NZ733006A - Rpe cell populations and methods of generating same - Google Patents

Abstract

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 same are also disclosed. me are also disclosed.

Description

/051269 RPE CELL POPULATIONS AND METHODS OF GENERATING SAME FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to retinal pigment epithelium cells and, more particularly, but not exclusively, to assessment of such cells as a eutic. The present invention also relates to generation of l pigment lium cells from embryonic stem cells.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells, which lies between the neural retina and the choriocapillaris. The RPE cells play l roles in the maintenance and function of the retina and its photoreceptors. These e the ion of the blood—retinal barrier, absorption of stray light, supply of nutrients to the neural retina, regeneration of Visual pigment, and uptake and recycling of shed outer segments of eceptors.

Retinal tissue may degenerate for a number of reasons. Among them are: artery or vein occlusion, diabetic retinopathy and retinopathy of prematurity, which are usually hereditary. Diseases such as retinitis pigmentosa, retinoschisis, lattice degeneration, Best disease, and age related r degeneration (AMD) are characterized by progressive types of retinal degeneration.

RPE cells may potentially be used for cell replacement therapy of the degenerating RPE in retinal diseases mentioned above. It may be also used as a vehicle for the introduction of genes for the treatment of retinal degeneration diseases. These cells may also serve as an in vitro model of retinal degeneration diseases, as a tool for high throughput screening for a therapeutic effect of small molecules, and for the discovery and testing of new drugs for retinal degeneration diseases. RPE cells could also be used for basic ch of RPE development, maturation, characteristics, ties, metabolism, immunogenicity, function and interaction with other cell types.

Human fetal and adult RPE has been used as an alternative donor source for allogeneic transplantation. However, practical problems in obtaining sufficient tissue supply and the ethical concerns regarding the use of tissues from aborted fetuses limit widespread use of these donor s. Given these limitations in supply of adult and fetal RPE grafts, the potential of alternative donor sources have been studied. Human 2015/051269 pluripotent stem cells provide significant advantages as a source of RPE cells for transplantation. Their pluripotent developmental potential may enable their differentiation into authentic functional RPE cells, and given their potential for infinite self l, they may serve as an unlimited donor source of RPE cells. Indeed, it has been demonstrated that human embryonic stem cells (hESCs) and human d pluripotent stem cells (iPS) differentiate into RPE cells in vitro, attenuate retinal ration and preserve visual function after subretinal transplantation to the Royal College of ns (RCS) rat model of l degeneration that is caused by RPE dysfunction. Therefore, otent stem cells may be an unlimited source for the production of RPE cells.

Current protocols for the derivation of RPE cells from pluripotent stem cells yields mixed populations of pigmented and non—pigmented cells. However, pure populations of pigmented cells are d for the usage of RPE cells in basic research, drug discovery and cell therapy. ound art includes WO 2013/114360, WO 2008/129554 and WO 2013/184809.

SUMMARY OF THE INVENTION According to an 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 thereof co—express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), wherein the trans—epithelial electrical resistance of the population of cells is greater than 100 ohms. ing to an aspect of some embodiments of the present invention there is provided a population of human RPE cells, wherein at least 80 % of the cells thereof co— express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP) and wherein cells of the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgpl30) and soluble form of the ubiquitous ne receptor 1 for tumor necrosis factor-0t (sTNF— R1).

According to ments of the invention, the cells of the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor—0c (sTNF—Rl).

According to embodiments of the invention, the cells secrete the angiogenin, the TIMPZ, the sgpl30 or the sTNF—Rl in a polarized manner.

According to embodiments of the invention, the cells secrete each of the angiogenin, the TIMPZ, the sgpl30 and the sTNF—Rl in a zed manner.

According to embodiments of the invention, the ratio of apical secretion of sgpl30: basal secretion of sgp130 is greater than 1.

According to embodiments of the invention, the ratio of apical secretion of sTNF—Rl: basal ion of sTNF—Rl is greater than 1.

According to embodiments of the invention, the ratio of basal secretion of angiogenin: apical secretion of angiogenin is r than 1.

According to ments of the invention, the ratio of apical secretion of TIMP2: basal secretion of TIMP2 is r than 1.

According to embodiments of the invention, the number of Oct4‘LTRA—l—60+ cells in the population is below l:250,000.

According to ments of the invention, at least 80 % of the cells express Bestrophin l, as measured by staining.

According to embodiments of the invention, at least 80 % of the cells express Microphthalmia—associated ription factor {MITF), as measured by staining.

According to embodiments of the invention, more than 50 % of the cells express paired box gene 6 (FAX-6) as ed by FACS.

According to embodiments of the invention, the cells secrete greater than 750 ng of Pigment epithelium—derived factor (PEDF) per ml per day.

According to embodiments of the invention, the cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner.

According to embodiments of the invention, the ratio of apical secretion of PEDF: basal secretion of PEDF is greater than 1. ing to embodiments of the invention, the ratio remains r than 1 following incubation for 8 hours at 2—8 0 C.

According to embodiments of the invention, the trans-epithelial electrical resistance of the population of cells is greater than 100 ohms.

According to embodiments of the invention, the trans—epithelial electrical resistance of the cells remains greater than 100 ohms following incubation for 8 hours at 2—8 0 C.

According to embodiments of the invention, the ratio of basal secretion of VEGF: apical secretion of VEGF is greater than 1.

According to embodiments of the invention, the ratio remains greater than 1 following tion for 8 hours at 2—8 0 C.

According to embodiments of the invention, the cell population is capable of rescuing visual acuity in the RC8 rat following subretinal administration.

According to embodiments of the invention, the cell population is capable of rescuing photoreceptors for at least 180 days ubretinal administration in the RC8 rat.

According to embodiments of the invention, the cell population is ted by ex—vivo differentiation of human embryonic stem cells.

According to embodiments of the invention, the cell population is generated by: (a) culturing human embryonic stem cells in a medium comprising nicotinamide so as to generate differentiating cells, wherein the medium is devoid of activin A; (b) culturing the differentiating cells in a medium comprising namide and activin A to generate cells which are r differentiated towards the RPE lineage; (c) culturing the cells which are r differentiated towards the RPE e in a medium comprising nicotinamide, wherein the medium is devoid of activin According to embodiments of the invention, the embryonic stem cells are propagated in a medium comprising bFGF and TGFB.

According to embodiments of the ion, the embryonic stem cells are cultured on human cord fibroblasts.

According to embodiments of the invention, the steps (a)-(c) are effected under conditions wherein the atmospheric oxygen level is less than about 10 %.

According to embodiments of the invention, the method further ses ing the differentiated cells in a medium under conditions wherein the atmospheric oxygen level is greater than about 10 % in the presence of nicotinamide ing step (c).

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the cell population described , as the active agent and a pharmaceutically acceptable carrier.

According to an aspect of some ments of the present invention there is provided a use of the cell population described herein, for treating a retinal degeneration.

According to an aspect of some embodiments of the present invention there is provided a method of generating RPE cells comprising: (a) culturing pluripotent stem cells in a medium comprising a differentiating agent so as to generate entiating cells, wherein the medium is devoid of a member of the transforming growth factor B (TGF [3) superfamily; (b) ing the differentiating cells in a medium comprising the member of the transforming growth factor [3 (TGF [3) superfamily and the differentiating agent to te cells which are further differentiated towards the RPE lineage; (c) culturing the cells which are further differentiated towards the RPE lineage in a medium comprising a differentiating agent so as to generate RPE cells, wherein the medium is devoid of a member of the orming growth factor B (TGF [3) superfamily, wherein steps (a)-(c) are effected under conditions wherein the atmospheric oxygen level is less than about 10 %.

According to embodiments of the invention, step (a) is effected under non— adherent conditions.

According to embodiments of the ion, the non-adherent conditions comprise a non—adherent culture plate. ing to embodiments of the invention, the step (a) comprises: i) culturing the cultured population of human pluripotent stem cells in a medium comprising nicotinamide, in the absence of activin A; under non—adherent ions to generate a cluster of cells comprising differentiating cells; and subsequently; ii) culturing the differentiating cells of (i) in a medium sing nicotinamide, in the absence of activin A under adherent conditions.

According to embodiments of the invention, the method. r comprises dissociating the cluster of cells prior to step (ii) to generate clumps of cells or a single cell suspension of cells.

According to embodiments of the invention, the method further comprises culturing the differentiated cells in a medium under conditions wherein the heric oxygen level is greater than about 10 % in the presence of a differentiating agent following step (c).

According to embodiments of the invention, the member of the transforming growth factor B (TGF [3) superfamily is selected from the group consisting of TGFBl, TGFB3 and n A.

According to embodiments of the invention, the differentiating agent of step (a) and the differentiating agent of step (c) are identical.

According to embodiments of the invention, the differentiating agent of step (a) is nicotinamide (NA) or 3-- aminobenzamide.

According to embodiments of the invention, the method further comprises selecting polygonal cells following step (c).

According to ments of the invention, the method further comprises propagating the polygonal cells.

According to embodiments of the invention, the propagating is effected on an adherent e or an extracellular matrix.

According to embodiments of the invention, the pluripotent stem cells comprise embryonic stem cell 3 According to embodiments of the invention, the embryonic stem cells are propagated in, a medium eomprising bFGF and TGFB.

According to ments of the invention, the embryonic stem cells are cultured on human cord fibroblasts.

Unless otherwise defined, all technical and/or scientific terms used herein have the same g as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE L VIEWS OF THE GS Some embodiments of the invention are herein bed, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative sion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the ion may be practiced.

In the drawings: is a graph illustrating the linearity of the data. is FACS analysis of negative control hESC cells stained with anti CRALBP and anti PMEL 17. is FACS analysis of positive control of the nce RPE line OpRegen® 5C cells stained with anti CRALBP and anti PMEL 17. is FACS analysis of 2.5% Spiked OpRegen® SC in hESCs d with anti CRALBP and anti PMEL 17. is FACS analysis of 50% Spiked OpRegen® SC in hESCs stained with anti CRALBP and anti PMELI7. is FACS is of 75% Spiked OpRegen® SC in hESCs stained with anti CRALBP and anti PMELi7. is FACS is of 95% Spiked OpRegen® SC in hESCs stained with anti CRALBP and anti PMEL 17. is FACS analysis of hESCs stained with Isotype Controls. is FACS analysis of OpRegen® 5C cells stained with the Isotype Controls.

: Co—immunostaining with PMEL17 differentiate RPE cells (CRALBP+PMEL17+) from non RPE pigmented cells (PMEL17+ CRALBP—; such as cytes).

: Morphology results for Mock 4 and 5 at In Process Control (H’C) points 5, and 8—10.

: Manufacturing Process, Steps 1—3: Generation of Human Cord Fibroblast Feeder Working Cell Bank.

: Manufacturing Process, Steps 4—5: Expansion of hESCs.

: Manufacturing Process, Steps 6—13: Differentiation into RPE (OpRegen®) cells.

: Manufacturing Process, Steps 14—17: Expansion of pigmented cells.

: Detailed OpRegen® manufacturing process and in process control points (yellow stars, lPCs 1—11). (NUTSPlus, Nutristem medium containing bFGF and TGFB; nus, Nutristem medium w/o bFGF and TGFB; NIC, Nicotinamide; SB s, Spheroid bodies).

: Level of CRALBP+PMEL17+ RPE cells along OpRegen® Mock production runs 4 and 5. Density plots of IPC points 8 and 11 (*IPC point 8 was tested post eservation) and representative density plots of positive l OpRegen® 5C and ve control HAD—C102 hESCs (range of % CRALBP+PMEL17+ in negative control was 0.02—0.17%). Numbers within each plot indicate t CRALBP+PMEL17+ cells out of the live single cell gated population. Analysis was done using the FCS express 4 software. : fluorescence staining of Mock 5 IPC points 7, 10 and 11 with antibodies specific for the RPE s Bestrophin 1, MITF, 20—1 and CRALBP.

FIGs. 19A—C: Representative color fundus photograph of group 2 (BSS+; Figure 19A), group 5 contra lateral untreated eyes (OD; Figure 19B) and group 5 treated eyes (OS; Figure 19C) at P60. The hyper and hypo-pigmented areas in the high dose treated eyes (OS) are presumed to be indicative of transplanted cells.

: Optokinetic tracking acuity thresholds measured at P60, P100, P150, and P200. Cell treated groups (group 3—25,000, group 4—100,000 and group 5—200,000) outperformed all controls with the group 4 (100,000) and 5 (200,000) dose achieving the best rescue. Contralateral unoperated eyes were equivalent to group 1 (untreated) and group 2 (vehicle control/BSS+) (not shown).

FIGs. 21A-B: Graphs illustrating the Focal (Figure 21A) and Full field (Figure 21B) s for a representative rat.

FIGs. 22A—B: Figure 22A illustrates a photomontage of individual images of cresyl violet d sections of a representative cell treated eye. Between the arrows illustrates the location of photoreceptor tion and presumed location of the d cells. Figure 22B illustrates the comparison between BSS+ (Group 2) injected eyes and representative cell injected eyes (multiple dosage groups represented) at post—natal day 60, 100, 150 and 200. GCL: Ganglion Cell Layer; ONL: Outer Nuclear Layer; RPE: Retinal Pigmented Epithelium.

: Outer r layer thickness measured in number of nuclei. Each dot represents the count from each animal from every dose group for all ages.

: Immunofluorescent images of positive control tissue and representative experimental cell treated animals at P60, P100, P150, and P200 stained with anti—human nuclei marker (H.N.M, green), anti-pre—melanosomal marker (PMEL17, red), anti- human proliferation marker (Ki67, red), and anti—rat cone arrestin (red). Dapi (blue) is used for background staining to highlight nuclear layers. Human ma was used as positive control tissue for PMELl7, human tonsil for Ki67, and juvenile RCS rat retina for cone arrestin. Downward arrows te outer nuclear layer; upward arrows indicate vely stained human RPE cells en®), generated as described herein. is a graph illustrating cone quantification following inal transplantation of OpRegen® cells into the RC8 rat. Cell d eyes were significantly higher than control eyes at all ages.

FIGS. 26A—J: Immunofluorescent staining of OpRegen® cells in the subretinal space. Figure 26A represents an area of retina with a number of RPE cells (red, arrows) central and no debris zone (viewed using anti-rat rhodopsin antibody, green; arrow), but where the cells are not (peripheral), the debris zone reconstitutes. At higher magnification (Figure 26B), some rhodopsin stained outer segments rest along the grafted cells. In addition, the debris zone reconstitutes as distance from transplanted cells increases. Figures 26C—J are individual slices through the section showing rhodopsin positive tissue within the transplanted cells (arrows).

FIGS. 27A—C are photographs illustrating the tribution of the cells following subretinal injection into NOD—SCID. Figure 27A illustrates the ability of OpRegen® cells to engraft in the ID subretinal space 9 months post transplant.

Pigmented cells stain positive for Human Nuclei and . Figure 27B is a photograph illustrating the clustered cells at the place of the bleb following injection.

Figure 27C is a photograph illustrating the subsequent spreading of the cells into a yer following injection. is a pictorial illustration of a transwell assay that may be used to assay the potency of RPE cells. is the results of the FACS analysis illustrating PAX6 expression in RPE cells generated as described herein (P2—DP, drug product: Mock IV, Mock V, OpRegen® batch 2A; HuRPE: normal human RPE from ScienCell) and along production (P0). is a graph illustrating PAX6 expression in OpRegen® cells, as assayed by FACS (HES, human embryonic stem cells used as negative control). is the results of the FACS analysis rating double staining of PAX6 and .

