RPE cell populations and methods for producing same

Abstract

To provide pure retinal pigment epithelium (RPE) cells that are not mixed populations of pigmented and non-pigmented cells, and provide methods of obtaining the same.SOLUTION: A population of human polygonal RPE cells is disclosed. At least 95% of the cells thereof co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), wherein the trans-epithelial electrical resistance of the cells is greater than 100 ohms. Methods of generating the same are also disclosed.SELECTED DRAWING: Figure 27A

Description

[Technical field]

[0001] The present invention, in some aspects thereof, relates to retinal pigment epithelial cells, and more particularly, but not exclusively, to the evaluation of such cells as a therapeutic. The present invention also relates to the generation of retinal pigment epithelial cells from embryonic stem cells. [Background technology]

[0002] The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and the choriocapillaris. RPE cells play a critical role in the maintenance and function of the retina and its photoreceptors. These functions include forming the blood-retinal barrier, absorbing stray light, providing nutrients to the neural retina, regenerating visual pigment, and incorporating and recycling shed photoreceptor outer segments.

[0003] Retinal tissue can degenerate for many reasons, including arterial or venous blockage, diabetic retinopathy, and retinopathy of prematurity, which are usually inherited. Diseases such as retinitis pigmentosa, retinoschisis, lattice degeneration, Best's disease, and age-related macular degeneration (AMD) are characterized by progressive retinal degeneration.

[0004] RPE cells could potentially be used for cell replacement therapy of degenerated RPE in the retinal diseases mentioned above. They could also be used as a vehicle to introduce genes to treat retinal degenerative diseases. These cells could also serve as in vitro models of retinal degenerative diseases, as tools for high-throughput screening of small molecules for therapeutic effects, and for discovering and testing new drugs for retinal degenerative diseases. RPE cells could also be used for basic research into the development, maturation, characteristics, properties, metabolism, immunogenicity, function, and interactions with other cell types of RPE.

[0005] Human fetal and adult RPE have been used as alternative donor sources for allogeneic transplantation. However, practical challenges in obtaining sufficient tissue supply and ethical concerns regarding the use of tissue from aborted fetuses have limited the widespread use of these donor sources. Given these constraints in the supply of adult and fetal RPE grafts, alternative donor sources have been investigated. Human pluripotent stem cells have significant advantages as a source of RPE cells for transplantation. Due to their multipotent developmental potential, human pluripotent stem cells have the potential to differentiate into bona fide functional RPE cells and, given their unlimited self-renewal potential, may serve as an unlimited donor source of RPE cells. Indeed, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) have been shown to differentiate into RPE cells in vitro and attenuate retinal degeneration and preserve visual function after subretinal transplantation in the Royal College of Surgeons (RCS) rat retinal degeneration model caused by RPE dysfunction. Thus, pluripotent stem cells may represent an unlimited source for producing RPE cells.

[0006] Current protocols for obtaining RPE cells from pluripotent stem cells result in mixed populations of pigmented and non-pigmented cells, however, a pure population of pigmented cells is desirable for the use of RPE cells in basic research, drug discovery, and cell therapy.

[0007] Background art includes WO2013 / 114360 (Patent Document 1), WO2008 / 129554 (Patent Document 2), and WO2013 / 184809 (Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2013 / 114360 [Patent Document 2] WO2008 / 129554 [Patent Document 3] WO2013 / 184809 Summary of the Invention

[0009] According to one aspect of some embodiments of the present invention, there is provided a population of human polygonal RPE cells, wherein at least 95% of the cells co-express a pre-melanosomal protein (PMEL17) and a cellular retinaldehyde binding protein (CRALBP), and wherein the population of cells has a transepithelial electrical resistance of greater than 100 ohms.

[0010] According to one aspect of some embodiments of the present invention, there is provided a population of human RPE cells, wherein at least 80% of the cells co-express pre-melanosomal protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP), and cells within the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

[0011] According to an embodiment of the invention, cells within the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

[0012] According to an embodiment of the invention, the cells secrete angiogenin, TIMP2, sgp130, or sTNF-R1 in a polarized manner.

[0013] According to an embodiment of the invention, the cells secrete each of angiogenin, TIMP2, sgp130, and sTNF-R1 in a polarized manner.

[0014] According to an embodiment of the invention, the ratio of apical to basolateral secretion of sgp130 is greater than one.

[0015] According to an embodiment of the present invention, the ratio of apical to basal secretion of sTNF-R1 is greater than one.

[0016] According to an embodiment of the invention, the ratio of basolateral secretion of angiogenin to apical secretion of angiogenin is greater than one.

[0017] According to an embodiment of the invention, the ratio of apical to basolateral secretion of TIMP2 is greater than one.

[0018] According to an embodiment of the present invention, Oct4 + TRA-1-60 + The cell count is below 1:250,000.

[0019] According to an embodiment of the invention, at least 80% of the cells express Bestrophin 1 as determined by immunostaining.

[0020] According to an embodiment of the invention, at least 80% of the cells express microphthalmia-associated transcription factor (MITF) as determined by immunostaining.

[0021] According to an embodiment of the invention, more than 50% of the cells express paired box gene 6 (PAX-6) as measured by FACS.

[0022] According to an embodiment of the invention, the cells secrete greater than 750 ng of pigment epithelium-derived factor (PEDF) per ml per day.

[0023] According to an embodiment of the invention, the cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner.

[0024] According to an embodiment of the present invention, the ratio of apical to basolateral secretion of PEDF is greater than one.

[0025] According to an embodiment of the invention, after 8 hours of incubation at 2-8° C., the ratio is still greater than 1.

[0026] According to an embodiment of the invention, the population of cells has a transepithelial electrical resistance of greater than 100 ohms.

[0027] According to an embodiment of the invention, after 8 hours of incubation at 2-8° C., the transepithelial electrical resistance of the cells is still greater than 100 ohms.

[0028] According to an embodiment of the invention, the ratio of basolateral secretion of VEGF to apical secretion of VEGF is greater than one.

[0029] According to an embodiment of the invention, after 8 hours of incubation at 2-8° C., the ratio is still greater than 1.

[0030] According to an embodiment of the present invention, the cell population is capable of rescuing vision in RCS rats following subretinal administration.

[0031] According to an embodiment of the invention, the cell population is capable of rescuing photoreceptors for at least 180 days following subretinal administration in RCS rats.

[0032] According to an aspect of the invention, the cell population is generated by ex vivo differentiation of human embryonic stem cells.

[0033] According to an aspect of the invention, the cell population comprises: (a) culturing human embryonic stem cells in a medium containing nicotinamide and lacking activin A to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to generate cells that are further differentiated into the RPE lineage; and (c) culturing the cells that have further differentiated into the RPE lineage in a medium that contains nicotinamide and lacks activin A. It is made by.

[0034] According to an embodiment of the invention, embryonic stem cells are grown in a medium comprising bFGF and TGFβ.

[0035] According to an embodiment of the invention, embryonic stem cells are cultured on human cord fibroblasts.

[0036] According to an embodiment of the invention, steps (a)-(c) are carried out under conditions where the atmospheric oxygen level is less than about 10%.

[0037] According to an embodiment of the invention, the method further comprises, after step (c), culturing the differentiated cells in a medium in the presence of nicotinamide and under conditions where the atmospheric oxygen level is greater than about 10%.

[0038] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising a cell population described herein as an active agent and a pharma- ceutically acceptable carrier.

[0039] According to an aspect of some embodiments of the present invention there is provided a use of the cell population described herein for treating retinal degeneration.

[0040] According to one aspect of some embodiments of the present invention, (a) culturing pluripotent stem cells in a medium containing a differentiation agent and lacking a transforming growth factor β (TGFβ) superfamily member to generate differentiated cells; (b) culturing the differentiated cells in a medium containing a member of the transforming growth factor β (TGFβ) superfamily and a differentiation agent to generate cells that are further differentiated into an RPE lineage; (c) culturing the cells that have further differentiated into the RPE lineage in a medium that contains a differentiation agent and lacks a member of the transforming growth factor beta (TGFβ) superfamily to generate RPE cells. The present invention provides a method for producing RPE cells, comprising the steps (a)-(c) being carried out under conditions where the atmospheric oxygen level is less than about 10%.

[0041] According to an embodiment of the invention, step (a) is carried out under non-stick conditions.

[0042] According to an embodiment of the invention, the non-adherent conditions include a non-adherent culture plate.

[0043] According to an embodiment of the invention, step (a) comprises: (i) culturing a population of cultured human pluripotent stem cells under non-adherent conditions in a medium containing nicotinamide in the absence of activin A to generate clusters of cells including differentiated cells; and thereafter (ii) culturing the differentiated cells of (i) under adherent conditions in a medium containing nicotinamide in the absence of activin A. Includes.

[0044] According to an embodiment of the invention, the method further comprises the step of dissociating clusters of cells prior to step (ii) to produce clumps of cells or a single cell suspension of cells.

[0045] According to an embodiment of the invention, the method further comprises, after step (c), culturing the differentiated cells in a medium in the presence of a differentiation agent and under conditions where the atmospheric oxygen level is greater than about 10%.

[0046] According to an embodiment of the invention, the member of the transforming growth factor β (TGFβ) superfamily is selected from the group consisting of TGFβ1, TGFβ3, and activin A.

[0047] According to an embodiment of the invention, the differentiation material of step (a) and the differentiation material of step (c) are identical.

[0048] According to an embodiment of the invention, the differentiation agent in step (a) is nicotinamide (NA) or 3-aminobenzamide.

[0049] According to an embodiment of the invention, the method further comprises, after step (c), a step of selecting polygonal cells.

[0050] According to an aspect of the invention, the method further comprises the step of expanding the polyhedral cells.

[0051] According to an embodiment of the invention, the growing step is carried out on an adherent surface or on an extracellular matrix.

[0052] According to an aspect of the invention, the pluripotent stem cells include embryonic stem cells.

[0053] According to an embodiment of the invention, embryonic stem cells are grown in a medium comprising bFGF and TGFβ.

[0054] According to an aspect of the invention, embryonic stem cells are cultured on human cord fibroblasts.

[0055] [The present invention 1001] A population of human polygonal RPE cells, wherein at least 95% of the cells co-express a pre-melanosome protein (PMEL17) and a cellular retinaldehyde binding protein (CRALBP), and wherein the population of cells has a transepithelial electrical resistance of greater than 100 ohms. [The present invention 1002] A population of human RPE cells, wherein at least 80% of the cells co-express pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP), and cells within the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1). [The present invention 1003] 1001. The cell population of the present invention, wherein cells within said population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1). [The present invention 1004] The cell population of invention 1002 or 1003, wherein said cells secrete said angiogenin, said TIMP2, said sgp130, or said sTNF-R1 in a polarized manner. [The present invention 1005] The cell population of invention 1002 or 1003, wherein said cells secrete each of said angiogenin, said TIMP2, said sgp130, and said sTNF-R1 in a polarized manner. [The present invention 1006] The cell population of the present invention 1004 or 1005, having a ratio of apical secretion of sgp130 to basolateral secretion of sgp130 of greater than 1. [The present invention 1007] The cell population of the present invention 1004 or 1005, having a ratio of apical to basal secretion of sTNF-R1 of greater than 1. [The present invention 1008] The cell population of the present invention 1004 or 1005, having a ratio of basolateral secretion of angiogenin to apical secretion of angiogenin greater than 1. [The present invention 1009] The cell population of the present invention 1004 or 1005, wherein the ratio of apical to basolateral secretion of TIMP2 is greater than 1. [The present invention 1010] Oct4 in the population + TRA-1-60 + The cell population of the present invention 1001 or 1002, wherein the number of cells is below 1:250,000. [The present invention 1011] The cell population of any of claims 1001 to 1010, wherein at least 80% of the cells express Bestrophin 1 as determined by immunostaining. [The present invention 1012] The cell population of any of claims 1001 to 1011, wherein at least 80% of the cells express microphthalmia-associated transcription factor (MITF) as determined by immunostaining. [The present invention 1013] The cell population of any of claims 1001 to 1012, wherein more than 50% of said cells express paired box gene 6 (PAX-6) as measured by FACS. [The present invention 1014] The cell population of any of claims 1001 to 1013, wherein the cells secrete more than 750 ng of pigment epithelium-derived factor (PEDF) per ml per day. [The present invention 1015] The cell population of any of claims 1001 to 1014, wherein said cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner. [The present invention 1016] The cell population of the present invention, 1015, having a ratio of apical to basolateral secretion of PEDF of greater than 1. [The present invention 1017] The cell population of the present invention, wherein the ratio is still greater than 1 after 8 hours of incubation at 2-8°C. [The present invention 1018] The cell population of the present invention 1002, wherein the transepithelial electrical resistance of said cell population is greater than 100 ohms. [The present invention 1019] The cell population of the present invention 1001 or 1018, wherein after 8 hours of incubation at 2-8°C, the transepithelial electrical resistance of the cells is still greater than 100 ohms. [The present invention 1020] The cell population of the present invention 1015 or 1016, having a ratio of basolateral secretion of VEGF to apical secretion of VEGF of greater than 1. [The present invention 1021] The cell population of the present invention 1020, wherein the ratio is still greater than 1 after 8 hours of incubation at 2-8°C. [The present invention 1022] The cell population of any of claims 1001 to 1021, which is capable of rescuing vision in RCS rats after subretinal administration. [The present invention 1023] A cell population of any of the present inventions 1001 to 1021, which is capable of rescuing photoreceptors for at least 180 days after subretinal administration in RCS rats. [The present invention 1024] A cell population according to any one of claims 1001 to 1023, produced by ex vivo differentiation of human embryonic stem cells. [The present invention 1025] (a) culturing human embryonic stem cells in a medium containing nicotinamide and lacking activin A to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to generate cells that are further differentiated into the RPE lineage; and (c) culturing the cells further differentiating into the RPE lineage in a medium containing nicotinamide and lacking activin A. The cell population of any one of 1001 to 1024 according to the present invention, produced by the above method. [The present invention 1026] The cell population of the present invention, wherein said embryonic stem cells are grown in a medium comprising bFGF and TGFβ. [The present invention 1027] The cell population of the present invention, wherein the embryonic stem cells are cultured on human cord fibroblasts. [The present invention 1028] The cell population of any of claims 1025 to 1027, wherein steps (a) to (c) are carried out under conditions where the atmospheric oxygen level is less than about 10%. [The present invention 1029] The cell population of claim 1028, further comprising, after step (c), culturing the differentiated cells in a medium in the presence of nicotinamide under conditions where the atmospheric oxygen level is greater than about 10%. [The present invention 1030] A pharmaceutical composition comprising any one of the cell populations of the present inventions 1001 to 1029 as an active substance and a pharma- ceutically acceptable carrier. [The present invention 1031] Use of the cell population of any of claims 1001 to 1030 for treating retinal degeneration. [The present invention 1032] (a) culturing pluripotent stem cells in a medium containing a differentiation agent and lacking a member of the transforming growth factor β (TGFβ) superfamily to generate differentiated cells; (b) culturing the differentiated cells in a medium containing the transforming growth factor β (TGFβ) superfamily member and the differentiation agent to generate cells that are further differentiated into an RPE lineage; (c) culturing the cells further differentiating into the RPE lineage in a medium containing a differentiation agent and lacking a member of the transforming growth factor beta (TGFβ) superfamily to generate RPE cells. A method for producing RPE cells, comprising: The method, wherein steps (a)-(c) are carried out under conditions where the atmospheric oxygen level is less than about 10%. [The present invention 1033] The method of claim 1032, wherein step (a) is carried out under non-adherent conditions. [The present invention 1034] The method of claim 1033, wherein said non-adherent conditions comprise a non-adherent culture plate. [The present invention 1035] Step (a) (i) culturing a population of cultured human pluripotent stem cells under non-adherent conditions in a medium containing nicotinamide in the absence of activin A to generate clusters of cells including differentiated cells; and thereafter (ii) culturing the differentiated cells of (i) under adherent conditions in a medium containing nicotinamide in the absence of activin A. The method of the present invention 1032, comprising: [The present invention 1036] The method of claim 1035, further comprising the step of dissociating said clusters of cells prior to step (ii) to produce clumps of cells or a single cell suspension of cells. [The present invention 1037] The method of claim 1032, further comprising, after step (c), culturing the differentiated cells in a medium in the presence of a differentiation agent under conditions where the atmospheric oxygen level is greater than about 10%. [The present invention 1038] The method of claim 1032, wherein said member of the transforming growth factor β (TGFβ) superfamily is selected from the group consisting of TGFβ1, TGFβ3, and activin A. [The present invention 1039] The method of claim 1032, wherein the differentiation material of step (a) and the differentiation material of step (c) are identical. [The present invention 1040] The method of claim 1032, wherein the differentiation agent in step (a) is nicotinamide (NA) or 3-aminobenzamide. [The present invention 1041] The method of claim 1032, further comprising, after step (c), a step of selecting the polyhedral cells. [The present invention 1042] The method of claim 1041, further comprising a step of proliferating said polygonal cells. [The present invention 1043] The method of claim 1042, wherein the growing step is carried out on an adherent surface. [The present invention 1044] The method of claim 1032, wherein said pluripotent stem cells comprise embryonic stem cells. [The present invention 1045] The method of claim 1044, wherein said embryonic stem cells are grown in a medium comprising bFGF and TGFβ. [The present invention 1046] The method of claim 1044, wherein said embryonic stem cells are cultured on human cord fibroblasts. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used in carrying out or testing embodiments of this invention, exemplary methods and / or materials are described below.In case of conflict, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting. [Brief description of the drawings]

