Methods for selecting cells for photoreceptor transplantation procedures - Patents.com

Abstract

The present disclosure provides methods for selecting retinal cells for use in cellular component transfer therapy, selected populations of retinal cells produced by such methods, and compositions comprising such selected populations of retinal cells.The present disclosure also provides the use of the selected populations of retina and compositions comprising the same for preventing and / or treating inherited retinal degenerative diseases.In certain embodiments, the present disclosure relates to methods, such as in vitro methods, for producing a selected population of retinal cells.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under Grants EY029157, EY007143, EY030872, EY001765 and EY033103 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 338,318, filed May 4, 2022, the contents of which are incorporated by reference in their entirety and priority is claimed.

[0003] Sequence Listing A sequence listing conforming to the rules of WIPO standard ST.26 is incorporated herein by reference. The sequence listing has been submitted as an electronic document via the Patent Center in ASCII format encoded as XML. The electronic document, created on May 2, 2023, is titled "088933.0113.xml" and is 8,708 bytes in size.

[0004] Introduction The present disclosure provides methods for sorting retinal cells for use in cellular component transfer therapy, sorted populations of retinal cells produced by such methods, and compositions comprising such sorted populations of retinal cells. The present disclosure also provides uses of the sorted populations of retinal cells and compositions comprising same for preventing and / or treating inherited or acquired retinal degenerative diseases. [Background technology]

[0005] background Photoreceptor cell transplantation is currently being developed as a treatment for blindness resulting from various inherited or acquired retinal degenerative diseases. In one approach, subretinal transplantation of retinal cells results in the therapeutic transfer of cytoplasm and other cellular components (including but not limited to macromolecules, mitochondria, endoplasmic reticulum, peroxisomes and lysosomes, nucleic acids, ciliary components, and membrane components) from donor cells to host cells. In contrast to the expectation that donor cells will remain independently functional photoreceptors with new synaptic connections with downstream neurons, cellular component transfer therapy ("CCTT") acts by repairing and / or promoting the health, lifespan, and / or functionality of dysfunctional photoreceptor cells already present in the recipient's retina. With appropriate donor cell preparations, this concept of intercellular component transfer is predicted to be therapeutically important in other neuronal targets such as dopaminergic neurons (Parkinson's disease), entorhinal cortex and hippocampus (Alzheimer's disease), and sensory hair cells of the inner ear (sensorineural hearing loss). Despite recent advances in cell culture strategies that allow the production of retinal organoids as a source of donor photoreceptor cells, there is still a need for improved methods to generate sorted populations of retinal cells suitable for efficiently and effectively treating inherited or acquired retinal degenerative diseases via CCTT. In particular, the goals of cell sorting include the depletion of potentially harmful or inactive cells and the enrichment of therapeutically active cells. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present disclosure provides methods for generating a sorted population of retinal cells for use in CCTT, sorted populations of retinal cells generated by such methods, and compositions comprising such sorted populations of retinal cells. The present disclosure also provides uses of the sorted populations of retinal cells and compositions comprising same for preventing and / or treating inherited or acquired retinal degenerative diseases.

[0007] In certain embodiments, the present disclosure relates to a method, such as an in vitro method, for producing a selected population of retinal cells.In certain embodiments, such a method comprises: generating three-dimensional retinal organoids; dissociating the three-dimensional retinal organoids; and positively selecting retinal cells based on one or more markers of photoreceptor cell identity and / or negatively selecting retinal cells based on one or more markers of non-photoreceptor cell identity to produce a selected population of retinal cells.In certain embodiments, the marker of photoreceptor cell identity is CD73.In certain embodiments, the marker of non-photoreceptor cell identity is one or more of CD24, CD302, CD9 and CD99. In certain embodiments, the markers of non-photoreceptor cell identity are ITM2B, CD63, ENO1, CALR, CANX, CLU, SLC3A2, BSG, GPM6B, ITGB1, PTTG1IP, TIMP1, PMEPA1, SSR2, DKK3, LRP1, ATRAID, HLA-A, HLA-C, EMP3, TMED9, GOLIM4, LTBP3, GALNT1, CD151, PLD3, CALU, LSAMP, CD59, SLC2A1, LAMP2, HLA-B, COL11A1, DPP7, DCBLD2, CD164, SLC1A3, F3, CTSD, FLNA, SLC39A10, FN1, TMEM106C, TMEM179B, ATP1B3, HLA-E, TMEM132A, FLT1, FGFR1, CAPNS1, FAT1, ANGPTL1, LRP10, CRELD2, SPPL2A, TSPAN4, PRSS35, ECE1, SYPL1, SORCS2, COL2A1, DNER, COL6A1, CD44 , GPC1, PCDH9, CRIM1, CHL1, TTYH3, IKBIP, NECTIN2, FBLN2, CCDC80, DAG1, PBXIP1, PRSS23, ACAA1, NTR K2, FSTL1, BCHE, TNFRSF1A, LGALS3BP, ITGB8, CP, ADGRG1, VCAN, OLFM1, NRP1, SCARB1, TSPAN6, PCDH17, PLTP, NECTIN3, PTPRD, CADM4, UNC5B, CSPG5, AXL, PLXNB2, PLPP3, NOTCH2, SLITRK2, AEBP1, ANGPTL4,COLEC12, VAMP5, NLGN4X, FGFRL1, EFNB2, COL5A1, LAMB2, LAMC1, IGFBP3, FNDC5, FCGRT, ADRA2C, SERPING1, EPHB2, CDH1 1, COL1A2, CNTFR, AGRN, ROBO1, LOX, MRC2, COL6A2, SLC6A11, DSC2, IGSF8, EMP1, ABI3BP, TTYH2, NOTCH1, ANO6, A2M, SORC In certain embodiments, the markers of non-photoreceptor cell identity are one or more of: S1, EFNA1, PTPRG, TF, EMP2, CEMIP2, SERPINE2, CDON, EGFR, PCDH7, MAN2A1, IL1R1, COL1A1, SEMA5A, ANTXR1, S1PR3, ITPRIP, MXRA8, PRELP, AQP1, CSF1, BCAN, ADGRA2, CA12, FAT3, HEPACAM, FGFR3, TRIL, HSD17B2, and HP. In certain embodiments, the markers of non-photoreceptor cell identity are one or more of: DKK3, LRP1, CLU, PMEPA1, ITGB1, and PTTG1IP.

[0008] In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more astrocyte markers. In certain embodiments, the one or more astrocyte markers are ITM2B, CD63, ENO1, CALR, CANX, CLU, SLC3A2, BSG, GPM6B, ITGB1, PTTG1IP, TIMP1, PMEPA1, SSR2, DKK3, LRP1, ATRAID, HLA-A, HLA-C, EMP3, TMED9, GOLIM4, LTBP3, GALNT1, CD151, PLD3, CALU, LSAMP, CD59, SLC2A1, LAMP2, HLA-B, COL11A1, DPP7, DCBLD 2, CD164, SLC1A3, F3, CTSD, FLNA, SLC39A10, FN1, TMEM106C, TMEM179B, ATP1B3, HLA-E, TMEM132A, FLT1, FGFR1, CAPNS1, FAT1, ANGPTL1, LRP10 , CRELD2, SPPL2A, TSPAN4, PRSS35, ECE1, SYPL1, SORCS2, COL2A1, DNER, COL6A1, CD44, GPC1, PCDH9, CRIM1, CHL1, TTYH3, IKBIP, NECTIN2, FBLN2 , CCDC80, DAG1, PBXIP1, PRSS23, ACAA1, NTRK2, FSTL1, BCHE, TNFRSF1A, LGALS3BP, ITGB8, CP, ADGRG1, VCAN, OLFM1, NRP1, SCARB1, TSPAN6, PCD H17, PLTP, NECTIN3, PTPRD, CADM4, UNC5B, CSPG5, AXL, PLXNB2, PLPP3, NOTCH2, SLITRK2, AEBP1, ANGPTL4, COLEC12, VAMP5, NLGN4X, FGFRL1, EF NB2, COL5A1, LAMB2, LAMC1, IGFBP3, FNDC5, FCGRT, ADRA2C, SERPING1, EPHB2, CDH11, COL1A2, CNTFR, AGRN, ROBO1, LOX, MRC2, COL6A2, SLC6A11 , DSC2, IGSF8, EMP1, ABI3BP, TTYH2, NOTCH1, ANO6, A2M, SORCS1, EFNA1, PTPRG, TF, EMP2, CEMIP2, SERPINE2, CDON, EGFR, PCDH7, MAN2A1, IL1R1,COL1A1, SEMA5A, ANTXR1, S1PR3, ITPRIP, MXRA8, PRELP, AQP1, CSF1, BCAN, ADGRA2, CA12, FAT3, HEPACAM, FGFR3, TRIL, HSD17B2 and HP. In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more astrocyte markers selected from ADGRL4, SERPINE2, BCHE, ABI3BP, NRP1, FSTL1, FAT1, NTRK2, FBLN2, PRSS35, SLC1A3, FCGRT, LAMC1, TF, SORCS2, DKK3, LRP1, PTPRD, ANGPTL1, LTBP3, CLU, CNTNAP2, CD151, PCDH9, CRIM1, CSPG5 and PMEPA1.

[0009] In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more cerebrospinal-like (BSL) cell markers. In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more cerebrospinal-like (BSL) cell markers. In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more markers of CLU, ITM2B, PTPRZ1, GPM6B, ATP1B2, CD63, BCAN, SLC1A3, SERPINE2, LRP1, PTPRA, ADGRG1, ENO1, CANX, SLC3A2, DNER, PTTG1IP, CALR, PCDH9, CCDC80, LSAMP, HEPACAM, F3, PLPP3, APLP2, FBLN2, TIMP1, SLC6A11, CSPG5, JAM2, FGFR3, DKK3, GOL IM4, NCAM1, CHL1, NRCAM, HLA-A, TMEM132A, PMEPA1, ITGAV, SSR2, ACAA1, BCHE, CD59, FAT3, PCDH17, ST3GAL5, PBXIP1, LAMP1, ITGB1, HP, ITGB 8, SGCB, LAMP2, CLDND1, TMEM106B, PTCH1, PLTP, RNF13, HLA-C, PTPRD, TMEM30A, TRIL, RAB5C, TTYH3, DAG1, CADM4, UBA1, SLC6A9, LRRC8A, ATP1 B3, SPPL2A, NTRK2, RNF130, LIFR, EMP3, PCDH7, NTRK3, COL6A1, IL17D, LRP10, ADAM19, SGCE, FAT1, SLC44A1, LTBP3, SLC39A10, ABCA1, SYPL1, SLITRK2, GNPTG, CD302, MRC2, LRP4, CALU, CD151, SORL1, TSPAN6, LRRN1, TENM2, CAPNS1, NLGN1, SLC15A2, NLGN4X, EGFR, ADORA1, SLC9A7, SIRP A, EFCAB14, ANGPTL1, FGFR1, VAMP5, CLDN12, LAMB2, GPR155, FGFR2, SLC44A2, LRP1B, PTGFRN, FNDC5, NOTCH1, DPY19L4, S1PR1, CD44, TNFRSF1 A, FAM234A, CDH4, HLA-E, COL11A1, NCAM2, AQP1, CPQ, EMP1, FCGRT, GPC5, ROBO1, VCAN, LGALS3BP, LDLR, LRRC4B, NOTCH2, ALCAM, RYR3, SLC9A9,The BSL cell marker is one or more selected from TMEM94, VCAM1, IGSF1, PCDHA10, CDH10, CACHD1, P2RX7, AEBP1, PLXNB1, AXL, ALPL, ST3GAL4, SERPINI1, ITGB5, CD58, FGFRL1, PLPP1, TTYH2, IL17RB, FAM171A1, IL17RD, ANO6, ADAM22, PTPRG, ANTXR1, ZDHHC23, AGRN, COL14A1, POSTN, CNTFR, SEMA5A, FLNA, EMP2, TFPI, ITGA7, MXRA8, TENM4, FSTL1, CD82, NRP2, GPC4, ARSF, LAMC1, KIT, SEMA4A, LTBP1 and CSF1. In certain embodiments, the one or more markers of non-photoreceptor cell identity are one or more BSL markers selected from HEPACAM, FGFR3, SERPINE2, BCAN, CCDC80, PLPP3, CHL1, ADGRG1, SLC6A11, LSAMP, FBLN2, F3, SLC1A3, DKK3, LRP1, DNER, CLU, PCDH9, and CSPG5.

[0010] In certain embodiments, the three-dimensional retinal organoid of the method disclosed herein is enzymatically dissociated.In certain embodiments, the enzyme is papain and / or trypsin.In certain embodiments, the retinal cell is contacted with the composition to ensure that the cell remains in dissociated cell suspension.In certain embodiments, the composition to ensure that the cell remains in dissociated cell suspension is an enzyme.In certain embodiments, the enzyme is DNAse.

[0011] In certain embodiments of the methods of the present disclosure, the three-dimensional retinal organoids reach about DD45 to DD300 before being dissociated. In certain embodiments, the three-dimensional retinal organoids reach about DD90 to about DD140 before being dissociated.

[0012] In certain embodiments of the methods of the present disclosure, the retinal cell population consists of at least about 70% single cells. In certain embodiments, the retinal cell population consists of at least about 80% single cells. In certain embodiments, the retinal cell population consists of at least about 90% single cells. In certain embodiments, the retinal cell population comprises about 55% to about 85% rod photoreceptor cells.

[0013] In certain embodiments of the method of the present disclosure, the stem cell is selected from non-embryonic stem cells of humans, non-human primates or rodents; embryonic stem cells of humans, non-human primates or rodents; induced pluripotent stem cells of humans, non-human primates or rodents; and recombinant pluripotent cells of humans, non-human primates or rodents. In certain embodiments, the stem cell is a human stem cell. In certain embodiments, the stem cell is a pluripotent stem cell or a multipotent stem cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is selected from embryonic stem cells, induced pluripotent stem cells and combinations thereof.

[0014] In certain embodiments, the present disclosure relates to a sorted population of in vitro differentiated retinal cells, wherein the in vitro differentiated and sorted retinal cells are obtained by the methods described herein.

[0015] In certain embodiments, the present disclosure relates to a composition comprising in vitro differentiated and sorted retinal cells, the in vitro differentiated and sorted retinal cells being obtained by the methods described herein, hi certain embodiments, the composition is a pharmaceutical composition comprising a sorted population of retinal cells and a pharma- ceutically acceptable carrier.

[0016] In certain embodiments, the present disclosure relates to a method for preventing and / or treating a genetic or acquired retinal degenerative disease in a subject, comprising administering to the subject an effective amount of one of the following: (a) a selected population of retinal cells as described herein; or (b) a composition comprising a selected population of retinal cells as described herein. In certain embodiments, the genetic retinal degenerative disease is selected from retinitis pigmentosa, total choroidal atrophy, Stargardt's disease, cone-rod dystrophy and Leber's congenital amaurosis. In certain embodiments, the acquired retinal degenerative disease is age-related macular degeneration. [Brief description of the drawings]

