a total engraftment. Total PRC engraftment was calculated from n=4. [Figure 10B]

[0243] Figure 10B is a graph showing quantification of the number of engrafted PRCs in four transplanted animals at P120, or 3 months after transplantation. Quantification of total HuNu+ PRCs in the subretinal grafts shows an average of 80,000 transplanted cells per eye, translating to an overall 80% engraftment rate compared to the initial injection of 100,000 cells per injection. [Figure 10C]

[0244] Figure 10C shows differential PRC engraftment in retinal degeneration models with varying disease onset and severity after treatment. The specific lots and doses of PRC used in this analysis are indicated in boxes at the top of the figure. Bar graph of relative subretinal engraftment: RCS rat and P23H hemizygous rat models of retinal degeneration demonstrate comparable GB2R-PRC engraftment, with maximum engraftment found to encompass 54 and 41% of the subretinal space, respectively. The rd10 mouse and P23H homozygous rat models of retinal degeneration demonstrate significantly lower engraftment, with PRC engraftment at 19% (rd10 mice) and 2% (P23H homozygous rats) of the subretinal space, respectively. Graph of relative retinal length graft coverage: Relative ratio calculations show coverage for different mouse and rat eye sizes, and maximum length plots represent absolute engraftment lengths. rd10 mice and P23H rats show reduced engraftment in both relative and absolute terms compared to both RCS and P23H hemizygous rats. [Figure 11A]

[0245] Figure 11A shows that proliferation of subretinal PRCs is downregulated by P120. HuNu (Millipore, MAB1281) and Ki67 (Abcam ab15580) double-positive cells in subretinal grafts were quantified in retinal cross sections from animals at P35, P60 (not shown), and P120. A total of three animals were used for each time point. Confocal images show Ki67 and HuNu double-positive cells in the subretinal space at P35. However, no proliferating (Ki67+) PRCs are observed until P120. Quantification reveals a significant downregulation of Ki67 and HuNu double-positive PRCs by P120, or 3 months after transplantation (bar graph in the lower left panel). Cell viability: P35: 43.5% and 52.3%; P60: 43.5% and 52.3% (cell counter); and P120: >70% (manual). [Figure 11B]

[0246] Figure 11B shows confocal images of engrafted PRCs. The absence of Oct4 (Abcam ab27985) expression in engrafted HuNu+ PRCs indicates a lack of pluripotency at all time points studied after transplantation (P35, P60, and P120). A total of three animals were used for each time point. Cultured GMP1 iPSCs were used as a positive control for Oct4 expression with WGA-647 (Thermo Scientific W32466) and DAPI staining for localization of subcellular compartments (small image in the middle panel). [Figure 12A]

[0247] Figure 12A is a schematic diagram of the protocol for generating PRC cells. Approximately 200 M cells are present at P4-PRC(DS), with a currently scalable maximum of 500-600 M cells at P4-PRC(DS). Prioritized process changes include: i) replacing culture dishes (with T-flasks) and ii) replacing mechanical dissociation with enzymatic dissociation for 2D to 3D lift-up (anticipated for GLP Tox and first-in-human trials). RIM: Rescue Induction Medium; NDM: Neural Differentiation Medium; ICC: Immunocytochemistry; IFA: Immunofluorescence Assay. [Figure 12B] Figure 12B is a schematic diagram of the protocol for generating PRC cells, including a cryopreservation step between P3 and P4. Approximately 200 M cells are present at P4-PRC(DS), with a currently scalable maximum of 500-600 M cells based on P4-PRC(DS). The implemented process will undergo process development with: i) the replacement of culture dishes (with T-flasks), and ii) the replacement of the 2D to 3D lift-up method from mechanical dissociation to enzymatic dissociation (expected to be implemented in GLP Tox and first-in-human trials), and iii) the implementation of intermediate cell stocks of P3 spheres. RIM: rescue induction medium; NDM: neural differentiation medium; ICC: immunocytochemistry; IFA: immunofluorescence assay. [Figure 12C]

[0249] Figure 12C is a schematic diagram of the manufacturing process of the PRC. [Figure 13]

[0250] Figure 13 is a schematic diagram of the production, reconstitution and injection of PRC cells. CZ: Crystal Zenith; CRF: controlled rate refrigerator. [Figure 14A]

[0251] Figure 14A is a graph showing the post-thaw cell viability of photoreceptor rescue cells (PRC) cells prepared in a formulation containing 2.5% rHA, DPBS containing Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO or 10% DMSO), and for comparison, PRC cells formulated in the commercially available CryoStor (registered trademark) CS10 cell freezing formulation containing 10% DMSO. [Figure 14B]

[0252] Figure 14B is a graph showing the percent viability of P4 PRC cells after cryopreservation under different conditions, shown as mean ± SD. [Figure 14C]

[0253] Figure 14C is a graph showing the percent viability of P4 PRC cells after cryopreservation under different conditions selected from those in Figure 14B, shown as mean ± SD. [Figure 15A]Figure 15A is a graph showing a comparison of OMR responses in animals subretinally injected with 100,000 or 200,000 GB2R PRCs. Twelve spatial frequencies (SPs) were performed, and OMR responses (tracking intensity) were recorded in animals subretinally injected with either 100K (left panel) or 200K (center panel) PRCs. A response of 1.2 on the Y-axis is the threshold for the presence of OMR (shown by the green dashed line). Any value below 1.2 indicates that the eye is unable to track. At P35, P42, and P50, the control eye was blind, and the eyes injected with 200K PRC cells consistently showed larger, broader amplitude curves than the 100K cohort. The shifted SP threshold to the right indicates better visual acuity in the 200K eye. Survival rate: 80.6% / 76.64% (manual, 2 vials). [Figure 15B]

[0255] Figure 15B is a schematic timeline for injections and analysis in rd10 mice. [Figure 15C]

[0256] Figure 15C shows that ICC analysis was used to demonstrate that ONL thickness in 200k eyes was thicker than in 100k, confirmed by ICC analysis. Quantification of ONL thickness at transplanted versus non-transplanted sites in animals receiving 100k and 200k cells. The data are consistent with the hypothesis that higher dosing results in greater efficacy. ONL thickness at P70: greater in the 200k vs. 100k group, consistent with a greater amplitude or wider OMR curve at P70 in the 200k vs. 100k group, consistent with greater ONL preservation in the 200k vs. 100k group. The data are consistent with the hypothesis that greater retinal coverage (with higher dosing) results in greater efficacy. [Figure 16A]

[0257] Figure 16A is a graph showing OMR recorded in rd10 mice injected with 300k cryopreserved PRC preparations (Cryo-PRCs) (same as previous rd10 mice, subretinal injection at P14). Cryo-PRCs were functionally effective at P35 and P42. However, at P49, OMR was absent in both vehicle and Cryo-PRC eyes. Cryopreserved preparations containing Cryo:PRC or vehicle. Cell viability: 69.56% (manual). N=8. [Figure 16B] Figure 16B is a graph showing OMR analysis of RCS rats injected with 100,000 cells / eye of GB2R-PRC (CN2 lot); GB2R-PRC (P3 INT DS); or GB2R-PRC at P25 (Cryo: cryopreserved preparation). Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT) with test article comparing uninjected eyes and GS2 vehicle-injected eyes. [Figure 16C] Figure 16C is a graph showing ERG analysis of RCS rats injected with 100,000 cells / eye of GB2R-PRC (CN2 lot); GB2R-PRC (P3 INT DS); or GB2R-PRC at P25 (Cryo: cryopreserved preparation). Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test (MCT) with test article comparing uninjected eyes and GS2 vehicle-injected eyes. [Figure 17A] Figures 17A and 17B are images showing OCT of eyes treated with GS2 buffer and supplemented IMT. Figure 17A shows the conditions: GS2+ / freeze buffer (1:4) without IMT (left column), and GS2+ / freeze buffer (1:4) with Dex (right column). Figure 17B shows GS2+ / freeze buffer (1:4) with Dex / CsA (left column), and BSS without IMT (right column). [Figure 17B] Figures 17A and 17B are images showing OCT of eyes treated with GS2 buffer and supplemented IMT. Figure 17A shows the conditions: GS2+ / freeze buffer (1:4) without IMT (left column) and GS2+ / freeze buffer (1:4) with Dex (right column). Figure 17B shows GS2+ / freeze buffer (1:4) with Dex / CsA (left column) and BSS without IMT (right column). [Figure 18A]18A-18C are graphs showing ERG responses for 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). FIG. 18A shows the a-wave response. FIG. 18B shows the scotopic b-wave response. FIG. 18C shows the photopic b-wave response. [Figure 18B] Figures 18A-18C are graphs showing ERG responses for 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). Figure 18A shows the a-wave response. Figure 18B shows the scotopic b-wave response. Figure 18C shows the photopic b-wave response. [Figure 18C] Figures 18A-18C are graphs showing ERG responses for 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). Figure 18A shows the a-wave response. Figure 18B shows the scotopic b-wave response. Figure 18C shows the photopic b-wave response. [Figure 19]

[0262] Figure 19 shows images showing retinal morphology for 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). [Figure 20]

[0263] Figure 20 shows OCT images for 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). [Figure 21]

[0264] Figure 21 is an image showing ONL thickness for 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). [Figure 22]

[0265] Figure 22 is an image showing intraretinal migration after injection with PRCs frozen at the intermediate step of P3 and further differentiated to P4 after thawing. [Figure 23]

[0266] Figure 23 is a graph showing OMR readings comparing P4(d) and P4(i) at time points P35, P42, and P49 after injection. [Figure 24]

[0267] Figure 24 is an image showing immunohistochemistry in the inner plexiform layer (IPL) showing intraretinal migration between P4(d) and P4(i) injections. [Figure 25]

[0268] Figure 25 is an image showing immunohistochemistry of the IPL showing enhanced migration into the IPL after injection of P4(i)PRC. [Figure 26]

[0269] Figure 26 is a graph showing OMR readings for Cohort 1 dosed with high doses of P4(d) or P4(i). [Figure 27]

[0270] Figure 27 is a graph showing OMR readings for Cohort 1 dosed with high doses of P4(d) or P4(i). [Figure 28]

[0271] Figure 28 is a graph showing OMR readings of eyes injected with PRCs prepared using CellSTACK® or PDL pre-coated flasks compared to CMC controls prepared using dishes as shown in Figures 12A-12B. [Figure 29]

[0272] Figure 29 shows OCT images of the retina at D7 and D38 (days post injection) compared to PRC preparations using CellSTACK® and PDL pre-coated flasks. [Figure 30A]30A-30B are images showing immunohistochemical staining of CAR / HuNu (FIGS. 30A and 30B) comparing PRCs prepared using CellSTACK® or PDL-precoated flasks compared to CMC controls. Both FIG. 30A and FIG. 30B show larger grafts and better cone preservation with PRCs prepared using CellSTACK®. [Figure 30B] Figures 30A-30B are images showing immunohistochemical staining of CAR / HuNu (Figures 30A and 30B) comparing PRCs prepared using CellSTACK® or PDL-precoated flasks compared to CMC controls. Both Figures 30A and 30B show larger grafts and better cone preservation with PRCs prepared using CellSTACK®. [Figure 31]

[0274] Figure 31 is an image showing immunohistochemical staining for GFAP and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks compared to CMC controls. [Figure 32]

[0275] Figure 32 is an image showing immunohistochemical staining for IBA1 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks compared to CMC controls. [Figure 33]

[0276] Figure 33 is an image showing immunohistochemical staining for Ki67 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks compared to CMC controls. [Figure 34]

[0277] Figure 34 is an image showing immunohistochemical staining for OCT4 and HuNu comparing PRCs prepared using CellSTACK® or PDL pre-coated flasks compared to CMC controls. [Figure 35]

[0278] Figure 35 is a schematic diagram of the experimental design for delayed injection of PRC into RCS rats. Injections were at P25, P45, or P60 after disease onset. OMR and ERG readings were taken from P60 to P150. [Figure 36A]

[0279] Figures 36A and 36B are graphs showing OMR (Figure 36A) and ERG (Figure 36B) readings at P60, P90, P120, and P150 after each injection time point. [Figure 36B] Figures 36A and 36B are graphs showing OMR (Figure 36A) and ERG (Figure 36B) readings at P60, P90, P120, and P150 after each injection time point. [Figure 37]

[0280] Figure 37 is an image showing immunohistochemical staining STEM21 of delayed injection of PRC at P45 in RCS rats. [Figure 38A]

[0281] Figures 38A and 38B are graphs showing quantification of subrentinal PRC engraftment for injection time points P25, P45, and P60. Figure 38A shows the maximum graft length, and Figure 38B shows the maximum graft / retina length ratio. [Figure 38B] Figures 38A and 38B are graphs showing quantification of subrentinal PRC engraftment for injection time points P25, P45, and P60. Figure 38A shows the maximum graft length, and Figure 38B shows the maximum graft / retina length ratio. [Figure 39A] Figures 39A and 39B are graphs showing quantification of ONL preservation for injection time points of P25, P45, and P60. Figure 39A shows the maximum preserved ONL length, and Figure 39B shows the maximum ONL / retina length ratio. Gb2R-GMP-MCB PRC-P4 RCS injected at P25 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P45 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P60 (P150). [Figure 39B]Figures 39A and 39B are graphs showing quantification of ONL preservation for injection time points of P25, P45, and P60. Figure 39A shows the maximum preserved ONL length, and Figure 39B shows the maximum ONL / retina length ratio. Gb2R-GMP-MCB PRC-P4 RCS injected at P25 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P45 (P150); Gb2R-GMP-MCB PRC-P4 RCS injected at P60 (P150). [Figure 40A] Optokinetic responses were preserved in PRC-injected eyes. OMR response assays were performed at postnatal ages P28 (Figure 40A), P35 (Figure 40B), P42 (Figure 40C), and P49 (Figure 40D). Statistically significant differences relative to control eyes (vehicle or uninjected) were observed in the OD of Group 1, while only a trend was observed in the OD of Group 2 relative to control eyes. No statistical differences were observed between cell-injected groups. *p<0.05, **p<0.01. [Figure 40B] Optokinetic responses were preserved in PRC-injected eyes. OMR response assays were performed at postnatal ages P28 (Figure 40A), P35 (Figure 40B), P42 (Figure 40C), and P49 (Figure 40D). Statistically significant differences relative to control eyes (vehicle or uninjected) were observed at OD for Group 1, while only a trend was observed relative to control eyes at OD for Group 2. No statistical differences were observed between cell-injected groups. *p<0.05, **p<0.01. [Figure 40C] Optokinetic responses were preserved in PRC-injected eyes. OMR response assays were performed at postnatal ages P28 (Figure 40A), P35 (Figure 40B), P42 (Figure 40C), and P49 (Figure 40D). Statistically significant differences relative to control eyes (vehicle or uninjected) were observed at OD for Group 1, while only a trend was observed relative to control eyes at OD for Group 2. No statistical differences were observed between cell-injected groups. *p<0.05, **p<0.01. [Figure 40D]Optokinetic responses were preserved in PRC-injected eyes. OMR response assays were performed at postnatal ages P28 (Figure 40A), P35 (Figure 40B), P42 (Figure 40C), and P49 (Figure 40D). Statistically significant differences relative to control eyes (vehicle or uninjected) were observed at OD for Group 1, while only a trend was observed relative to control eyes at OD for Group 2. No statistical differences were observed between cell-injected groups. *p<0.05, **p<0.01. [Figure 41A]