[0056] Certain aspects of the present invention are herein described, by way of example only, with reference to the accompanying drawings. Reference will now be made specifically to the drawings in detail, it being stressed that the matter shown is by way of example and for the purpose of illustratively discussing aspects of the present invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how aspects of the present invention may be practiced. [Figure 1] 1 is a graph showing the linearity of the data. [Diagram 2] FACS analysis of negative control hESC cells stained with anti-CRALBP and anti-PMEL17. [Diagram 3] FACS analysis of positive control reference RPE line OpRegen® 5C cells stained with anti-CRALBP and anti-PMEL17. [Figure 4] FACS analysis of 25% spiked OpRegen® 5C in hESCs stained with anti-CRALBP and anti-PMEL17. [Diagram 5] FACS analysis of 50% spiked OpRegen® 5C in hESCs stained with anti-CRALBP and anti-PMEL17. [Figure 6] FACS analysis of 75% spiked OpRegen® 5C in hESCs stained with anti-CRALBP and anti-PMEL17. [Figure 7]FACS analysis of 95% spiked OpRegen® 5C in hESCs stained with anti-CRALBP and anti-PMEL17. [Figure 8] FACS analysis of hESCs stained with isotype controls. [Figure 9] FACS analysis of OpRegen® 5C cells stained with isotype controls. [Figure 10] Co-immunostaining with PMEL17 differentiates between RPE cells (CRALBP+PMEL17+) and non-RPE pigment cells (PMEL17+CRALBP-; e.g., melanocytes). [Figure 11] Morphological results of mock exams 4 and 5 at in-process control (IPC) points 5 and 8-10. [Figure 12] Manufacturing process, steps 1-3: Creation of a human cord fibroblast feeder working cell bank. [Figure 13] Manufacturing process, steps 4 and 5: hESC expansion. [Figure 14] Manufacturing process, steps 6-13: Differentiation into RPE (OpRegen®) cells. [Figure 15] Manufacturing process, steps 14-17: Increase in pigment cells. [Figure 16] Detailed OpRegen® manufacturing process and in-process control points (yellow stars, IPC 1-11). (NUTSPlus. Nutristem medium with bFGF and TGFβ; NUTS Minus. Nutristem medium without bFGF and TGFβ; NIC, nicotinamide; SB, spheroids). [Figure 17]Levels of CRALBP+PMEL17+ RPE cells along runs 4 and 5 of OpRegen® mock production. Density plots of IPC points 8 and 11 (*IPC point 8 was tested after cryopreservation), as well as representative density plots of positive control OpRegen® 5C and negative control HAD-C 102 hESC (%CRALBP+PMEL17+ range in negative controls was 0.02-0.17%). Numbers within each plot indicate percent CRALBP+PMEL17+ cells from the single live cell gated population. Analysis was performed using FCS express 4 software. [Figure 18] Immunofluorescence staining of mock study 5 IPC points 7, 10, and 11 with antibodies specific for the RPE markers bestrophin 1, MITF, ZO-1, and CRALBP. [Figure 19] Representative color fundus photographs of Group 2 (BSS+; FIG. 19A), the contralateral untreated eye of Group 5 (OD; FIG. 19B), and the treated eye of Group 5 (OS; FIG. 19C) at P60. The hyperpigmented and hypopigmented areas in the high dose treated eye (OS) are presumed to represent transplanted cells. [Figure 20] Optokinetic tracking acuity thresholds measured at P60, P100, P150, and P200. Cell treatment groups (Group 3-25,000, Group 4-100,000, and Group 5-200,000) outperformed all controls, with Group 4 (100,000) and Group 5 (200,000) doses achieving the best rescue. Non-operated contralateral eyes performed comparable to Group 1 (untreated) and Group 2 (vehicle control / BSS+) (not shown). [Figure 21] Graphs showing local (FIG. 21A) and full-field (FIG. 21B) results for a representative rat. [Figure 22]Figure 22A shows a composite of individual images of cresyl violet stained sections of a representative cell treated eye. The arrows indicate the location of photoreceptor protection and the presumed location of transplanted cells. Figure 22B shows a comparison of BSS+ (group 2) injected eyes with representative cell injected eyes (representing multiple dose groups) at postnatal days 60, 100, 150, and 200. GCL: ganglion cell layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. [Figure 23] Outer nuclear layer thickness measured as number of nuclei. Each point represents the number from each animal from each dose group across all ages. [Figure 24-1] Immunofluorescence images of positive control tissues and representative animals treated with experimental cells at P60, P100, P150, and P200 stained with anti-human nuclear marker (HNM, green), anti-premelanosome marker (PMEL17, red), anti-human proliferation marker (Ki67, red), and anti-rat cone arrestin (red). Dapi (blue) was used for background staining to highlight the granular layer. Positive control tissues included human melanoma for PMEL17, human tonsil for Ki67, and young RCS rat retina for cone arrestin. Downward arrows indicate the outer nuclear layer. Upward arrows indicate positively stained human RPE cells (OpRegen®) generated as described herein. [Figure 24-2] See description of Figure 24-1. [Diagram 25] 1 is a graph showing cone quantification after subretinal transplantation of OpRegen® cells in RCS rats, with cell-treated eyes showing significantly more cones than control eyes at all ages. [Figure 26]Immunofluorescent staining of OpRegen® cells in the subretinal space. Figure 26A shows an area of ​​the retina with numerous RPE cells (red, arrows), a central compartment devoid of debris (probed with anti-rat rhodopsin antibody, green; arrows), but where there are no cells (periphery), the debris compartment reconstitutes. At higher magnification (Figure 26B), there are several outer segments stained for rhodopsin along with the transplanted cells. Furthermore, with increasing distance from the transplanted cells, the debris compartment reconstitutes. Figures 26C-J are individual slices through the section showing rhodopsin-positive tissue (arrows) within the transplanted cells. [Figure 27A] Photographs showing the biodistribution of cells after subretinal injection into NOD-SCID. Figure 27A shows the ability of OpRegen® cells to engraft into the NOD-SCID subretinal space 9 months after transplantation. Pigment cells stained positive for human nuclei and PMEL17. [Figure 27B] Figure 27B is a photograph showing the biodistribution of cells after subretinal injection in NOD-SCIDs, and Figure 27C is a photograph showing cells clustering at the site of the bleb after injection. [Figure 27C] Figure 27C shows the biodistribution of cells after subretinal injection in NOD-SCIDs, and Figure 27D shows subsequent cell spreading into a monolayer after injection. [Figure 28] FIG. 1 is a diagrammatic illustration of a transwell assay that can be used to assay the efficacy of RPE cells. [Figure 29] FIG. 1 shows the results of FACS analysis showing PAX6 expression in RPE cells (P2-DP, formulations: Mock IV, Mock V, OpRegen® Batch 2A; HuRPE: normal human RPE from ScienCell) generated as described herein and PAX6 expression along the production (P0). [Diagram 30] 1 is a graph showing PAX6 expression in OpRegen® cells as analyzed by FACS (HES, human embryonic stem cells were used as a negative control). [Diagram 31]1 shows the results of FACS analysis showing double staining of PAX6 and CRALBP. [Figure 32A] 1 is a graph showing ELISA assessment of angiogenin secretion by OpRegen® cells: A. Increase in angiogenin secretion along with mock V production. [Figure 32B] A. ELISA assessment of angiogenin secretion by OpRegen® cells. B. Angiogenin secretion by three different batches of OpRegen® cells (passage 3) and on Transwell over a three week period assessing apical and basolateral secretion (passage 4). [Figure 32C] C. Angiogenin secretion by RPE7 cells (passage 3). [Figure 33A] Secretion of TIMP-1 and TIMP-2 by OpRegen® cells. A. Relative TIMP-1 and TIMP-2 protein levels detected by protein array. [Figure 33B] A. ELISA TIMP-2 levels at mock V production QC points 3 and 4. B. Secretion of TIMP-1 and TIMP-2 by OpRegen® cells. [Figure 33C] C and D. ELISA TIMP-2 secretion levels by different batches of OpRegen® cells (passage 3) and ELISA TIMP-2 secretion levels on Transwell over a 3 week period assessing apical and basolateral secretion (passage 4). [Figure 33D] C and D. ELISA TIMP-2 secretion levels by different batches of OpRegen® cells (passage 3) and ELISA TIMP-2 secretion levels on Transwell over a 3 week period assessing apical and basolateral secretion (passage 4). [Figure 33E] Shows secretion of TIMP-1 and TIMP-2 by OpRegen® cells. E. Secreted TIMP-2 levels from RPE7 and HuRPE control cells (passage 3, day 4 and day 14). [Figure 34A] 1 shows sgp130 secretion by OpRegen® cells as measured by ELISA. A. sgp130 secretion levels at mock V production QC points 3 and 4. [Figure 34B] Shows sgp130 secretion by OpRegen® cells as measured by ELISA. B-C. Levels of secreted sgp130 by different batches of OpRegen® cells (passage 3) and levels of secreted sgp130 on Transwells over a 3 week period assessing apical and basolateral secretion (passage 4). [Figure 34C] Shows sgp130 secretion by OpRegen® cells as measured by ELISA. B-C. Levels of secreted sgp130 by different batches of OpRegen® cells (passage 3) and levels of secreted sgp130 on Transwells over a 3 week period assessing apical and basolateral secretion (passage 4). [Fig. 34D] Shown is sgp130 secretion by OpRegen® cells as measured by ELISA. D. Secreted sgp130 levels from RPE7 and HuRPE control cells (passage 3, day 4 and day 14). [Figure 35A] 1 shows sTNF-R1 protein levels in OpRegen® cell supernatants as measured by ELISA. A. sTNF-R1 levels in cell supernatants from mock test V production QC points 3 and 4. [Figure 35B]Shown are sTNF-R1 protein levels in OpRegen® cell supernatants as measured by ELISA. B-C. sTNF-R1 levels in the supernatants of OpRegen® batches (passage 3) and sTNF-R1 levels on Transwells over a 3 week period when apical and basolateral levels were assessed (passage 4). [Figure 35C] Shown are sTNF-R1 protein levels in OpRegen® cell supernatants as measured by ELISA. B-C. sTNF-R1 levels in the supernatants of OpRegen® batches (passage 3) and sTNF-R1 levels on Transwells over a 3 week period when apical and basolateral levels were assessed (passage 4). [Figure 35D] Shown are sTNF-R1 protein levels in OpRegen® cell supernatants as measured by ELISA.D. sTNF-R1 levels in RPE7 and control HuRPE cell cultures at days 4 and 14 (passage 3). [Diagram 36] Morphology of OpRegen® 5C (reference strain), RPE1, and RPE7 on Transwells. OpRegen® 5C, RPE1, and RPE7 were imaged weekly (weeks 1-4) after seeding on Transwells. OpRegen® 5C produced homogenous polygonal monolayers from week 1, whereas RPE1 and RPE7 produced heterogeneous and distinct morphologies after one week of seeding, and holes began to appear by week 2. After 3 weeks of culture, RPE1 cells detached from the Transwells. [Figure 37] Figure 1 shows that RPE1 and RPE7 cells co-express CRALBP and PMEL-17. FACS purity assay demonstrated that 99.91% of RPE1 cells and 96.29% of RPE7 cells were double positive for the RPE markers CRALBP and PMEL-17, similar to the levels observed in OpRegen® mock V cells (positive control). HAD-C 102 hESCs were used as a negative control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Description of Certain Aspects of the Invention The present invention, in some aspects thereof, relates to retinal pigment epithelial cells, and more particularly, but not exclusively, to the evaluation of such cells as therapeutic agents. The present invention also relates to the generation of retinal pigment epithelial cells from human embryonic stem cells.

[0058] Before describing at least one embodiment of the present invention in detail, it should be understood that the present application is not necessarily limited to the details set forth in the following description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0059] The neural retina initiates vision and is supported by the underlying retinal pigment epithelium (RPE). Dysfunction, degeneration, and loss of RPE cells are hallmarks of Best's disease, a subtype of retinitis pigmentosa (RP), and age-related macular degeneration (AMD), the number one cause of visual impairment in the Western world. These conditions lead to gradual loss of vision and often blindness.

[0060] Both the retina and the adjacent RPE arise from the neuroectoderm. In lower species, the RPE regenerates the retina, but in mammals, RPE-mediated regeneration is inhibited and occurs to a very limited extent via stem cells located in the peripheral retinal periphery.

[0061] Human embryonic stem cells (hESCs) have the potential to serve as an unlimited source of RPE cell donors for transplantation. Mouse ESCs, primate ESCs, and human ESCs have demonstrated the ability to differentiate into RPE-like cells, attenuate retinal degeneration, and preserve visual function after subretinal transplantation.

[0062] Various protocols have been developed for differentiating human embryonic stem cells into RPE cells (see, for example, WO2008 / 129554).

[0063] The inventors have now discovered a unique and simple method to qualify a cell population that has been successfully differentiated into RPE cells based on the expression of specific polypeptides. The inventors have discovered that successful differentiation can be demonstrated using a combination of two specific markers from among the myriad of potential polypeptides expressed on the surface of these differentiated cells.

[0064] The inventors also discovered that secretion of pigment epithelium-derived factor (PEDF) may be used as a marker to demonstrate early stages of the RPE differentiation process (see Table 4).

[0065] While further reducing the invention to practice, the inventors have identified additional proteins secreted by RPE cells that may be used as signatures to define RPE cells in some embodiments.

[0066] Thus, according to one aspect of the present invention, there is provided a method for determining whether a cell population is eligible for a suitable therapeutic agent for treating an ocular abnormality, the method comprising a step of analyzing the co-expression of pre-melanosome protein (PMEL17) and at least one polypeptide selected from the group consisting of cellular retinaldehyde binding protein (CRALBP), lecithin retinol acyltransferase (LRAT), and sex determining region Y-box 9 (SOX9) in the population of cells, and if the number of cells co-expressing PMEL17 and the at least one polypeptide is above a predetermined level, the cell population is determined to be eligible for a suitable therapeutic agent for treating a retinal disorder.

[0067] According to another aspect, there is provided a method for determining whether a cell population is eligible for a suitable therapeutic agent for treating an ocular abnormality, the method comprising a step of analyzing co-expression of cellular retinaldehyde binding protein (CRALBP) and at least one polypeptide selected from the group consisting of pre-melanosomal protein (PMEL17), lecithin retinol acyltransferase (LRAT), and sex determining region Y-box 9 (SOX9) in the population of cells, and the cell population is determined to be eligible for a suitable therapeutic agent for treating an ocular abnormality if the number of cells co-expressing CRALBP and the at least one polypeptide is above a predetermined level.

[0068] The phrase "suitable therapeutic agent" as used herein refers to the cell population being suitable for treating ocular abnormalities. The therapeutic agent cells can exert their effect through any one of several mechanisms. One exemplary mechanism is a supportive trophic effect that promotes the survival of degenerating photoreceptors or other cells in the retina. The therapeutic RPE cells may also exert their effect through a regenerative mechanism that replenishes dysfunctional and / or degenerating host RPE cells. According to one embodiment, the RPE cells are mature and have the functional ability to phagocytose shed outer segments of photoreceptors that contain rhodopsin. According to another embodiment, the RPE cells are not fully mature.

[0069] The ocular abnormalities for which the cell population serves as a therapeutic agent include, but are not limited to, retinal diseases or disorders generally associated with retinal dysfunction, retinal damage, and / or loss of retinal pigment epithelium.A non-limiting list of abnormalities that can be treated according to the present invention includes retinitis pigmentosa, Leber's congenital amaurosis, inherited or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy and other dystrophies of the RPE, Stargardt's disease, RPE damage and retinal damage caused by any one of the following damages: photoinjury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury, or traumatic injury.

[0070] As mentioned, the method of this aspect of the invention is carried out by measuring the amount (e.g., percent cells) expressing at least one polypeptide selected from the group consisting of pre-melanosome protein (PMEL17; SwissProt No. P40967), cellular retinaldehyde binding protein (CRALBP; SwissProt No. P12271), lecithin retinol acyltransferase (LRAT; SwissProt No. 095327), and sex determining region Y-box 9 (SOX9; P48436).

[0071] Alternatively, the method of this aspect is carried out by measuring at least one polypeptide selected from the group consisting of CRALBP (CRALBP; SwissProt No. P12271), and lecithin retinol acyltransferase (LRAT; SwissProt No. 095327), sex determining region Y-box 9 (SOX 9; P48436), and PMEL17 (SwissProt No. P40967).

[0072] Thus, for example, CRALBP and PMEL17 may be measured, or PMEL17 and LRAT may be measured, or PMEL17 and SOX9 may be measured, or CRALBP and LRAT may be measured, or CRALBP and SOX9 may be measured.

[0073] It will be appreciated that more than two of the polypeptides mentioned herein can be measured, for example, three of the aforementioned polypeptides, or even all four of the aforementioned polypeptides.

[0074] The method for analyzing the expression of the above-mentioned polypeptides typically involves the use of antibodies that specifically recognize the antigen. Commercially available antibodies that recognize CRALBP include, for example, antibodies manufactured by Abcam (e.g., ab15051 and ab189329, clone B2). Commercially available antibodies that recognize PMEL17 include, for example, antibodies manufactured by Abcam (e.g., ab137062 and ab189330, clone EPR4864). Commercially available antibodies that recognize LRAT include, for example, antibodies manufactured by Millipore (e.g., MABN644). Commercially available antibodies that recognize SOX9 include, for example, antibodies manufactured by Abcam (e.g., ab185230). Analysis can be performed using any method known in the art, including flow cytometry, Western blot, immunocytochemistry, radioimmunoassay, PCR, etc.

[0075] For flow cytometry, antibodies may be attached to a fluorescent moiety and analyzed using a fluorescence activated cell sorter (FACS), or the use of a secondary antibody bearing a fluorescent moiety is also envisioned.

[0076] It will be appreciated that since the analyzed polypeptide is an intracellular polypeptide, the cells are typically permeabilized to allow the antibody to bind to the target. First, the cells may be fixed to ensure the stability of soluble antigens or antigens with short half-lives. This should keep the target protein in its original cellular location. To ensure that the cells remain permeable, the antibody may be prepared in a permeabilization buffer. When gating cell populations, it will be appreciated that the light scattering profile of the cells in the flow cytometer changes significantly after permeabilization and fixation.