[0017] [Figure 1A-D] Figure 1 shows that human donor cells can migrate short or long distances from the subretinal space of recipient mice. Figure 1A shows immunohistochemistry (IHC) staining for human nuclei specific antibody (HNA) showing migrating (arrow) and non-migratory (open arrow) cells of donor human retinal organoids in the recipient retina. Figure 1B and Figure 1C show that migrating cells overlayed the graft (radial migration) and over the graft edge (tangential migration). Figure 1D shows negative staining of non-transplanted mouse eyes for HNA and CRX:tdTomato. Figure 1E shows the relative abundance of migratory human cells in different recipient retinal laminae (RGC, IPL, INL, RPE / C) (n=17 sections from 5 transplanted eyes). Figure 1F shows quantification of the distance of migratory human cell nuclei from the graft edge in different retinal laminae (RGC, IPL, INL, RPE / C) (n = 21 sections from 5 transplanted eyes). Figure 1G shows migratory Ku80+ human cells found in the area adjacent to the optic nerve (peripapillary migration). White arrows indicated representative migratory human cells. Abbreviations: RGC: retinal ganglion cells; IPL: inner plexiform layer; INL: inner nuclear layer; RPE / C: retinal pigment epithelium and choroid. Yellow lines in Figure 1B, Figure 1C, Figure 1D, and Figure 1G: border of recipient retina; green lines in Figure 1G: optic nerve. [Figure 1E-F]Figure 1 shows that human donor cells can migrate short or long distances from the subretinal space of recipient mice. Figure 1A shows immunohistochemistry (IHC) staining for human nuclei specific antibody (HNA) showing migrating (arrow) and non-migratory (open arrow) cells of donor human retinal organoids in the recipient retina. Figure 1B and Figure 1C show that migrating cells overlayed the graft (radial migration) and over the graft edge (tangential migration). Figure 1D shows negative staining of non-transplanted mouse eyes for HNA and CRX:tdTomato. Figure 1E shows the relative abundance of migratory human cells in different recipient retinal laminae (RGC, IPL, INL, RPE / C) (n=17 sections from 5 transplanted eyes). Figure 1F shows quantification of the distance of migratory human cell nuclei from the graft edge in different retinal laminae (RGC, IPL, INL, RPE / C) (n = 21 sections from 5 transplanted eyes). Figure 1G shows migratory Ku80+ human cells found in the area adjacent to the optic nerve (peripapillary migration). White arrows indicated representative migratory human cells. Abbreviations: RGC: retinal ganglion cells; IPL: inner plexiform layer; INL: inner nuclear layer; RPE / C: retinal pigment epithelium and choroid. Yellow lines in Figure 1B, Figure 1C, Figure 1D, and Figure 1G: border of recipient retina; green lines in Figure 1G: optic nerve. [Figure 1G]Figure 1 shows that human donor cells can migrate short or long distances from the subretinal space of recipient mice. Figure 1A shows immunohistochemistry (IHC) staining for human nuclei specific antibody (HNA) showing migrating (arrow) and non-migratory (open arrow) cells of donor human retinal organoids in the recipient retina. Figure 1B and Figure 1C show that migrating cells overlayed the graft (radial migration) and over the graft edge (tangential migration). Figure 1D shows negative staining of non-transplanted mouse eyes for HNA and CRX:tdTomato. Figure 1E shows the relative abundance of migratory human cells in different recipient retinal laminae (RGC, IPL, INL, RPE / C) (n=17 sections from 5 transplanted eyes). Figure 1F shows quantification of the distance of migratory human cell nuclei from the graft edge in different retinal laminae (RGC, IPL, INL, RPE / C) (n = 21 sections from 5 transplanted eyes). Figure 1G shows migratory Ku80+ human cells found in the area adjacent to the optic nerve (peripapillary migration). White arrows indicated representative migratory human cells. Abbreviations: RGC: retinal ganglion cells; IPL: inner plexiform layer; INL: inner nuclear layer; RPE / C: retinal pigment epithelium and choroid. Yellow lines in Figure 1B, Figure 1C, Figure 1D, and Figure 1G: border of recipient retina; green lines in Figure 1G: optic nerve. [Figure 2A]Figure 2 shows single-cell RNA sequencing analysis (scRNA-seq) revealing retinal- and non-retinal-derived cell fates of transplanted and cultured donor retinal organoid cells. Figure 2A is a schematic showing in vivo and in vitro control conditions of donor cells analyzed by scRNA-seq. CRX:tdTomato+hESC-derived retinal organoids (134 days old) were transplanted into Rd1 / NS mice or maintained in culture. After 4.5 months, single-cell suspensions of transplanted and cultured retinal organoids (age-matched) were collected by papain dissociation and analyzed by Chromium scRNA-seq. Figure 2B and Figure 2C show that scRNA-seq identified nine distinct cell clusters from a pool of transplanted and cultured retinal organoid cells (n=5,831 cells). Figure 2D shows dot plots of marker gene expression in the identified cell clusters. The color scale corresponds to the average gene expression, and the dot size corresponds to the percentage of positively expressing cells in each cluster. Figure 2E shows the relative abundance of each type of cell in transplanted and cultured retinal organoids. [Figure 2B-C]Figure 2 shows single-cell RNA sequencing analysis (scRNA-seq) revealing retinal- and non-retinal-derived cell fates of transplanted and cultured donor retinal organoid cells. Figure 2A is a schematic showing in vivo and in vitro control conditions of donor cells analyzed by scRNA-seq. CRX:tdTomato+hESC-derived retinal organoids (134 days old) were transplanted into Rd1 / NS mice or maintained in culture. After 4.5 months, single-cell suspensions of transplanted and cultured retinal organoids (age-matched) were collected by papain dissociation and analyzed by Chromium scRNA-seq. Figure 2B and Figure 2C show that scRNA-seq identified nine distinct cell clusters from a pool of transplanted and cultured retinal organoid cells (n=5,831 cells). Figure 2D shows dot plots of marker gene expression in the identified cell clusters. The color scale corresponds to the average gene expression, and the dot size corresponds to the percentage of positively expressing cells in each cluster. Figure 2E shows the relative abundance of each type of cell in transplanted and cultured retinal organoids. [Figure 2D]Figure 2 shows single-cell RNA sequencing analysis (scRNA-seq) revealing retinal- and non-retinal-derived cell fates of transplanted and cultured donor retinal organoid cells. Figure 2A is a schematic showing in vivo and in vitro control conditions of donor cells analyzed by scRNA-seq. CRX:tdTomato+hESC-derived retinal organoids (134 days old) were transplanted into Rd1 / NS mice or maintained in culture. After 4.5 months, single-cell suspensions of transplanted and cultured retinal organoids (age-matched) were collected by papain dissociation and analyzed by Chromium scRNA-seq. Figure 2B and Figure 2C show that scRNA-seq identified nine distinct cell clusters from a pool of transplanted and cultured retinal organoid cells (n=5,831 cells). Figure 2D shows dot plots of marker gene expression in the identified cell clusters. The color scale corresponds to the average gene expression, and the dot size corresponds to the percentage of positively expressing cells in each cluster. Figure 2E shows the relative abundance of each type of cell in transplanted and cultured retinal organoids. [Figure 2E]Figure 2 shows single-cell RNA sequencing analysis (scRNA-seq) revealing retinal- and non-retinal-derived cell fates of transplanted and cultured donor retinal organoid cells. Figure 2A is a schematic showing in vivo and in vitro control conditions of donor cells analyzed by scRNA-seq. CRX:tdTomato+hESC-derived retinal organoids (134 days old) were transplanted into Rd1 / NS mice or maintained in culture. After 4.5 months, single-cell suspensions of transplanted and cultured retinal organoids (age-matched) were collected by papain dissociation and analyzed by Chromium scRNA-seq. Figure 2B and Figure 2C show that scRNA-seq identified nine distinct cell clusters from a pool of transplanted and cultured retinal organoid cells (n=5,831 cells). Figure 2D shows dot plots of marker gene expression in the identified cell clusters. The color scale corresponds to the average gene expression, and the dot size corresponds to the percentage of positively expressing cells in each cluster. Figure 2E shows the relative abundance of each type of cell in transplanted and cultured retinal organoids. [Figure 3A]Figure 3 shows that donor retinal astrocytes and brain / spinal cord-like (BSL) cells exhibit long-distance migratory capacity. Figure 3A shows that scRNA-seq analysis showed the highest migration scores in astrocytes and BSL cells among the cell types identified in transplanted retinal organoids. Figure 3B shows that RNAscope staining showed that migratory cells expressed markers (red) for astrocytes (PAX2, HES6) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX). IHC counterstaining with human nuclear antibody Ku80 (white) was used to detect transplanted human cells. Figure 3C shows the relative abundance of migratory astrocytes and BSL cells in the retinal lamina of different recipients. Figure 3D shows the quantification of migratory astrocytes and BSL cells in migratory human cells (n=3–4 eyes). Figure 3E and Figure 3F show RNAscope staining (Figure 1E) and quantification (Figure 1F) of non-migratory astrocytes (PAX2) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX) that remained in the subretinal space. IHC for Ku80 (white) facilitated the detection of transplanted human cells (n = 6–9 areas of interest from four transplanted eyes). Figure 3G shows that migratory cells negatively express markers (green) of RGCs (RBPMS, NeuN), amacrine cells (NeuN), horizontal cells (calbindin), rod bipolar cells (PKCα), cone bipolar cells (SCGN), microglia (IBA1) and macrophages (CD68). DAPI staining (blue) was performed to identify nuclei in the recipient retinal lamina. [Figure 3B]Figure 3 shows that donor retinal astrocytes and brain / spinal cord-like (BSL) cells exhibit long-distance migratory capacity. Figure 3A shows that scRNA-seq analysis showed the highest migration scores in astrocytes and BSL cells among the cell types identified in transplanted retinal organoids. Figure 3B shows that RNAscope staining showed that migratory cells expressed markers (red) for astrocytes (PAX2, HES6) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX). IHC counterstaining with human nuclear antibody Ku80 (white) was used to detect transplanted human cells. Figure 3C shows the relative abundance of migratory astrocytes and BSL cells in the retinal lamina of different recipients. Figure 3D shows the quantification of migratory astrocytes and BSL cells in migratory human cells (n=3–4 eyes). Figure 3E and Figure 3F show RNAscope staining (Figure 1E) and quantification (Figure 1F) of non-migratory astrocytes (PAX2) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX) that remained in the subretinal space. IHC for Ku80 (white) facilitated the detection of transplanted human cells (n = 6–9 areas of interest from four transplanted eyes). Figure 3G shows that migratory cells negatively express markers (green) of RGCs (RBPMS, NeuN), amacrine cells (NeuN), horizontal cells (calbindin), rod bipolar cells (PKCα), cone bipolar cells (SCGN), microglia (IBA1) and macrophages (CD68). DAPI staining (blue) was performed to identify nuclei in the recipient retinal lamina. [Figure 3C-D]Figure 3 shows that donor retinal astrocytes and brain / spinal cord-like (BSL) cells exhibit long-distance migratory capacity. Figure 3A shows that scRNA-seq analysis showed the highest migration scores in astrocytes and BSL cells among the cell types identified in transplanted retinal organoids. Figure 3B shows that RNAscope staining showed that migratory cells expressed markers (red) for astrocytes (PAX2, HES6) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX). IHC counterstaining with human nuclear antibody Ku80 (white) was used to detect transplanted human cells. Figure 3C shows the relative abundance of migratory astrocytes and BSL cells in the retinal lamina of different recipients. Figure 3D shows the quantification of migratory astrocytes and BSL cells in migratory human cells (n=3–4 eyes). Figure 3E and Figure 3F show RNAscope staining (Figure 1E) and quantification (Figure 1F) of non-migratory astrocytes (PAX2) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX) that remained in the subretinal space. IHC for Ku80 (white) facilitated the detection of transplanted human cells (n = 6–9 areas of interest from four transplanted eyes). Figure 3G shows that migratory cells negatively express markers (green) of RGCs (RBPMS, NeuN), amacrine cells (NeuN), horizontal cells (calbindin), rod bipolar cells (PKCα), cone bipolar cells (SCGN), microglia (IBA1) and macrophages (CD68). DAPI staining (blue) was performed to identify nuclei in the recipient retinal lamina. [Figure 3E-F]Figure 3 shows that donor retinal astrocytes and brain / spinal cord-like (BSL) cells exhibit long-distance migratory capacity. Figure 3A shows that scRNA-seq analysis showed the highest migration scores in astrocytes and BSL cells among the cell types identified in transplanted retinal organoids. Figure 3B shows that RNAscope staining showed that migratory cells expressed markers (red) for astrocytes (PAX2, HES6) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX). IHC counterstaining with human nuclear antibody Ku80 (white) was used to detect transplanted human cells. Figure 3C shows the relative abundance of migratory astrocytes and BSL cells in the retinal lamina of different recipients. Figure 3D shows the quantification of migratory astrocytes and BSL cells in migratory human cells (n=3–4 eyes). Figure 3E and Figure 3F show RNAscope staining (Figure 1E) and quantification (Figure 1F) of non-migratory astrocytes (PAX2) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX) that remained in the subretinal space. IHC for Ku80 (white) facilitated the detection of transplanted human cells (n = 6–9 areas of interest from four transplanted eyes). Figure 3G shows that migratory cells negatively express markers (green) of RGCs (RBPMS, NeuN), amacrine cells (NeuN), horizontal cells (calbindin), rod bipolar cells (PKCα), cone bipolar cells (SCGN), microglia (IBA1) and macrophages (CD68). DAPI staining (blue) was performed to identify nuclei in the recipient retinal lamina. [Figure 3G]Figure 3 shows that donor retinal astrocytes and brain / spinal cord-like (BSL) cells exhibit long-distance migratory capacity. Figure 3A shows that scRNA-seq analysis showed the highest migration scores in astrocytes and BSL cells among the cell types identified in transplanted retinal organoids. Figure 3B shows that RNAscope staining showed that migratory cells expressed markers (red) for astrocytes (PAX2, HES6) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX). IHC counterstaining with human nuclear antibody Ku80 (white) was used to detect transplanted human cells. Figure 3C shows the relative abundance of migratory astrocytes and BSL cells in the retinal lamina of different recipients. Figure 3D shows the quantification of migratory astrocytes and BSL cells in migratory human cells (n=3–4 eyes). Figure 3E and Figure 3F show RNAscope staining (Figure 1E) and quantification (Figure 1F) of non-migratory astrocytes (PAX2) and BSL cells (ASCL1, HOXC8, NKX2-2, ARX) that remained in the subretinal space. IHC for Ku80 (white) facilitated the detection of transplanted human cells (n = 6–9 areas of interest from four transplanted eyes). Figure 3G shows that migratory cells negatively express markers (green) of RGCs (RBPMS, NeuN), amacrine cells (NeuN), horizontal cells (calbindin), rod bipolar cells (PKCα), cone bipolar cells (SCGN), microglia (IBA1) and macrophages (CD68). DAPI staining (blue) was performed to identify nuclei in the recipient retinal lamina. [Figure 4A-C]Figure 4 shows that actively proliferating cells are rare among migratory and non-migratory donor cells. Figure 4A shows that Ki-67+ proliferating cells were rare in cultured organoids (n=4 in one batch) and significantly less in transplanted organoids (n=3 eyes). Figure 4B shows rare Ki-67+ cells detected among non-migratory and migratory cells in transplanted retinal organoids (n=5 eyes). Figure 4C shows the relative abundance of migratory Ki-67+ cells in different retinal laminae (RGC, IPL, INL, RPE / C). Figure 4D shows that ScRNA-seq analysis showed the highest proliferation scores in Müller glia, RPC, astrocytes and BSL cells among the cell types identified in transplanted retinal organoids (n=3 eyes). FIG. 4E shows that RNAscope and IHC show very few Ki67+ cells in migratory astrocytes (PAX2+) and BSL cells (ASCL1+), as well as non-migratory retinal progenitor cells (VSX2+) and BSL cells (HOXC8+) (n=4 eyes). [Figure 4D] Figure 4 shows that actively proliferating cells are rare among migratory and non-migratory donor cells. Figure 4A shows that Ki-67+ proliferating cells were rare in cultured organoids (n=4 in one batch) and significantly less in transplanted organoids (n=3 eyes). Figure 4B shows rare Ki-67+ cells detected among non-migratory and migratory cells in transplanted retinal organoids (n=5 eyes). Figure 4C shows the relative abundance of migratory Ki-67+ cells in different retinal laminae (RGC, IPL, INL, RPE / C). Figure 4D shows that ScRNA-seq analysis showed the highest proliferation scores in Müller glia, RPC, astrocytes and BSL cells among the cell types identified in transplanted retinal organoids (n=3 eyes). FIG. 4E shows that RNAscope and IHC show very few Ki67+ cells in migratory astrocytes (PAX2+) and BSL cells (ASCL1+), as well as non-migratory retinal progenitor cells (VSX2+) and BSL cells (HOXC8+) (n=4 eyes). [Figure 4E]Figure 4 shows that actively proliferating cells are rare among migratory and non-migratory donor cells. Figure 4A shows that Ki-67+ proliferating cells were rare in cultured organoids (n=4 in one batch) and significantly less in transplanted organoids (n=3 eyes). Figure 4B shows rare Ki-67+ cells detected among non-migratory and migratory cells in transplanted retinal organoids (n=5 eyes). Figure 4C shows the relative abundance of migratory Ki-67+ cells in different retinal laminae (RGC, IPL, INL, RPE / C). Figure 4D shows that ScRNA-seq analysis showed the highest proliferation scores in Müller glia, RPC, astrocytes and BSL cells among the cell types identified in transplanted retinal organoids (n=3 eyes). FIG. 4E shows that RNAscope and IHC show very few Ki67+ cells in migratory astrocytes (PAX2+) and BSL cells (ASCL1+), as well as non-migratory retinal progenitor cells (VSX2+) and BSL cells (HOXC8+) (n=4 eyes). [Figure 5A]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5B-C]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5D]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5E-1]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5E-2]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5E-3]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 5F]Figure 5 shows that donor cone photoreceptors mature more rapidly in the recipient subretinal space. Figure 5A shows UMAPs embedding the maturation trajectory in pseudotime of cone photoreceptors in transplanted retinal organoids (n=3) and cultured retinal organoids (age-matched, n=2) compared to normal in vivo human cone development (ages from embryonic week 9 to adulthood). Cells are colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 5B shows ridge plots indicating that transcriptional maturation of transplanted cone photoreceptors resembles adult human cone photoreceptors, whereas cultured cone photoreceptors resemble embryonic human cone photoreceptors (aged 9-18 embryonic weeks). Figure 5C shows ScRNA-seq violin plots showing upregulation of OPN1LW, OPN1MW and OPN1SW in transplanted retinal organoids compared to cultured retinal organoids. Figure 5D shows IHC staining and quantification demonstrating significantly more L / M-opsin+ and S-opsin+ cone photoreceptors in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples per group). Figure 5E shows IHC images showing representative L / M-opsin+ or S-opsin+ cone photoreceptors with (OS+, yellow arrowhead) or without (OS-) outer segments. Histological quantification of the fraction of L / M-opsin+ or S-opsin+ cells with inner / outer segment formation (segment+) was significantly greater in transplanted retinal organoids than in cultured retinal organoids (n=16-20 sections from 4 individual samples / group). Figure 5F shows that single-cell patch clamp recordings of transplanted human cones showed large capacitance currents. [Figure 6A]Figure 6 shows that donor rod photoreceptors mature more rapidly in the recipient subretinal space. Figure 6A shows UMAPs embedding the maturation trajectory of rod photoreceptors in transplanted (n=3) and age-matched cultured retinal organoids (n=2) compared to in vivo human rod development (ages from 9 weeks to adulthood). Cells were colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 6B shows ridgeline plots indicating that transcriptional maturation of transplanted rod photoreceptors resembles adult human rod photoreceptors, whereas cultured rod photoreceptors transcriptionally resemble embryonic human rod photoreceptors (9-27 week old embryos). Figure 6C shows ScRNA-seq analysis demonstrating upregulation of RHO gene expression in transplanted retinal organoids compared to cultured retinal organoids. Figure 6D shows IHC staining and quantification demonstrating a greater percentage of Rho+ photoreceptors in transplanted than in cultured Crx:tdTomato+ photoreceptors (n=20 sections from 4 individual samples / group). Figure 6E shows IHC staining demonstrating representative Rho+ rod photoreceptors with (OS+, yellow arrowheads) or without (OS-) outer segments. Histological quantification showed a greater percentage of rods with inner / outer segment formation (segment+) in transplanted than in cultured retinal organoids (n=20 sections from 4 individual samples / group). [Figure 6B]Figure 6 shows that donor rod photoreceptors mature more rapidly in the recipient subretinal space. Figure 6A shows UMAPs embedding the maturation trajectory of rod photoreceptors in transplanted (n=3) and age-matched cultured retinal organoids (n=2) compared to in vivo human rod development (ages from 9 weeks to adulthood). Cells were colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 6B shows ridgeline plots indicating that transcriptional maturation of transplanted rod photoreceptors resembles adult human rod photoreceptors, whereas cultured rod photoreceptors transcriptionally resemble embryonic human rod photoreceptors (9-27 week old embryos). Figure 6C shows ScRNA-seq analysis demonstrating upregulation of RHO gene expression in transplanted retinal organoids compared to cultured retinal organoids. Figure 6D shows IHC staining and quantification demonstrating a greater percentage of Rho+ photoreceptors in transplanted than in cultured Crx:tdTomato+ photoreceptors (n=20 sections from 4 individual samples / group). Figure 6E shows IHC staining demonstrating representative Rho+ rod photoreceptors with (OS+, yellow arrowheads) or without (OS-) outer segments. Histological quantification showed a greater percentage of rods with inner / outer segment formation (segment+) in transplanted than in cultured retinal organoids (n=20 sections from 4 individual samples / group). [Figure 6C-D]Figure 6 shows that donor rod photoreceptors mature more rapidly in the recipient subretinal space. Figure 6A shows UMAPs embedding the maturation trajectory of rod photoreceptors in transplanted (n=3) and age-matched cultured retinal organoids (n=2) compared to in vivo human rod development (ages from 9 weeks to adulthood). Cells were colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 6B shows ridgeline plots indicating that transcriptional maturation of transplanted rod photoreceptors resembles adult human rod photoreceptors, whereas cultured rod photoreceptors transcriptionally resemble embryonic human rod photoreceptors (9-27 week old embryos). Figure 6C shows ScRNA-seq analysis demonstrating upregulation of RHO gene expression in transplanted retinal organoids compared to cultured retinal organoids. Figure 6D shows IHC staining and quantification demonstrating a greater percentage of Rho+ photoreceptors in transplanted than in cultured Crx:tdTomato+ photoreceptors (n=20 sections from 4 individual samples / group). Figure 6E shows IHC staining demonstrating representative Rho+ rod photoreceptors with (OS+, yellow arrowheads) or without (OS-) outer segments. Histological quantification showed a greater percentage of rods with inner / outer segment formation (segment+) in transplanted than in cultured retinal organoids (n=20 sections from 4 individual samples / group). [Figure 6E]Figure 6 shows that donor rod photoreceptors mature more rapidly in the recipient subretinal space. Figure 6A shows UMAPs embedding the maturation trajectory of rod photoreceptors in transplanted (n=3) and age-matched cultured retinal organoids (n=2) compared to in vivo human rod development (ages from 9 weeks to adulthood). Cells were colored by cell type (top UMAP) and pseudotime (bottom UMAP). Figure 6B shows ridgeline plots indicating that transcriptional maturation of transplanted rod photoreceptors resembles adult human rod photoreceptors, whereas cultured rod photoreceptors transcriptionally resemble embryonic human rod photoreceptors (9-27 week old embryos). Figure 6C shows ScRNA-seq analysis demonstrating upregulation of RHO gene expression in transplanted retinal organoids compared to cultured retinal organoids. Figure 6D shows IHC staining and quantification demonstrating a greater percentage of Rho+ photoreceptors in transplanted than in cultured Crx:tdTomato+ photoreceptors (n=20 sections from 4 individual samples / group). Figure 6E shows IHC staining demonstrating representative Rho+ rod photoreceptors with (OS+, yellow arrowheads) or without (OS-) outer segments. Histological quantification showed a greater percentage of rods with inner / outer segment formation (segment+) in transplanted than in cultured retinal organoids (n=20 sections from 4 individual samples / group). [Figure 7A-B] Figure 7 shows the breeding and phenotyping of recipient Rd1 / NS mice. Figure 7A shows a schematic diagram showing recipient Rd1 / NS mice generated by breeding Rd1 (Pde6brd1) mice with NOD / SCID (NOD.CB17-Prkdcscid / J) mice. Figure 7B shows IHC staining showing comparable phenotypes of retinal degeneration in age-matched Rd1 / NS and Rd1 mice: the outer nuclear layer was lost in both, and there was no expression of L / M-opsin, S-opsin, and rhodopsin (Rho). C57.BL / 6J mice served as wild-type controls. Figure 7C shows flow cytometry analysis showing a deficiency of CD3+ T cells and CD45R+ B cells in Rd1 / NS mice, corresponding to the NOD / SCID genotype. C57.BL / 6J mice served as wild-type controls. [Figure 7C-1] Figure 7 shows the breeding and phenotyping of recipient Rd1 / NS mice. Figure 7A shows a schematic diagram showing recipient Rd1 / NS mice generated by breeding Rd1 (Pde6brd1) mice with NOD / SCID (NOD.CB17-Prkdcscid / J) mice. Figure 7B shows IHC staining showing comparable phenotypes of retinal degeneration in age-matched Rd1 / NS and Rd1 mice: the outer nuclear layer was lost in both, and there was no expression of L / M-opsin, S-opsin, and rhodopsin (Rho). C57.BL / 6J mice served as wild-type controls. Figure 7C shows flow cytometry analysis showing a deficiency of CD3+ T cells and CD45R+ B cells in Rd1 / NS mice, corresponding to the NOD / SCID genotype. C57.BL / 6J mice served as wild-type controls. [Figure 7C-2] Figure 7 shows the breeding and phenotyping of recipient Rd1 / NS mice. Figure 7A shows a schematic diagram showing recipient Rd1 / NS mice generated by breeding Rd1 (Pde6brd1) mice with NOD / SCID (NOD.CB17-Prkdcscid / J) mice. Figure 7B shows IHC staining showing comparable phenotypes of retinal degeneration in age-matched Rd1 / NS and Rd1 mice: the outer nuclear layer was lost in both, and there was no expression of L / M-opsin, S-opsin, and rhodopsin (Rho). C57.BL / 6J mice served as wild-type controls. Figure 7C shows flow cytometry analysis showing a deficiency of CD3+ T cells and CD45R+ B cells in Rd1 / NS mice, corresponding to the NOD / SCID genotype. C57.BL / 6J mice served as wild-type controls. [Figure 7C-3]Figure 7 shows the breeding and phenotyping of recipient Rd1 / NS mice. Figure 7A shows a schematic diagram showing recipient Rd1 / NS mice generated by breeding Rd1 (Pde6brd1) mice with NOD / SCID (NOD.CB17-Prkdcscid / J) mice. Figure 7B shows IHC staining showing comparable phenotypes of retinal degeneration in age-matched Rd1 / NS and Rd1 mice: the outer nuclear layer was lost in both, and there was no expression of L / M-opsin, S-opsin, and rhodopsin (Rho). C57.BL / 6J mice served as wild-type controls. Figure 7C shows flow cytometry analysis showing a deficiency of CD3+ T cells and CD45R+ B cells in Rd1 / NS mice, corresponding to the NOD / SCID genotype. C57.BL / 6J mice served as wild-type controls. [Figure 8] Figure 8 shows RNAscope staining of positive and negative control probes. Frozen sections of non-transplanted Rd1 / NS mice and cultured retinal organoids were stained with 3-plex positive and negative control probes combined with TSA-Cy3 or TSA-Cy5 fluorophores. Positive staining indicated expression of the positive control genes PPIB (Cy3) and POLR2A (Cy5). [Figure 9A-B] Figure 9 shows quality control of scRNA-Seq data. Figures 9A and 9B show the number of genes and unique molecular identifiers per cell. Each bar is a cell, colored by sample library and ordered descending along the x-axis. Figure 9C shows a UMAP plot showing cells colored by sample library. Figure 9D shows a UMAP plot showing 10 (0-9) transcriptionally distinct cell clusters. [Figure 9C-D] Figure 9 shows quality control of scRNA-Seq data. Figures 9A and 9B show the number of genes and unique molecular identifiers per cell. Each bar is a cell, colored by sample library and ordered descending along the x-axis. Figure 9C shows a UMAP plot showing cells colored by sample library. Figure 9D shows a UMAP plot showing 10 (0-9) transcriptionally distinct cell clusters. [Figure 10A-B] Figure 10 shows UMAP of migrating and proliferating cell clusters. Figure 10A shows UMAP distinguishing cell clusters of cultured retinal organoids (gray) and transplanted retinal organoids (purple). Figure 10B shows UMAP stained cell clusters acquiring both migratory and proliferative transcriptomic characteristics. Figure 10C shows that UMAP showed expression of marker genes in different cell subpopulations of transplanted and cultured retinal organoids. [Figure 10C] Figure 10 shows UMAP of migrating and proliferating cell clusters. Figure 10A shows UMAP distinguishing cell clusters of cultured retinal organoids (gray) and transplanted retinal organoids (purple). Figure 10B shows UMAP stained cell clusters acquiring both migratory and proliferative transcriptomic characteristics. Figure 10C shows that UMAP showed expression of marker genes in different cell subpopulations of transplanted and cultured retinal organoids. [Figure 11] Figure 11 shows pseudo-temporal analysis of individual cell clusters and marker gene expression of cone and rod photoreceptors in cultured and transplanted retinal organoids. Figure 11A shows UMAPs identifying individual cell clusters in human retina (ages from embryonic week 9 to adult, cone developmental dataset: n=7,654 cells; rod developmental dataset: n=25,186 cells), cultured retinal organoids (cones: n=1,639 cells, rods: n=1,469 cells), and transplanted retinal organoids (cones: n=210 cells, rods: n=504 cells). Figure 11B shows a heatmap demonstrating the upregulation of marker genes specific for cone and rod photoreceptors in transplanted retinal organoids (including three independent repeats "transplant-1, transplant-2, transplant-3") compared to cultured retinal organoids (including two independent repeats "culture-1, culture-2"). [Figure 12A]Figure 12 shows the identification and quantification of presynaptic markers in cultured and transplanted retinal organoids. Figure 12A shows a heatmap showing that several synaptic genes were upregulated in transplanted retinal organoids (including cones and rods) compared to cultured retinal organoids. Figure 12B shows IHC staining and quantification demonstrating that CtBP2+ synaptic ribbons in photoreceptors (CRX:tdTomato+) were significantly more abundant in transplanted than in cultured retinal organoids. IHC staining of SCGN (green) was performed to show the recipient bipolar layer. Anti-human nuclear antibody (HNA, blue) was employed to label human cells. Abbreviations: INL: inner nuclear layer. [Figure 12B] Figure 12 shows the identification and quantification of presynaptic markers in cultured and transplanted retinal organoids. Figure 12A shows a heatmap showing that several synaptic genes were upregulated in transplanted retinal organoids (including cones and rods) compared to cultured retinal organoids. Figure 12B shows IHC staining and quantification demonstrating that CtBP2+ synaptic ribbons in photoreceptors (CRX:tdTomato+) were significantly more abundant in transplanted than in cultured retinal organoids. IHC staining of SCGN (green) was performed to show the recipient bipolar layer. Anti-human nuclear antibody (HNA, blue) was employed to label human cells. Abbreviations: INL: inner nuclear layer. [Figure 13A]Figure 13 shows single-cell RNA sequencing of prioritized CD markers. Figures 13A and 13B show CD302, which is highly expressed in astrocytes and brain and spinal cord-like cells (BSLCs). CD9 is highly expressed in non-neuronal cells and BSLCs. CD99 is highly expressed in all non-neuronal cells and BSLCs. CD24 is highly expressed in amacrine cells, photoreceptor progenitors, mature bipolar cells, horizontal cells and retinal ganglion cells. This data supports a strategy in which enrichment based on negative selection of CD24, CD302, CD9, CD99, alone or in combination, results in an enriched population of therapeutically competent photoreceptor cells for the transfer of cellular components into acceptor cells of the recipient retina. [Figure 13B] Figure 13 shows single-cell RNA sequencing of prioritized CD markers. Figures 13A and 13B show CD302, which is highly expressed in astrocytes and brain and spinal cord-like cells (BSLCs). CD9 is highly expressed in non-neuronal cells and BSLCs. CD99 is highly expressed in all non-neuronal cells and BSLCs. CD24 is highly expressed in amacrine cells, photoreceptor progenitors, mature bipolar cells, horizontal cells and retinal ganglion cells. This data supports a strategy in which enrichment based on negative selection of CD24, CD302, CD9, CD99, alone or in combination, results in an enriched population of therapeutically competent photoreceptor cells for the transfer of cellular components into acceptor cells of the recipient retina. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present disclosure provides methods for generating and selecting retinal cells for use in cellular component transfer therapy, sorted populations of retinal cells generated by such methods, and compositions comprising the sorted populations of retinal cells. The present disclosure also provides uses of the sorted populations of retinal cells and compositions comprising same for preventing and / or treating inherited or acquired retinal degenerative diseases.