[0285] Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 41B] Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 41C] Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 41D] Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 41E]Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 41F] Figures 41A-41F are OCT images of the retina for D0 group 1 (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); D35 group 1 (OD) ≧ 70% (Figure 41E); and D35 group 2 (OD) ≦ 60% (Figure 41F). [Figure 42A] Figure 42A is an image showing that positive HNA staining was observed in eyes injected with cells from Groups 1 and 2. Red = HNA, Blue = DAPI. Scale bar is 50 μm. Figure 42B shows Group 1 (OD) > 70% and Group 2 (OD) < 60%. [Figure 42B] Figure 42A is an image showing that positive HNA staining was observed in eyes injected with cells from groups 1 and 2. Red = HNA, blue = DAPI. Scale bar is 50 μm. Figure 42B shows group 1 (OD) > 70% and group 2 (OD) < 60%. [Figure 43A] Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≥70% viable PRC (Figure 43A), ≤60% viable PRC (Figure 43B), or vehicle (Figure 43C). Green = CAR, red = HNA. Scale bar is 50 μm. Figure 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See Figure 43E (Group 1) and Figure 43F (Group 2) for CAR staining of whole eyes. [Figure 43B]Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≧70% viable PRC (FIG. 43A), ≦60% viable PRC (FIG. 43B), or vehicle (FIG. 43C). Green=CAR, red=HNA. Scale bar is 50 μm. FIG. 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See FIG. 43E (Group 1) and FIG. 43F (Group 2) for CAR staining of whole eyes. [Figure 43C] Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≧70% viable PRC (FIG. 43A), ≦60% viable PRC (FIG. 43B), or vehicle (FIG. 43C). Green=CAR, red=HNA. Scale bar is 50 μm. FIG. 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See FIG. 43E (Group 1) and FIG. 43F (Group 2) for CAR staining of whole eyes. [Figure 43D] Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≧70% viable PRC (FIG. 43A), ≦60% viable PRC (FIG. 43B), or vehicle (FIG. 43C). Green=CAR, red=HNA. Scale bar is 50 μm. FIG. 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See FIG. 43E (Group 1) and FIG. 43F (Group 2) for CAR staining of whole eyes. [Figure 43E]Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≧70% viable PRC (FIG. 43A), ≦60% viable PRC (FIG. 43B), or vehicle (FIG. 43C). Green=CAR, red=HNA. Scale bar is 50 μm. FIG. 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See FIG. 43E (Group 1) and FIG. 43F (Group 2) for CAR staining of whole eyes. [Figure 43F] Examples of subretinal PRC grafts associated with elongated cone morphology are found in treated eyes from Groups 1 and 2. Exemplary images of cone morphology from eyes injected with ≧70% viable PRC (FIG. 43A), ≦60% viable PRC (FIG. 43B), or vehicle (FIG. 43C). Green=CAR, red=HNA. Scale bar is 50 μm. FIG. 43D shows quantification of cone length. One-way ANOVA (p<0.05). Error bars are SEM. See FIG. 43E (Group 1) and FIG. 43F (Group 2) for CAR staining of whole eyes. [Figure 44]

[0288] Figure 44 is a graph showing that subretinal PRC grafts were associated with thickened ONL at the graft site. One-way ANOVA (p=0.21). Error bars are SEM. [Figure 45]

[0289] GFAP immunoreactivity in Müller glia was substantial in all test eyes. Exemplary GFAP staining of test group eyes showing the strong vertical binding characteristic of GFAP morphology in diseased retina. Green = GFAP, red = HNA. Scale bar is 50 μm. [Figure 46]Figures 46A-46J are images showing that OCT imaging at D28 revealed surgery-related damage at the injection site. OCT images show the surgical damage and accumulation of subretinal material in injected eyes from Groups 1 (Figures 46A, 46B, and 46C), 2 (Figures 46D, 46E, and 46F), and 3 (Figures 46G, 46H, and 46I) compared with untreated Group 4 eyes (Figure 46J). The presence of subretinal material in Group 1 eyes suggested that the subretinal masses observed on OCT in cell-injected eyes (Groups 2 and 3) may contain significant numbers of host cells. In two eyes of separate female mice from Group 1, individual, medium-sized areas of retinal atrophy were observed at the center of the injection (red dashed line). [Figure 47A] 47A-47C are graphs showing that ERG recordings (D29-32) reveal no statistically significant differences between study groups. Figure 47A shows a-wave amplitude measurements via two-way analysis of variance; Figure 47B shows scotopic b-wave amplitude measurements via two-way analysis of variance; and Figure 47C shows photopic b-wave amplitude measurements via one-way analysis of variance. [Figure 47B] Figures 47A-47C are graphs showing that ERG recordings (D29-32) reveal no statistically significant differences between study groups. Figure 47A shows a-wave amplitude measurements via two-way analysis of variance; Figure 47B shows scotopic b-wave amplitude measurements via two-way analysis of variance; and Figure 47C shows photopic b-wave amplitude measurements via one-way analysis of variance. [Figure 47C] Figures 47A-47C are graphs showing that ERG recordings (D29-32) reveal no statistically significant differences between study groups. Figure 47A shows a-wave amplitude measurements via two-way analysis of variance; Figure 47B shows scotopic b-wave amplitude measurements via two-way analysis of variance; and Figure 47C shows photopic b-wave amplitude measurements via one-way analysis of variance. [Figure 48A]Figures 48A-48F are images showing HNA immunostaining and retinal layer structure at the center of the injection. Nuclear HNA staining was not detected in Group 1 (Figures 48A and 48B), Group 2 (Figures 48C and 48D), and Group 3 (Figures 48E and 48F) (HNA channel alone, HNA and DAPI overlay, bright field). However, subretinal graft-like structures (*) containing DAPI+ cells and autofluorescent debris were found in Groups 2 and 3 (Figures 48C, 48E, and 48F). Additionally, brighter autofluorescent debris (#) was observed across Groups 1-3, either subretinal or embedded in the outer retina (Figures 48B and 48D). Consistent with surgical injury, retinal layer structure was focally disrupted across the groups at the center of the injection (HNA and DAPI overlay in Figures 48A-48F). In many cases, the brightest autofluorescent structures were stained yellow / brightfield imaging (example in Figure 48B). Red = HNA, blue = DAPI. Scale bar is 100 μm. [Fig. 49A-D] Figures 49A-49H show images demonstrating that GFAP immunoreactivity in Müller glia was enriched at the injection site in injected eyes, consistent with injection-related damage. (A-H) Müller glial reactivity was assessed using GFAP immunostaining in Group 1 (Figures 49A and 49B), Group 2 (Figures 49C and 49D), Group 3 (Figures 49E and 49F), and Group 4 (Figures 49G and 49H) (GFAP channel alone; GFAP and DAPI overlay). GFAP in Figures 49C, 49E, and 49F also labeled subretinal cell clusters in the form of thin curves, as opposed to Müller glial hypertrophy (Figure 49A). Green = GFAP, Blue = DAPI. Scale bar = 100 μm. [Figure 49E-H]Figures 49A-49H show images demonstrating that GFAP immunoreactivity in Müller glia was enriched at the injection site in injected eyes, consistent with injection-related damage. (A-H) Müller glial reactivity was assessed using GFAP immunostaining in Group 1 (Figures 49A and 49B), Group 2 (Figures 49C and 49D), Group 3 (Figures 49E and 49F), and Group 4 (Figures 49G and 49H) (GFAP channel alone; GFAP and DAPI overlay). GFAP in Figures 49C, 49E, and 49F also labeled subretinal cell clusters in the form of thin curves, as opposed to Müller glial hypertrophy (Figure 49A). Green = GFAP, Blue = DAPI. Scale bar = 100 μm. [Figure 50A] 50A-50D are images showing that Iba1 immunoreactivity was elevated at the injection site in eyes from Groups 1 (FIG. 50A), 2 (FIG. 50B), and 3 (FIG. 50C) compared to untreated eyes from Group 4 (FIG. 50D), consistent with inflammation following injection-related damage. #Autofluorescent debris. Green = Iba1, Blue = DAPI. Scale bar is 100 μm. [Figure 50B] Figures 50A-50D are images showing that Iba1 immunoreactivity was elevated at the injection site in eyes from Groups 1 (Figure 50A), 2 (Figure 50B), and 3 (Figure 50C) compared to untreated eyes from Group 4 (Figure 50D), consistent with inflammation following injection-related damage. #Autofluorescent debris. Green = Iba1, Blue = DAPI. Scale bar is 100 μm. [Figure 50C] Figures 50A-50D are images showing that Iba1 immunoreactivity was elevated at the injection site in eyes from Groups 1 (Figure 50A), 2 (Figure 50B), and 3 (Figure 50C) compared to untreated eyes from Group 4 (Figure 50D), consistent with inflammation following injection-related damage. #Autofluorescent debris. Green = Iba1, Blue = DAPI. Scale bar is 100 μm. [Figure 50D] Figures 50A-50D are images showing that Iba1 immunoreactivity was elevated at the injection site in eyes from Groups 1 (Figure 50A), 2 (Figure 50B), and 3 (Figure 50C) compared to untreated eyes from Group 4 (Figure 50D), consistent with inflammation following injection-related damage. #Autofluorescent debris. Green = Iba1, Blue = DAPI. Scale bar is 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0193] Detailed Description

[0295] The present invention is directed to a photoreceptor rescue cell composition comprising a plurality of heterogeneous photoreceptor rescue cells with unique marker profiles. The heterogeneous photoreceptor rescue cell (PRC) composition of the present invention cumulatively expresses the following markers: FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA. The photoreceptor rescue cell composition of the present invention includes inhibitory neurons, excitatory neurons, precursors, astrocytes, and mixed neurons. PRCs can be phenotypically defined, for example, by intracellular or extracellular marker expression. Photoreceptor rescue cells can be further characterized by the expression or lack of expression of ocular precursor markers, neural markers, and / or rod / cone photoreceptor markers.

[0194]

[0296] These photoreceptor rescue cell compositions can be generated by in vitro differentiation from early precursors, including pluripotent stem cells, e.g., embryonic stem cells (ESCs), cells that have undergone transdifferentiation or partial reprogramming to a progenitor state, and induced pluripotent stem cells (iPSCs). The present invention provides compositions of photoreceptor rescue cells that are not accessible or reachable from primary sources and, as such, have a unique, non-native marker profile.

[0195]

[0297] Photoreceptor rescue cell compositions can be used in various in vivo and in vitro methods.For example, 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 wet and dry AMD, retinitis pigmentosa, and geographic atrophy secondary to AMD).Photoreceptor rescue cells can be used in vitro in screening assays to identify potential therapeutic or preventive treatment candidates.

[0196]

[0298] Functionally, PRCs demonstrate the ability to treat eye diseases and improve vision in subjects with retinal diseases or disorders by, for example, increasing the secretion of neuroprotective factors, preventing or slowing photoreceptor cell loss, increasing phagocytic activity (e.g., the ability to phagocytose isolated photoreceptor outer segments), inhibiting microglial activation (e.g., by increasing the expression of CNFT and / or MIF), reducing oxidative stress (e.g., by increasing the expression of CNFT), increasing the expression of anti-apoptotic factors, and / or preventing degeneration of the outer nuclear layer.

[0197] definition

[0299] As defined herein, the singular form is provided for descriptive purposes, but may also apply to plural versions of the words. The following definitions are meant to supplement the conventional definitions of terms as they would be understood by one of ordinary skill in the art.

[0198]

[0300] As used herein, the term "photoreceptor rescue cell composition" refers to a composition comprising a heterogeneous combination of photoreceptor rescue cells (PRCs). Photoreceptor rescue cell composition, as used herein, comprises heterogeneous cells, including, but not limited to, inhibitory neurons, excitatory neurons, mixed neurons, progenitors, and astrocytes. Cells in the photoreceptor rescue cell composition cumulatively express at least two, three, four, five, six, or seven of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and / or NFIA. In one embodiment, cells in the composition cumulatively express at least FOXG1 and MAP2. In another embodiment, cells in the composition cumulatively express at least each of the markers FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, and NFIA.

[0199]

[0301] Excitatory neurons in the photoreceptor rescue cell compositions of the present invention, as used herein, refer to cells that express one or more of the markers NEUROD2, NEUROD6, SLA, NELL2, and / or SATB2.

[0200]

[0302] Inhibitory neurons in the photoreceptor rescue cell compositions of the present invention, as used herein, refer to cells that express one or more of the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0201]

[0303] Replacement neurons, as used herein, refer to cells in the photoreceptor rescue cell composition that express one or more of the markers MEIS2, PBX3, GRIA2, and CACNA1C.

[0202]

[0304] As used herein, progenitor cells, when referring to the specific cell types contained in the photoreceptor rescue cell compositions of the present invention, refer to cells that express one or more of the markers VIM, MKI67, CLU, and GLI3.

[0203]

[0305] As used herein, astrocytes in the photoreceptor rescue cell compositions of the present invention express one or more of the markers GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0204]

[0306] The photoreceptor rescue cell composition may further contain photoreceptor rescue cells that express ocular progenitor markers, rod / cone photoreceptor markers, and / or neuronal markers.

[0205]

[0307] Exemplary ocular progenitor markers expressed by cells in the photoreceptor rescue cell compositions of the present invention include PAX6, LHX2, SIX3, NES, or SOX2. Some ocular progenitor markers, such as PAX6, LHX2, SOX2, and NES, are widely expressed in many neuronal progenitor cells and are also highly expressed in the PRCs of the present compositions. Thus, in one embodiment, the compositions of the present invention comprise heterogeneous photoreceptor rescue cells that cumulatively express at least PAX6, LHX2, SOX2, NES, and optionally SIX3. However, certain genes, such as RAX, SIX6, and TBX3, which are specific ocular progenitor markers in neuronal progenitor cells, have little to no expression in the PRCs of the present compositions. Thus, in one embodiment, the compositions of the present invention are substantially free of photoreceptor rescue cells, or cells that generally express RAX, SIX6, or TBX3. In one embodiment, the compositions of the invention are substantially free of photoreceptor rescue cells, or cells that generally express either RAX and / or TBX3.

[0206]

[0308] Exemplary rod / cone photoreceptor markers expressed by cells in the photoreceptor rescue cell composition 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 precursors and are also highly expressed in PRCs of the present composition. Thus, in one embodiment, the composition of the present invention comprises heterogeneous photoreceptor rescue cells that cumulatively express at least Mash1 / ASCL1 and RORB. However, certain genes, such as NRL and NR2E3, which are specific rod / cone photoreceptor markers in neuroectoderm and neuroectoderm-derived neural precursors, have little to no expression in PRCs of the present composition. Thus, in one embodiment, the compositions of the invention are substantially free of photoreceptor rescue cells, or cells generally expressing CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0207]

[0309] Exemplary neuronal markers expressed by cells in the photoreceptor rescue cell composition 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 reliably expressed in the PRCs of the composition. Therefore, in one embodiment, the composition of the present invention contains heterogeneous photoreceptor rescue cells that cumulatively express TUBB3, NFIA, DCX, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. However, certain genes, such as OTX2, which are specific neuronal markers in neuronal progenitor cells, have low expression in the PRCs of the composition. Therefore, in one embodiment, the composition of the present invention is substantially free of photoreceptor rescue cells, or cells that generally express OTX2.

[0208]

[0310] In a further embodiment, the compositions of the invention are substantially free of photoreceptor rescue cells, or cells generally expressing any of the pluripotency markers SSEA4 and / or OCT4.

[0209]

[0311] "Retinal cells" refers to the neural cells of the eye, which are layered into three granular layers composed of photoreceptors, horizontal cells, bipolar cells, amacrine cells, Müller glial cells, and ganglion cells. Retinal cells include neural retinal cells (also referred to herein as photoreceptor cells), retinal pigment epithelial (RPE) cells, iris epithelial cells, and their precursors. "Retinal pigment epithelial (RPE) cells," as used herein, refer to the cells in the outermost layer of the retina. RPE cells function to provide support for retinal photoreceptors and are responsible for metabolic digestion of discarded outer segments of the neural retina. "Neural retinal cells," as used herein, refer to the layer of photoreceptor cells (i.e., rod and cone cells) in the retina below the RPE cell layer. Neural retinal (NR) cells are modified light-sensitive neurons. As used herein, the term "cumulatively" and its grammatical equivalents refer to the expression of a marker across a heterogeneous population of cells in a composition. Specifically, cumulative expression refers to a composition in which at least one cell in the composition expresses one of the markers, such that the entirety of the cells in the composition expresses all of the listed genes. For example, the statement "multiple heterogeneous cells cumulatively express FOXG1 and MAP2" can 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 a single cell expresses FOXG1 and MAP2, such that multiple cells in the composition cumulatively express both FOXG1 and MAP2. As a further example, the statement "multiple heterogeneous cells cumulatively express FOXG1, MAP2, STMN2, and DCX" can 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, compositions in which at least one cell expresses FOXG1 and MAP2 and at least one other cell expresses STMN2 and DCX are intended to be encompassed by such phrases.As a further example, 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 is intended to be encompassed by such a term.