[0077] Methods for permeabilizing cell membranes are known in the art and include, for example, 1. Formaldehyde followed by detergent: fixation with formaldehyde (e.g., up to 4.5% for 10-15 minutes (this stabilizes the proteins) followed by disruption of the membrane with detergents such as Triton or NP-40 (0.1-1% in PBS), Tween 20 (0.1-1% in PBS), saponin, digitonin, and Leucoperm (e.g., 0.5% v / v in PBS); 2. Formaldehyde (e.g., 4.5% or less), followed by methanol; 3. Methanol followed by detergent (e.g., 80% methanol then 0.1% Tween 20); 4. Acetone Fixation and Permeabilization Includes.

[0078] As used herein, the term "flow cytometry" refers to an assay method in which the proportion of material in a sample (e.g., RPE cells containing a particular marker) is determined by labeling the material (e.g., by binding the material with a labeled antibody), passing a stream of fluid containing the material through a beam of light, separating the light emitted from the sample into its constituent wavelengths by a series of filters and mirrors, and detecting the light.

[0079] Numerous flow cytometers are commercially available, including, for example, the Becton Dickinson FACScan, the Navios Flow Cytometer (Beckman Coulter serial number AT15119 RHE9266, and the FACScalibur (BD Biosciences, Mountain View, Calif.). Antibodies that can be used for FACS analysis are disclosed in Schlossman S, Boumell L, et al., [Leucocyte Typing V. New York: Oxford University Press; 1995] and are widely available commercially.

[0080] It will be understood that the expression level of the above-mentioned polypeptide may be performed at the RNA level as well as the protein level.Exemplary methods for confirming the expression of the polypeptide based on the RNA level include, but are not limited to, PCR, RT-PCR, Northern blot, etc.

[0081] To qualify the cells as useful as a therapeutic, the amounts of at least two of the co-expressed polypeptides in the cells must be increased above a statistically significant level when compared to non-RPE cells (e.g., undifferentiated embryonic stem cells).

[0082] According to a particular embodiment, in order to determine the eligibility of the cells to be useful as a therapeutic agent, at least 80% of the cells in the population must express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., CRALBP) when assayed by methods known to those skilled in the art (e.g., FACS), more preferably, at least 85% of the cells in the population must express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., CRALBP), more preferably, at least 90% of the cells in the population must express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., CRALBP), more preferably, at least 95% of the cells in the population must express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., CRALBP). More preferably, at least 100% of the cells in the population must express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., CRALBP).

[0083] In another aspect, to qualify the cells as useful as a therapeutic agent, the level of co-expression of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17) (e.g., as measured by mean fluorescence intensity) must be increased by at least 2-fold, more preferably at least 3-fold, more preferably at least 4-fold, even more preferably at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold compared to undifferentiated ESCs.

[0084] According to certain aspects, to determine the eligibility of the cells to be useful as a therapeutic agent, when assayed by methods known to those skilled in the art (e.g., FACS), at least 80% of the cells in the population must express detectable levels of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17); more preferably, at least 85% of the cells in the population must express detectable levels of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17); more preferably, at least 90% of the cells in the population must express detectable levels of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17); more preferably, at least 95% of the cells in the population must express detectable levels of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17); more preferably, 100% of the cells in the population must express detectable levels of CRALBP and one of the above-mentioned polypeptides (e.g., PMEL17).

[0085] Furthermore, the cells can be qualified in an animal model in vivo. One such model is the Royal College of Surgeons (RCS) rat model. After transplantation, the therapeutic effect of cells can be analyzed using methods including fundus imaging, optokinetic tracking threshold (OKT), electroretinogram (ERG), histology, cone counting, and rhodopsin uptake. These methods are further described in Example 5 below herein.

[0086] The cells may be qualified or characterized by further techniques including, for example, karyotype analysis, morphology, cell number, and viability, efficacy (barrier function and polarized secretion of PEDF and VEGF), levels of residual hESC, Gram stain, and sterility. Exemplary assays that may be performed are described in Example 4.

[0087] Additionally, the cells may be analyzed for barrier function and levels of secretion of growth factors (e.g., pigment epithelium-derived factor (PEDF) or VEGF, cytokines, interleukins, and / or chemokines) in a polarized manner.

[0088] To analyze secreted PEDF, the supernatant is collected from the cell culture, and the cells are harvested and counted. The amount of PEDF in the cell culture supernatant may be quantified using a PEDF ELISA assay (e.g., ELISAquant™ PEDF Sandwich ELISA Antigen Detection Kit, BioProductsMD, PED613) according to the manufacturer's protocol.

[0089] Furthermore, the direction of secretion of PEDF and VEGF in the cells may be analyzed. This may be done using a Transwell assay as shown in Figure 28. Before or after the qualification, the cells may be preserved (e.g., frozen or cryopreserved) according to methods known in the art, or may be directly administered to a subject.

[0090] The present invention intends to analyze cell populations comprising retinal pigment epithelial (RPE) cells from any source.Thus, this cell population may comprise RPE cells obtained from donors (i.e., natural RPE cells of the pigmented layer of the retina), or may comprise RPE cells differentiated ex vivo from a population of stem cells (hSC-derived RPE cells, e.g., pluripotent stem cells, e.g., human embryonic stem cells).In another embodiment, RPE cells are obtained by transdifferentiation.See, for example, Zhang et al., Protein Cell 2014, 5(1): 48-58, the contents of which are incorporated herein by reference.

[0091] According to one embodiment, the RPE cells analyzed do not express Pax6.

[0092] According to another embodiment, the RPE cells being analyzed express Pax6.

[0093] "Retinal pigment epithelial cells," "RPE cells," and "RPE," may be used interchangeably when the context permits, and refer to a cell type that is functionally similar to native RPE cells that form the pigment epithelial cell layer of the retina (e.g., when transplanted into the eye, they exhibit functional activity similar to native RPE cells).

[0094] According to one embodiment, RPE cell expresses at least one, two, three, four or five mature RPE cell markers.Such markers include but are not limited to CARLBP, RPE65, PEDF, PMEL17, bestrophin and tyrosinase.Optionally, RPE cell can also express RPE progenitor cell markers, such as MITF.In another embodiment, RPE cell expresses PAX-6.In another embodiment, RPE cell expresses at least one retinal progenitor cell marker, including but are not limited to OTX2, SIX3, SIX6 and LHX2.

[0095] In yet another embodiment, the RPE cells are RPE cells differentiated from embryonic stem cells according to the methods described in the Examples section herein below, the content of which is incorporated herein by reference.

[0096] As used herein, the phrase "mature RPE cell marker" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in mature RPE cells relative to non-RPE cells or immature RPE cells.

[0097] As used herein, the phrase "RPE progenitor cell marker" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in RPE progenitor cells relative to non-RPE cells.

[0098] According to another embodiment, the RPE cells have a morphology similar to that of natural RPE cells that form the pigmented epithelial cell layer of the retina, ie, they are pigmented cells and / or have a characteristic polygonal shape.

[0099] According to yet another embodiment, the RPE cells can treat diseases such as macular degeneration.

[0100] According to yet another embodiment, the RPE cells meet at least one, two, three, four, or all of the requirements listed herein above.

[0101] The term "hSC-derived RPE cells" is used herein to refer to RPE cells obtained by directed differentiation from hSCs. According to a preferred embodiment, the hSC-derived RPE cells are functional RPE cells as indicated by the parameters defined below. The term "directed differentiation" is used synonymously with the term "RPE-induced differentiation" and should be understood to mean the process of manipulating hSCs under culture conditions that induce / promote differentiation into RPE cell types.

[0102] According to a particular embodiment, the RPE cells are obtained by directed differentiation of hSCs in the presence of one or more members of the TGFβ superfamily and exhibit at least one of the following characteristics: - that cultured cells respond to TGFβ signaling during differentiation; - that the RPE cells express markers indicative of terminal differentiation, e.g., bestrophin 1, CRALBP, and / or RPE65; - after transplantation (i.e. in situ), RPE cells exhibit a trophic effect supporting photoreceptors adjacent to the RPE cells; -Furthermore, that in situ, RPE cells can function with the phagocytosis of shed photoreceptor outer segments as part of the normal regeneration process of these photoreceptors; -Furthermore, in situ, RPE cells are able to create the retinal barrier and function in the visual cycle.

[0103] As used herein, the phrase "stem cell" refers to a cell (e.g., a pluripotent or multipotent stem cell) that can remain in an undifferentiated state in culture for an extended period of time until induced to differentiate into another cell type (e.g., a fully differentiated cell) with a specific specialized function. Preferably, the phrase "stem cell" encompasses embryonic stem cells (ESCs), induced pluripotent stem cells (iPS), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells.

[0104] According to certain embodiments, the RPE cells are derived from pluripotent stem cells, including human embryonic stem cells or induced pluripotent stem cells.

[0105] The phrase "embryonic stem cells" refers to embryonic cells that can differentiate into all three germ layers (i.e., endoderm, ectoderm, and mesoderm) or remain undifferentiated. The phrase "embryonic stem cells" may include cells obtained from embryonic tissues (e.g., blastocysts) formed after conception before embryo implantation (i.e., pre-implantation blastocysts), extended blastocyst cells (EBCs) obtained from blastocysts at post-implantation / pre-gastrulation stages (see WO2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissues at any time during pregnancy, preferably before 10 weeks of pregnancy. Embryonic stem cells of some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo pre-implantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. To isolate human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by surgery. In this case, trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in tissue culture flasks containing an appropriate medium that allows for its outgrowth. After 9-15 days, the ICM-derived outgrowths are dissociated into clumps by mechanical dissociation or enzymatic degradation, and the cells are then replated on fresh tissue culture medium. Individual colonies that show undifferentiated morphology are selected by micropipette / stem cell tool, mechanically dissected into fragments / clumps, and replated. The resulting ES cells are then periodically split every 4-7 days.For further details regarding methods for preparing human ES cells, see Reubinoff et al., Nat Biotechnol 2000, May: 18(5): 559; Thomson et al., [U.S. Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

[0106] It will be appreciated that commercially available stem cells may also be used according to some embodiments of the present invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry [Hypertext Transfer Protocol: / / grants(dot)nih(dot)gov / stem_cells / registry / current(dot)htm] and other European registries. A non-limiting example of a commercially available embryonic stem cell line is HAD-C. 102, ESI, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES1, HUES2, HUES3, HUES4, HUES5, HU ES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, H UES27, HUES28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES45, Shef3, Shef6, BJNhem19, BJNhem20, SA001, and SA001.

[0107] Furthermore, ES cells have been successfully used in mouse (Mills and Bradley, 2001), golden hamster (Doetschman et al., 1988, Dev Biol. 127: 224-7), rat (Iannaccone et al., 1994, Dev Biol. 163: 288-92), rabbit (Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33), and several livestock species (Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67], as well as from other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0108] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days after fertilization, a stage prior to gastrulation. Prior to culturing the blastocysts, the zona pellucida is digested [e.g., by Tyrode's acid solution (Sigma Aldrich, St Louis, MO, USA)] to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos at least 9 days and no more than 14 days after fertilization (i.e., prior to the gastrulation event) using standard embryonic stem cell culture methods.

[0109] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during an in vitro fertilization process, where the embryo is not destroyed in the process.

[0110] Yet another method for preparing ES cells is by parthenogenesis, a process that also does not destroy the embryo.

[0111] Current ES culture methods are mainly based on the use of feeder cell layers that secrete factors necessary for stem cell proliferation but at the same time inhibit stem cell differentiation. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEFs), mouse embryonic fibroblasts (MEFs), mouse fetal fibroblasts (MFFs), human embryonic fibroblasts (HEFs), human fibroblasts obtained from differentiation of human embryonic stem cells, human fetal muscle cells (HFMs), human fetal skin cells (HFSs), human adult skin cells, human foreskin fibroblasts (HFFs), human umbilical cord fibroblasts, human cells obtained from umbilical cord or placenta, and human bone marrow stromal cells (hMSCs). Growth factors may be added to the medium to maintain ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGFβ. In another embodiment, agents may be added to the medium to maintain hESCs in a naive undifferentiated state. See, e.g., Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

[0112] Feeder-free systems have also been used in ES cell culture. In these systems, matrices supplemented with serum replacements, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimeras) are used as substitutes for feeder cell layers. Stem cells can be grown on solid surfaces such as extracellular matrices (e.g., Matrigel® or laminin) in the presence of culture media, e.g., Lonza L7 system, mTeSR, StemPro, XFKSR, E8. Unlike feeder-based cultures, which require feeder cells and stem cells to be grown simultaneously and may result in mixed cell populations, stem cells grown on feeder-free systems are easily separated from the surface. The culture media used to grow stem cells contain factors that effectively inhibit differentiation and promote stem cell growth, e.g., MEF conditioned medium and bFGF. However, commonly used feeder-free culture systems use animal-based matrices (e.g., Matrigel®) supplemented with mouse or bovine serum or MEF-conditioned medium [Xu C, et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19: 971-4]. Animal-based matrices pose the risk of cross-transfer of animal pathogens to human ES cells, thus compromising future clinical applications.

[0113] Numerous methods are known for differentiating ESCs into the RPE lineage, including both directed differentiation protocols, e.g., those described in WO2008 / 129554, 2013 / 184809, and spontaneous differentiation protocols, e.g., those described in U.S. Patent No. 8,268,303 and U.S. Patent Application No. 20130196369, the contents of each of which are incorporated by reference.

[0114] According to a particular embodiment, RPE cells are generated from ESC cells using a directed differentiation protocol, for example according to the protocol disclosed in the Examples section.

[0115] In one exemplary differentiation protocol, embryonic stem cells are differentiated into an RPE cell lineage using a first differentiation agent and then further differentiated into RPE cells using members of the transforming growth factor-β (TGFβ) superfamily (e.g., TGFβ1, TGFβ2, and TGFβ3 subtypes, as well as homologous ligands including activins (e.g., activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), several bone morphogenetic proteins (BMPs), such as BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth differentiation factors (GDFs).

[0116] According to a particular embodiment, the TGFβ superfamily member is selected from the group consisting of TGFβ1, activin A, and TGFβ3.

[0117] According to a particular embodiment, the member of the transforming growth factor-β (TGFβ) superfamily is activin A, for example, 20-200 ng / ml, for example, 100-180 ng / ml activin A.

[0118] The first differentiation agent promotes differentiation into the RPE lineage. For example, the first differentiation agent may promote differentiation of pluripotent stem cells into neural progenitors. Such cells may express a neural progenitor marker, such as PAX6.

[0119] According to a particular embodiment, the first differentiation agent is nicotinamide (NA), for example, 1 to 100 mM, 5 to 50 mM, 5 to 20 mM, for example, 10 mM nicotinamide (NA).

[0120] NA, also known as "niacinamide," is an amide derivative form of vitamin B3 (niacin) that is believed to protect and improve beta cell function. The chemical formula of NA is C6H6N2O. NA is essential for growth and the conversion of food to energy and has been used in the treatment of arthritis and the treatment and prevention of diabetes. TIFF0007608192000001.tif43128

[0121] According to certain embodiments, the nicotinamide is a nicotinamide derivative or a nicotinamide mimetic. The term "nicotinamide (NA) derivative" as used herein refers to a compound that is a chemically modified derivative of natural NA. In one embodiment, the chemical modification may be the substitution of the pyridine ring of the basic NA structure (through the carbon or nitrogen member of the ring) through the nitrogen or oxygen atom of the amide moiety. During the substitution, one or more hydrogen atoms may be replaced by a substituent and / or a substituent may be attached to the N atom to form a quadrivalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In another embodiment, the chemical modification may be the deletion or exchange of a single group, for example, to form a thiobenzamide analog of NA. All of these are as understood by those familiar with organic chemistry. Derivatives in the context of the present invention also include nucleoside derivatives of NA (e.g., nicotinamide adenine).

[0122] Various NA derivatives have been described, some of which are also described in relation to the inhibitory activity of PDE4 enzyme (WO03 / 068233; WO02 / 060875; GB2327675A) or as VEGF-receptor tyrosine kinase inhibitor (WO01 / 55114).For example, the process for preparing 4-aryl-nicotinamide derivatives (WO05 / 014549).Other exemplary nicotinamide derivatives are disclosed in WO01 / 55114 and EP2128244.

[0123] Nicotinamide mimetics include modified nicotinamide and chemical analogs of nicotinamide that reproduce the action of nicotinamide in the differentiation and maturation of RPE cells from pluripotent cells.Exemplary nicotinamide mimetics include benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide.Another class of compounds that can act as nicotinamide mimetics are poly(ADP-ribose) polymerase (PARP) inhibitors.Exemplary PARP inhibitors include 3-aminobenzamide, Iniparib (BSI201), Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP9722, MK4827, and BMN-673.

[0124] According to a particular embodiment, differentiation is performed as follows: (a) culturing the ESCs in a medium containing a first differentiation agent (e.g., nicotinamide); and (b) culturing the cells obtained from step (a) in a medium containing a member of the TGFβ superfamily (eg, activin A) and a first differentiation agent (eg, nicotinamide).

[0125] Preferably, step (a) is carried out in the absence of a member of the TGFβ superfamily.

[0126] The above protocol may be continued by culturing the cells obtained in step (b) in a medium containing the first differentiation agent (e.g., nicotinamide) but lacking a member of the TGFβ superfamily (e.g., activin A), this step being referred to herein as step (c).

[0127] The above protocol will now be explained in more detail with further aspects.