[0019] Non-limiting embodiments of the presently disclosed subject matter are illustrated herein and by way of example. For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections. 1. Definition; 2. Methods for generating retinal cells; 3. Retinal cell populations and compositions; and 4. Methods for treating inherited retinal degenerative diseases.

[0020] 1.Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and within the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0021] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, such as up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold of a value.

[0022] As used herein, the term "cell population" or "cell population" refers to a group of at least two cells. In non-limiting examples, the cell population may include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population may be a pure population that includes one cell type, for example, a population of photoreceptor cells, or a population of undifferentiated stem cells. Alternatively, the population may include more than one cell type, for example, a mixed cell population. In certain embodiments, the cells in the population of cells are completely dissociated from each other, for example, the population of cells is a suspension of individual cells. In certain embodiments, the population of cells includes non-dissociated cell clusters. For example, and without limitation, such cell populations may comprise up to about 1%, up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, or up to about 10% of the cells in the population that are present as undissociated clusters containing up to about 10 cells. In certain embodiments, such cell populations may comprise up to about 1%, up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, or up to about 10% of the cells in the population that are present as undissociated clusters containing up to about 25 cells.

[0023] As used herein, the term "stem cell" refers to a cell that has the capacity to divide indefinitely in culture and give rise to specialized cells.

[0024] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from preimplantation stage embryos that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a type of pluripotent stem cell derived from early stage human embryos up to the blastocyst stage that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers.

[0025] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured under in vitro conditions that allow proliferation without differentiation for up to several days, months to years.

[0026] As used herein, the term "totipotency" refers to the ability to give rise to all cell types of the body, as well as all of the cell types that make up extraembryonic tissues such as the placenta.

[0027] As used herein, the term "multipotency" refers to the capacity to develop into more than one cell type of the body.

[0028] As used herein, the term "pluripotency" refers to the capacity to develop into an organism's three developmental germ layers, including endoderm, mesoderm and ectoderm.

[0029] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell that is formed by introducing certain embryonic genes (e.g., but not limited to, OCT4, SOX2 and KLF4 transgenes) (see, e.g., Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference) into somatic cells.

[0030] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm), and is sometimes referred to as an "adult" cell.

[0031] As used herein, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues, which have limited capacity for both self-renewal (in the laboratory) and differentiation.

[0032] As used herein, the term "proliferation" refers to an increase in cell number.

[0033] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0034] As used herein, the term "differentiation" refers to the process by which unspecialized embryonic cells acquire the characteristics of specialized cells, such as retinal, heart, liver, or muscle cells. Differentiation is usually controlled by the interaction of the cell's genes with extracellular physical and chemical conditions through signaling pathways involving proteins embedded in the cell surface.

[0035] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a particular (e.g., desired) cell type, such as a retinal cell. In the context of stem cells, "directed differentiation" refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of stem cells from a pluripotent state to a more mature or specialized cell fate.

[0036] As used herein, the term "induce differentiation" with respect to cells refers to changing a default cell type (gene expression profile and / or phenotype) to a non-default cell type (gene expression profile and / or phenotype). Thus, "induce differentiation in stem cells" refers to inducing stem cells (e.g., human stem cells) to divide into progeny cells that have characteristics different from stem cells, such as gene expression profile (e.g., changes in gene expression determined by genetic analysis such as microarrays) and / or phenotype (e.g., changes in the number or presence of protein markers, e.g., cell surface markers, of rod or cone photoreceptor cells, such as CRX, RCVRN, CNGA3, CNGB3, ARR3, THRB, OPN1S2, OPN1MW, NRL, NR2E3, PDE6B, CNGA1, and RHO).

[0037] As used herein, the term "sorting" refers to positive sorting, i.e., sorting in which the presence of a particular feature results in the inclusion of a cell in the sorted population, and negative sorting, i.e., sorting in which the presence of a particular feature results in the exclusion of a cell from the sorted population. Exemplary features associated with positive sorting disclosed herein include, but are not limited to, cell markers for photoreceptor cells, such as CD73. Exemplary features associated with negative sorting disclosed herein include, but are not limited to, cell markers for non-photoreceptor cells, such as CD24, CD302, CD9, and CD99. Exemplary features associated with negative sorting disclosed herein may include, but are not limited to, cell markers for astrocytes, cell markers for brain spinal cord-like (BSL) cells, and both markers for astrocytes and markers for BSL cells. The sorted population may include positively sorted cells, negatively sorted cells, or a combination of positively and negatively sorted cells.

[0038] As used herein, the term "cell culture" refers to the growth of cells in vitro in an artificial medium for research or medical treatment.

[0039] As used herein, the term "culture medium" refers to a liquid containing nutrients to cover, nourish and support cells in a culture vessel such as a petri dish, multi-well plate, spinner flask, etc. Culture medium may also contain growth factors that are added to produce desired changes in the cells.

[0040] As used herein, the term "contacting" one or more cells with a compound (e.g., at least one inhibitor, activator and / or inducer) refers to providing the compound in a location that allows one or more cells to access the compound. Contacting can be achieved using any suitable method. For example, contacting can be achieved by adding a concentrated form of the compound to a cell or cell population, e.g., in the context of a cell culture, to achieve a desired concentration. Contacting can also be achieved by including the compound as a component of a formulated culture medium.

[0041] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[0042] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or a cell), as well as processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, retinogenesis, and the like.

[0043] As used herein, the term "express" with respect to a gene or protein refers to producing mRNA or protein that can be observed using an assay such as a microarray assay, an antibody staining assay, or the like.

[0044] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. A cell marker may not be limited to one marker, and may refer to a "pattern" of markers, such that a specified group of markers may make a cell or cell type identical to another cell or cell type.

[0045] As used herein, the terms "derived from" or "established from" or "differentiated from" when made with reference to any cell disclosed herein refer to a cell line, a tissue (such as a dissociated embryo), or a cell obtained (e.g., isolated, purified, etc.) from an ultimate parent cell in a fluid using any manipulation, such as, but not limited to, single cell isolation, in vitro culture, treatment and / or mutagenesis, such as with proteins, chemicals, radiation, infection with viruses, transfection with DNA sequences, such as morphogens, selection (such as by continuous culture) of any cells contained within the cultured parent cell. Derived cells may be selected from a mixed population by response to growth factors, cytokines, a selected course of cytokine treatment, adherence, lack of adherence, sorting procedures, etc.

[0046] An "individual" or "subject" herein is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sports animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys.

[0047] As used herein, the term "disease" refers to any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ.

[0048] As used herein, the term "treating" or "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, prevention of disease occurrence or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing down the rate of disease progression, improving or alleviating disease state, and remission or improved prognosis. By preventing disease progression, treatment can prevent disease deterioration in affected or diagnosed subjects or subjects suspected of having disease, but treatment can also prevent disease onset or disease symptoms in subjects at risk of disease or suspected of having disease.

[0049] 2. Methods for generating and selecting retinal cells 2.1. 3D cell culture of retinal cells The present disclosure provides an in vitro method for inducing differentiation of stem cells (e.g., human stem cells) and then selecting the resulting differentiated cells. For example, the subject matter of the present disclosure provides an in vitro method for inducing differentiation of stem cells to produce retinal cells, e.g., rod and / or cone photoreceptor cells, and then selecting the retinal cells. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and combinations thereof. In certain embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the methods of the present disclosure include human, non-human primate, or rodent non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and engineered pluripotent cells. In certain embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human pluripotent stem cells (hPSCs) (including but not limited to human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In certain embodiments, the stem cells are embryonic stem cells (ESCs). In certain embodiments, the stem cells are human embryonic stem cells (hESCs). In certain embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In certain embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs).