FIGs. 32A—C are graphs illustrating ELISA assessment of Angiogenin secretion by OpRegen® cells. A. Increased secretion of angiogenin along Mock V production. B.

Secretion of angiogenin by three different batches of n® cells (Passage 3) and on a transwell for 3 weeks (Passage 4) during which apical and basal ion was assessed. C. ion of angiogenin by RPE 7 cells (Passage 3).

FIGs. 33A—E illustrate TIMP—1 and TIMP—2 Secretion by OpRegen® cells. A. ve TIMP—1 and TIMP—2 protein levels detected by protein array. B. ELISA TIMP— 2 levels in Mock V production QC points 3 and 4. C—D. ELISA TIMP—2 secretion levels by different batches of OpRegen® cells (Passage 3) and on a ell for 3 weeks during which apical and basal secretion was assessed (Passage 4). E. TIMP-2 levels secreted from RPE 7 and HuRPE control cells (Passage 3, Days 4 & 14).

FIGS. 34A—D illustrate sgpl30 Secretion by OpRegen® Cells as measured by ELISA. A. sgpl30 secretion levels in Mock V production QC points 3 and 4. B—C.

Levels of secreted sgpl30 by various batches of OpRegen® cells (Passage 3) and on a transwell for 3 weeks during which apical and basal secretion was assessed (Passage 4).

D. sgp130 levels secreted from RPE 7 and HuRPE control cells (Passage 3, Days 4 & 14).

FIGS. 35A-D rate sTNF-Rl protein levels in OpRegen® cell supernatant as measured by ELISA. A. sTNF—Rl levels in cell supernatant from Mock V tion QC points 3 and 4. B—C. Levels of sTNF—Rl in the supernatant of OpRegen® s (Passage 3) and on a transwell for 3 weeks during which apical and basal levels were assessed (Passage 4). D. sTNF—Rl levels in day 4 and day 14 RPE7 and control HuRPE cell cultures (Passage 3). illustrates the morphology of OpRegen® 5C (Reference Line), RPEl and RPE7 on Transwell. OpRegen® 5C, RPEl and RPE7 were imaged weekly (week 1— 4) following their seeding on transwell. OpRegen® 5C generated a homogeneous polygonal monolayer from week 1 while RPEl and RPE7 generated a different non— homogeneous logy one week post seeding and holes started to appear at week 2.

RPEl cells detached from the transwell after 3 weeks in culture. illustrates that RPEl and RPE7 cells co—express CRALBP and PMEL— 17. FACS Purity assay demonstrated that 99.91% and 96.29% of RPEl and RPE7 cells, respectively, are double positive for the RPE markers CRALBP and PMEL—l7, similar to the levels seen in OpRegen® Mock V cells (Positive Control). HAD—C 102 hESCs were used as the negative l.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE ION The present invention, in some ments thereof, relates to retinal t epithelium cells and, more particularly, but not exclusively, to assessment of such cells as a therapeutic. The present invention also relates to generation of retinal pigment epithelium cells from human embryonic stem cells.

Before ning at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The neural retina initiates vision and is supported by the underlying retinal pigment epithelium (RPE). Dysfunction, ration, and loss of RPE cells are ent features of Best disease, subtypes of retinitis pigmentosa (RP), and age— related macular degeneration (AMD), which is the leading cause of Visual disability in the western world. In these conditions, there is progressive visual loss that often leads to blindness.

The retina and adjacent RPE both arise from neural ectoderm. In lower s, RPE rates retina but in mammals, RPE—mediated regeneration is inhibited and renewal occurs to a very limited extent via stem cells located at the eral retinal Human embryonic stem cells (hESC) may serve as an unlimited donor source of RPE cells for transplantation. The potential of mouse, e, and human ESCS to differentiate into RPE—like cells, to attenuate retinal degeneration, and to preserve visual function after subretinal transplantation has been demonstrated.

Various protocols for the differentiation of human embryonic stem cells into RPE cells have been developed (see for example WC 2008/129554).

The present inventors have now discovered a unique and simple way of qualifying cell populations which have been successfully differentiated into RPE cells based on expression of particular polypeptides. Of the myriad of ial polypeptides expressed on these differentiated cells, the present inventors have found that a combination of two particular markers can be used to ntiate successful entiation.

The present inventors have also discovered that secretion of Pigment epithelium— derived factor (PEDF) may be used as a marker to substantiate early stages of the RPE differentiation process (see Table 4).

Whilst further reducing the present invention to practice, the present inventors identified additional proteins which are secreted by RPE cells which may be used, in some embodiments, as a signature to define the cells.

Thus, according to one aspect of the t invention there is provided a method of qualifying whether a cell population is a suitable therapeutic for treating an eye condition, comprising analyzing ression of premelanosome protein (PMEL l7) and at least one ptide selected from the group consisting of cellular retinaldehyde binding protein (CRALBP), lecithin retinol acyltransferase {LRAT) and sex determining region Y—box 9 (SOX 9) in the population of cells, wherein when the number of cells that coexpress the PMELl7 and the at least one polypeptide is above a predetermined level, the cell population is qualified as being a suitable therapeutic for treating a retinal disorder.

According to another aspect, there is provided a method of qualifying whether a cell tion is a suitable therapeutic for treating an eye condition, comprising analyzing co-expression of cellular retinaldehyde binding protein (CRALBP) and at least one polypeptide selected from the group consisting of premelanosome protein (PMEL17), lecithin retinol ansferase (LRAT) and sex determining region Y—box 9 (SOX 9) in the population of cells, wherein when the number of cells that ress the CRALBP and the at least one polypeptide is above a predetermined level, the cell population is qualified as being a suitable therapeutic for treating an eye condition.

As used herein, the phrase “suitable therapeutic” refers to the ility of the cell population for treating eye conditions. Cells which are therapeutic may exert their effect through any one of a multiple mechanisms. One exemplary ism is trophic supportive effect promoting the survival of degenerating photoreceptors or other cells within the retina. Therapeutic RPE cells may also exert their effect h a ration mechanism replenishing mal—functioning and/or degenerating host RPE cells. According to one embodiment, the RPE cells are mature and have the functional capability of phagocytosing outer shedded segments of photoreceptors which include rhodopsin. According to another embodiment, the RPE cells are not fully mature.

Eye conditions for which the cell populations serve as therapeutics e, but are not limited to retinal diseases or disorders lly associated with l dysfunction, retinal injury, and/or loss of retinal t epithelium. A non—limiting list of conditions which may be treated in accordance with the invention comprises retinitis pigmentosa, lebers congenital amaurosis, hereditary or acquired macular degeneration, age related macular degeneration (AMD), Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy as well as other dystrophies of the RPE, Stargardt disease, RPE and retinal damage due to damage caused by any one of photic, laser, inflammatory, ious, radiation, neo vascular or tic injury.

As mentioned, the method of this aspect of the invention is carried out by measuring the amount (e.g. percent cells) sing premelanosome protein (PMELl7; SwissProt No. P40967) and at least one polypeptide selected from the group consisting of cellular retinaldehyde g protein (CRALBP; SwissProt No. P1227l), lecithin retinol acyltransferase (LRAT; SwissProt No. ) and sex determining region Y- box 9 (SOX 9; P48436).

Alternatively, the method of this aspect is carried out by measuring CRALBP (CRALBP; SwissProt No. Pl227l) and at least one polypeptide selected from the group consisting of lecithin l acyltransferase (LRAT; SwissProt No. 095327), sex determining region Y—box 9 (SOX 9; ) and PMEL17 (SwissProt No. P40967).

Thus, for example, CRALBP and PMEL17 may be measured; PMEL17 and LRAT may be measured, or PMEL17 and SOX9 may be measured. Alternatively, CRALBP and LRAT may be measured, or CRALBP and SOX9 may be measured.

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

Methods for analyzing for expression of the above mentioned polypeptides typically involve the use of antibodies which specifically recognize the antigen.

Commercially available antibodies that recognize CRALBP include for example those manufactured by Abcam (e.g. ab15051 and abl89329, clone B2). Commercially available antibodies that recognize PMEL17 include for example those ctured by Abcam (e.g. abl37062 and abl89330, clone EPR4864). Commercially available antibodies that recognize LRAT include for example those manufactured by Millipore (e.g. MABN644). cially available dies that recognize SOX9 include for example those ctured by Abcam (e.g. ab185230). The analyzing may be d out using any method known in the art including flow try, Western Blot, immunocytochemistry, radioimmunoassay, PCR, etc.

For flow cytometry, the antibody may be attached to a fluorescent moiety and analyzed using a fluorescence-activated cell sorter (FACS). Alternatively, the use of secondary antibodies with fluorescent moieties is envisioned.

It will be appreciated that since the polypeptides which are analyzed are intracellular polypeptides, lly the cells are permeabilized so that the antibodies are capable of binding to their targets. Cells may be fixed first to ensure stability of soluble ns or antigens with a short half—life. This should retain the target protein in the original ar location. Antibodies may be prepared in permeabilization buffer to ensure the cells remain permeable. It will be appreciated that when gating on cell populations, the light r profiles of the cells on the flow cytometer will change considerably after permeabilization and fixation.

Methods of permeabilizing the cell membrane are known in the art and include for example: 1. Formaldehyde followed by ent: Fixation in formaldehyde (e.g. no more than 4.5 % for 10—15 min (this will ize proteins), followed by disruption of membrane by detergent such as Triton or NP—40 (0.1 to 1% in PBS), Tween 20 (0.1 to 1% in PBS), Saponin, nin and Leucoperm (e.g. 0.5% v/v in PBS); 2. dehyde (e.g. no more than 4.5 %) followed by methanol; 3. Methanol followed by detergent (e.g. 80 % methanol and then 0.1 % Tween ); 4. Acetone fixation and permeabilization.

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

A multitude of flow cytometers are commercially available ing for e.g.

Becton Dickinson FACScan, Navios Flow Cytometer (Beckman Coulter serial#AT15119 RHE9266 and FACScalibur (BD Biosciences, Mountain View, CA).

Antibodies that may be used for FACS analysis are taught in Schlossman S, l L, et al., [Leucocyte Typing V. New York: Oxford University Press; 1995] and are widely commercially available.

It will be appreciated that the expression level of the above mentioned polypeptides may be effected on the RNA level as well as the protein level. Exemplary methods for determining the expression of a polypeptide based on the RNA level include but are not limited to PCR, RT—PCR, Northern Blot etc.

In order to qualify that the cells are useful as a therapeutic, the amount of at least two of the polypeptides ressed in the cells should be increased above a statistically significant level as compared to non—RPE cells (e.g. non—differentiated embryonic stem cells).

According to a ular embodiment, in order to qualify that the cells are useful as a eutic, at least 80 % of the cells of the population should s detectable levels of PMEL17 and one of the above ned polypeptides (e.g.

CRALBP), more preferably at least 85 % of the cells of the population should s detectable levels of PMEL17 and one of the above mentioned polypeptides (e.g.

CRALBP), more preferably at least 90 % of the cells of the population should express detectable levels of PMEL17 and one of the above mentioned polypeptides (e.g.

CRALBP), more preferably at least 95 % of the cells of the population should express able levels of PMEL17 and one of the above mentioned polypeptides (e.g.

CRALBP), more preferably 100 % of the cells of the population should express detectable levels of PMEL17 and one of the above mentioned polypeptides (e.g.

CRALBP as assayed by a method known to those of skill in the art (e.g. FACS).

According to another embodiment, in order to qualify that the cells are useful as a therapeutic, the level of CRALBP and one of the above mentioned polypeptides (e.g.

PMEL17) coexpression (e.g. as ed by the mean fluorescent intensity) should be increased by at least two fold, more preferably at least 3 fold, more preferably at least 4 fold and even more preferably by at least 5 fold, at least 10 fold, at least 20 fold, at least fold, at least 40 fold, at least 50 as compared to non—differentiated ESCs.

According to a particular embodiment, in order to qualify that the cells are useful as a therapeutic, at least 80 % of the cells of the population should express detectable levels of CRALBP and one of the above mentioned polypeptides (e.g. ), more preferably at least 85 % of the cells of the population should express detectable levels of CRALBP and one of the above mentioned polypeptides (e.g.

PMEL17), more preferably at least 90 % of the cells of the population should s detectable levels of CRALBP and one of the above mentioned polypeptides (e.g.

PMEL17), more preferably at least 95 % of the cells of the tion should express detectable levels of CRALBP and one of the above mentioned polypeptides (e.g.

PMEL17), more preferably 100 % of the cells of the population should express detectable levels of CRALBP and one of the above mentioned polypeptides (e.g.

PMEL17 as assayed by a method known to those of skill in the art (e.g. FACS).

In addition, the cell may be qualified in vivo in animal models. One such model is the Royal College of Surgeons (RCS) rat model. Following transplantation, the WO 08239 therapeutic effect of the cells may be analyzed using methods which include fundus imaging, optokinetic tracking thresholds (OKT), electroretinogram (ERG), ogy, cone counting and rhodopsin ingestion. These methods are further described in Example , herein below.

The cells may be qualified or characterized in additional ways including for example karyotype analysis, morphology, cell number and viability, potency (barrier function and polarized secretion of PEDF and VEGF), level of residual hESCs, gram staining and ity. Exemplary assays which may be performed are described in Example 4.

In addition, the cells may be analyzed for barrier on and their level of growth factor secretion in a polarized manner (e.g. t epithelium—derived factor (PEDF) or VEGF, cytokines, interleukins and/or chemokines).

For is of secreted PEDF, supernatant is ted from es of the cells, and cells are harvested and counted. The amount of PEDF in the cell’s culture supematants may be quantified by using a PEDF ELISA assay (such as ELISAquantTM PEDF Sandwich ELISA Antigen Detection Kit, BioProductsMD, PED613) according to the manufacturer's protocol.

In addition, the direction of ion of PEDF and VEGF may be analyzed in the cells. This may be effected using a ell assay as illustrated in Figure 28. Prior to or following qualification, the cells may be preserved according to methods known in the art (e. g. frozen or cryopreserved) or may be directly administered to the subject.

The present invention contemplates analyzing cell populations which comprise retinal pigment epithelial (RPE) cells from any source. Thus, the cell tions may comprise RPE cells obtained from a donor (i.e. native RPE cells of the pigmented layer of the retina) or may comprise RPE cells which were ex—vivo differentiated from a population of stem cells (hSC—derived RPE cells, such as pluripotent stem cells — e.g. human embryonic stem cells). According to another embodiment, the RPE cells are obtained by transdifferentiation — see for example Zhang et al., Protein Cell 2014, (1):48—58, the contents of which are incorporated herein by reference.

According to one ment, the RPE cells that are analyzed do not express Pax6.

According to another embodiment, the RPE cells that are analyzed express Pax6.

“Retinal pigment epithelium cells”, “RPE cells”, “RPEs”, which may be used hangeably as the context allows, refers to cells of a cell type functionally similar to that of native RPE cells which form the pigment epithelium cell layer of the retina (e.g. upon transplantation within an eye, they exhibit functional activities r to those of native RPE cells).