[0210]

[0312] The term "human neural stem cells" or "hNSCs" is used herein to refer to cells that self-renew and are generated throughout adult life via neurogenesis. These multipotent adult stem cells generate the major phenotypes of the nervous system, differentiating into neurons, astrocytes, and oligodendrocytes.

[0211]

[0313] The term "neural progenitor cell" or "NPC" is used herein to refer to the progenitor cells of the central nervous system (CNS) that give rise to many, if not all, of the glial and neuronal cell types found in the CNS.

[0212]

[0314] The term "plurality" is used herein to refer to a condition of more than one, ie, at least two, eg, cell types, eg, multiple heterogeneous photoreceptor rescue cells.

[0213]

[0315] The terms "substantially free" or "essentially free" are used herein to refer to greater than about 95%, 96%, 97%, 98%, 99%, or 100% free. By way of example, the phrase "a composition substantially free of cells expressing progenitor markers RAX, SIX6, and / or TBX3" refers to a composition in which at least 95%, 96%, 97%, 98%, 99%, or 100% of the cells do not express any of the aforementioned markers.

[0214]

[0316] The compositions of the invention may be characterized as comprising 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 being PRCs. In certain embodiments, the methods described herein comprise methods for determining whether at least about 50% to about 100%, at least 50% to about 95%, at least 50% to about 90%, 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 100%, at least 55% to about 95%, or at least about 100% of the cells in the composition are PRCs. Approximately 55% to approximately 90%, 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%, approximately 55% to approximately 60%, 60% to approximately 100%, approximately 60% to approximately 95%, approximately 60% to approximately 90%, approximately 60% to approximately 85%, approximately 60% to approximately 80%, approximately 60% to approximately 75%, approximately 60% to approximately 70%, approximately 60% to approximately 65%, 65% to approximately 1 00%, 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 100%, about 75% to about 95%, about 75% to about 90%, about 7 Compositions of the invention can be produced that can be characterized as being 5% 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% PRC.

[0215]

[0317] The compositions of the invention may be characterized as having 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 that are viable. In certain embodiments, the methods described herein provide for the production of at least about 50% to about 85%, at least about 50% to about 80%, at least about 50% to about 75%, at least about 50% to about 70%, at least about 50% to about 65%, at least about 50% to about 60%, at least about 50% to about 55%, at least about 55% to about 85%, at least about 55% to about 80%, at least about 55% to about 75%, at least about 55% to about 70%, at least about 55% to about 65%, at least about 55% to about 60%, at least about 60% to about 85 ... Compositions of the invention can be produced that can be characterized as being 0% 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 80%, about 70% to about 75%, about 75% to about 85%, about 75% to about 80%, or about 80% to about 85% viable.

[0216]

[0318] As used herein, the phrase "substantially pure photoreceptor rescue cell composition" refers to a heterogeneous photoreceptor rescue cell composition (e.g., a cell-containing composition) in which the composition contains a substantial percentage of cells, e.g., 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%, that share identical expression as regards a particular marker profile. The ability of all or a majority of PRC cells to contact their surrounding medium, and therefore the factors in such medium, to an approximately equal degree, results in these progenitor cells differentiating at similar times and to similar degrees. This similar differentiation timeline for a population of PRC cells indicates that such cells are synchronized. PRCs may also, in some instances, be cell-cycle synchronized. Such synchrony results in a subpopulation of cells that is homogeneous or nearly homogeneous as regards a particular marker expression profile.

[0217]

[0319] For example, purity can refer to the percentage of photoreceptor cells in a composition that exhibit a particular expression profile. For example, a photoreceptor rescue cell composition that is substantially pure with respect to expression of FOXG1 and MAP2 can refer to a heterogeneous photoreceptor rescue cell composition 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 in the composition express FOXG1 and MAP2. In some embodiments, the heterogeneous 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 about 50% pure to about 95% pure, about 55% pure to about 95% pure, about 60% pure to about 95% pure, about 70% pure to about 95% pure, about 75% pure to about 95% pure, about 80% pure to about 95% pure, about 85% pure to about 95% pure, about 90% pure to about 95% pure, about 50% pure to about 90% pure, about 55% pure to about 90% pure, about 60% 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, about 80 %pure to about 85% pure, about 50% pure to about 80% pure, about 55% pure to about 80% pure, about 60% pure to about 80% pure, about 65% pure to about 80% pure, about 70% pure to about 80% pure, about 75% pure to about 80% pure, about 50% pure to about 75% pure, about 55% pure to about 75% pure, about 60% pure to about 75% pure, about 65% pure to about 75% pure, about 70% pure to 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.

[0218]

[0320] As used herein, a majority of the cells means at least 50%, and depending on the embodiment, 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 the cells.

[0219]

[0321] The degree of purity that can be achieved using the methods of the present invention is particularly important when such cell populations are to be used in vivo for therapeutic or prophylactic purposes. The ability to obtain highly pure populations of cells avoids performing additional manipulations, such as enrichment or selection steps, that may result in unnecessary cell loss. This is particularly important when cell populations may be small or when cell numbers may be limited.

[0220]

[0322] For example, the level of purity can be quantified by determining the proportion of cells in a preparation that express one or more markers, such as PRC markers (including those markers identified in this application), relative to the total number of cells in the preparation, for example, by detecting cells that do or do not express the one or more markers. Optionally, the expression of markers indicative of non-PRC cells can also be detected, thereby facilitating the detection and / or quantification of the cells. Exemplary methods that can be used to detect and / or quantify marker expression include, but are not limited to, flow cytometry, fluorescence-activated cell sorting (FACS), immunohistochemistry, in situ hybridization, scRNA sequencing, immunofluorescence, single-cell proteomics (e.g., via LC-MS), possibly single-cell metabolomics, lipidomics, scPCR, and other suitable methods known in the art. Optionally, the purity of a composition can be determined by the percentage of viable cells present in the composition.

[0221]

[0323] "Embryoid bodies" refer to aggregates or clusters of pluripotent cells (e.g., iPSCs or ESCs) that can be formed by culturing pluripotent cells under non-adherent conditions, e.g., on a low-adhesion substrate or in "hanging drops." In these cultures, pluripotent cells can form aggregates or clusters of cells designated as embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb;6(2):88-95, which is hereby incorporated by reference in its entirety. Typically, embryoid bodies initially form as solid aggregates or clusters of pluripotent cells; over time, some embryoid bodies contain fluid-filled cavities; the former are referred to in the literature as "simple" EBs, and the latter as "cystic" embryoid bodies.

[0222]

[0324] The present disclosure provides heterogeneous PRC compositions based on the ability of the disclosed methods to directly differentiate progenitor cells, such as, but not limited to, pluripotent stem cells (e.g., ESCs and iPSCs). As used herein, directed differentiation refers to the differentiation of a progenitor cell population into or toward a desired lineage, due in part to factors or other stimuli provided to such progenitor cells, thereby avoiding differentiation into other undesirable, and therefore potentially contaminating, lineages. In some embodiments, the methods provided herein drive the differentiation of, for example, pluripotent stem cells into PRCs without generating embryoid bodies (EBs). EBs, as described below, are three-dimensional cell clusters that can form during the differentiation of pluripotent stem cells, including, but not limited to, embryonic stem (ES) cells and iPSCs, and typically contain cells including precursors of mesodermal, ectodermal, and endodermal lineages. The three-dimensional nature of EBs may create a different environment, involving different cell-cell interactions and different cell-cell signaling, than occurs in the non-EB-based methods described herein. In addition, cells within EBs may not receive similar doses of exogenously added agents, such as differentiation factors, present in the surrounding medium, which can lead to different differentiation events and decisions during EB development.

[0223]

[0325] In contrast, the PRC cell culture methods of the present invention do not require EB formation, and preferably avoid EB formation. Instead, these methods culture cells under conditions that provide equal contact between the cells and the surrounding medium, including the factors in such medium. In certain embodiments, PRCs can adhere to the culture surface and grow, for example, as a monolayer or near-monolayer under adherent conditions. In some embodiments, the culture methods disclosed herein provide that PRCs are cultured under non-adherent or low-adherent conditions, for example, in suspension.

[0224]

[0326] The term "embryonic stem cells" (ES cells or ESCs) is used herein as it is used in the art. This term includes cells derived from the inner cell mass of a human blastocyst or morula, including those serially passaged as cell lines. ES cells can be derived from the fertilization of an egg cell with sperm, as well as using DNA, nuclear transfer, parthenogenesis, or other means to generate ES cells homozygous for the HLA region. ES cells are also cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, generated by the fusion of sperm and egg cells, nuclear transfer, parthenogenesis, androgenesis, or chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the cell membrane to generate cells. Regardless of their source or the specific method used to generate them, embryonic stem cells can be identified based on (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least OCT4 and alkaline phosphatase, and (iii) their ability to generate teratomas when transplanted into immunodeficient animals. Embryonic stem cells that may be used in embodiments of the present invention include, but are not limited to, human embryonic stem cells ("ESCs" or "hES cells"), such as MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional 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 may be used is MA09 cells. The isolation and preparation of MA09 cells was previously described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell Lines Derived from Single Blastomeres," Nature 444:481-485. Human ES cells used in accordance with exemplary embodiments of the present invention may be derived and maintained in accordance with GMP standards.

[0225]

[0327] The term "ES cells" does not imply, and should not be inferred to imply, that the cells were generated by the destruction of an embryo. Conversely, various methods are available that can be used to generate ES cells without the destruction of an embryo, such as a human embryo. As an example, ES cells may be generated from a single blastomere derived from an embryo in a manner similar to the extraction of blastomeres for preimplantation genetic diagnosis (PGD). Examples of such cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. An exemplary human embryonic stem cell line that can be used is MA09 cells. The isolation and preparation of MA09 cells was 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 were generated without embryo destruction. As used herein, the term "pluripotent stem cells" includes, but is not limited to, tissue-derived stem cells, embryonic stem cells, embryo-derived stem cells, induced pluripotent stem cells, and stimulus-induced acquisition of pluripotency (STAP) cells, regardless of the method by which the pluripotent stem cells are derived. This term also includes pluripotent stem cells that have the functional and phenotypic characteristics of the aforementioned cells, regardless of the method used to generate such cells. Pluripotent stem cells are functionally defined as stem cells that: (a) can induce teratomas when transplanted into immunodeficient (SCID) mice; (b) can differentiate into cell types of all three germ layers (e.g., ectoderm, mesoderm, and endoderm); (c) express one or more markers of embryonic stem cells (e.g., express OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.); and (d) are capable of self-renewal. The term "pluripotency" refers to the ability of a cell to form all lineages of the body or neural cell body (ie, the embryo itself).In certain embodiments, the pluripotent stem cells express one or more markers selected from the group consisting of 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 a blastocyst-stage embryo and embryonic stem cells derived from one or more blastomeres of a cleavage-stage or morula-stage embryo (optionally without destroying the remainder of the embryo). Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPSCs) generated by reprogramming somatic cells by expressing 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, the terms "pluripotent stem cells," "PS cells," or "PSCs" include embryonic stem cells, induced pluripotent stem cells, and embryo-derived pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. For example, embryonic stem cells and induced pluripotent stem cells are types of pluripotent stem cells that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous series of developmental potentials ranging from incomplete or partial pluripotent cells that cannot give rise to a complete organism to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism. Exemplary pluripotent stem cells can be produced, for example, using methods known in the art. Exemplary pluripotent stem cells include, but are not limited to, embryonic stem cells induced from the inner cell mass of the lung at the blastocyst stage, embryonic stem cells induced from one or more blastomeres of a cleavage or morula stage embryo (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 germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF). Such 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.

[0226]

[0328] In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of OCT4 (sometimes referred to as OCT3 / 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, a combination of OCT4, SOX2, Nanog, and Lin28. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, additional 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 that have been reprogrammed using any of a variety of methods (e.g., integrative vectors, non-integrative vectors, chemical means, etc.). Pluripotent stem cells may be genetically modified or otherwise modified to increase lifespan, efficacy, homing, prevent or reduce allogeneic immune responses, or deliver desired factors via cells differentiated from such pluripotent cells (e.g., photoreceptor rescue cells, photoreceptor progenitor cells, rods, cones, etc., and other cell types described herein, e.g., in the Examples). In certain embodiments, pluripotent stem cells may be genetically modified or otherwise modified, e.g., to increase lifespan, efficacy, homing, prevent or reduce immune responses, or deliver desired factors into cells obtained from such pluripotent cells (e.g., photoreceptor rescue cells or cells present in a composition of photoreceptor rescue cells).For example, pluripotent stem cells, and thus the resulting differentiated cells, can be engineered or otherwise modified to lack or reduce expression of beta-2 microglobulin, class I 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, which are incorporated by reference in their entireties, in some embodiments, cells, e.g., pluripotent stem cells, and resulting differentiated cells, e.g., photoreceptor rescue cells or cells present in a composition of photoreceptor rescue cells, comprise a genetically engineered disruption in the beta-2 microglobulin (B2M) gene. In some embodiments, the cells further comprise a polynucleotide capable of encoding a single-chain fusion human leukocyte antigen (HLA) class I protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of an HLA-1 alpha chain. In some embodiments, the HLA-1 alpha chain is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the cells comprise a genetically engineered disruption in a human leukocyte antigen (HLA) class II-associated gene. In some embodiments, the HLA class II-associated gene is selected from regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory 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 comprises one or more polynucleotides encoding a single-chain fusion HLA class II protein or an HLA class II protein.In some embodiments, the cells further comprise one or more factors selected from the group consisting of CD200, CD24, PD-L1, HLAG or H2-M3, Cd47, FASLG or Fas1, Ccl21 or Ccl21b, Mfge8, serpin B9 or Spi6 or DUX4.

[0227]

[0329] "Induced pluripotent stem cells" (iPS cells or iPSCs) can be generated by protein transduction of reprogramming factors in somatic cells. In certain embodiments, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram the somatic cells. In other embodiments, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram the somatic cells. In other embodiments, at least four reprogramming proteins are transduced into somatic cells to successfully reprogram the somatic cells.

[0228]

[0330] Pluripotent stem cells can be derived from any species. Embryonic stem cells have been successfully derived from, for example, mice, several species of non-human primates, and humans, and embryonic stem-like cells have been produced from numerous additional species. Therefore, those skilled in the art can produce embryonic stem cells and embryo-derived stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, for example, lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.). In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0229]

[0331] 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 easily generated using donor cells from any species. Therefore, iPS cells can be generated from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild), cats (domestic and wild, e.g., lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0230]

[0332] Induced pluripotent stem cells can be generated using virtually any somatic cell at any developmental stage as a starting point. For example, cells can be derived from embryonic, placental, fetal, neonatal, juvenile, or adult donors. Exemplary somatic cells that can be used include fibroblasts, such as skin fibroblasts obtained by skin samples or biopsies, synovial cells from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. In some embodiments, the somatic cells are placental cells. Although skin and cheek cells provide a readily available and easily obtainable source of suitable cells, virtually any cell can be used. In certain embodiments, the somatic cells are not fibroblasts. In certain embodiments, the somatic cells are derived from the placenta.

[0231]

[0333] 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-fibroblastic somatic cells. Somatic cells can be reprogrammed by inducing the expression of at least one, two, three, four, or five reprogramming factors (e.g., by a viral transduction vector, an integrating vector, or a non-integrating vector, etc.) and / or by contacting at least one, two, three, four, or five reprogramming factors (e.g., using a protein transduction domain, electroporation, microinjection, cationic amphiphiles, fusion with a lipid bilayer containing a reprogramming factor, detergent permeabilization, etc.). The reprogramming factors can be selected from OCT3 / 4, SOX2, NANOG, LIN28, C-MYC, and KLF4. The expression of the reprogramming factors can be induced by contacting the somatic cells with at least one agent, such as a small organic molecule drug, that induces the expression of the reprogramming factors.

