Generation, identification, and enrichment of retinal pigment epithelial cells and their derivatives

Abstract

The compositions and methods disclosed herein relate to retinal pigment epithelial (RPE) cells, isolated or generated from the retinal pigment epithelium (RPE) of adult mammals, from RPESCs or from other stem cells, which may be selected, identified, or enriched based on expression of one or more genes or cell surface markers. Accordingly, the disclosure provides methods of generating a non-native retinal pigment epithelial (RPE) cell product in vitro, the methods including: obtaining a starting cell population comprising RPE cells; and generating from the starting cell population a culture product of RPE cells, wherein one or more cells of the culture product of RPE cells expresses one or more genes or cell surface markers disclosed herein.

Description

[0001] Attorney Docket No. 27562-0031WO1

[0002] GENERATION, IDENTIFICATION, AND ENRICHMENT OF RETINAL PIGMENT EPITHELIAL CELLS AND THEIR DERIVATIVES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 733.325, filed on December 12, 2024, the contents of which are incorporated herein by reference in their entirety.

[0005] TECHNICAL FIELD

[0006] The compositions and methods disclosed herein relate to retinal pigment epithelial (RPE) cells or their derivatives isolated from the retinal pigment epithelium (RPE) of prenatal or postnatal mammals or from stem cells, which may be selected, enriched, or cultured to generate non-naturally occurring subpopulations or mixtures of subpopulations based on expression of one or more genes or cell surface markers. The compositions and methods disclosed herein also include pharmaceutical compositions comprising RPE cells or their derivatives that have been selected, enriched, or cultured and which may be used to restore vision lost due to diseases, disorders, or abnormal physical states of the retina, other neurological, non- neurological diseases, and / or tissue injuries that benefit from cell therapy.

[0007] BACKGROUND

[0008] The retina is a light-sensitive layer of tissue that lines the inner surface of the eye. Photoreceptor cells, either rods or cones, in the retina are directly sensitive to light and transform chemical light signals into electrical events that trigger nerve impulses. The retinal pigment epithelium (RPE) is a layer of pigmented cells directly underneath the neural retina that forms the blood-retinal barrier. The RPE cells play important roles in the maintenance of photoreceptor cells, visual function and the transport of ions, water, and metabolic end products between the subretinal space and the blood (Strauss et al., 2005).

[0009] Further. RPE cells establish immune privilege of the eye by secreting immunosuppressive factors. A disorder or injury to the RPE cells can result in degeneration of the retina, loss of visual function, and blindness. Several disorders of Attorney Docket No. 27562-0031WO1 the retina, including acute and age-related macular degeneration and Best disease, involve degeneration of the RPE; therefore, cell replacement therapy is a possible therapeutic option for preservation of the RPE layer and vision (Buchholz et al., 2009).

[0010] The therapeutic strategies for treating loss of vision caused by retinal cell damage vary, but they are all directed to controlling the illness causing the damage, rather than reversing the damage caused by an illness by restoring or regenerating retinal cells. Patients with eye diseases remain vulnerable to sustaining permanent damage to the retinal cells, even if drug treatments to control the illness are available. Thus, successful treatments of retinal cell damage will include approaches that aid in the regeneration and / or replacement of damaged retinal cells without causing the harmful side effects caused by cunent treatment methods.

[0011] Thus, there is a need for new treatment options for the treatment of diseases of the retina and of many other tissues where cell regeneration and / or replacement would be beneficial. There remains a need for methods and compositions including RPE cells or stem cells and their RPE-related derivatives that may be generated from adult tissue, and that may be enriched for increased therapeutic efficacy and increased success at engraftment into the RPE monolayer. The present disclosure describes such cells and methods for generating, isolating, and enriching populations of such cells. The disclosure describes ex vivo activation of RPE stem cells (RPESC) and their expansion to produce clinically useful quantities of RPE progeny (RPESC-RPE). Specific properties describing the therapeutic potency of RPESC-RPE subpopulations are disclosed.

[0012] The citation and / or discussion of cited references in this section and throughout the specification is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the present invention. All cited references are incorporated herein by reference in their entirety.

[0013] SUMMARY

[0014] The compositions and methods disclosed herein relate to retinal pigment epithelial (RPE) cells, isolated or generated from the retinal pigment epithelium (RPE) of adult mammals, from RPESCs or from other stem cells, which may be Attorney Docket No. 27562-0031WO1 selected, identified or enriched based on expression of one or more genes or cell surface markers. The RPE layer contains a subpopulation of cells that can be activated in vitro by addition of culture medium factors into a dividing RPE stem cell (RPESC). The RPESC can proliferate and differentiate to produce RPESC-derived RPE progeny (RPESC-RPE). In some embodiments, the isolated RPE layer cells are enriched based on expression of one or more genes and / or one or more cell surface markers.

[0015] In a first aspect, the disclosure provides methods of generating a non-native retinal pigment epithelial (RPE) cell product in vitro, the method including: obtaining a starting cell population including RPE cells; and generating from the starting cell population a culture product of RPE cells, wherein one or more cells of the culture product of RPE cells expresses PDPN and ITGA6 and one or more cell surface markers selected from the group consisting of CD63, CD24, CD82, NECTIN2, CD151, CD9, ITGB1, F3, and CD81, thereby generating a non-native RPE cell product. In some embodiments, each RPE cell of the starting population of RPE cells expresses one or more of RPE65, MITF. PAX6, CRALBP, OTX2, Bestrophin, CD82, CD81, CD63, F3, 1TGA6, ITGB1, and PDPN.

[0016] In some embodiments, the methods further include determining a level of enrichment of the culture product of RPE cells as compared to the starting cell population. In some embodiments, the starting cell population includes RPE cells derived from pluripotent stem cells (PSCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs). In some embodiments, the starting cell population includes in vv / ro-generated adult RPE stem cells (RPESCs). In some embodiments, the starting cell population includes RPESC progeny (RPESC-RPEs). In some embodiments, the starting cell population includes human RPE cells.

[0017] In another aspect, the disclosure provides compositions including a non-native retinal pigment epithelial (RPE) cell product generated by the method described above and disclosed herein.

[0018] In another aspect, the disclosure provide compositions including a therapeutically effective amount of RPE cell derivatives, wherein the RPE cell derivatives include one or more subpopulations of RPE cell derivatives that have been enriched from a starting population of RPE cells, wherein the one or more subpopulations are selected from subpopulations of RPE cell derivatives expressing Attorney Docket No. 27562-0031WO1 cell surface markers: DPP4; or ENTPD1; or ITGA1; or ICAM1; or CSF1R; or TFRC; or NCRl; or ITGB3; or PDPN; or ITGA6; or SIGLEC1.

[0019] In some embodiments, the RPE cells are derived from pluripotent stems cells (PSCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs). In some embodiments, the starting cell population includes adult RPE stem cells (RPESCs). In some embodiments, the starting cell population includes RPESC progeny (RPESC-RPEs). In some embodiments, the RPESC-RPEs have been cultured prior to enrichment.

[0020] In some embodiments, the RPE cell product or RPE cell derivatives are in suspension. In some embodiments, the RPE cell product or RPE cell derivatives are suspended in an injection vehicle. In some embodiments, the injection vehicle includes at least one component selected from the group consisting of glucose, sodium pyruvate, human serum albumin (HSA), and water. In some embodiments, the injection vehicle includes glucose, sodium pyruvate, human serum albumin (HSA), and water. In some embodiments, the injection vehicle includes sodium chloride, potassium chlonde, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water.

[0021] In some embodiments, the RPE cell product or RPE cell derivatives are on a scaffold. In some embodiments, the scaffold is a polymeric scaffold including collagen, albumin, fibrin, alginate, hyaluronic acid, polyactic acid (PLA), poly glycolic acid (PGA), polylactic acid-glycolic acid (PGLA), polyorthoester, poly caprolactone (PCL), polyanhydride, polyphosphazene, poly acrylate, polymethacrylate, ethylene vinyl acetate, polyvinyl alcohol, or combinations thereof.

[0022] In another aspect, the disclosure provides dosage forms including the compositions described above and disclosed herein. In some embodiments, the dosage form is frozen. In some embodiments, the dosage form has been thawed after having previously been frozen.

[0023] In some embodiments, the RPE cell derivatives of the dosage form have been diluted in an injection vehicle. In some embodiments, the injection vehicle includes at least one component selected from the group consisting of glucose, sodium pyruvate, human serum albumin (HSA), and water. In some embodiments, the injection vehicle Attorney Docket No. 27562-0031WO1 includes glucose, sodium pyruvate, human serum albumin (HSA), and water. In some embodiments, the injection vehicle includes sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water. In some embodiments, prior to dilution in the injection vehicle, the RPE cell derivatives of the dosage form have been washed with a balanced salt solution (BSS) or BSS including HSA.

[0024] In another aspect, the disclosure provides aqueous formulations for use as a storage medium or an injection vehicle for a tissue culture or a suspension of cells, the aqueous formulation including: glucose; and sodium pyruvate. In some embodiments, the aqueous formulations further include human serum albumin (HSA). In some embodiments, the aqueous formulations further including one or more of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hy droxide and hydrochloric acid.

[0025] In some embodiments, the aqueous formulations include: 0.1 g / L to 1.1 g / L glucose; 1.0 g / L to 5.0 g / L sodium pyruvate; and 0.01% to 1.0% HSA. In some embodiments, the aqueous formulations include: about 1.0 g / L glucose; about 2.2 g / L sodium pyruvate; and about 0.1% HSA.

[0026] In another aspect, the disclosure provide methods of treating a disease in a subject in need thereof, the methods including: administering to the subject a therapeutically effective amount of RPE cell derivatives, wherein the RPE cell derivatives include one or more subpopulations of RPE cell derivatives that have been enriched from a starting population of RPE cells, wherein the one or more subpopulations are selected from subpopulations of RPE cell derivatives expressing cell surface markers: DPP4; or ENTPD1; or ITGA1 ; or ICAM1; or CSF1R; or TFRC; or NCRl ; or ITGB3; or PDPN; or ITGA6; or SIGLEC1.

[0027] In some embodiments, the RPE cells are derived from pluripotent stem cells (PSCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs). In some embodiments, the starting cell population includes adult RPE stem cells (RPESCs). In some embodiments, the starting cell population includes RPESC progeny (RPESC-RPEs). In some embodiments, the RPESC-RPEs have been cultured Attorney Docket No. 27562-0031WO1 prior to enrichment. In some embodiments, the starting cell population are human RPE cells.

[0028] In some embodiments, the subject has been diagnosed with age-related macular degeneration (AMD). In some embodiments, the AMD is dry AMD. In some embodiments, the AMD is intermediate AMD. In some embodiments, the AMD is wet AMD.

[0029] In some embodiments, the subject has been diagnosed with an ophthalmic disorder. In some embodiments, the subject has been diagnosed with an ophthalmic disorder characterized by RPE layer and / or photoreceptor layer dysfunction or degeneration. In some embodiments, the subject has been diagnosed with diabetic retinopathy, Stargardt disease, or Choroideremia.

[0030] In some embodiments, the composition is administered to the eye. In some embodiments, the composition is administered by intravitreal, suprachoroidal, or subretinal injection. In some embodiments, the RPE cell derivatives are in suspension. In some embodiments, the RPE cell derivatives are on a scaffold. In some embodiments, the scaffold is a polymeric scaffold including collagen, albumin, fibrin, alginate, hyaluronic acid, polyactic acid (PLA), poly glycolic acid (PGA), polylactic acid-glycolic acid (PGLA), polyorthoester, polycaprolactone (PCL), polyanhydride, polyphosphazene, polyacrylate, polymethacrylate, ethylene vinyl acetate, polyvinyl alcohol, or combinations thereof.

[0031] In another aspect, the disclosure provides pharmaceutical compositions including the compositions described above and disclosed herein.

[0032] As used herein, the term “retinal pigment epithelial (RPE) cells'’ refers to pigmented cells expressing RPE65 that form a single layer of tightly joined cells forming a honeycomb pattern that in vivo form a barrier between the choroid and retina. RPE cells can exist in vivo or as cells isolated from a subject.

[0033] As used herein, “retinal pigment epithelial stem cells (RPESC)’' refers to a sub-population of RPE cells that is not present in vivo and can be activated in vitro to undergo extended proliferation and expansion to generate RPE cell progeny.

[0034] As used herein, “RPESC-RPE” refers to a non-naturally occurring cell product generated by expansion of RPESC and differentiation to RPE under particular cell culture conditions. Attorney Docket No. 27562-0031WO1

[0035] As used herein, “enriched’' refers to cells sorted by particular features including cell size, expression of particular cell surface proteins, or other characteristics as described herein.

[0036] As used herein, “generate” or “generated” refers to production of a cell product using the methods disclosed herein.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0038] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0039] DESCRIPTION OF DRAWINGS

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee

[0041] FIG. 1 A shows an outline of experimental plan for RNA-seq experiments involving retinal pigment epithelial stem cell-derived (RPE) cells (RPESC-RPE).

[0042] FIG. IB shows a dot plot illustrating the time points RNA was collected, the cell lines used, and the known and predicted transplant status.

[0043] FIG. 1C shows a singular value decomposition (SVD) analysis of the 4.000 most variable genes by time.

[0044] FIG. ID shows a singular value decomposition (SVD) analysis of the 4,000 most variable genes by transplant.

[0045] FIG. IE show s a pie chart of distribution of significantly different features by RNA type.

[0046] FIG. IF shows a pie chart of distribution of significantly different features by transplant group for coding genes. Attorney Docket No. 27562-0031WO1

[0047] FIG. 1G shows a pie chart of distribution of significantly different features for long noncoding genes.

[0048] FIG. 1H shows a display of GO enrichment analysis followed by a semantic similarity7analysis visualized by treemap.

[0049] FIG. II shows a plot of selected pathways from a REACTOME pathway enrichment analysis.

[0050] FIG. 2A shows a plot of dimensionality reduction followed by clustering that was used to identify differing clusters of RPESC-RPE revealing considerable heterogeneity7.

[0051] FIG. 2B shows a plot indicating that the clusters depicted in FIG. 2A appeared to be well mixed when looking at time of collection.

[0052] FIG. 2C shows a plot indicating that all clusters had cells from each time period with some clusters showing more variation in their composition.

[0053] FIG. 2D shows a dot plot demonstrating the top five gene markers for each cluster as identified by a Wilcoxon Rank Sum test.

[0054] FIG. 2E shows a display of GO enrichment followed by a semantic similarity analysis using the marker genes for each cluster.

[0055] FIG. 2F shows a display of REACTOME pathway enrichment analysis using the marker genes for each cluster. The numbers above the dots show' the number of enriched pathway s in each REACTOME tree.

[0056] FIG. 3 A is an upset plot showing the overlap between significantly changing genes (DEGs) in the bulk RNA-seq data (Bulk-EFF) and the marker genes in the scRNA-seq data based on cluster (Cluster-SC) or time (Time-SC).

[0057] FIG. 3B is a plot of membership by time of Bulk-EFF genes and single-cell marker genes classified by time in the single-cell data.

[0058] FIG. 3C shows a plot of the cluster membership of single-cell cluster marker genes found in the Bulk-EFF data by counts and percentage of all marker genes in cluster.

[0059] FIG. 3D shows a display of GO enrichment follow ed by a semantic similarity analysis using the genes shared between the Bulk-EFF and clusters 2, 6, and 10. Attorney Docket No. 27562-0031WO1

[0060] FIG. 3E shows a display of REACTOME pathway enrichment analysis of the genes shared between the Bulk-EFF and clusters 2, 6, and 10. The numbers above the dots show the number of enriched pathways in each REACTOME tree.

[0061] FIG. 4A shows a plot of TREX expression data versus know n optokinetic (OKT) results from transplants.

[0062] FIG. 4B is a plot of expression of TREX across transplantation groups.

[0063] FIG. 4C is a plot of TREX expression across multiple lines (n = 9) over time using qPCR with 18s as an internal control. The threshold of TREX expression for a successful transplant is calculated based on the midpoint of TREX expression from the efficacious cells and non-efficacious cells.

[0064] FIG. 4D is a plot of TREX expression using qPCR with HPRT as the internal control.

[0065] FIG. 4E is a plot of TREX expression normalized to 18s as measured by qPCR for cytoplasmic and nuclear fractions of RPESC-RPE cells (n = 2).

[0066] FIG. 4F is a plot of TREX expression normalized to 18S as measured by qPCR for four gapmers against TREX used to induce TREX knockdown (KD). Gapmer G2 was used for all further KT) experiments.

[0067] FIG. 4G is a plot of results of integration assay for TREX-KD by gapmer (n = 4, control n = 3).

[0068] FIG. 4H is a plot of results of integration assay for TREX overexpression (n = 4 per condition).

[0069] FIG. 41 is a plot showing a “region of practical equivalence” (ROPE, a range of values representing no effect) based on the highest density interval (HDI, range of values that 95% of the distribution lays under) of 92 sham transplants. Densities were plotted for OKT measurements taken 90 days after RPESC-RPE transplants were made into RCS rats using empty vector control RPESC-RPE (n = 8).

[0070] FIG. 4J is a plot showing a ROPE based on the HDI of 92 sham transplants. Densities were plotted for OKT measurements taken 90 days after RPESC transplants were made into RCS rats using TREX overexpressing RPESC-RPE (n = 8)

[0071] FIG. 4K is a plot showing a ROPE based on the HDI of 92 sham transplants. Densities were plotted for OKT measurements taken 90 days after RPESC transplants were made into RCS rats using RPESC-RPE from line 255, which exhibits high Attorney Docket No. 27562-0031WO1

[0072] TREX expression at the passage 2 (P2) 2 Week culture timepoint as measured by qPCR (n = 5).

[0073] FIG. 5 A is a schematic of a cellular indexing of trans criptomes and epitopes by sequencing (CITE-Seq) study in human RPE tissues.

[0074] FIG. 5B is a uniform manifold approximation and projection (UMAP) visualization of acutely isolated native human RPE subpopulations integrated across modalities (cell surface protein and RNA expression) by weighted-nearest neighbor (WNN) analysis and then clustered.

[0075] FIG. 5C is a plot of expression of RPE specific markers in all clusters.

[0076] FIG. 5D is a dot plot of gene markers of RPE clusters.

[0077] FIG. 5E is a dot plot of surface protein markers enriched in each cluster.

[0078] FIG. 5F is a graph showing the enrichment of select Gene Ontology categories.

[0079] FIG. 6A is a schematic of human RPE showing spatial distribution of B3GAT1, CD24 and TNFRSF14 expressing cells in five concentric, morphologically different regions of the native RPE layer.

[0080] FIG. 6B shows representative immunofluorescence images showing expression of B3GAT1, CD24, TNFRSF14 and ITGB3 in subpopulations of adult RPE cells in different regions of the human RPE tissue.

[0081] FIG. 6C is a series of five box plots showing the cell size by area of B3GAT1, CD24 and TNFRSF14 expressing cells in different regions of the human RPE layer.

[0082] FIG. 6D is a series of density7plots show ing the cell area distribution for RPE subpopulations. B3GAT1 expressing cells in Pl had a larger area than B3GAT1- cells, while in P5 B3GAT1+ cells were smaller than the negative population. CD24 and TNFRSF14 expressing cells were found to be larger than their non-expressing counterparts.

[0083] FIG. 7A is a schematic of a CITE-Seq study of both primary7RPE cells and cultured RPESC-RPE cells.

[0084] FIG. 7B is a UMAP figure visualization of combined native and cultured subpopulations with modalities (cell surface protein and RNA expression) integrated by WNN analysis followed by clustering. Attorney Docket No. 27562-0031WO1

[0085] FIG. 7C shows a Riverplot illustrating the contribution of the native RPE clusters to the integrated data clusters.

[0086] FIG. 7D is a dot plot of surface protein markers of RPE clusters.

[0087] FIG. 7E is a UMAP figure showing distribution of cells from different timepoints.

[0088] FIG. 7F shows a stacked bar plot illustrating the relative cell proportions across various time-points within RPE clusters.

[0089] FIG. 7G is a dot plot of surface protein markers enriched in different time points.

[0090] FIG. 7H is a dot plot of changes in the expression of RPE-specific genes in native RPE and cultured RPESC-RPE from tw o weeks to ten weeks.