[0128] Once a sufficient amount of ESCs is obtained, the differentiation process is started. ESCs are typically removed from the adherent cell culture (e.g., by using collagenase A, dispase, TrypLE select, EDTA) and plated onto a non-adherent substrate (e.g., Hydrocell non-adherent cell culture plate) in the presence of nicotinamide (and in the absence of activin A). Exemplary nicotinamide concentrations are 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM. Once the cells are plated onto the non-adherent substrate, the cell culture may be referred to as a cell suspension, preferably floating clusters in suspension culture, i.e., aggregates of cells derived from human embryonic stem cells (hESCs). The cell clusters do not adhere to any substrate (e.g., culture plate, carrier). Sources of floating stem cells have been previously described in WO06 / 070370, which is incorporated herein by reference in its entirety. This step may be carried out for a minimum of 1 day, more preferably 2 days, 3 days, 1 week or even 10 days. Preferably, the cells are not cultured in suspension with nicotinamide (and in the absence of a TGFβ superfamily member, e.g., activin A) for longer than 2 weeks.

[0129] According to preferred embodiments, when cells are cultured on a non-adherent substrate, atmospheric oxygen conditions are manipulated to have a percentage of about 20%, 15%, 10%, or less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, and more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%).

[0130] Examples of non-adherent cell culture plates include non-adherent cell culture plates manufactured by Hydrocell (eg, catalog number 174912), Nunc, and the like.

[0131] Typically, the cluster comprises at least 50-500,000, 50-100,000, 50-50,000, 50-10,000, 50-5000, 50-1000 cells. According to one embodiment, the cells in the cluster are not organized into layers and form irregular shapes. In one embodiment, the cluster is devoid of pluripotent embryonic stem cells. In another embodiment, the cluster comprises a small number of pluripotent embryonic stem cells (e.g., 5% or less or 3% or less (e.g., 0.01-2.7%) of cells co-expressing OCT4 and TRA1-60 at the protein level). Typically, the cluster comprises cells that are partially differentiated under the influence of nicotinamide. Such cells may express neural progenitor markers, such as PAX6. The cells may also express other lineage progenitor markers, such as alpha-fetoprotein, MIXL1, and Brachyuri.

[0132] The clusters may be dissociated using enzymatic or non-enzymatic methods (e.g., mechanical methods) known in the art. According to one embodiment, the cells are dissociated such that they are no longer in the form of clusters, e.g., aggregates or clumps of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells, 2-5000 cells, 2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells. According to a particular embodiment, the cells are present in a single cell suspension.

[0133] The cells (e.g., dissociated cells) are then plated onto an adherent substrate and cultured in the presence of nicotinamide, e.g., 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM nicotinamide (and in the absence of activin A). This step may be carried out for a minimum of 1 day, more preferably 2 days, 3 days, 1 week, or even 14 days. Preferably, the cells are not cultured in the presence of nicotinamide (and in the absence of activin A) in adherent cell culture for more than 1 week.

[0134] Generally speaking, the cells are typically exposed to nicotinamide (at a concentration of 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM) for about 2-3 weeks, preferably no more than 4 weeks, before a second differentiation factor (e.g., activin A) is added.

[0135] Examples of adherent substrates include, but are not limited to, collagen, fibronectin, laminin, (eg, laminin 521).

[0136] After the first stage of directed differentiation (i.e., culturing in the presence of nicotinamide (e.g., 10 mM) under non-adherent culture conditions, under hypoxic atmospheric conditions, followed by culturing on an adherent substrate in the presence of nicotinamide under hypoxic atmospheric conditions), the semi-differentiated cells are then cultured on an adherent substrate in the presence of nicotinamide (e.g., 10 mM) and activin A (e.g., 20-200 ng / ml, 100-200 ng / ml, e.g., 140 ng / ml, 150 ng / ml, 160 ng / ml, or 180 ng / ml). This stage may be performed for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, 1 week to 8 weeks, 1 week to 4 weeks, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or even 8 weeks. Preferably, this step is carried out for about 2 weeks. According to one embodiment, this differentiation step is also carried out under low atmospheric oxygen conditions, i.e., less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%).

[0137] After the second stage of directed differentiation (i.e., culturing on the adherent substrate in the presence of nicotinamide and activin A), the further differentiated cells may optionally be cultured on the adherent substrate for a subsequent differentiation stage, i.e., in the presence of nicotinamide (e.g., 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM) and in the absence of activin A. This stage may be performed for at least 1 day, 2 days, 3 days, 1 week, at least 2 weeks, at least 3 weeks, or even 4 weeks. Preferably, this stage is performed for about 1 week. This differentiation step may be performed under low atmospheric oxygen conditions (i.e., less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%) or normoxic conditions, or a combination of both (i.e., initially under low atmospheric oxygen conditions, followed by normoxic conditions if faintly pigmented cells are observed).

[0138] According to certain embodiments, when atmospheric oxygen conditions are returned to normal atmospheric conditions, the cells are cultured in the presence of nicotinamide (e.g., 10 mM) and in the absence of activin A for at least another day (e.g., up to two weeks).

[0139] The basal medium according to the present invention is any known cell culture medium known in the art for supporting cell growth in vitro, typically a medium that includes a defined base solution that includes salts, sugars, amino acids, and any other nutrients necessary to maintain cells in a viable state in culture. Non-limiting examples of commercially available basal media that can be used according to the present invention include Nuristem (without bFGF and TGFβ for ESC differentiation, and with bFGF and TGFβ for ESC expansion), Neurobasal™, KO-DMEM, DMEM, DMEM / F12, Lonza L7 system, mTeSR, StemPro, XF KSR, E8, Cellgro™ Stem Cell Growth Medium, or X-Vivo™. The basal medium may be supplemented with various agents known in the art dealing with cell culture. The following is a non-limiting reference to various supplements that can be included in the culture system used according to the present disclosure. - Media containing serum or serum replacements, such as, but not limited to, knock out serum replacement (KOSR), Nutridoma-CS, TCH™, N2, N2 derivatives, or B27, or a combination. - Extracellular matrix (ECM) components, including but not limited to fibronectin, laminin, collagen, and gelatin. The ECM may be used to carry one or more members of the TGFβ superfamily of growth factors. -Antibacterial agents, such as, but not limited to, penicillin and streptomycin. - Non-essential amino acids (NEAAs) Neurotrophins known to play a role in promoting survival of SCs in culture, including but not limited to BDNF, NT3, NT4.

[0140] In a preferred embodiment, the medium used to differentiate the ESCs is Nuristem medium (Biological Industries, 05-102-1A or 05-100-1A).

[0141] According to a particular embodiment, differentiation of the ESCs is performed under xeno-free conditions.

[0142] According to one embodiment, the proliferation / growth medium is free of xenogeneic contaminants, i.e. free of animal-derived components such as serum, animal-derived growth factors, and albumin, etc. Thus, according to this embodiment, the culturing step is carried out in the absence of xenogeneic contaminants.

[0143] Other methods for culturing ESCs under xeno-free conditions are provided in U.S. Patent Application Publication No. 20130196369, the contents of which are incorporated in their entirety.

[0144] During the differentiation step, the embryonic stem cells may be monitored for their differentiation state. Cell differentiation can be determined by examining cell- or tissue-specific markers known to indicate differentiation.

[0145] Tissue / cell specific markers can be detected using immunological techniques well known in the art [Thomson JA et al., (1998). Science 282: 1145-7]. Examples include, but are not limited to, flow cytometry for membrane-bound or intracellular markers, immunohistochemistry for extracellular and intracellular markers, and enzyme immunoassay for secreted molecular markers (e.g., PEDF).

[0146] Thus, according to another aspect of the present invention, (a) culturing pluripotent stem cells in a medium containing a differentiation agent and lacking a member of the transforming growth factor β (TGFβ) superfamily to generate differentiated cells; (b) culturing the differentiated cells in a medium containing a member of the transforming growth factor β (TGFβ) superfamily and a differentiation agent to generate cells that are further differentiated into an RPE lineage; (c) analyzing the secretion of pigment epithelium-derived factor (PEDF) from cells further differentiating into the RPE lineage; and (d) culturing the cells that have further differentiated into the RPE lineage in a medium that contains a differentiation agent and lacks a member of the transforming growth factor beta (TGFβ) superfamily to generate RPE cells. Including, Step (d) is performed if the amount of PEDF is above a predetermined level. A method for producing retinal epithelial cells is provided.

[0147] Preferably, step (d) is performed when the level of PEDF is greater than 100 ng / ml / day, 200 ng / ml / day, 300 ng / ml / day, 400 ng / ml / day, or 500 ng / ml / day.

[0148] Another method for determining the effectiveness of the cells during or after the differentiation process is by analyzing the barrier function and polarized secretion of PEDF and VEGF, as shown in Example 4 herein below.

[0149] The cells may be selected and / or expanded once they have been induced to become RPE cells.

[0150] According to a particular embodiment, the selection is based on negative selection, i.e., on removing non-RPE cells, which may be performed mechanically, by removing non-pigmented cells, or by removing non-polygonal cells, or by using surface markers.

[0151] According to another embodiment, the selection is based on positive selection, i.e., selection based on morphology (e.g., pigmented and / or polygonal cells), which may be performed by visual analysis or by using surface markers.

[0152] According to yet another embodiment, the selection is first based on a negative selection and then based on a positive selection.

[0153] Expansion of RPE cells may be performed on extracellular matrices, such as gelatin, collagen, or poly-D-lysine, and laminin. For expansion, the cells may be cultured in serum-free KOM, serum-containing medium (e.g., DMEM+20%), or Nuristhem medium (06-5102-01-1A Biological Industries). Optionally, the cells may be exposed to nicotinamide at concentrations of 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM, during the expansion phase. Under these culture conditions, the pigment cells lose pigmentation and acquire a morphology similar to uterine fibroids. After further long-term culture and expansion to high-density cultures, the cells regain the characteristic polygonal morphology and, preferably, the pigmentation of RPE cells.

[0154] RPE cells may be grown in suspension or in monolayers. Growth of RPE cells in monolayer cultures may be modified for large scale growth in bioreactors by methods well known to those of skill in the art.

[0155] The populations of RPE cells generated according to the methods described herein may be characterized according to a number of different parameters.

[0156] Thus, for example, the resulting RPE cells are polygonal in shape and pigmented.

[0157] According to one embodiment, at least 70%, 75%, 80%, 85%, 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% of the cells within the resulting RPE cell population co-express pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP).

[0158] Following administration, the cells described herein can form a monolayer (as shown in FIG. 27C).

[0159] According to one embodiment, the transepithelial electrical resistance of the cells in the monolayer is greater than 100 ohms.

[0160] Preferably, the transepithelial electrical resistance of the cells is greater than 150 ohms, 200 ohms, 250 ohms, 300 ohms, 300 ohms, 400 ohms, 500 ohms, 600 ohms, 700 ohms, 800 ohms or even greater than 900 ohms.

[0161] According to certain embodiments, the TEER is between 100 and 1000 ohms, more preferably between 100 and 900 ohms, for example, between 200 and 900 ohms, between 300 and 800 ohms, between 300 and 700 ohms, between 400 and 800 ohms, or between 400 and 700 ohms.

[0162] Devices for measuring transepithelial electrical resistance (TEER) are known in the art. An exemplary setup for measuring TEER is shown in FIG.

[0163] It will be understood that the cell populations disclosed herein are free of undifferentiated human embryonic stem cells. In one embodiment, for example, fewer than 1:250,000 cells are Oct4+, as measured by FACS. + TRA-1-60 + The cells also do not express or down-regulate hESC-associated GDF3 or TDGF as measured by PCR.

[0164] Another way to characterize the cell populations disclosed herein is by marker expression, such that at least 80%, 85%, or 90% of the cells express bestrophin 1, for example, as measured by immunostaining. In one embodiment, 90-95% of the cells express bestrophin.

[0165] According to another embodiment, at least 80%, 85%, 87%, 89%, or 90% of the cells express microphthalmia-associated transcription factor (MITF) as measured by immunostaining, e.g., 85-95% of the cells express MITF.

[0166] According to another embodiment, at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 87%, 89%, or 90% of the cells express paired box gene 6 (PAX-6) as measured by FACS.

[0167] The cells described herein can also be characterized according to the amount and / or type of factor they secrete. Thus, according to one embodiment, the cells preferably secrete more than 500, 750, 1000 ng, or even 2000 ng of pigment epithelium-derived factor (PEDF) per ml per day (e.g., after 14 days in culture) as measured by ELISA.

[0168] It will be understood that the RPE cells prepared herein secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner. According to certain embodiments, the ratio of apical secretion of PEDF to basal secretion of PEDF is greater than 1. According to certain embodiments, the ratio of apical secretion of PEDF to basal secretion of PEDF is greater than 2. According to certain embodiments, the ratio of apical secretion of PEDF to basal secretion of PEDF is greater than 3. Furthermore, the ratio of basal secretion of VEGF to apical secretion of VEGF is greater than 1. According to certain embodiments, the ratio of basal secretion of VEGF to apical secretion of VEGF is greater than 1.5, 2 or 2.5.

[0169] The cells of the invention secrete additional factors including, for example, angiogenin, the immunomodulatory factors IL-6, sgp130, MIF, sTNF-R1, sTRAIL-R3, MCP-1 and osteoprotegerin, the extracellular matrix regulators TIMP-1 and TIMP-2, and the protein Ax1.

[0170] According to another aspect, at least 80% of the cells in the cell population co-express pre-melanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), and further, a portion of the cells (at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) secrete / release each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

[0171] In some cases, it will be appreciated that all cells co-expressing pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) also secrete / release angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

[0172] In other cases, the majority (>50%, 60%, 70%, 80, 90%) of cells co-expressing premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) also secrete / release angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

[0173] The RPE cells generated herein preferably secrete angiogenin, TIMP2, sgp130, and sTNF-R1 in a polarized manner.

[0174] According to a particular embodiment, the ratio of apical to basal secretion of sgp130 is greater than 1. According to a particular embodiment, the ratio of apical to basal secretion of sgp130 is greater than 2. According to a particular embodiment, the ratio of apical to basal secretion of sgp130 is greater than 3.

[0175] Furthermore, the ratio of apical to basal sTNF-R1 is greater than 1. According to a particular embodiment, the ratio of apical to basal sTNF-R1 is greater than 2. According to a particular embodiment, the ratio of apical to basal sTNF-R1 is greater than 3.

[0176] Additionally, the ratio of basolateral secretion of angiogenin to apical secretion of angiogenin is greater than 1. According to particular embodiments, the ratio of basolateral secretion of angiogenin to apical secretion of angiogenin is greater than 1.5, 2, 2.5, or 3.

[0177] Furthermore, the ratio of apical to basal secretion of TIMP2 is greater than 1. According to a particular embodiment, the ratio of apical to basal secretion of TIMP2 is greater than 2. According to a particular embodiment, the ratio of apical to basal secretion of TIMP2 is greater than 3.

[0178] The stability of the cells is another feature. Thus, for example, the amount of PEDF secretion remains stable in the cells after 6, 8, 10, 12, or even 24 hours of incubation at 2-8°C. Moreover, the polarized secretion of PEDF and VEGF remains stable after 6, 8, 10, 12, or even 24 hours of incubation at 2-8°C. Moreover, the TEER of the cells remains stable in the cells after 6, 8, 10, 12, or even 24 hours of incubation at 2-8°C.

[0179] In another embodiment, the cells are characterized as having a therapeutic effect.Thus, for example, the present inventors have shown that the cell population can rescue vision in RCS rats after subretinal administration.Furthermore, the cell population can rescue photoreceptors (e.g., cone photoreceptors) in RCS rats for up to 180 days (in some embodiments, at least 180 days) after subretinal administration.

[0180] It will be well understood by those skilled in the art that obtaining RPE cells would be of great benefit. RPE cells may be used as an in vitro model to develop new drugs that promote RPE cell survival, regeneration, and function. RPE cells may be useful for high-throughput screening of compounds that have toxic or regenerative effects on RPE cells. RPE cells may be used to reveal mechanisms, new genes, soluble factors, or membrane-bound factors that are important for the development, differentiation, maintenance, survival, and function of photoreceptor cells.

[0181] RPE cells may also serve as an unlimited source of RPE cells to transplant, replenish, and support dysfunctional or altered RPE cells in retinal degeneration. Furthermore, genetically modified RPE cells may serve as vectors to deliver and express genes in the eye and retina after transplantation.

[0182] The RPE cells produced by the method of the present disclosure may be used for large-scale and / or long-term culture of such cells.For this purpose, the method of the present invention must be carried out in a bioreactor and / or cell culture system suitable for large-scale production of cells, and in this case, undifferentiated hSC must be cultured according to the present invention.The general requirements for culturing cells in a bioreactor and / or cell culture system are well known to those skilled in the art.

[0183] The cells can be harvested by various methods known in the art. Non-limiting examples include mechanical dissection and dissociation using papain or trypsin (e.g., TrypLE select). Other methods known in the art are also applicable.

[0184] The RPE cells prepared as described herein can be transplanted into various target sites in the eye of a subject.According to one embodiment, the transplantation of RPE cells is into the subretinal space of the eye (between the photoreceptor outer segment and the choroid), which is the normal anatomical location of RPE.Furthermore, depending on the migration ability and / or positive paracrine effect of said cells, transplantation into other eye compartments can be considered, including the inner or outer retina, the retinal periphery, and the choroid.

[0185] Retinal diseases that may be treated using the RPE cells described herein include, but are not limited to, retinitis pigmentosa, retinoschisis, lattice degeneration, Best's disease, and age-related macular degeneration (AMD).

[0186] Moreover, transplantation can be carried out by various techniques known in the art.The method for carrying out RPE transplantation is described in, for example, U.S. Patent No. 5,962,027, U.S. Patent No. 6,045,791 and U.S. Patent No. 5,941,250, and Eye Graefes Arch Clin Exp Opthalmol March 1997; 235(3): 149-58; Biochem Biophys Res Commun Feb. 24, 2000; 268(3): 842-6; Opthalmic Surg February 1991; 22(2): 102-8. Methods for performing corneal transplants are described, for example, in U.S. Pat. No. 5,755,785, as well as in Eye 1995; 9 (Pt 6 Su): 6-12; Curr Opin Opthalmol August 1992; 3 (4): 473-81; Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; Ophthalmology April 2000; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999; 43(6): 502-8. If a mainly paracrine effect is used, cells can also be delivered to and maintained in a semipermeable container in the eye, which also reduces exposure of the cells to the host immune system (Neurotech USA CNTF delivery system; PNAS March 7, 2006 vol. 103(10) 3896-3901).