[0232]

[0334] Further exemplary pluripotent stem cells include induced pluripotent stem cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors ("reprogramming factors"). iPS cells may be obtained from cell banks. The generation of iPS cells may be an initial step in the generation of differentiated cells. iPS cells may be specifically generated using material from a particular patient or matched donor, with the goal of generating tissue-matched photoreceptor rescue cells. iPSCs may be generated from cells that are substantially non-immunogenic in the intended recipient, for example, from autologous cells or from cells that are histocompatible with the intended recipient.

[0233]

[0335] Somatic cells can also be reprogrammed using a combination approach in which reprogramming factors are expressed (e.g., using a viral vector, a plasmid, etc.) and the expression of the reprogramming factors is induced (e.g., using a small organic molecule). For example, reprogramming factors can be expressed in somatic cells by infection with a viral vector, such as a retroviral vector or a lentiviral vector. Reprogramming factors can also be expressed in somatic cells using a non-integrating vector, such as an episomal plasmid. See, for example, Yu et al., Science. 2009, May 8; 324 (5928): 797-801, which is hereby incorporated by reference in its entirety. When reprogramming factors are expressed using a non-integrating vector, the factors can be expressed in cells using 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 integrating viral vector is sufficient to reprogram somatic cells. In human cells, expression of four factors (OCT3 / 4, SOX2, NANOG, and LIN28) using integrating viral vectors is sufficient to reprogram somatic cells.

[0234]

[0336] Once the reprogramming factors are expressed in the cells, the cells can be cultured. Over time, cells with ES characteristics appear in the culture dish. The cells can be selected and subcultured, for example, based on ES morphology or based on the expression of selectable or detectable markers. The cells can be cultured to generate a culture of cells that resemble ES cells.

[0235]

[0337] To confirm the pluripotency of iPS cells, cells can be tested in one or more assays of pluripotency.For example, cells can be tested for the expression of ES cell markers; when cells are transplanted into SCID mice, cells can be evaluated for the ability to generate teratomas; cells can be evaluated for the ability to differentiate to generate the cell types of all three germ layers.Once pluripotent iPSCs are obtained, they can be used to generate the cell types disclosed herein, such as photoreceptor rescue cells.

[0236]

[0338] Stimulus-induced acquisition of pluripotency (STAP) cells are pluripotent stem cells generated by reprogramming somatic cells using sublethal stimuli, such as low pH exposure. Reprogramming does not require nuclear transfer or genetic manipulation of somatic cells. See Obokata et al., Nature, 505:676-680, 2014.

[0237]

[0339] As used herein, the term "stem cell" refers to a master cell that can indefinitely reproduce to form the specialized cells of tissues and organs. Stem cells are developmentally pluripotent or multipotent cells. Stem cells can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor ("transient") cell, which then proliferate to become the mature, fully formed cells of the tissue.

[0238]

[0340] As used herein, the term "adult stem cells" refers to stem cells (e.g., bone marrow stem cells, umbilical cord blood stem cells, and adipose stem cells) isolated from tissues or organs of animals (e.g., humans) at a stage of development beyond the embryonic stage. In one aspect, the stem cells of the present invention can be isolated at a postnatal stage. The cells are preferably isolated from mammals, e.g., humans. Adult stem cells differ from embryonic stem cells, which are defined by the inner cell mass of the blastocyst from which they originate. Adult stem cells according to the present invention can be isolated from any non-embryonic tissue, including neonatal, juvenile, adolescent, and adult patients. Generally, the stem cells of the present invention can be isolated from non-neonatal mammals, more preferably from non-neonatal humans. These adult stem cells are characterized in their undifferentiated state by expressing telomerase, not exhibiting gap junctional intercellular communication (GJIC), and not having a transformed phenotype.

[0239]

[0341] A "sign" of disease, as used herein, refers broadly to any abnormality indicative of disease that is detectable upon examination of a patient; an objective indicator of disease, as opposed to a symptom, which is a subjective indicator of disease.

[0240]

[0342] A "symptom" of disease, as used herein, refers broadly to any morbid phenomenon or deviation from the normal in structure, function, or sensation experienced by a patient and that is indicative of disease.

[0241]

[0343] "Therapeutic," "therapeutic," "treating," "treat," or "treatment," as used herein, broadly refer to treating a disease, halting or reducing the occurrence of a disease or its clinical symptoms, and / or alleviating a disease, causing regression of a disease or its clinical symptoms. Treatment includes prevention, prevention, treatment, cure, remediation, reduction, relief, and / or providing relief from a disease, signs and / or symptoms of a disease. Treatment includes the alleviation of signs and / or symptoms in a patient with ongoing signs and / or symptoms of a disease. Treatment also encompasses "prophylaxis" and "prevention." Prophylaxis includes preventing a disease from occurring after treatment of a disease in a patient, or reducing the incidence or severity of a disease in a patient. The term "reduced," for purposes of therapy, broadly refers to a clinically significant reduction in signs and / or symptoms. Treatment includes treating signs and / or symptoms of relapse or recurrence. Treatment includes, but is not limited to, reducing existing signs and / or symptoms, as well as preventing the appearance of signs and / or symptoms at any time. Treatment includes treating chronic diseases ("maintenance") and treating acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms.

[0242]

[0344] Conditions that can be treated by the present invention, and therefore by using one or more of the preparations provided herein, include, but are not limited to, macular degeneration, including age-related macular degeneration, and such macular degeneration can be in early or late stages.Other conditions that can 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, rod-cone dystrophy, Leber's congenital amaurosis, Stargardt's disease, retinal detachment, glaucoma, optic neuropathy, and trauma affecting the eye.

[0243] Cell markers:

[0345] Photoreceptor rescue cell compositions comprising a plurality of heterogeneous photoreceptor rescue cells exhibit a unique marker profile that distinguishes them from naturally occurring cells. In particular, the compositions of the present invention are characterized by the expression of FOXG1, MAP2, STMN2, DCX, LINC00461, NEUROD2, GAD1, NFIA, or a combination thereof. In some embodiments, the plurality of 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 plurality of photoreceptor rescue cells in the compositions of the present invention are characterized by the cumulative expression of FOXG1 and MAP2.

[0244]

[0346] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise inhibitory neurons and are therefore characterized by expression of DLX5, TUBB3, SCGN, ERBB4, CALB2, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of DLX5, TUBB3, SCGN, ERBB4, and CALB2.

[0245]

[0347] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise excitatory neurons and are therefore characterized by expression of NEUROD2, NEUROD6, SLA, NELL2, SATB2, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of NEUROD2, NEUROD6, SLA, NELL2, and SATB2.

[0246]

[0348] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise precursors and are therefore characterized by expression of VIM, MKI67, CLU, GLI3, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of VIM, MKI67, CLU, and GLI3.

[0247]

[0349] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise astrocytes and are therefore characterized by expression of GFAP, LUCAT1, MIR99AHG, FBXL7, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of GFAP, LUCAT1, MIR99AHG, and FBXL7.

[0248]

[0350] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention comprise replacement neurons and are therefore characterized by expression of MEIS2, PBX3, GRIA2, CACNA1C, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of MEIS2, PBX3, GRIA2, and CACNA1C.

[0249]

[0351] In some embodiments, cells in the photoreceptor rescue cell compositions of the present invention are characterized by expression of an ocular progenitor marker selected from the group consisting of PAX6, LHX2, SIX3, NES, SOX2, or a combination thereof, hi some embodiments, a plurality of photoreceptor rescue cells in the compositions of the present invention are characterized by cumulative expression of PAX6, LHX2, SIX3, NES, and SOX2.

[0250]

[0352] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention are substantially free of cells expressing ocular progenitor markers RAX, SIX6, and / or TBX3, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by little to no cumulative expression of RAX, SIX6, and TBX3.

[0251]

[0353] In some embodiments, the cells in the photoreceptor rescue cell compositions of the invention are characterized by expression of a rod / cone photoreceptor marker selected from the group consisting of ASCL1, RORB, NR2E3, NRL, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the invention are characterized by cumulative expression of ASCL1, RORB, NR2E3, and NRL.

[0252]

[0354] In some embodiments, the cells in the photoreceptor rescue cell compositions of the invention are characterized by expression of a neuronal marker selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, HES5, or a combination thereof. In some embodiments, the photoreceptor rescue cells in the compositions of the invention are characterized by cumulative expression of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5.

[0253]

[0355] In some embodiments, the cells in the photoreceptor rescue cell compositions of the invention are substantially free of cells expressing CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, GNAT2, or combinations thereof. In some embodiments, the plurality of photoreceptor rescue cells in the compositions of the invention are characterized by little to no cumulative expression of CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, and GNAT2.

[0254]

[0356] In some embodiments, the cells in the photoreceptor rescue cell compositions of the invention are substantially free of cells expressing VSX2, POU5F1, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the invention are characterized by little to no cumulative expression of VSX2 and POU5F1.

[0255]

[0357] In some embodiments, the cells in the photoreceptor rescue cell compositions of the present invention are substantially free of cells expressing OCT4, SSEA4, or a combination thereof, hi some embodiments, the photoreceptor rescue cells in the compositions of the present invention are characterized by little to no cumulative expression of OCT4 and SSEA4.

[0256]

[0358] In some embodiments, the cells in the photoreceptor rescue cell compositions of the invention are selected from the group consisting of 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, TR beta 2, MASH1, ROR beta, MAP2, ELAVL3, NFIA, DCX, LHX2, SL They are characterized by the expression of C1A2, ELAVL4, PAX6, EMX2, ASCL1, DLL1, NFIB, ENOX1, 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, which can be assessed at the protein and / or mRNA level (Fischer et al., 2014). See 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, each of which is hereby incorporated by reference in its entirety.

[0257]

[0359] In some embodiments, cells in the photoreceptor rescue cell compositions of the present invention are characterized by assessing the expression of cellular markers compared to expression in pluripotent stem cells, e.g., iPSCs or embryo-derived PSCs. In some embodiments, cells in the photoreceptor rescue cell compositions of the present invention are characterized 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, e.g., iPSCs or embryo-derived PSCs. In some embodiments, cells in the photoreceptor rescue cell compositions of the present invention are characterized by increased expression of AGT, ACBLN2, DCH7, DNA11, and EGR1 compared to ESCs or iPSCs. Markers are generally human, unless, for example, the context indicates otherwise. Cellular markers can be identified using conventional immunocytochemistry methods, conventional PCR methods, transcriptomics analysis including RNAseq, quantitative real-time PCR, flow cytometry, FACS, scRNAseq, bulk RNAseq, single cell or bulk qRT-PCR, immunocytochemistry, immunofluorescence, single cell or bulk proteomics (e.g., by LC-MS), possibly metabolomics, lipidomics, and other suitable methods known in the art.

[0258]

[0360] As used herein, the term "scRNAseq" refers to single-cell RNA sequencing. In some embodiments, scRNAseq provides data for clustering single cells in a population of cells based on the expression of gene markers. In some embodiments, scRNAseq provides data for determining the percentage of single cells in a population, such as a population of PRC cells, that express gene markers. The sequence data mapped to scRNAseq is filtered for quality control measures, reduced for visualization, and clustered using a shared nearest neighbor method using the Louvain algorithm. From these, differential expression is performed on the clusters, and the most strongly differentially expressed genes are compared between the clusters and several public data sources. Numerous human developmental biology reviews, as well as datasets for 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), are used to assign best guess cell identities to clusters based on differentially expressed genes and expression patterns during development and in the reviewed datasets.

[0259]

[0361] As used herein, the term "bulk" refers to RNA sequencing analysis of a population of cells. Bulk RNA seq provides transcripts per million (TPM), where for every 1,000,000 RNA molecules in the RNA-seq sample, the amount is obtained from the gene of interest.

[0260] Cell culture medium:

[0362] In embodiments of the present invention, cells are stored, expanded, or differentiated in various cell culture media. Rescue induction medium is utilized for the differentiation of stem cells into early neuronal progenitor cells. Rescue induction medium (RIM) may contain D-glucose, N2 supplement (e.g., 0.1-5%), B27 supplement (e.g., 0.005-0.2%), MEM non-essential amino acid solution, and optionally insulin and / or noggin, in DMEM / F12 (Invitrogen) or a similar basal medium. For example, rescue induction medium may contain at least insulin. In addition, insulin concentrations may be varied or increased to promote cell survival and / or differentiated cell yield. For example, insulin concentrations may be varied over a range, and survival and / or differentiation may be monitored to identify insulin concentrations that improve either or both of these attributes. The addition of noggin, while not believed to be necessary, has been observed to increase the expression of neural progenitor-associated transcription factors.

[0261]

[0363] The components of DMEM / F12, Neurobasal Medium, N2 Serum Supplement, and B27 Serum Supplement are provided in Tables 1-4. It should be understood that the present invention contemplates the use of these particular media and supplements, or media or supplements that comprise, consist essentially of, or consist of these components.

[0262] [Table 1-1]

[0263] [Table 1-2]

[0264] [Table 2]

[0265] [Table 3]

[0266] [Table 4]

[0267]

[0364] The methods described herein may use human factors, such as human noggin, human insulin, and the like.

[0268]

[0365] Noggin is a secreted bone morphogenetic protein (BMP) inhibitor that reportedly binds with high affinity to BMP2, BMP4, and BMP7, blocking the activity of the TGFβ family. SB431542 is a small molecule that reportedly inhibits TGFβ / activin / Nodal by blocking the phosphorylation of ACTRIB, TGFβR1, and ACTRIC receptors. SB431542 is thought to not only destabilize the activin- and Nanog-mediated pluripotency network by blocking endogenous activin and BMP signals, but also suppress BMP-induced trophoblast, mesoderm, and endoderm cell fates. It is expected that agents with one or more of the aforementioned activities, for example, when used in the context of the disclosed methods, can replace or enhance the function of one or both of Noggin and SB431542. For example, the applicants envision that the protein Noggin and / or the small molecule SB4312542 can be replaced or augmented by one or more inhibitors that affect any or all of the following three target areas: 1) preventing the binding of a ligand (e.g., bone morphogenetic protein (BMP), e.g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14) to a receptor (e.g., a bone morphogenetic protein receptor); 2) blocking the activation of a receptor (e.g., dorsomorphin); and 3) inhibiting SMAD intracellular proteins / transcription factors. Exemplary potentially suitable agents include the naturally secreted BMP inhibitors Chordin (which blocks BMP4) and Follistatin (which blocks activin), as well as analogs or mimetics thereof. Additional exemplary agents that can mimic the effects of Noggin include the use of dominant-negative receptors or blocking antibodies that sequester BMP2, BMP4, and / or BMP7. Additionally, dorsomorphin (or Compound C) has been reported to have similar effects on stem cells in terms of blocking receptor phosphorylation.Inhibition of SMAD proteins can also be achieved using 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 inhibitor SMADs (e.g., SMAD6, SMAD7, SMAD10), or RNAi against one of the receptor SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8 / 9). Another combination of factors expected to be suitable for generating neural progenitors includes a cocktail of leukemia inhibitory factor (LIF), a GSK3 inhibitor (CHIR 99021), compound E (γ-secretase inhibitor XXI), and the TGFβ inhibitor SB431542, which has previously been 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). Additional exemplary factors may include derivatives of SB431542, e.g., molecules containing one or more additional or different substituents, similar functional groups, etc., that have similar inhibitory effects on one or more SMAD proteins. Suitable factors or combinations of factors can be identified, for example, by contacting pluripotent cells with the factors and monitoring for adoption of an early neuronal precursor phenotype, e.g., characteristic gene expression (including expression of markers described herein, expression of a reporter gene coupled to an early neuronal precursor promoter, etc.) or the ability to form a cell type disclosed herein, e.g., early neuronal progenitor cells, late neuronal progenitor cells, retinal neural progenitor cells, photoreceptor precursors, rod precursors, cones, rods and / or photoreceptor rescue cells.