[0091] FIG. 8A shows representative immunofluorescence images of RPESC-RPE- 4W cultures showing expression of cultured RPE surface markers identified through CITE-Seq analysis. Subpopulations of RPE cells were detected in RPESC-RPE-4W cells expressing B3GAT1, CD24 and TNFRSF14 surface proteins that were observed to be upregulated in RPESC-RPE-4W cells according to CITE-Seq analysis (results shown in FIG. 5F). ITGB3, a marker of cluster 9, was also found to be expressed in a subset of RPESC-RPE-4W cultures.

[0092] FIG. 8B is a series of plots showing morphological characterization of cells expressing B3GAT1. CD24, and ITGB3. CD24+ and ITGB3+ cells exhibited larger cellular areas, whereas TNFRSF 14+ cells displayed a smaller area compared to their negative counterparts. ITGB3-positive cells demonstrated the largest cellular area among the four subpopulations.

[0093] FIG. 9A shows representative immunofluorescence images of human RPE flat mount confirming the distribution of expression of ITGB3 in human RPE tissue. The spatial distribution of ITGB3+ cells was analyzed as described in FIG. 6A.

[0094] FIG. 9B show s a dot plot representing functional specialization of RPE subpopulations. The dot plot visualizes Gene ontology database analysis using screp package in R. The dot size reflects the percentage of genes in each category expressed by the cluster and the y-axis represents the log of the false discovery rate (FDR) for the enrichment of each category. Attorney Docket No. 27562-0031WO1

[0095] FIG. 9C shows representative images of automated cell tracking results showing cells with low- and high- migratory capacity.

[0096] FIG. 9D shows a series of density plots showing the distribution of migration distance for cells expressing markers of clusters 5 and 7, ITGB3 and CSF1R respectively in native RPE and RPESC-RPE-4W cells. CSF1R expressing cells had a lower migration distance compared to CSF1R- cells in both native and RPESC-RPE- 4W cultures. ITGB3+ cells of the native and RPESC-RPE-4W cultures traveled a longer distance compared to their negative counterparts.

[0097] FIG. 10A is a dot plot of gene markers of cultured RPESC-RPE cells at two weeks (2W), four weeks (4W), and ten weeks (10W), iPSC-RPE cells at ten weeks (ipse), and native RPE (P0) cells, detected through CITE-Seq analysis.

[0098] FIG. 10B is a dot plot of surface protein markers of cultured RPESC-RPE cells at two weeks (2W), four weeks (4W), and ten weeks (10W), iPSC-RPE cells at ten weeks (ipse), and native RPE (P0) cells, detected through CITE-Seq analysis.

[0099] FIG. 10C is a heatmap showing differential expression of RPE signature genes in native RPE, cultured adult RPESC-RPE and iPSC-RPE cells.

[0100] FIG. 11 A shows a schematic of RPESC-RPE-4W investigational therapy manufacture from isolation of donor tissue through transplantation.

[0101] FIG. 11B shows a schematic of proposed process modification.

[0102] FIG. 11C shows improvements due to cryopreserved product. This strategy is estimated to reduce manufacturing costs while also enhancing reproducibility' and patient safety7. MCB, Master Cell Bank; WCB, Working Cell Bank.

[0103] FIG. 12 is a plot of Cry opreserved RPESC-RPE provide vision rescue comparable to fresh product in the RCS rat model. OKT results from thawed RPESC- RPE (0 weeks in culture) are significantly improved over injection of Balanced Salt Solution (BSS) alone at P90. ** p<0.01, *** p<0.001, **** pO.OOOl.

[0104] FIG. 13A shows schematics of two strategies for utilizing cryopreserved RPESC-RPE-4W cell product.

[0105] FIG. 13B shows a plot of cell viability measured by trypan blue exclusion for donor cell lines fresh and post-thaw, either with (TWI) or without (TDI) a wash step. Data indicate greater than 90% viability even after 4 hours using a simple dilution step. Attorney Docket No. 27562-0031WO1

[0106] FIG. 13C shows donor cells (red) prepared using the TDI method, appearing to integrate into a pre-established RPESC-RPE monolayer, comparable to fresh and cells that were washed in TAB2 + 10% HI-FBS (‘thaw-wash’).

[0107] FIG. 14A is a table listing comparability criteria for releasing RPESC-RPE- 4W cell products.

[0108] FIG. 14B is a Phase image of 4-week cells grown in a T25 flask. Scale bar = 200um.

[0109] FIG. 14C shows a plot of viability of clinical (red) and scale-up (blue) cells at harvest by automated count with propidium iodide exclusion.

[0110] FIG. 14D shows a plot of quantification of RPE identity (MITF, OTX2, BEST- 1) and purity (KI67, proliferation; SMA. epithelial -to-mesenchymal transition) markers by immunocytochemistry.

[0111] FIG. 14E shows a plot of potency measure by ELISA for PEDF release, normalized to media volume and cell number.

[0112] FIG. 15 shows a plot of osmolarity for three technical replicates measured for various cry opreservation agents and cell culture media. DMEM / F12 alone (“DMEM / F12”) and supplemented with HI-FBS (’‘+10%HI-FBS) were tested as approximating TAB2+10%HI-FBS. IV is custom injection vehicle developed at the Regenerative Research Foundation (RRF).

[0113] FIG. 16A shows a plot of viability and percentage of input cells recovered in TAB 2+ 10% HI-FBS post-thaw facilitated by newly developed parameters for cell concentration, total volume and freezing method. Bars indicated mean + / - SD for 3 experimental replicates.

[0114] FIG. 16B shows a plot of manual post-thaw cell counts after successive washes and formulation, in which cell retention is facilitated by addition of human serum albumin (HSA) to BSS and IV. At least 3 biological replicates were assessed in each condition. “BSS+ / IV+” indicates 0.1% HSA was used post-thaw in both the wash and vehicle formulation.

[0115] FIG. 17A is a plot of % viability of fresh and post-thaw RPESC-RPE-4W cells by manual counts using try pan blue exclusion, n = 5 biological replicates (donor lines), triangles represent 3 experimental replicates for one donor line and squares Attorney Docket No. 27562-0031WO1 represent 2 experimental replicates for another donor line. Dashed lines indicate release thresholds. Bars indicate mean + / - standard deviation.

[0116] FIG. 17B is a plot of % positive fresh and post-thaw RPESC-RPE-4W cells as quantified by immunocytochemistry. Bars indicate mean + / - standard deviation.

[0117] FIG. 17C is a plot of PEDF release at 3 days after replating of 4 week cells, either fresh or post-thaw, in 96-well plates, n = 4 biological replicates, one donor line was tested in 2 experimental replicates (represented by squares). Dashed lines indicate release thresholds. Bars indicate mean + / - standard deviation.

[0118] FIG. 17D is a series of phase contrast images of RPESC-RPE-4W before harvest and after replating, fresh and post-thaw. Scale bars lOOum.

[0119] FIG. 18A is a plot of manual cell counts showing viability of three donor cell lines at harvest (Live) and following thaw and resuspension in either the original vehicle formulation (Thawed) or modified vehicle (Thawed (IV+)). All cell suspensions were maintained at 4°C for the 72-hour test period.

[0120] FIG. 18B is a plot of % viability after recovery of RPESC-RPE-4W cells from long-term storage in liquid nitrogen. Bars indicated mean + / - standard deviation for 3 biological replicates.

[0121] FIG. 18C is a plot of potency as indicated by PEDF release within 3 days after recovery of RPESC-RPE-4W cells from long-term storage. Bars indicated mean + / - standard deviation for 3 biological replicates.

[0122] FIG. 18D is a plot of % of cells positive for several identity and purify markers after recovery of RPESC-RPE-4W cells from long-term storage. Bars indicated mean + / - standard deviation for 3 biological replicates.

[0123] FIG. 19A is a series of phase contrast pictures of hRPE-022 cells (Passage 2, 3.5 weeks) cultured in parallel in a 24-well plate (left) and T25 flask (right).

[0124] FIG. 19B is a table of results for release assays performed in parallel to the experiments of FIG 19 A.

[0125] FIG. 20A is a series of dimplots of RPESC-RPE-4W before and after cryopreservation analyzed by single cell RNA-seq, split by condition.

[0126] FIG. 20B is a series of violin plots of the genes utilized for identify and purity evaluation for RPESC-RPE-4W analyzed by single cell RNA-seq. Attorney Docket No. 27562-0031WO1

[0127] FIG. 21 A is a schematic of an integration assay used to measure integration of cryopreserved RPESC-RPE-4W cells in an RPESC-RPE monolayer.

[0128] FIG. 21 B is a plot of % integration for fresh and thawed donor cells integrating in a mature RPESC-RPE recipient monolayer.

[0129] FIG. 21C shows confocal imaging of fresh and thawed cells, donor cells labeled with Cell Tracker Red, all cells labeled with phalloidin for cell membranes (green) and DAPI (blue) for nuclei. Scale bars 20 = pm.

[0130] FIG. 22 shows a schematic of RPESC-RPE-4W investigational therapy manufacture, from isolation of donor tissue through transplantation. After formulation, the drug product is shipped live to the clinic. The drug product can be produced, e.g, in Los Angeles, CA and surgery can be performed in, e.g.. Ann Arbor. MI. A timeline of DP formulation and usage in a Phase l / 2a clinical trial is shown, presented in Central Time (CT). DP, drug product; MCB, Master Cell Bank.

[0131] FIG. 23 shows a plot of vision rescue by RPESC-RPE-4W up to 20 hours in 4°C storage when formulated in BSS. RCS rats were injected subretinally with at least 50,000 cells at ~P30. OKT measurements were performed at P60 and P90. N = 3 donor cell lines. Each point represents one injected eye. BSS, Balanced Salt Solution; OKT, optokinetic tracking; P30, postnatal day 30. **** p < 0.0001; ns, not significant.

[0132] FIG. 24A shows a plot of results from OKT testing of RCS rats at P60, P90, P150 and P180. RPESC-RPE-4W were stored in IV at 4°C for 24 hours prior to transplantation (“Cells in Vehicle”). ** p < 0.01; **** p < 0.0001; ns, not significant.

[0133] FIG. 24B shows a plot of OKT testing of RCS rats at P60, P90, Pl 50 and Pl 80. RPESC-RPE-4W were stored in BSS supplemented with 1 g / L glucose (glu) at 4°C for 24 hours prior to injection. ** p < 0.01; **** p < 0.0001; ns, not significant.

[0134] FIG. 24C shows a plot of OKT testing of RCS rats at P60 and P90. Rats were injected w ith BSS alone or with RPESC-RPE-4W that had been stored in BSS supplemented with glucose and 0.22 g / L sodium pyruvate for 24 hours prior to transplantation. ** p < 0.01; **** p < 0.0001; ns, not significant.

[0135] FIG. 25A is a plot of cell viability7and recovery for RPESC-RPE-4W cells thawed and diluted in IV+ injection vehicle. Attorney Docket No. 27562-0031WO1

[0136] FIG. 25B is a plot of cell viability and recovery for iPSC-RPE cells thawed and diluted in IV+.

[0137] FIG. 26A shows a plot of spatial frequency (cycles / degree) for RPESC-RPE- 4W cells formulated in vehicle, stored at 4 °C for 48 hours, then injected subretinally in the RCS rat. OKT testing was performed at postnatal day 60 (P60), postnatal day 90 (P90) and postnatal day 150 (P150). Vehicle alone and unoperated rats served as controls.

[0138] FIG. 26B shows a plot of spatial frequency (cycles / degree) for RPESC-RPE- 4W cells formulated in vehicle, stored at 4 °C or 12 °C for 24 hours, then injected subretinally in the RCS rat. OKT testing was performed at postnatal day 60 (P60), postnatal day 90 (P90). postnatal day 150 (Pl 50) and postnatal day 180 (Pl 80). Unoperated rats served as control.

[0139] FIG. 26C shows a plot of percent viability of cells from complex neural tissue and cell ty pes stored in custom vehicle at 4 °C for 48 hours.

[0140] DETAILED DESCRIPTION

[0141] The compositions and methods disclosed herein relate to retinal pigment epithelial (RPE) cells, isolated or generated from the retinal pigment epithelium (RPE) of adult mammals, from RPESCs or from other stem cells, which may be selected, identified or enriched based on expression of one or more genes or cell surface markers. The RPE layer contains a subpopulation of cells that can be activated in vitro by addition of culture medium factors into a dividing RPE stem cell (RPESC). The RPESC can proliferate and differentiate to produce RPESC-derived RPE progeny (RPESC-RPE). In some embodiments, the isolated RPE layer cells are enriched based on expression of one or more genes and / or one or more cell surface markers.

[0142] In some embodiments, the generated RPE cells express one or more cell surface markers selected from the group consisting of ICOSLG, ITGA2, CSF1R, TFRC, DPP4, CD200, ENTPD1, IFNGR1, ICAM1, MCAM, CD55, CD44, TNFRSF14, ITGA1, NT5E, PVR, B3GAT1, HLA, FAS, CD58, CD47, CD63, CD24, CD82, NECTIN2, CD151. CD9. ITGB1, F3, CD81. ITGA6, and PDPN.

[0143] In some embodiments, the generated RPE cells include one or more subpopulations of RPE cell derivatives defined by7expression of cell surface markers. In some embodiments, the generated RPE cells include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Attorney Docket No. 27562-0031WO1 or more subpopulations of RPE cell derivatives defined by expression of cell surface markers. In some embodiments, the subpopulations of RPE cell derivatives are defined by the expression of, for example, CD81, CD82, and F3; or CD74, FCER2, CX3CR1, and CD163; or NT5E2, DPP4, and CD200; or ENTPD1, HLA, and CD9; or ITGA1; or ICAM1, CD47, and B2GAT1; or CSF1R and CD63; or TFRC; or NCR1; or ITGB3; or PDPN; or SIGLEC1.

[0144] In some embodiments, the generated RPE cells express one or more cell surface markers selected from the group consisting of PDPN, CD24, TNFRSF14, and B3GAT1. In some embodiments, the RPE layer cells express the cell surface markers PDPN, CD24, TNFRSF14, and B3GAT1. In some embodiments, the RPE cells also express the cell surface marker ITGB3. In some embodiments, the RPE cells also express the long noncoding RNA (IncRNA) TREX (transplanted RPE expressed). The compositions and methods disclosed herein also include pharmaceutical compositions including RPE cells that have been generated, identified, selected or enriched based on expression of one or more genes and / or one or more cell surface markers and which may be used to restore vision lost due to diseases, disorders or abnormal physical states of the retina, other neurological, non-neurological diseases, such as Parkinson’s Disease, and / or tissues injuries that benefit from cell therapy.

[0145] While developing the compositions and methods disclosed herein, the inventors have surprisingly discovered gene expression signatures and cell surface markers unique to RPESC cells and to their progeny, including the RPESC-RPE-4W that are particularly efficacious in the context of cell replacement therapies and vision rescue. These genes and cell surface markers are described below in the Examples and include, among others, cell surface markers CD24, TNFRSF14, B3GAT1, ITGB3, and IncRNA TREX.

[0146] The compositions and methods disclosed herein improve the efficacy of current cell replacement and cell transplantation technologies by providing methods for generating and identifying a cell population from the RPE layer or from the RPESC or other stem cells. RPE cells can be derived from fetal or adult retinal tissues, cultured RPESC cell lines, or pluripotent stem cells such as embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). RPESC-RPE cells that have been found to have characteristics that enhance their therapeutic efficacy have Attorney Docket No. 27562-0031WO1 specific cell surface antigens which can be used to define or to enrich the RPESC- derived cell population to be used in the context of a therapeutic cell transfer. Certain methodologies, such as Magnetic Activated Cell Sorting (MACS®), Fluorescent Activated Cell Sorting (FACS), or single cell sorting, and the like, are known in the art to separate various cell populations depending on their surface antigens. As described in further detail below, RPE cells can be cultured to generate RPE cell derivatives that enhance their therapeutic efficacy in the context of a therapeutic cell transfer. In some embodiments, RPE cells are cultured to enrich for RPE cell derivatives that express one or more cell surface markers selected from the group consisting of ICOSLG, ITGA2, CSF1R, TFRC, DPP4, CD200, ENTPD1, IFNGR1, ICAM1, MCAM, CD55. CD44, TNFRSF14, ITGA1, NT5E, PVR, B3GAT1. HLA, FAS, CD58, CD47, CD63, CD24, CD82, NECTIN2, CD151, CD9, ITGB1, F3, CD81, ITGA6, and PDPN.

[0147] In some embodiments, RPE cells are cultured to enrich for RPE cell derivatives that include one or more subpopulations of RPE cell derivatives defined by expression of, for example, CD81, CD82, and F3; or CD74, FCER2, CX3CR1, and CD163; or NT5E2, DPP4, and CD200; or ENTPD1, HLA, and CD9; or ITGA1; or ICAM1, CD47, and B2GAT1; or CSF1R and CD63; or TFRC; orNCRl; or ITGB3; or PDPN; or SIGLEC1.

[0148] In some embodiments, the present disclosure includes a method to use these sorting methodologies to enrich for PDPN, CD24, TNFRSF14, B3GAT1, and / or ITGB3 positive cells from a starting RPE, RPESC or RPESC-RPE cell population to obtain an enriched cell subpopulation. Thus, the present methods can enable manufacture of enriched cell populations for therapeutic compositions and methods.

[0149] As is shown herein below and in the Examples section which follows, the inventors have shown that the isolated RPE-derived cells having the gene expression signatures and cell surface markers disclosed herein display unique morphological characteristics, increased motility, increased success in integration into a mature RPE monolayer, and increased efficacy for restoring vision in an animal model. Thus, isolated RPE-derived cells having the gene expression signatures and cell surface markers disclosed herein can be used in the context of a pharmacal composition for transplantation and treatment of diseases of the retina, e.g., AMD. Attorney Docket No. 27562-0031WO1

[0150] Retinal Pigment Epithelial Cells

[0151] The compositions and method disclosed herein relate to generation, identification, isolation, enrichment, and transplant of retinal pigment epithelial (RPE) cells. The retinal pigment epithelium acts as a barrier between the bloodstream and the retina and closely interacts with photoreceptors in the maintenance of visual function. The retinal pigment epithelium is composed of a single layer of hexagonal cells that are densely packed with granules of melanin that absorbs light energy' that arrives to the retina. The main functions of the specialized RPE cells include: transport of nutrients such as glucose, retinol, and fatty' acids from the blood to the photoreceptors: transport of water, metabolic end products, and ions from the subretinal space to the blood; absorption of light and protection against photooxidation; reisomerization of all-trans-retinol into 11-cis-retinal; phagocytosis of shed photoreceptor membranes; and secretion of various essential factors for the structural integrity of the retina.

[0152] RPE cell markers may be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot-blot hybridization analysis using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANK®). Expression of tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least or about 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-fold, and more particularly more than 10- , 20-, 30, 40-, 50-fold or higher above that of a control cell, such as an undifferentiated pluripotent stem cell or other unrelated cell ty pe.

[0153] RPE cell surface markers can be detected by immunodetection of cell surface antigens or by any means well known in the art.

[0154] Dysfunction, injury', and loss of RPE cells are factors of many eye diseases and disorders including age-related macular degeneration (AMD), hereditary macular degenerations including Best disease, and retinitis pigmentosa. A potential treatment for such diseases is the transplantation of RPE cells into the retina of those in need of such treatment. It is speculated that the replenishment of RPE cells by their Attorney Docket No. 27562-0031WO1 transplantation may delay, halt or reverse degradation, improve retinal function and prevent blindness stemming from such conditions. However, obtaining RPE cells directly from human donors and embryos is a challenge.