[0050] In certain embodiments, the in vitro method for inducing the differentiation of stem cells to produce retinal cells of the present disclosure comprises the use of factors that promote the fate specification and survival of rod and cone photoreceptors. In certain embodiments, the in vitro method for inducing the differentiation of stem cells to produce retinal cells of the present disclosure comprises the use of factors that suppress the fate specification and survival of retinal interneurons, such as bipolar cells and retinal ganglion cells. In certain embodiments, the in vitro method for inducing the differentiation of stem cells to produce retinal cells of the present disclosure comprises the use of factors that suppress the fate specification and survival of retinal glia, such as Müller glia. In certain embodiments, the in vitro method for inducing the differentiation of stem cells to produce retinal cells of the present disclosure comprises the use of factors that (a) promote the fate specification and survival of rod and cone photoreceptors; suppress the fate specification and survival of retinal interneurons, such as bipolar cells and retinal ganglion cells; and / or (c) suppress the fate specification and survival of retinal glia, such as Müller glia.

[0051] In certain embodiments, the present disclosure relates to the generation of three-dimensional retinal organoids, for example, three-dimensional human retinal organoids.For example, but not limited to, the strategy for generating three-dimensional human retinal organoids can be used as described in Eldred et al., Science, 362:6411 (2018); et al., Nat Commun., 5:4047 (2014); Reichman et al., Stem Cells, 35:1176-88 (2017); Wahlin et al., Sci Rep., 7:766 (2017); Hallam et al., Stem Cells, 36:1535-51 (2018); Kaya et al., Mol.Vis., 25:663-678 (2019); or Regent et al., Mol Vis., 26:97-105 (2020), each of which is incorporated herein by reference in its entirety. In certain embodiments, human retinal organoids are differentiated to achieve a certain ratio of cone subtypes (red / long, green / mid, and blue / short). For example, but not limited to, culturing organoids in the presence of low retinoic acid (RA), e.g., less than about 1 μM RA, results in organoids with high red cones. In certain exemplary embodiments, culturing organoids in high RA, e.g., greater than about 1 μM to about 20 μM RA (or knockout of CYP26a1), results in organoids with high blue and green cones. In certain exemplary embodiments, culturing organoids in RA until day 80 results in a surrounding mixture of red, green, and blue cones. In certain exemplary embodiments, culturing organoids in high thyroid hormone (T3), e.g., greater than about 1 nM to about 1 μM T3, high RA, e.g., greater than about 1 μM to about 20 μM RA, results in organoids with high green cones. In certain exemplary embodiments, culturing organoid with high T3, for example, about 1 nM or more to about 1 μM T3, and low RA, for example, about 1 μM or less RA, results in organoid with high red cone.In certain exemplary embodiments, knocking out thyroid hormone receptor in organoid results in high blue cone.

[0052] In certain embodiments, the differentiation of stem cells into retinal organoids comprises the in vitro differentiation of stem cells into cells that express at least one retinal organoid marker. In certain embodiments, the differentiation of stem cells into retinal organoids comprises the in vitro differentiation of stem cells into cells that show at least one morphological characteristic associated with retinal organoid differentiation. In certain embodiments, the differentiation of stem cells into retinal organoids comprises the in vitro differentiation of stem cells into cells that express at least one retinal organoid marker and show at least one morphological characteristic associated with retinal organoid differentiation. Non-limiting examples of retinal organoid markers include Nrl, Rho, Arr3, and combinations thereof. Non-limiting examples of retinal organoid morphological characteristics include (a) the development of multi-layered retinal organoid anatomical structures, including, for example, photoreceptor outer nuclear layer and nascent outer segment; and (b) retinal pigment epithelium (RPE) pigmentation development.

[0053] In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 45 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 50 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 55 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 60 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 70 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 75 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of retinal organoids for at least about 80 days to about 300 days. In certain embodiments, the stem cells can be differentiated to achieve a target differentiation stage of cells of the retinal organoid for at least about 85 days to about 300 days.In certain embodiments, the stem cells are differentiated to produce a culture medium for at least about 90 days, at least about 91 days, at least about 93 days, at least about 94 days, at least about 95 days, at least about 96 days, at least about 97 days, at least about 98 days, at least about 99 days, at least about 100 days, at least about 101 days, at least about 102 days, at least about 103 days, at least about 104 days, at least about 105 days, at least about 106 days, at least about 107 days, at least about 108 days, at least about 109 days, at least about 110 days, at least about 111 days, at least about 112 days, at least about 113 days, at least about 114 days, at least about 115 days, at least about 116 days, at least about 117 days, at least about 118 days, at least about 119 days, at least about 200 days, at least about 201 days, at least about 202 days, at least about 203 days, at least about 204 days, at least about 205 days, at least about 206 days, at least about 207 days, at least about 208 days, at least about 209 days, at least about 210 days, at least about 211 days, at least about 212 days, at least about 213 days, at least about 214 days, at least about 215 days, at least about 216 days, at least about 217 days, at least about 218 days, at least about 219 days, at least about 220 days, at least about 221 days, at least about 222 days, at least about 223 days, at least about 224 days, at least about 225 days, at least about 226 days, The target differentiation stage of the cells of the retinal organoid can be achieved for about 116 days, at least about 117 days, at least about 118 days, at least about 119 days, at least about 120 days, at least about 121 days, at least about 122 days, at least about 123 days, at least about 124 days, at least about 125 days, at least about 126 days, at least about 128 days, at least about 129 days, at least about 130 days, at least about 131 days, at least about 132 days, at least about 133 days, at least about 134 days, at least about 135 days, at least about 136 days, at least about 137 days, at least about 138 days, at least about 139 days, or at least about 140 days. The period of differentiation can be indicated as "DD", for example, the cells can be differentiated to achieve the target differentiation stage of the cells of the retinal organoid for at least about 50 days ("DD50") to about 300 days ("DD300").

[0054] 2.2. Dissociation of retinal organoids In certain embodiments, the present disclosure relates to the generation of a population of retinal cells through the dissociation of the above-mentioned retinal organoids. In certain embodiments, such dissociation includes the disruption of the layered organization of cells within the organoids. In certain embodiments, such retinal organoids are dissociated by the addition of certain enzymes and / or additives that ensure that cells remain in the dissociated cell suspension and not as aggregates. For example, but not limited to, enzymes useful in connection with the dissociation of retinal organoids include papain and trypsin. Compositions useful for ensuring that cells remain in the dissociated cell suspension include compositions that include DNAse. Compositions useful for enhancing the survival of cells in dissociated cell suspension include compositions that include B-27 cell culture supplement (Thermo Fisher Scientific) or N-2 cell culture supplement (Thermo Fisher Scientific).

[0055] In certain embodiments, the population of retinal cells resulting from the dissociation of the retinal organoids of the present disclosure comprises at least 70% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 70%-80% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 70%-85% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 70%-90% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell populations of the present disclosure comprise between 70%-95% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell populations of the present disclosure comprise between 70%-100% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells).

[0056] In certain embodiments, the retinal cell population resulting from the dissociation of the retinal organoids of the present disclosure comprises at least 80% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 80%-85% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 80%-90% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 80%-95% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells). In certain embodiments, the cell populations of the present disclosure comprise between 80%-100% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order undissociated clusters of cells).

[0057] In certain embodiments, the retinal cell population resulting from the dissociation of the retinal organoids of the present disclosure comprises at least 85% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher-order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 85%-90% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher-order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 85%-95% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher-order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 85%-100% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher-order non-dissociated clusters of cells).

[0058] In certain embodiments, the retinal cell population resulting from the dissociation of the retinal organoids of the present disclosure comprises at least 90% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 90%-95% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 90%-100% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher non-dissociated clusters of cells).

[0059] In certain embodiments, the retinal cell population resulting from the dissociation of the retinal organoids of the present disclosure comprises at least 95% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells). In certain embodiments, the cell population of the present disclosure comprises between 95% and 100% single cells relative to the total number of cells (including doublet cells, triplet cells, and higher order non-dissociated clusters of cells).

[0060] 3. Retinal Cell Populations and Retinal Cell Composition In certain embodiments, the present disclosure relates to the generation and sorting of retinal cell populations. In certain embodiments, retinal cell populations are sorted, for example via fluorescence activated cell sorting, to selectively enrich and / or negatively select for specific cell types.

[0061] In certain embodiments, positively selected cells of the sorted retinal cell population of the present disclosure express markers of photoreceptor cells, such as CD73. In certain embodiments, negatively selected cells excluded from the sorted retinal cell population of the present disclosure express markers of non-photoreceptor cells, such as CD24, CD302, CD9 and CD99.

[0062] In certain embodiments, the negatively sorted cells excluded from the sorted retinal cell population of the present disclosure express a marker that distinguishes non-photoreceptor cells from photoreceptor cells. For example, but not limited to, such negative sorting can be performed based on the expression of one or more cell surface markers. In certain embodiments, such cell surface marker(s) can be selected from CD markers and surface receptors. In certain embodiments, such marker(s) exhibit greater than 75% expression in non-photoreceptor cells and less than 25% expression in photoreceptor cells. For example, but not limited to, such negative sorting can be performed based on the expression of one or more of the following: ITM2B; CD63; ENO1; CALR; CANX; CLU; SLC3A2; BSG; GPM6B; ITGB1; PTTG1IP; TIMP1; PMEPA1; SSR2; DKK3; LRP1; ATRAID; HLA-A; HLA-C; EMP3; TMED9; GOLIM4; LT BP3;GALNT1;CD151;PLD3;CALU;LSAMP;CD59;SLC2A1;LAMP2;HLA-B;COL11A1;DPP7;DCBLD2;CD164;SLC1A3;F3 ;CTSD;FLNA;SLC39A10;FN1;TMEM106C;TMEM179B;ATP1B3;HLA-E;TMEM132A;FLT1;FGFR1;CAPNS1;FAT1;ANGPTL 1;LRP10;CRELD2;SPPL2A;TSPAN4;PRSS35;ECE1;SYPL1;SORCS2;COL2A1;DNER;COL6A1;CD44;GPC1;PCDH9;CRI M1;CHL1;TTYH3;IKBIP;NECTIN2;FBLN2;CCDC80;DAG1;PBXIP1;PRSS23;ACAA1;NTRK2;FSTL1;BCHE;TNFRSF1A;L GALS3BP;ITGB8;CP;ADGRG1;VCAN;OLFM1;NRP1;SCARB1;TSPAN6;PCDH17;PLTP;NECTIN3;PTPRD;CADM4;UNC5B; CSPG5;AXL;PLXNB2;PLPP3;NOTCH2;SLITRK2;AEBP1;ANGPTL4;COLEC12;VAMP5;NLGN4X;FGFRL1;EFNB2;COL5A1;LAMB2;LAMC1;IGFBP3;FNDC5;FCGRT;ADRA2C;SERPING1;EPHB2;CDH11;COL1A2;CNTFR;AGRN;ROBO1; LOX;MRC2;COL6A2;SLC6A11;DSC2;IGSF8;EMP1;ABI3BP;TTYH2;NOTCH1;ANO6;A2M;SORCS1;EFNA1;PT PRG;TF;EMP2;CEMIP2;SERPINE2;CDON;EGFR;PCDH7;MAN2A1;IL1R1;COL1A1;SEMA5A;ANTXR1;S1PR3; ITPRIP;MXRA8;PRELP;AQP1;CSF1;BCAN;ADGRA2;CA12;FAT3;HEPACAM;FGFR3;TRIL;HSD17B2; and HP. In certain embodiments, the particular subset of non-photoreceptor cell markers used in negatively sorting non-photoreceptor cells from photoreceptor cells is one or more of the following markers: DKK3; LRP1; CLU; PMEPA1; ITGB1; and PTTG1IP.

[0063] In certain embodiments, the negatively sorted cells excluded from the sorted retinal cell population of the present disclosure express a marker that distinguishes astrocytes from photoreceptor cells. For example, but not limited to, such negative sorting can be performed based on the expression of one or more cell surface markers. In certain embodiments, such cell surface marker(s) can be selected from CD markers and surface receptors. In certain embodiments, such marker(s) show greater than 75% expression in astrocytes and less than 25% expression in photoreceptor cells. For example, but not limited to, such negative sorting can be performed based on the expression of one or more of the following: ITM2B; CD63; ENO1; CALR; CANX; CLU; SLC3A2; BSG; GPM6B; ITGB1; PTTG1IP; TIMP1; PMEPA1; SSR2; DKK3; LRP1; ATRAID; HLA-A; HLA-C; EMP3; TMED9; GOLIM4; LTBP. 3;GALNT1;CD151;PLD3;CALU;LSAMP;CD59;SLC2A1;LAMP2;HLA-B;COL11A1;DPP7;DCBLD2;CD164;SLC1A3;F3;CT SD;FLNA;SLC39A10;FN1;TMEM106C;TMEM179B;ATP1B3;HLA-E;TMEM132A;FLT1;FGFR1;CAPNS1;FAT1;ANGPTL1;L RP10;CRELD2;SPPL2A;TSPAN4;PRSS35;ECE1;SYPL1;SORCS2;COL2A1;DNER;COL6A1;CD44;GPC1;PCDH9;CRIM1;C HL1;TTYH3;IKBIP;NECTIN2;FBLN2;CCDC80;DAG1;PBXIP1;PRSS23;ACAA1;NTRK2;FSTL1;BCHE;TNFRSF1A;LGALS 3BP;ITGB8;CP;ADGRG1;VCAN;OLFM1;NRP1;SCARB1;TSPAN6;PCDH17;PLTP;NECTIN3;PTPRD;CADM4;UNC5B;CSPG5 ;AXL;PLXNB2;PLPP3;NOTCH2;SLITRK2;AEBP1;ANGPTL4;COLEC12;VAMP5;NLGN4X;FGFRL1;EFNB2;COL5A1;LAMB2;LAMC1;IGFBP3;FNDC5;FCGRT;ADRA2C;SERPING1;EPHB2;CDH11;COL1A2;CNTFR;AGRN;ROBO1;LOX;M RC2;COL6A2;SLC6A11;DSC2;IGSF8;EMP1;ABI3BP;TTYH2;NOTCH1;ANO6;A2M;SORCS1;EFNA1;PTPRG ;TF;EMP2;CEMIP2;SERPINE2;CDON;EGFR;PCDH7;MAN2A1;IL1R1;COL1A1;SEMA5A;ANTXR1;S1PR3;ITPRIP;MXRA8;PRELP;AQP1;CSF1;BCAN;ADGRA2;CA12;FAT3;HEPACAM;FGFR3;TRIL;HSD17B2; and HP. In certain embodiments, the particular subset of astrocyte markers used in negatively sorting astrocytes from photoreceptor cells are one or more of the following markers: ADGRL4; SERPINE2; BCHE; ABI3BP; NRP1; FSTL1; FAT1; NTRK2; FBLN2; PRSS35; SLC1A3; FCGRT; LAMC1; TF; SORCS2; DKK3; LRP1; PTPRD; ANGPTL1; LTBP3; CLU; CNTNAP2; CD151; PCDH9; CRIM1; CSPG5; and PMEPA1.

[0064] In certain embodiments, the negatively sorted cells excluded from the sorted retinal cell population of the present disclosure express markers that distinguish "brain spinal cord-like" (BSL) cells, i.e., BSL cells as described in Example 1, from photoreceptor cells. For example, but not by way of limitation, such negative sorting can be based on the expression of one or more cell surface markers. In certain embodiments, such cell surface marker(s) can be selected from CD markers and surface receptors. In certain embodiments, such marker(s) exhibit greater than 75% expression in BSL cells and less than 25% expression in photoreceptor cells. For example, and without limitation, such negative selection may be performed based on expression of one or more of the following: CLU; ITM2B; PTPRZ1; GPM6B; ATP1B2; CD63; BCAN; SLC1A3; SERPINE2; LRP1; PTPRA; ADGRG1; ENO1; CANX; SLC3A2; DNER; PTTG1IP; CALR; PCDH9; CCDC80; LSAMP; HEPACAM; F3; PLPP3; APLP2; FBLN2; TIMP1; SLC6A11; CSPG5; JAM2; FGFR3; DKK3; GOLIM4; NCAM1; CHL1; NRCAM; HLA-A; TMEM132A; PMEPA1; ITGAV; SSR2; ACAA1; BCHE; CD59; FAT3; PCDH17; ST3GAL5; PBXIP1; LAMP1; IT GB1;HP;ITGB8;SGCB;LAMP2;CLDND1;TMEM106B;PTCH1;PLTP;RNF13;HLA-C;PTPRD;TMEM30A;TRIL;RAB5C;T TYH3;DAG1;CADM4;UBA1;SLC6A9;LRRC8A;ATP1B3;SPPL2A;NTRK2;RNF130;LIFR;EMP3;PCDH7;NTRK3;COL6A 1;IL17D;LRP10;ADAM19;SGCE;FAT1;SLC44A1;LTBP3;SLC39A10;ABCA1;SYPL1;SLITRK2;GNPTG;CD302;MRC 2;LRP4;CALU;CD151;SORL1;TSPAN6;LRRN1;TENM2;CAPNS1;NLGN1;SLC15A2;NLGN4X;EGFR;ADORA1;SLC9A7;SIRPA;EFCAB14;ANGPTL1;FGFR1;VAMP5;CLDN12;LAMB2;GPR155;FGFR2;SLC44A2;LRP1B;PTGFRN;FNDC5;NOTC H1;DPY19L4;S1PR1;CD44;TNFRSF1A;FAM234A;CDH4;HLA-E;COL11A1;NCAM2;AQP1;CPQ;EMP1;FCGRT;GPC5;RO BO1;VCAN;LGALS3BP;LDLR;LRRC4B;NOTCH2;ALCAM;RYR3;SLC9A9;TMEM94;VCAM1;IGSF1;PCDHA10;CDH10;CAC HD1;P2RX7;AEBP1;PLXNB1;AXL;ALPL;ST3GAL4;SERPINI1;ITGB5;CD58;FGFRL1;PLPP1;TTYH2;IL17RB;FAM17 1A1;IL17RD;ANO6;ADAM22;PTPRG;ANTXR1;ZDHHC23;AGRN;COL14A1;POSTN;CNTFR;SEMA5A;FLNA;EMP2;TFPI;ITGA7;MXRA8;TENM4;FSTL1;CD82;NRP2;GPC4;ARSF;LAMC1;KIT;SEMA4A;LTBP1;and CSF1. In certain embodiments, a particular subset of BSL cell markers used in negatively sorting BSL cells from photoreceptor cells is one or more of the following markers:HEPACAM;FGFR3;SERPINE2;BCAN;CCDC80;PLPP3;CHL1;ADGRG1;SLC6A11;LSAMP;FBLN2;F3;SLC1A3;DKK3;LRP1;DNER;CLU;PCDH9;and CSPG5.;