According to one embodiment, the RPE cell expresses at least one, two, three, four or five markers of mature RPE cells. Such s include, but are not limited to CARLBP, RPE65, PEDF, PMEL17, Bestrophin and tyrosinase. Optionally, RPE cells may also express a marker of an RPE progenitor — e. g. MITF. In another embodiment, the RPE cells express FAX—6. In another embodiment, the RPE cells express at least one marker of a retinal progenitor cell including, but not d to OTX2, SIX3, SIX6 and LHX2.

According to yet another embodiment, the RPE cells are those that are differentiated from nic stem cells according to the method described in the Examples section herein below, the ts of the Examples being as if included in the specification itself.

As used herein, the phrase “markers of mature RPE cells” refers to antigens (e.g. proteins) that are elevated (e.g. at least 2 fold, at least 5 fold, at least 10 fold) in mature RPE cells with respect to non RPE cells or immature RPE cells.

As used herein the phrase “markers of RPE progenitor cells” refers to antigens (e.g. ns) that are elevated (e.g. at least 2 fold, at least 5 fold, at least 10 fold) in RPE progenitor cells with respect to non RPE cells. ing to another ment, the RPE cells have a morphology similar to that of native RPE cells which form the pigment epithelium cell layer of the retina i.e. pigmented and/or have a characteristic polygonal shape.

According to still another embodiment, the RPE cells are capable of treating diseases such as macular degeneration. ing to still another embodiment, the RPE cells fulfill at least 1, 2, 3, 4 or all of the requirements listed herein above.

The term "hSC—derived RPE cells" is used herein to denote RPE cells that are obtained by directed differentiation from hSCs. In accordance with a preferred embodiment, the hSC—derived RPE cells are functional RPE cells as exhibited by parameters defined hereinbelow. The term ”directed differentiation" is used interchangeably with the term "RPE induced differentiation" and is to be understood as meaning the process of manipulating hSCs under culture conditions which induce/promote differentiation into RPE cell type.

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 TGFB superfamily, and exhibit at least one of the following characteristics: — during differentiation, the cultured cells respond to TGFB signaling; — the RPE cells express markers tive of terminal entiation, e.g. bestrophin 1, CRALBP and/or RPE65; — following transplantation (i.e. in situ), the RPE cells exhibit trophic effect supporting eceptors adjacent to RPE cells; — further, in situ the RPE cells are capable of functioning with phagocytosis of shed photoreceptor outer segments as part of the normal renewal process of these eceptors; — further, in situ the RPE cells are capable of generating a retinal barrier and functioning in the visual cycle.

As used herein, the phrase “stem cells” refers to cells which are capable of ing in an undifferentiated state (e.g., pluripotent or multipotent stem cells) for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (e.g., fully differentiated cells). Preferably, the phrase “stem cells” encompasses embryonic stem cells (ESCs), d pluripotent stem cells (iPS), adult stem cells, hymal stem cells and hematopoietic stem cells.

According to a ular embodiment, the RPE cells are d from pluripotent stem cells ing human embryonic stem cells or induced otent stem cells.

The phrase “embryonic stem cells” refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i. 6., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state. The phrase “embryonic stem cells” may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre—implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post— implantation/pre—gastrulation stage blastocyst (see WO2006/040763) and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation. The embryonic stem cells of some embodiments of the ion can be obtained using well—known ulture s.

For e, human embryonic stem cells can be isolated from human blastocysts.

Human blastocysts are typically obtained from human in viva preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells, the zona pellucida is d from the cyst and the inner cell mass (ICM) is isolated by surgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. es demonstrating undifferentiated morphology are individually ed by micropipette/stem cell tool, mechanically dissected into fragments/clumps, and re- plated. Resulting ES cells are then routinely split every 4—7 days. For further details on methods of preparation human ES cells see Reubinoff et al., Nat Biotechnol 2000, May: 18(5): 559; Thomson et al., [US 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].

It will be appreciated that commercially available stem cells can also be used according to some embodiments of the invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry text Transfer Protocol://grants(dot)nih(dot)gov/stem_cells/registry/current(dot)htm] and other European registries. Non—limiting examples of cially available embryonic stem cell lines are HAD-C102, ESI, BGOl, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-ll, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUESl, NYUES2, NYUES3, , NYUESS, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CTl, CT2, CT3, CT4, MA135, Eneavour—2, WIBRl, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES 45, Shef 3, Shef 6, BJNhem19, BJNhem20, SA001, SA001.

In addition, ES cells can be obtained from other species as well, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224—7], rat [Iannaccone et al., 1994, Dev Biol. 163: ] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130—8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424—33], several domestic animal 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] and non—human primate species s monkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci U S A. 92: 7844—8; Thomson et al., 1996, Biol Reprod. 55: 254—9].

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for e by Tyrode’s acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass. The blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the lation event) in vitro using standard embryonic stem cell culturing methods. r 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 comprises removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in this process.

Yet another method for preparing ES cells is by parthenogenesis. The embryo is also not destroyed in the process.

Currently practiced ES culturing methods are mainly based on the use of feeder cell layers which e factors needed for stem cell proliferation, While at the same time, inhibit their differentiation. Exemplary feeder layers include Human embryonic fibroblasts, adult fallopian epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), murine fetal fibroblasts (MFF), human nic last (HEF), human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human umbilical cord fibroblasts, human cells obtained from the umbilical cord or ta, and human marrow stromal cells (hMSCs). Growth s may be added to the medium to maintain the ESCs in an undifferentiated state. Such growth factors include bFGF and/or TGFB. In another embodiment, agents may be added to the medium to maintain the hESCs in a naive undifferentiated state — see for e Kalkan et al., 2014, Phil.

Trans. R. Soc. B, 369: 20130540.

Feeder cell free systems have also been used in ES cell culturing, such systems utilize matrices supplemented with serum replacement, cytokines and growth factors (including IL6 and e 1L6 receptor chimera) as a replacement for the feeder cell layer. Stem cells can be grown on a solid surface such as an extracellular matrix (e.g., MatrigelRTM or laminin) in the presence of a culture medium — for example the Lonza L7 system, mTeSR, StemPro, XFKSR, E8). Unlike feeder-based cultures which require the simultaneous growth of feeder cells and stem cells and which may result in mixed cell populations, stem cells grown on feeder—free systems are easily separated from the surface. The culture medium used for growing the stem cells ns factors that effectively inhibit entiation and promote their growth such as MEF—conditioned medium and bFGF. However, commonly used feeder—free culturing systems e an animal—based matrix (e.g., MatrigelRTM) supplemented with mouse or bovine serum, or with MEF conditioned medium [Xu C, et al. (2001). Feeder—free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19: 971—4] which present the risk of animal pathogen cross—transfer to the human ES cells, thus compromising future clinical applications.

Numerous methods are known for differentiating ESCs towards the RPE lineage and include both directed entiation protocols such as those described in WO 2008/129554, 2013/184809 and spontaneous differentiation ols such as those described in U.S. Patent No. 8,268,303 and U.S. Patent application 20130196369, the contents of each being incorporated by reference.

According to a ular embodiment, the RPE cells are generated from ESC cells using a directed differentiation ol — for example according to that disclosed in the Example section.

In one exemplary differentiation protocol, the embryonic stem cells are differentiated towards the RPE cell lineage using a first differentiating agent and then further differentiated towards RPE cells using a member of the transforming growth factor—B (TGFB) amily, (e.g. TGFBl, TGFBZ, and TGFB3 subtypes, as well as homologous ligands ing activin (e.g., activin A, activin B, and activin AB), nodal, anti—mullerian hormone (AMH), some bone morphogenetic proteins (BMP), e.g. BMP2, BMP3, BMP4, BMPS, BMP6, and BMP7, and growth and differentiation factors (GDF)).

According to a ular embodiment, the TGFB superfamily member is selected from the group ting of TGFBI, activin A and TGFB3.

According to a specific embodiment, the member of the transforming growth —B (TGFB) superfamily is activin A — e.g. between 20—200 ng/ml, e.g. 100—180 ng/ml.

The first differentiating agent promotes differentiation towards the RPE lineage.

For e, the first differentiating agent may promote differentiation of the pluripotent stem cells into neural progenitors. Such cells may express neural precursor markers such as PAX6.

According to a particular embodiment, the first differentiating agent is nicotinamide (NA) — e.g. between l—lOO mM, 5—50 mM, 5—20 mM, e.g. 10 mM.

NA, also known as “niacinamide”, is the amide derivative form of Vitamin B3 (niacin) which is thought to preserve and improve beta cell function. NA has the chemical formula C6H6N20. NA is essential for growth and the sion of foods to energy, and it has been used in arthritis ent and diabetes treatment and prevention.

\ \O N 25 Nicotinamide (NA) According to a particular embodiment, the namide is a namide derivative or a namide mimic. The term ative of nicotinamide (NA)" as used herein denotes a compound which is a chemically modified derivative of the natural NA. In one embodiment, the chemical modification may be a substitution of the pyridine ring of the basic NA structure (via the carbon or nitrogen member of the ring), via the nitrogen or the oxygen atoms of the amide moiety. When substituted, one or more hydrogen atoms may be replaced by a substituent and/or a substituent may be attached to a N atom to form a tetravalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes a tuted or non—substituted nicotinamide. In another embodiment, the al modification may be a deletion or replacement of a single group, e.g. to form a thiobenzamide analog of NA, all of which being as appreciated by those versed in organic chemistry. The derivative in the context of the invention also includes the nucleoside derivative of NA (e.g. nicotinamide adenine).

A variety of derivatives of NA are described, some also in tion with an inhibitory activity of the PDE4 enzyme 068233; WOO2/060875; GB2327675A), or as VEGF—receptor tyrosine kinase inhibitors (WOOl/55114). For example, the process of preparing 4—aryl—nicotinamide derivatives (W005/014549).

Other exemplary nicotinamide derivatives are disclosed in WOOl/55114 and EP2128244.

Nicotinamide mimics include modified forms of nicotinamide, and chemical s of nicotinamide which recapitulate the effects of nicotinamide in the differentiation and tion of RPE cells from pluripotent cells. Exemplary nicotinamide mimics include benzoic acid, 3—aminobenzoic acid, and 6— aminonicotinamide. Another class of compounds that may act as nicotinamide mimics are inhibitors of poly(ADP—ribose) polymerase (PARP). Exemplary PARP inhibitors include 3-aminobenzamide, Iniparib (B31 201), Olaparib (AZD-2281), rib (AG014699, PF- 01367338), Veliparib (ABT-888), CEP 9722, MK 4827, and BMN- 673.

According to a particular ment, the differentiation is effected as follows: a) culture of ESCs in a medium comprising a first differentiating agent (e.g. nicotinamide); and b) culture of cells obtained from step a) in a medium comprising a member of the TGFB superfamily (e.g. activin A) and the first differentiating agent (e.g. nicotinamide).

Preferably step (a) is effected in the absence of the member of the TGFB superfamily.

The above described protocol may be continued by culturing the cells obtained in step (b) in a medium comprising the first differentiating agent (e.g. nicotinamide), but devoid of a member of the TGFB superfamily (e.g. activin A). This step is referred to herein as step (c).

The above described protocol is now described in further detail, with additional embodiments.

The differentiation process is d once sufficient quantities of ESCs are obtained. They are typically removed from the adherent cell culture (e.g. by using collagenase A, dispase, TrprE select, EDTA) and plated onto a non-adherent substrate (e.g. Hydrocell non—adherent cell culture plate) in the presence of nicotinamide (and the absence of activin A). Exemplary concentrations of nicotinamide are between 1—100 mM, 5—50 mM, 5—20 mM, e.g. 10 mM, Once the cells are plated onto the non—adherent ate, the cell culture may be referred to as a cell suspension, preferably free floating clusters in a sion culture, i.e. aggregates of cells derived from human embryonic stem cells (hESCs). The cell clusters do not adhere to any ate (e.g. culture plate, carrier). Sources of free floating stem cells were previously described in WC 06/070370, which is herein incorporated by reference in its entirety. This stage may be effected for a minimum of 1 day, more preferably two days, three days, 1 week or even 10 days. Preferably, the cells are not ed for more than 2 weeks in suspension together with the nicotinamide (and in the absence of the TGFB superfamily member e. g. activin A).

According to a preferred embodiment, when the cells are cultured on the non- adherent substrate, the heric oxygen conditions are manipulated such that the percentage is equal or less than about 20 %, 15 %, 10 %, more ably less than about 9 %, less than about 8 %, less than about 7 %, less than about 6 % and more preferably about 5 % (e.g. between 1 % — 20 %, l %—10 % or 0-5 %).

Examples of non—adherent cell culture plates include those manufactured by Hydrocell (e.g. Cat No. 174912), Nunc etc.

Typically, the clusters comprise at least 50-500,000, 50-100,000, 50-50,000, 50- ,000, 50-5000, 50—1000 cells. According to one embodiment, the cells in the clusters are not organized into layers and form irregular shapes. In one embodiment, the clusters are devoid of pluripotent embryonic stem cells. In another embodiment, the clusters comprise small amounts of pluripotent embryonic stem cells (e.g. no more than 5 %, or no more than 3 % (e.g. 0.01—2.7%) cells that co—express OCT4 and TRA 1—60 at the protein level). Typically, the clusters comprise cells that have been partially differentiated under the influence of nicotinamide. Such cells may express neural precursor markers such as PAX6. The cells may also express markers of progenitors of other lineages such as for example alpha—feto protein, MIXLl and Brachyuri.

The clusters may be dissociated using enzymatic or non—enzymatic methods (e.g., ical) known in the art. According to one ment, the cells are dissociated such that they are no longer in clusters — e.g. aggregates or clumps of 2— 0 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 in a single cell suspension.

The cells (e.g. iated cells) are then plated on an adherent substrate and cultured in the presence of nicotinamide e.g. between l—100 mM, 5—50 mM, 5—20 mM, e.g. 10 mM (and the absence of activin A). This stage may be effected for a minimum of 1 day, more preferably two days, three days, 1 week or even 14 days. Preferably, the cells are not cultured for more than 1 week in the presence of nicotinamide on the adherent cell e (and in the absence of activin).

Altogether, the cells are typically exposed to namide, (at concentrations between l-lOO mM, 5—50 mM, 5-20 mM, e.g. 10 mM), for about 2—3 weeks, and preferably not more than 4 weeks prior to the addition of the second differentiating factor (e. g. Activin A). es of nt substrates include but are not limited to collagen, fibronectin, n, (e.g. laminin 521).

Following the first stage of directed entiation (i.e. culture in the ce of nicotinamide (e.g. 10 mM) under non—adherent culture conditions under low oxygen atmospheric conditions ed by ing on an adherent substrate in the presence of nicotinamide under low oxygen atmospheric conditions), the semi—differentiated cells are then subjected to a further stage of differentiation on the adherent substrate - culturing 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 effected for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, one week to eight weeks, one week to four weeks, for example for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks or even eight weeks. Preferably this stage is effected for about two weeks. According to one embodiment, this stage of differentiation is also effected at 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 % and more preferably about 5 % (e.g. between 1 % — 20 %, l %—10 % or 0-5 Following the second stage of directed differentiation (i.e. e in the presence of nicotinamide and activin A on an adherent substrate), the further differentiated cells may optionally be subjected to a subsequent stage of differentiation on the adherent substrate — ing in the presence of nicotinamide (e.g. between 1—100 mM, 5—50 mM, 5—20 mM, e.g. 10 mM), in the e of activin A. This stage may be effected for at least one day, 2 days, 3 days, 1 week, at least two weeks, at least three weeks or even four weeks. Preferably this stage is effected for about one week. This stage of differentiation may be effected at low (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 % and more preferably about 5 % (e.g. between 1 % — 20 %, l %—10 % or 0-5 %) or normal atmospheric oxygen conditions or a combination of both (i.e. initially at low atmospheric oxygen ions and uently when lightly pigmented cells are ed, at normal oxygen conditions).