[0269]

[0366] Preferably, cells are treated with or cultured in a rescue induction medium before culturing 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. The neural differentiation medium can contain D-glucose, penicillin, streptomycin, GlutaMAX™, N2 supplement, B27 supplement, 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 according to the transcriptional signatures of excitatory or inhibitory neurons, and neurons or "photoreceptor rescue cells," but does not contain noggin. In certain embodiments, noggin is not required once PSCs and PSC subpopulations are no longer present in the maturation of the cell product.

[0270]

[0367] The composition of the neural differentiation medium is as follows: N2: 1% (1 ml of N2 per 100 ml), B27: 2% (2 ml of B27 per 100 ml), and Noggin: 50 ng / ml.

[0271]

[0368] Noggin is not required after the cells have all become early neuronal precursors.

[0272] Stem cells, embryonic stem cells (ESC) or adult stem cells or induced pluripotent stem cells (iPS):

[0369] The ESCs, or adult stem cells or iPS cells utilized herein can be propagated in a feeder-free system, for example, in Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) or another matrix. Additionally or alternatively, the pluripotent cells can be propagated in a matrix containing laminin (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™-511), laminin-521, laminin-213, laminin-432, laminin- The cells may be cultured on a matrix selected from the group consisting of collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The matrix may comprise, consist of, or consist essentially of Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). In some embodiments, the stem cells do not form embryoid bodies in culture, which is an improvement over the prior art. In certain embodiments, stem cells, such as ESCs or iPSCs, differentiate into photoreceptor rescue cells in the presence of noggin.

[0273] stem cells

[0370] In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult tissues, stem and progenitor cells act as a repair system for the body, replacing specialized cells, but also maintaining the normal turnover of regenerative organs, such as blood, skin, or intestinal tissue.

[0274]

[0371] Pluripotent stem cells, such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can be proliferated in vitro for long periods while retaining the potential to differentiate into all cell types of the body, including photoreceptor rescue cells. Therefore, these cells potentially provide an unlimited supply of patient-specific functional photoreceptor rescue cells for both drug development and transplantation therapy. Differentiation of pluripotent stem cells into photoreceptor rescue cells in vitro may involve the addition of different growth factors at different stages of differentiation and may require approximately 10–30 days of differentiation (see, for example, Figure 12). Pluripotent stem cells, with their unlimited proliferation capacity, offer advantages over somatic cells as a starting cell population for the generation of photoreceptor rescue cells.

[0275]

[0372] Pluripotent stem cells, such as embryonic stem (ES) cells or iPS cells, can be the starting material for the disclosed methods. In any of the embodiments herein, the pluripotent stem cells can be human pluripotent stem cells (hPSCs). Pluripotent stem cells (PSCs) can be cultured by any method known in the art, for example, with or without feeder cells. In addition, PSCs generated using any method can be used as starting material for generating photoreceptor rescue cells. For example, hES cells can be derived from a blastocyst-stage embryo that was the product of in vitro fertilization of an egg and sperm. Alternatively, hES cells can be derived from one or more blastomeres removed from an early cleavage-stage embryo, optionally without destroying the remainder of the embryo. In yet other embodiments, hES cells can be generated using nuclear transfer. In further embodiments, iPSCs can be used. Previously cryopreserved PSCs can be used as starting material. In another embodiment, PSCs that have not previously been cryopreserved can be used.

[0276]

[0373] In one aspect of the present invention, PSCs are plated on an extracellular matrix under feeder or feeder-free conditions. In certain embodiments, PSCs can be cultured on an extracellular matrix, including, but not limited to, laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the extracellular matrix is ​​laminin with or without e-cadherin. In some embodiments, the laminin can be selected from the group including laminin 521, laminin 511, or iMatrix 511. In some embodiments, the feeder cells are human feeder cells, such as human dermal fibroblasts (HDFs). In other embodiments, the feeder cells are mouse embryonic fibroblasts (MEFs).

[0277]

[0374] In certain embodiments, the medium used for culturing PSCs can be selected from any medium suitable for culturing PSCs. In some embodiments, any medium that can support PSC culture can be used. For example, those skilled in the art can select from commercially available or proprietary media.

[0278]

[0375] The pluripotency-supporting medium can be any such medium known in the art. In some embodiments, the pluripotency-supporting medium is Nutristem™. In some embodiments, the pluripotency-supporting medium is TeSR™. In some embodiments, the pluripotency-supporting medium is StemFit™. In other embodiments, the pluripotency-supporting medium is Knockout™ DMEM (Gibco), which may be supplemented with Knockout™ Serum Replacement (Gibco), LIF, bFGF, or any other factor. Each of these exemplary media is known in the art and commercially available. In further embodiments, the pluripotency-supporting medium may be supplemented with a ROCK inhibitor, bFGF, or any other factor. In certain embodiments, 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 may prime PSCs for differentiation.

[0279]

[0376] The concentration of PSCs used in the production method of the present invention is not particularly limited. For example, when a 10 cm dish is used, 1 × 10 per dish 4 ~1×10 8 cells, preferably 5 x 10 per dish 4 ~5×10 6 cells, more preferably 1 x 10 per dish 5 ~1×10 7 For example, if CellSTACK® vessels are used, 1 x 10 cells per vessel are used. 4 ~1×10 8 cells, preferably 5 x 10 per vessel 4 ~5×10 6 1.5 x 10 cells or per vessel 6 ~2.2×10 6 cells, more preferably 1 x 10 per vessel 5 ~1×10 7For example, if flasks (e.g., PDL pre-coated T175 flasks) are used, 1 x 10 cells per flask 4 ~1×10 8 cells, preferably 5 x 10 per flask 4 ~5×10 6 4.3 x 10 cells per flask 5 ~6.2×10 6 cells, more preferably 1 x 10 per flask 5 ~1×10 7 cells are used.

[0280]

[0377] In some embodiments, the hESCs are 3×10 3 and 6×10 3 cells / cm 2 In some embodiments, hESCs are preferably grown in 6-well plates (3 x 10 per vessel) using a target seeding density of 10 x 10. 4 ~6×10 4 cells, e.g., 2 x 10 per vessel 4 ~7×10 4 cells) or T75 flasks (2 x 10 cells per vessel) 5 ~5×10 5 cells, e.g., 1 x 10 per vessel 5 and 6×10 5 In some embodiments, CellSTACK® vessels or T175 flasks are used to expand the hESCs.

[0281]

[0378] In some embodiments, hESCs are seeded to initiate the differentiation process towards PRCs. In some embodiments, the target seeding density is 3,000 cells / cm. 2 This target density is approximately 1.5 x 10 per CellSTACK® vessel. 6 ~2.2×10 6 cells, and 4.3 x 10 per T175 flask 5 ~6.2×10 6 Each cell is an individual cell.

[0282]

[0379] In some embodiments, the PSCs are present at about 1,000-100,000 cells / cm. 2 In some embodiments, PSCs are plated at a cell density of about 5,000-100,000 cells / cm. 2 , about 5000~50,000 cells / cm 2 , or approximately 5,000–15,000 cells / cm 2 In other embodiments, the PSCs are plated at a cell density of about 10,000 cells / cm. 2 are sown at a density of

[0283]

[0380] In some embodiments, pluripotency-supporting medium, such as StemFit™ or other similar medium, is replaced with differentiation medium to differentiate the cells into photoreceptor rescue cells. In some embodiments, the replacement of medium from pluripotency-supporting medium to differentiation medium can occur at different time points during cell culture of PSCs and may also depend on the initial plating density of the PSCs. In some embodiments, medium replacement can occur after 2-14 days of culture of PSCs in pluripotency medium. In some embodiments, medium replacement can occur on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0284]

[0381] In some embodiments, 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, epithelial stem cells, gastrointestinal stem cells, neural stem cells, hepatic stem cells, adipose-derived mesenchymal stem cells, pancreatic progenitor cells, hair follicle stem cells, endothelial progenitor cells, and smooth muscle progenitor cells.

[0285]

[0382] In some embodiments, the stem cells used for the methods described herein are isolated from umbilical cord, placenta, amniotic fluid, chorionic villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, gastrointestinal tract, umbilical cord blood, blood vessels, skeletal muscle, skin, liver, and menstrual blood.

[0286]

[0383] Detailed procedures for isolating human stem cells from various sources are described in Current Protocols in Stem Cell Biology (2007), which is incorporated herein by reference in its entirety. Methods for isolating and culturing stem cells from various 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, each of which is incorporated herein by reference in its entirety.

[0287] somatic cells

[0384] Certain aspects of the invention may also provide methods of transdifferentiation, ie, the direct conversion of one somatic cell type into another, for example, deriving photoreceptor rescue cells from other somatic cells.

[0288]

[0385] However, human somatic cells may be in limited supply, particularly from living donors. To provide an unlimited supply of starting cells for photoreceptor rescue cell differentiation, somatic cells may be immortalized by the introduction of immortalizing genes or proteins, such as hTERT and / or other oncogenes. Cell immortalization can be reversible (e.g., using a removable expression cassette) or inducible (e.g., using an inducible promoter).

[0289]

[0386] Somatic cells in certain aspects of the invention may be primary cells (non-immortalized cells), e.g., cells freshly isolated from an animal, or may be derived from a cell line (immortalized cells). Cells may be maintained in cell culture after their isolation from a subject. In certain embodiments, cells are passaged one or more times (e.g., between 2-5, 5-10, 10-20, 20-50, 50-100, or more) before their use in the methods of the invention. In some embodiments, cells have been passaged no more than 1, 2, 5, 10, 20, or 50 times before their use in the methods of the invention.

[0290]

[0387] The somatic cells used or described herein can be native somatic cells or engineered somatic cells, i.e., genetically altered somatic cells. The somatic cells of the present invention are typically mammalian cells, such as human cells, primate cells, or mouse cells. They may 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, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs.

[0291]

[0388] Mammalian somatic cells useful in the present invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac myocytes, and other muscle cells.

[0292]

[0389] The methods described herein can be used to program one or more somatic cells, for example, a colony or population of somatic cells, into photoreceptor rescue cells. In some embodiments, the population of cells of the present invention is substantially homogeneous in that at least 90% of the cells exhibit a desired phenotype or characteristic. In some embodiments, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9, 99.95% or more of the cells exhibit a desired phenotype or characteristic. In certain embodiments of the present invention, the somatic cells have the ability to divide, i.e., the somatic cells are not post-mitotic.

[0293]

[0390] Somatic cells can be partially or fully differentiated. As described herein, both partially and fully differentiated somatic cells can be differentiated to generate photoreceptor rescue cells.

[0294] Photoreceptor rescue cells (PRCs):

[0391] PRCs can be differentiated from pluripotent stem cells (e.g., ESCs or iPSCs in neural differentiation medium in the absence of noggin). 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 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 may be grown as spheres or neurospheres (eg, on low-attachment plates or optionally in hanging drop cultures, in a low-gravity environment, aggrewell, or other suitable culture conditions).

[0295]

[0392] Schematics of an alternative PRC production process are shown in Figures 12A and 12B. In some embodiments, hESCs or pluripotent cells are expanded prior to PRC differentiation. Expansion of hESCs or pluripotent cells can be performed using a culture chamber (e.g., a culture dish, a culture flask, e.g., an iMatrix-coated T75 flask, or a culture vessel, e.g., a TC-coated CellSTACK®). In some embodiments, expansion of hESCs or pluripotent cells involves thawing hESCs or pluripotent cells on day -10, counting and seeding them onto iMatrix (laminin-511, Matrixome)-coated culture vessels, and culturing them under feeder-free conditions in StemFit™ medium (Ajinomoto Healthy Supply Co., Ltd.) supplemented with 100 ng / mL bFGF (Peprotech) and 10 μM ROCK inhibitor (Y-27632; Fujifilm Wako Pure Chemical Corporation). After 4 days of daily medium change (StemFit+bFGF without ROCK inhibitor), hESCs or pluripotent cells are harvested using cell dissociation buffer (Gibco) and re-plated using the culture conditions described above.

[0296]

[0393] After an additional 4 days of culture (day -2), hESCs were harvested as described above, percent viability and viable cell counts were obtained, and hESCs were plated at 3,000 cells / cm for PRC differentiation in culture chambers (e.g., culture dishes, culture flasks, e.g., iMatrix-coated culture T75 flasks, or culture vessels, e.g., TC-coated CellSTACK®) in StemFit+bFGF medium supplemented with ROCK inhibitor (Y-27632). 2After seeding on day 2, cultures are fed with: (1) Day 1 - StemFit + bFGF (without ROCK inhibitor); (2) Days 0-3 (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) Days 4-19 (every 2-3 days) - Neural Differentiation Medium + Noggin (NDM+: Neurobasal medium + B27 + N2 + non-essential amino acids + glucose + glutamax (Gibco) + Noggin).

[0297]

[0394] On day 19, the cultures are "lifted" to suspension culture (2D → 3D) through incubation with a combination of Liberase and Thermolysin enzymes (Roche Custom Labs) and seeded into culture chambers (e.g., culture dishes, culture flasks, e.g., ultra-low attachment T75 flasks, or culture vessels, e.g., ultra-low attachment CellSTACK®). The 3D cultures are maintained using NDM-Noggin (NDM-) for 3-4 days to allow the formation of neural spheroids ("spheres"). The spheres in suspension are then seeded (3D → 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virally inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to initiate passage 0 (P0d0) and cultured under 2D conditions for 14 days (until P0d14) using NDM- (fed every 2-3 days).

[0298]

[0395] The 2D → 3D → 2D transition is repeated three times (through P1, P2, and P3, with 3-4 days of 3D and 14 days of 2D culture) until P3d14 is reached, 90 days after differentiation. P3d14 cultures are harvested using Accutase (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 hours. P3 spheres are then cryopreserved by resuspending in Cryostor CS10 (Stemcell Technologies), freezing at -80°C, and then transferring to the vapor phase of liquid nitrogen for storage. Cryopreserved P3 spheres are referred to as cell stocks (CS). In some embodiments, cells in the composition are expanded by growing in culture under adherent and then low-attachment conditions. In some embodiments, the cycle of culturing under adherent and then low-attachment conditions 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 some embodiments, the cells in the composition are harvested after a third iteration of adhesion followed by culturing under low-adhesion conditions, a fourth iteration of adhesion followed by culturing under low-adhesion conditions, or a fifth iteration of adhesion followed by culturing under low-adhesion conditions.

[0299]

[0396] In some embodiments, the cells in the composition are harvested after the fifth iteration of adhesion followed by culturing under low-adhesion conditions. In some embodiments, the harvested cells in the composition are cryopreserved.

[0300]

[0397] In some embodiments, suspension culture to adherent culture (2D to 3D) is grown in culture dishes, culture flasks, or culture vessels. In some embodiments, the culture vessel is a Corning CellSTACK® vessel. In some embodiments, the CellSTACK® is treated with a low-adhesion or adherent coating. In one embodiment, cells are cultured in low-adhesion conditions and the CellSTACK® is coated with an ultra-low-adhesion coating. In one embodiment, cells are cultured in adherent conditions and the CellSTACK® is coated with a TC-treated coating.

[0301]

[0398] Examples of methods for "removing" cells into suspension include, but are not limited to, those shown in Table 13.

[0302] [Table 5]

[0303]

[0399] To generate the compositions of the present invention, vials of CS are thawed in a 37°C water bath, resuspended in NDM-, transferred to ultra-low attachment T75 flasks, and cultured in 3D suspension for 2-3 days, followed by replated onto poly-D-lysine / fibronectin / laminin-521-coated T75 flasks and cultured in 2D conditions in NDM- for 14 days to complete passage 4. On P4d14, cells are harvested using Accutase, resuspended in NDM-, triturated, and filtered through a 40 μm cell strainer to obtain single-cell suspensions that constitute the PRC compositions of the present invention, which can then be formulated with cryopreservatives.

[0304]

[0400] The PRC manufacturing protocol can be modified to allow for larger scale. For example, the flasks used can be varied from T75 flasks to T225 flasks or cell stacks (e.g., Corning® CellSTACK®).