[0065] In certain embodiments, the negatively sorted cells excluded from the sorted retinal cell population of the present disclosure express a marker that distinguishes astrocytes from photoreceptor cells, and the cells are further negatively sorted (before, after, or in conjunction with negative astrocyte sorting) based on the expression of a marker that distinguishes BLS cells from photoreceptor cells. For example, without limitation, such negative sorting can be performed based on the expression of one or more cell surface markers. In certain embodiments, such cell surface marker(s) can be selected from CD markers and surface receptors. In certain embodiments, such marker(s) show greater than 75% expression in astrocytes and less than 25% expression in photoreceptor cells for the marker(s) used for negative astrocyte sorting, and such marker(s) show greater than 75% expression in BSL cells and less than 25% expression in photoreceptor cells for the marker(s) used for negative BSL sorting. For example, and without limitation, such negative selection can be performed using the following astrocyte markers: ITM2B; CD63; ENO1; CALR; CANX; CLU; SLC3A2; BSG; GPM6B; ITGB1; PTTG1IP; TIMP1; PMEPA1; SSR2; DKK3; LRP1; ATRAID; HLA-A; HLA-C; EMP3; TMED9; GOLIM4; LTBP3; GALNT1; CD151; PLD3; CALU; LSAMP; CD59; SLC2A1; LAMP2; HLA-B; COL11A1; DPP7; DCBLD2; CD164; SLC1A3; F3; CTSD; FLNA; SLC39A 10;FN1;TMEM106C;TMEM179B;ATP1B3;HLA-E;TMEM132A;FLT1;FGFR1;CAPNS1;FAT1;ANGPTL1;LRP10;CRELD2;SPPL2A;TSPAN4;PRSS35;ECE1;SYPL1;SORCS2;COL2A1;DN ER;COL6A1;CD44;GPC1;PCDH9;CRIM1;CHL1;TTYH3;IKBIP;NECTIN2;FBLN2;CCDC80;DAG1;PBXIP1;PRSS23;ACAA1;NTRK2;FSTL1;BCHE;TNFRSF1A;LGALS3BP;ITGB8;CP;ADGRG1;VCAN;OLFM1;NRP1;SCARB1;TSPAN6;PCDH17;PLTP;NECTIN3;PTPRD;CADM4;UNC5B;CSPG5;AXL;PLXNB2;PLPP3;NOTCH2;SLITRK2;AEBP1;ANGPT L4;COLEC12;VAMP5;NLGN4X;FGFRL1;EFNB2;COL5A1;LAMB2;LAMC1;IGFBP3;FNDC5;FCGRT;ADRA2C;SERPING1;EPHB2;CDH11;COL1A2;CNTFR;AGRN;ROBO 1;LOX;MRC2;COL6A2;SLC6A11;DSC2;IGSF8;EMP1;ABI3BP;TTYH2;NOTCH1;ANO6;A2M;SORCS1;EFNA1;PTPRG;TF;EMP2;CEMIP2;SERPINE2;CDON;EGFR;P CDH7;MAN2A1;IL1R1;COL1A1;SEMA5A;ANTXR1;S1PR3;ITPRIP;MXRA8;PRELP;AQP1;CSF1;BCAN;ADGRA2;CA12;FAT3;HEPACAM;FGFR3;TRIL;HSD17B2; and HP, and the following BSL cell markers: CLU; ITM2B; PTPRZ1; GPM6B; ATP1B2; CD63; BCAN; SLC1A3; SERPINE2; LRP1; PTPRA; ADGRG1; ENO1; CANX; SLC3A2; DNER; PTTG1IP; CALR; PCDH9; CCDC80; LSAMP; HEPACAM; F3; PLPP3; APLP2; FBLN2; TIMP1; SLC6A11; CSPG5; JAM2; FGFR3; DKK3; GOLIM4; NCAM1; CHL1; N RCAM;HLA-A;TMEM132A;PMEPA1;ITGAV;SSR2;ACAA1;BCHE;CD59;FAT3;PCDH17;ST3GAL5;PBXIP1;LAMP1;ITGB1;HP;ITGB8;SGCB;LAMP2;CLDND1;TMEM1 06B;PTCH1;PLTP;RNF13;HLA-C;PTPRD;TMEM30A;TRIL;RAB5C;TTYH3;DAG1;CADM4;UBA1;SLC6A9;LRRC8A;ATP1B3;SPPL2A;NTRK2;RNF130;LIFR;EMP3;PCDH7;NTRK3;COL6A1;IL17D;LRP10;ADAM19;SGCE;FAT1;SLC44A1;LTBP3;SLC39A10;ABCA1;SYPL1;SLITRK2;GNP TG;CD302;MRC2;LRP4;CALU;CD151;SORL1;TSPAN6;LRRN1;TENM2;CAPNS1;NLGN1;SLC15A2;NLGN4X;EGFR;ADORA1 ;SLC9A7;SIRPA;EFCAB14;ANGPTL1;FGFR1;VAMP5;CLDN12;LAMB2;GPR155;FGFR2;SLC44A2;LRP1B;PTGFRN;FNDC5 ;NOTCH1;DPY19L4;S1PR1;CD44;TNFRSF1A;FAM234A;CDH4;HLA-E;COL11A1;NCAM2;AQP1;CPQ;EMP1;FCGRT;GPC5; ROBO1;VCAN;LGALS3BP;LDLR;LRRC4B;NOTCH2;ALCAM;RYR3;SLC9A9;TMEM94;VCAM1;IGSF1;PCDHA10;CDH10;CACH D1;P2RX7;AEBP1;PLXNB1;AXL;ALPL;ST3GAL4;SERPINI1;ITGB5;CD58;FGFRL1;PLPP1;TTYH2;IL17RB;FAM171A1; This can be based on expression of one or more of: IL17RD; ANO6; ADAM22; PTPRG; ANTXR1; ZDHHC23; AGRN; COL14A1; POSTN; CNTFR; SEMA5A; FLNA; EMP2; TFPI; ITGA7; MXRA8; TENM4; FSTL1; CD82; NRP2; GPC4; ARSF; LAMC1; KIT; SEMA4A; LTBP1; and CSF1. In certain embodiments, the specific subset of astrocyte markers used in negatively sorting astrocytes from photoreceptor cells includes the following markers: ADGRL4; SERPINE2; BCHE; ABI3BP; NRP1; FSTL1; FAT1; NTRK2; FBLN2; PRSS35; SLC1A3; FCGRT; LAMC1; TF; SORCS2; DKK3; LRP1; PTPRD; ANGPTL1; LTBP3; CLU; CNTNAP2; CD151; PCDH9; CRIM1; CSPG5;and PMEPA1, and a specific subset of BSL cell markers used in negatively sorting BSL cells from photoreceptor cells are one or more of the following markers: HEPACAM; FGFR3; SERPINE2; BCAN; CCDC80; PLPP3; CHL1; ADGRG1; SLC6A11; LSAMP; FBLN2; F3; SLC1A3; DKK3; LRP1; DNER; CLU; PCDH9; and CSPG5.

[0066] In certain embodiments, at least about 60% of the cells of the sorted retinal cell population of the present disclosure express a marker of photoreceptor cell identity. For example, and without limitation, the marker of photoreceptor cell identity is CRX or RCVRN. In certain embodiments, at least about 65% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 70% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 75% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 80% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 85% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 90% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 90% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 95% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, up to about 100% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity.

[0067] In certain embodiments, at least about 15% to about 45% of the cells of the retinal cell population of the present disclosure express at least one marker of cone photoreceptor cell identity. For example, and without limitation, the marker of cone photoreceptor cell identity can be CNGA3, CNGB3, ARR3, THRB, or S-opsin. In certain embodiments, at least about 20% to about 45% of the cells of the retinal cell population of the present disclosure express a marker of cone photoreceptor cell identity. In certain embodiments, at least about 25% to about 45% of the cells of the retinal cell population of the present disclosure express a marker of photoreceptor cell identity. In certain embodiments, at least about 30% to about 45% of the cells of the retinal cell population of the present disclosure express a marker of cone photoreceptor cell identity. In certain embodiments, at least about 35% to about 45% of the cells of the retinal cell population of the present disclosure express a marker of cone photoreceptor cell identity. In certain embodiments, at least about 40% to about 45% of the cells in the retinal cell populations of the present disclosure express markers of cone photoreceptor cell identity.

[0068] In certain embodiments, at least about 30% of the cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express CNGA3. In certain embodiments, at least about 30% of the cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express CNGB3. In certain embodiments, at least about 20% of the cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express ARR3. In certain embodiments, at least about 3% of the cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express THRB. In certain embodiments, at least one cell of the retinal cell population expressing at least one marker of cone photoreceptor cell identity expresses S-opsin.

[0069] In certain embodiments, at least about 30% of cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express CNGA3, at least about 30% of cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express CNGB3, at least about 20% of cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express ARR3, at least about 3% of cells of the retinal cell population expressing at least one marker of cone photoreceptor cell identity express THRB, and at least one cell of the retinal cell population expressing at least one marker of cone photoreceptor cell identity expresses S-opsin.

[0070] In certain embodiments, at least about 55% to about 85% of the cells of the retinal cell population of the present disclosure express at least one marker of rod photoreceptor cell identity. For example, but not limited to, the marker of rod photoreceptor cell identity can be NRL, NR2E3, PDE6B, CNGA1 or RHO. In certain embodiments, at least about 60% to about 85% of the cells of the retinal cell population of the present disclosure express a marker of rod photoreceptor cell identity. In certain embodiments, at least about 65% to about 85% of the cells of the retinal cell population of the present disclosure express a marker of rod photoreceptor cell identity. In certain embodiments, at least about 70% to about 85% of the cells of the retinal cell population of the present disclosure express a marker of rod photoreceptor cell identity. In certain embodiments, at least about 75% to about 85% of the cells of the retinal cell population of the present disclosure express a marker of rod photoreceptor cell identity. In certain embodiments, at least about 80% to about 85% of the cells in the retinal cell populations of the present disclosure express markers of rod photoreceptor cell identity.

[0071] In certain embodiments, at least about 50% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express NRL. In certain embodiments, at least about 40% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express NR2E3. In certain embodiments, at least about 20% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express PDE6B. In certain embodiments, at least about 30% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express CNGA1. In certain embodiments, at least one cell of the retinal cell cluster expressing at least one marker of rod photoreceptor cell identity expresses RHO.

[0072] In certain embodiments, at least about 50% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express NRL, at least about 40% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express NR2E3, at least about 20% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express PDE6B, at least about 30% of the cells of the retinal cell population expressing at least one marker of rod photoreceptor cell identity express CNGA1, and at least one cell of the retinal cell population expressing at least one marker of rod photoreceptor cell identity expresses RHO.

[0073] In certain embodiments, cells of the retinal cell populations of the present disclosure are selected to contain no more than about 40% of cells expressing markers of non-photoreceptor cell identity, such as, but not limited to, markers of non-photoreceptor cell identity associated with bipolar cells, Muller glial cells, retinal microglia, forebrain neural progenitor cells, retinal progenitor cells, horizontal cells, ganglion cells, retinal amacrine cells, and retinal pigment epithelial cells.

[0074] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they comprise less than about 10% bipolar cells. In certain embodiments, the markers associated with bipolar cell identity are one or more of ISL1, SEBOX, CAPB5, BHLHE23, GRM6, SCGN, NRN1L, GRIK1, KLHDC8A and PROX.

[0075] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected to contain less than about 20% Müller glial cells. In certain embodiments, the markers associated with Müller glial cell identity are one or more of AQP4, PRDX6, VIM, HES1, SLC1A3, GLUL, CLU, RLBP1 and LHX2.

[0076] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they comprise less than about 10% retinal microglial cells. In certain embodiments, the markers associated with retinal microglial cell identity are one or more of PTPRC, MPEG1, and CXCR1.

[0077] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected to comprise less than about 5% forebrain neural progenitor cells.In certain embodiments, the markers related to the identity of forebrain neural progenitor cells are one or more of NKX2.2, RGCC, NEUROD1, BTG2, GADD45A and GADD45G.

[0078] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected so that they contain less than about 3% retinal progenitor cells.In certain embodiments, the markers associated with the identity of retinal progenitor cells are one or more of HOPX, CDK4, CCND2, VSX2, FGF19, SFRP2, CCNB2, and CCND1.

[0079] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they comprise less than about 10% horizontal cells. In certain embodiments, the marker associated with horizontal cell identity is one or more of ONECUT2, ONECUT1 and LHX1.

[0080] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they comprise less than about 10% retinal ganglion cells. In certain embodiments, the markers associated with retinal ganglion cell identity are one or more of POU4F1, THY1, BRN3B, POU4F2, POU4F3, ISL2, RBPMS, and SNCG.

[0081] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they contain less than about 5% retinal amacrine cells. In certain embodiments, the markers associated with retinal amacrine cell identity are one or more of TFAP2A, TFAP2B, ELAVL3, NeuN, and ELAVL4.

[0082] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that they contain less than about 10% retinal pigment epithelial cells. In certain embodiments, the markers associated with retinal pigment epithelial cell identity are one or more of BEST1, TIMP3, GRAMD3, and PITPNA.

[0083] In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that less than 30% of the cells express a marker associated with inflammatory cell identity. For example, but not limited to, the markers of inflammatory cell identity are CD15, CD133, A2B5 and CD38. In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that less than about 30% of the cells express A2B5 and / or CD38. In certain embodiments, the cells of the retinal cell population of the present disclosure are selected such that no more than one cell expresses CD15 or CD133.

[0084] The present disclosure provides sorted cell populations of in vitro differentiated retinal cells, in which at least about 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the differentiated cells express at least one marker of photoreceptor cell identity.

[0085] In certain embodiments, the present disclosure provides sorted cell populations of in vitro differentiated retinal cells, in which at least less than about 40% (e.g., less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%) of the differentiated cells express at least one marker of non-photoreceptor cell identity.

[0086] In certain embodiments, the sorted population of in vitro differentiated retinal cells comprises about 1×10 4 ~Approx. 1×10 10 pieces, about 1×10 4 ~Approx. 1×10 5 pieces, about 1×10 5 ~Approx. 1×10 9 pieces, about 1×10 5 ~Approx. 1×10 6 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 6 ~Approx. 1×10 7 pieces, about 1×10 6 ~Approx. 1×10 8 pieces, about 1×10 7 ~Approx. 1×10 8 pieces, about 1×10 8 ~Approx. 1×10 9 pieces, about 1×10 8 ~Approx. 1×10 10 1 x 10 pieces or approx. 9 ~Approx. 1×10 10 10 in vitro differentiated photoreceptor cells.

[0087] The present disclosure also provides a composition comprising such a sorted population of in vitro differentiated retinal cells. In certain embodiments, the sorted population of in vitro differentiated retinal cells is obtained by the differentiation method described herein. In certain embodiments, the composition is frozen. In certain embodiments, the composition further comprises at least one cryoprotectant, such as, but not limited to, dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0088] In certain embodiments, the composition is a pharmaceutical composition that includes pharma- ceutical acceptable carrier.The composition can be used to prevent and / or treat hereditary or acquired retinal degenerative disease, such as retinitis pigmentosa, total choroidal atrophy, Stargardt's disease, cone-rod dystrophy, Leber's congenital amaurosis, and age-related macular degeneration, including but not limited to "dry" age-related macular degeneration and "wet" age-related macular degeneration.

[0089] 4. Methods for treating inherited retinal degenerative diseases The selected retinal cell populations and compositions disclosed herein can be used to prevent and / or treat inherited and / or acquired retinal degenerative diseases. For example, but not limited to, the selected retinal cell populations and compositions disclosed herein can be used in CCTT, which is understood to act, without being bound by theory, by repairing dysfunctional photoreceptor cells present in the recipient's retina. Again, without being bound by theory, it is understood that the selected retinal cell populations and compositions disclosed herein exert their therapeutic effect, at least in part, by transferring healthy cellular components, such as organelles including mitochondria, together with other nuclear, cell membrane-bound, and / or cytoplasmic components, such as therapeutic proteins. Thus, the subject matter of the present disclosure provides a method of preventing and / or treating inherited and / or acquired retinal degenerative diseases. In certain embodiments, the method comprises administering the presently disclosed selected population of retinal cells, such as stem cell-derived retinal cells, or a composition comprising the same, to a subject suffering from inherited or acquired retinal degenerative diseases. In certain embodiments, the compositions described herein are pharmaceutical compositions that further comprise a pharma- ceutically acceptable carrier.

[0090] CCTT is effective in multiple mutation classes. For example, CCT is effective in X-linked mutations, autosomal dominant (AD) mutations, autosomal recessive (AR) mutations, and non-Mendelian mutations, such as mitochondrial mutations. In addition, for AD mutations, CCTT is effective in haploinsufficient or dominant negative mutations (e.g., dominant negative interfering mutations and dominant negative toxic mutations). CCTT has also been shown to be effective in transferring multiple types of cellular components, such as membrane-bound proteins, nuclear localized proteins, and cytoplasmic proteins. CCTT is also effective in transferring cellular components to both types of photoreceptor cells, i.e., rods and cones.

[0091] Non-limiting examples of inherited retinal degenerative diseases include retinitis pigmentosa, total choroidal atrophy, Stargardt's disease, cone-rod dystrophy and Leber's congenital amaurosis. Non-limiting examples of acquired retinal degenerative diseases include age-related macular degeneration, including but not limited to "dry" age-related macular degeneration and "wet" age-related macular degeneration.

[0092] The sorted population of retinal cells or compositions described herein can be administered in any physiologically acceptable vehicle. The cells or compositions of the present disclosure can be administered via local injection or subretinal transplantation. In certain embodiments, the sorted population of cells or compositions are resuspended in medium and transplanted into the subretinal space using a device that preserves their biological activity and ensures on-target placement. In certain embodiments, the device is composed of a biocompatible material. In certain embodiments, the device achieves transplantation with limited shear stress on the cells, for example, includes a low-friction passage. An exemplary device for subretinal transplantation is described in International Patent Application No. PCT / US2019 / 045074 (published as International Publication No. WO2020028892), which is incorporated herein by reference in its entirety.

[0093] The cells or compositions described herein can be conveniently provided as a sterile liquid formulation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition that can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. A liquid or viscous composition can include a carrier, which can be, for example, a solvent or dispersion medium containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. A sterile injectable solution can be prepared by incorporating a composition of the subject matter of the present disclosure, for example, a composition comprising stem cell-derived retinal cells of the present disclosure, in a required amount of an appropriate solvent, along with various amounts of other ingredients as needed. Such compositions can be mixed with an appropriate carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, and the like. The composition can also be lyophilized.The composition can contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the preparation desired.Suitable preparations can be prepared without undue experimentation by referring to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985, which is incorporated herein by reference.

[0094] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum inurn monostearate and gelatin.

[0095] The viscosity of the composition can be maintained at a selected level using a pharma- ceutically acceptable thickening agent if desired. Methylcellulose can be used because it is readily and economically available and easy to handle. Other suitable thickening agents include, for example, hyaluronic acid, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. The selection of suitable carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or other liquid form, for example, a time-release form or a liquid-filled form).