According to a particular embodiment, when the atmospheric oxygen conditions are returned to normal atmospheric conditions the cells are cultured for at least one more day (e.g. up to two weeks) in the presence of nicotinamide (e.g. 10 mM) and in the e of activin A. 2015/051269 The basic medium in accordance with the invention is any known cell culture medium known in the art for supporting cells growth in vitro, typically, a medium comprising a defined base solution, which includes salts, sugars, amino acids and any other nutrients required for the maintenance of the cells in the culture in a Viable state.

Non-limiting examples of commercially available basic media that may be utilized in accordance with the invention comprise Nuristem (without bFGF and TGFB for ESC entiation, with bFGF and TGFB for ESC expansion) NeurobasalTM, KO—DMEM, DMEM, DMEM/Fl2, Lonza L7 system, mTeSR, StemPro, XF KSR, E8, oTM Stem Cell Growth Medium, or X—VivoTM. The basic medium may be supplemented with a variety of agents as known in the art dealing with cell cultures. The following is a non—limiting nce to various supplements that may be ed in the culture system to be used in accordance with the present disclosure: — serum or with a serum replacement containing , such as, without being limited thereto, knock out serum replacement (KOSR), Nutridoma-CS, TCHTM, N2, N2 derivative, or B27 or a combination; — an extracellular matrix (ECM) component, such as, without being limited thereto, fibronectin, laminin, collagen and gelatin. The ECM may them be used to carry the one or more s of the TGFB superfamily of growth s; — an antibacterial agent, such as, without being limited thereto, penicillin and streptomycin; — non—essential amino acids , neurotrophins which are known to play a role in promoting the survival of SCs in culture, such as, without being limited thereto, BDNF, NT3, NT4.

According to a preferred embodiment, the medium used for differentiating the ESCs is Nuristem medium gical Industries, 05—102—1A or 05—100—1A).

According to a particular embodiment, differentiation of ESCs is effected under xeno free conditions.

According to one embodiment, the proliferation/growth medium is devoid of xeno contaminants i.e. free of animal derived components such as serum, animal derived growth factors and albumin. Thus, according to this embodiment, the culturing is performed in the absence of xeno contaminants.

WO 08239 Other methods for culturing ESCs under xeno free conditions are provided in U.S. Patent Application Publication No. 20130196369, the contents of which are orated in their entirety.

During differentiation steps, the embryonic stem cells may be monitored for their differentiation state. Cell differentiation can be determined upon examination of cell or tissue—specific markers which are known to be indicative of differentiation.

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

Thus, according to another aspect of the present invention there is provided a method of generating retinal epithelial cells sing: (a) ing pluripotent stem cells in a medium sing a differentiating agent so as to generate differentiating cells, wherein the medium is devoid of a member of the transforming growth factor B (TGF [3) superfamily; (b) culturing the differentiating cells in a medium comprising the member of the transforming growth factor [3 (TGF [3) superfamily and the entiating agent to generate cells which are further differentiated towards the RPE lineage; (c) analyzing the secretion of Pigment epithelium—derived factor (PEDF) from the cells which are further differentiated towards the RPE lineage; and (d) ing the cells which are further differentiated towards the RPE lineage in a medium comprising a differentiating agent so as to generate RPE cells, wherein the medium is devoid of a member of the transforming growth factor B (TGF [3) superfamily, wherein step (d) is effected when the amount of the PEDF is above a predetermined level.

Preferably, step (d) is effected when the level of PEDF is above 100 ng/ml/day, 200 ng/ml/day, 300 ng/ml/day, 400 ng/ml/day, or 500 ng/ml/day.

Another method for determining potency of the cells during or following the entiation process is by analyzing barrier function and polarized PEDF and VEGF secretion, as illustrated in Example 4, herein below.

Once the cells are promoted into RPE cells, they may be selected and/or expanded.

According to a particular embodiment, the selection is based on a negative selection — i.e. removal of non—RPE cells. This may be done mechanically by removal of non-pigmented cells or l of non—polygonal cells or by use of e markers.

According to another embodiment, the selection is based on a positive selection i.e. selection based on morphology (e.g. pigmented cells and/or polygonal cells). This may be done by visual analysis or use of surface markers.

According to still another embodiment, the selection is based first on a negative selection and then on a positive selection.

Expansion of RPE cells may be effected on an extra cellular matrix, e.g. gelatin, collagen or poly—D—lysine and laminin. For expansion, the cells may be cultured in serum—free KOM, serum comprising medium (e.g. DMEM + 20 %) or Nuristem medium (06-5 1021A ical Industries). ally, the cells may be exposed to nicotinamide during the expansion phase — at concentrations between l—lOO mM, 5-50 mM, 5—20 mM, e.g. 10 mM. Under these e conditions, the pigmented cells reduce pigmentation and e a fibroid—like morphology. Following further ged culture and proliferation into high—density es, the cells re—acquire the characteristic polygonal shape morphology and preferably also pigmentation of RPE cells.

The RPE cells may be expanded in suspension or in a monolayer. The expansion of the RPE cells in yer cultures may be modified to large scale expansion in bioreactors by methods well known to those versed in the art.

The population of RPE cells generated according to the s described herein may be characterized according to a number of different parameters.

Thus, for example, the RPE cells obtained are nal in shape and are pigmented.

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 of the RPE cell populations obtained co—express both premelanosome protein 7) and cellular retinaldehyde binding protein (CRALBP).

Following administration, the cells described herein are capable of forming a monolayer (as illustrated in Figure 27C).

WO 08239 According to one ment, the trans—epithelial electrical resistance of the cells in a monolayer is greater than 100 ohms.

Preferably, the trans-epithelial electrical ance of the cells is greater than 150, 200, 250, 300, 300, 400, 500, 600, 700, 800 or even greater than 900 ohms. ing to a particular embodiment, the TEER is n 100—1000 ohms, more preferably between 100—900 ohms for e between 200—900 ohms, 300-800 ohms, 300—700 ohms, 400—800 ohms or 400—700 ohms.

Devices for measuring trans—epithelial electrical resistance (TEER) are known in the art. An exemplary set—up for measuring TEER is illustrated in Figure 28.

It will be appreciated that the cell populations disclosed herein are devoid of undifferentiated human embryonic stem cells. According to one embodiment, less than l:250,000 cells are Oct4J'TRA—1—60+ cells, as measured for e by FACS. The cells also do not express or downregulate expression of GDF3 or TDGF relative to hESCs as measured by PCR.

Another way of characterizing the cell populations disclosed herein is by marker sion. Thus, for example, at least 80 %, 85 %, or 90 % of the cells express Bestrophin l, as measured by immunostaining. According to one embodiment, n 90—95 % of the cells express bestrophin. ing to another embodiment, at least 80 %, 85 %, 87 %, 89 % or 90 % of the cells express Microphthalmia—associated transcription factor {MITF), as measured by immunostaining. For example, between 85—95 % of the cells express MITF.

According to r embodiment, at least 50 %, 55 %, 60 %, 70 %, 75 % 80 % 85 %, 87 %, 89 % or 90 % of the cells express paired box gene 6 (FAX—6) as measured by FACS.

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

It will be appreciated that the RPE cells generated herein secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner. According to particular embodiments, the ratio of apical secretion of PEDF: basal secretion of PEDF is greater than 1. According to particular embodiments, the ratio of apical secretion of PEDF: basal secretion of PEDF is greater than 2. According to ular embodiments, the ratio of apical secretion of PEDF: basal secretion of PEDF is greater than 3. In addition, the ratio of basal ion of VEGF: apical secretion of VEGF is greater than 1. According to particular embodiments, the ratio of basal ion of VEGF: apical secretion of VEGF is greater than 1.5, 2 or 2.5.

The cells of the present invention secrete additional factors including for example angiogenin, the immunomodulatory factors IL—6, sgpl30, MIF, sTNF—Rl, sTRAIL—R3, MCP—l and Osteoprotegerin, the ellular matrix regulators TIMP—1 and TIMP—2 and the protein Axl.

According to another aspect, at least 80 % of the cells of the cell population co— express premelanosome protein (PMELl7) and cellular retinaldehyde binding protein (CRALBP) and r a portion (at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %) of the cells secrete/shed each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 0) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor-0t (sTNF-Rl).

It will be appreciated that in some cases all the cells that co—express premelanosome protein (PMELl7) and cellular retinaldehyde binding n (CRALBP) also secrete/shed angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgpl30) and soluble form of the tous ne receptor 1 for tumor necrosis factor—0t (sTNF—Rl).

In other cases the majority (more than 50 %, 60 %, 70 %, 80, 90 % of the cells that co—express premelanosome protein (PMELl7) and cellular retinaldehyde binding n (CRALBP) also secrete/shed angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgpl30) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis —0t (sTNF—Rl).

The RPE cells generated herein preferably secrete enin, TIMP2, sgpl30 and sTNF—Rl in a polarized manner.

According to particular embodiments, the ratio of apical secretion of sgpl30: basal secretion of sgpl30 is greater than 1. According to particular embodiments, the ratio of apical ion of sgpl30: basal secretion of sgpl30 is greater than 2.

WO 08239 ing to particular embodiments, the ratio of apical secretion of sgp130: basal secretion of sgpl30 is greater than 3.

Furthermore, the ratio of apical sTNF-Rl: basal sTNF-Rl is greater than 1.

According to particular embodiments, the ratio of apical sTNF—Rl: basal sTNF—Rl is greater than 2. ing to particular embodiments, the ratio of apical sTNF—Rl: basal sTNF—Rlis greater than 3.

In addition, the ratio of basal secretion of angiogenin: apical secretion of angiogenin is greater than 1. According to particular embodiments, the ratio of basal secretion of angiogenin: apical secretion of angiogenin is greater than 1.5, 2, 2.5 or 3.

Furthermore, the ratio of apical secretion of TIMP2: basal secretion of TllVIP2 is greater than 1. According to particular embodiments, the ratio of apical secretion of TIMP2: basal secretion of TIMP2 is r than 2. ing to ular embodiments, the ratio of apical secretion of TIMP2: basal secretion of TIMP2 is greater than 3.

The stability of the cells is another characterizing feature. Thus, for example the amount of PEDF secretion remains stable in the cells following their incubation at 2-8 °C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours. Further, the polarized secretion of PEDF and VEGF s stable following incubation of the cells at 2—8 ”C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours. Further, the TEER of the cells remains stable in the cells following their tion at 2—8 ”C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours.

In another embodiment, the cells are characterized by their therapeutic effect.

Thus, for example the present inventors have shown that the cell tions are capable of rescuing visual acuity in the RC3 rat following subretinal administration. In on, the cell tions are capable of rescuing photoreceptors (e.g. cone photoreceptors) for up to 180 days (in some embodiments at least 180 days) post— subretinal administration in the RC3 rat.

It would be well appreciated by those versed in the art that the derivation of RPE cells is of great benefit. They may be used as an in vitro model for the development of new drugs to promote RPE cell survival, regeneration and function. RPE cells may serve for high throughput screening for compounds that have a toxic or regenerative effect on RPE cells. They may be used to uncover mechanisms, new genes, soluble or membrane—bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.

The RPE cells may also serve as an unlimited source of RPE cells for transplantation, replenishment and support of malfunctioning or degenerated RPE cells in retinal degenerations. Furthermore, cally modified RPE cells may serve as a vector to carry and express genes in the eye and retina after transplantation.

The RPE cells produced by the method of the present disclosure may be used for large scale and/or long term ation of such cells. To this end, the method of the invention is to be performed in bioreactors and or cell culture s suitable for large scale production of cells, and in which undifferentiated hSCs are to be cultivated in accordance with the invention. General requirements for cultivation of cells in bioreactors and or cell culture systems are well known to those versed in the art.

Harvesting of the cells may be performed by various methods known in the art.

Non-limiting examples include mechanical dissection and dissociation with papain or trypsin (e.g. TrprE select). Other methods known in the art are also applicable.

The RPE cells generated as described herein may be transplanted to various target sites within a subject's eye. In accordance with one embodiment, the transplantation of the RPE cells is to the subretinal space of the eye, which is the normal anatomical location of the RPE (between the photoreceptor outer segments and the choroid). In addition, dependent upon migratory ability and/or positive paracrine s of the cells, transplantation into additional ocular compartments can be considered including the inner or outer retina, the l periphery and within the ds.

Retinal diseases which may be treated using the RPE cells described herein include, but are not d to retinitis pigmentosa, retinoschisis, lattice degeneration, Best disease, and age related macular degeneration (AMD).

Further, transplantation may be performed by various techniques known in the art. Methods for performing RPE lants are described in, for example, U.S. Pat.

Nos. 027, 6,045,791, and 5,941,250 and in Eye s Arch Clin Exp Opthalmol March 1997; :149—58; Biochem s Res Commun Feb. 24, 2000; 268(3): 842—6; Opthalmic Surg February 1991; 22(2): 102—8. s for performing corneal transplants are described in, for example, US. Pat. No. 5,755,785, and 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 mainly paracrine s are to be ed, cells may also be delivered and maintained in the eye encapsulated within a semi—permeable ner, which will also decrease re of the cells to the host immune system (Neurotech USA CNTF delivery system; PNAS March 7, 2006 vol. ) 3896—3901).

In accordance with one embodiment, transplantation is performed via pars plana Vitrectomy surgery followed by delivery of the cells through a small retinal opening into the sub—retinal space or by direct injection. Alternatively, cells may be delivered into the subretinal space via a trans—scleral, choroidal approach. In addition, direct trans— scleral injection into the vitreal space or delivery to the anterior retinal periphery in proximity to the ciliary body can be performed.

The RPE cells may be transplanted in various forms. For example, the RPE cells may be introduced into the target site in the form of cell suspension, or adhered onto a matrix, extracellular matrix or substrate such as a biodegradable polymer or a combination. The RPE cells may also be lanted together ansplantation) with other retinal cells, such as with photoreceptors.

Thus, the invention also pertains to pharmaceutical compositions of RPE cells described herein. The composition is preferably such suitable for transplantation into the eye. Thus, for example, the RPE cells may be formulated in an intraocular irrigating solution such as BSS plusTM.

It is expected that during the life of a patent maturing from this application many relevant technologies will be developed for the generation of RPE cells, and the term RPE cells is intended to include all such new technologies a priori.

As used herein the term “about” refers to i- 10 %.

The terms "comprises", "comprising", "includes", "including", g” and their conjugates mean "including but not limited to".

The term “consisting of" means “including and d to”.