[0155] Surgical transplantation (Algvere et al., 1997) or translocation (van Meurs and Van Den Biesen, 2003) of RPE sheets into the macula preserves central vision, providing proof of concept that RPE transplantation can be beneficial in AMD (reviewed in Binder et al., 2007). Stem cell technology now provides ample sources of RPE cells for transplantation to counteract RPE cell loss in AMD. Pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells have been successfully differentiated into RPE (Buchholz et al., 2009, Klimanskaya et al., 2004), and early-stage clinical trials transplanting ESC-derived RPE suspensions report safety and preliminary benefit (Schwartz et al., 2012, Schwartz et al., 2015, Song et al., 2015). An important concern with PSC-derived RPE is the possibility' of overgrowth and mis-differentiation due to residual undifferentiated source cells; this has been addressed by extensive differentiation into the RPE phenotype prior to transplantation (Kanemura et al., 2014). The influence of RPE differentiation stage on transplant efficacy, however, has not been described. We used an adult RPE stem cell (RPESC), a non-native tissue culture derivative of the RPE cell layer, which is less plastic than PSC and does not form tumors, as an alternative source of RPE cells for transplantation.

[0156] The human RPE layer contains a minor subpopulation of cells that can be activated in vitro into a stem cell state. Stringent clonal analyses and other tests show that these in vitro-generated RPESCs fulfill the criteria of stem cells, namely they can self-renew and produce differentiated progeny; these are adult RPE stem cells (RPESCs) (Salero et al., 2012). RPESCs are poised to generate highly pure cultures of RPE progeny7(RPESC-RPE) displaying characteristics of native RPE cells (Blenkinsop et al., 2015) but being otherwise distinct. We previously reported that subretinal transplantation of RPESC-RPE in the Royal College of Surgeons (RCS) rat prevents the loss of photoreceptor cells that occurs in these animals (Davis et al., 2016). Transplantation of RPESC-RPE can effectively rescue vision (i.e., prevent vision loss that normally occurs in the RCS rat) in a differentiation stage-dependent Attorney Docket No. 27562-0031WO1 manner. Specifically, transplantation of an intermediate 4-week stage of RPE differentiation (RPESC-RPE-4W) + / - 1 week most consistently preserves vision.

[0157] RPE Cell Transplantation

[0158] The human RPE cells described herein, or a pharmaceutical composition including these cells, can be used for the manufacture of a medicament to treat a condition in a patient in need thereof. The RPE cells can be previously cryopreserved. In certain aspects, the disclosed RPE cells are derived from iPSCs, and thus can be used to provide "personalized medicine" for patients with eye diseases. In some embodiments, somatic cells obtained from patients can be genetically engineered to correct the disease-causing mutation, differentiated into RPE, and engineered to form an RPE tissue. This RPE tissue can be used to replace the endogenous degenerated RPE of the same patient. Alternatively, iPSCs generated from a healthy donor or from HLA homozy gous "super- donors" or immune-cloaked lines can be used. RPE cells can be treated in vitro with certain factors, such as pigment epithelium-derived factor (PEDF), transforming growth factor (TGF)-beta, and / or retinoic acid to generate an anti-inflammatory and immunosuppressive environment in vivo.

[0159] In some embodiments, the RPE cells can be used for autologous RPE grafts to those subjects suitable for receiving regenerative medicine. The RPE cells may be transplanted in combination with other retinal cells, such as with photoreceptors.

[0160] Transplantation of the RPE cells produced by the disclosed methods can be performed by various techniques known in the art. For example, methods for performed RPE transplants are described in U.S. Patent No. 5,962,027 and U.S. Patent No. 6,045,791, each of which is incorporated herein by reference in its entirety’. In accordance with one embodiment, the transplantation is performed via pars plana vitrectomy surgery’ followed by delivery’ of the cells through a small retinal opening into the sub-retinal space or by direct injection. The RPE cells can be introduced into the target site in the form of cell suspension, adhered onto a matrix, such as extracellular matrix, or provided on substrate such as a persistent or biodegradable polymer. The RPE cells can also be transplanted together (co-transplantation) with other cells, such as photoreceptor cells. Thus, a composition including RPE cells obtained by the methods disclosed herein is provided. Attorney Docket No. 27562-0031WO1

[0161] Depending on the application, the method may be effected using cells which are syngeneic or non-syngeneic with the subject. The compositions and methods disclosed herein can include xenogeneic cells derived from a variety of species. Thus, in some embodiments, the cells may be derived from any mammal. Suitable species origins for the cells include the major domesticated or livestock animals and primates. Such animals include, but are not limited to, porcines (e.g. pig), bovines (e.g., cow), equines (e.g.. horse), ovines (e.g., goat, sheep), felines (e.g.. Felis domestica), canines (e.g., Canis domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil, hamster), and primates (e.g., chimpanzee, rhesus monkey, macaque monkey, marmoset).

[0162] In some embodiments, the subject is a human being and the cells are from a human origin. In some embodiments, the cells are non-syngeneic with the subject. According to one embodiment, the cells are allogeneic with the subject. According to one embodiment, the cells are xenogeneic with the subj ect. According to one embodiment, the cells are syngeneic with the subject (e.g. autologous).

[0163] Depending on the application and available sources, the cells of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Such determinations are well within the ability of one of ordinary skill in the art.

[0164] Any method known in the art may be employed to obtain cells for transplantation. Thus, for example, RPE cells can be obtained by collecting cells from a donor. According to another example, cells may be obtained from an organ or tissue. Likewise, various methods may be employed to obtain an organ or tissue from an adult organism (e.g. live or cadaver). Thus, for example, obtaining a tissue (e.g. retina tissue) may be effected by harvesting the tissue from an organ donor by a surgical procedure. Alternatively, a tissue may be obtained by in vitro or ex vivo culture of cells, organs or tissues. Such controlled in vitro differentiation of cells, tissues or organs is routinely performed, for example, using culturing of induced pluripotent stem cell lines to generate cultures containing cells / tissues / organs of desired lineages.

[0165] According to one embodiment, the cells of the present invention are ex vivo differentiated from adult stem cells or pluripotent stem cells such as embryonic stem cells (ES cells) or iPS cells. Attorney Docket No. 27562-0031WO1

[0166] The cell suspension of the invention may be obtained by any mechanical or chemical (e.g. enzymatic) means. Several methods exist for dissociating cell clusters to form cell suspensions (e.g. single cell suspension) from primary tissues, attached cells in culture, and aggregates, e.g., physical forces (mechanical dissociation such as cell scraper, trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Accutase and the like) or a combination of both.

[0167] According to the present invention, the cell suspension of differentiated cells includes viable cells. Cell viability may be monitored using any method known in the art, as for example, using a cell viability assay (e.g. MultiTox Multiplex Assay available from Promega), Flow cytometry, Tr pan blue, etc.

[0168] Typically, the differentiated cells are immediately used for transplantation. However, in situations in which the cells are to be maintained in suspension prior to transplantation, e.g., for 1-72 hours, the cells may be placed in a culture medium, suspension medium, or vehicle that is capable of supporting their viability and / or efficacy. Such a medium can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which can benefit maintaining the isolated population of differentiated cells in a viable state. For example, a suspension medium according to this aspect of the present invention can be a salt solution such as Balanced Salt Solution Combination 2 or a synthetic tissue culture medium such as RPMI-1640 (Life Technologies, Israel), Ko-DMEM (Gibco- Invitrogen Corporation products, Grand Island, NY. USA), DMEM / F12 (Biological Industries, Beit Haemek, Israel), Mab ADCB medium (HyClone, Utah, USA) or DMEM / F12 (Biological Industries, Beit Haemek, Israel) supplemented with the necessary’ additives.

[0169] Thus, according to one aspect of the present invention there is provided a method of transplantation, the method including administering to a subject in need of transplantation of cells in suspension (e.g., enriched RPE cells).

[0170] According to one embodiment, the method further includes administering cells in suspension to the subject (e.g., enriched RPE cells). Attorney Docket No. 27562-0031WO1

[0171] For any preparation used in the methods disclosed herein, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays or from studies involving animal models, for example, the RCS rat. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0172] Determination of a therapeutically effective amount is well within the capability' of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0173] Toxicity' and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0174] In some embodiments, the compositions are formulated with a pharmaceutically' acceptable carrier. In general, the pharmaceutical compositions and / or formulations are formulated to be administered to the eye. For example, the pharmaceutical compositions and / or formulations can be administered by intraocular injection. In some embodiments, the pharmaceutical compositions and / or formulations are administered by intravitreal or suprachoroidal injection. In some embodiments, administration of the injectable dosage form includes administration of a sterile aqueous solutions or dispersions including the cell-based compositions disclosed herein. In some embodiments administration of the injectable dosage form includes administration of a sterile solution including the cell-based compositions disclosed herein in combination with a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol. In some embodiments, the Attorney Docket No. 27562-0031WO1 compositions are formulated on a carrier matrix or substrate, adhered onto a matrix, such as extracellular matrix, or provided on substrate such as a persistent or biodegradable polymer.

[0175] In some embodiments, the pharmaceutical compositions and / or formulations, e.g., RPESC-RPE-4W, are suspended in an injection vehicle prior to administration to a subject, e g., prior to administration to the eye of a subject. In some embodiments, the injection vehicle is a balanced salt solution. In some embodiments, the injection vehicle is a modified balanced salt solution. The injection vehicle can include one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), and water. In some embodiments, the injection vehicle further includes one or more of glucose, sodium pyruvate, and human serum albumin (HSA).

[0176] In some embodiments, the injection vehicle includes 0.3 mg / ml magnesium chloride, 0.48 mg / ml calcium chloride, 0.75 mg / ml potassium chloride, 1.7 mg / ml sodium citrate, 3.9 mg / ml sodium acetate, 6.4 mg / ml sodium chloride, sodium hydroxide and / or hydrochloric acid (to adjust pH), and water. In some embodiments, the injection vehicle includes 0.3 mg / ml magnesium chloride, 0.48 mg / ml calcium chloride, 0.75 mg / ml potassium chloride, 1.7 mg / ml sodium citrate, 3.9 mg / ml sodium acetate, 6.4 mg / ml sodium chloride, sodium hydroxide and / or hydrochloric acid (to adjust pH), 1.0 g / L glucose, 2.2 g / L sodium pyruvate, and water. In some embodiments, the injection vehicle includes 0.01% to 1%, 0.02% to 0.9%, 0.03% to 0.8%, 0.04 to 0.7%, 0.04% to 0.6%, 0.05% to 0.5%, 0.06% to 0.4%, 0.07% to 0.3%, 0.08% to 0.3%, 0.09% to 0.2%, or about 0.1% HSA. In some embodiments, the injection vehicle includes 0.3 mg / ml magnesium chloride. 0.48 mg / ml calcium chloride, 0.75 mg / ml potassium chloride, 1.7 mg / ml sodium citrate, 3.9 mg / ml sodium acetate, 6.4 mg / ml sodium chloride, sodium hydroxide and / or hydrochloric acid (to adjust pH), 1.0 g / L glucose, 2.2 g / L sodium pyruvate, 0.1% HSA, and water.

[0177] In some embodiments, between the generation of components of the pharmaceutical compositions and / or formulations, e.g., generation of the RPESC- RPE-4W drug substance and generation of the injection vehicle, and administration of the pharmaceutical compositions and / or formulations to a subject, the components of the pharmaceutical composition and / or formulation is cryopreserved either alone or in Attorney Docket No. 27562-0031WO1 combination. The cry opreservation can be of any length of time between generation of the components of the pharmaceutical composition and administration of the composition to a subject.

[0178] In some embodiments, cells are cryopreserved as a suspension in commercially available cryopreservative agents. In some embodiments, the cryopreservative agent is supplemented with an injection vehicle. In some embodiments, the injection vehicle includes one or more of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, sodium citrate dihydrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, human serum albumin (HSA), and water.

[0179] The cells can be cryopreserved at a concentration of IxlO5to 5xl07cells / ml, 4.5xl05to IxlO7cells / ml, or IxlO6to 4.5xl06cells / ml. In some embodiments, the cells are cryopreserved at a concentration of 4.5xl06cells / ml.

[0180] In some embodiments, all components of the cry opreservation process are preconditioned to 4°C followed by immediate transfer to -80°C.

[0181] Thawing of cryopreserved cells can include washing and dilution steps. In some embodiments, cryopreserved RPESC-RPE-4W are thawed, washed and formulated in an injection vehicle. In some embodiments, cryopreserved RPESC-RPE-4W vials are thawed and directly diluted to the appropriate dose concentration in an injection vehicle. In some embodiments, the injection vehicle includes one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, human serum albumin (HSA), and water.

[0182] Generation, Enrichment and Identification of RPE Cells

[0183] Provided herein are methods to produce an RPE cell population which can be used in the context of a therapy for disease of the retina and associated vision loss. The methods disclosed herein can include generation, identification and enrichment of a starting population of RPE cells based on the expression of one or more cell surface markers. The resulting population of RPE cells is an identified, enriched population of RPE cells. The generated population of RPE cells can be used for downstream therapeutic applications, e.g., transplantation to treat a disorder of the retina in a Attorney Docket No. 27562-0031WO1 patient in need thereof. In some embodiments, the RPE cells express one or more cell surface markers selected from the group consisting of PDPN, CD24, TNFRSF14, and B3GAT1. In some embodiments, the RPE cells express the cell surface markers PDPN, CD24, TNFRSF14, and B3GAT1. In some embodiments, the RPE cells also express the cell surface marker ITGB3. In some embodiments, the starting population of RPE cells are adult RPE cells. In some embodiments, the starting population of RPE cells are from RPE tissue. In some embodiments, the starting population of RPE cells are RPE cells derived from pluripotent stems cells (PSCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs). In some embodiments, the starting population of RPE cells are derived from adult RPE stem cells (RPESCs). In some embodiments, the starting population of RPE cells are RPESC progeny (RPESC- RPE). In some embodiments, the starting population of RPE cells are RPESC-RPEs that have been cultured for some time, e.g. at least three weeks prior to enrichment.

[0184] In some aspects, the methods disclosed herein result in a cell population of at least or about 106, 107, 108, SxlO8, 109, IO10, 1011cells (or any range derivable therein) including at least or about 90% (for example, at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein) RPE-derived cells expressing one or more of cell surface markers PDPN, B3GAT1, CD24, TNFRSF14, and / or ITGB3. In certain aspects, starting cells for the present methods may include the use of at least or about 104, 105, 106, 107, 108, 109, 1010, 1011, 1012. 1013cells or any range derivable therein. The starting cell population may have a seeding densify of at least or about 101, 102, 103, 104, 103, 106, 107, 108cells / cm2or any range derivable therein.

[0185] In some embodiments, the starting population of RPE cells is generated using Fluorescence-Activated Cell Sorting (FACS). FACS utilizes flow cytometry to separate cells based on morphological parameters and the expression of multiple extracellular and intracellular proteins, for example, cell surface markers. This method allows multiparameter cell sorting and involves encapsulating cells into small liquid droplets which are selectively given electric charges and sorted by an external electric field. Fluorescence activated cell sorting utilizes fluidic, optical, and electrostatic systems. The fluidic system has to establish a precisely-timed break off from the liquid stream in small uniform droplets, so that droplets containing individual cells can then be deflected electrostatically. Droplet formation of the liquid Attorney Docket No. 27562-0031WO1 jet of a cell sorter is stabilized by vibrations of an ultrasonic transducer at the exit of the nozzle orifice. The disturbances grow exponentially and lead to break up of the jet in droplets with precise timing. A cell of interest, e.g., a cell displaying PDPN, B3GAT1, CD24, TNFRSF14, and / or ITGB3 on its surface, that should be sorted is measured at the sensing zone and moves down the stream to the breakoff point. During the separation of the droplet with the cell in it from the intact liquid jet, a voltage pulse is given to the liquid jet so that droplets containing the cells of interest can be deflected in an electric field between two deflection plates for sorting. The droplets are then caught by collection tubes or vessels placed below the deflection plates.

[0186] In some embodiments, the starting population of RPE cells is generated using magnetic-activated cell sorting (MACS™). MACS is a method for separation of various cell populations depending on their surface antigens, for example, PDPN, B3GAT1, CD24, TNFRSF14, and / or ITGB3.

[0187] MACS uses superparamagnetic nanoparticles to tag the targeted cells in order to capture them inside a column. The column is placed between permanent magnets so that when the magnetic particle-cell complex passes through it, the tagged cells can be captured. The column consists of steel wool which increases the magnetic field gradient to maximize separation efficiency when the column is placed between the permanent magnets. MACS allows cells to be separated by using magnetic nanoparticles coated with antibodies against a particular surface antigen for example, PDPN, B3GAT1, CD24, TNFRSF14, and / or ITGB3. This causes the cells expressing this antigen to attach to the magnetic nanoparticles. After incubating the beads and cells, the solution is transferred to a column in a strong magnetic field. In this step, the cells attached to the nanoparticles (expressing the antigen) stay on the column, while other cells (not expressing the antigen) flow through. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s).With positive selection, the cells expressing the antigen(s) of interest, which attached to the magnetic column, are washed out to a separate vessel, after removing the column from the magnetic field. This method is useful for isolation of a particular cell type, for example, PDPN, B3GAT1, CD24, TNFRSF14, and / or ITGB3. Attorney Docket No. 27562-0031WO1

[0188] In some embodiments, the RPE cell derivatives are generated by the cell culture conditions, for example, providing culture media with mitogens and survival factors that enable the proliferation and differentiation of one or more RPE subpopulations. Conditions that can be used to generate RPE cell derivatives by extended culture are described in further detail in Example 12 below.

[0189] Pharmaceutical Compositions

[0190] Also provided herein are pharmaceutical compositions of the RPE cells or their derivatives generated by the methods disclosed herein. These compositions can include at least about 1 x 103RPE cells, about 1 x 104RPE cells, about 1 x 105RPE cells, about 1 x 106RPE cells, about 1 x 107RPE cells, about 1 x 108RPE cells, or about 1 x 109RPE cells. In certain embodiments, the compositions are substantially enriched preparations including RPE cells produced by the methods disclosed herein, e.g., RPE cells that express one or more of cell surface markers PDPN, CD24, TNFRSF14, B3GAT1. 1TGB3, and / or IncRNA TREX.

[0191] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0192] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manners using one or more physiologically acceptable carriers including excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0193] The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, and the resulting method of administration.

[0194] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers Attorney Docket No. 27562-0031WO1 such as Hank's solution, Ringer's solution, or physiological salt buffer. Such penetrants are generally known in the art. As discussed above, in some embodiments, the pharmaceutical composition is formulated in an injection vehicle including sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water.

[0195] Compositions are also provided that include a scaffold, such as a polymeric carrier and / or an extracellular matrix, and an effective amount of the RPE cells generated by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in in vivo applications. For example, the physiologically acceptable materials include, but are not limited to, solid matrix materials that are absorbable and / or nonabsorbable, such as small intestine submucosa (SIS), crosslinked or non-crosslinked alginate, hydrocolloid, foams, collagen gel, collagen sponge, polyglycolic acid (PGA) mesh, fleeces and bioadhesives. Suitable polymenc carriers also include porous meshes or sponges formed of synthetic or natural polymers, as well as polymer solutions. For example, the matrix is a polymeric mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polysaccharides such as alginate and polymers of hyaluronic acid. Synthetic polymers include both biodegradable and nonbiodegradable polymers. For example, biodegradable polymers include polymers of hydroxyl acids such as polyactic acid (PLA), polyglycolic acid (PGA) and polylactic acid-glycolic acid (PGLA), polyorthoesters, polycaprolactone (PCL), polyanhydrides, polyphosphazenes, and combinations thereof. Nonbiodegradable polymers include poly acrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohols.

[0196] Polymers that can form ionic or covalently crosslinked hydrogels which are malleable can be used. A hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross- linked via covalent, ionic, or hydrogen bonds to create a three dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are Attorney Docket No. 27562-0031WO1 crosslinked ionically, or block copolymers such as PLURON1CS™ or TETRON1CS™, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or H, respectively. Other matenals include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.

[0197] One aspect of the present invention concerns the therapeutic use of the pharmaceutical compositions of this invention to treat patients having degenerative diseases, such as age-related macular degeneration, or disorders or abnormal physiological states of the eye, which includes an acceptable carrier, auxiliary or excipient. The compositions can be for topical, parenteral, local, intraocular or intraretinal use.

[0198] The pharmaceutical composition can be administered to humans or animals. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration.

[0199] The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the cells is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions could include an active compound or substance, such as growth factors, genetically engineered stem cells or retinal cells which secrete growth factor or other substances, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pEI and isosmotic with the physiological fluids. The methods of combining growth factor or cells with the vehicles or combining them with diluents are well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound or cells to specified sites within the eye, such as specific cells, tissues or organs.