[0187] In one embodiment, transplantation is performed by delivering the cells through a small retinal opening into the subretinal space or by direct injection after pars plana vitrectomy. Alternatively, the cells may be delivered to the subretinal space via a transscleral, transchoroidal approach. Additionally, direct transscleral injection into the vitreous space or delivery to the anterior retinal periphery near the ciliary body can be performed.

[0188] RPE cells can be transplanted in various forms.For example, RPE cells can be introduced into target site in the form of cell suspension, or can be attached on matrix, extracellular matrix, or substrate, for example, on biodegradable polymer, or combination.RPE cells can also be transplanted together with other retinal cells, for example, photoreceptors (co-transplantation).

[0189] Therefore, the present invention also relates to pharmaceutical compositions of RPE cells as described herein.The compositions are preferably suitable for transplantation into the eye.Thus, for example, RPE cells may be formulated in intraocular irrigation fluid such as BSS plus™.

[0190] It is expected that many related technologies for generating RPE cells will be developed during the life of the patent proceeding from this application, and the term RPE cells is intended a priori to include all such new technologies.

[0191] As used herein, the term "about" refers to ±10%.

[0192] The terms "comprises," "comprising," "includes," "including," "having," and their conjugations mean "including but not limited to."

[0193] The term "consisting of" means "including and limited to."

[0194] The term "consisting essentially of" means that the composition, method, or structure may include additional ingredients, steps, and / or moieties, so long as the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed compositions, methods, and structures.

[0195] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0196] Throughout this application, various aspects of the invention may be presented in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0197] The term "method" as used herein refers to methods, means, techniques, and procedures for accomplishing a given task, including but not limited to methods, means, techniques, and procedures known to those of skill in the art of chemistry, pharmacology, biology, biochemistry, and medicine, or readily developed from known methods, means, techniques, and procedures by those of skill in the art of chemistry, pharmacology, biology, biochemistry, and medicine.

[0198] As used herein, the term "treating" includes preventing, substantially inhibiting, slowing, or reversing the progression of the abnormality, substantially ameliorating a clinical or cosmetic symptom associated with the abnormality, or substantially preventing the appearance of a clinical or cosmetic symptom associated with the abnormality.

[0199] It is understood that certain features of the invention that are described in the context of separate embodiments for ease of understanding may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination, or as appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0200] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0201] Reference is now made to the following examples, which together with the above descriptions illustrate certain aspects of the invention in a non-limiting manner.

[0202] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques which are thoroughly explained in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vol. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent No. 4,666,828; Nos. 5,192,659 and 5,272,057; Cell Biology: A Laboratory Handbook, Volume I-III Cellis, JE, ed. (1994); Culture of Animal Cells - A Manual of Basic Technique by Freshney, Wiley-Liss, NY (1994), Third Edition; Current Protocols in Immunology, Volume I-III Coligan JE, ed.(1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). The patent and scientific literature provides extensive descriptions of available immunoassay methods. See, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771, and 5,281,521. “Oligonucleotide Synthesis” Gait, MJ, ed. (1984); “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985); “Transcription and Translation” Hames, BD, and Higgins SJ, eds. (1984); “Animal Cell Culture” Freshney, RI, ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984), and “Methods in Enzymology” Vol.1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. The information contained therein is entirely incorporated herein by reference.

[0203] Example 1 Qualification of CRALBP / PMEL17 double staining FACS method The aim of this study was to qualify the CRALBP / PMEL17 dual staining FACS method by demonstrating its accuracy and precision in at least six independent spiking assays over at least three test days. The assay was qualified using OpRegen® batch 5C as positive control cells and HAD-C 102-hESC as negative control cells. A calibration curve of known amounts of RPE (OpRegen® 5C) spiked into hESC was used to test the accuracy and precision of different spiking points. The expected accuracy and precision was up to 25% across all points.

[0204] Staining protocol: Negative control hESC cells obtained from a cryopreserved hESC bank (HAD-C 102 p48 4.5.2014) were thawed into Nutristem (containing HSA) according to sponsor protocol. Positive control RPE cell stock: OpRegen® Batch 5C cells (reference line) were thawed into 20% HS-DMEM according to sponsor protocol. Thawed OpRegen® 5C and HAD-C 102 hESC were spun down, resuspended in 1 ml PBS(-), filtered through a 35 μM cell strainer, and counted using trypan blue. Cell concentration was 0.73 x 10 per ml in PBS(-). 6 ~10 6 The cells were adjusted to 100,000 cells / ml. 1 μl / ml FVS450 was added to each cell suspension, followed by vortexing and incubation at 37°C for 6 min. FVS450 was washed with 0.1% BSA and resuspended in 0.1% BSA-Fc-block (5 min at RT) to block all Fc epitopes on the cells. Cells were then washed with PBS(-) and fixed with 80% methanol (5 min at 4°C). Fixed cells were washed once with PBS(-) and once with 0.1% PBS-T and permeabilized with 0.1% PBS-T (20 min at RT). The permeabilization solution was replaced with 10% normal goat serum (NGS) blocking solution (200,000 cells / 50 μl) for at least 30 min (maximum 1 h) at RT. Quality sample tubes (QS) were prepared during the incubation time and at the end of blocking the cells were divided and immunostained. Cells were incubated with primary antibody for 30 min, followed by washing three times with 0.1% PBS-T, incubation with secondary antibody for 30 min, and washing three times with 0.1% PBS-T.

[0205] Negative and positive control cells were stained with viability stain FVS450, fixed, blocked, and permeabilized. A calibration curve of known amounts of positive control RPE (OpRegen® 5C) cells in negative control hESCs at four concentrations (25%, 50%, 75%, and 95% RPE in hESCs) was then generated based on the cell counts by trypan blue viability for each population. Negative and positive control cells and mixed populations were immunostained with primary monoclonal antibodies specific for RPE markers CRALBP and PMEL17, followed by staining with matched secondary antibodies (anti-mouse-FITC and anti-rabbit-Alexa Fluor647, respectively). Stained cells were FACS analyzed to measure the percent single live cell gated CRALBP+PMEL17+ cells.

[0206] result Accuracy: The accuracy of the assay was determined by testing four levels of spiked RPE (25%, 50%, 75%, and 95%). Accuracy was determined for RPE stock (OpRegen® 5C) against a potential of 100% RPE cells. Each level value was analyzed from six independent runs / measurements.

[0207] The 50% concentration level was considered to be the lower limit of quantification with an expected accuracy of up to 25% (50% levels were −8.41 to 20.14; 75% and 99.5% levels were −5.32 to 6.88).

[0208] These results met the expected relative bias outcome of up to 25%, indicating that the assay is accurate for determining concentrations of CRALBP+PMEL17+ double positive cells between 50 and 99.5%. Because OpRegen® 5C yields 99.5% of CRALBP+PMEL17+ double positive RPE cells, a relative bias of less than 25% cannot be guaranteed for results greater than 99.5%.

[0209] (Table 1) TIFF0007608192000002.tif232111

[0210] Intermediate Precision: The intermediate precision of the assay was determined from six assays performed by one operator. Percentage single raw RPE was determined for each assay, from which the %CY was calculated. Table 2 summarizes the test results. As shown, the %CY was less than 20% for all concentration levels and can be measured with sufficient precision. The %CY for concentration levels of 25%, 50%, 75%, 95%, and 100% RPE were 16.14%, 10.61%, 5.10%, 1.17%, and 0.34%, respectively. These results meet the precision expectations. The measured values ​​of percent RPE at all concentrations are within 20% of the expected values. These results indicate that the assay is precise to measure RPE at concentrations from 25 to 99.5%.

[0211] (Table 2) TIFF0007608192000003.tif230105

[0212] Repeatability: Sample repeatability was tested in three runs (#2, #3, and #4) where two identical OpRegen® SC samples were stained and acquired in parallel. Results confirmed that sample identity obtained within an experiment was reproducible and consistent across samples.

[0213] Linearity / Range: Linearity was measured using data that was found to be accurate and precise as shown in Figure 1. The regression coefficient between the target value (spiked) and the measured results over the assay range tested (50%-100%) was found to be 0.99. Thus, the range of this method, which demonstrated acceptable accuracy and precision as well as linearity, is in the range of 50%-99.5% of RPE cells, covering the expected range of test samples.

[0214] Positive control cells: the provisional level of CRALBP / PMEL17 double positive cells was set at 95% or higher.

[0215] Negative control cells: The provisional level of CRALBP / PMEL17 double positive cells for hESCs was set at 2% or less.

[0216] Stability: Results show that the stained samples are stable at 4°C after 1 and 4 days and accuracy remains within expected acceptance criteria, therefore data can be obtained within 96 hours of sample preparation.

[0217] conclusion The results presented herein qualify the disclosed method and demonstrate its suitability for its intended use in vitro determination of RPE purity in the final OpRegen® product and at different stages along the OpRegen® production process. Over the range of 50%-99.5% RPE cells, the accuracy of the relative bias was <25% and the precision of the %CV was <20%.

[0218] Example 2 Evaluation of OpRegen® Purity Levels We developed a FACS-based method to assess the purity of human retinal pigment epithelial cells (RPE) as well as the level of non-RPE cell impurities in RPE cells. A visual cycle component, cellular retinaldehyde-binding protein (CRALBP), was identified using bioinformatics as a unique marker for mature RPE cells. Preliminary studies using a CRALBP-specific monoclonal antibody have shown greater than 98% purity in RPE cells generated according to the methods described herein. Furthermore, these results were supported by immunostaining for PMEL17, a melanosome marker found in RPE. Furthermore, unlike some RPE-specific markers, CRALBP is not expressed in melanocytes, a possible neural crest cell contaminant.

[0219] Test samples and controls: Human primary melanocytes (ATCC, PCS-200-013) were used as negative control cells for CRALBP and positive control cells for PMEL17, a type I transmembrane glycoprotein abundant in melanosomes (melanin granules). HADC102-hESCs (OpRegen® parent line) at P29 were used as negative control cells for both CRALBP and PMEL17. Clinical grade OpRegen® cells (Batch 2A) and research grade OpRegen® (produced in mock production similar to GMP; mock IV D16) were used as test samples. These cells were generated as described in Example 3.

[0220] Immunostaining and FACS analysis: Cells were thawed, stained with Fixable Viability Stain (FVS450) (BD 562247), fixed in 80% methanol and immunostained with primary mouse anti-CRALBP (clone B2, Abcam ab15051) or its isotype control to mouse IgG2a (Abcam ab170191) and rabbit anti-human PMEL17 (clone EPR4864, Abcam ab137062), followed by secondary antibodies goat anti-mouse (Dako F0479) and goat anti-rabbit (Jackson 111-606-144), respectively.

[0221] FACS data acquisition was performed using a validated Navios flow cytometer (Beckman Coulter) and analysis was performed using FlowJo 7.6.

[0222] result Initial FACS data using anti-CRALBP monoclonal antibody showed OpRegen® purity levels to be greater than 98%. Melanocytes, potential neural crest cell contaminants, were found to be negative for CRALBP, a unique RPE-specific marker (1.7%). The parental line HADC102-hESC was CRALBP negative (0.2%) as expected.

[0223] The purity level of OpRegen® remained above 98% even after double staining with CRALBP and PMEL17 (Figure 10). Melanocytes showed PMEL17 positive staining as expected but were negative for the double marked population (~1%). HADC102-hESCs showed negative staining for CRALBP and PMEL17 (0.07%).

[0224] Example 3 Description of manufacturing process and process control OpRegen® is produced from xeno-free GMP grade HAD-C 102 hESC line grown on irradiated, xeno-free GMP grade human umbilical cord fibroblast feeders. Clinical grade human fibroblast feeder cell line (CRD008; MCB) and working cell bank (WCB) were produced under Good Manufacturing Practice (GMP) and xeno-free conditions, properly tested, characterized and banked. They were then used to obtain clinical grade hESC HAD-C 102 line from surplus human blastocysts under GMP and xeno-free conditions.

[0225] In the initial stages of production, hESCs are expanded as colonies on irradiated feeders. They are then transferred to suspension culture to initiate directed differentiation. Spheroids (SBs) are formed and then plated as adherent cultures under conditions for continued directed differentiation towards neural fates and then towards RPE cells. At the end of the differentiation phase, non-pigmented areas are physically excised and pigmented cells are enzymatically harvested, seeded and expanded. Purified hESC-derived RPE cells (DS) are harvested at passage 2 and immediately processed into DP. The duration of the manufacturing process depends on the proliferation rate of hESCs (approximately 2 months from thawing) and typically spans a total of 4-5 months or more.

[0226] Each step of the manufacturing process, including in-process quality control (QC) testing, is briefly described below.

[0227] Steps 1-3: Creation of a human zona fibroblast feeder working cell bank (WCB). Vials of human zona feeder master cell bank (MCB) (CRD008-MCB) at passages 3 and 4 were thawed, expanded in Dulbecco's Modified Eagle Medium (DMEM, SH30081.01, Hyclone) supplemented with 20% human serum (14-498E, Lonza), irradiated (Gamma cell, 220 Exel, MDS Nordion 3,500 rad), and cryopreserved at passages 7 and 8 to create a working cell bank (WCB). Prior to cryopreservation, samples from the feeder cell cultures were tested for sterility, mycoplasma, and Limulus Amebocyte Lysate (LAL), morphology, karyotype, cell number, and viability. Furthermore, after thawing, the identity with the MCB, inability to proliferate, and ability to support undifferentiated HAD-C 102-hESC growth were confirmed. If the WCB passed all QC tests, the bank was released for hESC expansion.

[0228] Production steps 1 to 3 are illustrated in FIG.

[0229] Stages 4 and 5: Expansion of hECS. One vial of human cord fibroblast WCB (CRD008-WCB8 or CRD008-WCB9) was thawed and plated in a center-well plate covered with recombinant human gelatin (RhG 100-001, Fibrogen) at a concentration of 70,000–100,000 cells / ml / plate in DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (14-498E, Lonza). To allow fibroblast attachment, cells were incubated overnight at 37°C, 5% CO2. After 1-4 days, samples from the HAD-C 102-hESC MCB were thawed and plated on top of feeder cells in Nutristem "Plus" medium (GMP grade and xeno-free) containing the growth factors bFGF and TGF-β (05-102-1A, Biological Industries, Israel) at 37°C and 5% CO2 for 6-7 days. On days 6-7, the hESC cultures were mechanically disrupted (with a sterile tip or a disposable sterile stem cell tool; 14602 Swemed) and passaged to additional freshly prepared plates containing feeder cells at a concentration of 70,000-100,000 cells / plate. This was repeated weekly for several passages to reach the amount of hESC required to initiate differentiation (Figure 13, stages 4 and 5). Prior to use, the expanded HAD-C 102-hESCs were tested for sterility, mycoplasma, LAL, karyotype, and identity with MCB. Additionally, pluripotent morphological appearance and integrated expression of pluripotent markers (TRA-1-60, Oct4, and alkaline phosphatase) were confirmed (FIG. 2, stage 5). Production stages 4 and 5 are illustrated in FIG. 13.

[0230] Steps 6-13: Differentiation into RPE cells. The expanded HAD-C 102-hESCs were enzymatically treated with collagenase (4152, Worthington) in 6 cm cell culture plates for further expansion (FIG. 14, step 6). The expanded HAD-C 102-hESCs were then used in obtaining OpRegen® DS.

[0231] Differentiation of each OpRegen® batch was initiated by mechanical transfer of HAD-C 102-hESC clusters harvested by collagenase A from stage 6 cultures onto feeder-free, non-adherent 6 cm Hydrocell culture dishes in the presence of Nutristem "Minus" medium (without growth factors bFGF and TGF-β; 06-5102-01-1A Biological Industries, special order) supplemented with 10 mM nicotinamide (N-5535, Sigma) (Figure 14, stage 7). Plates were then cultured under low oxygen (5%) atmospheric conditions (37°C, 5% CO2) for up to 1 week to allow spheroids to form. Age-old spheroids were then collected in suspension, gently dissociated by pipetting, and transferred to human laminin (511, Biolamina)-coated 6-well plates for further growth in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide under a low oxygen atmosphere (5%) for one more week (Figure 14, step 8). Cells continued to grow under a low oxygen (5%) atmosphere for up to four more weeks; for two weeks in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide and 140 ng / ml activin A (G-120-14E, Peprotech) (Figure 14, step 9), followed by up to two weeks in the presence of Nutristem "Minus" medium supplemented with only 10 mM nicotinamide (Figure 14, step 10). When areas of light pigmentation were clearly visible in the polygonal cell patches, the plates were returned to a normoxic (20%) atmosphere (37°C, 5% CO2) and grown for up to 2 weeks in the presence of Nutristem "Minus" medium containing 10 mM nicotinamide (Figure 14, step 11). By 2 weeks, enlarged polygonal patches with distinctive pigmentation were clearly visible amongst areas of non-pigmented cells (Figure 14, step 12). The remaining pigmented cells were peeled off and manually harvested after treatment with TrypLE Select (12563-011, Invitrogen) for 15 min at 37°C (Figure 14, step 13). Production steps 6-13 are illustrated in Figure 14.