The term "consisting ially of“ means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the onal ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form ll H Y a ‘an” and "the" include plural nces 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.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for ience and brevity and should not be construed as an ble limitation on the scope of the invention. Accordingly, the ption of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies less of the breadth of the range.

As used herein, the term d" refers to manners, means, techniques and procedures for lishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, ical, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially rating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is iated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, bed in the t of a single embodiment, may also be provided separately or in any suitable subcombination or as le in any other described embodiment of the ion. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as d in the claims section below find experimental support in the following examples.

EXAMPLES Reference is now made to the following es, which er with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I—III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, ore, 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", Vols. 1—4, Cold Spring Harbor Laboratory Press, New York ; methodologies as set forth in US. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory ok", Volumes I—III Cellis, J. E., ed. (1994); "Culture of Animal Cells — A Manual of Basic Technique" by Freshney, Wiley— Liss, N. Y. (1994), Third n; "Current Protocols in Immunology" Volumes I—III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in ar Immunology", W. H. Freeman and Co., New York ; available immunoassays are extensively described in the patent and ific literature, see, for e, US Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 517; 3,879,262; 3,901,654; 3,935,074; 533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; nucleotide Synthesis" Gait, M.

J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. 1., 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, ic 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 " CSHL Press (1996); all of which are orated by reference as if fully set forth herein. Other l references are provided throughout this nt. The procedures therein are ed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Qualification 0fthe CRALBP/PM’EL I 7 double ng FACS method The aim of this study was to qualify the CRALBP/PMEL 17 double staining FACS method. by demonstrating the methods accuracy and precision in a minimum of 6 independent spiking assays over at least 3 testing days. The assay qualification was performed using OpRegen® batch 5C as the positive control cells and HAD—C 102- hESCs, as the negative control cells. A, calibration curve of known quantities of RPE (OpRegenCFD 5C) spiked into hESCs was used for testing the accuracy and precision at different spiking points. The expected cy and precision were up to 25% at all points.

Staining Protocol: Negative Control hlESC cells taken from a cryopreserved hESC hank (HAD~C l02 p48 4.5.2014) were thawed in tem (containing BSA) according to sponsor protocols. Positive Control RPE cell stock: OpRegen® batch 5C cells (reference line) were thawed into in 20%HS—DMEM according to sponsor protocols. Thawed OpRegen® 5C and HAD—C102 hESC were spun down, re— ded in l, nil PBS (9), filtered h a 35 uM cell strainer and counted with Trypan Blue. The cell concentration was adjusted to 0.73x106 «106' cells."ml in PBS (—). 1 nlinil FVS450 was added to each cell suspension followed by vortexing and incubation for 6 minutes at 37 3C. FVS450 was washed with 0.1% BSA, and re—suspended in 0.1% BSA—Pc—block (5 min at RT) to block all Fc—epitopes on the cells. Cells were then washed with PBS (—) and fixed in 80% Methanol (5 min at 4°C). Fixed cells were washed once with PBS (—), once with 0.1% PBS—T, and permeabilized with 0.1% PBS—T (20 minutes at RT). Permeabilization solution. was replaced with 10% Normal goat serum (NGS) Blocking Solution (2009000 cells/50 ill) for at least 30 minutes {max one hour) at. RT. During incubation time quality sample tubes (QSs) were prepared and at. the end of blocking, cells were divided and lininunostained. Cells were incubated with y antibodies for 30 minutes followed by 3 washes with 0.1% PBS—T and 30 min incubation with secondary antibodies and 3 washes with 0.1% PBS—T.

Negative and positive control cells were stained with the viability stain FVS450, fixed, blocked and bilized. A calibration curve of known quantities of positive control RPE (QpRegenQ‘) 5C) cells in negative control hESCs, at 4 concentrations (25%“, 50%, 75%, and 95% RPE in thSC), was then generated based on the Trypan Blue viability cell count of each population. Negative and positive control cells and the mixed tions were noslained with primary monoclonal antibodies specific to the RPE s CRALBP and Plv’llila 17, followed by staining with d secondary antibodies (anti-niouse—FITC and anti—rabbit-Alexa Fluor 647;, respectively). d cells were FACS ed to measure the percent viable single cell gated CRALBP~s~PMELl7+ cells, RESULTS Accuracy: Accuracy of the assay was determined from test results of 4 levels of spiked RPEs (25%, 5.0%, 75% and 95%). The accuracy of the RPE stock (OpRegen® 5C) was determined with respect to it being ially 100% RPE cells. Each level values were analyzed by six independent runs/detenninations.

The 50% concentration level was considered to be the lower limit of quantitation with an expected accuracy of up to 25% (50% level ranged from —8,4l to 2014; 75% and 99.5% levels ranged from 6.32 to 6.88).

These results meet the expected outcomes for relative bias of up to 25%;, and indicate that the assay is accurate for determination of CRALBP+PMELl7+ double positive cells in concentrations ranging from 50—995%. Since OpRegen® 5C yields 99.5% CRAl_,BP+PMELl,'7+ double positive RPE cells, a relative bias of less than 25% for a result >99.5% cannot be assured.

W0 2016/108239 2015/051269 Table .1 Run Assigned Concentration (%) Measured Concentration (%) Relative Bias (%) .88 - 16.48 31.61 26.44 32.20 28.80 32.01 28.04 .71 2.84 26.87 7.48 45.93 —8. 14 60.08 20.16 56.87 13.74 58.51 17.02 50.56 1.12 49.52 -0.96 71.01 -5.32 79.64 6.19 78.41 4.55 80.16 6.88 73.85 -1.53 72.94 -2.75 93.94 -1 . 12 96.14 1.20 95.11 0.12 95.59 1.01 93.81 -1.25 93.70 -1.37 98.79 —1.21 99.69 -0.31 99.62 -0.38 100% 99.59 -0.41 99.60 -0.40 99.48 -0.52 Intermediate Precision: The intermediate precision of the assay was determined from s of 6 assays carried out by one operator. In each assay the percent single viable RPEs was determined and from that. the %CY was calculated. Table 2 summarizes the test results. As shown, %CY for all concentration levels was below % and can be measured with adequate precision. %CY for the concentration levels %., 50%., 75%. 95% and l,()()% 'RPEs, were 16.14%, 10.61%, 5.10%, 1.l§7%, and 0.34%., respectively. These results meet the expected values for precision. The ed percent RPES is within 20% of the expected value at all concentrations. These results indicate that the assay is e for determination of RPES in concentrations ranging from 25~99.5%.

Table .2 Assigned Concentration (%) Run Measured Concentration (%RPE) —Mean %RPE —%CV -Ln Mean %RPE -L11 Mean %RPE WO 08239 l 98.79 2 99.69 3 99.62 4 99.59 99.60 99.48 Mean %RPE 99.46 SD 0.34 %CV 0.34 Repeatability: Sample repeatability was tested in 3 runs {#2, #3 and #4) in which duplicate OpRegen® SC samples were stained and acquired side by side. The results confirmed that sample identity obtained within an experiment is able and consistent across samples.

Linearity/range: As shown in Figure l, linearity was measured using data that were found to be both accurate and precise. The coefficient of regression between the target (spiked) and ed results across the tested assay range (5095-10093) was found to be 0.99, Thus, the range of the method which demonstrates acceptable accuracy and precision and ity is the range between 50% and 99.5% RPE cells, which covers the expected range of tested samples.

Positive control cells: The provisional level of CRALBP/PMEL17 double positive cells was set at equal to or r than 95 %.

Negative control cells: The provisional level of CRALBP/PMEL17 double positive cells for hESCs was set at equal to or less than 2 %.

Stability: The results show that stained samples are stable at 4°C also after one and 4 days and accuracy is kept within expected acceptance ia therefore the data acquisition can be performed within 96 hours of sample preparation.

Conclusion The results presented herein indicate that the disclosed method is qualified and suitable for its intended use of in vitro determination of RPE purity in OpRegen® final product and at different stages along the production process of ()pRegent’E, with Accuracy of Relative Bias ot"< 25% and precision of QECV < 20% in the range of 50%— 99.5% RPE cells.

EXAMPLE 2 Assessing the level of OpRegen® purity A FACS based method for assessing the level of human retinal pigment epithelial cells (RPE) purity as well as non-RPE cellular impurities in RPE cells was developed. Cellular retinaldehyde—binding protein P), one of the visual cycle ents, was bioinformatically identified as a unique marker for mature RPE cells.

Preliminary studies using CRALBP specific onal antibody have shown purity of above 98% in RPE cells generated according to methods described herein. These s were further supported by immunostaining for PMELl7, a melanosome marker found in RPE. In addition, different from some RPE specific markers, CRALBP is not expressed in melanocytes, a possible neural crest cellular contamination.

Test Sample and Controls: Human primary melanocytes (ATCC, PCS—200—013) were used as negative control cells for CRALBP and as positive control cells for PMELl7, type I transmembrane rotein enriched in melanosomes (melanin granules). HADClO2—hESCs at P29 (OpRegen® parental line), were used as negative control cells for both CRALBP and PMELl7, Clinical grade OpRegen® cells (batch 2A), and research grade OpRegen® (produced in GMP like Mock production; Mock IV D16) were used as the tested samples. The cells were generated as described in Example 3. staining and FACS analysis: cells were thawed and stained using the e Viability Stain (FVS450) (BD 562247), fixed with 80% Methanol, stained with the primary mouse anti CRALBP (Clone B2, Abcam ab15051), or its e control for mouse lgG2a (Abcam abl70l9l) and rabbit anti human PMELl7 (Clone EPR4864, Abcam 62) followed by secondary antibodies goat anti mouse (Dako F0479) and goat anti rabbit (Jackson 111—606—144), respectively.

Acquisition of FACS data was performed using a validated Navios flow cytometer (Beckman Coulter) and analysis was performed using FlowJo 7.6.

WO 08239 RESULTS Initial FACS data using anti CRALBP onal antibody and showed that the purity level of OpRegen® is above 98%. Melanocytes which are a le neural crest ar contaminant were found negative for the unique RPE specific marker CRALBP (1.7%). The parental line HADC102—hESCs were negative to CRALBP (0.2%), as expected.

The purity level of OpRegen® stayed above 98% following double staining with CRALBP and PMEL17 (Figure 10). Melanocytes stained positive for PMEL17, as expected, but were negative for the double marked population (~l%). HADC102— hESCs were negative stained for CRALBP and PMEL17 (0.07%).

EXAMPLE 3 Description ofmanufacturing process andprocess controls OpRegen® is manufactured from the 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 banks (WCBs) were produced under Good Manufacturing ce (GMP) and xeno— free conditions, appropriately tested, characterized and banked. These were then used in the derivation of clinical—grade hESC line HAD—C 102 from surplus human blastocysts under GMP and xeno—free conditions.

At the initial phase of production hESCs are expanded on irradiated feeders as colonies. They are then transferred to suspension culture to initiate differentiation in a directed . Spheroid bodies (SBs) are formed and then plated as an adherent culture under continued ed differentiation conditions towards a neural fate and subsequently towards RPE cells. At the end of the differentiation phase non—pigmented areas are physically excised and pigmented cells are tically collected, seeded and expanded. Purified hESC-derived RPE cells (D8) are harvested at passage 2 and immediately sed to the DP. Duration of the manufacturing s depends on the hESCs growth rate (~2 months from thawing) and in total usually spans over 4-5 months.

Each step of the manufacturing process, including the in—process quality control (QC) tests is briefly described below.

Steps 1-3: Generation of human cord fibroblast feeder Working Cell Bank (WCB). A Vial of human cord feeder Master Cell Bank (MCB) 8—MCB) at passage 3-4 was thawed, expanded in Dulbecco's Modified s Medium (DMEM, SH30081.01, Hyclone) supplemented with 20% human serum (l4—498E, Lonza), ated (Gamma cell, 220 Exel, MDS Nordion 3,500 rads) and eserved at passages 7—8 to generate the working cell banks (WCBs). Prior to cryopreservation, samples from the feeder cell es were tested for sterility, mycoplasma and Limulus Amebocyte Lysate (LAL), morphology, karyotype, cell number, and ity. In addition, post thawing, their identity to the MCB, their inability to proliferate and their ability to support un—differentiated HAD—C102—hESC growth were confirmed. If the WCB passed all QC testing, the bank was released for expansion of hESCs.

Production Steps 1—3 are depicted in Figure 12.

Steps 4-5: Expansion of hECSs. A single Vial of the human cord fibroblast WCB (either CRD008-WCB8 or CRDOOS-WCB9) was thawed and plated in center well plates d with recombinant human gelatin (RhG100—001, Fibrogen) at a concentration of 70,000—100,000 ml/plate in DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (l4—498E, Lonza). The cells were incubated over night at 37 °C 5% C02 to allow the fibroblasts to attach. 1—4 days later, a sample from HAD—ClO2—hESC MCB was thawed and plated for 6—7 days at 37 OC 5% C02 on top of the feeder cells in Nutristem "Plus" Medium (which is GMP—grade and xeno—free) that contains the growth factors bFGF and TGF—B (05—102—1A, ical Industries, Israel).

On day 6—7 hESC culture was mechanically disrupted (using a sterile tip or a disposable sterile stem cell tool; 14602 Swemed) and passaged into 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 necessary amount of hESC to initiate differentiation (Figure 13, Steps 4—5). Prior to their use, expanded HAD—C102—hESCs were tested for sterility, asma, LAL, karyotype, and identity to the MCB. In addition, their pluripotent morphological appearance as well as d expression of pluripotency markers (TRA-l—60, Oct4, and alkaline phosphatase) were confirmed (Figure 2, Step 5). Production Steps 4—5 are depicted in Figure 13.

Steps 6-13: Differentiation into RPE cells. Expanded 02—hESCs were enzymatically treated with enase (4152, Worthington) for additional expansion in 6 cm cell culture plates (Figure 14, Step 6). ed HAD—C102—hESCs were then used in the derivation of the OpRegen® DS. entiation of each n® batch was initiated by mechanical transfer of collagenase A harvested clusters of HAD—ClO2—hESCs from Step 6 culture to a feeder— free non—adherent 6 cm Hydrocell culture dishes in the presence of Nutristem "Minus" Medium (that does not contain the growth factors bFGF and TGF—B; 06—5102—Ol—lA Biological Industries, Special Order) supplemented with 10 mM Nicotinamide (N—5535, Sigma) e 14, Step 7). The plates were then cultured for up to one week under low oxygen atmosphere (5%) conditions (37 ”C, 5% C02) to allow the generation of spheroid bodies. Week old id bodies in suspension were then collected, dissociated gently by pipetting, and erred to human laminin (5ll, Biolamina)— coated 6—well plates for an additional week of growth under a low oxygen atmosphere (5%) in the H presence of Nutristem "Minus Medium supplemented with 10 mM Nicotinamide (Figure 14, Step 8). The cells continued to grow under low oxygen (5%) atmosphere for an additional up to 4 weeks; two weeks in the presence Nutristem " Medium supplemented with 10 mM nicotinamide and 140 ng/ml Activin A (G- l20—l4E, Peprotech) (Figure 14, Step 9), followed by up to 2 weeks in the presence of Nutristem "Minus" Medium supplemented with only 10 mM nicotinamide (Figure 14, Step 10). When areas of light pigmentation became apparent in s of polygonal cells, plates were transferred back to normal oxygen (20%) atmosphere (37°C, 5% C02) and were grown for up to 2 weeks in the presence of Nutristem "Minus" Medium with mM Nicotinamide (Figure 14, Step 11). After up to 2 weeks, expanded nal patches with distinctive tation were apparent within areas of non—pigmented cells (Figure 14, Step 12) and remaining pigmented cells were detached and manually collected following 15 minutes TrprE Select (12563—011, Invitrogen) treatment at 37 0C (Figure 14, Step 13). Production Steps 6—13 are depicted in Figure 14.