[0305]

[0401] Additionally, the PRC manufacturing protocol may include an intermediate cryopreservation step at any one or more of P0-P1, P1-P2, P2-P3, and / or P3-P4. In some embodiments, the PRC manufacturing protocol may eliminate an intermediate cryopreservation step.

[0306]

[0402] 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.

[0307]

[0403] Intermediate cryopreservation steps may include cryopreservation formulations as described herein. PRC production protocols may include overnight incubation with thermolysin and liberase and Accutase at day 19, followed by a ROCK inhibitor. The harvested cells or cell clusters can be seeded into Aggrewell™ media to allow for uniformly sized spheroids to develop. On day 19, the cultures are "removed" to suspension culture (2D → 3D) through incubation with Accutase (Innovative Cell Technologies) and seeded into ultra-low attachment T75 flasks using NDM-Noggin (NDM-) medium supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako Pure Chemical Corporation). After 24 hours, the growth medium is replaced with NDM- without Y-27632, and the 3D cultures are maintained using NDM- for an additional 2–3 days to allow for the formation of neural spheroids ("spheres"). The spheres in suspension were then seeded (3D → 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virally inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to initiate passage 0 (P0d0) and cultured under 2D conditions using NDM (fed every 2–3 days) for 14 days (until P0d14). The 2D → 3D → 2D transition was repeated three times (through P1, P2, and P3, with 3–4 days of 3D and 14 days of 2D culture) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Accutase (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 h. The P3 spheres are then cryopreserved by resuspending them in a Cryostor CS10 (Stemcell Technologies), freezing them to -80°C, and then transferring them to the vapor phase of liquid nitrogen for storage. Cryopreserved P3 spheres are called cell stocks (CS).

[0308]

[0404] In some embodiments, the PRC production protocol uses Aggrewell. According to this method, on day 19, the culture is "ejected" to suspension culture (2D → 3D) through incubation with Accutase (Innovative Cell Technologies) and seeded into Aggrewell plates (Stemcell Technologies) using NDM-Noggin (NDM-) medium supplemented with Y-27632 (ROCK inhibitor, Fujifilm Wako Pure Chemical Corporation) to allow the formation of neural spheroids ("spheres"). After 24 hours, the cells are harvested from the Aggrewell plates and transferred to ultra-low attachment T75 flasks where the 3D culture is maintained for an additional 2-3 days using NDM- without Y-27632. The spheres in suspension were then seeded (3D → 2D) onto culture vessels coated with poly-D-lysine (Advanced Biomatrix), virally inactivated human fibronectin (Akron Biotech), and laminin-521 (Biolamina) to initiate passage 0 (P0d0) and cultured under 2D conditions using NDM (fed every 2–3 days) for 14 days (until P0d14). The 2D → 3D → 2D transition was repeated three times (through P1, P2, and P3, with 3–4 days of 3D and 14 days of 2D culture) until P3d14 was reached after 90 days of differentiation. P3d14 cultures were harvested using Accutase (Innovative Cell Technologies) and cultured in ultra-low attachment T75 flasks for 24 h. The P3 spheres are then cryopreserved by resuspending them in a Cryostor CS10 (Stemcell Technologies), freezing them to -80°C, and then transferring them to the vapor phase of liquid nitrogen for storage. Cryopreserved P3 spheres are called cell stocks (CS). The PRC composition can be pretreated with sucrose, for example, at D14 and P4, before being formulated with a cryoprotectant.

[0309]

[0405] Finally, the cryopreservation step of the final PRC preparation may include poloxamer 188, a non-ionic block linear copolymer and / or sucrose.

[0310] Extracellular vesicles (EVs)

[0406] The present invention also provides extracellular vesicles secreted from photoreceptor rescue cells and their use in methods of treating eye diseases in a subject. Extracellular vesicles secreted from photoreceptor rescue cells

[0311]

[0407] The present invention also provides extracellular vesicles isolated from, derived from, secreted from, or released from cells, eg, photoreceptor rescue cells of the present invention.

[0312]

[0408] As used herein, the term "extracellular vesicles" or "EVs" refers to lipid-bound vesicles secreted by cells into the extracellular space. Three major subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, contents, and function (Zaborowski MP et al. Bioscience. 2015; 65:783-797). Generally, extracellular vesicles range in diameter from 20 nm to 5000 nm and can contain various macromolecular payloads either within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The payloads can include nucleic acids, e.g., microRNAs (miRNAs), long non-coding RNAs (lncRNAs), mRNAs, DNA fragments; proteins; carbohydrates; lipids; small molecules; and / or combinations thereof. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles induced / secreted from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solution), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be induced / secreted from living or dead organs, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0313]

[0409] As used herein, the term "exosome" refers to small cell-derived vesicles containing a membrane enclosing an internal space (i.e., lumen) that are formed from the cell by direct plasma membrane budding or by fusion of a late endosome with the plasma membrane (Yanez-MoM. et al. J. Extracell. Vesicles. 2015;4:27066). Specifically, exosomes are involved in protein sorting, recycling, storage, transport, and release. Exosomes generally range in diameter from 20 to 300 nm. Exosomes 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.

[0314]

[0410] Exosomes have been found to participate in cell-cell communication, cell maintenance, and tumor progression. In addition, exosomes have been found to stimulate immune responses by acting as antigen-presenting vesicles (Bobrie A. et al., Traffic. 2011; 12: 1659-1668). In the nervous system, exosomes have been found to help promote myelination, neurite outgrowth, and neuronal survival, thus playing a role in tissue repair and regeneration (Faure J. et al., Mol. Cell. Neurosci. 2006; 31: 642-648). At the same time, exosomes in the central nervous system (CNS) have been found to contain pathogenic proteins, such as beta-amyloid peptide, superoxide dismutase, and alpha-synuclein, which may contribute to disease progression (Fevrier B. et al., Proc. Natl. Acad. Sci. USA. 2004; 101: 9683-9688). Exosomes have also been shown to be carriers for disease markers. The use of exosomes as carriers of biomarkers is ideal because these vesicles are found in body fluids, such as blood and urine, which allows for minimally invasive "liquid biopsy"-type methods for diagnosing and further monitoring patient response to treatment.

[0315]

[0411] In addition to their natural role in cell-cell interaction, exosomes can be loaded with different cargoes, such as drugs and exogenous nucleic acids or proteins, and deliver these cargoes to different cells.Cargoes can be conjugated to extracellular vesicles, embedded in extracellular vesicles, encapsulated in extracellular vesicles, or carried by extracellular vesicles in other ways, or any combination thereof.Therefore, as used herein, reference to cargoes "present" in extracellular vesicles or their lumen is understood to include any of the aforementioned means of transporting cargoes.

[0316]

[0412] The cargo may be endogenous cargo, exogenous cargo, or a combination thereof. Examples of cargoes that can be conjugated, embedded, enclosed, or otherwise carried by the extracellular vesicles described herein include, but are not limited to, nucleic acid molecules (e.g., DNA, cDNA, antisense oligonucleotides, mRNA, inhibitory 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), polypeptides (e.g., enzymes, antibodies), lipids, hormones, vitamins, minerals, small molecules, and pharmaceuticals, or any combination thereof. Importantly, exosomes are natural carriers of miRNA and other non-coding RNA, allowing them to directly deliver their contents into the cytosol through direct membrane fusion with target cells. This makes exosomes an excellent delivery system for small molecules (Lai RC et al. Biotechnol. Adv. 2013;31:543-551).

[0317]

[0413] Microvesicles are EVs formed by budding directly outward from or pinching the plasma membrane of cells. Microvesicles typically range in size from 100 nm to a maximum of 1000 nm in diameter. The pathway of microvesicle formation is not fully understood, but is thought to require cytoskeletal components such as actin and microtubules, along with molecular motors (kinesin and myosin), and the fusion machinery (SNAREs 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 cells (Zaborowski MP et al. Bioscience. 2015;65:783-797). Similarly, it has previously been demonstrated that the number of microvesicles consumed depends on the physiological state and microenvironment of the recipient cells. Like exosomes, microvesicles are involved in cell-cell communication between nearby and distant cells. The ability of these EVs to modify recipient cells has been well documented (Harding CV et al. J. Cell Biol. 2013; 200:367-371; White IJ et al. EMBO J. 2006; 25:1-12). The uniqueness of EVs is their ability to package active cargo (proteins, nucleic acids, and lipids) and deliver them to nearby or distant cells, thereby modifying the function of the recipient cells.

[0318]

[0414] Apoptotic bodies are released into the extracellular space by dying cells. They range in size from 50 nm to a maximum of 5000 nm in diameter, with most apoptotic bodies tending to be larger in size (Borges F. et al., Braz. J. Med. Biol. Res. 2013; 46: 824-830). These bodies form by the separation of the cell's plasma membrane from the cytoskeleton as a result of increased hydrostatic pressure following cell contact (Wickman G. et al., Cell Death Differ. 2012; 19: 735-742). The composition of apoptotic bodies is in stark contrast to exosomes and microvesicles. Unlike exosomes and microvesicles, apoptotic bodies contain intact organelles, chromatin, and small amounts 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).

[0319] Methods for isolating extracellular vesicles

[0415] The EVs of the present invention can be isolated, secreted, derived, or separated from culture media or other source material, e.g., photoreceptor rescue cells of the present invention, using routine methods known in the art (see, e.g., 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, Vol. 56 (2012), pp. 293-304, and the techniques described in the references cited therein), and methods described in the Examples section below. The most commonly used methods involve multiple centrifugation and ultracentrifugation steps.

[0320]

[0416] Physical properties of EVs can be used to isolate, purify, or enrich EVs, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation), or Svedberg content (e.g., sedimentation with or without external forces, etc.). Alternatively, or in addition, isolation may be based on one or more biological properties, including methods that may use surface markers (e.g., precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, nonspecific ligand binding, immunomagnetic capture of EVs using magnetic beads coated with antibodies against proteins exposed on the EV membrane, etc.).

[0321]

[0417] Methods based on the use of volume-exclusion polymers, such as PEG, have recently been described by many different groups (US Patent Application Publication No. 20130273544, US Patent Application Publication No. 20130337440). Two such products are ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). These polymers act by tethering water molecules and pushing less soluble components, such as extracellular vesicles and proteins, out of solution, allowing them to be collected by short, low-speed centrifugation.

[0322]

[0418] In some embodiments, isolation, purification, and enrichment can be performed in a general, non-selective manner (typically involving sequential centrifugation). Alternatively, isolation, purification, and enrichment can be performed in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers can be used in immunoprecipitation, FACS sorting, affinity purification, or bead-bound ligands for magnetic separation.

[0323]

[0419] In some embodiments, tangential flow filtration may be used to isolate or purify EVs.

[0324]

[0420] In some embodiments, size-exclusion chromatography can be used to isolate or purify EVs. Size-exclusion chromatography techniques are known in the art. In some embodiments, density gradient centrifugation can be used to isolate EVs. In some embodiments, EV isolation can involve ion chromatography, such as anion exchange, cation exchange, or mixed-mode chromatography. In some embodiments, EV isolation can involve desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some embodiments, EV isolation can involve 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 isolation can involve one or more centrifugation steps. Centrifugation can be performed at approximately 50,000 to 150,000 x g. Centrifugation can be performed at approximately 50,000 x g, 75,000 x g, 100,000 x g, 125,000 x g, or 150,000 x g. In another embodiment, EVs are separated from non-membranous particles by utilizing their relatively low buoyant density (Raposo et al., 1996; Escola et al., 1998; van Niel et al., 2003; Wubbolts et al., 2003). Kits for such isolation are commercially available, for example, from Qiagen, InVitrogen, and SBI. Methods for loading therapeutic agents into EVs are known in the art and include lipofection, electroporation, and any standard transfection method.

[0325] Application and Use Screening assays

[0421] The present invention provides a method for screening various agents that modulate the differentiation of retinal progenitor cells. It can also be used to discover therapeutic agents that support and / or rescue mature photoreceptors produced in culture from retinal progenitor cells. For purposes of the present invention, "agent" 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 molecules), or ribozymes. Numerous compounds, such as polymers such as polypeptides and polynucleotides, as well as synthetic organic compounds based on various core structures, can be synthesized and are also included in the term "agent." In addition, various natural sources can provide compounds for screening, such as plant or animal extracts. Although not always explicitly stated, it should be understood that agents may be used alone or in combination with other agents that have the same or different biological activity as the agent identified by the screening methods of the present invention.

[0326]

[0422] To carry out the screening method in vitro, an isolated population of cells can be obtained as described herein.If the agent is a composition other than DNA or RNA, such as a small molecule as described above, the agent can be added directly to cells or added to culture medium for addition.As will be clear to those skilled in the art, an "effective" amount must be added, which can be empirically determined.If the agent is a polynucleotide, it can be added directly by using a gene gun or electroporation.Alternatively, it can be inserted into cells using a gene delivery vehicle or other methods described above.Positive and negative controls can be assayed to confirm the claimed activity of drugs or other agents. Biocompatible supports for photoreceptor rescue cells

[0327]

[0423] The biocompatible support for cells can be a biodegradable polyester film support for photoreceptor rescue cells. The biodegradable polyester can be any biodegradable polyester suitable for use as a substrate or scaffold. The polyester must be capable of forming a thin film, preferably a microtextured film, and must be biodegradable if used for tissue or cell transplantation. Suitable biodegradable polyesters for use in the present invention include polylactic acid (PLA), polylactide, polyhydroxyalkanoates, both 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), polyesteramide (PEA), aliphatic copolyesters, such as polybutylene succinate (PBS) and polybutylene succinate / adipate (PBSA), and aromatic copolyesters. Both 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).

[0328]

[0424] In certain embodiments, the biocompatible support is a poly(p-xylylene) polymer, such as Parylene N, Parylene D, Parylene-C, Parylene AF-4, Parylene SF, Parylene HT, Parylene VT-4, and Parylene CF, most preferably Parylene-C.

[0329]

[0425] The polymeric support can typically be formed into a thin film using known techniques. The film thickness is advantageously about 1 micron to about 50 microns, preferably about 5 microns thick. The surface of the film can be smooth, or the film surface can be partially or completely microtextured. Suitable surface textures include, for example, microgrooves or micropillars. The film can be cut and shaped to form shapes suitable for implantation.

[0330]

[0426] Photoreceptor rescue cells can be seeded directly onto the film to form a biocompatible scaffold. Alternatively, the polymer film can be coated with a suitable coating material, such as poly-D-lysine, poly-L-lysine, fibronectin, laminin (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™-511), laminin-521, laminin-213, laminin-432, laminin-522, laminin-532, and / or laminin fragments), collagen I, collagen IV, vitronectin, and Matrigel™. The cells can be plated to any desired density, although a single layer of RPE cells (RPE monolayer) is preferred.

[0331]

[0427] Alternatively, photoreceptor rescue cells (PRCs) may be administered together with other cell types, including, but not limited to, other retinal cell types such as retinal ganglion cells, retinal ganglion progenitor cells, retinal pigment epithelial (RPE) cells, or RPE precursors. Photoreceptor rescue cells (PRCs) may be administered together with one or any combination of these different cell types, for example, corneal endothelial cells. The cells may be administered on a matrix or scaffold or membrane, as described above, or they may be administered as cell aggregates, or they may be administered as a dissociated cell suspension. In some embodiments, the cells may be administered on top of a monolayer of RPE cells, which may or may not themselves be located on a matrix or substrate. The administered cells may all be derived from in vitro differentiation of hES cells or iPS cells, or in some instances, they may be derived or obtained from other sources. Certain cells may be derived from the in vitro differentiation of hES cells or iPS cells, while other cells may be derived or obtained from other sources. At a minimum, photoreceptor rescue cells are derived from the in vitro differentiation of pluripotent stem cells, such as hES cells or iPS cells. Any of these various cell combinations may be administered in conjunction with other therapeutic agents, such as those described herein.

[0332]

[0428] Photoreceptor rescue cells, either alone or in combination with another cell type, e.g., retinal pigment epithelial cells, can be administered via a device, e.g., a syringe, Oxulumis Illuminated Suprachoroidal Microcatheter (Oxular), POD3 Gold (Oxular), Oxuspheres (Oxular), SCS microinjector (Clearside Biomedical), Orbit SDS (Gyroscope), via an implant, via a cell-seeded substrate or implantable membrane.