[0096] Those skilled in the art will recognize that the non-cell-derived components of the composition should generally, but not exclusively, be selected to be chemically inert and therefore not affect the viability or efficacy of the retinal cells of the present disclosure.This does not present a problem to those skilled in the art of chemical and pharmaceutical principles, or can be easily avoided from the present disclosure and the literature cited herein, by referring to standard textbooks, or by simple experimentation (without undue experimentation).

[0097] In certain embodiments, the compositions described herein comprise an effective amount of selected retinal cells. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to affect beneficial or desired clinical outcomes upon treatment. An effective amount can be administered to a subject in at least one dose. In terms of treatment, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse or delay the progression of an inherited or acquired retinal degenerative disease, or otherwise alleviate the pathological consequences of an inherited or acquired retinal degenerative disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. When determining the appropriate dosage to achieve an effective amount, several factors are usually considered. These factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells being administered.

[0098] In certain embodiments, the effective amount of cells is sufficient to improve the retinal function of the subject suffering from hereditary or acquired retinal degenerative disease.In certain embodiments, the effective amount of cells is sufficient to improve the retinal function of the subject suffering from hereditary or acquired retinal degenerative disease, for example, the improved function can be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or about 100% of the retinal function of the individual who does not suffer from hereditary or acquired retinal degenerative disease.

[0099] The amount of selected cells administered will vary depending on the subject being treated. In certain embodiments, the amount is about 1×10 4 ~Approx. 1×10 10 pieces, about 1×10 4 ~Approx. 1×10 5 pieces, about 1×10 5 ~Approx. 1×10 9 pieces, about 1×10 5 ~Approx. 1×10 6 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 6 ~Approx. 1×10 7 pieces, about 1×106 ~Approx. 1×10 8 pieces, about 1×10 7 ~Approx. 1×10 8 pieces, about 1×10 8 ~Approx. 1×10 9 pieces, about 1×10 8 ~Approx. 1×10 10 pcs or approx. 1×10 9 ~Approx. 1×10 10 In certain embodiments, about 1×10 selected cells are administered to the subject. 5 pieces~approx. 1×10 7 The selected cells are administered to a subject suffering from an inherited or acquired retinal degenerative disease. In certain embodiments, about 1×10 6 pieces~approx. 1×10 7 The selected cells are administered to a subject suffering from an inherited or acquired retinal degenerative disease. In certain embodiments, about 1×10 6 pieces~approx. 4×10 6 The selected cells are administered to the subject suffering from inherited or acquired retinal degenerative disease.The exact determination of what is considered to be an effective dose may be based on the individual factors of each subject, including the size, age, sex, weight and condition of the particular subject.The dosage can be easily ascertained by those skilled in the art from this disclosure and the knowledge of those skilled in the art.

[0100] Exemplary embodiments A. In certain non-limiting embodiments, the presently disclosed subject matter provides an in vitro method of producing a sorted population of retinal cells, comprising generating three-dimensional retinal organoids, dissociating the three-dimensional retinal organoids, and positively sorting retinal cells based on one or more markers of photoreceptor cell identity and / or negatively sorting retinal cells based on one or more markers of non-photoreceptor cell identity to produce a sorted population of retinal cells.

[0101] A1. The marker of photoreceptor cell identity is CD73; said markers of non-photoreceptor cell identity being one or more of CD24, CD302, CD9 and CD99; ITM2B, CD63, ENO1, CALR, CANX, CLU, SLC3A2, BSG, and GP M6B, ITGB1, PTTG1IP, TIMP1, PMEPA1, SSR2, DKK3, LRP1, ATRAID, HLA-A, HLA -C、EMP3、TMED9、GOLIM4、LTBP3、GALNT1、CD151、PLD3、CALU、LSAMP、CD59、S LC2A1, LAMP2, HLA-B, COL11A1, DPP7, DCBLD2, CD164, SLC1A3, F3, CTSD, FLNA SLC39A10, FN1, TMEM106C, TMEM179B, ATP1B3, HLA-E, TMEM132A, FLT1, FGF R1, CAPNS1, FAT1, ANGPTL1, LRP10, CRELD2, SPPL2A, TSPAN4, PRSS35, ECE1, S YPL1, SORCS2, COL2A1, DNER, COL6A1, CD44, GPC1, PCDH9, CRIM1, CHL1, TTYH 3. IKBIP, NECTIN2, FBLN2, CCDC80, DAG1, PBXIP1, PRSS23, ACAA1, NTRK2, FST L1, BCHE, TNFRSF1A, LGALS3BP, ITGB8, CP, ADGRG1, VCAN, OLFM1, NRP1, SCAR B1, TSPAN6, PCDH17, PLTP, NECTIN3, PTPRD, CADM4, UNC5B, CSPG5, AXL, PLXNB 2. PLPP3, NOTCH2, SLITRK2, AEBP1, ANGPTL4, COLEC12, VAMP5, NLGN4X, and FGFR L1, EFNB2, COL5A1, LAMB2, LAMC1, IGFBP3, FNDC5, FCGRT, ADRA2C, SERPING1 EPHB2, CDH11, COL1A2, CNTFR, AGRN, ROBO1, LOX, MRC2, COL6A2, SLC6A11, DS C2, IGSF8, EMP1, ABI3BP, TTYH2, NOTCH1, ANO6, A2M, SORCS1, EFNA1, PTPRG, T F. EMP2, CEMIP2, SERPINE2, CDON, EGFR, PCDH7, MAN2A1, IL1R1, COL1A1, SEM A5A, ANTXR1, S1PR3, ITPRIP, MXRA8, PRELP, AQP1, CSF1, BCAN, ADGRA2, CA12.A. The aforementioned method of claim 1, wherein one or more of FAT3, HEPACAM, FGFR3, TRIL, HSD17B2 and HP are selected from the group consisting of FAT3, HEPACAM, FGFR3, TRIL, HSD17B2 and HP.

[0102] A2. The aforementioned method of A, wherein said markers of non-photoreceptor cell identity are one or more of DKK3, LRP1, CLU, PMEPA1, ITGB1, and PTTG1IP.

[0103] A3. The one or more markers of non-photoreceptor cell identity are: ITM2B, CD63, ENO1, CALR, CANX, CLU, SLC3A2, BSG, GPM6B, ITGB1, PTTG1IP, TIMP1, PMEPA1, SSR2, DKK3, LRP1, ATRAID, HLA-A, HLA-C, EMP3, TMED9, GOLIM4, LTBP3, GALNT1, CD151, PLD3, CALU, LSAMP, CD59, SLC2A1, LAMP2, HLA-B, COL11A1, DPP7, DCBLD2, CD164, SLC1A3, F3, CTSD, FLNA, SLC39A10, FN1, TMEM106C, TMEM179B, ATP1B3, HLA-E, TMEM132A, FLT1, FGFR1, CAPNS1, FAT1, ANGPTL1, LRP10, CRELD2, SPPL2A, TSPAN4, P RSS35, ECE1, SYPL1, SORCS2, COL2A1, DNER, COL6A1, CD44, GPC1, PCDH9, CRIM1, CHL1, TTYH3, IKBIP, NECTIN2, FBLN2, CCDC80, DAG1, PBXIP1, PRSS23, ACA A1, NTRK2, FSTL1, BCHE, TNFRSF1A, LGALS3BP, ITGB8, CP, ADGRG1, VCAN, OLFM1, NRP1, SCARB1, TSPAN6, PCDH17, PLTP, NECTIN3, PTPRD, CADM4, UNC5B, CSP G5, AXL, PLXNB2, PLPP3, NOTCH2, SLITRK2, AEBP1, ANGPTL4, COLEC12, VAMP5, NLGN4X, FGFRL1, EFNB2, COL5A1, LAMB2, LAMC1, IGFBP3, FNDC5, FCGRT, ADRA 2C, SERPING1, EPHB2, CDH11, COL1A2, CNTFR, AGRN, ROBO1, LOX, MRC2, COL6A2, SLC6A11, DSC2, IGSF8, EMP1, ABI3BP, TTYH2, NOTCH1, ANO6, A2M, SORCS1, E FNA1, PTPRG, TF, EMP2, CEMIP2, SERPINE2, CDON, EGFR, PCDH7, MAN2A1, IL1R1, COL1A1, SEMA5A, ANTXR1, S1PR3, ITPRIP, MXRA8, PRELP, AQP1, CSF1, BCAN,The aforementioned method of A, wherein the one or more astrocyte markers are selected from ADGRA2, CA12, FAT3, HEPACAM, FGFR3, TRIL, HSD17B2 and HP.

[0104] A4. The aforementioned method of A3, wherein said one or more markers of non-photoreceptor cell identity are one or more astrocyte markers selected from ADGRL4, SERPINE2, BCHE, ABI3BP, NRP1, FSTL1, FAT1, NTRK2, FBLN2, PRSS35, SLC1A3, FCGRT, LAMC1, TF, SORCS2, DKK3, LRP1, PTPRD, ANGPTL1, LTBP3, CLU, CNTNAP2, CD151, PCDH9, CRIM1, CSPG5 and PMEPA1.

[0105] A5. One or more of the markers of non-photoreceptor cell identity are: CLU, ITM2B, PTPRZ1, GPM6B, ATP1B2, CD63, BCAN, SLC1A3, SERPINE2, LRP1, PTPRA, ADGRG1, ENO1, CANX, SLC3A2, DNER, PTTG1IP, CALR, PCDH9, CCDC80, LSAMP, HEPACAM, F3, PLPP3, APLP2, FBLN2, TIMP1, SLC6A11, CSPG5, JAM2, FGFR3, DKK3, GOLIM4, NCAM1, CHL1, NRCAM , HLA-A, TMEM132A, PMEPA1, ITGAV, SSR2, ACAA1, BCHE, CD59, FAT3, PCDH17, ST3GAL5, PBXIP1, LAMP1, ITGB1, HP, ITGB8, SGCB, LAMP2, CLDND1, TMEM106B , PTCH1, PLTP, RNF13, HLA-C, PTPRD, TMEM30A, TRIL, RAB5C, TTYH3, DAG1, CADM4, UBA1, SLC6A9, LRRC8A, ATP1B3, SPPL2A, NTRK2, RNF130, LIFR, EMP3, PCD H7, NTRK3, COL6A1, IL17D, LRP10, ADAM19, SGCE, FAT1, SLC44A1, LTBP3, SLC39A10, ABCA1, SYPL1, SLITRK2, GNPTG, CD302, MRC2, LRP4, CALU, CD151, SOR L1, TSPAN6, LRRN1, TENM2, CAPNS1, NLGN1, SLC15A2, NLGN4X, EGFR, ADORA1, SLC9A7, SIRPA, EFCAB14, ANGPTL1, FGFR1, VAMP5, CLDN12, LAMB2, GPR155, FG FR2, SLC44A2, LRP1B, PTGFRN, FNDC5, NOTCH1, DPY19L4, S1PR1, CD44, TNFRSF1A, FAM234A, CDH4, HLA-E, COL11A1, NCAM2, AQP1, CPQ, EMP1, FCGRT, GPC5, ROBO1, VCAN, LGALS3BP, LDLR, LRRC4B, NOTCH2, ALCAM, RYR3, SLC9A9, TMEM94, VCAM1, IGSF1, PCDHA10, CDH10, CACHD1, P2RX7, AEBP1, PLXNB1, AXL, ALPL,The aforementioned method of A, wherein the marker is one or more cerebrospinal-like (BSL) cell markers selected from ST3GAL4, SERPINI1, ITGB5, CD58, FGFRL1, PLPP1, TTYH2, IL17RB, FAM171A1, IL17RD, ANO6, ADAM22, PTPRG, ANTXR1, ZDHHC23, AGRN, COL14A1, POSTN, CNTFR, SEMA5A, FLNA, EMP2, TFPI, ITGA7, MXRA8, TENM4, FSTL1, CD82, NRP2, GPC4, ARSF, LAMC1, KIT, SEMA4A, LTBP1 and CSF1.

[0106] A6. The aforementioned method of A5, wherein said one or more markers of non-photoreceptor cell identity are one or more BSL markers selected from HEPACAM, FGFR3, SERPINE2, BCAN, CCDC80, PLPP3, CHL1, ADGRG1, SLC6A11, LSAMP, FBLN2, F3, SLC1A3, DKK3, LRP1, DNER, CLU, PCDH9 and CSPG5.

[0107] A7. The method of A-A1, wherein the three-dimensional retinal organoids are enzymatically dissociated.

[0108] A8. The aforementioned method of A7, wherein said enzyme is papain and / or trypsin.

[0109] A9. The method of A7, wherein the retinal cells are contacted with a composition to ensure that the cells remain in a dissociated cell suspension.

[0110] A10. The aforementioned method of A9, wherein said composition is an enzyme.

[0111] A11. The aforementioned method of A10, wherein the enzyme is a DNAse.

[0112] A12. The aforementioned method of A-A1, wherein the three-dimensional retinal organoids reach about DD45-DD300 before being dissociated.

[0113] A13. The aforementioned method of A12, wherein the three-dimensional retinal organoids reach about DD90 to about DD140 before being dissociated.

[0114] A14. The method of any one of A to A13, wherein the retinal cell population consists of at least about 70% single cells.

[0115] A15. The aforementioned method of A14, wherein said retinal cell population consists of at least about 80% single cells.

[0116] A16. The aforementioned method of A14, wherein said retinal cell population consists of at least about 90% single cells.

[0117] A17. The method of any one of A to A16, wherein the retinal cell population comprises about 55% to about 85% rod photoreceptor cells.

[0118] A18. The aforementioned method of A-A17, wherein said stem cells are selected from human, non-human primate or rodent non-embryonic stem cells; human, non-human primate or rodent embryonic stem cells; human, non-human primate or rodent induced pluripotent stem cells; and human, non-human primate or rodent recombinant pluripotent cells.

[0119] A19. The aforementioned method of A to A18, wherein the stem cells are human stem cells.

[0120] A20. The aforementioned method of A to A19, wherein the stem cells are pluripotent stem cells or multipotent stem cells.

[0121] A21. The aforementioned method of A to A20, wherein the stem cells are pluripotent stem cells.

[0122] A22. The aforementioned method of A-A21, wherein said pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells and combinations thereof.

[0123] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a sorted population of in vitro differentiated retinal cells, wherein the in vitro differentiated retinal cells are obtained by the aforementioned methods of A-A22.

[0124] C. In certain non-limiting embodiments, the presently disclosed subject matter provides compositions comprising a population of B cells.

[0125] C1. The aforementioned composition of C, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0126] D. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of preventing and / or treating an inherited or acquired retinal degenerative disease in a subject, comprising administering to the subject an effective amount of: (a) the selected population of in vitro differentiated retinal cells of B, or (b) one of the compositions of C-C1.

[0127] D1. The aforementioned method of D, wherein said inherited retinal degenerative disease is selected from retinitis pigmentosa, total choroidal atrophy, Stargardt's disease, cone-rod dystrophy and Leber's congenital amaurosis.

[0128] D2. The aforementioned method of D, wherein said acquired retinal degenerative disease is age-related macular degeneration.

[0129] E. In certain non-limiting embodiments, the presently disclosed subject matter provides a selected population of in vitro differentiated retinal cells of claim A18, or a composition of C-C1, for use in preventing and / or treating an inherited or acquired retinal degenerative disease in a subject.

[0130] E1. The aforementioned sorted population or composition of in vitro differentiated retinal cells for use in the prevention and / or treatment of an inherited retinal degenerative disease in a subject of E, wherein said inherited retinal degenerative disease is retinitis pigmentosa, total choroidal atrophy, Stargardt's disease, cone-rod dystrophy, or Leber's congenital amaurosis.

[0131] E2. The aforementioned selected population or composition of in vitro differentiated retinal cells for use in the prevention and / or treatment of an acquired retinal degenerative disease in a subject of claim E, wherein said acquired retinal degenerative disease is age-related macular degeneration. EXAMPLES

[0132] Working Example 1. Human donor cells migrate out of or remain in the subretinal space To determine how recipient subretinal space affects donor cells, we differentiated human retinal organoids and transplanted them into recipient mice, and then evaluated the location, fate and maturation of donor cells. To generate recipient mice, we cross-bred mice with immune deficiency and retinal degeneration. These C3H / HeJ-Pde6bRd1 / Rd1 (Rd1) and NOD.CB17-Prkdcscid / J (NOD / Scid) double mutant mice are called Rd1 / NS. To generate donor cells, we used H9 human embryonic stem cells (hESCs) carrying a reporter expressed in all photoreceptors (CRX:tdTomato). We used a gravity aggregation approach to differentiate stem cells into retinal organoids with robust generation of photoreceptors. On day 134 of organoid culture, we microdissected human retinal organoids and transplanted fragments into the subretinal space of recipient eyes (n=16 eyes). Four and a half months later, we evaluated the transplants. Because homozygosity for the Rd1 allele causes virtually all photoreceptors to be altered in mice by adulthood, separate recipient outer nuclear and outer plexiform layers were not observed, but the inner nuclear layer, inner plexiform layer, retinal ganglion cells (RGCs) and retinal nerve fiber layer (collectively, the "inner retina") were present. We determined the location of donor cells relative to the subretinal transplantation site. We identified all human donor cells based on immunolabeling for human nuclear antigen (HNA) or human ATP-dependent DNA helicase 2 subunit (Ku80 protein). We identified human donor photoreceptors based on transgenic expression of CRX:tdTomato. We observed two major classes of donor cells: (1) human cells in the recipient subretinal space that were either photoreceptor cells or non-photoreceptor cells ("non-migratory cells") (Figure 1A); (2) human cells in the recipient inner retina that were not photoreceptor cells ("migratory cells") (Figure 1A), suggesting that this population had migrated from the graft.The migrating cells migrated to all retinal layers, including the RGC, inner plexiform layer (IPL), inner nuclear layer (INL), and retinal pigment epithelium / choroid (RPE / C) layers (Figure 1E). While a subset of migrating cells was observed in the inner retina of the recipient overlying the graft ("radial migration"), including the area adjacent to the optic nerve, others migrated tangentially beyond the edge of the graft ("tangential migration") ("peripapillary migration" Figure 1B). 98.9% (n=2,378 cells) of the tangentially migrating cells were within 1500 μm of the edge of the graft. The remaining 1.1% migrated beyond 1500 μm and were located exclusively in the retinal ganglion cell (RGC) layer (Figure 1D). Next, we sought to molecularly classify the fates of these non-migratory and migratory cells.