Steps 14-17: Expansion of OpRegen® cells. Pigmented cells were then transferred to 6—well gelatin—coated plates (0.5—lx106 cells/plate; P0) for a 2—3 days of growth in the presence of DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (l4—498E, Lonza) (Figure 15, Step 14). DMEM was then ed with Nutristem “Minus" Medium and cells were grown for 2—3 weeks until the plate was covered with lightly pigmented nal cells (Figure 15, Step 14). These PO cells were then ed in n—covered flasks for an additional two passages (P1, P2).

Cells at P0 and at P1 were harvested following TrprE Select treatment at 37 °C, washed and cultured for 2-3 days on gelatin-coated flasks in the presence of DMEM supplemented with 20% human serum. DMEM was replaced with Nutristem "Minus" Medium and the cells were grown for 2—3 weeks until the plate was covered with lightly pigmented polygonal cells (Figure 15, Steps 15—16). Cells at P2 grown in T175 flasks were then harvested following TrprE Select treatment at 37 0C, re—suspended in DMEM supplemented with 20% human serum, pooled and counted.

A sample of growth medium from each batch was taken for sterility, asma, and LAL testing. The cells morphology was observed and documented (Figure 15, Step 17). Production steps 14—17 are depicted in Figure 15.

EXAMPLE 4 Process Control Points IPC points are depicted in Figure 16. The sampling points chosen to assess hESC impurity and RPE purity along the production process are bed below: IPC point 1: Mechanically expanded HAD—C 102 hESCs prior to their differentiation that have normal karyotype. This is the starting material in which the highest level of hESCs is expected. This point was added to evaluate the maximal hESC level prior to entiation.

IPC point 2: Collagenase expanded HAD—C 102 hESCs prior to their differentiation. At this stage, some differentiation is expected, and thereby a reduction in the level of cells sing Oct4 and 60 as well as in the expression level of GDF3 and TDGF. This point was added to evaluate hESC impurity during the phase of non—directed differentiation.

IPC point 3: Spheroid Bodies produced one week post induction of hESC differentiation under feeder free conditions in the presence of namide. At this earlier stage of differentiation, hESC impurity during differentiation is expected at the maximal level and thereby this assessment is expected to give an indication for the highest level of safety concern.

IPC point 4: Cells at the end of Activin A treatment. Activin A directs the differentiation towards RPE cells. At this point, a major decrease in hESC impurity and a high increase in expression of RPE markers are expected. This point was added to monitor hESC differentiation to RPE.

IPC points 5-7: Cells at the end of the differentiation s prior and post separation of the non—pigmented areas (IPC point 6) from the pigmented areas (IPC point 7). IPC points 5 and 6 are expected to contain cellular ties, while sample 7 ents the product at the end of the differentiation process prior to its expansion.

Cellular contaminations found in sample 6, may be found is small quantities in sample 7, and in r quantities in the product.

IPC point 8: Pigmented cells at P0. Pigmented cells at the end of the entiation process that were expanded for 2—3 weeks. These cells represent the product two stages prior to the end of the production process.

IPC point 9: Pigmented cells at P1. P0 cells that were expanded for 2—3 weeks.

These cells represent the product one stage prior to the end of the production process.

IPC point 10: Pigmented cells at P2 prior to cryopreservation. P1 cells that were expanded for 2—3 weeks are harvested and pooled. These cells represent the drug substance (DS) prior to cryopreservation.

IPC point 11: Cryopreserved ted cells at P2. These cells represent the drug product (DP). Throughout production, at all sampling points, cell culture medium was collected for assessment of pigment epithelium derived factor (PEDF) secretion, known to be ed from RPE cells.

RESULTS Quantification of TRA60+0ct4+hESCs: The level of hESCs in the various samples ted along the production process was determined using a highly sensitive, robust Oct4/TRA—1—6O double staining FACS method. A week following removal of feeders and growth factors that supports pluripotent cell growth (TGFB and bFGF), at growth conditions that supports early neural/eye field differentiation, there were only 0.0106—2.7% TRA—l—60+Oct4+ cells (IPC point 3, Spheroid Bodies). ing on of Activin A that promotes RPE differentiation, the level of TRA—l—60+Oct4+ cells was further deceased to 0.00048—0.0168% (IPC point 4, end of actiVin), and at the end of differentiation following excision of non—pigmented cells, the level of TRA—l-60+Oct4+ cells was 0.00033—0.03754% (IPC point 7, pigmented cells). At P0, two stages prior to the end of the production process, TRA—l— 60+Oct4+ cells in levels of 0.00009— 0.00108% (below LOD—close to LLOQ) were detected (IPC point 8). The levels of TRA60+Oct4+ cells at P1 (IPC point 9), P2 prior to cryopreservation (Drug Substance; IPC point 10), and P2 post cryopreservation (DP; IPC point 11) were below assay LLOQ (i.e. 0.00004-0.00047%, 0.00000—0.00016% and 0.00000-0.00020% respectively).

Relative expression of the pluripotency hESC markers GDF3 and TDGF: The relative expression of the pluripotency genes GDF3 and TDGF at the various IPC points along the production s was analyzed. There was a l reduction in the expression level of GDF3 and TDGF, which was correlated with the gradual reduction in the s of TRA—l—60+Oct4+ cells, along the differentiation process. At the end of P0, two stages prior to the end of the production process, P1, and P2 prior (Drug Substance) and post (Drug t) cryopreservation, the expression levels of GDF3 and TDGF were r to the level of expression seen in the negative control OpRegen® 5C cells.

Quantification of CRALBP+PMEL17+ cells: Assessment of CRALBP+PMEL17+ cells for measurement of RPE purity was ed at the end of the differentiation phase, at P0 and P2 (IPC points 8 and 11), respectively), were assessed.

As can be seen in Table 3 and in Figure 17, the level of CRALBP+PMEL17+ RPE purity at P0 (IPC point 8), two stages prior to the end of the production process, was in the range of 98.53—98.83%. Similar level of RPE purity was ed at P2 post eservation (99.61—99.76%; IPC point 11) (Table 3).

Table 3 0"MCRALBP PMM‘H+ 1 + (,ellsV IPC Point Sampling Time and Stage n-———— DP, Drug Product. *IPC point 8 was tested post cryopreservation. Internal assay controls of RPE cells (OpRegen® 5C, positive control) spiked into hESCs (HAD-C 102, negative control) demonstrated accuracy error of 325%.

Confocal imaging of Bestrophin 1, MITF, and CRALBP immunostained cells along Mock production runs 4 and 5: Cells were immunostained for the RPE markers WO 08239 Bestrophin 1, MITF, 20-1 and CRALBP at the end of the differentiation phase (IPC point 7), at the end of the expansion phase (IPC point 10, DS), and post eservation (IPC point 11, DP). Manually isolated non-pigmented cells (IPC point 6) were plated for immunostaining, but during fixation were detached from the plate and thereby could not be stained. Selected pigmented cells (IPC point 7) plated for 12 days (in mock 5 only, in parallel to cells at P0 from the ongoing production) and for 28 days were positively d for all tested RPE markers and the percent cells expressing Bestrophin 1 and MITF were 93% and 93.3—96.5%, respectively. Similar levels of Bestrophin 1 and MITF positive cells were detected at P0 (94.9% and 95.9%, respectively; tested only in mock 4), P2 prior cryopreservation, Drug Substance (92.2— 92.75% and 5.5%, respectively), and P2 post cryopreservation, Drug Product (91.1—95.7% and 83.8—94.9%, respectively; sed MITF immunostaining in mock 5 demonstrate an outlier of the randomly ed area for analysis). CRALBP (as well as ZO-l) expression was detected in all IPC 7, 10 and 11 samples (Figure 18).

Relative expression ofthe RPE markers Bestrophin 1, CRALBP and RPE65 along Mock productions 2, 4 and 5: The relative expression of the RPE genes phin 1, CRALBP and RPE65 at the s IPC points along the production process was measured. There was a gradual increase in the relative expression level of Bestrophin 1, CRALBP and RPE65 along the production process. At the end of Activin A treatment (IPC point 4), that directs the differentiation towards RPE cells, the relative levels of Bestrophin 1, CRALBP and RPE65 were 685, 36, and 325, respectively, fold higher as compared to their ve levels in mechanically passaged hESCs prior to differentiation (IPC point 1; mock 4). The relative expression levels of Bestrophin 1, CRALBP and RPE65 reached a peak from the end of the differentiation stage (IPC points 5) to the P1 stage (IPC point 9). At these stages the respective levels of expression were 5,838—11,841, 9, and 5,708—8,687, fold higher as compared to the levels in mechanically passaged hESCs prior to differentiation (IPC point 1).

Morphology assessment along Mock productions 4 and 5: Cells were analyzed for morphology at the end of the differentiation phase (IPC point 5) for estimation of the relative area of ted cells, and at the expansion phases P0—P2 (IPC points 8—10), to verify confluent polygonal morphology. The relative pigmented cellular area estimated at the end of the differentiation phase prior to excision of the non—pigmented areas (IPC point 5), was 32.5% i 13.5% (average 1 SD, n=7 wells of a 6 well plate) in mock 4 and 60% i 13% in mock 5 (average i SD, n=7 wells of a 6 well plate) (see representative images in Figure 11). Areas of pigmented cells were selected and expanded.

Morphology at the end of the ion phases PO (IPC point 8), Pl (IPC point 9), and P2 (IPC point 10) demonstrated a densely packed culture with a typical polygonal— shaped epithelial monolayer morphology (Figure 11).

PEDF secretion and potency measurement along Mock productions 4 and 5: Pigment epithelium—derived factor (PEDF), known to be secreted from RPE cells, was measured in the cell culture medium at various IPC points along mock productions 4 and 5. As can be seen in Table 4, very low levels of PEDF, in the range of 4—79 ng/mL/day, were secreted by hESCs (IPC points 1 and 2) and by spheroid bodies (IPC point 3; end of the first week with Nicotinamide). At the end of Activin A treatment (IPC point 4), that directs the differentiation s RPE cells, the level of secreted PEDF was in the range of 682-1,038 day, 31-37 fold higher ed to the level ed by spheroid bodies. Following incubation of cells at normal oxygen conditions with Nicotinamide (IPC point 5), further increase (2.2—4.6 fold) in PEDF secretion to l,482—4,746 ng/mL/day, was observed. During the expansion phase (PO—P2, IPCs 8—10, respectively), PEDF secreted levels were in the range of 2,187—8,68l day, peaking at PO—Pl.

Table 4: PEDF ion along mock productions 4 and 5.

IPC Sampling Time and Stage PEDF secretion (ng/mL/day) Range Mock 4 Mock 5 nMechanically passagcd hESCs — l Mechanically passagcd hESCs D D Mechanically passaged hESCs 4 D 22 Collagenase passaged hESCs —21 79 21-79 28 22-28 Cells at the end of Act1v1n A 682 1 038 682-1,038 treatment “——7523-7951 “——zzsv-sasl 1187-1147 OpRegen® (P2); DP 2,462 3,936 2,462-3,936 ND, Not done; NA, Not able; DS, Drug Substance; DP, Drug Product.

Tight junctions generated between RPE cells enable the generation of the blood— l barrier and a polarized PEDF and VEGF secretion. PEDF is secreted to the apical side where it acts as an anti angiogenic and neurotropic growth factor. VEGF is mainly secreted to the basal side, where it acts as a proangiogenic growth factor on the choroidal endothelium. RPE polarization (barrier on and polarized PEDF and VEGF secretion) was measured in a transwell system at the end of P0 (IPC point 8), end of P2 prior to cryopreservation (IPC point 10), and end of P2 post eservation (IPC point 11). As can be seen in Table 5, barrier function/trans-epithelial electrical resistance (TEER) and polarized secretion of PEDF and VEGF were demonstrated at all IPC points.

Table 5 Polarization Transwell- ell- . . . Transwell IPC Pomt Sampling Tlme PEDF ratio at VEGF ratio PEDF Ba 14 -TEER and Stage y Week 3 at Week 3 (ng/mL/day) 6521?; (Apical/Basal (Basal/Apical ) ) 3,229 6.72 4,55 4.72 Polarization Transwell- Transwell- Transwell IPC Point ng Time PEDF ratio at VEGF ratio PEDF Day 14 -TEER and Stage Week 3 at Week 3 (ng/mL/day)-‘21!2.£; (Apical/Basal (Basal/Apical ) ) ratio: 2.54— 2.73 2:6 3693 ND, Not Done; DS, Drug Substance; DP, Drug Product. PEDF and VEGF were measured by ELISA. PEDF day 14 was collected from the cells during their culture in a 12-well plate. Cells were then passaged onto a transwell and ed for 6 weeks, during which TEER, and secretion of VEGF and PEDF from the basal and apical sides of the transwell were measured Batch Release Testing ofRPE cells produced in Mock runs 4 and 5: To verify that n® produced in mock runs 4 and 5, is comparable to GMP produced OpRegen®, iated OpRegen® batch release testing was carried out that included morphology testing at the end of P2 prior to cryopreservation (IPC point 10, DS), and Viability, total cell number/cryovial, identity ssion of Bestrophin l and MITF), hESC impurity, and karyotyping at the end of P2 post cryopreservation (IPC point 11, DP). OpRegen® produced in Mock runs 4 and 5 passed batch release criteria.

OpRegen® produced in mock run 2 was not eserved, and thereby could not be tested.

CONCLUSION Three mock production runs (mock runs 2, 4, and 5) were carried out under research grade conditions using the same GMP—production methods, XCI’IO-fI'CC GMP— grade cells (HAD-C 102 hESCs grown on irradiated CRD008 feeders), xeno-free GMP grade reagents and GMP grade lab—ware that were used in the GMP tion of the clinical batches. Mock productions 2, 4 and 5 aimed at assessing the level of hESC impurity along the production and Mock productions 4 and 5, also aimed at identifying important in process quality ls.

Using a qualified TRA—l-60/Oct4 double ng FACS method (LOD 0.0004%, 1/250,000 and LLOQ of 0.001%, 1/ 100,000) and a qualified flow cytometer, hESC impurity in level below assay LOD was observed at the end of the differentiation phase, in the negatively selected pigmented cells, three stages prior to the end of Mock production s. In mock runs 2 and 4, performed prior to assay ication using core facility flow cytometer, the level of hESC impurity was below assay LOD two stages prior to the end of the production s. In support with this data, quantitative RT—PCR analysis demonstrated down regulated expression of the pluripotent hESC genes GDF3 and TDGF to levels similar to the negative control (OpRegen® 5C cells) two stages prior to the end of the production process.