[0333] therapeutic use

[0429] The present invention also provides a method for improving photoreceptor cell activity / function in a patient in need of such treatment, comprising administering to the patient a pharmaceutical preparation comprising, derived from, or a combination thereof, a plurality of heterogeneous photoreceptor rescue cells of the present invention. As described herein, the pharmaceutical preparation can be a suspension of cells or cells formed in vitro into an implantable tissue or matrix. In many instances, the cells are administered to the subretinal or suprachoroidal space of a diseased or degenerated retina. However, because the plurality of heterogeneous photoreceptor rescue cells of the present invention also have neuroprotective effects, the cells can be administered locally but outside the retina (e.g., the vitreous), suprachoroidally, or via depot or systemic delivery to other parts of the body. For example, the photoreceptor rescue cells of the present invention can be administered via a patch or other implantable device in which a population of cells secretes neuroprotective factors. In one embodiment, such a device can be reloaded with photoreceptor cells for repeated administration to improve the longevity of the therapeutic effect.

[0334]

[0430] The pharmaceutical preparations of the present invention can be used for a wide range of diseases and disorders that result in deterioration of the visual system, including retinal degeneration-related diseases. Because such diseases and disorders can be caused by aging, it is clear that there is no recognizable injury or disease as the substantial source of the deterioration. Those skilled in the art will understand established methods for diagnosing such disease states and / or examining known signs of such injuries. In addition, there is a wealth of literature on age-related decline or deterioration in aspects of the animal visual system. The term "retinal degeneration-related disease" is intended to refer to any disease resulting from congenital or postnatal retinal degeneration or abnormality. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration (wet or dry), geographic atrophy secondary to AMD, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber's congenital amaurosis, Stargardt's disease, retinal detachment, glaucoma, optic neuropathy, and trauma.

[0335]

[0431] Additionally or alternatively, deterioration of visual system components such as the neurosensory retina may be caused by damage, such as trauma, to the visual system itself (e.g., eye), the head or brain, or more generally to the body.Certain such damage is known to be age-related damage, i.e., its likelihood or frequency increases with age.Examples of such damage include retinal tears, macular holes, epiretinal membranes, and retinal detachments, each of which can occur in animals of any age, but are more likely or occur more frequently in aged animals, including otherwise healthy aged animals.

[0336]

[0432] Deterioration of the visual system or its components can also be due to disease. Included diseases are various age-related diseases that affect the visual system. Such diseases occur with a higher likelihood and / or frequency in aging animals than in young animals. Examples of diseases that can affect and cause deterioration of the visual system, including the neurosensory retinal layer, 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, retinal inflammation, retinal edema, retinoblastoma, or retinitis pigmentosa.

[0337]

[0433] Some of the above-mentioned diseases tend to be specific to certain animals, such as companion animals, for example, dogs and / or cats.Some of the diseases are listed generically, i.e., they can be present in many types of retinitis or retinal hemorrhage; thus, some of the diseases are not attributed to one specific etiological agent, but are more descriptive of the type of disease or outcome.Many of the diseases that can cause the deterioration or degradation of one or more components of the visual system can have both primary and secondary or more distant effects on the visual system of animals.

[0338]

[0434] Advantageously, the pharmaceutical preparations of the present invention can be used to compensate for the absence or decline of photoreceptor cell function. As described in the Examples, the cells of the present invention, including photoreceptor rescue cells, can be used as cell replacement therapy in subjects who have lost all or part of their photoreceptor function. Such subjects may have vision characterized as 20 / 60 or worse, including 20 / 80 or worse, 20 / 100 or worse, 20 / 120 or worse, 20 / 140 or worse, 20 / 160 or worse, 20 / 180 or worse, or 20 / 200 or worse in humans. Thus, the present disclosure contemplates the treatment of subjects with a certain level of vision as well as subjects who do not have discernible vision.

[0339]

[0435] The photoreceptor rescue cells of the present disclosure can be characterized by their ability to restore a certain level of vision in an animal model, such as a mouse model. In some examples, a suitable animal model can be a model with visual impairment manifested as an optokinetic response that is 10% or less, 20% or less, 30% or less, 40% or less, or 50% or less than the wild-type response. Optokinetic response can be measured using an assay such as that described in the Examples. After transplantation of the photoreceptor rescue cells of the present invention, such optokinetic response is preferably increased by a statistically significant amount, as shown in the Examples.

[0340]

[0436] Thus, the present disclosure contemplates the administration of a composition comprising a plurality of heterogeneous photoreceptor rescue cells described herein for the purpose of preventing disease progression, in whole or in part, or improving the recipient's vision, or some combination thereof. The extent to which either mechanism contributes to an improved outcome will depend on the extent of the recipient's retinal degeneration.

[0341]

[0437] Preferably, the photoreceptor rescue cell compositions of the present invention are administered to subjects with residual functional photoreceptors at the time of administration to preserve / protect them. Thus, the target patient population is those with intermediate-stage disease. In certain embodiments, the subjects do not have substantial or near-total loss of photoreceptors.

[0342]

[0438] Examples of retinal dysfunctions and methods of the present invention that can be treated with the retinal stem cell populations and photoreceptor rescue cell compositions include, but are not limited to, partial or complete photoreceptor degeneration (e.g., occurring in retinitis pigmentosa, cone dystrophies, cone-rod and / or rod-cone dystrophies, and macular degeneration); retinal detachment and retinal trauma; photopathologies caused by lasers or sunlight; macular holes; macular edema; night blindness and color vision disorders; ischemic retinopathy caused by diabetes or vascular occlusion; retinopathy due to prematurity / premature birth; infectious conditions, such as CMV retinitis and toxoplasmosis; inflammatory conditions, such as uveitidies; tumors, such as retinoblastoma and ocular melanoma; and those affected by ocular neuropathies, including glaucoma, traumatic optic neuropathy, and radioactive optic neuropathy and retinopathies, due to replacement of inner retinal neurons.

[0343]

[0439] In one embodiment, the photoreceptor rescue cells can treat or alleviate the symptoms of retinitis pigmentosa in a patient in need of treatment. In another embodiment, the cells can treat or alleviate the symptoms of macular degeneration in a patient in need of treatment, such as age-related macular degeneration (wet or dry), Stargardt's disease, myopic macular degeneration, etc. For all of these treatments, the cells can be autologous or allogeneic to the patient. In a further embodiment, the cells of the present invention can be administered in combination with other treatments.

[0344]

[0440] Retinitis pigmentosa (RP) refers to a heterogeneous group of genetic disorders of the eye characterized by progressive blindness due to gradual degeneration of photoreceptors. An estimated 100,000 people in the United States have RP. Classification of this group of disorders under a single rubric is based on the clinical features most commonly observed in these patients. RP is characterized by night blindness and reduced peripheral vision, narrowing of retinal blood vessels, and migration of pigment from the destroyed retinal pigment epithelium into the retina, often forming clumps of various sizes adjacent to retinal blood vessels.

[0345]

[0441] Typically, patients first notice difficulty seeing at night due to loss of rod photoreceptors; then, the remaining cone photoreceptors become the mainstay of visual function. However, over years and decades, the cones also degenerate, resulting in a progressive loss of vision. In many patients with RP, visual field defects begin in the mid-periphery, between 30° and 50° from fixation. The area of ​​loss gradually expands, leaving islands of vision in the periphery and narrowed central vision (so-called tunnel vision). Patients become legally blind when the visual field shrinks to below 200 and / or central vision is 20 / 200 or worse.

[0346]

[0442] Inheritance patterns indicate that RP can be transmitted in an X-linked (XLRP), autosomal dominant (ADRP), or recessive (ARRP) manner. Of the three genotypes of RP, ADRP is the mildest. These patients often retain good central vision beyond the age of 60. In contrast, patients with the XLRP form of the disease usually become legally blind by the age of 30–40. However, the severity and age of onset of symptoms vary greatly among patients with the same genotype of RP. This variation is evident even within the same family, perhaps when all affected members share the same genetic mutation. Many RP-causing mutations have now been described. Of the genes identified to date, many encode photoreceptor-specific proteins, and several are involved in rod phototransduction, such as rhodopsin, a subunit of cGMP phosphodiesterase, and cGMP-gated Ca2+ channels. Numerous mutations in each of the cloned genes have been found. For example, 90 different mutations in the rhodopsin gene have been identified in ADRP patients.

[0347]

[0443] Regardless of the specific mutation, the most significant blindness in RP patients is due to the mild degeneration of cones. In many cases, the proteins affected by RP-causing mutations are not expressed in cones; a prime example is rhodopsin, a rod-specific visual pigment. Thus, cone loss may be an indirect consequence of rod-specific mutations. The ability to replace damaged photoreceptors offers an approach to treating this disease.

[0348]

[0444] In certain embodiments, the subject is diagnosed with RP, for example, by genotyping. Specifically, the subject is diagnosed with RP based on the identification of a mutation affecting the RPE gene or photoreceptor before treatment. In a specific embodiment, the patient is visually impaired, but is not yet completely blind or without light perception (NLP). Preferably, the subject suitable for treatment has a best-corrected visual acuity (BCVA) ranging from 20 / 50 (visually impaired) to 20 / 200 (legally blind, not NLP). In other embodiments, the subject suitable for treatment has a visual acuity worse than 20 / 200, but remains without light perception.

[0349]

[0445] Age-related macular degeneration (AMD) causes progressive loss of central vision and is the most common cause of blindness in people over the age of 55. The underlying pathology is photoreceptor degeneration. Various studies have implicated genetic factors, cardiovascular disease, environmental factors such as smoking and light exposure, and nutritional causes as contributing to the risk of developing AMD. RPE degeneration is accompanied by a variable loss of both the overlying photoreceptors and the underlying choroidal perfusion. Vision loss or visual field loss occurs when the RPE atrophies, resulting in a secondary loss of the overlying photoreceptor cells that supply it. The ability to replace the RPE and / or photoreceptor cells provides a means to treat established AMD.

[0350]

[0446] AMD is diagnosed by fundus examination, and early, intermediate, or late AMD is determined based on the anatomical characteristics of the disease stage observed on fundus images. Intermediate AMD is defined by the presence of at least one large drusen (>125 μm) and / or pigmentary abnormalities. AMD pigmentary abnormalities are defined as hyperpigmentation or hypopigmentation within two disc diameters of the center of the macula in eyes with drusen ≥63 μm in diameter, without any known underlying retinal disease or other cause of such abnormalities. (See Garcia-Layana A, Cabrera-Lopez F, Garcia-Arumi 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 encompasses a wide range of clinical manifestations of intermediate AMD, including only one large drusen (>125 μm) or many large drusen and abundant pigmentary abnormalities, as long as the criteria for geographic atrophy or neovascular AMD, two versions of late AMD, are not met.

[0351]

[0447] Macular degeneration is broadly classified into two types. In the exudative neovascular form, or "wet" AMD, which accounts for 10% of all cases, abnormal blood vessel growth occurs under the macula. There is the formation of a subretinal network of choroidal neovascularization, often associated with intraretinal hemorrhage, subretinal fluid, pigment epithelial detachment, and hyperpigmentation. Eventually, this complex contracts, leaving a distinct, raised scar at the posterior pole. These blood vessels leak fluid and blood into the retina, causing damage to photoreceptors. Wet AMD tends to progress rapidly and can cause severe damage; rapid loss of central vision can occur in just a few months.

[0352]

[0448] The remaining 90% of AMD cases are atrophic macular degeneration (dry type), which involves pigmentary damage in the macula but without elevated macular scars and hemorrhage or exudation in the macular region. In these patients, the retinal pigment epithelium (RPE) gradually disappears, resulting in focal areas of atrophy. Because loss of photoreceptors leads to loss of the RPE, the affected retinal areas have little or no visual function. Blindness from dry AMD occurs more gradually over many years. These patients usually retain some central vision, but the loss can be severe enough to impair the performance of tasks requiring detailed vision.

[0353]

[0449] When age-matched and clinical findings are present with loss of vision, visual field, or other visual functions, the condition is often classified as AMD. Sometimes, even before the onset of vision loss, the condition is classified as AMD if the patient has characteristic drusen and a relevant family history.

[0354]

[0450] Occasionally, macular degeneration occurs at a fairly young age. Many of these cases result from genetic mutations. There are many forms of hereditary macular degeneration, each with its own clinical manifestations and genetic causes. The most common form of early-onset macular degeneration, known as Stargardt disease, is inherited as an autosomal recessive disorder. Patients are usually diagnosed before the age of 20. Although the progression of blindness varies, many of these patients are legally blind by the age of 50. Mutations that cause Stargardt disease have been identified in the ABCR gene, which encodes a protein that transports retinoids across photoreceptor membranes.

[0355]

[0451] The photoreceptor rescue cell compositions of the present invention find use in the treatment of degenerative diseases. The cells are administered in a manner that allows the cells to be transplanted or transferred to a desired retinal location, such as the outer nucleated layer, to reconstitute or regenerate functionally deficient areas.

[0356]

[0452] The examples demonstrate the ability of the photoreceptor rescue cells disclosed herein to restore vision in a mouse model of blindness due to photoreceptor degeneration, where vision can be assessed using the optokinetic response (or optokinetic nystagmus response).

[0357]

[0453] In another aspect, the present disclosure provides a method of drug delivery comprising administering to said patient a photoreceptor rescue cell composition described herein or produced by any method described herein, wherein said photoreceptor rescue cell composition delivers said drug. A wide range of drugs can be used. The engineered photoreceptor rescue cell compositions may be prepared to contain one or more compounds selected from the group consisting of drugs acting on synapses and neuroeffector junctions; drugs acting on the central nervous system; drugs that modulate the inflammatory response, e.g., anti-inflammatory agents including nonsteroidal anti-inflammatory agents; drugs that affect renal and / or cardiovascular function; drugs that affect gastrointestinal function; antibiotics; antiviral, anti-neoplastic and anti-cancer agents; immunomodulatory agents; anesthetics, steroids, antigens, vaccines, antibodies, decongestants, antihypertensives, sedatives, birth control agents, progestational agents, anticholinergic agents, analgesics, antidepressants, antipsychotics, beta-adrenergic blockers, diuretics, cardiovascular active agents, vasoactive agents, nutritional supplements, drugs acting on the blood and / or hematopoietic organs; hormones; hormone antagonists; drugs that affect calcification and bone turnover, vitamins, gene therapy agents; or other agents such as targeting agents.

[0358]

[0454] For example, the photoreceptor rescue cell composition may be used in combination with drugs that act at synapses and neuroeffector junctions (e.g., acetylcholine, methacholine, pilocarpine, atropine, scopolamine, physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine, dobutamine, isoproterenol, albuterol, propranolol, serotonin); drugs that act in the central nervous system (e.g., clonazepam, diazepam, lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine, amitriptyline); drugs that modulate the inflammatory response (e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, cromolyn sodium, theophylline); drugs that affect renal and / or cardiovascular function (e.g., furosemide, thiazide, amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine, verapamil, thiazide ... mil, digoxin, isordil, dobutamine, lidocaine, quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, mevalonate); drugs that affect gastrointestinal function (e.g., omeprazole, sucralfate); antibiotics (e.g., tetracycline, clindamycin, amphotericin 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, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, decarbazine);Immunomodulators (e.g., interleukins, interferons, GM-CSF, TNFα, TNFβ, cyclosporine, FK506, azathioprine, steroids); drugs acting on the blood and / or blood-forming 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, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgen, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodothyronine, thiazolinone ... dothyronine); hormone antagonists; drugs affecting mineralization and bone turnover (e.g., calcium, phosphate, parathyroid hormone (PTH), vitamin D, biphosphonates, calcitonin, fluoride), vitamins (e.g., riboflavin, nicotinic acid, pyridoxine, pantothenic acid, biotin, choline, inositol, carnitine, vitamin C, vitamin A, vitamin E, vitamin K), gene therapy agents (e.g., viral vectors, nucleic acid-carrying liposomes, 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 contain one or more therapeutic agents that are released or secreted by these cells in either a passive manner (diffuses from the cells over time) or an active manner (upon intentional destruction or lysis of the cells). hESCs and / or hiPSCs can be genetically modified and used to generate photoreceptor rescue cells (PRCs) that express desired drugs for the treatment of disease. In one embodiment, hESCs, hiPSCs, and / or multilymphoid progenitors (MLPs) can be genetically modified to express anti-ulcer agents. Photoreceptor rescue cells (PRCs), adult stem cells, and multilymphoid progenitors (MLPs) generated from such genetically modified hESCs and hiPSCs can be used to deliver such anti-tumor agents to tumors for the treatment of neoplastic diseases, including, for example, retinoblastoma.