[0133] 2. Donor cells adopt retinal and non-retinal cell fates To determine how the recipient subretinal microenvironment influences gene expression and cell fate specification of migratory and non-migratory donor cells, we performed single-cell RNA sequencing on cells derived from human retinal organoids transplanted and matured in vivo ("transplanted organoids") and cells derived from age-matched organoids maintained in vitro ("cultured organoids") (Figure 2A). We analyzed a total of 5,831 human cells recovered from transplanted organoids (1,561 cells) and cultured organoids (4,270 cells). We identified retinal cell types based on their gene expression profiles, including retinal progenitor cells (RPCs), photoreceptor cell precursors, rods, cones, bipolar cells, horizontal cells, and Müller glia (Figure 2B-D). The amounts of cones, bipolar cells, and horizontal cells were similar in transplanted and cultured organoids. In contrast, retinal progenitor cells (RPCs), photoreceptor progenitors, and Müller glia were more abundant in cultured organoids, whereas rods were more abundant in transplanted organoids (Figure 2E). The smaller populations of RPCs and photoreceptor progenitors and the larger population of rods in transplanted organoids suggests that the recipient microenvironment promotes retinal cell fate specification and maturation.

[0134] In addition to these cell types, we identified two cell clusters that could not be ascribed solely to known retina-derived cell fates. Cells in one cluster expressed genes that are broadly expressed in retina and other CNS progenitors, such as ASCL1 and HES6 (Figure 2D, Supplementary Figure S4). They also expressed genes that are not normally detected in the developing retina, including NKX2-2 and ARX, both of which are prominently expressed in ventral telencephalic and diencephalic neural progenitors, and HOXC8, whose expression is normally restricted to the developing spinal cord (Figure 2D). Based on this gene expression profile, we named the cells in this cluster "brain-spinal cord-like" (BSL) cells. BSL cells constituted approximately 1% of the cells in cultured organoids, but were more than 30-fold more abundant in transplanted organoids (Figure 2E). Cells in the second cluster expressed markers characteristic of retinal astrocytes, such as PAX2 and S100B (Figure 2D). Normally, retinal astrocytes are born in the optic nerve head and migrate to the retina. Surprisingly, astrocytes were completely absent from cultured organoids, but comprised approximately 8% of the cells in transplanted organoids (Figure 2E). These data suggest that the recipient microenvironment instructs some donor cells to assume fates not normally acquired by retinal progenitor cells.

[0135] 3. Actively proliferating cells are rare among migratory and nonmigratory donor cells Migratory cells, especially if they are proliferative, can have a negative effect on the recipient. To determine the effect of the recipient microenvironment on the proliferation of migratory and non-migratory donor cells, we examined the expression of the proliferation marker protein Ki-67. As expected, Ki-67 expression was significantly higher in cultured organoids than in CRX:tdTomato. +It was hardly observed in photoreceptor precursors and was significantly less in transplanted organoids (Figure 4A). In eyes transplanted with organoids, 0.7% of non-migrating cells and 1.4% of migrating cells expressed Ki-67 (Figure 4B), and the difference between these values ​​was not statistically significant. We found that a small number of Ki-67 + We observed that migrating cells occupied all retinal laminae of the recipients (Figure 4C).

[0136] To identify proliferating cells, we developed a proliferation scoring system by computationally aggregating the expression levels of proliferation-related genes (Supplementary Data File S2). We found that astrocytes, Müller glia, RPCs and BSL cells showed the highest proliferation scores (Figure 4D), suggesting that these cells were proliferating. To test this hypothesis, we used PAX2+ (astrocytes), VSX2 + (RPC), and ASCL1 + and HOXC8 + (BSL cells) We examined the expression of Ki-67 in cells. Accurate quantification was not practical due to the rarity of double-positive cells. Nevertheless, we found that a small number of migratory PAX2 cells were Ki-67+. + We found astrocytes and very few migratory BSLs. + / VSX2 + RPCs remained in the subretinal space ( Fig. 4E ).

[0137] Taken together, these data suggest that migratory proliferative donor human cells are rare and are mostly astrocytes, and that non-migratory proliferative cells are rare and are mostly RPCs.

[0138] 4. Donor cones and rods mature more rapidly in the recipient subretinal space than in culture Our scRNA-seq analysis suggested that the recipient subretinal space promotes photoreceptor fate and possibly maturation (Figure 2D). To test this hypothesis, we first assessed cone maturation. We used pseudotime analysis to assess gene expression profiles of cones from transplanted and cultured organoids and compared these cells to published datasets of embryonic, postnatal, and adult cones isolated directly from human retina (42). Transcriptional profiles suggested that cones from transplanted organoids resembled adult cones, while cones from cultured organoids more closely resembled embryonic cones (Figure 5A-B). Expression of mature cone-specific genes was consistently higher in transplanted cones (Figure 5C) than in cultured cones (Supplementary Figure S5), including all three cone opsins (OPN1LW, OPN1MW, and OPN1SW). CRX:tdTomato expressing L / M opsin or S opsin + The ratio of cells in cultured organoids (L / M-opsin + : 2.7%, S-opsin + : 1.3%) compared with transplanted organoids (L / M-opsin + : 26.4%, S-opsin + : 28.7%) (Figure 5D). + or S-opsin cells + The percentage of α-terminal β-terminal endothelial cells was significantly higher in transplanted organoids than in cultured organoids (Figure 5E). We measured the intrinsic electrical properties of transplanted human pyramidal cells and found large capacitive currents (~2 nA), indicating the relatively large plasma membrane area typically observed in mature cones (Figure 5F).

[0139] Next, we assessed rod maturation in transplanted and cultured organoids. Similar to cones, gene expression and pseudotime analysis suggested that rods derived from transplanted organoids resembled adult rods, whereas cultured rods derived from cultured organoids resembled embryonic rods (Figure 6A-B). Expression of RHO (Figure 6C) and other rod-specific genes (Supplementary Figure S5) was higher in rods derived from transplanted organoids than in cultured organoids. Rho expressed CRX:tdTomato + The percentage of Rho cells was significantly higher in transplanted organoids (61.5%) compared to cultured organoids (45.5%) (Figure 6D). + The percentage of cells was significantly higher in transplanted organoids (89.6%) than in cultured organoids (29.8%) (Figure 6E).

[0140] Finally, we investigated general features of photoreceptor maturation. Expression of specific synaptic proteins was upregulated in cones and rods in transplanted retinal organoids compared to cultured retinal organoids (Supplementary Figure S6A). CRX:tdTomato + In donor photoreceptors, CtBP2 shows synaptic ribbons + The number of puncta was significantly higher in cells derived from transplanted organoids compared to cultured retinal organoids (Supplementary Figure S6 B). These data suggest that the recipient subretinal space promotes rod and cone maturation compared to the in vitro environment of cultured organoids.

[0141] 5. CD Markers for Selection / Purification of Therapeutic Cells Digestion. Recognizing that donor cell viability is critical for transplantation outcomes, we constructed a single cell dissociation system optimized for the generation of single cell suspensions from retinal organoids. Digestion efficiency and cell viability were compared using papain and Accumax solutions. Single cells from papain digestion showed higher viability (90%) than Accumax solution (10%), although papain is much less efficient than Accumax (yield: 0.6 × 10 papain 5Cells / h vs Accumax 2×10 5 Agitation is used to approximately double the dissociation efficiency. Optimal agitation settings are 400 rpm for 3 h at 37 °C.

[0142] Storage conditions before transplantation. Storage conditions of dissociated donor cells prior to cell sorting and transplantation were optimized. More than 85% of the cells in suspension survived for at least 4 hours when maintained at 4° C., whereas cell viability was lower (approximately 70%) after storage at 37° C.

[0143] Magnetic Assisted Cell Sorting (MACS). Donor retinal organoids are dissociated from relatively mature organoids (>120 days of differentiation, up to 180 days or more). Less mature organoids (<55 days) containing only rare photoreceptors were used as negative controls. In mature retinal organoids, 12.5% ​​of the cells were CD24 + CD99 + and 87.5% of the cells were CD24 - CD99 - In less mature retinal organoids, 12.3% of the cells were CD24 + and CD99 + To verify the identity of the sorted cells, we performed immunocytochemistry (ICC) using markers expressed in glial cells, including photoreceptor cells (anti-recoverin, REC) and astrocytes (anti-glial fibrillary acid protein, GFAP). - CD99 - In cell suspension, REC + Photoreceptors account for 48% of cells, and GFAP + CD24 cells accounted for 37% of the cells. + CD99 + In cell suspensions, we REC + No photoreceptor was detected. CD24 derived from less mature organoids + CD99 + In cell suspensions, we REC + No photoreceptors were detected.

[0144] 6. Discussion In these studies, we observed two main differences between cells derived from donor retinal organoids transplanted into mice and cells derived from time-equivalent retinal organoids maintained in culture. The transplanted cells were maintained for months in the subretinal space of the degenerated recipients, thus mimicking conditions directly relevant for cell-based therapies for photoreceptor dystrophies. The most striking and unexpected difference was the observation of migratory donor astrocytes and BSL cells in the transplanted cell population. Astrocytes and BSL cells underwent radial migration into and long-distance tangential migration along all retinal laminae (except for the outer nuclear layer of photoreceptor cells, which were absent in the degenerated recipients). Migratory astrocytes and BSL cells were generally non-proliferative, whereas graft-derived retinal progenitor cells showed proliferation without migration. In contrast to these migratory cells, transplanted photoreceptors, inner retinal neurons and Müller glia were non-migratory and remained at the subretinal transplant site. The second major difference between transplanted and cultured organoids was related to photoreceptor maturation. Based on gene expression and morphology, transplanted rods and cones were more mature than photoreceptors from cultured organoids. These data expand our understanding of photoreceptor and non-photoreceptor development in transplanted retinal organoids and highlight the importance of an unbiased approach to cell fate identification and spatial tracking after organoid transplantation.

[0145] Migratory astrocytes and BSL cells derived from transplanted organoids display a molecular profile distinct from cells in mature cultured organoids. Astrocytes express PAX2, which normally delineates the optic stalk in vivo. PAX2 is detected in retinal progenitor cells in early stage retinal organoids but not in later stages. Furthermore, cultured retinal organoids have not been reported to generate astrocytes in vitro. BSL cells express ASCL1, HOXC8, NKX2-2 and ARX. ARX and NKX2-2 expressing cells are found in very early stage retinal organoids but not after 60 days in culture. HOXC8 expression is normally restricted to the posterior spinal cord and is absent in the developing human retina and retinal organoids. PAX2 + Astrocytes and ARX + Although telencephalic interneurons undergo long-distance tangential migration in vivo, astrocyte or BSL identity was not sufficient to induce migration of graft-derived cells, as many astrocytes and BSL cells remained localized in the subretinal space. Our experiments lacked the temporal resolution to determine whether transplantation induced transdifferentiation of cells that had initially adopted a retinal identity or selectively promoted the proliferation of the few remaining BSL cells.

[0146] Previous publications have shown migratory transplanted cells, but their proliferation and long-distance migration capabilities were unknown. Seiler and colleagues noted migratory human donor cells 6 months after transplantation of early-stage hESC-derived retinal organoids in nude rats with retinal degeneration. Using LMNB2 to identify human donor cells, Lamba and colleagues found that human induced pluripotent stem cell (iPSC)-derived PAX6 occasionally migrated at 2 months. + and GFAP + Another study reported that human fetal CD29 + / SSEA1 +Migratory cells were seen as soon as 7 days after subretinal delivery of donor cells, suggesting that migration occurs soon after transplantation. Whether the early migratory cells are the same as those observed several months later remains to be seen. In wild-type cats, increased immunosuppression appears to result in greater cell migration, suggesting a role for immune cells. It is not known whether migratory donor cells adversely affect recipient retinal function and whether depletion is required prior to transplantation.

[0147] The cues in the host environment that promote migratory cell fate and photoreceptor maturation are unknown. Multiple extracellular cues regulate cell specification in human retinal organoids. Dynamic regulation of thyroid hormone and retinoic acid signaling specifies cone subtypes in human retinal organoids. Although the role of these cues in the subretinal environment after transplantation is not understood, they potentially regulate photoreceptor specification and maturation.

[0148] In conclusion, we found that human stem cell-derived retinal organoid cells are influenced by the mouse host graft environment in two distinct ways. First, the host environment promotes a population of organoid-derived astrocytes capable of radial and tangential migration. Second, the host environment promotes maturation of organoid-derived rod and cone photoreceptors that reside in the subretinal space.

[0149] 7. Materials and Methods Research plan This study was designed to investigate the influence of the recipient retinal microenvironment on the migration, fate specification, and maturation of human donor cells. CRX:tdTomato H9 human embryonic stem cell (hESC-H9)-derived retinal organoids were employed as donor cells and transplanted into Rd1 / NS mice with retinal degeneration and immunodeficiency. Four and a half months after transplantation, migratory and non-migratory human donor cells were identified by single-cell RNA sequencing (scRNA-seq) and histological analysis (i.e., RNAscope and immunohistochemical counterstaining). Lineage specification and maturation of donor human cells were characterized by scRNA-seq, pseudo-chronological reconstruction, histological analysis, and electrophysiological recordings. Age-matched human retinal organoids cultured in vitro served as controls.

[0150] b. Cell culture and retinal organoid differentiation The use of human stem cells was approved by Johns Hopkins ISCRO (ISCRO00000249). CRX:tdTomato H9 human embryonic stem cell line (hESC) was cultured following the gravity aggregation approach to differentiate retinal organoids as previously described. On day 134, retinal organoids were used for transplantation.

[0151] The use of human stem cells was approved by Johns Hopkins ISCRO (ISCRO00000249). H9 CRX:tdTomato human embryonic stem cell line (hESC) was a kind gift from Dr. David M. Gamm (University of Wisconsin Hospital, USA). Stem cells were maintained in mTeSR1 (Stem Cell Technologies, Cambridge, MA, USA) on 1% (vol / vol) Matrigel-GFR™ (BD Biosciences, USA, no. 354230) coated dishes and grown at 37°C in a HERAcell 150i incubator with 10% CO2 and 5% O2 incubator (Thermo Fisher Scientific, MA, USA). Cells were passaged at confluence (every 3–6 days) using Accutase (Sigma-Aldrich, MO, USA, no. SCR005) for 7–10 min and dissociated into single cells. Cells in Accutase were added 1:2 to mTeSR1 + 5 μM blebbistatin (Bleb; B0560, Sigma), pelleted at 80 g for 5 min, suspended in mTeSR1 + Blebbistatin, and seeded at 5,000 cells per well in 6-well plates. After 48 h, cells were fed with mTeSR1 (without Bleb) every 24 h until the next passage. No antibiotics were used in RPMI (Gibco, USA) and supplemented medium (10% fetal bovine serum (FBS), 2.5% penicillin) to minimize cell stress. Cells were maintained at 37 °C and 5% CO2 and cultured at approximately 1 × 10 5 ~2×10 6 Cells were passaged every 3–4 days at 100 cells / mL. Cells were routinely tested for mycoplasma using MycoAlert (Lonza, Switzerland, No. LT07).

[0152] H9 CRX:tdtomato hESCs were dissociated with Accutase for 12 min at 37 °C and seeded into 96-well ultra-low attachment round-bottom Lipidure-coated plates (AMSBIO, MA, USA, No. 51011610) at 3,000 cells / well in 50 μl mTeSR1. Cells were placed in hypoxic conditions (10% CO2 and 5% O2) for 24 h to enhance viability. Cells were allowed to naturally aggregate by gravity over 24 h. On day 1, cells were transferred to normoxic conditions (5% CO2). On days 1–3, 50 μl of BE6.2 medium containing 3 μM Wnt inhibitor (IWR1e, EMD Millipore, MA, USA, No. 681669) and 1% (v / v) Matrigel (Supplementary Table 1) was added to each well.

[0153] [Table 1-1] [Table 1-2]

[0154] On days 4–9, 100 μl of medium was removed from each well and 100 uL of medium was added. On days 4–5, BE6.2 medium containing 3 μM Wnt inhibitor and 1% Matrigel was added. On days 6–7, BE6.2 medium containing 1% Matrigel was added. On days 8–9, BE6.2 medium containing 1% Matrigel and 100 nM Smoothened agonist (SAG, EMD Millipore, No. 566660) was added. On day 10, aggregates were transferred to a 15 mL tube, rinsed three times with DMEM (Gibco, No. 11885084), and resuspended in BE6.2 containing 100 nM SAG in an untreated 10 cm polystyrene Petri dish. From this point on, medium was changed every other day. Aggregates were monitored and manually separated if they were together or attached to the bottom of the plate. On day 11, retinal vesicles were manually dissected using a sharp tungsten needle. After dissection, cells were transferred to a 15 mL tube and washed twice with 5 mL of DMEM. On days 14–17, long-term retinal (LTR, Supplementary Table 2) medium containing 100 nM SAG was added.

[0155] [Table 2]

[0156] On days 18–21, cells were maintained in LTR and washed twice with 5 mL of DMEM before being transferred to a new plate to wash away dead cells. To increase survival and differentiation, 1 μM all-trans retinoic acid (ATRA; R2625; Sigma) was added to the LTR medium from days 22–138. 10 μM γ-secretase inhibitor (DAPT, EMD Millipore, No. 565770) was added to the LTR from days 28–42. Retinal organoids were grown at low density (10–20 per 10 cm dish) to reduce aggregation.

[0157] C. Recipient mice All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee (approval M016M17). We generated a recipient mouse model (termed Rd1 / NS) with immune deficiency and retinal degeneration by crossing Rd1 mice with Nod-Scid (NS) mice as previously reported. Mice were genotyped by the Transnetyx Tag Center (Cordova, TN, USA) and characterized by immunohistochemistry (IHC) staining and flow cytometry analysis as previously reported.

[0158] C3H / HeJ-Pde6brd1 (referred to as rd1) and NOD.CB17-Prkdcscid / J (referred to as NS) mice of either sex (6–8 weeks of age) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in cages under a 12:12 h light / dark cycle with free access to water and food.

[0159] We generated a recipient mouse model (called Rd1 / NS) with immune deficiency and retinal degeneration by crossing Rd1 mice with Nod-Scid (NS) mice (8 weeks old). The breeding strategy was performed as previously reported. Genomic DNA of the third generation offspring was extracted from ear biopsies and genotyped by the Transnetyx Tag Center (Cordova, TN, USA). Primers were listed in Supplementary Table 3.

[0160] [Table 3]

[0161] Eyes of adult Rd1 / NS mice (n=3) were collected to characterize photoreceptor degeneration using immunohistochemistry (IHC) staining as previously reported. Flow cytometry was performed using spleen biopsies of adult Rd1 / NS mice (n=3) to confirm T and B cell deficiency as previously described. Phenotyping data for Rd1 / NS mice are shown in Supplementary Figure S1.

[0162] d.Transplantation Donor retinal organoid cells (harvested as microdissected multi-layered retinal fragments) were cultured in Crx:tdTomato + hESC-derived retinal organoids (134 days old, n=4) were obtained. Cultured human retinal organoids were imaged using a fluorescent microscope (Carl Zeiss, Jena, Germany). Images were used as references to image Crx-tdTomato from donor retinal organoids. + The cluster was then isolated. + Retinal organoid clusters were cut into 1 × 1 mm pieces using 27-gauge horizontal curved scissors (VitreQ, Kingston, NC, USA) under a dissecting microscope. 2 or 1×2mm 2 The donor cells were transplanted within 2 hours of isolation.