Identity g med three stages prior to the end of production (isolation of pigmented cells) demonstrated sion of Bestrophin 1 and MITF by 93% and 96.5% of the immunostained cells, tively, as well as expression of CRALBP and ZO-l (not quantified). RPE purity testing performed one stage later (i.e. P0, 2 stages prior to the end of the production process), following one expansion cycle of the negatively selected pigmented cells, showed that > 98.5% of the cells were CRALBP+PMEL17+ double positive by FACS. Similar level of RPE purity (i.e. > 99.6%) was also detected in the drug product. These results were supported by morphology testing demonstrating typical polygonal shaped epithelial monolayer morphology and by quantitative RT—PCR analysis demonstrating upregulated expression of the RPE genes Bestrophin l, CRALBP, and RPE65 to levels similar to the positive control (OpRegen® 5C cells).

PEDF, known to be secreted from RPE cells, was measured in the cell culture medium at various stages along the production process of mock runs 4 and 5. At the end of the n A treatment (IPC point 4), previously shown by Idelson et al. 2009) to direct the differentiation towards RPE cells, the level of secreted PEDF was highly increased (31 fold in mock 4 and 37 fold in mock 5) relative to the us production step (induction of spheroid bodies). PEDF secretion levels continued to increase and peaked at P0—P1 (1.7—5.8 fold increase relative to the levels after Activin A).

Assessment of the relative area of pigmented cells at the end of the differentiation process (IPC point 5) was fied as r important quality control e for assessment of RPE differentiation. Using this measure, a 2 fold difference in the yield of pigmented cells in mock 4 and 5 runs (32.5% in mock 4 and 60% in mock 5) was observed, that was correlated with a similar difference seen in PEDF secretion at this stage (1,482 ng/ml/day in mock 4 and 4,746 ng/ml/day in mock 5).

In conclusion, no TRA—1—60+Oct4+ hESC impurity observed as early as 3 stages prior to the end of the production process. This was correlated with low expression levels of GDF3 and TDGF, high expression levels of Bestrophin 1, CRALBP and RPE65, and high levels of Bestrophin 1 and MITF single positive cells, as well as high CRALBP+PMEL17+ double positive cells (tested one stage later). Important safety and efficacy IPCs were fied at critical production stages.

EXAMPLE 5 Efficacy Assessment Experimental set-up: The present inventors examined whether subretinal transplantation of the RPE cells generated as described in Example 4 could delay the progression of RDD in the Royal College of Surgeons (RCS) rat model. ,000, 100,000 or 200,000 RPE cells were transplanted into the subretinal space of one eye of RCS rats on post—natal day (P)21—23 (prior to photoreceptor death onset); BSS+(Alcon) treated and naive untreated animals served as controls. Groups were separated into 4 survival ages: post—natal day P60, P100, P150 and P200. Fundus photography was used to identify bleb formation and monitor injection y.

Funduscopy was also performed at P60, P100, P150 and P200. Optomotor tracking was used to measure Visual acuity of all animals at all time points (P60, P100, P150, P200).

Focal and full field ERGs were ed in all study groups at P60 and P100. At the assigned sacrifice date for each , both eyes were removed, fixed in 4% paraformaldehyde, cryopreserved, ed in Optimum g Temperature compound (OCT) and cryosectioned. Cresyl Violet staining was used to fy and ate photoreceptor structural rescue. Immunofluorescent ng (IF) was used to identify transplanted cells, assess their fate, their state of proliferation, and their ability to phagocytose photoreceptor outer segments. In addition immunofluorescene was used in measurement of host cones rescue.

The study design is summarized in Table 6 herein below.

Table 6 TIME OF SACRIFICE POST INJECTION TREATMENT GROUPS Number of Mice (male and female) # at Study Initiation III-MI [Mm IIII I’ PE High Dose IIII MATERIALS AND METHODS Cell counts: Cells were counted before being aliquoted into appropriate dosage concentrations. Pre—injection cell viability for all injection time points averaged 94.0%i 0.03. Post ion cell viability ed 92.4%i0.02.

Surgery: A small incision was made through the conjunctiva and sclera using incrementally smaller gauge s: 18, 22, 25, and 30. A lateral margin puncture of the cornea was used to reduce cular pressure, to reduced egress of the injected cells. The glass e was then inserted into the subretinal space and 2 ul of suspension injected. The sclerotomy was then sutured closed. Successful injection of the cells or buffer alone (BSS+) was med first by manual visualization of a subretinal bleb, which was subsequently raphed through the use of a fundus camera (Micron III).

Optokinetic tracking thresholds: Optokinetic tracking thresholds were measured and recorded in a blinded fashion. Repeated measures ANOVA or one—way ANOVA with Fisher’s LSD post hoc analysis was used to analyze OKT data.

Electroretinagram (ERG): Two forms of ERGs were ed: an exploratory form of focal ERG where a small spot of light is used to stimulate a localized area of retina, and a standard style of full field ERG where the entire visual field is stimulated.

Histology and Immunohistochemistry: Both eyes from each animal were harvested, fixed, cryoprotected, embedded, and . Frozen blocks were cryosectioned at 12 um. Approximately 60 slides containing 4 sections per slide were obtained.

Cresyl : Cresyl violet stained sections were examined for: 1) injection site and suture, 2) evidence of photoreceptor rescue, 3) evidence of transplanted cells, 4) untoward pathology. For each slide, maximum outer nuclear layer thickness was also recorded for quantification of rescue. fluorescence (IF): RPE cell treated eye slides ed for IF were chosen from cresyl violet stained sections that contained cells in the subretinal space tent with the size and morphology of the lanted human cells. In on, protection of the host ONL was used as a secondary criterion. All IF ng was performed as dual stains with DAPI serving as a background nuclear stain. At least one slide from every cell treated animal was used for each run.

Run #1 was performed using rabbit onal Anti—Melanoma gplOO (PMEL17, Clone EPR4864; human specific, Abcam cat#ab137062) co—stained with mouse monoclonal Anti—Nuclei Marker (HuNu, Clone 3El.3, Millipore, cat#MAB4383) for detecting human RPE and non—RPE cells.

Run #2 was performed using rabbit monoclonal Anti—Ki67 (Ki67; Clone EPR3610, human specific, Abcam, cat#ab92742) and uclei Marker for detecting human proliferating cells.

Run #3 was performed using rabbit polyclonal at Cone Arrestin (Millipore cat#ab15282) to evaluate sections for cone counting (see Section 6.8.3). In addition, selected slides were stained using mouse monoclonal Anti Rhodopsin (Clone Rho 1D4, Millipore, MAB5356) in combination with PMEL17 to identify transplanted human cells containing host rhodopsin/outersegments as a measure of their phagocytic activity.

Cone Counting: Confocal z-stack images were acquired from sections of retina obtained from all cell transplanted eyes and from age—matched saline injected controls. ns from cell injected eyes were chosen in the area of photoreceptor rescue as defined using the previously evaluated cresyl violet stained sections. Cones were counted by 3 observers in a blinded fashion. The three counts were then averaged and counts compared between dosage groups and age.

Rhodopsin ingestion: A potential mechanism of rescue employed by the transplanted cells is to ingest photoreceptor outer segments and shed debris. Removal of the debris zone reduces the toxic stress on the photoreceptors and thus, aids in sustaining photoreceptor survival. Here, the present inventors selected specific animals for evaluation of sin ingestion by the RPE cells based on the cell survival and photoreceptor protection indices. This evaluation was performed using immunofluorescence.

RESULTS Fundus Imaging: Fundus images ted at necropsy of cell treated eyes revealed hyper and hypo—pigmented areas of the retina that corresponded to the location where subretinal blebs were formed during surgery; the location at which cells were deposited in the subretinal space (Figures l9A—C). These patchy areas were not evident in BSS+ injected or non—injected eyes.

Optokinetic ng thresholds: OKT thresholds were rescued in all cell treated groups at all ages (Figure 20). Cell—treated groups outperformed un—operated or saline injected eyes at all ages. There was a significant dose dependent effect between the low dose (25K) and the two larger doses (100K (p<0.0001) and 200K (p<0.0001)), especially at the later ages, but no clear benefit to the OKT from the high dose (200K) over the intermediate (100K) dose was observed 646). While OKT thresholds were rescued in all cell treated groups, the te visual acuity values slowly declined with time. Untreated and saline injected animals’ OKT thresholds continue 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.

Focal ERG: Focal ERG’s were measured in all (n=252) mental rats at ~P60. Individual animals treated with RPE cells performed well and significantly formed controls, as rated in Figure 21A.

Fullfield ERG: Full field ERG’s were measured from 125 RCS rats at P60 and from 63 RCS rats at P100. Individual animals treated with RPE cells med well and icantly outperformed controls, as illustrated in Figure 21B.

Cresyl Violet staining: An ary photomontage of a cresyl violet stained section is presented in Figure 22A. Representative images from BSS+ injected and cell treated s from multiple groups) eyes are presented in Figure 22B.

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

Immunofluorescence: Transplanted RPE cells were positively identified by immunofluorescence in s of each survival age (Figure 24), however, the number of animals with identified cells decreased as age increased. Repeat staining of additional slides in animals that did not originally reveal transplanted cells resulted in additional s identified with positive cells, but not in all cases.

Despite not g transplanted cells in all s by IF is, ONL thickness measurement results indicated 70-90% of cell treated animals had significant photoreceptor , confirmed with OKT rescue, suggesting that most treated eyes contained transplanted cells at some point. The proliferation marker Ki67 was used to identify proliferating human cells. Ki67 positive human cells were not observed (Figure 24).

Cone Counting: Cone counts in animals that received cell transplants were significantly better than control eyes (Figure 25; p=<0.0001 for each comparison). In general, there was no difference n cone counts across the low, middle and high dosage of cells. A representative image from each age is presented in Figure 24.

Rhodopsin ingestion: In each case tested (n26), fluorescently labeled rhodopsin was observed within the transplanted RPE cells (Figures 26A—J). This ms the transplanted cells do ingest outer segment debris post lantation.

Conclusion When transplanted into the subretinal space of RCS rats, RPE cells rescued Visual acuity in the RC8 rat over that of controls at all ages tested. ERG responses were protected when the graft was large enough or in an area of retina accessible for assessment. Rod and cone photoreceptors were rescued in the area of the grafts for up to 180 days post—transplantation. Collectively, this data demonstrates that OpRegen® maintain the functional and structural integrity of the host retina for ed periods.

Thus, OpRegen® hold significant potential for the treatment of human RPE cell disorders such as RP and AMD.

EXAMPLE 6 Stability ofRPE cells Short-term stability Formulated RPE cells (generated as described in Example 4) in BSS plus were prepared at a final volume of 600—1000 ul per vial. Short term stability was tested at time points 0, 4, 8 and 24 hours. Cells were found stable at all time points.

RPE cell viability and cell tration were stable at the 8 hour incubation time point for all dose formulations; percent average ity (i SD) for the ing concentrations: - Low tration (70 x103 per 100 ul BSS plus) changed from 93% i 5 at time point 0 hours to 91 % i 1 at time point 8 hours, a non-significant decrease.

- High concentration (70 x103 per 100 ul BSS plus) changed from 92% i 3 at time point 0 hours to 91 % i 2 at time point 8 hours, a non—significant decrease.

For the medium concentration (250 x103 per 100 pl BSS plus) that was tested there was no significant change throughout the time points.

The overall range for all time points and formulated doses was between 88% — 97% from time point 0 hours to 8 hours, when ing all results for time point 0 hours (93% i 3) and time point 8 hours (91 % i 1) a decrease of 2% was found.

No significant changes in the cell concentration were observed, in either time points or formulated doses. Cell concentration did not change in all 3 studies other than a small decrease seen in one batch in the high dose (2%).

Appearance of the different dose formulations did not change throughout the tested time points; cell suspension was free of foreign particles and non—dissociated aggregates.

Identity and purity of each formulated RPE cell dose at all tested time points were stable up to 24 hours and were within the batch e criteria. At 8 hours (for all formulated RPE cell doses), the level of MITF and Bestrophin positive cells was in the range of 86—97% and 90—94%, respectively, and the level of +PMEL17+ double positive cells was in the range of 98.35—99.64%.

WO 08239 2015/051269 Formulated RPE cell doses maintained their potency in all tested time points (4, 8, 24 hours), both secreting high levels of PEDF and forming a polarized RPE monolayer with a polarized secretion of PEDF predominantly to the apical side and VEGF to the basal side. Results for the tested time points 8 hours: TEER was in the range of 376 — 724 ohms, PEDF apical to basal ratio in the range of 2.77 - 5.70 and VEGF basal to apical ratio in the range of 2.04 — 3.88.

Sterility was kept at all incubation time points for all cell dose formulations.

These results support OpRegen® cell stability in final formulation at all clinical doses for at least 8 hours when kept at 2—8°C. A safety margin of up to 24 hours exists based on l data collected (identity, sterility, and medium dose potency).

Results of the short term stability assay are summarized in Table 7 below.

Table 7 MID DOSE HIGHDOSE ANCE DOSE 250,000 700,000 CRITERIA 70,000 cells/100 ml cells/100 ml cells/100 ml Cell Viability 2 70% 91 i1 (11:3) 92 (11:1) 91 i 1.5 (11:3) i 40%from initial 91.3 i 30 Cell Dose 103 (n—1)_ 104 i 5.7 (n—3)_ dose (11:3) MITF Positive Cells 2 80% 90 (n:2) 93 (n=1) 96 (n:2) phin 1 Positive Cells 2 80% 94 (n:2) 92 (n=l) 92 (11:2) CRALBP+PMEL17+ Cells 2 95% 9951:3215 99.5 (11:1) 99 i 0.65 (11:3) Barrier Function, TER ((2) 605 (n:2) 724 (n=1) 410 (n:2) Polarized PEDF Secretion 3501—1)_ 4'5 ("—2)_ (Apical/Basal) For Information Only Polarized VEGF Secretion 2’2 ("—1)_ 2'3 ("—2)_ (Basal/Apical) Sterility USP<71> Negative Negative No foreign particles Appearance and/or non-dissociated Pass Pass Pass aggregates Long-term stability: Three s of RPE cells were frozen in vapor phase liquid nitrogen. Testing of the long—term stability in eservation started after the ng date. Results provided are following three years of freezing. The following parameters are being tested: Viability, cell number, RPE identity (% Bestrophin l and % MITF ve cells), RPE purity (FACS% CRALBP+PMEL17+ RPE cells), potency (polarization and PEDF secretion), karyotype analysis and sterility. At each time point, the ed number of Vials are thawed and the cells are ed for the assays as bed herein.

Results of the long term stability assay are summarized in Table 8 below.

Table 8 TEST 0-3 Months 19-21 Months 34-36 Months Cell Viability 86 i 2 (n=3) 87 i- 4 (n=5) 89 i- 2 (n=6) Total Cells/Vial 1.44 i 0.13 (n=3) 1.13 i 0.2 (n=5) 1.13 i 0.2 (n=6) —--86(11-2)Identity: MITF Positive _ Bestrophin 1 . CRALBP PMEL17- + + 99.8 NA 994 Cells Pgtency: Barner Function, TER 616 368 396 i 200 (n=3) Polarized PEDF Secretion 3.93 3.86 3.05 i 0.04 (n—3)_ (Apical/Basal) Polarized VEGF Secretion Safety: Karyotyping ity USP<71> RESULTS Viability, total cell number/vial and RPE identity were ined throughout the three year period. In addition, as indicated, data demonstrated potency and purity at levels similar to the ones collected prior to preservation.