[0200] The pharmaceutical compositions of the present invention could also include the active compound or substance, such as the generated subpopulation or mixture of subpopulations of RPE cells disclosed herein, or retinal progenitor cells or Attorney Docket No. 27562-0031WO1 differentiated cells derived from those stem cells, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The methods of combining cells with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within the eye. such as specific cells, tissues or organs.

[0201] The pharmaceutical compositions can be optionally packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of RPE cell function to improve a disease or abnormality of the retinal tissue.

[0202] Age-Related Macular Degeneration

[0203] In some embodiments, the methods disclosed herein are used to generate a subpopulation or mixture of subpopulations of RPE cells from a starting population of RPE cells. The generated subpopulations of RPE cells having the gene expression signatures and cell surface markers disclosed herein can be used in the context of a pharmaceutical composition for transplantation and treatment of diseases of the retina, e.g., AMD.

[0204] Vision loss may be caused by disease or damage to the retina of the eye. The retina consists of a specialized layer of cells at the back of the eye where light entering the eye is sensed as an image. These cells normally respond to all aspects of the light emitted from an object and allow perception of color, shape and intensity. When normal retinal function is impaired, it may lead to a loss of color perception, blind spots, reduced peripheral vision, night blindness, photophobia, decreased visual acuity’ or blindness.

[0205] Macular degeneration, also know n as age-related macular degeneration (AMD), and related disorders such as Stargardt or Best Disease are medical conditions that may result in blurred or no vision in the center of the visual field. Symptoms are usually progressive, with some patients experiencing a gradual w orsening of vision that may affect one or both eyes. While AMD does not result in complete blindness, loss of central vision can make it hard to recognize faces, drive, Attorney Docket No. 27562-0031WO1 read, or perform other activities of daily life. Visual hallucinations may also occur. Hence patients can be designated as legally blind.

[0206] Age-related macular degeneration (AMD) typically occurs in advanced age, and is caused by damage to the macula of the retina. Genetic factors, diet and smoking may play a role. The condition is diagnosed through a complete eye exam. Severity is divided into early, intermediate, and late types. The late type is additionally divided into "dry" and "wet" forms, with the dry form making up c. 90% of cases. The difference between the two forms is categorized by the changes present in the macula. Those with dry form AMD have drusen, made up primarily of protein and lipid deposits located under the RPE layer on top of Bruch’s membrane. Drusen adversely impact RPE functions and the photoreceptors the RPE support, leading to vision loss. In wet form AMD, drusen are also present or preceding, but what differentiates it from the dry form is that blood vessels grow into and through Bruch’s membrane, causing blood and fluid to leak into the retina.

[0207] Signs and symptoms of macular degeneration can include:

[0208] • Distorted vision in the form of metamorphopsia, in which a grid of straight lines appears wavy and parts of the grid may appear blank: Patients often first notice this when looking at things like miniblinds in their home or telephone poles while driving. There may also be central scotomas, shadows or missing areas of vision

[0209] • Slow recovery of visual function after exposure to bright light (photostress test)

[0210] • Visual acuity drastically decreasing (two levels or more), e.g.: 20 / 20 to 20 / 80

[0211] • Blurred vision: Those with dry AMD may be asymptomatic or notice a gradual loss of central vision, whereas those with wet AMD often notice a rapid onset of vision loss (often caused by leakage and bleeding of abnormal blood vessels)

[0212] • Trouble discerning colors, specifically dark ones from dark ones and light ones from light ones

[0213] • A loss in contrast sensitivity Attorney Docket No. 27562-0031WO1

[0214] • Formed visual hallucinations and flashing lights have also been associated with severe visual loss secondary to wet AMD.

[0215] Only a limited number of AMD patients are amenable to treatment despite the high incidence and severity of vision impairment (Ciulla et al. (1998) Surv. Ophthalmol. 43, 134-146). To date, there is a great need and effort to develop effective treatments or preventative measures, and to slow down, halt or reverse the progression of AMD would be a major achievement.

[0216] "Treating" or "treatment" of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical or sub-clinical symptoms of the state, disorder or condition developing in a mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub- clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0217] A "therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treating a state, disorder or condition, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity’ and the age, weight, physical condition and responsiveness of the mammal to be treated.

[0218] EXAMPLES

[0219] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0220] The practice of the methods and compositions of the disclosure employs, unless otherwise indicated, conventional techniques of molecular biology7(including recombinant techniques), cell culture, microbiology, cell biology, biochemistry, immunology, and neuroscience, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Attorney Docket No. 27562-0031WO1

[0221] Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait. 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology " (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the methods and compositions of the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. The following materials, reagents, and methods are used for the Examples described herein.

[0222] Materials and Methods

[0223] RPE cell culture arid subject details

[0224] All RPESC lines were generated from donor eyes obtained from certified eye banks with consent for research use. The donor details are listed in Table S13. RPE cells were cultured in a 24-well plate at 100,000 cells per well. Different RPE lines were used for different experiments in this study (Table SI 3) due to the limited availability7of adult RPESC-derived RPE cells (RPESC-RPE). RPESC-RPE cells used in scRNA-seq experiments were cultured on Transwell inserts (Coming). Cultures were maintained in RPE medium: DMEM F12 50 / 50 medium (Coming), MEM alpha modification medium (Sigma-Aldrich), 1.25 ml Glutamax (Gibco), 2.5ml sodium pyruvate (Gibco), 2.5 ml niacinamide (1 M; Spectmm Chemical Inc.), 2.5 ml MEM non-essential amino acid solution (Gibco), 10% heat-inactivated fetal bovine serum (FBS). supplemented with THT (taurine, hydrocortisone, triiodo-thyronin), and 1.25 ml N1 medium supplement (Sigma- Aldrich). Cells were incubated in a humidified incubator at 37°C and 5% CO2. RPE medium with 10% FBS was used until the cultures were confluent and 2% FBS was used after confluency. The medium was replaced every 3 days.

[0225] Bulk and scRNA-seq

[0226] Bulk and scRNA-seq were performed on cultured RPE cells collected at 2, 4, and 8 w eeks after plating. Single-cell suspensions were prepared using 0.25% Try psin Attorney Docket No. 27562-0031WO1

[0227] (Thermo Fisher Scientific) and processed for either bulk or scRNA-seq. For bulk sequencing, RNA was isolated with Direct-zol RNA kit (Zymo Research). Library preparation was then carried out with the TruSeq Stranded Total RNA kit (Illumina) ribo-depleted by the University of Rochester Genomics Research Center and sequenced using aNextSeq550 high-output flow cell generating 2 x 151 -bp read lengths. For scRNA-seq, the RPE single cell suspension was stained with 1 pl SYTO64 dye (Invitrogen) in 1 ml PBS for 20 min at room temperature, then washed twice in fresh PBS. Next, cells were diluted to 25,000 cells / ml and dispensed into ICELL8 3’ DE chips (Takara Bio) using an MultiSample NanoDispenser device (Takara Bio). Cell dispensing and in-chip reverse transcription PCR was performed using a 3' DE Chip and Reagent kit (Takara) Bio) according to the manufacturer's instructions. Following the extraction of PCR products, cDNA samples were concentrated and purified using a DNA Clean & Concentrator-5 kit (Zymo Research) and purified using a 0.6x proportion of AMPure XP magnetic beads (Beckman Coulter) according to manufacturer protocols. Library preparation was performed using a Nextera XT DNA Library Preparation Kit (Illumina) according to the Takara’s 39 DE chip and Reagent kit instructions. The quantification and quality checks of the cDNA products were carried out at the University7at Albany’s NextGen Sequencing core facility.

[0228] The concentration of cDNA products was quantified using a Qubit Fluorometer and the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). The quality of the cDNA product was checked using an Agilent High Sensitivity DNA Kit and Agilent 2100 Bioanalyzer (Agilent Technologies) to ensure the complete removal of contaminants. Libraries were sequenced using a NovaSeq 6000 high-output flowcell generating 2 x 150-bp read lengths (GeneWiz).

[0229] Data processing and analysis

[0230] For bulk sequencing, raw Illumina BCL files were process and provided as fastq files. Files were then mapped to hgl9 and converted to bam files using STAR (v2.4). Bam files were then read into R using the GenomicAlignments package to generate a counts matrix for further analysis. For single-cell sequencing, raw Illumina read BCL files were converted to fastq files using the bcl2fastq2 software (bcl2Fastq Attorney Docket No. 27562-0031WO1 v2. 19.1; Illumina™, Inc.). For scRNA-seq, the fastq files were then merged into readl (ICELL8® barcode sequence) and read2 (transcript sequence) fastq files. Using the metadata file from the ICELL8® system containing single-cell well information, their specific nanowell barcodes, readl, and read2 files were de-multipl exed based on nanowell barcodes. The sequence reads were then converted to bam files and mapped to hgl9 using STAR (v2.5). The final transcript read counts Rdata file was used as the input reads matrix for further analysis in R. Briefly, bulk data was analyzed with the EdgeR and DESeq2 packages to identify DEGs. Single-cell data was analyzed with the Seurat package (V3.1). Strict criteria were used for the qualify control of the single-cell dataset, including a minimum feature cutoff of 200 and a minimum cell cutoff of 3. Different clusters were identified according to Seurat’s clustering workflow. scRNA-seq enrichment analysis was performed with the screp package.

[0231] Image processing

[0232] Morphological analysis was performed by analyzing phalloidin staining images from RPE318, RPE319, and RPE322 lines cultured for 2, 4, and 8 weeks. Images were converted to binary and cell-cell junctions were detected using the Ridge Detection plugin for ImageJ. Object identification andmorphological analysis were performed using the EBImage package (Pau et al., 2010) in R.

[0233] Lentiviral vectors production and infection

[0234] The exonic TREX sequence (TCONS_00005049) was downloaded from the UCSC genome browser (hgl9) and a TREX insert with 15 bp overhangs was synthesized with GeneArt Gene Synthesis (Thermo Fisher Scientific). The insert was then cloned into a EcoRI cut TetO-FUWvector (Addgene) using an In-Fusion HD Cloning kit (Takara Bio) and sequenced (GeneWiz) to verify the plasmid. Lentivirus was generated in 75% confluent 293FT cells by co-transfecting the packaging plasmids pCMVpLNV and pCMV-pVSVG, as well as either the TetO-FUWTREX or TetO-FUWplasmids into the cells with the XtremeGene HP DNA transfection reagent at a ratio of 1:2.5 according to manufacturer’s protocol. Supernatant was collected 24 and 72 h after transfection followed by centrifugation at 21,700 g for 2.5 h to Attorney Docket No. 27562-0031WO1 concentrate the viral particles. Viral particleswere tittered via qPCR (ABM qPCR Lentivirus Titration Kit) before storage at -80°C.

[0235] Animal maintenance, transplantation, and statistical analysis

[0236] RCS rats and Long Evans rats were maintained under a 12-h light / dark cycle according to Institutional Animal Care and Use Committee-approved procedures (University at Albany Institutional Animal Care and Use Committee approval #722). Transplantations were carried out as previously described (Zhao et al., 2017). Briefly, RCS rats at postnatal days 28-32 (P28-P32 d) were treated with cyclosporine (210 mg / liter), then a 33-gauge needle was used to inject 1.5 pl of RPESC-RPE cell suspension or balanced salt solution vehicle control under the retina under isoflurane anesthesia. Surgical success was confirmed by visualization of a subretinal bleb using optical coherence tomography. The spatial frequency threshold for OKT was measured (Table SI) by observers masked to treatment group using a device (CerebralMechanics) and methods previously described (Douglas et al., 2005; Prusky et al., 2004; Table S13). The results were characterized by a non-normal bimodal distribution leading us to assign transplants as either efficacious or not efficacious. In addition to binomial testing, a Bayesian approach was utilized to compare groups using R. Briefly, a ROPE was established using the results from 92 sham experiments. Each group was then compared with this ROPE to determine if a treatment demonstrated an unambiguous difference from the sham experiments.

[0237] In vitro integration assay

[0238] A “receiver” monolayer of RPE (lines 270 and 322) was transfected with an RFP lentiviral maker for visualization and grown on Transwell inserts (6.5 mm diameter) for 8 wk. The integrity' and maturation of the monolayer was examined by immunostaining for RPE65 and phalloidin. “Donor” RPESC-RPE cells at P2 (lines 270 and 323) were transfected with a GFP lentiviral maker and grown for 4 weeks, made into a single-cell suspension, and 12,000 RPESC-RPE cells were transplanted onto the receiver RPE monolayer (FIG. 4E). After 1 week. Transwells were washed, fixed with 4% paraformaldehyde, and imaged using an LSM780 Zeiss confocal microscope (Zeiss). 20 images, whose locations were distributed across the Transwell Attorney Docket No. 27562-0031WO1 and in the same relative position to each other, were taken for each Transwell. The images covered ~4% of the wells’ surface. Confocal images were analyzed using the Zeiss ZEN software (V3. 1). Integrated cells were identified by determining if a nucleus from a donor cell was in the same z-plane as receiver nuclei using the 2.5D view. The percent of integrated cells for each sample was calculated using the following equation: Number of integrated cells plated (donor cells x covered well surface) x 100% integrated RPESC-RPE

[0239] Immunofluorescence staining

[0240] Fixed Transwell membranes (in 4% paraformaldehyde for 10 min at room temperature) were immunostained for RPE65. EZH2, and YEATS2 to determine the identity7of integrated cells. Cells were permeabilized using 1% Triton X-100 in Dulbecco’s PBS for 1 h at room temperature. After removing Triton X-100 and rinsing two times with DPBS, cells were incubated with RPE65, EZH2, and YEATS2 primary antibodies (Thermo Fisher Scientific) according to the manufacturer’s instructions. Next, primary antibodies were removed and cells were washed with DPBS twice and incubated with secondary donkey anti-rabbit or goat anti-mouse antibody conjugated with Alexafluor 647 or Alexafluor 555 dyes and DAPI (1:1,000) diluted in DPBS with 0.5% BSA for 1 h at room temperature. Immunostained cells were then washed two times with DPBS for 5 min at room temperature and mounted on glass slides using Fluoromount G (Thermo Fisher Scientific) and a coverslip. Samples were imaged using an LSM780 Zeiss confocal microscope (Zeiss).

[0241] Quantitative RT-PCR

[0242] RNA was isolated with the Direct-zol RNA kit (Zymo Research) and cDNA was generated using the Superscript VILO kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Quantitative RT-PCR was performed using TaqMan gene expression assays and TaqMan Universal Master Mix (Thermo Fisher Scientific). Either 18s-VIC or HPRT-VIC was used as an internal control for all reactions.

[0243] CITE-Seq Experimental Model and Subject Details Attorney Docket No. 27562-0031WO1

[0244] CITE-Seq was performed on primary' RPE cells from three adult human donor eyes and three cultured adult RPE cell lines at Passage 2. All donor eyes were obtained from approved eye banks with consent for research use. RPE cells were then dissociated from donor tissues and CITE-Seq was performed on acutely isolated and cultured cells.

[0245] Eye dissection and RPE dissociation

[0246] Human eyes were cut at the ora serrata, and the RPE layer was exposed by removing the anterior segment, the vitreous and retina. RPE dissection and was performed with care taken to avoid the edge of the posterior eyecup or to puncture Bruch’s membrane. 16 RPE cells were dissociated by incubating the RPE with collagenase IV (170 Units / mL) at 37 C in a humidified incubator. Cultured RPE lines were prepared by culturing dissociated cells for 7-8 weeks followed by cr oprescrvation using CS2 medium at Pl. RPE cultures used in CITE-Seq experiments were generated by thawed Pl cells and culturing them on Transwell inserts for 2-10 weeks. RPE cultures were maintained in RPE medium: DMEM F 12 50 / 50 medium (Coming), MEM alpha modification medium (Sigma- Aldrich), 1.25 ml Glutamax (Gibco), 2.5 ml sodium pyruvate (Gibco), 2.5 ml niacinamide (1 M; Spectrum Chemical Inc.), 2.5 ml MEM non-essential amino acid solution (Gibco), 10% heat-inactivated fetal bovine serum (FBS), supplemented with THT (taurine, hydrocortisone, triiodo-thyronin), and 1.25 ml N1 medium supplement (Sigma- Aldrich). Cells were incubated in a humidified incubator at 37°C and 5% CO2. RPE medium with 10% FBS was used until the cultures were confluent and 2% FBS was used after confluency. The medium was replaced every 3 days.

[0247] CITE-Seq

[0248] After dissociation of adult RPE cells from human donor eyes or cultured cells from transwell inserts, the resulting single RPE cells were tagged with the TotalSeq-A human universal cocktail, V1.0 (Biolegend), containing 154 unique cell surface antigens and 9 isoform control antibodies. Cells were then washed three times with DPBS and stained with 1 pl SYTO64 dye (Invitrogen, Carlsbad, CA) in IrnL DPBS for 20 minutes at room temperature, then washed twice in fresh DPBS. Next, cells Attorney Docket No. 27562-0031WO1 were diluted to 25,000 cells / mL and dispensed into ICELL8® 3’ DE chips (Takara Bio, CA) using an MSND device (Takara Bio). Cell dispensing, and in-chip reverse transcription PCR were performed using a 3’ DE Chip and Reagent kit (Takara Bio) according to the manufacturer’s instructions.

[0249] Library preparation and sequencing

[0250] To prepare ADT libraries, an ADT additive primer (0.1 ng) was introduced into the RT-PCR reaction. Following PCR product extraction, cDNA samples underwent concentration and purification steps using a DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA) and were further purified with a 0.6X proportion of AMPure XP magnetic beads (Beckman Coulter. Brea. CA) following manufacturer protocols. Subsequently, ADT libraries were constructed from the supernatant of the initial AMPure XP purification utilizing 2X AMPure XP magnetic beads. Amplification and purification of the purified cDNA samples were carried out using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) in accordance with the instructions provided in Takara's 3’ DE chip and reagent kit (Biolegend). ADT libraries underwent amplification using RPI-X primers containing the P7 sequence and were then purified using 2X AMPure XP beads. The concentration of cDNA products was determined using a Qubit Fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA). Quality assessment of the cDNA and ADT product was conducted using an Agilent High Sensitivity DNA Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to ensure the complete removal of contaminants. Finally, libraries were sequenced on a NovaSeq 6000 high-output flow cell, generating 2 x 150-bp read lengths (GeneWiz).

[0251] Data processing and analysis

[0252] Raw Illumina read BCL Files were converted to fastq files using the bcl2fastq2 software (bcl2Fastq v2.19.1, Illumina™, Inc). Fastq files were then merged into readl (ICELL8® barcode sequence) and read2 (transcript sequence) fastq files. Read were mapped to human hg38 genome using the Cogent NGS Analysis Pipeline (VI.5, Takara Bio). The resulting transcript and ADT read count matrices were used as the input for further analysis with Seurat (V4 and V5) package for R. Attorney Docket No. 27562-0031WO1

[0253] First, the counts matrix was converted to a Seurat object using CreateSeuratObject function with a minimum cell cutoff of 3 and a minimum feature cutoff of 200. Data integration and Weighted Nearest Neighbor Analysis were performed using the Seurat package to combine different datasets and cluster cells using both transcriptomic and surfaceome data.

[0254] RPE Flat mount preparation and immunofluorescence staining

[0255] Human donor eyes were dissected as described in earlier. After removing the retina, the eyecup was filled with 4% paraformaldehyde to fix the RPE-choroid for 1 hour at room temperature. RPE-choroid was flattened by cutting the RPE-choroid- sclera from the edge towards the optic nerve. Flat mount pieces containing peripheral and central RPE were moved to glass slides and immunostained with conjugated primary antibodies diluted in 2% BSA according to the manufacturer instructions. Tissues were then washed with DBPS three times and covered with glass coverslips using Fluoromount-G (Thermofisher) mounting media and imaged using a fluorescence microscope.