[0163] Isolated retinal fragments were transplanted into the subretinal space of Rd1 / NS mice (6-8 weeks old, n = 16 eyes) as previously reported. Briefly, recipient mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg body weight) and xylazine hydrochloride (20 mg / kg body weight). Mouse pupils were dilated with 1% (wt / vol) tropicamide (Bausch & Lomb, Rochester, NY, USA). Mouse corneas were covered with sodium hyaluronate (Healon GV, Abbott Medical Optics Inc., CA, USA) and a cover glass (Deckglaser, USA) to facilitate transpupillary visualization. Donor retinal organoid sheets were mounted, photoreceptor side down, on the bevel of a 26 G microneedle, gently aspirated into an attached microsyringe (Hamilton, Reno, CT, USA), and then injected tangentially through the sclera of recipient mice into the subretinal space. Successful injection was verified by direct visualization through the recipient's dilated pupils under a surgical microscope (Leica, Wetzlar, Germany).

[0164] e. Single-cell RNA sequencing Four and a half months after transplantation, single-cell RNA sequencing (scRNA-seq) was performed on dissociated single cells from transplanted (n=3 eyes) and cultured retinal organoids (n=2) (age-matched) using the Chromium platform (10X Genomics). ScRNA-Seq was performed on dissociated cells from transplanted and cultured retinal organoids using the Chromium platform (10X Genomics). Briefly, retinal organoid cells were dissociated into a single-cell suspension using a papain dissociation system (Worthington) at 37 °C for 60 min31, with gentle mixing every 5 min, before the reaction was stopped using ovomucoid protease inhibitor. Cells were centrifuged, resuspended in ice-cold PBS containing 0.04% bovine serum albumin (BSA) and 0.5 U / μl RNase inhibitor, and filtered through a 40 μm Flowmi cell strainer (Bel-Art SP). Cell number and viability were assessed by trypan blue staining before loading 6000 cells into a chromium single cell line using the Next GEM 3' Reagent v3.1 kit. Libraries were pooled and sequenced on an Illumina NextSeq 500 with approximately 50,000 reads per cell. The Cell Ranger 4 (10X Genomics) pipeline was used to process raw sequencing reads for demultiplexing, alignment to the GRCh38 human reference genome, and generation of cell-specific gene count matrices for downstream analysis. The generated cell-gene count matrices were analyzed using the Seurat ver3 R package. We excluded cells with UMIs less than 300 or more than 50000 and with a mitochondrial fraction of more than 20%. Doublets were identified and removed using the DoubletFinder R package. Log-normalization, scaling, UMAP dimensionality reduction and clustering were performed using the standard Seurat pipeline. The major retinal cell types were identified using previously identified cell type markers.Enriched genes from brain / spinal cord-like cell clusters were compared to the ASCOT gene expression overview of publicly available RNA-Seq data to determine their classification. Differential gene testing was performed by the FindMarkers function in Seurat using the Wilcoxon rank sum test with default parameters (52). Hierarchical clustering was used to group differentially expressed genes. The UCell R package was used to calculate the migratory or proliferation scores. Gene sets were constructed by identifying enriched genes within gene ontology terms (cell migration and cell motility for migratory scores, and cell division for proliferation scores, respectively). Seurat integration functions (SelectIntegrationFeatures, FindIntegrationAnchors and IntegrateData) were used to integrate the organoid data into the human retinal development dataset. Pseudotemporal analysis was performed to identify trajectory pathways within the data using Monocle 3.

[0165] f. Histological analysis Four and a half months after transplantation, recipient mouse eyes and cultured retinal organoids were fixed in 4% paraformaldehyde (PFA) in PBS (Electron Microscopy Sciences, Hartfield, PA, USA), dehydrated in a sucrose gradient (10%, 20%, 30%), and then blocked in optimal cutting temperature compound (OCT) (Sakura Finetek, Torrance, CA, USA). Seven to 10 micrometer sections of recipient eyes and cultured organoids were used for RNAscope and IHC counterstaining.

[0166] Recipient mice were sacrificed by overdose anesthesia and prefixed by cardiac perfusion with 4% paraformaldehyde (PFA) in PBS (Electron Microscopy Sciences, Hatfield, PA, USA). Eyes were gently removed, postfixed in 4% PFA / PBS at room temperature (RT) for 1 h, dehydrated in a sucrose gradient (10%, 20%, 30%), and then blocked in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Cultured retinal organoids were fixed in 4% PFA at room temperature for 15 min (min), dehydrated in gradient sucrose (10%, 20%, 30%), and blocked in OCT compound. OCT-blocked recipient mouse eyes and cultured retinal organoids were cut into 7–10 μm thick frozen sections using a microtome (CM 1850; Leica) for histological staining. RNAscope and IHC counterstaining were performed according to the manufacturer's protocol (Advanced Cell Diagnostics (ACD), protocol no. MK 51-150, see Appendix D). Briefly, frozen sections of recipient mouse eyes and cultured retinal organoids were rinsed in PBS, baked in a HybEZ™ oven (ACD, USA) at 60°C for 30 min, and post-fixed in pre-chilled 4% PFA in PBS at 4°C for 15 min. Slides were dehydrated in gradient ethanol (50%, 70%, 100%), treated with hydrogen peroxide (10 min at RT), and then target retrieved at 98-102°C for 5 min using Co-detection Target Retrieval solution (ACD, Cat. no. 323180). After rinsing with distilled water (2 min × 2) and PBS-T (5 min × 1), slides were incubated with diluted primary antibodies at 4°C overnight. On day 2, slides were post-fixed in 4% PFA for 30 min at RT, treated with protease III for 30 min at 40°C, and subjected to RNAscope staining using the RNAscope Multiplex 32 Fluorescent V2 assay according to the manufacturer's protocol (ACD, RNAscope USM-323100, see "Fixed frozen tissue samples protocol").Briefly, RNA probe hybridization was performed at 40 °C for 2 h using a HybEZ™ oven. Slides were then assigned to three series of amplifications, fluorescent dye combinations, and HRP blocking. After the RNAscope procedure, slides were incubated with secondary antibodies for 1 h at RT, counterstained with DAPI, and mounted with Prolong Diamond (Life Technology, Carlsbad, CA, USA). The RNA probes, fluorophores, primary and secondary antibodies used are listed in Supplementary Table 4.

[0167] [Table 4-1] [Table 4-2]

[0168] Negative and positive multiplex control probe staining was performed in parallel with the target probes following the same protocol (data shown in Supplementary Figure S2). IHC staining was performed as previously described. Briefly, cryosections of transplanted Rd1 / NS mice and cultured retinal organoids were rinsed in PBS (5 min × 1), permeabilized, and blocked with a mixture of 0.1% Triton®-X100 and 5% goat serum in PBS for 1 h at RT. Slides were rinsed in PBS (5 min × 3), incubated with primary antibodies overnight at 4 °C, incubated with secondary antibodies for 1 h at RT, then counterstained with DAPI and mounted using ProLong Diamond mounting medium. Primary and secondary antibodies used are listed in Supplementary Table 5.

[0169] [Table 5]

[0170] For migration distance quantification of transplanted retinal organoid cells, retinal sections from recipient mice were stained with human nucleus-specific antibody HNA (Sigma-Aldrich, MO, USA) or Ku80 (Thermo Fisher Scientific, MA, USA). Tile scan images were collected using a confocal LSM 880 (Zeiss, Oberkochen, Germany) for distance quantification. The migration distance of transplanted retinal organoids was defined as the shortest distance between the migrating cells and the nearest graft edge (i.e., graft left migrating cells to the left end point of the graft; graft right migrating cells to the right end point of the graft). We used a mathematical method to facilitate the quantification of distance. Specifically, we defined the graft edge as the "starting point" and manually targeted migrating cells in different retinal laminae (RGC, IPL, INL, RPE / C), both of which were processed with the "Cell Counter" plugin of ImageJ. Cell coordinates were automatically collected and the X and Y axial distances of individual cells were quantified by Cell Counter. The axial distances at the ends (origins) of the graft were designated as "X start" and "Y start". The axial distances of migrating cells were termed "X migration" and "Y migration". Migration distances were calculated in the R platform according to the following formula:

number

[0171] G. Electrophysiology Electrophysiological recordings were performed on the transplanted photoreceptors 8 months after transplantation to measure their physiological properties. We were able to test only one recipient mouse (the second recipient mouse died before the assay during long-term observation). The recipient eye was gently pulled out from the recipient mouse and placed in Ames' medium (Sigma No. A1420). The retina transplanted with retinal organoids was dissected under infrared light, sectioned into 200 μm slices, and then transferred to a recording chamber. Crx:tdTomato of the transplanted retinal organoids + Photoreceptors were targeted under an epifluorescence microscope for the resulting whole-cell patch clamp recordings. Fluorescent signals were imaged by a Nikon CCD camera with 20 ms flashes of epifluorescence excitation light and synchronized data acquisition. Total exposure time to excitation light before recording was less than 500 ms. During recordings, retinas were perfused with Ames medium bubbled with 95% O2 / 5% CO2. Patch electrodes (5–7 MΩ) were pulled from borosilicate capillaries (GC150-10, Harvard Apparatus) and filled with an internal solution typically containing (in mM): 120 K-gluconate, 5 NaCl, 4 KCl, 10 HEPES, 2 EGTA, 4 ATP-Mg, 0.3 GTP-Na2, and 7 phosphocreatine-Tris, pH adjusted to 7.3 with KOH. Whole-cell patch clamp recordings were performed at 30–32 °C with an Axon Instruments Multiclamp 700B amplifier. The series resistance of the patch electrodes was 10–30 MΩ. The liquid junction potential (measured as -13 mV) was compensated. In voltage-clamp mode, recorded cells were held at -40 mV, followed by 100 ms voltage steps (-70 mV to -10 mV). All procedures were performed in a dark room to avoid bleaching of photoreceptors.

[0172] h. Statistical analysis Quantitative histological data were analyzed using two-way ANOVA. Sidak test or Tukey test were employed for multiple comparisons (two-tailed). Independent T-test or Mann-Whitney U test11 were used for comparison of two variants. Statistical analysis was performed using SPSS software (version 25, IL, USA). p<0.05 was considered significant. Graphs were drawn using GraphPad Prism software (version 8, CA, USA).

[0173] Although the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, means, methods and steps described herein. Those skilled in the art will readily appreciate from this disclosure that the subject matter of the present disclosure can be utilized in accordance with any currently existing or hereafter developed subject matter, process, machine, manufacture, composition of matter, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0174] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. An in vitro method for producing a selected population of retinal cells, To generate three-dimensional retinal organoids, Dissociating the aforementioned three-dimensional retinal organoid, To produce the aforementioned selected population of retinal cells, the retinal cells are positively selected based on one or more markers of photoreceptor cell identity, and / or negatively selected based on one or more markers of non-photoreceptor cell identity. Methods that include...

2. a) The marker for photoreceptor cell identity is CD73, b) The marker for non-photoreceptor cell identity is one or more of CD24, CD302, CD9, and CD99, 2) The snow-white snowflake snowflake 、DY1、SYSY、SYSY、SYSY2、SYSYS、 CHRIST、SHA1、SHAS10、SHAS1、SHAS 1. SH2、SHAK3、SHA1、SHASHKH、SHASH、N4 DASH、DASH3、SHAD3、DASH4、DASH3、FIC RO11、SIC151、SIC33、SICSU、SICYS、SIC59 SHY11、SYSY2、SYSYS、SYS111 、KDY2、DY164、SYS13、D3、SYSH、0 2000.0010.0010.0010.000.000 THIS、SHY3、HHHHH、HHHH320、HHHH1 19. CHEEK1、SHEEK1、SHEEK10、SHEEK10、5 ROSHY2、SAXY22、SASY44、SYS35、SYS1、 LOVE1、SHASH2、SHASH21、SHASHY、SHASH1 、SHR44、SHR1、SHR09、SHR1、SHR1、S3 N3、DYS2、DYS22、SHYS80、5 H11、SHASH1、SHAS23、SHAS1、SHAS2、S CHA1、SHARE、SHASHS1S、SHASHSHAS、SHAS 8. SHAH、DASHK1、SHASH、DASH1、SHA1 SH1、SHASH06、SHASH017、SHASH、SHASHY3、 THIS IS THIS IS THIS IS THIS IS THIS IS THIS IS THIS IS THIS 22、SHARE3、SHAS2、SHASHA2、SHAS1、Z DASH44、DASH12、DASH55、9447 LOVE1、LOVE2、LOVE21、LOVE2、LOVE10 CHEEKS3、SHEYS、SHEYSYS1 、SHY2、SHORE11、SHOSE12、SHOSEY、SHEYS、 LOVE1、LOVE2、LOVE22、LOVE11、5 TH2、DYS8、DYS1、DYS30、SYS2、 CHA1、HYS、SYS1、SYS1、SHY1 THIS、SHY2、SHYH2、SHY2、SHY0 ROCK、SH07、HYD21、HYH11、SYS 15. THIS IS THE ONLY THING CHRIST、SAR1、SAR1、SARKY、SAR22、SAR12、One or more of FAT3, HEPACAM, FGFR3, TRIL, HSD17B2, and HP; d) The marker for non-photoreceptor cell identity is one or more of DKK3, LRP1, CLU, PMEPA1, ITGB1, and PTTG1IP; e) The marker for non-photoreceptor cell identity is one or more astrocyte markers selected from ADGRL4, SERPINE2, BCHE, ABI3BP, NRP1, FSTL1, FAT1, NTRK2, FBLN2, PRSS35, SLC1A3, FCGRT, LAMC1, TF, SORCS2, DKK3, LRP1, PTPRD, ANGPTL1, LTBP3, CLU, CNTNAP2, CD151, PCDH9, CRIM1, CSPG5, and PMEPA1; f) The markers of non-photoreceptor cell identity are CLU, ITM2B, PTPRZ1, GPM6B, ATP1B2, CD63, BCAN, SLC1A3, SERPINE2, LRP1, PTPRA, ADGRG1, ENO1, CANX, SLC3A2, DNER, PTTG1IP, CALR, PCDH9, CCDC80, LSAMP, HEPACAM, F3, PLPP3, APLP2, FBLN2, TIMP1, SLC6A11, CSPG5, JAM2, FGFR3, DKK3, GOLIM4, NCAM1, CHL1, NRCAM, HLA-A, TMEM132A, PMEPA1, ITGAV, SSR2, ACAA1, BCHE, CD59, FAT3, PCDH17, ST3GAL5, PBXIP1, LAMP1, ITGB1, HP, ITGB8, SGCB, LAMP2, CLDND1, TMEM106B, PTCH1, PLTP, RNF13, HLA-C, PTPRD, TMEM30A, TRIL, RAB5C, TTYH3, DAG1, CADM4, UBA1, SLC6A9, LRRRC8A, ATP1B3, SPPL2A, NTRK2, RNF130, LIFR, EMP3, PCDH7, NTRK3, COL6A1, IL17D, LRP10, ADAM19, SGCE, FAT1, SLC44A1, LTBP3, SLC39A10, ABCA1, SYPL1, SLITRK2, GNPTG, CD302, MRC2, LRP4, CALU, CD151, SORL1, TSPAN6, LRRRN1, TENM2, CAPNS1, NLGN1, SLC15A2, NLGN4X, EGFR, ADORA1, SLC9A7, SIRPA, EFCCAB14, ANGPTL1, FGFR1, VAMP5, CLDN12, LAMB2, GPR155, FGFR2, SLC44A2, LRP1B, PTGFRN, FNDCC5, NOTCH1, DPY19L4, SPR1, CD44, TNFRSF1A, FAM234A, CDH4, HLA-E, COL11A1, NCAM2, AQP1, CPQ, EMP1, FCGRT, GPC5, ROBO1, VCAM, LGALS3BP, LDLR, LRRRC4B, NOTCH2, ALCAM, RYR3, SLC9A9, TMEM94, VCAM1, IGSF1, PCDHA10, CDH10, CACH1, P2RX7, AEBP1, PLXNB1, AXL, ALPL, ST3GAL4, SERPINI1,One or more brain and spinal cord-like (BSL) cell markers selected from ITGB5, CD58, FGFRL1, PLPP1, TTYH2, IL17RB, FAM171A1, IL17RD, ANO6, ADAM22, PTPRG, ANTXR1, ZDHHC23, AGRN, COL14A1, POSTN, CNTFR, SEMA5A, FLNA, EMP2, TFPI, ITGA7, MXRA8, TENM4, FSTL1, CD82, NRP2, GPC4, ARSF, LAMC1, KIT, SEMA4A, LTBP1, and CSF1; or, g) The method according to claim 1, wherein the marker for non-photoreceptor cell identity is one or more BSL markers selected from HEPACAM, FGFR3, SERPINE2, BCAN, CCDC80, PLPP3, CHL1, ADGRG1, SLC6A11, LSAMP, FBLN2, F3, SLC1A3, DKK3, LRP1, DNER, CLU, PCDH9, and CSPG5.

3. The method according to claim 1, wherein the three-dimensional retinal organoid is enzymatically dissociated.

4. The method according to claim 3, wherein the enzyme is papain and / or trypsin.

5. The method according to claim 3, wherein the retinal cells are brought into contact with the composition to ensure that the cells remain in the dissociated cell suspension.

6. The method according to claim 5, wherein the composition is an enzyme.

7. The method according to claim 1, wherein the three-dimensional retinal organoid reaches approximately DD45 to DD300 before dissociation.

8. The method according to claim 7, wherein the three-dimensional retinal organoid reaches approximately DD90 to approximately DD140 before dissociation.

9. The method according to claim 1, wherein the retinal cell population consists of at least about 70% single cells.

10. The method according to claim 1, wherein the retinal cell population comprises about 55% to about 85% rod photoreceptor cells.

11. The method according to claim 1, wherein the stem cells are selected from non-embryonic stem cells of humans, non-human primates, or rodents; embryonic stem cells of humans, non-human primates, or rodents; induced pluripotent stem cells of humans, non-human primates, or rodents; and recombinant pluripotent cells of humans, non-human primates, or rodents.

12. The method according to claim 1, wherein the stem cells are human stem cells.

13. The method according to claim 1, wherein the stem cells are pluripotent stem cells or multipotent stem cells.

14. A selected population of in vitro differentiated retinal cells, wherein the in vitro differentiated retinal cells are obtained by the method described in any one of claims 1 to 13.

15. A pharmaceutical composition comprising the cell population described in claim 14, and further comprising a pharmaceutically acceptable carrier.

16. A composition for preventing and / or treating hereditary or acquired retinal degenerative disease in a subject, (a) The selected population of in vitro differentiated retinal cells according to claim 14, or (b) A pharmaceutical composition comprising the cell population described in claim 14 and further comprising a pharmaceutically acceptable carrier. A composition containing the following:

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