[0232] Steps 14-17: Expansion of OpRegen® cells. Pigment cells were then plated in 6-well gelatin-coated plates (0.5-1 × 10 cells) for proliferation in the presence of DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (14-498E, Lonza) for 2-3 days. 6 The cells were then transferred to a gelatin-coated flask and expanded for two more passages (P1, P2). After treatment with TrypLE Select at 37°C, the cells in P0 and P1 were collected, washed, and placed on gelatin-coated flasks and cultured for 2-3 days in the presence of DMEM supplemented with 20% human serum. The DMEM was then replaced with Nutristem "Minus" medium and the cells were expanded for two to three weeks until the plates were covered with lightly pigmented polygonal cells (Figure 15, step 14). These P0 cells were then expanded for two more passages (P1, P2) in gelatin-coated flasks. The cells in P0 and P1 were collected after treatment with TrypLE Select at 37°C, washed, and placed on gelatin-coated flasks and cultured for 2-3 days in the presence of DMEM supplemented with 20% human serum. The DMEM was replaced with Nutristem "Minus" medium and the cells were expanded for two to three weeks until the plates were covered with lightly pigmented polygonal cells (Figure 15, step 15-16). Cells at P2 grown in T175 flasks were then harvested after treatment with TrypLE Select at 37° C., resuspended in DMEM supplemented with 20% human serum, pooled and counted.

[0233] Growth medium samples were taken from each batch for sterility, mycoplasma, and LAL testing. Cell morphology was observed and recorded (Figure 15, step 17). Production steps 14-17 are illustrated in Figure 15.

[0234] Example 4 Process control points The IPC points are illustrated in Figure 16. The sampling points selected to assess hESC impurities and RPE purity along the production process are described below.

[0235] IPC point 1: Mechanically expanded HAD-C 102 hESCs with normal karyotype before differentiation. This is the starting material where the highest levels of hESCs are expected. This point was added to assess the maximum hESC levels before differentiation.

[0236] IPC point 2: HAD-C 102 hESCs expanded with collagenase prior to differentiation. At this stage, some differentiation is expected, which leads to a decrease in the levels of cells expressing Oct4 and TRA-1-60, as well as a decrease in the expression levels of GDF3 and TDGF. This point was added to assess hESC impurities during the non-directed differentiation stage.

[0237] IPC Point 3: Spheroids produced one week after induction of hESC differentiation in the presence of nicotinamide under feeder-free conditions. At this early stage of differentiation, hESC impurities during differentiation are expected to be at the highest levels, and therefore this assessment is expected to present the highest level of safety concerns.

[0238] IPC point 4: Cells at the end of Activin A treatment. Activin A directs differentiation towards RPE cells. At this point, a large decrease in hESC impurities and a large increase in RPE marker expression are expected. This point was added to monitor hESC differentiation towards RPE.

[0239] IPC points 5-7: cells at the end of the differentiation process before and after separation of the non-pigmented areas (IPC point 6) from the pigmented areas (IPC point 7). IPC points 5 and 6 contain cellular impurities, whereas sample 7 corresponds to the product at the end of the differentiation process, before the increase. The cellular contaminants found in sample 6 are in small amounts in sample 7 and even less in the product.

[0240] IPC point 8: Pigment cells at P0. Pigment cells at the end of the differentiation process, increased by 2-3 weeks. These cells represent the product two steps before the end of the production process.

[0241] IPC point 9: Pigment cells at P1. P0 cells expanded for 2-3 weeks. These cells represent the final product, one step before the end of the production process.

[0242] IPC point 10: Pigmented cells in P2 before cryopreservation. Collect and pool P1 cells that have been expanded for 2-3 weeks. These cells represent the bulk material (DS) before cryopreservation.

[0243] IPC point 11: Cryopreserved pigment cells in P2. These cells represent the formulation (DP). Throughout the production, at all sampling points, cell culture medium was collected to assess the secretion of pigment epithelium-derived factor (PEDF), which is known to be secreted by RPE cells.

[0244] result TRA-1-60 + Oct4 + hESC Quantification: The levels of hESCs in various samples collected along the production process were determined using a sensitive and robust Oct4 / TRA-1-60 dual staining FACS method. TRA-1-60 was assayed 1 week after removal of feeders and growth factors (TGFβ and bFGF) that support pluripotent cell growth under growth conditions that support early neural / eye field differentiation. + Oct4 + The percentage of cells was only 0.0106-2.7% (IPC point 3, spheroids). After adding activin A, which promotes RPE differentiation, TRA-1-60 + Oct4 + The levels of TRA-1-60 cells further decreased to 0.00048–0.0168% (IPC point 4, end of Activin). At the end of differentiation, after ablation of non-pigmented cells, + Oct4 + The levels of TRA-1-60 in cells ranged from 0.00033 to 0.03754% (IPC point 7, pigment cells). At P0, two steps before the end of the production process, the levels of TRA-1-60 in cells ranged from 0.00009 to 0.00108% (below the LOD - close to the LLOQ). + Oct4 +cells were detected (IPC point 8). TRA-1-60 in P1 (IPC point 9), P2 before cryopreservation (drug prod.; IPC point 10), and P2 after cryopreservation (DP; IPC point 11). + Oct4 + The levels in the cells are below the assay LLOQ (i.e., 0.00004-0.00047%, 0.00000-0.00016%, and 0.00000-0.00020%, respectively).

[0245] Relative expression of pluripotent hESC markers GDF3 and TDGF: The relative expression of the pluripotency genes GDF3 and TDGF was analyzed at various IPC points along the production process. The expression levels of GDF3 and TDGF gradually decreased. This indicates that TRA-1-60+Oct4 cells were more efficient at expressing pluripotent hESCs along the differentiation process. + This correlated with a gradual decline in cell numbers. At the end of the two stages prior to the end of the production process, P0, P1, and at the end of pre-cryopreservation P2 (bulk product) and post-cryopreservation P2 (formulation), GDF3 and TDGF expression levels were similar to those seen in the negative control OpRegen® 5C cells.

[0246] CRALBP + PMEL17 + Cell quantification: At the end of the differentiation phase at P0 and P2 (IPC points 8 and 11, respectively), CRALBP was used to measure RPE purity. + PMEL17 + As can be seen from Table 3 and Figure 17, CRALBP at P0 (IPC point 8), which is two steps before the end of the production process, + PMEL17 + The levels of RPE purity ranged from 98.53 to 98.83%. Similar RPE purity levels were detected in P2 after cryopreservation (99.61 to 99.76%; IPC point 11) (Table 3).

[0247] (Table 3) TIFF0007608192000004.tif25128DP, Drug Product.* IPC point 8 was tested after cryopreservation and demonstrated an accuracy error of ≦25% from an internal standard of RPE cells (OpRegen® 5C, positive control) spiked into hESCs (HAD-C 102, negative control).

[0248] Confocal imaging of cells immunostained for bestrophin 1, MITF, and CRALBP along mock production runs 4 and 5: Cells were immunostained for the RPE markers bestrophin 1, MITF, ZO-1, and CRALBP at the end of the differentiation phase (IPC point 7), at the end of the expansion phase (IPC point 10, DS), and after cryopreservation (IPC point 11, DP). For immunostaining, manually isolated non-pigmented cells (IPC point 6) were plated but detached from the plate during fixation and therefore could not be stained. Selected pigmented cells plated for 12 days (mock run 5 only; in parallel with cells in P0 from ongoing production) and 28 days (IPC point 7) stained positive for all RPE markers tested, with the percentage of cells expressing bestrophin 1 and cells expressing MITF ranging from 93% and 93.3 to 96.5%, respectively. Similar levels of bestrophin 1- and MITF-positive cells were detected in P0 (94.9% and 95.9%, respectively; only mock test 4 was tested), pre-cryopreservation P2 bulk (92.2-92.75% and 93.7-95.5%, respectively), and post-cryopreservation P2 formulation (91.1-95.7% and 83.8-94.9%, respectively; reduced MITF immunostaining in mock test 5 demonstrated outliers in areas randomly selected for analysis). CRALBP (as well as ZO-1) expression was detected in all IPC7, 10, and 11 samples (Figure 18).

[0249] Relative expression of RPE markers bestrophin 1, CRALBP, and RPE65 along mock production 2, 4, and 5: Relative expression of the RPE genes bestrophin 1, CRALBP, and RPE65 was measured at various IPC points along the production run. Along the production run, the relative expression levels of bestrophin 1, CRALBP, and RPE65 increased gradually. At the end of Activin A treatment to direct differentiation towards RPE cells (IPC point 4), the relative levels of bestrophin 1, CRALBP, and RPE65 were 685-, 36-, and 325-fold, respectively, above their respective relative levels in pre-differentiated mechanically passaged hESCs (IPC point 1; mock production 4). The relative expression levels of bestrophin 1, CRALBP, and RPE65 peaked at the end of the differentiation stage (IPC point 5) through the P1 stage (IPC point 9). At these stages, the respective expression levels were 5,838-11,841-fold, 211-299-fold, and 5,708-8,687-fold higher than the levels in mechanically passaged hESCs before differentiation (IPC point 1).

[0250] Morphological assessment along mock trials production 4 and 5: Cell morphology was analyzed at the end of the differentiation phase (IPC point 5) to estimate the relative area of ​​pigmented cells, and during the increase phase P0-P2 (IPC points 8-10) to verify a confluent polygonal morphology. At the end of the differentiation phase (IPC point 5), the relative area of ​​pigmented cells was estimated before excising the non-pigmented areas. The relative area of ​​pigmented cells was 32.5% ± 13.5% (mean ± SD, n = 7 wells of 6-well plates) in mock trial 4 and 60% ± 13% (mean ± SD, n = 7 wells of 6-well plates) in mock trial 5 (see representative images in Figure 11). Pigmented cell areas were selected and expanded. Morphology at the end of the increase phase P0 (IPC point 8), P1 (IPC point 9), and P2 (IPC point 10) evidenced densely packed cultures with a typical polygonal epithelial monolayer morphology (Figure 11).

[0251] PEDF secretion and efficacy measurements along mock productions 4 and 5: Pigment epithelium-derived factor (PEDF), known to be secreted by RPE cells, was measured in the cell culture medium at various IPC points along mock productions 4 and 5. As can be seen from Table 4, very low levels of PEDF, ranging from 4 to 79 ng / mL / day, were secreted by hESCs (IPC points 1 and 2) as well as by spheroids (IPC point 3; end of the first week with nicotinamide). At the end of Activin A treatment to direct differentiation towards RPE cells (IPC point 4), the levels of secreted PEDF ranged from 682 to 1,038 ng / mL / day, 31-37 times more compared to the levels secreted by spheroids. After incubation of cells in normoxic conditions with nicotinamide (IPC point 5), a further increase (2.2-4.6 times) in PEDF secretion was observed to be from 1,482 to 4,746 ng / mL / day. During the increasing phase (P0–P2, IPC8–10, respectively), PEDF secretion levels ranged from 2,187 to 8,681 ng / mL / day and peaked at P0–P1.

[0252] Table 4. PEDF secretion along simulated trials production 4 and 5 TIFF0007608192000005.tif61154ND, not performed; NA, not applicable; DS, drug substance; DP, formulation.

[0253] The tight junctions formed between RPE cells allow the blood-retinal barrier and the polarized secretion of PEDF and VEGF to occur. PEDF is secreted apically and acts as an antiangiogenic and neurotropic growth factor at the apical side. VEGF is mainly secreted basally and acts as a proangiogenic growth factor at the basal side for the choroid plexus endothelium. RPE polarization (barrier function and polarized secretion of PEDF and VEGF) was measured in a Transwell system at the end of P0 (IPC point 8), at the end of P2 before cryopreservation (IPC point 10), and at the end of P2 after cryopreservation (IPC point 11). As can be seen from Table 5, barrier function / transepithelial electrical resistance (TEER) and polarized secretion of PEDF and VEGF were demonstrated at all IPC points.

[0254] (Table 5) TIFF0007608192000006.tif233166ND, not performed; DS, bulk drug; DP, formulation. PEDF and VEGF were measured by ELISA. PEDF was collected from cells on day 14 while cultured in 12-well plates. Cells were then passaged onto Transwell and cultured for 6 weeks. During this time, TEER and secretion of VEGF and PEDF from the basolateral and apical sides of Transwell were measured.

[0255] Batch release testing of RPE cells produced in mock runs 4 and 5: To verify that OpRegen® produced in mock runs 4 and 5 was equivalent to GMP produced OpRegen®, an abbreviated OpRegen® batch release test was performed including morphology testing at the end of P2 before cryopreservation (IPC point 10, DS), and viability, total cell number / cryovial, identity (bestrophin 1 and MITF expression), hESC impurities, and karyotype analysis at the end of P2 after cryopreservation (IPC point 11, DP). OpRegen® produced in mock runs 4 and 5 passed the batch release criteria. OpRegen® produced in mock run 2 was not cryopreserved and therefore could not be tested.

[0256] conclusion Three mock production runs (mock runs 2, 4, and 5) were performed under research grade conditions using the same GMP production method, xeno-free GMP grade cells (HAD-C 102 hESCs grown on irradiated CRD008 feeders), xeno-free GMP grade reagents, and GMP grade laboratory equipment used in the GMP production of clinical batches. Mock runs 2, 4, and 5 were aimed at assessing the levels of hESC impurities along the production, and mock runs 4 and 5 were also aimed at identifying those critical in process quality control.

[0257] Using a qualified TRA-1-60 / Oct4 dual staining FACS method (LOD 0.0004%, 1 / 250,000, and LLOQ 0.001%, 1 / 100,000) and a qualified flow cytometer, levels of hESC impurities below the assay LOD were observed in the negatively selected pigmented cells at the end of the differentiation stage, three stages before the end of the mock 5 production run. In mock runs 2 and 4, performed using the core flow cytometer prior to assay qualification, levels of hESC impurities were below the assay LOD two stages before the end of the production run. Consistent with this data, quantitative RT-PCR analysis demonstrated that expression of pluripotent hESC genes GDF3 and TDGF was downregulated to levels similar to the negative control (OpRegen® 5C cells) two stages before the end of the production run.

[0258] Identity testing performed three steps before the end of production (isolation of pigment cells) demonstrated that bestrophin 1 and MITF were expressed in 93% and 96.5% of immunostained cells, respectively, and that CRALBP and ZO-1 were expressed (not quantified). RPE purity testing performed one step beyond (i.e., P0, two steps before the end of the production process) and after one cycle of negatively selected pigment cell expansion demonstrated that >98.5% of cells were expressed by FACS with CRALBP + PMEL17 + A double positive result was found. Similar levels of RPE purity (i.e., >99.6%) were also detected in the preparations. These results were supported by morphological studies that demonstrated a typical polygonal epithelial monolayer morphology and by quantitative RT-PCR analysis that demonstrated that expression of the RPE genes bestrophin 1, CRALBP, and RPE65 was upregulated to levels similar to the positive control (OpRegen® 5C cells).

[0259] PEDF, known to be secreted by RPE cells, was measured in the cell culture medium at various stages along the production process in mock runs 4 and 5. At the end of Activin A treatment (IPC point 4), which was previously shown by Idelson et al., 2009, to direct differentiation of RPE cells, the levels of secreted PEDF were greatly increased compared to the previous production stage (induction of spheroids) (31-fold in mock 4 and 37-fold in mock 5). PEDF secretion levels continued to increase, peaking at P0–P1 (1.7–5.8-fold increase compared to levels after Activin A). Evaluation of the relative area of ​​pigment cells at the end of the differentiation process (IPC point 5) was identified as another important quality control measure for assessing RPE differentiation. Using this measure, a two-fold difference in pigment cell yield was observed in mock runs 4 and 5 (32.5% in mock 4 and 60% in mock 5). This correlated with similar differences observed in PEDF secretion during this phase (1,482 ng / ml / day in sham 4 and 4,746 ng / ml / day in sham 5).

[0260] In conclusion, TRA-1-60 is used in three stages at the end of the production process and in the early stages. + Oct4 + No hESC impurities were observed, as evidenced by low expression levels of GDF3 and TDGF, high expression levels of bestrophin 1, CRALBP, and RPE65, as well as high levels of bestrophin 1 single positive cells and MITF single positive cells, and abundant CRALBP. + PMEL17 + Correlated with double positive cells (tested one stage past) Important safety and efficacy IPCs were identified at critical production stages.

[0261] Example 5 Effectiveness evaluation Experimental Design: We investigated whether subretinal transplantation of RPE cells, generated as described in Example 4, could slow the progression of RDD in the Royal College of Surgeons (RCS) rat model.

[0262] On postnatal days (P) 21–23 (before photoreceptor death begins), 25,000, 100,000, or 200,000 RPE cells were transplanted into the subretinal space of one eye of RCS rats. Animals treated with BSS+ (Alcon) and naive untreated animals served as controls. Groups were divided into four life ages: postnatal days P60, P100, P150, and P200. Fundus photography was used to identify bleb formation and monitor the quality of the injection. Fundus examinations were also performed on days P60, P100, P150, and P200. Visual acuity was measured in all animals at all time points (P60, P100, P150, P200) using optokinetic tracking.

[0263] Focal and full-field ERGs were assessed in all study groups at P60 and P100. On each animal's assigned sacrifice date, both eyes were removed, fixed in 4% paraformaldehyde, cryopreserved, embedded in Optimum Cutting Temperature compound (OCT), and cryosectioned. Cresyl violet staining was used to identify and enumerate structural rescue of photoreceptors. Immunofluorescence staining (IF) was used to identify transplanted cells and assess their fate, proliferation status, and ability to phagocytose photoreceptor outer segments. Additionally, immunofluorescence was used to measure rescue of host cones.

[0264] The study design is summarized in Table 6 herein below.

[0265] (Table 6) TIFF0007608192000007.tif67152

[0266] material and method Cell count: Cells were counted and then aliquoted to appropriate dosing concentrations. The average pre-injection cell viability for all injection time points was 94.0% ± 0.03. The average post-injection cell viability was 92.4% ± 0.02.