[0256] Cell sorting

[0257] Magnetic activated cell sorting (MACS) and florescence activated cell sorting (FACS) were performed for positive selection of RPE subpopulations. For MACS sorting, RPE cells were immunostained using PE- or APC-conjugated antibodies and MACS anti PE of anti APC magnetic microbeads were used to capture positively labeled cells using MACS MS Columns (Miltenyi Biotec). Briefly, cells were incubated with PE- or APC-conjugated primary antibodies for 20 minutes, rinsed twice with DPBS+2% Bovine Serum Albumin (BSA) and captured in MS columns attached to a MACS separator magnet (Miltenyi Biotec). Unlabeled cells were collected by washing the column twice with RPE medium containing 10% FBS and labeled cells were released by removing the column from the magnetic stand and washing the column with fresh RPE medium with 10% FBS. Flow cytometry and FACS sorting was performed using an ARIA I system (BD Biosciences).

[0258] Time-lapse imaging and cell migration analysis Attorney Docket No. 27562-0031WO1

[0259] After cell sorting, labeled and unlabeled cells were cultured in 12-well plates at -10,000 cells per well. Time-lapse microscopy was performed on cultured cells using a Zeiss Axio Observer Z 1 microscope equipped with a humidified incubator to maintained the cells at 37oC and 5% CO2. Time-lapse videos were analyzed in Python (V 3.12.2) using pyclesperanto and napari-pyclesperanto-assistant plugin for segmentation, and btrack for cell tracking. The analysis of tracking data and figure generation were performed in R (V 4.3.2).

[0260] Image processing

[0261] Morphological analysis was performed by analyzing phase contrast and phalloidin staining images from human RPE or cultured RPE. Images were converted to binary and cell-cell junctions were detected using the Ridge Detection plugin for ImageJ (https: / / ieeexplore.ieee.org / abstract / document / 659930). Object identification and morphological analysis were performed using the EBImage package in R. 17.

[0262] Statistical Analysis

[0263] Analysis of single cell transcriptomic data was carried out in R utilizing the following packages: Seurat (V4 and V5), ggplot2, hypeR, rrvgo. GOfuncR, igraph, and riverplot packages. Differences in the distribution of cell area in morphologican analysis and migration distance in cell tracking were analyzed with a two-sample test based on Wasserstein’s distance using the twosamples package (V 2.0.1) in R. All code and session info written for this study can be found at github.

[0264] EXAMPLE 1: Bulk RNA-seq uncovers a transplant efficacy gene signature

[0265] We sought to determine genes associated with efficient RPE cell transplantation by comparing the transcriptome of RPESC-RPE cultures over an 8-wk time course. We utilized RPESC-RPE lines with previously established transplantation effects or a transplant status that could be predicted based on the transplant status of neighboring samples in the timeline (lines 228, 229, and 230; FIG. 1A and FIG IB). In addition, we isolated RPESC-RPE from a fourth donor (line 233) for the experiments. During culture of each RPE line, we collected RNA at tire 2-, 3-, 4-, 5-, 7-, and 8-week time points for library preparation and sequencing. The data were then mapped using STAR aligner (Dobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1:29(1): 15-21.). We accounted for cell-line-to-cell-line variance by batch correction using Attorney Docket No. 27562-0031WO1 combat-seq (Zhang Y, et al. ComBat-seq: batch effect adjustment for RNA-seq count data. NAR Genom Bioinform. 2020 Sep;2(3):lqaa078.) (note that tire RPE line 233 at the 4-wk time point had much lower counts and that time point was discarded from subsequent analysis).

[0266] We selected the 4,000 most variable genes in the whole bulk RNA-seq dataset and utilized the singular value decomposition (SVD) approach to examine the relationship of samples to each other. Utilizing the known and predicted transplant status of the samples clearly separated them into three groupings based on transplantation status: 2-wk non-efficacious cultured RPE cells (W2-NE), efficacious RPE (EFF-RPE), or non- efficacious RPE (NE-RPE; FIG. ID).

[0267] Using this grouping, we proceeded to look for differential gene expression to identify potential biomarkers of transplant efficacy. We used the general linear model approaches in the edgeR and DESeq2 packages to identify differentially expressed genes (DEGs) and included genes identified by both approaches for additional confidence. There were 1,465 DEGs with ~14% being long-noncoding RNAs (IncRNAs; FIG. IE). We next determined the extent to which these genes were associated with a transplant group based on their maximum expression and found that the majority of coding DEGs were associated with theW2-NE group (FIG. IF) while the IncRNA DEGs were associated with each group in roughly equal numbers (FIG. 1G). Proportionately, the IncRNAs made up a much larger fraction of the DEGs in both the EFF-RPE and the NE- RPE. These results indicate that these genes distinguish between groups based on transplant efficacy.

[0268] To gain insight into the biological processes differing between RPE cultures from the W2-NE, EFF-RPE, and NE-RPE groups, we performed enrichment analysis using the goseq package (Young MD. et al. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010; 11(2):R14.). Following the gene ontology (GO) enrichment testing, semantic similarity analysis was used to group terms (FIG. 1H). Similarly, we performed enrichment for REACTOME pathways (FIG. II). Some of the most over-represented GO parent terms and REACTOME pathways were associated with proliferation and developmental processes, supporting tire concept that the RPESCs are undergoing developmental processes during cell culture and that the cells reach a specific point of intermediate maturation that is ideal for transplantation. The SVD analysis (FIG. 1C and FIG ID) suggests that as tire RPESC-RPE cultures develop, multiple Attorney Docket No. 27562-0031WO1 subpopulations arise, with one or more subpopulations more effectively conferring transplantation efficacy.

[0269] EXAMPLE 2: Single-cell sequencing reveals changes in RPESC-RPE heterogeneity over time

[0270] We next sought to identify if changes in RPE subpopulations influence transplant efficacy. For these experiments, P2 RPESC-dcrivcd cells were cultured for 2, 4, and 8 weeks using the same methodology as for the bulk RNA-seq experiments described above. The ICELL8® platform (Takara Bio) was used to isolate single RPE cells and generate libraries for sequencing with Illumina™ NovaSeq™ 6000. Data were processed and mapped with the Cogent™ N GS Analysis Pipeline (V 1 ; T akara Bio) utilizing the STAR aligner. The mapped data were then analyzed using the Seurat (v3) package in R and normalization was performed using the SCTransform pipeline (Hafemeister C, Satija R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 2019 Dec 23;20(l):296 ). The dataset was analyzed and 13 clusters (decreasing in size from 0 to 12) representing subpopulations of RPE cells (FIG. 2A) were discovered. All clusters contained cells from all time points (FIG. 2B), but tire subpopulation composition of the whole RPE population changed with time in culture (FIG. 2C).

[0271] Subpopulations were identified as RPESC-RPE cells based on expression of previously identified human RPE cell signature genes (Bennis A, et al. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration. PLoS One. 2015 Oct 30; 10(10):e0141597.); all RPESC-RPE cell clusters demonstrated expression of the majority (136-163 / 171. 79-95%) of RPE signature genes, underscoring the RPE identity of the subpopulations.

[0272] To identify genes associated with each cluster, we used the FindAllMarkers function from the Seurat package in R (FIG. 2D). Each cluster was associated with between 6 (cluster 11) and 1,967 (cluster 10) genes with a median of 300 genes across clusters (FIG. 2D). RPE-associated genes such as RPE65 and BEST1 demonstrated differential expression across the subpopulations. BEST1 has higher expression in clusters 2, 3, 8, and 9. and RPE65 showed a similar expression pattern but with increased expression in cluster 7 rather than cluster 9. Transthyretin (TTR) also demonstrated variable expression, with cluster 3 having the highest expression followed by cluster 8. Attorney Docket No. 27562-0031WO1

[0273] CXCL14 demonstrated higher expression in clusters 7 and 5, suggesting a potential macular phenotype for these clusters.

[0274] EXAMPLE 3: Enrichment analysis reveals RPE subpopulation functional specialization

[0275] GO and REACTOME pathway enrichment with the hyper package were utilized to uncover high-level functional differences among the RPE subpopulations. The number of enriched GO categories per cluster ranged from 18 categories in cluster 4 to 465 categories in cluster 10, with a median of 76 enriched categories per cluster. After GO enrichments were calculated, a semantic similarity analysis for tire enriched terms was performed to group the terms together and assist in visualizing and summarizing the analysis (FIG. 2E). For visualizing the signaling pathway enrichments, we utilized the REACTOME pathways database (v72), which is arranged into multiple hierarchical trees composed of related pathways. For our analysis, we took the top-level terms in the trees and counted the number of enriched pathways under those terms (FIG. 2F). The number of enriched pathways for each cluster ranged from 0 in cluster 9 to 329 in cluster 10 with a median of 12 enriched pathways per cluster. Most clusters showed enrichment for "homeostatic processes ’ ‘"intracellular transport,” and “sensory organ development” GO categories as well as metabolism- and signaling-related REACTOME pathways (FIG. 2E and FIG. 2F), as expected given the role of RPE to maintain the retinal microenvironment. Cluster 0 (~20% of cells) demonstrated strong enrichments for metabolic pathways and pathways involved in protein and ion transport. Cluster 1 (~ 17% of cells) also demonstrated enrichment for pathways associated with metabolism and GO terms associated with metabolism and molecule transport.

[0276] Cluster 2 (~9% of cells) showed enrichment in a broader number of GO categories ranging from “homeostatic process,” and “ion transport” to “growth,” “neurogenesis,” and a variety of development-related terms. Cluster 2 is enriched for many REACTOME pathways associated with “signal transduction,” indicating these RPE cells are likely reactive to environmental challenges and may have a particularly active role in regulating the retinal microenvironment. Cluster 3 (~8% of cells) had a similar enrichment profile to cluster 2; however, cluster 3 had less enrichment in developmental pathways and an increased enrichment for metabolic and response to stress functions. In contrast, cluster 4 (~8% of cells) was enriched for fewer GO terms and pathways and the enriched functions were focused on metabolic activities. Together these five clusters Attorney Docket No. 27562-0031WO1 make up more than 60% of the RPE cells sequenced; they show a spectrum of functions, with cluster 4 being highly metabolic, cluster 2 being highly reactive, and clusters 0, 1, and 3 falling between these categories of function.

[0277] The pathway enrichments in the remaining clusters also revealed specialization in function. Clusters 5 and 7 showed very similar patterns of enrichment in the GO and pathway analysis for functions associated with locomotion and morphogenesis. The enrichment profile of these clusters could be indicative of subpopulations that could successfully integrate into an RPE monolayer. Notably, cell cycle was substantially enriched in clusters 6 and 10, indicating these may be proliferative subpopulations. Clusters 6 and 10 were also enriched in signal transduction and immune and cytokine responses. These clusters are similar to cluster 2 in that they have enrichment profiles with a significant developmental component that could be indicative of populations that benefit cell manufacture and transplantation. Cluster 8 was enriched for functions of molecule transport and stress response. Clusters 9 and 11 were not enriched for any functions and both had few marker genes. Cluster 12, which also had a small number of marker genes, did show enrichment for functions associated with metabolic activities and cation transport. Overall, the RPE subpopulations have overlapping, but distinct functional profiles. Based on the enrichment data, several clusters are candidate subpopulations for efficacious transplantation.

[0278] EXAMPLE 4: Morphological analysis showed significant morphological heterogeneity in RPESC-RPE cultures

[0279] We also performed morphological analysis on single P2 RPESC-RPE cells that were cultured for 2, 4, and 8 weeks. Microscopy images of RPE cells stained with ActinRed™ 555 were used to visualize cell membrane and quantify area, shape, and number of neighbors for individual cells. A total of 3,990 2-wk-old, 3,817 4-wk-old, and 3,623 8-wk-old cells were analyzed. Cells were classified according to cell area into four different size categories (A1-A4) based on statistically different RPE cell sizes in human eyes. The percentage of tire smallest size category (Al : 0-150 pm2) decreased while the percentage of A2 (150-250 pm2) and A3 (250-350 pm2) cells increased with time. The percentage of the largest cell category (A4: >350 pm2) remained unchanged from 2 to 8 weeks. The compactness of the cells was used as a measure of cell elongation, with compact round cells having a value close to 1 and decreased values for elongated cells. The compactness of the cells increased and the number of neighbors decreased with time. Attorney Docket No. 27562-0031WO1

[0280] EXAMPLE 5: Intersection of scRNA-seq and bulk RNA-seq data implicates three clusters that are more likely to confer transplantation efficacy

[0281] To gain a better understanding of which subpopulations may play a role in transplantation efficacy, we intersected the DEGs correlating positively or negatively with efficiency (Bulk-Eff) from the bulk RNA-seq data with the scRNA-seq data. Two sets of DEGs were determined using the FindAllMarkcrs function in Seurat: the first set was DEGs across the 2-, 4-, and 8-week culture time points (Time-SC) and the second set was DEGs across the clusters (Cluster-SC). 45% of the Bulk-Eff DEGs intersected with at least one of the two gene sets from the scRNA-seq data (FIG. 3A). The Bulk-Eff DEGs were most abundantly expressed in the single-cell data at the 4-week time point (FIG. 3B). When we compared the Bulk-Eff DEGs to the Cluster-SC data, we found that the genes associated with efficacy were most abundantly expressed in clusters 2, 6, and 10 (FIG. 3C). As noted in the previous section, clusters 2, 6, and 10 were enriched for a variety of functions related to development, environmental reactivity, and, notable in cluster 10, proliferation. This enrichment profile along with the large number of marker genes associated with transplant efficacy make these three subpopulations the lead candidates for playing a key role in successful engraftment and vision rescue after transplantation.

[0282] Following up on this discovery, we intersected the markers of clusters 2, 6, and 10 with the Bulk-Eff DEGs and performed GO (FIG. 3D) and REACTOME pathway (FIG. 3E) enrichment analyses on the intersected gene list for each cluster. The GO analysis revealed differences in the intersected genes from the selected subpopulations (FIG. 3D and FIG. 3E) when compared with tire overall cluster GO and REACTOME data (FIG. 2E and FIG. 2F). The intersected gene set from cluster 10 was highly enriched for terms relating to proliferation and cell organization. The cluster 2 intersected gene set was enriched for several terms relating to metabolism and homeostatic processes. The intersected genes from cluster 6 were more specifically enriched for terms related to development and cell differentiation than the total cluster 6 marker genes. In tire REACTOME analysis, the cluster 2 intersected gene set was enriched for either pathways related to vision or sensing external stimuli. The cluster 6 intersected gene set was enriched for developmental pathways and pathways relating to the extracellular matrix, and specifically for pathways interacting with extracellular matrix components and for degrading the matrix. This cluster 6 enrichment profile may suggest a subpopulation Attorney Docket No. 27562-0031WO1 capable of breaking down and integrating into the RPE monolayer. The cluster 10 intersected gene set was again highly enriched for proliferation pathways. Overall, this data analysis indicates that three subpopulations of RPESC-RPE cells, clusters 2, 6, and 10, have gene expression correlating with properties that can contribute to efficacious transplantation.

[0283] EXAMPLE 6: Cluster 10, but not cluster 2, RPESC-RPE subpopulations can integrate into an RPE monolayer

[0284] RPESC-RPE cells can integrate into the existing RPE monolayer in vivo. As a surrogate of the cell integration process, we developed an in vitro assay. The assay workflow begins by culturing RPE test cells and labeling these with GFP. Then ~ 12,000 GFP+ test cells are plated onto a preexisting 8-week-old RPE monolayer grown in 24- well Transwell® format; at the 8-week stage, the monolayers are highly polarized and exhibit typical mature RPE cobblestone morphology that serves as an in vitro model of the native RPE layer. 7 days after plating tire GFP+ labeled test cells on tire mature RPE monolayer, the cultures are fixed and imaged by confocal microscopy over a pre-set 20- position grid covering ~4% of the Transwell® surface. GFP+ cells that integrate into the mature GFP-negative monolayer are then counted and the percentage of integrated cells is calculated.

[0285] We performed this in vitro integration assay by plating 4-week old GFP-labeled RPESC-RPE test cultures on mature monolayers. To determine if integrating cells came from a particular subpopulation, we identified markers for our candidate cluster 2 (YEATS2) and cluster 10 (EZH2) subpopulations and used confocal immunofluorescence to determine if the integrated cells expressed either marker along with GFP. Our results showed that ~90% of EZH2+ cells present in the original suspension had integrated into the RPE monolayer. Cluster 10 only7makes up ~3% of the original 4-week RPESC-RPE population, yet EZH2+ cells included 22% of all integrated cells (FIG. 3F). This demonstrates that cluster 10 cells will successfully integrate and establish in a preformed RPE monolayer. None of the integrated RPESC-RPE cells exhibited YEATS2 staining, indicating that tire cluster 2 subpopulation, which represents ~9% of the original isolate, did not successfully establish within the monolayer. These results indicate that specific RPE subpopulations contribute to an efficacious transplant.

[0286] EXAMPLE 7: An IncRNA as a biomarker of efficacious transplantation Attorney Docket No. 27562-0031WO1

[0287] After probing for a possible connection between transplantation and changes in the RPE subpopulation composition, we sought to identify a biomarker of efficacious transplantation. Potential efficacy marker candidates were identified by selecting the genes that had a maximum expression of at least 100 counts in EFF-RPE and at least a two-fold increase in expression over the W2-NE and NE-RPE groups in our bulk sequencing data. This yielded a total of 36 candidate markers. The most consistent and differentially expressed candidate gene is an IncRNA, TCONS_00005049, referred to as TREX (transplanted RPE expressed). We examined the level of TREX in the bulk RNA- seq data of RPESC-RPE samples that had available in vivo efficacy data in the RCS rat quantified by optokinetic tracking (OKT) measures of visual acuity. There was a positive correlation between OKT data indicating improved vision and increased levels of TREX (FIG. 4A). We next verified TREX levels in these samples by performing quantitative PCR (qPCR), and again EFF-RPE demonstrated higher levels of TREX (FIG. 4B) than the non-efficacious groups. These data suggest that a threshold level of TREX expression was associated with effective transplantation and improved vision.

[0288] We next undertook qPCR for nine RPESC-RPE lines including clinical-gradc cultures obtained under GMP conditions at different cell culture times (FIG. 4C). Most of the lines follow a similar trend with a low level at two weeks peaking at four weeks. Based on the known transplant data outcomes described previously, we were then able to determine a minimal level of TREX expression (compared to control 18s levels) that 'as associated with a successful transplant. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) w as also used as an internal control for future use, using RPE samples that had demonstrated in vivo efficacy (229 at 7 w eek) versus not efficacious (230 at 8 week; FIG. 4D).

[0289] We next sought to manipulate the level of TREX. As a starting point, we determined if TREX was primarily in the cytoplasm or the nucleus. Using two different RPESC-RPE cell lines cultured for 4 weeks, we performed qPCR for TREX on the cytoplasmic fraction or on isolated nuclei and found a 40%:60% distribution, respectively (FIG. 4E). Based on this distribution, we used a gapmer-based approach to knock down TREX levels. Gapmer knockdow n (KD) relies on RNase H to cleave the targets and can be effective in both the nucleus and cytoplasm. Wc designed and tested four gapmers against TREX and achieved over 50% KD with one of the gapmers compared with scrambled and untransfected controls (FIG. 4F). Attorney Docket No. 27562-0031WO1

[0290] To assess the functional effect of TREX levels on RPE transplantation, we utilized our in vitro integration assay. Using the gapmer-based approach, we knocked down TREX in RPESC-RPE cells that had been cultured for 4 weeks and then plated the TREX KD cells on the established mature RPE monolayers used for the integration assay. The scrambled control cells had an integration rate of ~30% while significantly fewer TREX-KD cells were integrated (~12%; FIG. 4G).

[0291] We next used a lentiviral overexpression system to overexpress TREX in RPESC- RPE cells cultured for 4 weeks and then perfomied the integration assay. The empty- vector control cells had an integration rate of ~20% whereas tire TREX overexpressing cells demonstrated a marked increase in integration with ~45% or double the number of cells integrating into the mature monolayer (FIG. 4H). These results indicate that TREX is associated with an increase in engraftment and transplant efficacy, supporting an important role of TREX to mediate RPE cell integration into a mature RPE monolayer.