[0359]

[0455] In some embodiments, the photoreceptor rescue cells of the invention are generated by increasing the expression of one or more tra...

Claims

1. A photoreceptor rescue cell composition comprising a plurality of heterogeneous photoreceptor rescue cells, wherein the plurality of heterogeneous photoreceptor rescue cells cumulatively express (i) the markers FOXG1 and MAP2, and (ii) at least one marker selected from the group consisting of STMN2, LINC00461, NEUROD2, and NFIA, and (iii) optionally express DCX and / or GAD1, and further comprising a culture medium suitable for maintaining cell viability.

2. (i) Cells are produced by in vitro differentiation of pluripotent cells, wherein the pluripotent cells are embryonic cells (ESCs) or induced pluripotent stem cells (iPSCs), (ii) At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition are photoreceptor rescue cells. (iii) Multiple heterogeneous cells cumulatively express at least two additional markers selected from the group consisting of STMN2, LINC00461, NEUROD2, and NFIA, and / or (iv) Multiple heterogeneous cells cumulatively express at least three additional markers selected from the group consisting of STMN2, LINC00461, NEUROD2, and NFIA. The photoreceptor rescue cell composition according to claim 1.

3. Multiple heterogeneous photoreceptor rescue cells comprise one or more cell types selected from the group consisting of inhibitory neurons, excitatory neurons, precursors, astrocytes, and alternative neurons. In some cases, multiple heterogeneous photoreceptor rescue cells include inhibitory neurons, excitatory neurons, precursors, astrocytes, and alternative neurons, respectively. The photoreceptor rescue cell composition according to claim 1 or 2.

4. (i) A plurality of heterogeneous photoreceptor rescue cells include inhibitory neurons expressing one or more markers selected from the group consisting of DLX5, TUBB3, SCGN, ERBB4, and CALB2, (ii) Multiple heterogeneous photoreceptor rescue cells include multiple inhibitory neurons that cumulatively express the markers DLX5, TUBB3, SCGN, ERBB4, and CALB2, respectively. (iii) Multiple heterogeneous photoreceptor rescue cells include excitatory neurons expressing one or more markers selected from the group consisting of NEUROD2, NEUROD6, SLA, NELL2, and SATB2, (iv) Multiple heterogeneous photoreceptor rescue cells contain multiple excitatory neurons that cumulatively express the markers NEUROD2, NEUROD6, SLA, NELL2, and SATB2, respectively. (v) Multiple heterogeneous photoreceptor rescue cells comprising precursors expressing one or more markers selected from the group consisting of VIM, MKI67, CLU, and GLI3, (vi) Multiple heterogeneous photoreceptor rescue cells comprising multiple precursors that cumulatively express the markers VIM, MKI67, CLU, and GLI3, (vii) Multiple heterogeneous photoreceptor rescue cells include astrocytes expressing one or more markers selected from the group consisting of GFAP, LUCAT1, MIR99AHG, and FBXL7. (viiii) Multiple heterogeneous photoreceptor rescue cells comprising multiple astrocytes that cumulatively express the markers GFAP, LUCAT1, MIR99AHG, and FBXL7, respectively (ix) Multiple heterogeneous photoreceptor rescue cells comprising alternative neurons expressing one or more markers selected from the group consisting of MEIS2, PBX3, GRIA2, and CACNA1C, and / or (x) Multiple heterogeneous photoreceptor rescue cells comprising multiple surrogate neurons that cumulatively express the markers MEIS2, PBX3, GRIA2, and CACNA1C, respectively. The photoreceptor rescue cell composition according to claim 3.

5. The cells in the composition further express one or more eye region precursor markers, rod / cone photoreceptor markers, and / or neuron markers. Depending on the circumstances, (i) The precursor marker for the ophthalmological field is selected from the group consisting of PAX6, LHX2, SIX3, NES, and SOX2, and / or, a plurality of heterogeneous cells cumulatively express at least one, two, three, or four of the precursor markers for the ophthalmological field PAX6, LHX2, SIX3, NES, or SOX2, and / or, a plurality of heterogeneous cells cumulatively express at least each of the precursor markers for the ophthalmological field PAX6, LHX2, SIX3, NES, and SOX2, and / or, a plurality of heterogeneous cells cumulatively express SOX2, and / or, the composition substantially does not contain cells expressing the precursor markers for the ophthalmological field RAX, SIX6, and / or TBX3. Furthermore / or, (ii) The rod / cone photoreceptor marker is selected from the group consisting of ASCL1, RORB, NR2E3, and NRL, and optionally, multiple heterogeneous cells cumulatively express each of the rod / cone photoreceptor markers ASCL1, RORB, NR2E3, and NRL, and / or the composition is substantially free of cells expressing the rod / cone photoreceptor markers CRX, RHO, OPN1SW, PDE6B, RCVRN, ARR3, CNGB1, GNAT1, and GNAT2. Furthermore / or, (iii) Neuronal markers are selected from the group consisting of TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5, and in some cases, multiple heterogeneous cells cumulatively express each of the neuronal markers TUBB3, NFIA, NFIB, OTX2, ELAVL3, ELAVL4, SLC1A2, SLC1A3, HCN1, and HES5. The photoreceptor rescue cell composition according to claim 1 or 2.

6. (i) The composition substantially does not contain at least one cell type selected from the group consisting of pluripotent stem cells, retinal ganglion cells, photoreceptors and amacrine cells. (ii) The composition is substantially free of pluripotent stem cells, retinal ganglion cells, photoreceptors and amacrine cells. (iii) The composition is substantially free of retinal precursors expressing VSX2 and / or POU5F1. (iv) The cells in the composition have phagocytic activity, and optionally have the ability to phagocytose isolated photoreceptor outer segments, pigment conjugates, or both. (v) Cells in the composition secrete one or more neuroprotective factors, wherein the neuroprotective factor is selected from the group consisting of CNTF, MIF, S100B, GFAP, TAU, NCAM1, and TNC. (vi) 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, and / or (vii) 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. The photoreceptor rescue cell composition according to claim 1.

7. A pharmaceutical preparation comprising the photoreceptor rescue cell composition according to claim 1 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is optionally suitable for ocular delivery.

8. a) The composition according to claim 1 or 2, or the pharmaceutical preparation according to claim 7; and b) Approximately 4–10% (v / v) of cryoprotective agent, approximately 2–3% (w / v) of albumin, approximately 0–1.5% (w / v) of glucose, and buffer solution. A formulation containing, The preparation, which may be stored by freezing in some cases.

9. a) The composition according to claim 1 or 2, or the pharmaceutical preparation according to claim 7; and b) (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) Osmotic agents that maintain the solution at a physiological volume molar osmotic pressure concentration. Solution containing A formulation containing, Here, the solution, (a) It can 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 precipitation of the solute and / or measurable loss of the solution's ability to support the viability and viability of the cells stored in the solution, and / or (b) Can 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 precipitation of solutes and / or measurable loss of the solution's ability to support the viability and viability of cells stored in the solution. Furthermore / or, The aforementioned solution (a) 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) Essentially pyrogen-free, and / or (c) sterile, and / or (d) For irrigation, cell reconstitution, cell storage, cell transport, and / or administration to a subject, The aforementioned formulation.

10. a) The composition according to claim 1 or 2, or the pharmaceutical preparation according to claim 7; and b) (1) A buffer that maintains a solution at a physiological pH, but is not a dicarbonate buffer; and (2) glucose; and (3) Osmotic agents that maintain the solution at a physiological volume molar osmotic concentration; and (4) Sources of divalent cations Solution containing A formulation containing, Optionally, the divalent cation source includes a calcium source and / or a magnesium source, and / or the buffer includes an acetate buffer and / or a citrate buffer. The aforementioned formulation.

11. A method for producing the composition described in claim 1, 1) The step of culturing pluripotent stem cells in rescue induction medium (RIM) and noggin; 2) The step of culturing the cells from step 1 in neuronal differentiation medium (NDM) and noggin; 3) A step of expanding and growing the cells from step 2 in nogging-free NDM, comprising: a) culturing the cells in nogging-free NDM under low-adhesion or non-adhesion conditions; and b) culturing the cells in nogging-free NDM under non-adhesion conditions, thereby differentiating the pluripotent stem cells into photoreceptor rescue cells. A method that includes this.

12. (i) The pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), (ii) 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. (iii) Cells in the composition are collected after the third iteration of step 3, the fourth iteration of step 3, or the fifth iteration of step 3, and optionally the collected cells in the composition are cryopreserved. (iv) The composition is cryopreserved during the first and second iterations of step 3, during the second and third iterations of step 3, during the third and fourth iterations of step 3, or during the fourth and fifth iterations of step 3. (v) 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 after cryopreservation and thawing. (vi) The composition includes cell spheres, (vii) Between steps 1, 2 and / or 3, the cells of the composition are cultured in a cell culture vessel selected from the group consisting of culture dishes, culture flasks and culture chambers, wherein the culture chamber is a stackable rectangular chamber, and / or the cell culture vessel is coated for low-adhesion or non-adhesion cell culture for step 3a and / or adherent cell culture for step 3b, and / or (viiii) The cells are enzymatically dissociated from the plate into a cell suspension, and in some cases the enzymes used to dissociate the cells are thermolysin, liberase, and / or accutase, and in some cases the dissociation of cells from the plate does not involve manual scraping. The method according to claim 11.

13. A photoreceptor rescue cell composition produced by the method described in claim 11.

14. A photoreceptor rescue cell composition according to claim 6 or 13, or a pharmaceutical preparation according to claim 7, for use in a method of treating an eye disease or disorder in a subject.

15. The formulation according to claim 8, for use in a method of treating an eye disease or disorder in a subject.

16. A photoreceptor rescue cell composition according to claim 6 or 13, or a pharmaceutical preparation according to claim 7, for use in a method for increasing the secretion of neuroprotective factors in the eye of a subject having an eye disease or disorder, 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. The photoreceptor rescue cell composition or the pharmaceutical preparation.

17. A formulation according to claim 8, for use in a method for increasing the secretion of neuroprotective factors in the eye of a subject having an eye disease or disorder, 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. The aforementioned formulation.

18. A photoreceptor rescue cell composition according to claim 6 or 13, or a pharmaceutical preparation according to claim 7, for use in a method for improving visual acuity in subjects with retinal disease or impairment, Depending on the circumstances, the method may increase 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. In some cases, visual acuity is measured by optokinetic response (OMR) and / or electroretinography (ERG). The photoreceptor rescue cell composition or the pharmaceutical preparation.

19. A formulation according to claim 8 for use in a method for improving visual acuity in a subject having a retinal disease or disorder, Depending on the circumstances, the method may increase 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. In some cases, visual acuity is measured by optokinetic response (OMR) and / or electroretinography (ERG). The aforementioned formulation.

20. The treated eye has an increased spatial frequency threshold, as measured by OMR, of 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, and / or The treated eye has an increased dark adaptation b-wave amplitude, as measured by ERG, of 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 dark adaptation b-wave amplitude before administration. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 18.

21. The treated eye has an increased spatial frequency threshold, as measured by OMR, of 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, and / or The treated eye has an increased dark adaptation b-wave amplitude, as measured by ERG, of 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 dark adaptation b-wave amplitude before administration. The formulation according to claim 19.

22. (i) For use in a method for preventing or slowing the loss of photoreceptor cells in a subject having a retinal disease or disorder, wherein the prevention of photoreceptor cell loss is measured by the expression of CNFT, and 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 before administration, for use (ii) For use in a method for increasing phagocytic activity in the eye of a subject having a retinal disease or disorder, 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, for use (iii) For use in a method for inhibiting microglial activation in the eye of a subject having retinal disease or impairment, wherein the inhibition of microglial activation is measured by the expression of CNFT and / or MIF, 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, and 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, for use in the above. (vi) For use in a method for reducing oxidative stress in the eye of a subject having retinal disease or impairment, wherein the reduction in oxidative stress is measured by the expression of CNFT, for the use thereof (v) For use in a method for increasing the expression of anti-apoptotic factors in the eyes of subjects with retinal disease or impairment, wherein the method optionally increases the expression of anti-apoptotic 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 expression of anti-apoptotic factors before administration, wherein the anti-apoptotic factor is S100B or (vi) For use in a method for preventing degeneration of the outer granular layer (ONL) in the eye of a subject with retinal disease or impairment, A photoreceptor rescue cell composition according to claim 6 or 13, or a pharmaceutical preparation according to claim 7.

23. (i) For use in a method for preventing or slowing the loss of photoreceptor cells in a subject having a retinal disease or disorder, wherein the prevention of photoreceptor cell loss is measured by the expression of CNFT, and 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 before administration, for use (ii) For use in a method for increasing phagocytic activity in the eye of a subject having a retinal disease or disorder, 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, for use (iii) For use in a method for inhibiting microglial activation in the eye of a subject having retinal disease or impairment, wherein the inhibition of microglial activation is measured by the expression of CNFT and / or MIF, 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, and 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, for use in the above. (vi) For use in a method for reducing oxidative stress in the eye of a subject having retinal disease or impairment, wherein the reduction in oxidative stress is measured by the expression of CNFT, for the use thereof (v) For use in a method for increasing the expression of anti-apoptotic factors in the eyes of subjects with retinal disease or impairment, wherein the method optionally increases the expression of anti-apoptotic 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 expression of anti-apoptotic factors before administration, wherein the anti-apoptotic factor is S100B or (vi) For use in a method for preventing degeneration of the outer granular layer (ONL) in the eye of a subject with retinal disease or impairment, The formulation according to claim 8.

24. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 14.

25. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The formulation according to claim 15.

26. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 16.

27. ​​(i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The formulation according to claim 17.

28. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 18.

29. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The formulation according to claim 19.

30. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 20.

31. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The formulation according to claim 21.

32. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The photoreceptor rescue cell composition or pharmaceutical preparation according to claim 22.

33. (i) The disease or disorder is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, geographic atrophy secondary to macular degeneration, intermediate dry age-related macular degeneration (AMD), Leber congenital amaurosis, or Stargardt disease. (ii) The disease is associated with the loss of photoreceptor cells, (iii) The photoreceptor rescue cell composition, formulation, or pharmaceutical preparation is administered into the subretinal space, the suprachoroid space, by depot into the eye, or by systemic delivery to another part of the body of the subject, and optionally the cell preparation or formulation is administered by injection or implantation, and optionally intraocular administration includes injecting an aqueous solution, optionally an isotonic solution and / or saline solution into the subretinal space to form a prebleb, and removing the aqueous solution before administering the photoreceptor rescue cell composition to the same subretinal space as the aqueous solution. (iv) The cell preparation or formulation is administered within at least one week, at least one month, at least six months, at least one year, at least two years, at least three years, at least four years, or at least five years from the onset of symptoms. (v) One or more anti-inflammatory agents are administered to the subject, and optionally one or more anti-inflammatory agents are administered simultaneously or separately, and / or one or more anti-inflammatory agents are administered before the cell preparation or formulation, or the cell preparation or formulation is administered before one or more anti-inflammatory agents, and / or one or more anti-inflammatory agents are administered before and after the administration of the cell preparation or formulation, and optionally one or more anti-inflammatory agents include dexamethasone and / or cyclosporine. (vi) The administration of the cell preparation or formulation is without one or more anti-inflammatory agents. The formulation according to claim 23.

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