[0267] Surgery: Small incisions were made through the conjunctiva and sclera using progressively smaller gauge needles: 18, 22, 25, and 30. Intraocular pressure was reduced using a puncture in the outer peripheral part of the cornea to reduce the escape of injected cells. A glass pipette was then inserted into the subretinal space and 2 μl of suspension was injected. The scleral incision was then closed with sutures. Successful injection of cells or buffer only (BSS+) was confirmed first by manual visualization of the subretinal bleb followed by photography using a fundus camera (Micron III).

[0268] Optokinetic tracking threshold: Optokinetic tracking threshold was measured and recorded in a blinded manner. OKT data were analyzed using repeated measures ANOVA or one-way ANOVA with Fisher's LSD post-hoc analysis.

[0269] Electroretinagrams (ERGs): Two types of ERGs were measured: an exploratory style focal ERG, in which a small dot of light is used to stimulate a localized area of ​​the retina, and a standard style full-field ERG, in which the entire visual field is stimulated.

[0270] Histology and Immunohistochemistry: Both eyes from each animal were collected, fixed, cryoprotected, embedded, and frozen. Frozen blocks were cryosectioned at 12 μm. Approximately 60 slides were obtained with 4 sections per slide.

[0271] Cresyl violet: Cresyl violet stained sections were examined for (1) injection sites and sutures, (2) evidence of photoreceptor rescue, (3) evidence of transplanted cells, and (4) inappropriate pathology. For each slide, the maximum thickness of the outer nuclear layer was also recorded to quantify rescue.

[0272] Immunofluorescence (IF): Slides of RPE cell-treated eyes selected for IF were selected from cresyl violet stained sections that contained cells in the subretinal space consistent with the size and morphology of transplanted human cells. In addition, preservation of the host ONL was used as a secondary criterion. All IF staining was performed as double staining. DAPI served as background nuclear stain. At least one slide from every animal treated with cells was used for each run.

[0273] Run #1 was performed using rabbit monoclonal anti-melanoma gp100 (PMEL17, clone EPR4864; human specific, Abcam catalog number ab137062) co-stained with mouse monoclonal anti-nuclear marker (HuNu, clone 3E1.3, Millipore, catalog number MAB4383) to detect human RPE and non-RPE cells.

[0274] Run #2 was performed using rabbit monoclonal anti-Ki67 (Ki67; clone EPR3610, human specific, Abcam, catalog no. ab92742) and an anti-nuclear marker to detect human proliferating cells.

[0275] Run #3 was performed using rabbit polyclonal anti-rat cone arrestin (Millipore catalog no. ab15282) to assess cone counts in sections (see section 6.8.3). Additionally, selected slides were stained with a combination of mouse monoclonal anti-rhodopsin (clone Rho1D4, Millipore, MAB5356) and PMEL17 to identify transplanted human cells containing host rhodopsin / outer segments as a measure of phagocytic activity.

[0276] Cone counts: Confocal z-stack images were taken from retinal sections obtained from all cell-implanted eyes and from age-matched saline-injected controls. Sections from cell-injected eyes were selected in areas of photoreceptor rescue previously assessed using cresyl violet-stained sections. Cones were counted in a blinded fashion by three observers. Triplicate counts were then averaged and counts were compared between treatment groups and ages.

[0277] Rhodopsin uptake: A potential rescue mechanism used by transplanted cells is that of ingesting photoreceptor outer segments and sloughed debris. Removal of the debris compartment reduces toxic stress on the photoreceptors and thus helps sustain their survival. Here, we selected specific animals to evaluate rhodopsin uptake by RPE cells based on cell viability and photoreceptor protection index. We performed this evaluation using immunofluorescence.

[0278] result Fundus imaging: Fundus images collected at necropsy of cell-treated eyes revealed hyperpigmented and hypopigmented areas of the retina corresponding to where subretinal blebs formed during surgery; where cells were deposited in the subretinal space (FIG. 19A-C). These patchy areas were not evident in either BSS+-injected or uninjected eyes.

[0279] Optokinetic tracking threshold: OKT threshold was rescued in all cell treatment groups at all ages (Figure 20). Cell treatment groups outperformed unoperated or saline injected eyes at all ages. There was a significant dose-dependent effect between the low dose (25K) and the two higher doses (100K (p<0.0001) and 200K (p<0.0001)), especially at older ages, but no clear benefit on OKT was observed from the high dose (200K) past the mid dose (100K) (p=0.5646). OKT threshold was rescued in all cell treatment groups, whereas absolute visual acuity slowly declined over time. OKT thresholds in untreated and saline injected animals continued to decline over the course of the study. BSS+ injected eyes were not different from naive untreated group (p=0.6068) and untreated fellow eyes.

[0280] Local ERG: Local ERG was measured in all experimental rats (n=252) at approximately P60. As shown in Figure 21A, individual animals treated with RPE cells performed well and significantly outperformed controls.

[0281] Full-field ERGs: Full-field ERGs were measured from 125 RCS rats at P60 and 63 RCS rats at P100. As shown in Figure 21B, individual animals treated with RPE cells performed well and significantly outperformed controls.

[0282] Cresyl violet staining: An exemplary composite photograph of a cresyl violet stained section is shown in Figure 22 A. Representative images from BSS+ injected eyes and cell treated eyes (images from multiple groups) are shown in Figure 22B.

[0283] Outer nuclear layer thickness (ONL) was measured as the primary indicator of photoreceptor rescue. Data were recorded as the maximum number of photoreceptor nuclei present in each dose group across ages (Figure 23). ONL thickness in cell-treated groups at P60, P100, and P150 was significantly thicker than BSS+ treated eyes (all p<0.0001). In terms of the percentage of animals with evidence of photoreceptor rescue, 76-92% of animals at P60, 80-90% at P100, 72-86% at P150, and 0-18% at P200 had evidence of photoreceptors.

[0284] Immunofluorescence: Transplanted RPE cells were identified as positive by immunofluorescence in animals at each life age (Figure 24). However, with increasing age, the number of animals with identified cells decreased. Repeated staining of additional slides of animals that initially showed no transplanted cells identified positive cells in additional animals, but not in all cases.

[0285] Although IF analysis did not find transplanted cells in all animals, measurements of ONL thickness indicated that 70-90% of animals treated with cells had significant photoreceptor rescue confirmed by OKT rescue. This suggested that most treated eyes had transplanted cells at some point. The proliferation marker Ki67 was used to identify proliferating human cells. No Ki67-positive human cells were observed (Figure 24).

[0286] Cone counts: The number of cones in animals receiving cell transplants was significantly higher than in control eyes (Figure 25; p=<0.0001 for each comparison). In general, there was no difference in cone numbers across low, medium, and high dose cells. Representative images from each age are shown in Figure 24.

[0287] Rhodopsin uptake: In each case tested (n=6), fluorescently labeled rhodopsin was observed in the transplanted RPE cells (Figure 26A-J), confirming that the transplanted cells ingested outer segment debris after transplantation.

[0288] conclusion When transplanted into the subretinal space of RCS rats, RPE cells rescued the visual acuity of RCS rats above that of controls at all ages tested. ERG responses were preserved when the graft was in a retinal area large enough or accessible for evaluation. Rod and cone photoreceptors were rescued in the transplanted area by 180 days after transplantation. Collectively, this data demonstrates that OpRegen® maintains the functional and structural integrity of the host retina over the long term. Thus, OpRegen® has great potential for the treatment of human RPE cell disorders such as RP and AMD.

[0289] Example 6 Stability of RPE cells short term stability Formulated RPE cells in BSS plus (produced as described in Example 4) were prepared in a final volume of 600-1000 μl per vial. Short-term stability was tested at 0, 4, 8, and 24 hours. Cells were found to be stable at all time points.

[0290] RPE cell viability and cell concentration were stable at 8 hours of incubation for all dose formulations; percent mean viability (±SD) for the following concentrations: Low concentration (70 x 10 per 100 μl of BSS plus 3 The percentage of patients with pulmonary circulation disorders (P<0.05) decreased from 93% ± 5 at time 0 to 91% ± 1 at time 8, which was not a significant decrease. High concentration (70 x 10 per 100 μl of BSS plus 3 (individuals) changed from 92% ± 3 at time 0 to 91% ± 2 at time 8, which was not a significant decrease.

[0291] The medium concentration tested (250 × 10 per 100 μl of BSS plus 3 There were no significant changes across time points for the number of

[0292] The overall range for all time points and prescribed doses was 88% to 97% from time point 0 to time point 8. A 2% reduction was found when averaging all results for time point 0 (93% ± 3) and time point 8 (91% ± 1).

[0293] No significant changes in cell concentration were observed at any time point or dose prescribed. Cell concentration remained unchanged in all three studies except for a slight decrease (2%) seen in one batch at the higher dose.

[0294] The appearance of the various dosage formulations did not change across the time points tested. The cell suspensions were free of foreign particles and undissociated aggregates.

[0295] At all time points tested, the identity and purity of each formulated RPE cell dose was stable and within batch release standards up to 24 hours. At 8 hours (for all formulated RPE cell doses), the levels of MITF-positive cells and bestrophin-positive cells were 86-97% and 90-94%, respectively, and CRALBP-positive cells were 86-97% and 90-94%, respectively. + PMEL17 + The levels of double positive cells ranged from 98.35% to 99.64%.

[0296] The formulated RPE cell doses maintained efficacy at all time points tested (4 h, 8 h, 24 h) and both secreted high levels of PEDF and formed polarized RPE monolayers with polarized secretion of PEDF primarily apically and VEGF basolaterally. Results at the 8-h time point tested: TEER ranged from 376 to 724 ohms, apical to basal PEDF ratios ranged from 2.77 to 5.70, and basal to apical VEGF ratios ranged from 2.04 to 3.88.

[0297] Sterility was maintained at all incubation time points for all cell dose formulations.

[0298] These results support OpRegen® cell stability for at least 8 hours in the final formulation for all clinical doses when kept at 2-8° C. Based on the partial data collected (identity, sterility, and mid-dose efficacy), a safety margin exists for up to 24 hours.

[0299] The results of the short term stability assay are summarized in Table 7 below.

[0300] (Table 7) TIFF0007608192000008.tif96151

[0301] Long-term stability: Three batches of RPE cells were frozen in vapor phase liquid nitrogen. Testing of long-term stability during cryopreservation began after the day of freezing. Results obtained are 3 years after freezing. The following parameters were evaluated: viability, cell number, RPE identity (% bestrophin 1 positive cells and % MITF positive cells), RPE purity (FACS% CRALBP + PMEL17 + RPE cells), potency (polarization and PEDF secretion), karyotyping, and sterility are tested. At each time point, the required number of vials are thawed and the cells are prepared for the assay as described herein.

[0302] The results of the long term stability assay are summarized in Table 8 below.

[0303] (Table 8) TIFF0007608192000009.tif90151

[0304] result Viability, total cell number / vial, and RPE identity were maintained over the three-year period, and as shown, the data demonstrated potency and purity at levels similar to those collected prior to storage.

[0305] A normal karyotype was observed after 4 years of cryopreservation, indicating that long-term storage in vapor phase has not thus far had a detrimental effect on RPE genomic stability.

[0306] Sample sterility was demonstrated by testing all clinical batches for the absence of bacterial / fungal growth at 3 months. Another batch tested negative after 4 years of frozen storage. Based on these uniformly acceptable stability results thus far, spanning 3 years of stability testing, it can be concluded that the RPE cell product is stable for at least 3 years when stored in the vapor phase of liquid nitrogen at temperatures below -180°C.

[0307] Example 7 Safety and Biodistribution The objective of this study was to evaluate the survival, biodistribution, and safety of RPE cells (generated as described in Example 4) following subretinal administration in male and female NOD-SCID mice over a 6 month study period.

[0308] NOD-SCID mice (NOD.CB17-Prkdcscid), 5–6 weeks old at the time of injection, were injected with either BSS Plus (vehicle control) or two doses of RPE cells suspended in 1 μL of BSS Plus: 50 × 10 3 cells or 100 x 10 3 cells (maximum feasible dose). RPE was administered subretinal via the transvitreal route (proposed clinical route of administration) using a 33G Hamilton needle. A single dose of 50 × 10 3 cells or 100 x 10 3Cells were injected into one eye, whereas the fellow eye served as an internal control. Each dosing run contained mice (male and female) from each group. Mice entered into the study after the pre-test were randomly assigned to the various test groups. Two randomizations were performed. A weight-based measured randomization procedure was used for placement into treatment groups prior to administration of vehicle / test article. After administration, animals suitable for use in the study were transferred to the target study using sequential randomization for placement into the final treatment group. Mice with ocular abnormalities, abnormal clinical observations, or body weights less than 16 grams in the pre-test and mice in which the subretinal RPE injection was unsuccessful were excluded from the study.

[0309] Study Measurements: Evaluation of RPE safety in this study was based on animal mortality, clinical observations, body weight, ophthalmic examination, clinical pathology (hematology and blood chemistry), gross evaluation with gross pathology, organ weights (absolute and relative to body and brain weight), histopathological evaluation of eyes and various organs. Evaluation of RPE viability and biodistribution was performed by histopathological and immunofluorescence staining evaluation of eyes and various organs as well as qPCR analysis. The following measurements were performed: · Clinical observation; ·body weight; Ophthalmological examination (including gross and biomicroscopic examination); · Surgical microscopic examination of the quality of the subretinal injection using a LEICA M80 Stereo microscope (fundus examination); Complete blood count and blood chemistry; · Autopsy and gross pathology; · Organ weights (absolute and relative to body and brain weight); Collection, fixation, and paraffin blocking of the contralateral treated and untreated eyes, including the optic nerve; · Blinded hematoxylin-eosin histopathology of eyes and tissues (sternum with bone marrow, brain, heart, kidney, liver, lung, mandibular lymph nodes, spinal cord, spleen, thymus, masses, and gross lesions); Blinded semi-quantification of pigmented cells in hematoxylin-eosin stained slides; · Blinded immunostaining of selected slides adjacent to representative hematoxylin-eosin slides demonstrating pigment cell grafts in the eye for human marker (human nuclei) + RPE marker (human PMEL17) and assessment of human RPE cells and non-RPE cells, human marker (human nuclei) + proliferation marker (human Ki67) and assessment of human proliferating cells and non-human proliferating cells, RPE marker (RPE65) + proliferation marker (human Ki67) and assessment of RPE human proliferating cells and non-RPE human proliferating cells; · Blinded immunostaining of selected slides adjacent to representative hematoxylin-eosin slides demonstrating teratomas, tumors, abnormal cells, and lesions for human markers (human nuclei) to exclude human origin; Collection and extraction of genomic DNA from blood, bone marrow (collected from the thigh), brain, left and right eyes with optic nerve, heart, left and right kidneys, liver, lung, mandibular lymph nodes, ovaries, skeletal biceps femoris, spinal cord, spleen, testis, and thymus, and qPCR analysis of human β-globin; Hematoxylin and eosin histopathology for tissues (other than those mentioned above) found to be positive for human β-globin in animals from the same group and time point.

[0310] result There were no RPE-related toxicological findings during in-life examination, including detailed clinical observations, body weights, ophthalmologic examinations, and clinical pathology consisting of hematology and serum clinical chemistry. Detailed clinical observations and ophthalmologic examinations found that mice treated with RPE pigment cells at both dose levels exhibited a dark eye discoloration in the left eye with an albino background. Ophthalmologic examination of surviving animals showed that this consisted of mid-vitreal dark pigmented foci. The pigmented foci were randomly distributed along a line extending from the temporal posterior lens capsule to the nasal retinal surface. These foci were interpreted as RPE cells leaking from the injection cannula when it was removed from the eye after injection, as evidenced by vitreous reflux seen during injection, or RPE cells leaking into the vitreous humor after subretinal implantation.

[0311] All ocular lesions observed in this study were believed to be secondary to anesthesia, surgical injection procedures, or incidentally age-related changes. Multiple pigmented foci were found in the vitreous humor, suggesting that RPE cells may be able to survive within the vitreous. Pigmented cells were microscopically present in the vitreous in some of the RPE-treated animals.

[0312] In terms of biodistribution, as assessed by qPCR using a set of human β-globin gene probes / primers at intervals of 2 weeks, 2 months, and 6 months, 3Left eyes treated with OpRegen® cells were RPE DNA positive in 8 / 12, 11 / 12, and 16 / 16 animals with group mean levels of 38, 47, and 249 copies per μg of total ocular DNA, respectively, indicating a trend toward increasing over time. There were no significant differences between males and females. In these animals, RPE DNA was not detected in the untreated right eye and all non-ocular tissues, including blood, femoral bone marrow, brain, heart, kidney, liver, lung, mandibular lymph node, ovary, skeletal biceps femoris, spinal cord, spleen, testis, and thymus, except for spinal cord (27 copies / μg DNA) from one 2-week male animal and skeletal muscle (16 copies / μg DNA) and spinal cord (below the level of eligibility) from one 2-week female animal (presumably due to inadvertent contamination with exogenous human DNA during DNA extraction from these tissues).

[0313] Macroscopic changes related to the RPE were limited to black discoloration or black foci in the left eyes of some animals at the 2- and 6-month intervals, which were consistent with clinical observations and / or ophthalmologic examinations during life. These changes correlated with pigment cells and were not considered adverse as determined by microscopic examination of surviving animals in the high-dose group and animals euthanized near death and found dead in both dose groups. Pigment cells were present in the treated left eyes in nearly all of the surviving mice examined at each time point in the high-dose group (in the subretinal space of 11 / 12, 12 / 12, and 16 / 16 at the 2-week, 2-month, and 6-month intervals) and in animals euthanized near death or found dead in the low- and high-dose groups. The most common locations of pigment cells were the subretinal space and vitreous, as confirmed by immunostaining for human cell- and RPE-specific biomarkers. In the subretinal space, pigment cells tended to be confined to the injection site at early time points, whereas at later time points, they were present at locations distant from the injection site, suggesting that the cells had spread locally. In males, the mean total number of pigment cells per eye at 6 months was slightly increased compared with that at 2 weeks or 2 months. This increase in the number of human-derived pigment cells was supported by qPCR analysis.