[0292] Based on our in vitro results, we proceeded to assess the role of TREX in RPE transplant efficacy at vision rescue. As previously observ ed, RPE line 230 cells cultured for 7 weeks were not efficacious after subretinal transplantation in the RCS rat model of retinal degeneration (FIG. IB). We transfected RPE 230 cells with either an empty control vector or a dox-inducible TREX overexpression (TREX-OE) virus and cultured them for 7 weeks prior to transplantation. Vision was measured by OKT at 60 days after transplantation. Eight rats were transplanted for each condition and five sham (vehicle only) subretinal injections were used as control.

[0293] The OKT results generally have a bimodal distribution indicative of cither a positive effect on vision rescue or a lack of vision rescue. An animal’s vision was considered "‘rescued” if the OKT measure was above 0.4 cycles / degree and considered “not rescued” if below this threshold. We took a Bayesian approach to the analysis and identified a “region of practical equivalence” (ROPE, a range of values representing no effect) based on the highest density interval (HD1, range of values that 95% of the distribution lays under) from the OKT results at p90 of the cumulative sham (vehicle injected; n = 92). Using the sham results as a prior probability , we applied a Bernoulli likelihood function to calculate the posterior distribution of the probability of an efficacious transplant of our empty vector control (FIG. 41) and of TREX-OE cells (FIG. 4 J). We then sampled from the posterior probabilities to identify the HDI of each condition. Attorney Docket No. 27562-0031WO1

[0294] In the case of the empty vector control cells, tire HDI completely encompassed the ROPE, indicating that the control RPE cells performed similarly to the sham as previously observed with the inefficacious 7-week-old RPE 230 cultures. However, in the case of the TREX-OE cells, the HDI laid completely to the right of the ROPE, indicating an increase in efficacy associated with RPESC-RPE cells overexpressing TREX. In addition to our Bayesian approach, we also performed binomial tests for each comparison and found that while the control cells were not significantly different from sham (P = 0.09157), the TREX-OE cells were different from both sham (P = 1.92e-06) and the control cells (P = 0.004227). Hence, there was a positive effect of creating high TREX levels on cell transplant efficacy; we essentially converted a non-efficacious RPESC-RPE cell preparation to one that effectively rescued vision in this animal model.

[0295] We next sought to determine if having endogenous high levels of TREX was sufficient to demonstrate transplant efficacy. RPE line 255 has exceptionally high levels of TREX after only 2 weeks of culture (FIG. 4C), a time point in RPESC-RPE culture that is usually ineffective at vision rescue compared with 4 weeks of culture prior to transplantation. We cultured RPE 255 cells for 2 w eeks without modification and transplanted them into the RCS rat model at p30, followed by OKT 60 days later.

[0296] The results demonstrated that the RPE 225 cells with high endogenous TREX were indeed efficacious for transplantation (five out of five animals: FIG. 4K). This result indicates that TREX level was more accurate than time in culture as a biomarker of RPE255 transplantation efficacy. The HDI of the posterior probability of a successful transplant w'as outside our ROPE and the binomial test also supports a significant change (P = 1. 18e-06). Overall, these results provide strong evidence that TREX is not only a biomarker of efficacious RPE, but that TREX also functions to mediate RPE transplantation efficacy.

[0297] EXAMPLE 8: Identification of human RPE Subpopulations with distinct Surface Protein Expression

[0298] Three human donor eyes (2 males, 1 female) were obtained from a local eye bank, and RPE cells w ere dissociated for analysis. Utilizing a TotalSeq™-A antibody cocktail, acutely isolated cells were labeled with 156 uniquely barcoded antibodies, followed by single-cell library preparation using the ICELL8® system. After sequencing, the transcript reads were mapped to the human genome (hg38) and antibody derived tag (ADT) reads were mapped to an antibody / barcode sequence list. Dimensionality Attorney Docket No. 27562-0031WO1 reduction and clustering for RNA sequencing data were performed using the Seurat Package in R (schematic of workflow depicted in FIG. 5A). The ADT counts were ArcSinh transformed and normalized.

[0299] After clustering, seven distinct RPE subpopulations were identified, each characterized by unique surface protein and transcript markers (FIG. 5B). To verily that each cluster was composed of RPE cells, we examined the expression of a subset of 171 genes designated to be ‘RPE signature genes’ by bulk-RNA-seq analysis of acutely isolated RPE cells. 140 of tire analyzed genes were found to be expressed by primary RPE cells, with 95 of them being shared betw een all clusters. Each of the seven RPE cell clusters exhibited expression of at least 101 signature genes (FIG. 5C, Table 1), verifying the RPE identify of the revealed subpopulations.

[0300] Table 1 : Genes of Seven Distinct Subpopulations of RPE Cells Derived from Human Donor Eyes Attorney Docket No. 27562-0031WO1 Attorney Docket No. 27562-0031WO1 Attorney Docket No. 27562-0031WO1

[0301] To characterize the RPE subpopulations through our CITE-Scq analysis, we searched for differentially expressed genes and surface proteins across all seven clusters. Our results revealed unique gene and surface protein expression profdes for each cluster (FIG. 5D and FIG. 5E).

[0302] Cluster 1 exhibited higher expression of HTRA1, which has been associated with an increased risk of AMD development. Clusters 2 and 3 showed elevated expression of PLIN2, a gene involved in the formation of lipid droplets by RPE cells, contributing to drusen formation. Increased exposure to oxidative stress has also been shown to elevate PLIN2 expression in RPE cells. Cluster 4 displayed higher expression of HNRNPH3, which encodes an RNA-binding protein involved in RNA splicing. Reduced HNRNPH3 expression has been linked to an increased risk of AMD. Cluster 5 expressed a wide range of genes, including SLC39A6, a zinc transporter involved in lipid metabolism, and SIX3, a regulator of Wnt / p-catenin signaling that maintains neuroretinal progenitors. Cluster 6 exhibited higher levels of RB 1 CC 1 , an essential factor for the autophagic activity of RPE cells. Attorney Docket No. 27562-0031WO1

[0303] The CITE-Seq data also enabled us to identify distinct surface protein markers for each cluster. Cluster 1 exhibited higher expression of NCR1 and CD24. NCR1 is a marker of natural killer (NK) cells, and CD24 is a marker of neural progenitor cells and has been found to regulate cellular autophagy. Cluster 2 was characterized by elevated B3GAT1 expression. In addition to B3GAT1, Cluster 3 expressed CD58, TNFRSF14, ITGA1, and CD55. Cluster 4 showed significantly higher levels of CCR5, and Cluster 5 was enriched with TFRC. Although Cluster 6 expressed a diverse range of surface proteins, it exhibited exclusive expression of KIR3DL1 and SIGLEC7.

[0304] The expression of the majority of detected surface markers has not been previously reported in RPE cells. We proceeded to evaluate the expression of these proteins directly within human RPE tissue. We chose markers of Clusters 2 and 3, specifically B3GAT 1, CD24, and TNFRSF14, for this purpose. Immunostaining conducted on human donor RPE tissues revealed positive expression for all three markers. Subsequently, we investigated the spatial distribution of cells expressing these markers within the human RPE. For this purpose, we used a previously established classification system, categorizing RPE cells into five concentric regions, ranging from macular RPE (Pl) to peripheral RPE cells (P5) (FIG. 6A and FIG. 6B). Quantitative analysis revealed that B3GAT1 was expressed in cells across all five regions, with notably higher concentration observed in P2 and P5. CD24 expression was detected in regions P3, P4, and P5, while TNFRSF14 was exclusively localized to region P5. Interestingly, ITGB3 expression was limited to the midzone between the macula and periphery (P3 region). This result reveals that RPE subpopulations can have distinct regional distributions.

[0305] We next sought to determine if the positive cells in the different zones of tire native tissue demonstrated size differences to their neighbors (FIG. 6C and FIG. 6D). B3GAT1+ cells in Pl or P5 showed a similar distribution in size, however compared to the negative cells within each zone the B3GAT1+ cells were larger in Pl but smaller in P5 due to general regional size differences (FIG. 6D). Likewise, CD24+ cells showed a similar size distribution in both P3 and P5, however in both regions the CD24+ cells were smaller than the negative cells, contrary to tire results of our analysis in the cultured RPESC-RPE. TNFRSF 14+ cells in the P5 zone were smaller than their negative neighbors.

[0306] To investigate whether adult RPE subpopulations are recapitulated in RPE cells derived from the proliferative RPESC-RPE subpopulation, we also performed CITE-seq Attorney Docket No. 27562-0031WO1 experiments on passage 2 RPE cells that had been cultured for approximately 4 months and cryopreserved at passage 1, then thawed as passage 2 and cultured for 2, 4 and 10 weeks (FIG. 7A). After sequencing and initial QC filtering, cultured and primary CITE- Seq data were merged and clustered using the Seurat package in R. This resulted in the identification of 12 RPE subpopulations (FIG. 7B). Besides the initially analyzed 3706 acutely isolated (primary) RPE cells, we obtained 2586, 3354 and 2431 cells from the 2- week, 4-week and 10-week cultures, respectively. The average read depth was 221,231 per cell for the transcriptomic profiles and 272,903 per cell for the surfaceome profile.

[0307] Each of the 12 RPE cell clusters exhibited expression of at least 110 RPE signature genes, with 91 genes being shared across all clusters. In total, the expression of 140 RPE signature genes were detected in the combined dataset. Clusters 7, 8 and 11 displayed the highest number of RPE signature genes (129, 128 and 126 respectively), while clusters 4 and 0 exhibited the lowest number expression (1 10 and 1 15 genes, see Table 2). This analysis verifies the RPE identity of the revealed subpopulations in both native and cultured cell populations. Comparing tire combined dataset with the CITE-Seq data from only primary cells showed that most of the clusters found in primary RPE cells were preserved in the combined dataset, suggesting tire recapitulation of native RPE subtypes in cultured RPESC-RPE (FIG. 7C). The majority of cells in clusters 0 and 4 of the native datasets were gathered in cluster 1 of the combined dataset and native clusters 1, 2, 5 and 6 were assigned to clusters 8, 10, 7 and 8 of the merged dataset, respectively.

[0308] Table 2: Genes of 12 Distinct Subpopulations of RPE Cells Derived from RPE Cells Derived From the Proliferative RPE Subpopulation

[0309]

[0310]

[0311]

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[0313] Attorney Docket No. 27562-0031WO1

[0314] We next sought to determine cell surface proteins capable of isolating each subpopulation. We identified combinations of positive and negative surface markers for subpopulations that could be used in magnetic and FACS based isolation methods (FIG. 7D). For instance, CSF1R and ITGB3 can be used to isolate the cluster 6 and 9 subpopulations respectively.

[0315] To identify differences in the subpopulation composition of the acutely isolated native RPE and the cultured RPESC-RPE, we quantified the contribution of each timepoint to the size of each cluster (FIG. 7E and FIG. 7F). Except for cluster 10 which was primary constituted of primary cells, all subpopulations appeared to have cells from both native RPE cells and cultured RPESC-RPE cells. However, we noticed a time-point variability in some subpopulations. The predominant composition of clusters 1, 7, 8 and 10 and 11 was native RPE cells, while clusters 0 and 4 were mainly constituted by cultured RPESC-RPE cells, with 94 and 96 percent of the cluster sizes being accounted for by cultured cells (FIG. 7F). The comparison between cultured and native RPE showed a distinct surface protein signature in our CITE-seq data (FIG. 7G). In total, 43 surface proteins showed differential expression among different time points (Table 3). 2W and 4W cultured RPESC-RPE cells showed an increased expression of surface proteins, CD24, FAS, MCAM, CD151, PVR, F3 NECTIN2, CD81 and CD9. 4W RPESC-RPE cells had a unique surface protein signature with an elevated expression of HLA, CD47, CD58, NT5E, B3GAT1, ITGA1, CD44, CD55 and TNFRSF14. As expected, these markers were primarily associated with clusters with a higher proportion of cultured cells (FIGs. 7D-7F). The expression levels of all these markers decreased at 10W, reaching similar levels to those observed in primary cells (FIG. 7G). In contrast, primary RPE cells had a higher expression of CD74, CD200, FCER2, CD 163, CX3CR1, OLR1, and ICAM1, CCR6, IGHM and TFRC proteins. We also observed an increase in the expression of key RPE signature genes from 2 to 10 weeks in cultured RPESC-RPE cells (FIG. 7H), suggesting the in vitro maturation of cultured RPESC-RPE cells.

[0316] Table 3: Surface Proteins Showing Differential Expression Among Different Time Points Attorney Docket No. 27562-0031 WO 1 Attorney Docket No. 27562-0031 WO 1

[0317] Given our finding of surface markers that distinguish different clusters, these findings were confirmed by immunofluorescence microscopy of cultured RPESC-RPE cells. Immunostaining was performed on markers of RPESC-RPE -4W cells, targeting specific surface proteins B3GAT1, CD24, and TNFRSF14, as well as the cluster 9 marker, ITGB3 (FIG. 8A). In cultured RPESc-RPE cells, all four markers were detected, yet a diversity was evident among the cells expressing them. According to the CITE-Seq data, B3GAT1, CD24, TNFRSF14, and ITGB3 were expressed in 77.8%, 91.6%, 50.7%, and 4.7% of the RPESC- RPE-4W cells, respectively. Our immunostaining analysis revealed similar proportions for TNFRSF14 and ITGB3 populations (55.6% and 6.8%, respectively), while fewer B3GAT1 and CD24 expressing cells were observed (36% and 52%, respectively).

[0318] Morphological analysis showed that CD24+ and ITGB3+ cells were slightly larger and TNFRSF14+ cells were slightly smaller than their negative counterparts (FIG. 8B). Further analysis of cell morphology revealed that ITGB3+ cells had a significantly larger area than others (FIG. 8B). This observation underscores the morphological heterogeneity within cultured adult RPE cell populations and demonstrates a distinct morphological feature of the ITGB3+ subpopulation (cluster 9). Overall, these results illustrate distinct morphological and spatial distributions of the cell surface markers and subpopulations identified in our CITE-seq analysis in human RPE. Attorney Docket No. 27562-0031 WO 1

[0319] EXAMPLE 9: Expression of Surface Proteins in Human RPE Tissue

[0320] Our CITE-seq analysis revealed less expression of B3GAT1, CD24, TNFRSF14, and 1TGB3 in human RPE cells compared to 4W cultured RPESC-RPE cells. Specifically, approximately 36% of native RPE cells exhibited B3GAT1 expression, contrasting with 78% observed in 4-week-cultured RPESC-RPE cells. Similarly, CD24 expression was detected in 23% of native cells, whereas 92% of 4-week-cultured cells showed CD24 expression. TNFRSF14 expression was present in 17% of primary RPE cells, whereas 51% of 4-week- cultured cells exhibited TNFRSF14 expression. Notably, ITGB3 demonstrated a more consistent expression pattern, with 4% of primary RPE cells and 4.7% of RPESC-RPE-4W cells expressing this marker. To validate the expression of ITGB3 in primary cells, we performed immunofluorescence imaging on human RPE tissue and detected the expression of ITGB3 in a small subpopulation of native RPE cells located within the region 3 of the RPE tissue (FIG. 9A).

[0321] EXAMPLE 10: Functional Diversity Among RPE Subpopulations

[0322] To gain a deeper insight into the functional diversity among these RPE subpopulations, we first identified gene expression associated with each cluster using the FindAllMarkers function in the Seurat package. We then performed gene ontology pathway enrichment analysis utilizing the genes associated with each cluster (FIG. 9B). We employed a semantic similarity approach provided by the screp package in R. Our analysis revealed a significant diversity in pathway enrichment across different RPE subpopulations. Notably, cluster 0 and 3 and 4, predominantly including cultured RPESC-RPE cells, exhibited highly significant enrichment for pathways associated with cell cycle regulation, cell division. Despite the lower expression levels of RPE-specific markers within these subpopulations, likely indicative of their less mature state, they remained enriched for pathways crucial to fundamental RPE functions, such as pathways involved in pigmentation, establishment or maintenance of cell polarity and autophagy (FIG. 9B), highlighting the preservation of essential cellular processes despite the less mature stage of these cells.

[0323] In contrast, clusters 1 and 10, characterized by a higher proportion of native RPE cells, exhibited enrichment in pathways associated with metabolic processes and stress Attorney Docket No. 27562-0031 WO 1 response mechanisms. The fact that these clusters were also enriched for homeostasis pathways suggests a specialized functional profile within these clusters, emphasizing their potential role in lipid homeostasis and stress management as well as responding to external or internal stresses by activating specific molecular mechanisms to maintain cellular homeostasis and survival within the human RPE Clusters 2 and 3 had enrichment for a variety of pathways including cell cycle, migration, cytoskeleton organization and response to stress. Cluster 5 was enriched for pathways such as, response to organic cyclic and nitrogen compounds, response to endogenous stimulation and homeostatic process. Clusters 7, 8 and 11 showed enrichment for “sensory perception of light stimulus” and “vascular process in circulatory system”.

[0324] Cluster 9, marked by expression of ITGB3, exhibited predominant and substantial enrichment in pathways associated with cell morphogenesis, migration, locomotion, and regulation of organelle organization, suggesting a potential emphasis on cell motility function within this cluster. To investigate this observation further, we sought to investigate whether ITGB3+ cells exhibited the higher motility predicted by our GO pathway enrichment analysis.

[0325] EXAMPLE 11: ITGB3+ cells are more migratory than CSFR+ RPE cells

[0326] Magnetic-activated cell sorting (MACS) was used for the positive and negative selection of ITGB3- and CSFIR-expressing native RPE cells. We utilized the same MACS approach for sorting cells expressing ITGB3 or CSF1R in cultured RPESC-RPE. After isolation, cells were plated in culture plates and followed by time-lapse microscopy for 24 hours. Time-lapse videos were analyzed to quantify cell migration distances using the Bayesian Tracker (btrack) package in Python (FIG. 9C). This approach allowed us to explore the dynamic behaviors of ITGB3-expressing cells of both the cultured and native cluster 9 RPE subpopulation, shedding light on their migratory capabilities and potential functional significance.

[0327] CSF1R+ cells exhibited a slightly lower migratory capacity compared to CSF1R- cells within both RPESC-RPE -4W and native RPE. Overall, CSF1R+ cells migrated at a slower rate compared to CSF1R- cells in both cultured and primary RPE cells. However, primary CSF1R+ RPE cells migrated a longer distance compared to cultured cells (FIG. 9D). Attorney Docket No. 27562-0031 WO 1

[0328] The motility of ITGB3+ cells within both native and cultured RPE cells showed a bimodal distribution, indicating two distinct migratory potentials within the cluster 9 subpopulation. The larger population demonstrated high motility, with a peak migration distance of approximately 150 pm, while the smaller population exhibited low motility, with a peak migration distance of around 12 pm. Interestingly, in native RPE cells, the ITGB3+ cell population exhibited a slightly lower migration distance compared to the ITGB3- population due to a larger portion of the low-motility ITGB3+ population in primary cells. However, in cultured RPESC-RPE cells, we observed a different pattern, with the ITGB3+ cell population exhibiting higher motility compared to the ITGB3- population due to an increase in the higher motility ITGB3+ population and a general downward shift in the motility of the ITGB3- cells. The comparison of ITGB3+ cells in native and RPESC-RPE-4W settings did not show a significant difference between their migratory capacity, but a more prominent peak at approximately 12 pm was observed for native ITGB3+ cells (FIG. 9D). This observation showed a similarity in motility characteristic of native and cultured ITGB3+ cells. ITGB3+ cells maintained a bimodal motility profile in both settings, indicating a migration potential of the cluster 9 subpopulation. These results suggest that RPE subpopulation characteristics are preserved even after long-term in vitro culturing.

[0329] Finally, to compare the adult primary RPE and cultured RPESC-RPE cells with those derived from iPSC cells, we performed CITE-Seq on IPSC-derived RPE cells (iPSC-RPE) differentiated with a previously described method. These cells were cultured for 10 weeks prior to CITE-Seq experiments to match the 10W time point of the adult RPESC cultures. Our analysis showed a unique gene (FIG. 6A) and surface protein expression (FIG. 6B) for iPSC-RPE cells. These cells exhibited elevated expression of genes, such as ALDH1 Al, a regulator of VEGF expression in RPE cells, and PCDH9, a gene marker of cone bipolar cells, suggesting a less mature state for iPSC-RPE cells compared to primary RPE and cultured adult RPESC-RPE cells. A list of gene and surface protein markers expressed differentially between native RPE and cultured adult and iPSC-RPE cells can be found in Tables 6 and 7.

[0330] Table 6: Genes Differentially Express between Native RPE and Cultured Adult and iPSC-RPE Cells Attorney Docket No. 27562-0031 WO 1 Attorney Docket No. 27562-0031 WO 1 Attorney Docket No. 27562-0031 WO 1 Attorney Docket No. 27562-0031 WO 1

[0331] Table 7: RPESC-RPE Cell Surface markers verified by Flow Cytometry Attorney Docket No. 27562-0031 WO 1

[0332] EXAMPLE 12: Generating RPE Cell Derivatives by Extended Culture

[0333] RPESC cultures were generated from donated whole globes meeting previously established criteria. In-process monitoring metrics included total RPE cells isolated (> IxlO6cells per pair of eyes), attachment, and emergence of cobblestone morphology. RPE cells were seeded at a density of 100,000 to 132,000 cells per well of a 24-well plate, or 1,300,000 to 1,400,000 cells per T25 flask. After passaging once to achieve further expansion, RPESC were cryopreserved into master cell banks (MCBs) at 6 to 8 weeks in culture, with an average increase of 20-fold over the original isolate. Concurrent with banking, release testing was performed for viability, identity (expression of RPE markers), purity (lack of alpha- SMOOTH MUSCLE ACTIN / aSMA and KI67), potency (secretion of Pigment Epithelium- Derived Factor / PEDF), lack of adventitious viruses, genotyping for known AMD risk alleles and normal karyotype by G-banding (Table 8 below). The RPESC-RPE-4W drug substance was produced by thawing one or more MCB vials and culturing for 3.5 to 5 weeks when cells are largely post-mitotic but not fully differentiated RPESC-RPE. Up to 72 hours before harvest of ‘clinical’ wells, a ‘sister’ well was collected and tested for identity / punty, potency and sterility release criteria. Conditioned medium was collected before harvest and the concentration of PEDF was tested by Enzyme-linked Immunosorbent Assay (ELISA) as a measure of potency (>1,000 ng / ml). At times, PEDF measurement was normalized to sample volume and cell number. Cells were lifted and reseeded to terasaki micro-trays, incubated for 2 hours and fixed for immunostaining. Expression of MITF (report), OTX2 (>90% of cells positive) and BEST1 (>85% of cells positive) were measured by immunocytochemistry (ICC) as diagnostic of RPE identity. Purity was assessed by ICC for aSMA (<5% positive), an indicator of epitheli al -to-mesenchymal transition (EMT), and proliferation marker KI67 (<10% positive). Clinical wells were then harvested, measured for viability by manual cell count with trypan blue exclusion or automated cell count with propidium iodide exclusion (>80% viable), washed in ophthalmic Balanced Salt Solution (BSS) and formulated as a live cell suspension for transplantation. Cell surface markers expressed on RPESC-RPE-4W cells Attorney Docket No. 27562-0031 WO 1 after generation by culturing according to the methods described in this Example are provided in Table 9 below. Additional details of the process for generating RPESC-RPE-4W are discussed below.

[0334] RPESC isolation from donor eyes

[0335] Primary RPE cells were isolated from donor eyes obtained from approved eye banks with consent for research use. Pre-screened cadaveric eyes were procured within 12 hours post-death and transported to the GMP manufacturing facility within 35 hours post-death of the donor. Exclusion criteria were donor age over 80 years, chemotherapy treatment within the preceding 6 months, history of proliferative retinal disease or blindness, viremia, bacteremia and endophthalmitis. Donor eyes met criteria for cornea transplantation including viral screening but not including age (70 year upper limit), history of intravenous drug addiction, international travel, blood dyscrasias such as melanoma, lymphoma or leukemia, or the use of excessive colloid infusion. Determination of donor eligibility included but was not limited to testing for HIV, hepatitis and screening for spongiform encephalitis. The donors were assessed for common AMD-associated single nucleotide polymorphisms. The RPE layer was isolated and dissociated from the posterior eye cup. Eyes were fully submerged in betadine for 4-5 minutes to sterilize them. Transfer to gauze-filled dish and using scissors, cut and remove the anterior portion of the eye cup. Neural retinas were removed, collected in a microcentrifuge tube and stored at -80°C for donor genotyping.

[0336] Eye cup was rinsed with calcium- and magnesium-free Dulbecco’s phosphate buffered saline (CMF-DPBS) for 4 minutes, Trypsin-EDTA / DNase I was added. Eye cup was incubated for 40 minutes in incubator set at 37°C and 5% CO2 while dissecting second eye. EH-FBS was added to eye cup once it was ready to neutralize trypsin (RPE cells should come off by pipetting). Eye cup was transferred to microscope ready BSC. Under the dissection microscope, a sterile brush was used to gently brush the RPE cells off the membrane. A Pl 000 pipet was used to periodically remove trypsin to a conical tube and fresh TAB2+10% HI-FBS was added to maintain clear visibility. Brushing was continued until all visible patches of RPE were removed. Alternatively, eye cups were rinsed with CMF-DPBS, filled with Collagenase IV (170 units / ml) and incubated at 37oC and 5% CO2 for Attorney Docket No. 27562-0031 WO 1 approximately 4 - 5 hours. RPE cells were collected by pipetting without use of brushes, and transferred to conical tubes containing TAB2 + 10% HI-FBS.

[0337] Conical tubes were centrifuged at 286 x g for 5 minutes at room temperature. Supernatant was removed and cells resuspended in 5mL TAB2+10% HI-FBS. Cells were counted with a hemocytometer using trypan blue exclusion to identify live cells. Cells were spun again and resuspended in calculated amount of TAB2+10% HI-FBS to seed at least 100,000 cells per well in 600uL. 600uL of cell suspension was added to each well of a synthemax coated 24 well plate using a Pl 000. After 5 minutes plate was placed in incubator set at 37° C and 5% CO2. Plate was incubated without disturbance for 48 - 72 hours. Attachment was assessed between day 3 to day 5.

[0338] Expansion

[0339] Media changes were performed 3 times per week. On Mondays and Fridays, 50% of the medium (eg: 300 pL from one well of a 24-well plate) was removed and replaced with the same volume of pre-warmed TAB2 + HI-FBS medium. On Wednesdays, 1 / 3 volume (eg: 200 pL in one well of a 24-well plate) was added without removing any medium (“boost”). TAB2 + 10% HI-FBS was used for 3 media changes after seeding, followed by TAB2 + 2% HI-FBS for the remainder of the culture period.

[0340] Passaging

[0341] CMF-DPBS, TAB2+10% HI-FBS, and Tiypsin-EDTA were prewarmed in a bead bath set at 37°C. Trypsin-EDTA / DNasel was prepared by adding 3uL DNasel for every 1ml of trypsin. Media was aspirated from each well and cells were rinsed twice by adding 0.5mL CMF-PBS. 0.3mL of trypsin was added per well and incubated for 4 minutes. If cell edges were not yet rounded, cells were incubated for a maximum of two 2 minutes. Trypsin was removed and cells were added to conical tube containing an equal amount of TAB2+10% HI-FBS. Cells were washed twice with 0.3mL CMF-PBS by gently pipetting up and down around the edges of the wells. Cells were centrifuged at 286 x g for 5 minutes at room temperature. Cells were resuspended in 0.5mL - ImL TAB2+10% HI- FBS for every well. Cells were counted using a hemocytometer with trypan blue exclusion. Volume of cell suspension was adjusted with TAB2+10% HI-FBS and seeded Attorney Docket No. 27562-0031 WO 1 to appropriate cell culture vessel at at least 100,000 cells / well of a 24-well plate or at least 1,300,0000 cells / T25 flask. Vessels were left in the BSC for 5 minutes before being transferred to an incubator and were then left undisturbed for 48 - 72 hours. Cells were cultured for an additional 6 - 8 weeks as described above, followed by cryopreservation or were passaged for an additional expansion.

[0342] Cryopreservation of Master Cell Banks

[0343] Conditioned medium was drawn from each well and pooled into a 15mL conical tube labeled ‘supernatant’. Cells were harvested as described above. After counting, an aliquot of cell suspension was seeded to terasaki plates for ICC. Remaining cell suspension was centrifuged and resuspended in Cryostor CS2 cry opreservation medium to achieve 260,000 - 500,000 cells / vial in 500uL. 500uL was transferred into each prechilled cryovial and place into ice bucket. One vial was reserved as a control. Cells were allowed to sit on ice for 5-10 minutes. Cryovials were transferred to control rate freezer. After the cycle was complete, cryovials were transferred to LN2 storage.

[0344] RPESC-RPE Production

[0345] At least lOmL of TAB2+10% HI-FBS was prewarned in a 37°C bead bath. In the BSC 3mL TAB2+10% HI-FBS was added to a 15mL conical tube. Cryovial was removed from LN2 storage and caps were loosened to prevent pressure build up. Cryovial was immersed in beaker of water at 37°C, ensuring the O-ring stayed above the water. Vial was swirled and checked every 30 seconds until thawed. Outside of the cryovial was wiped down and transfered to BSC. Cells were transferred to conical tube using a P1000 pipette and rinsed with an additional 0.5mL of TAB2+10% HI-FBS. Conical tube was centrifuged at 286 x g for 5 minutes at room temperature. Supernatant was removed and cells were resuspended in TAB2+10% HI-FBS. Cells were counted with a hemocytometer. Cells were diluted witMh warm TAB2+10% HI-FBS and at least 100,000 live cells / well were plated in 0.6mL per well onto synthemax coated wells. After 5 minutes, plate was transferred to incubator and was not disturbed for at least 48 hours. Attorney Docket No. 27562-0031 WO 1

[0346] Conditioned medium was collected from each well and pooled into a 15mL conical tube labeled supernatant. RPE cells were harvested from incubated plate. Once cells were spun and resuspended in TAB2+10% HI-FBS, and counted with a hemocytometer. An aliquot of cell suspension was taken for seeding to terasakis for ICC. Cells were spun again at 286 x g for 5 minutes at room temperature. Cells were resuspended in 2mL of room temperature BSS (or 0.5mL / well harvested). Cells were centrifuged again and resuspend in cool injection vehicle. Cells were counted with a hemocytometer. Cells were diluted with injection vehicle to the dose concentration (500 live cells / pL). Cell suspension was transferred to externally-threaded screw cap vials.

[0347] Product Release Criteria

[0348] Product release criteria for MCB and drug substance (DS) are enumerated in Table 8 below. MCB release tests were performed concurrent with banking. DS release tests were performed up to 48 hours prior to harvest of product release, using a sister well to evaluate identity / purity and potency.

[0349] Table 8: Product Release Criteria for Master Cell Banks and Drug Substance Attorney Docket No. 27562-0031 WO 1 Attorney Docket No. 27562-0031 WO 1

[0350] Table 9: Cell Surface Markers Expressed on RPESC-RPE-4W Cells After Generation by Culturing

[0351] EXAMPLE 13: Cryopreservation of RPESC-RPE-4W Cells

[0352] We sought to develop a cryopreserved final cell product (RPESC-RPE-4W) for multiple reasons, including 1) increasing the number of doses that could be used effectively per donor line; 2) facilitating distribution of doses of RPESC-RPE-4W cell product to more people and more geographic regions; 3) reducing the expense of manufacture per dose (see, e.g., Figure 1). Properties of an effective cryopreserved RPESC-RPE-4W cell product can include high cell recovery and viability post-thaw, retention of identity and / or purity and potency characteristics. Additionally, these properties can be stably retained even with longterm storage in liquid nitrogen (LN2). As exemplified herein, we developed a stable formulation of cryopreserved RPESC-RPE-4W. This product can be implemented in at least two ways. In one scenario, cryopreserved RPESC-RPE-4W are thawed, washed and formulated in an Injection Vehicle (IV+). Alternatively, cryopreserved RPESC-RPE-4W vials can be thawed and directly diluted to the appropriate dose concentration with IV+.

[0353] In vivo experiments provided proof-of-principle that cryopreserved RPESC-RPE can deliver effective vision rescue immediately post-thaw, without additional culture. RPESC- RPE produced according to the methods disclosed herein were thawed and injected Attorney Docket No. 27562-0031 WO 1 subretinally in Royal College of Surgeons (RCS) rats. Visual acuity was measured at postnatal day 60 (P60) and P90 by optokinetic tracking (OKT), with vision rescue comparable to fresh RPESC-RPE-4W. Based on this result, we worked to increase recovery and viability.

[0354] We evaluated a variety of strategies for maintaining high viability of RPESC-RPE- 4W post-thaw and providing consistently high recovery post-thaw (Table 10 below). Viability refers to the percentage of live cells upon thaw, while recovery is the yield of live cells at thaw relative to the number of live cells frozen.

[0355] Table 10: Strategies tested for cryopreservation of RPESC-RPE-4W Cell Product

[0356] Advantageous modifications to the cryopreservation and thaw protocol identified included:

[0357] (1) pre-conditioning of all materials to 4°C followed by immediate transfer to -80°C, rather than slower transfer recommended by the CryoStor CS2 manufacturer (BioLife Solutions);

[0358] (2) the cell product is frozen at a significantly higher concentration of cells than previously used, such as 4.5xl06cells / mL; (3) custom wash steps before and after cryopreservation of cells.

[0359] After achieving consistently high recovery (>85% of input), we performed additional in-depth validation of post-thaw cell product as detailed below.

[0360] We measured the comparability and stability of cryopreserved RPESC-RPE-4W working cell banks (WCB). Comparison of fresh to frozen-thawed product demonstrated similar levels of positive (OTX2, BEST1, MITF) and negative (KI67) identity markers. Attorney Docket No. 27562-0031 WO 1

[0361] Thawed cells were also seeded to cell culture plates and left undisturbed for 3 days before testing PEDF production, a measure of potency. These results indicate that thawed product exceeds our release criterion of 125 ng PEDF / 100,000 cells. Finally, staining for SMA indicated that the freeze-thaw process did not increase epithelial-to-mesenchymal transition (EMT) characteristics. Moreover, cells seeded to culture plates did not exhibit increased fibroblastic morphology at 3 days or 4 weeks post-thaw. Finally, we demonstrated that these comparability criteria are stable in three donor lines stored for up to 12 months in liquid nitrogen.

[0362] Additionally, we measured the stability of thawed RPESC-RPE-4W in injection vehicle at 4°C. A previous injection vehicle (IV) extended the stable cold storage of formulated RPESC-RPE-4W doses to 48 hours when stored at 4-12°C. We found that while the new cryopreserved product was stable until 24 hours in IV, viability declined by 48 hours. We supplemented the original IV with a low concentration of human serum albumin and demonstrated that the new “IV+” formulation supported high viability of new cryopreserved RPESC-RPE-4W product from three donor cell lines through at least 72 hours after thaw.

[0363] Further, two strategies were developed for cryopreserving the RPESC-RPE-4W cell product (see, e.g., FIGs 13A-13B). Comparability data, scRNA-seq data, and integration data disclosed herein are applicable to either strategy. We therefore tested the viability of RPESC- RPE-4W cells after thawing and diluting in IV+ without a wash step. We found that cell viability was >90% across three donor lines, and that high viability was maintained to at least 4 hours post-thaw. These data suggest that a thaw-dilute-inject (TDI) product is achievable and that there will be sufficient flexibility to accommodate preparation and surgery.

[0364] Next, we quantified the ability of thawed RPESC-RPE-4W to integrate into an RPE monolayer in vitro. The ability of RPESC-RPE-4W cells to engraft into the existing RPE monolayer can be an important function for restoration of vision of a subject in need thereof. We used our established in vitro integration assay (Farjood F, et al. Identifying biomarkers of heterogeneity and transplantation efficacy in retinal pigment epithelial cells. J Exp Med. 2023 Dec 4;220(12):e20230913., incorporated herein by reference) to assess the ability of the new cryopreserved product to engraft as compared to the original fresh product. We found that cryopreserved RPESC-RPE-4W integration is comparable to fresh product. Further, we Attorney Docket No. 27562-0031 WO 1 tested two thawing approaches (Stage 1, thaw-wash-inject / TWI and Stage 2, thaw-dilute- inject / TDI). Collectively, these data indicate that the new cryopreserved product is functionally comparable to the fresh product, with equivalent ability to integrate into an established RPE monolayer.

[0365] Next, we developed a standard operating procedure (SOP) for RPESC-RPE-4W cryopreservation. We have verified that the cryopreservation method is both rigorous and readily transferrable to new operators of varying experience. Parameters and standards for standard operating procedures (SOPs) are provided in FIG. 14A and experimental results confirming that scaled-up larger format cell culture vessels perform similarly to experimental conditions (see, e.g., FIGs. 14B-14E).

[0366] EXAMPLE 14: Characterizing and Measuring Performance of Cryopreservation Methods

[0367] Features of Cryopreservation Method and Working Cell Banks (WCB)

[0368] Initial expenments indicated that RPESC-RPE-4W cells were lost upon resuspension in cryopreservative agents (CPAs). These experiments suggested that osmotic shock was a significant driver of cell loss. We determined empirically that the osmolarities of Cryostor CS2 and CS5 solutions are substantially higher than those of cell culture media or our custom injection vehicle (see, e.g., FIG. 15). Therefore, the cryopreservation protocol disclosed herein can include lowering the temperature of cells and CPA to reduce osmotic shock. Materials that come into contact with the cells after washing in Balanced Salt Solution (BSS) should be pre-conditioned and maintained at 4°C. This includes cryovials, Cryostor CS2 (CS2) solution and Mr. Frosty™ controlled rate freezing devices.

[0369] We therefore performed a direct comparison: a single suspension of RPESC-RPE-4W was made and 50 pl aliquots dispensed to multiple cryovials (FIG. 16A). Vials were transferred to separate Mr. Frosty™ devices, one was placed at 4°C for 10 minutes (“Manuf. Method”) and the other was moved immediately to -80°C (“NSCI Method”). Both sets were left at -80°C overnight, then all vials were transferred to liquid nitrogen (LN2) storage. Subsequently, cells were thawed and manually counted in TAB2 + 10% HI-FBS medium to determine viability and recovery. This experiment was repeated three separate times. Postthaw recovery was consistently higher using the NSCI method. We concluded that once cells Attorney Docket No. 27562-0031 WO 1 have been resuspended in CS2, cell suspension should be distributed to cryovials and moved to controlled rate freezer as quickly as possible. However, while these data suggested that the extended incubation at 4°C was detrimental to recovery, the maximum time frame has not been determined.

[0370] A BSS wash is performed at room temperature before resuspension in cold CS2. This is consistent with the progression of washes (warm TAB2+10% HI-FBS, room temperature BSS, cold IV) in the protocol for harvest and formulation of the current fresh RPESC-RPE- 4W product. We have found this BSS wash to be critical for efficient recovery of cells postthaw. Additionally, we performed manual cell counts using trypan blue exclusion at multiple post-thaw steps including in TAB2 + 10% HI-FBS wash and after formulation in our custom injection vehicle (IV; BSS supplemented with 1 g / L glucose and 2.2 g / L sodium pyruvate). We found that cells were lost during the Balanced Salt Solution (BSS) wash and formulation in IV (FIG. 16B). We therefore tested whether the addition of human serum albumin (HSA; Irvine Scientific, cat# 9988-100ML) to the BSS wash and IV would improve recovery. Cell counts in TAB2 +10% HI-FBS were normalized to the number of cells cryopreserved and cell counts in IV or IV + 0.1% HSA were normalized to the corresponding TAB2 + 10% HI- FBS count. This minor process modification enhanced recovery as shown in FIG. 16B.

[0371] Comparability Data

[0372] RPESC-RPE-4W release criteria (viability, identity / purity, potency) were used to compare fresh and cryopreserved cells (results shown in FIGs. 17A-17D). Cryopreserved RPESC-RPE-4W were thawed, washed and resuspended in TAB2 + 10% HI-FBS. Post-thaw viability was measured by manual cell counts with trypan blue exclusion. Thawed cells were seeded to Terasaki plates for immunocytochemistry (ICC) and to Synthemax-coated 96-well plates for PEDF release measurements by Enzyme-Linked Immunosorbent Assay (ELISA).