[0314] Long-term engraftment of RPE cells is shown in Figure 27A. Nine months after transplantation, pigment cells in the NOD-SCID subretinal space stained positive for human nuclei and PMEL17.

[0315] Figure 27B is a photograph showing cells confluent at the site of the bleb after injection, and Figure 27C is a photograph showing subsequent cell spreading into a monolayer after injection.

[0316] RPE was not associated with any organ weight changes. There were no gross or microscopic changes in the untreated right eye and non-ocular organs examined in this study, including the brain, heart, kidney, liver, lung, mandibular lymph node, spinal cord, spleen, and thymus. In animals examined in the high-dose group, anti-human nuclear biomarker antibody staining (human nuclei) was detected in 64% of the left eyes examined at 2 weeks, 36% at 2 months, and 73% at 6 months.

[0317] The highest levels of human nuclei were found in the pigment cell population in the subretinal space, followed by the vitreous. Anti-human RPE-specific biomarker PMEL17 staining was observed in most animals tested, whereas another RPE-specific biomarker, RPE65, was found at various levels at different time points. These RPE-specific biomarkers were detected mainly in the subretinal space and to a lesser extent in the vitreous. Ki67, a human cell proliferation biomarker, was detected in only a few cells in a minority of animals, mainly in pigment cells in the vitreous and to a lesser extent in the subretinal space. The incidence of Ki67 positivity decreased over time, to only one animal at 6 months. Ki67-positive cells were not associated with any abnormal morphology.

[0318] Several microscopic changes were noted at the injection sites across all time points and study groups and were believed to be related to the surgical injection procedure. Some of these changes were slightly more common in animals examined in the high-dose group at 6 months. For example, retinal detachment was noted in one animal, and the incidence or severity of retinal degeneration / atrophy or fibroplasia was slightly increased compared to the vehicle control group.

[0319] There were no RPE-dependent effects on animal mortality or survival.

[0320] conclusion No local or systemic toxicological, lethal, or tumorigenic effects were observed in the NOD / SCID animal model after a single injection of RPE at dose levels up to 100,000 cells / μl / eye over the 6-month study period. Biodistribution of RPE cells was restricted to the treated left eye, with subretinal cells spreading locally from the subretinal injection site as a function of time. At 2-week, 2-month, and 6-month intervals, RPE cells were primarily present in the subretinal space in most of the animals examined in the high-dose group, followed by the vitreous, with variable positivity for immunostaining with antibodies against human nuclear-specific and / or human RPE-specific biomarkers. Persistence of RPE cells in the eye was estimated for at least 6 months, with very limited cell proliferation. The limited proliferation occurred primarily in the vitreous and had no adverse effects. There was evidence of an increase in RPE cell numbers over time in treated eyes, but this was accompanied by a reduced proliferation occurrence in the subretinal population examined. The RPE-specific markers RPE65 and PMEL17 were expressed primarily in RPE cells within the subretinal space, as opposed to within the vitreous. The majority of Ki67-positive cell occurrence was found within the vitreous. The latter suggests that the increase in RPE cells over time was restricted to the vitreous space and that expression of specific RPE65 and PMEL17 RPE markers may be regulated by the microenvironment. In conclusion, based on the data presented above, there were no significant safety issues associated with the injection of RPE cells described herein when compared to the vehicle control group.

[0321] Example 8 Pax-6 expression in RPE cells Objective: To develop a FACS-based method for assessing PAX-6 levels in human retinal pigment epithelial (RPE) cells.

[0322] material and method Frozen RPE cells (prepared as described in Example 4) were thawed, spun down, resuspended in 1 ml of PBS minus, filtered through a 35 μM cell strainer, and counted using an NC-200 cell counter. The cell concentration was approximately 1×10 per ml in PBS minus.6 The cells were adjusted to 100x the volume of cells. 1μl / ml of FVS450 was added per ml of cell suspension, followed by vortexing and incubation at 37℃ for 6 min. FVS450 was quenched with 0.1%BSA(-Ig)-PBS minus and resuspended in 0.1%BSA(-Ig)-Fc-block (RT for 5 min) to block all Fc epitopes on the cells. Cells were then fixed and stained with anti-Pax-6 antibody (AF647 Cat. No. 562249).

[0323] result As can be seen from Figure 29, cells in P0 and P2 are PAX6 positive (81.5%-82.5% for P0 and 91.3%-96.1% for P2). P2 was passaged at the end of the production run, while P0 was two expansion steps prior. As shown in Figures 29 and 30, the data showed to be consistent across batches. Furthermore, we showed by FACS analysis that RPE cells were double stained for PAX-6 and CRALBP (Figure 31).

[0324] Example 9 Identification of proteins secreted by RPE cells Objective: To identify a signature of proteins (known and novel) secreted by OpRegen® (RPE cells) that can be used as a batch release efficacy assay as well as a process control assay.

[0325] Supernatants were collected from RPE cells (generated as described in Example 3) cultured under various culture conditions as shown below. Supernatants were then screened using G6 and G7 RayBiotech arrays following the manufacturer's instructions after screening the relevant arrays and supernatants overnight. 1. Thawed RPE preparation cells (0.5 × 10 at passage 3) cultured for 4 and 14 days on 12-well plates 6 cells / well) (referred to herein as OpRegen®). 2. Thawed RPE preparation cells cultured for 14 days on 12-well plates and then cultured for 3 weeks on Transwell (according to AM-RPE-15) and exhibiting a TEER of over 500Ω. Supernatants were collected from the apical and basolateral chambers. 3. Cells generated according to the protocol described in Example 3, before (QC3) and after (QC4) activin A treatment. 4. Nutristem medium without added TGFβ and FGF (Nut-).

[0326] Supernatants were also collected from the following cell cultures and tested by ELISA. 1. Thawed OpRegen® formulated cells cultured for 14 days on 12-well plates and then for 3 weeks on Transwell (according to AM-RPE-15), exhibiting TEER of 355Ω and 505Ω, respectively. Supernatants were taken from day 14 (passage 3) and from the apical and basolateral chambers. 2. Thawed RPE7 cells (passage 3, 0.5 × 10 cells) cultured for 14 days on a 12-well plate. 6 cells / well). 3. Mock test VI cells at the end of passage 1 of the production run, isolated enzymatically or mechanically (as described in Example 3) and then grown on laminin 521. These cells were tested for efficacy according to AM-RPE-15, with supernatants collected from cells at day 14 (passage 2) on 12-well plates and from cells harvested from the apical and basolateral chambers after 3 weeks on Transwell. 4. Fetal HuRPE cells (0.5 × 10) at passage 3 on days 4 and 14 6 cells / well).

[0327] Validation of the ELISA test was performed according to the manufacturer's instructions associated with each ELISA kit. Incubation with supernatant in each protocol was overnight.

[0328] Study design: Supernatants were collected from cells grown under various culture conditions and kept at -80° C. After protein array analysis, validation of hits was measured by ELISA.

[0329] result The G7 array results are shown in Table 9 herein below.

[0330] (Table 9) TIFF0007608192000010.tif212147TIFF0007608192000011.tif163153

[0331] The G6 array results are shown in Table 10 herein below.

[0332] (Table 10) TIFF0007608192000012.tif51145TIFF0007608192000013.tif235145TIFF0007608192000014.tif91145

[0333] RPE secreted proteins can be divided into three functional groups: (1) angiogenic proteins, e.g., VEGF and angiogenin, (2) extracellular matrix regulators, e.g., TIMP-1 and TIMP-2, and (3) immunomodulatory proteins, e.g., IL-6, MIF, sgp130, sTNF-R1, sTRAIL-R3, MCP-1, and osteoprotegerin. The receptor tyrosine kinase Ax1 was also found to be secreted by RPE cells. Six proteins (angiogenin, TIMP-2, MIF, sgp130, sTNF-R1, and sTRAIL-R3) that showed high levels of secretion and / or a polarized secretion (apical / basolateral) pattern were selected for validation by ELISA. Array data also demonstrated VEGF secretion as seen in the polarized assay.

[0334] Angiogenin: Protein array data demonstrated an increase in angiogenin secretion along the production run (Tables 9 and 10). These results were confirmed by ELISA, which demonstrated that differentiated cells treated with nicotinamide prior to the addition of activin A secreted 0.52 ng / mL angiogenin, whereas after 2 weeks of treatment with nicotinamide and activin A, the secretion level of angiogenin increased to 0.91 ng / mL (Figure 32A). RPE cells (0.5x10 cells) cultured in 12-well plates for 2 weeks after thawing were 6 cells / well; passage 3) secreted angiogenin (Figure 32B). Polarized RPE cells (week 3 on Transwell; TEER>350Ω, PEDF apical / basal and VEGF basal / apical ratios>1) secreted angiogenin in a polarized manner to the basolateral side with little or no apical secretion (basolateral angiogenin levels ranged from 0.1-0.25ng / mL and apical angiogenin levels ranged from 0.05-0.12ng / mL; Figure 32B). RPE7 cells generated according to Idelson et al., 2009 were unable to produce barrier function in the Transwell system (TEER <100Ω) but were able to secrete VEGF and PEDF. The ability of RPE7 cells to secrete angiogenin was tested when plated in 12-well plates for 14 days. After 14 days in culture, RPE7 secreted angiogenin at levels within the range of RPE cells generated as described herein (Figure 32C).

[0335] Secretion of TIMP-1 and TIMP-2: Protein array screening demonstrated the secretion of TIMP-1 and TIMP-2 from polarized and non-polarized RPE cells (Figure 33A-E). Interestingly, the array data showed a polarized secretion of TIMP-2 apically and TIMP-1 basolaterally (Figure 33A). ELISA data confirmed that TIMP-2 was primarily secreted apically by all RPE batches tested to date (Figure 33C-D, apical range of 69.9-113.3 ng / mL and basolateral range of 11.9-43.7 ng / mL). TIMP-2 was also secreted by non-polarized OpRegen® cells at levels similar to those secreted by normal human fetal RPE cells (HuRPE, ScienCell) (Figure 33C-E). RPE7 cells also secreted TIMP-2 at levels similar to OpRegen® cells (Figures 33C-E).Interestingly, very low levels of TIMP-2 were detected at the QC3 and QC4 checkpoints along the production process (Figure 33B).

[0336] sgp130 secretion by OpRegen® cells: Protein array data also demonstrated increased sgp130 secretion along the OpRegen® production run, as seen at IPC / QC checkpoints 3 and 4 (Tables 9 and 10). ELISA data confirmed higher levels of sgp130 secretion after 2 weeks of treatment with activin A (IPC / QC4; 1.64 ng / mL) compared to levels secreted by cells after treatment with nicotinamide and prior to the addition of activin A (IPC / QC3; 0.68 ng / mL) (FIG. 34A). OpRegen® cells (0.5×10 6 cells / well; passage 3) secreted sgp130 (Figures 34B-C). RPE7 cells cultured under similar conditions secreted levels of sgp130 within the range of OpRegen® cells (1.0 ng / mL on day 14; Figure 34D). Fetal HuRPE cells secreted low levels of sgp130 on days 4 and 14.

[0337] Polarized OpRegen® cells secreted sgp130 in a polarized manner to the apical side and little or no secretion to the basal side (apical sgp130 secretion levels ranged from 0.93 to 2.06 ng / mL, and basal sgp130 levels ranged from 0 to 0.2 ng / mL; Figures 34B-C).

[0338] Released sTNF-R1: Very low levels of released sTNF-R1 were detected in the supernatants of differentiated cells by ELISA before (IPC / QC3 0.01 ng / mL) and after (IPC / QC4 0.02 ng / mL) 2-week treatment with nicotinamide and activin A (Figure 35A). OpRegen® cells (0.5×10 6 HuRPE cells cultured under similar conditions had similar levels of sTNF-R1 in their culture supernatants, whereas RPE7 cells showed relatively low levels of sTNF-R1 (Figure 35D).

[0339] Polarized OpRegen® cells secreted high levels of released sTNF-R1 apically (apical sTNF-R1 levels ranged from 0.22 to 1.83 ng / mL and basolateral sTNF-R1 levels ranged from 0.01 to 0.11 ng / mL; Figures 35C-D).

[0340] sTRAIL-R3: Protein array data detected sTRAIL-R3 in the supernatants of OpRegen® cells (Tables 9 and 10). ELISA confirmed the presence of sTRAIL-R3 along the OpRegen® production run (493 pg / mL in QC3 and 238 pg / mL in QC4). There was no sTRAIL-R3 in fetal HuRPE cultures and very low levels of sTRAIL-R3 (4 pg / mL) in RPE7 cultures.

[0341] Detection of MIF: Protein array data detected MIF in the supernatants of OpRegen® cells (Tables 9 and 10). ELISA confirmed the presence of MIF along the OpRegen® production process (100.3 ng / mL in QC3 and 44.7 ng / mL in QC4). Polarized OpRegen® cells demonstrated high levels of MIF on the apical side (apical MIF levels ranged from 26.6-138.3 ng / mL and basolateral MIF levels ranged from 1.9-30.5 ng / mL).

[0342] Example 10 OpRegen® vs. RPE1 and RPE7 Objective: To compare OpRegen® (RPE cells) with RPE cells generated according to the protocol of Idelson et al., 2009.

[0343] material and method OpRegen® (RPE cells) were generated as described in Example 3.

[0344] RPE cells were generated according to the protocol of Idelson et al., 2009, and designated RPE1 and RPE7.

[0345] Using the Transwell system (as shown in Figure 28), we were able to develop polarized RPE monolayers with stable barrier properties and polarized secretion of PEDF and VEGF. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of the RPE monolayer, and enzyme-linked immunosorbent assays (ELISAs) were used to assess polarized secretion of PEDF and VEGF. Cells were thawed and cultured in the presence of nicotinamide for 14 days. PEDF secretion was tested on days 7 and 14. Cells were then transferred to Transwells (Costar 3460, 0.4 μm) for an additional 4 weeks. During this time, TEER was measured and media was collected weekly from the upper and lower Transwell chambers (to assess cytokine secretion) for up to 4 weeks. If cells are polarized, TEER should be greater than 100 Ω and the ratio of apical to basolateral PEDF secretion and the ratio of basolateral to apical VEGF secretion should be greater than 1.

[0346] All OpRegen® batches tested demonstrated the ability to produce barrier function (TEER ranged from 368-688Ω) and secrete PEDF and VEGF in a polarized manner (apical PEDF / basolateral PEDF ratios ranged from 3.47-8.75 and basolateral VEGF / apical VEGF ratios ranged from 1.39-2.74) (see Table 11).

[0347] (Table 11) TIFF0007608192000015.tif90154ND: TEER was less than 100 Ω and was not measured. Large holes were observed in the culture.

[0348] RPE1 and RP7 produced under GMP conditions according to Idelson et al. (2009) failed to produce barrier function in three independent studies (TEER<100Ω). Cells seeded on Transwells failed to produce a homogenous closed polygonal monolayer and large holes were observed (Figure 36). Although these cells were unable to produce barrier function, RPE1 and RPE7 were able to secrete PEDF (see Table 11) and VEGF (not shown) at levels similar to OpRegen®. These CRALBPs + PMEL17 + Purity levels were 99.91% and 96.29%, respectively, similar to OpRegen® (Figure 37).

[0349] Based on these data, it can be concluded that RPE1 and RPE7 are defective in their ability to generate tight junctions.

[0350] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0351] All publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

Claims

1. A population of human retinal pigment epithelial (RPE) cells, at least 95% of the cells co-express a pre-melanosomal protein (PMEL17) and a cellular retinaldehyde binding protein (CRALBP), and the population of cells in a monolayer has a transepithelial electrical resistance of greater than 100 ohms; and Here, the ratio of apical to basal secretion of pigment epithelium-derived growth factor (PEDF) of a population of RPE cells in a monolayer is greater than 1, a population of human polygonal RPE cells.

2. 2. The population of cells of claim 1, wherein cells within the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor alpha ubiquitous membrane receptor 1 (sTNF-R1).

3. The population of cells of claim 2 , wherein the cells secrete the angiogenin, the TIMP2, the sgp130, or the sTNF-R1 in a polarized manner.

4. The population of cells of claim 3, wherein the ratio of apical secretion of sgp130 to basal secretion of sgp130 is greater than 1.

5. The population of cells of claim 3, wherein the ratio of basolateral secretion of angiogenin to apical secretion of angiogenin is greater than 1.

6. The population of cells of claim 3, wherein the ratio of apical to basal secretion of TIMP2 is greater than 1.

7. Oct4 in the population + TRA-1-60 + 2. The population of cells of claim 1, wherein the number of cells is below 1:250,000.

8. (i) at least 80% of the cells express Bestrophin 1, as determined by immunostaining; (ii) at least 80% of the cells express microphthalmia-associated transcription factor (MITF) as measured by immunostaining; (iii) more than 50% of the cells express paired box gene 6 (PAX-6) as measured by FACS; and / or (iv) the cells secrete greater than 750 ng of pigment epithelium-derived factor (PEDF) per ml per day; A population of cells according to any one of claims 1 to 7.

9. (a) culturing human embryonic stem cells in a medium containing nicotinamide and lacking activin A to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to generate cells that are further differentiated into the RPE lineage; and (c) culturing the cells further differentiating into the RPE lineage in a medium containing nicotinamide and lacking activin A.

9. The population of cells according to any one of claims 1 to 8, produced by

10. 10. The population of cells of claim 9, wherein steps (a)-(c) are performed under conditions where the atmospheric oxygen level is less than about 10%.

11. A pharmaceutical composition comprising a population of cells according to any one of claims 1 to 10 as an active substance and a pharma- ceutically acceptable carrier.

12. A pharmaceutical composition comprising the population of cells according to any one of claims 1 to 10 for treating retinal degeneration.

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