[0373] To measure PEDF release post-thaw, we seeded 150,000 cells in 150 pl TAB2 + 10% HI-FBS medium to a single Synthemax-coated well of a 96-well plate. These parameters were selected because the surface area of a well in a 96-well plate is similar to that of the human macula. Plates were incubated at 37°C / 5% CO2 for 3 days undisturbed, followed by collection of the supernatant media. PEDF concentration was measured by ELISA and normalized to supernatant volume and total cells using the established method. 150,000 cells Attorney Docket No. 27562-0031 WO 1 was used for total cell count, as re-seeded cells are 100% confluent at the density used here and little to no proliferation is expected. Fresh cells, harvested at 4 weeks of culture and replated without cryopreservation, exhibited similar 3-day release.

[0374] Stability’ Testing

[0375] Two aspects of stability were assessed: 1) viability in injection vehicle at 4°C after thaw and 2) viability, identity / purity and potency after long-term storage of WCB vials in LN2. Following thaw, wash, and resuspension in IV, viability was determined by manual cell counts using trypan blue exclusion (results shown in FIG. 18A). We found that post-thaw viability was sustained for approximately 24 hours but declined at later time points. We therefore tested a modification to the IV formula, adding 0.1% human serum albumin (HSA; Irvine Scientific, cat# 9988-100ML). In HSA-modified vehicle (“IV+”), post-thaw cell viability was restored to levels observed for fresh product (see, e.g., FIG. 18A).

[0376] The amount of albumin used in the modified injection vehicle disclosed herein is at least 30 times lower than the albumin level in normal human serum, 50 times lower than the eye drops, and 250 times lower than in an approved blood infusion product for a variety of indications.

[0377] Having established 72 hour viability of thawed RPESC-RPE-4W in IV+, we further tested identity / purity, potency, and 72 hour viability of thawed cells after intervals in LN2 storage (see, e g., FIGs. 18B-18D). As before, cells were seeded for ICC after TAB2 + 10% HI-FBS wash during the thaw process and PEDF release was measured after three days in culture. Thawed cells were resuspended in IV+ and stored at 4°C for manual cell counts and viability measurements. We found that thawed RPESC-RPE-4W met established release criteria after LN2 storage at each time point tested (3, 6, 9 and 12 months).

[0378] Process Scale

[0379] We previously performed culture of Passage 1 RPESC-RPE in multiple T25 flasks to produce master cell banks (MCBs) (FIGs. 19A-19B), including those used in preclinical safety and efficacy studies. To pilot this method for Passage 2 culture and production of RPESC-RPE-4W, we thawed 3 vials (500,000 cells / vial) of hRPE-022 MCB simultaneously. Cells were transferred to TAB2 + 10% HI-FBS medium, counted and seeded to a T25 flask Attorney Docket No. 27562-0031 WO 1

[0380] (1.3 x 106cells) and a 24-well plate (1 11,000 cells / well) following existing procedures. Both T25 flask and 24-well plate were maintained for 4 weeks. T25 flask and 24-well plate were harvested separately and QC tests performed for comparison. RPESC-RPE-4W cells were cryopreserved in 10, 13 and 25 vial quantities. At thaw, these vials had 90%, 95% and 83% viability, respectively.

[0381] Additional Comparisons

[0382] Additional assays were performed to further establish the identity and potency of cryopreserved RPESC-RPE-4W. To generate additional deep molecular identity data for cryopreserved RPESC-RPE-4W compared to fresh cells, we performed scRNA-seq on three donor lines (results shown in FIGs. 20A-20B). Each line was thawed from MCB and cultured for 4 weeks according to current manufacturing protocols. Cells from each line were harvested and loaded fresh to 3’ microwell chips using the Takara (Wafergen) iCELL8 single cell platform. Remaining cells were cryopreserved and transferred to LN2 storage, then thawed and loaded onto 3’ chips. Illumina sequencing libraries were prepared for each sample and sequenced. Bioinformatic comparison using reciprocal principal correlation analysis (RPCA) integration and DA-seq analysis indicated that RPESC-RPE-4W subpopulations are comparable in fresh and post-thaw samples.

[0383] In addition, we evaluated the ability of RPESC-RPE-4W cells to engraft into the existing RPE monolayer, which is believed to be an important function for restoration of vision (see, e.g., FIGs. 21 A-21C). We therefore aimed to test this function using an in vitro integration assay (schematic of assay shown in FIG. 21 A). Briefly, RPESC-RPE from two cell lines were cultured for 4 weeks in 24-well plates. Cells were labeled with Cell Tracker Red live cell stain, then a subset of cells were harvested and cryopreserved. These cells were then thawed and seeded to 8-week mature RPE monolayers. Additional labeled cells were seeded onto separate wells of the 8-week RPE monolayers without cryopreservation (fresh). Ten thousand cells, either fresh or thawed, were seeded per well in duplicate. All cultures were maintained for one additional week. Cultures were then rinsed, washing away nonadherent cells, and fixed. To determine whether remaining donor cells had truly integrated, cells were further stained with phalloidin to identify cell borders and DAPI to identify cell nuclei, and imaged by confocal microscopy. The number of well-integrated cells was Attorney Docket No. 27562-0031 WO 1 quantified by manual counts, and reported as a percentage of the 10,000 donor cells that were initially distributed per well, as described in FIG. 21B. Two different donor cell lines were tested. A single RPE cell line was used to form the recipient layers for all tests, allowing for direct comparison within and between donor samples. These results indicate that cryopreservation does not affect the integration efficiency of a given donor cell line (see, e.g., FIR. 21B) and that integrated cells are phenotypically normal (see, e.g., FIG. 21C).

[0384] EXAMPLE 15: Vehicle for Cell and Tissue Therapies

[0385] The description below exemplifies development of an injection vehicle formulation that stabilizes the viability and vision rescue efficacy of RPESC-RPE-4W cells (drug substance, “DS”) for more than 24 hours, promotes stability of cryopreserved DS, and enhances yield of DS for transplantation. RPESC-RPE-4W doses can be produced at a Good Manufacturing Practice (GMP) cell manufacturing facility (see, e.g., FIG. 22A). One or more Master Cell Bank (MCB) vials can be thawed, cells can be cultured for 3.5 - 5 weeks, then harvested and formulated as a cell suspension. The drug product (DP; RPESC-RPE-4W formulated in injection vehicle) can then be shipped live to the clinical site for subretinal transplantation (see, e.g., FIGs. 22B-22C).

[0386] In preclinical efficacy studies, we used the Royal College of Surgeons (RCS) rat model, which exhibits progressive vision loss due to a genetic mutation. RPESC-RPE cells were injected subretinally and visual acuity was measured by tracking head movement in response to a rotating field of bars. These experiments establish the stage of RPESC-RPE differentiation, dose and immunosuppressive strategy. In previous experiments, RPESC-RPE cells were suspended in a recognized ophthalmological Balanced Salt Solution (BSS; McKesson) and injected within 3 hours of formulation. Subsequently, we evaluated the stability of RPESC-RPE-4W efficacy when suspended in BSS and stored at 4°C (results shown in FIG. 23). We found that while cells stored in BSS for less than 20 hours provided vision rescue at post-natal day 90 (P90), cells stored for longer did not. We therefore sought to identify a formula that would sustain RPESC-RPE-4W efficacy while also remaining safe for subretinal injection. Further in vivo experiments demonstrated that a combination of 1.0 g / L glucose and 2.2 g / L sodium pyruvate in BSS was sufficient to extend the stability of RPESC-RPE-4W (results shown in FIG. 24A), while glucose alone or glucose with 0.22 g / L Attorney Docket No. 27562-0031 WO 1 sodium pyruvate (the concentration in RPE medium) were not sufficient (results shown in FIGs. 24B-24C).

[0387] Subsequently, we developed a cryopreservation method to freeze RPESC-RPE-4W for improved scale, stability and logistics in production of the final drug substance. We found that a modification of the injection vehicle, addition of human serum albumin (HSA), promoted post-thaw viability and that this new “IV+” also supported fresh RPESC-RPE-4W. In addition, the IV+ formulation enhanced recovery of RPESC-RPE-4W post-thaw (see, e.g., FIGs. 16A-16B and 18A-18D).

[0388] Cell culture media such as alpha MEM (aMEM), DMEM / F12 and RPMI contain many ingredients to support cell viability, growth and function in vitro. Therefore, it was surprising that only three ingredients (glucose, sodium pyruvate, and HSA) were sufficient to stabilize the viability and vision rescue efficacy of RPESC-RPE-4W cells. Moreover, the concentration of glucose in most culture media is much higher than in our IV+, while the reverse is true for sodium pyruvate. HSA is not an ingredient typically used in commercial cell culture media, nor is it added in more complex mixtures (eg: RPESC medium). Therefore, it was unexpected that a minimalist mixture of 3 ingredients in BSS, each at very different concentrations than in other well-known media, would significantly enhance the stability of viable, efficacious RPESC-RPE-4W.

[0389] FIGs. 25A and 25B shows results of experiments evaluating cell viability and recovery for RPESC-RPE-4W cells thawed and diluted in IV+ injection vehicle (FIG. 25A) or iPSC-RPE cells thawed and diluted in IV+ (FIG. 25B). RPESC-RPE-4W were cryopreserved with 75,000 cells in 50 pl of CryoStor CS2. After storage in liquid nitrogen, vials were thawed and diluted in IV+. Cells were manually counted immediately and after 4 °C storage for 4 hours. Percent viability and recovery after 4 hours is shown in FIG. 25A.

[0390] RPE cells derived from induced pluripotent stem cells (iPSC-RPE) were cryopreserved, then thawed and counted for viability and recovery (manual counts with trypan blue exclusion). Two iPSC-RPE lines were each tested twice. Cells were washed with BSS, resuspended in 0.05 ml CryoStor CS2, and transferred to cryovials. Cryovials were frozen at -1 °C / minute overnight, then transferred to liquid nitrogen (LN2) storage. Following storage in LN2, vials were thawed and cell suspension was diluted with 0.1 ml Attorney Docket No. 27562-0031 WO 1

[0391] IV+. Cells were counted within one hour of dilution. Percent viability and recovery is shown in FIG. 25B.

[0392] Additional experiments demonstrated that custom vehicle (IV+) preserves cell therapy efficacy for 48 hours after harvest (FIG. 26A), cells can be stored from 4 °C to 12 °C for at least 24 hours (FIG. 26B), and custom vehicle supports health of complex neural tissue and cell types (FIG. 26C).

[0393] RPESC-RPE-4W cells were harvested and formulated in custom vehicle, then stored at 4 °C for 48 hours prior to subretinal injection in the RCS rat. OKT testing was performed at postnatal day 60 (P60), postnatal day 90 (P90) and postnatal day 150 (Pl 50). As shown in FIG. 26 A, cell therapy efficacy was preserved for 48 hours after harvest.

[0394] RPESC-RPE-4W cells were harvested and formulated in vehicle, then stored at either 4 °C or 12 °C for 24 hours prior to subretinal injection in the RCS rat. OKT testing was performed at postnatal day 60 (P60), postnatal day 90 (P90), postnatal day 150 (Pl 50) and postnatal day 180 (Pl 80). As shown in FIG. 26B, cell therapy efficacy was preserved for 24 hours after harvest for cells stored at both 4 °C and 12 °C.

[0395] Cerebral organoids composed of neurons, astrocytes, oligodendrocytes and progenitor cells were generated from two iPSC lines and aged to 6 months and 10 months. (Whole Organoids) 6-month organoids were transferred to vehicle and stored at 4 °C for 48 hours. Control organoids were maintained in organoid medium at 37 °C and 5% CO2 during the same period. At 48 hours, all sets were dissociated and cell viability was quantified by manual cell counts with trypan blue exclusion. (Single Cell Suspension) 10-month organoids were dissociated to single cell suspensions and immediately counted for cell viability (control). Cells were resuspended in vehicle and stored at 4 °C, followed by quantification of viability at 24 hours and 48 hours. These results demonstrate that custom vehicle supports health of complex neural tissue and cell types for at least 48 hours.

[0396] OTHER EMBODIMENTS

[0397] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not Attorney Docket No. 27562-0031 WO 1 limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 27562-0031WO1WHAT IS CLAIMED IS:

1. A method of generating a non-native retinal pigment epithelial (RPE) cell product in vitro, the method comprising: obtaining a starting cell population comprising RPE cells; and generating from the starting cell population a culture product of RPE cells, wherein one or more cells of the culture product of RPE cells expresses PDPN and ITGA6 and one or more cell surface markers selected from the group consisting of CD63, CD24, CD82, NECTIN2, CD151, CD9, ITGB1, F3, and CD81, thereby generating a non-native RPE cell product.

2. The method of claim 1, wherein each RPE cell of the starting population of RPE cells expresses one or more of RPE65, MITF, PAX6, CRALBP, OTX2, Bestrophin, CD82, CD81, CD63, F3, ITGA6, ITGB1. and PDPN.

3. The method of claim 1, further comprising determining a level of enrichment of the culture product of RPE cells as compared to the starting cell population.

4. The method of any one of claims 1-3, wherein the starting cell population comprises RPE cells derived from pluripotent stem cells (PSCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs).

5. The method of any one of claims 1-4, wherein the starting cell population comprises in vitro-genQva adult RPE stem cells (RPESCs).

6. The method of any one of claims 1-3, wherein the starting cell population comprises RPESC progeny (RPESC-RPEs).

7. The method of any one of claims 1 -6, wherein the starting cell population comprises human RPE cells.

8. A composition comprising a non-native retinal pigment epithelial (RPE) cell product generated by the method of any one of claims 1-7.Attorney Docket No. 27562-0031WO19. A composition comprising a therapeutically effective amount of RPE cell derivatives, wherein the RPE cell derivatives comprise one or more subpopulations of RPE cell derivatives that have been enriched from a starting population of RPE cells, wherein the one or more subpopulations are selected from subpopulations of RPE cell derivatives expressing cell surface markers:DPP4; orENTPD1 ; orITGA1; orICAM1; orCSF1R; orTFRC; orNCR1; orITGB3; orPDPN; orITGA6; orSIGLEC1.

10. The composition of claim 9, wherein the RPE cells are derived from pluripotent stems cells (PSCs). embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs).

11. The composition of claim 9 or 10, wherein the starting cell population comprises adult RPE stem cells (RPESCs).

12. The composition of claim 9 or 10, wherein the starting cell population comprises RPESC progeny (RPESC-RPEs).

13. The composition of claim 12, wherein the RPESC-RPEs have been cultured prior to enrichment.

14. The composition of any one of claims 8-13, wherein the RPE cell product or RPE cell derivatives are in suspension.Attorney Docket No. 27562-0031WO115. The composition of claim 14, wherein the RPE cell product or RPE cell derivatives are suspended in an injection vehicle.

16. The composition of claim 15, wherein the injection vehicle comprises at least one component selected from the group consisting of glucose, sodium pyruvate, human serum albumin (EISA), and water.

17. The composition of claim 15, wherein the injection vehicle comprises glucose, sodium pyruvate, human serum albumin (HSA), and water.

18. The composition of claim 15, wherein the injection vehicle comprises sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water.

19. The composition of any one of claims 8-13, wherein the RPE cell product or RPE cell derivatives are on a scaffold.

20. The composition of claim 19, wherein the scaffold is a polymeric scaffold comprising collagen, albumin, fibrin, alginate, hyaluronic acid, polyactic acid (PLA), poly glycolic acid (PGA), polylactic acid-glycolic acid (PGLA), polyorthoester, poly caprolactone (PCL), polyanhydride, polyphosphazene, polyacrylate, polymethacrylate, ethylene vinyl acetate, polyvinyl alcohol, or combinations thereof.

21. A dosage form comprising the composition of any one of claims 8-20.

22. The dosage form of claim 21, wherein the dosage form is frozen.

23. The dosage form of claim 21, wherein the dosage form has been thawed after having previously been frozen.

24. The dosage form of claim 23, wherein the RPE cell derivatives of the dosage form have been diluted in an injection vehicle.Attorney Docket No. 27562-0031WO125. The dosage form of claim 24, wherein the injection vehicle comprises at least one component selected from the group consisting of glucose, sodium pyruvate, human serum albumin (HSA), and water.

26. The dosage form of claim 25, wherein the injection vehicle comprises glucose, sodium pyruvate, human serum albumin (HSA), and water.

27. The dosage form of claim 25, wherein the injection vehicle comprises sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and / or hydrochloric acid (to adjust pH), glucose, sodium pyruvate, HSA, and water.

28. The dosage form of any one of claims 24-27, wherein prior to dilution in the injection vehicle, the RPE cell derivatives of the dosage form have been washed with a balanced salt solution (BSS) or BSS comprising HSA.

29. An aqueous formulation for use as a storage medium or an injection vehicle for a tissue culture or a suspension of cells, the aqueous formulation comprising: glucose; sodium pyruvate.

30. The aqueous formulation of claim 29, further comprising human serum albumin (HSA).

31. The aqueous formulation of claim 29 or 30, further comprising one or more of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride, sodium acetate, sodium citrate, sodium hydroxide and hydrochloric acid.

32. The aqueous formulation of claim 30 or 31, comprising:0.1 g / L to 1.1 g / L glucose;1.0 g / L to 5.0 g / L sodium pyruvate; and0.01% to 1.0% HSA.Attorney Docket No. 27562-0031WO133. The aqueous formulation of claim 32, comprising: about 1.0 g / L glucose; about 2.2 g / L sodium pyruvate; and about 0.1% HSA.

34. A method of treating a disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of RPE cell derivatives, wherein the RPE cell derivatives comprise one or more subpopulations of RPE cell derivatives that have been enriched from a starting population of RPE cells, wherein the one or more subpopulations are selected from subpopulations of RPE cell derivatives expressing cell surface markers:DPP4; orENTPD1; orITGA1 ; orICAM1; orCSF1R; orTFRC; orNCR1; orITGB3; orPDPN; orITGA6; orS1GLEC1.

35. The method of claim 34, wherein the RPE cells are derived from pluripotent stem cells (PSCs), embry onic stem cells (ESCs), or induced pluripotent stem cells (iPSCs).

36. The method of claim 34, wherein the starting cell population comprises adult RPE stem cells (RPESCs).Attorney Docket No. 27562-0031WO137. The method of claim 34, wherein the starting cell population comprises RPESC progeny (RPESC-RPEs).

38. The method of claim 37, wherein the RPESC-RPEs have been cultured prior to enrichment.

39. The method of any one of claims 34-38, wherein the starting cell population are human RPE cells.

40. The method of any one of claims 34-39, wherein the subject has been diagnosed with age-related macular degeneration (AMD).

41. The method of claim 40, wherein the AMD is dry AMD.

42. The method of claim 40, wherein the AMD is intermediate AMD.

43. The method of claim 40, wherein the AMD is wet AMD.

44. The method of any one of claims 34-39, wherein the subject has been diagnosed with an ophthalmic disorder.

45. The method of claim 44, wherein the subject has been diagnosed with an ophthalmic disorder characterized by RPE layer and / or photoreceptor layer dysfunction or degeneration.

46. The method of claim 44, wherein the subj ect has been diagnosed with diabetic retinopathy, Stargardt disease, or Choroideremia.

47. The method of any one of claims 34-36, wherein the composition is administered to the eye.

48. The method of claim 47, wherein the composition is administered by intravitreal, suprachoroidal, or subretinal injection.Attorney Docket No. 27562-0031WO149. The method of any one of claims 34-48, wherein the RPE cell derivatives are in suspension.

50. The method of any one of claims 34-48, wherein the RPE cell derivatives are on a scaffold.

51. The method of claim 50, wherein the scaffold is a polymeric scaffold comprising collagen, albumin, fibrin, alginate, hyaluronic acid, polyactic acid (PLA), poly glycolic acid (PGA), polylactic acid-glycolic acid (PGLA), poly orthoester, poly caprolactone (PCL), polyanhydride, polyphosphazene, polyacrylate, polymethacrylate, ethylene vinyl acetate, polyvinyl alcohol, or combinations thereof.

52. A pharmaceutical composition comprising the composition of any one of claims 8-20.

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