A normal karyotype was observed 4 years post cryopreservation. This indicates that long—terrn storage in vapor phase thus far did not have any deleterious effects on RPE genomic stability.

Sample sterility was demonstrated by testing for the absence of bacterial/fungal growth in all clinical batches at 3 months. Another batch was tested negative 4 years post cryopreservation. Based on these uniformly acceptable stability results, covering a period of three years of ity testing thus far, it is concluded that the RPE cellular product is stable for at least three years when stored at a temperature 3 —180°C in the vapor phase of liquid nitrogen.

EXAMPLE 7 Safety and Biodistribution The objectives of the study were to evaluate survival, biodistribution, and safety of RPE cells ated as described in Example 4) following subretinal stration in male and female ID mice over a 6-month study duration.

NOD—SCID mice (NOD.CBl7—Prkdcscid), 5—6 weeks of age at the time of injection, were injected with either BSS Plus (Vehicle l) or with two doses of RPE cells: 50x103 cells or 100x103 cells (maximal feasible dose), suspended in l uL BSS Plus. RPE was administered into the subretina via the transvitreal route (the proposed clinical route of stration) using a 33G Hamilton needle. A single dose of 50x103 cells or 100x103 cells was ed to one eye, while the fellow eye served as an internal control. Each dosing session contained mice (males and females) from each group. Mice included in the study after pretest, were randomly assigned to the various test . Two randomizations were performed. A measured value randomization procedure, by weight, was used for placement into treatment groups prior to vehicle/test article administration. Following administration, s suitable for use on study were transferred to the target study using a sequential randomization for placement into the final treatment groups. Mice with ocular abnormalities, al clinical observations or weighing less than 16 gram at pretest and mice undergoing non—successful subretinal RPE injection were excluded from the study.

Study Measurements: Assessment of RPE safety in this study was based on animal mortality, clinical observations, body weight, ophthalmologic examinations, clinical pathology (hematology and blood chemistry), gross ogical macroscopic evaluations, organ weights ute and relative to body and brain weights), histopathological evaluation of eyes and various . Assessment of survival and biodistribution of RPE was performed by histopathological and fluorescence immunostaining evaluations of eyes and various organs and qPCR analysis. The following measurements were performed: - Clinical observation; - Body weight; - Ophthalmologic examinations (including macroscopic and biomicroscopic examinations); WO 08239 - Surgical microscopic examination of subretinal injection y using the LEICA M80 Stereo microscope (funduscopy); 0 Complete blood count and blood chemistry; - Necropsy and gross pathology; - Organ weight (absolute and relative to body and brain weights); - Collection, fixation, and paraffin blocking of d and non—treated lateral eyes including optic nerve; - d H&E histopathology of eyes and tissues (sternum bone with bone marrow, brain, heart, kidneys, liver, lung, mandibular lymph nodes, spinal cord, spleen, thymus, masses and gross lesions); - Blinded semi quantitation of pigmented cells in H&E stained slides; - Blinded immunostaining of selected slides nt to a representative H&E slide demonstrating pigmented cell graft in the eye for a human marker (human nuclei) plus an RPE marker (human PMEL17) and assessment of human RPE and non-RPE cells, human marker (human nuclei) plus a proliferation marker (human Ki67) and assessment of human and non-human proliferating cells, and RPE marker ) plus proliferation marker (human Ki67) and assessment of RPE and non—RPE human proliferating cells; - Blinded immunostaining of selected slides adjacent to a representative H&E slide demonstrating teratoma, tumor, al cells and lesions for a human marker (human nuclei) to exclude human origin; - Collection and extraction of genomic DNA from blood, bone marrow (collected from femurs), brain, left and right eyes with optic nerves, heart, left and right kidneys, liver, lung, mandibular lymph nodes, ovaries, skeletal biceps femoris muscle, spinal cord, spleen, testes, and thymus and qPCR analysis of human beta globin; - H&E histopathology on tissues (other than the above) found positive for human beta globin in animals from the same group and time point.

RESULTS There were no RPE—related toxicologic findings in the e examinations which included detailed clinical observation, body weight, ophthalmologic examination and clinical ogy sed of hematology and serum clinical chemistry. The WO 08239 ation of “Eye discolored, dark” in the left eye with an albino background was found in mice treated with pigmented RPE cells at both dose levels in the detailed clinical observation and ophthalmologic examination. lmologic examination of the surviving animals indicated that this ation consisted of treal, darkly pigmented foci. The pigmented foci were distributed randomly along a line extending from the temporal ior lens capsule to the nasal retinal surface. These foci were reted to be RPE cells ng from the injection a upon its removal from the eye following injection, as supported by the vitreal reflux seen during injection or RPE cells leaking into the vitreous humor subsequent to subretinal implantation.

All of the ocular lesions observed on this study were considered to arise secondary to anesthesia, the surgical injection procedure, or incidentally as age—related changes. The finding of multiple pigmented foci within the vitreous humor suggests that RPE cells may be viable within the vitreous body. The presence of pigmented cells in the vitreous body in some of the RPE-treated animals was confirmed at the microscopic level.

In terms of biodistribution as evaluated by qPCR using a set of human beta- globin gene probe/primers, at the 2-week, 2—month, and 6—month intervals, the left eyes treated with 100x103 OpRegen® cells were positive for RPE DNA in 8/12, 11/12, and 16/16 animals with group mean levels at 38, 47 and 249 copies/ug total eye DNA, respectively, ting a trend of increase over time. There was no significant difference between males and females. In these animals, RPE DNA was not detected in the untreated right eyes and all the non—eye tissues, which included blood, femoral bone marrow, brain, heart, kidneys, liver, lung, mandibular lymph nodes, ovaries, skeletal biceps femoris muscle, spinal cord, spleen, testes, and thymus, except for the spinal cord (27 copies/ug DNA) from one 2—week male animal and the skeletal muscle (16 copies/ug DNA) and spinal cord (below level of qualification) from one 2—week female animal (probably due to inadvertent contamination by ous human DNA during DNA tion from these tissues).

RPE-related macroscopic changes were limited to black discoloration or black foci in the left eye of a few animals at the 2 and 6—month intervals, consistent with in— life clinical observation and/or ophthalmologic examination. These changes correlated to pigmented cells and were not considered adverse as determined by microscopic WO 08239 examination of surviving animals in the high—dose group and of the animals euthanized in extremis and found dead in both dose groups. Pigmented cells were present in the treated left eye in nearly all of the surviving mice examined at each time point in the high dose group (at the subretinal space in 11/12, 12/12 and 16/16 in the 2—week, 2— month, and 6—month intervals), as well as the animals euthanized in extremis or found dead in both low and high dose groups. The most common locations of the pigmented cells were the subretinal space and the vitreous body as confirmed by immunostaining of human cell— and RPE—specific kers. In the subretinal space, pigmented cells tended to be restricted to the injection site at the earlier time points, whereas at the later time points they were present at locations distant from the injection sites, suggesting local cell spreading. There was a slight increase in average total number of pigmented cells per eye at the h time point compared to 2—week or 2—month time points in males. This sed number of pigmented cells of human origin was supported by the qPCR analysis.

Long—term engraftment of the RPE cells is illustrated in Figure 27A. ted cells stain ve for Human Nuclei and PMEL17 in NOD-SCID subretinal space 9 months post transplant.

Figure 27B is a photograph illustrating the clustered at the place bleb following injection. Figure 27C is a photograph illustrating the subsequent spreading of the cells into a monolayer following ion.

RPE was not associated with any organ weight changes. There were no macroscopic and microscopic changes in the untreated right eyes and the non—eye organs examined in this study which included brain, heart, kidneys, liver, lung, mandibular lymph nodes, spinal cord, spleen, and thymus. uman nuclei ker antibody stain (Human Nuclei) was observed in 64%, 36%, and 73% of the tested left eyes at 2—week, h, and 6—month time points, respectively, in the animals examined in the high dose group.

The highest detection level for Human Nuclei was noted in pigmented cell populations within the subretinal space followed by the vitreous body. Anti—human RPE—specific biomarker PMEL17 staining was observed in most of the s tested s another RPE—specific biomarker, RPE65, had various levels of detection at the different time points. These RPE—specific biomarkers were mostly detected in the subretinal space and less in the vitreous body. Human cell proliferation biomarker Ki67 was detected in only a few cells in a small number of animals, mainly in ted cells within the vitreous body and less within the subretinal space. The incidence of Ki67 positivity decreased over time with only one animal at 6 month. The Ki67—positive cells were not associated with any abnormal morphology.

Several microscopic changes were noted at the injection site across all the time points and all the study groups and considered related to the surgical injection procedure. Some of these changes were slightly more prominent in s examined in the high dose group at 6 months. For example, retinal detachment was noted in one animal and the incidence or ty of retinal degeneration/atrophy or fibroplasia was slightly increased compared to the vehicle control group.

There were no RPE—dependent effects on animal mortality rate and survival.

Conclusion No local or systemic toxicologic, lethal, or tumorigenic effects were observed in the NOD/SCID animal model during the 6—month study period following single injection of RPE at dose levels of up to 100,000 cells/ul/eye. Biodistribution of RPE cells was restricted to the d left eye with local subretinal cell spreading from the subretinal injection site as a function of time. RPE cells were present predominantly in the subretinal space followed by the vitreous body in most of the animals examined in the high dose group at 2—week, 2—month, and 6—month intervals, with variable vity in staining by antibodies against the human nuclei and/or human RPE—specific biomarkers. The persistence of RPE cells in the eye was ted to be at least 6 months with very limited cell proliferation. The limited proliferation took place mostly in the vitreous body and had no adverse effects. There was evidence that the number of RPE cells increased in the treated eye over time, although this was accompanied by decreased proliferation incidence in the inal population examined. Expression of both RPE specific s RPE65 and PMEL17 was predominantly in RPE cells within the subretinal space as opposed to those within the us body, where most of Ki67— positive cell nces were found. The latter suggests that the increase in RPE cells over time is limited to the vitreous space and that the expression of specific RPE65 and PMEL17 RPE markers may be regulated by the microenvironment. In conclusion, based on the data presented above, there are no serious safety concerns d to the injection of the presently described RPE cells as compared to vehicle control group.

EXAMPLE 8 Expression 0fPax-6 in the RPE cells Objective: Development of a FACS based method for assessing the level of PAX—6 in human retinal pigment epithelial (RPE) cells.

MATERIALS AND METHODS Frozen RPE cells (generated as described in Example 4, were thawed spun down, re—suspended in 1 ml PBS minus, filtered through a 35pM cell strainer and counted with the NC—2OO cell counter. The cell concentration was adjusted to ~lx106 cells/ml in PBS minus. 1 ill/ml FVS450 was added to each ml cell sion followed by vortexing and tion for 6 minutes at 37 0C. FVS450 was quenched with 0.1% BSA(-Ig)-PBS minus, and pended in 0.1% BSA(-Ig)-Fc-block (5 min at RT) to block all Fc—epitopes on the cells. Cells were then fixed and stained with anti—Pax—6 antibody (AF647 Cat#562249).

RESULTS As can be seen in Figure 29, cells at P0 and P2 are positive for PAX6 (81.5%- 82.5% at PO and 91.3%—96.l% at P2). P2 is the passage at the end of the production process and P0 is two expansion stages earlier. The data was shown to be consistent across batches, as shown in Figures 29 and 30. In on, the present inventors showed by FACS analysis that the RPE cells double stained for PAX—6 and CRALBP (Figure 31).

EXAMPLE 9 Identification ofproteins secreted by the RPE cells Objective: To identify a signature of proteins (known and new) ed by the n® (RPE cells) that can be used as a batch release potency assay as well as a process control assay.

Supernatants were collected from RPE cells (generated as described in Example 3) that were cultured under different culture conditions indicated below. Supernatants were then screened using the G6 and G7 RayBiotech arrays according to manufacturer’s ctions after an overnight tion of the supematants with the related array. 1. RPE drug product cells post thawing cultured for 4 and 14 days on 12—well plate 06 cells/well at Passage 3) (referred to herein as OpRegen®). 2. RPE drug product cells post thawing cultured for 14 days on 12-well plate and then cultured for 3 weeks on a Transwell (as per AM—RPE—15) and demonstrated TEER >500Q. Supernatants were taken from the apical and basal chambers. 3. Cells generated according to the protocol bed in Example 3, prior (QC3) and post (QC4) Activin A ent. 4. Nutristem medium (Nut—) without addition of TGFB and FGF.

Supernatants were also collected from the ing cell cultures and tested by ELISA: l. OpRegen® drug product cells post thawing that were each cultured for 14 days on 12—well plate and then ed for 3 weeks on a Transwell (as per AM—RPE— ) and demonstrated TEER of 355Q and 5059, respectively. Supernatants were taken from day 14 (passage 3) and from the apical and basal chambers. 2. RPE 7 cells post thawing that were cultured for 14 days on 12—well plate (0.5x106 cells/well at Passage 3). 3. Mock VI cells at the end of Passage 1 of the production process that were grown on 1aminin52l following Enzymatic or Mechanical isolation (as described in Example 3). These cells were tested for potency as per AM—RPE—15 and supematants were collected from cells at Day 14 on 12 well plate (passage 2) and cells after 3 weeks on transwell from the apical and basal chambers. 4. Fetal HuRPE cells at Passage 3 Days 4 and 14 106 cells/well).

ELISA test validation was performed according to manufacturer’s instructions related to each ELISA kit. In each protocol, tion with the supernatants was overnight.

Study design: Supernatants were ted from the cells that were cultured under different culture conditions and kept at -80°C. Following protein array analysis, tion of the hits was measured by ELISA. 2015/051269 RESULTS The G7 array results are provided in Table 9 herein below.

Table9 —-—-G7 Nut (-) Day4 Day14 Alical TWBasal QC3 QC4 POS 185132185132 AgRP 56 62 72 94 Axl 15 100 365 103 BTC 41 46 47 127 59 —-_—m73 Dtk 16 17 21 24 GITR-Ligand 47 52 50 56 GRO-alha 65 79 64 85 ICAM-l 13 24 27 106 56 IGFBP-6 13 172 39 167 107 66 IL-11 54 51 60 n- 64 IL-1270 15 27 19 20 2015/051269 \] 1.1 -—-—«69 IL-8 107 113 237 135 226 I-TAC 14 23 1s 24 3,736 MIP-lbeta 18 20 171,056 —-_—n15 Osteo-rote_erin 16 4,622 191 830 593 sTNFRII 13 12 13 10 1,793 TIMP—2 15 571 621 1,937 753 483 776 uPAR 68 161 67 148 276 87 546 592 VEGF-D 20 23 20 19 The G6 array results are provided in Table 10 herein below.

Table 10 W W .- G6 14 Apical Basal QC3 QC4 POS 12,843 12,843 12,843 12,84312,843 NEG 20 Anuioenin 3,152 423 1,749 2,838 3,574 BDNF 12 nun BLC 18 11 17 BMP—4 9 null BMP-6 4 CK beta 8-1 CNTF Eotaxin E0taxin—2 E0taxin—3 FGF-6 FGF-7 Flt-3 Ligand Fractalkjne 6 3 6 GCP-2 8 8 9 GDNF 10 11 12 GM-CSF 63 52 58 I-309 5 7 9 IFN-gamma 96 77 72 IGFBP—1 7 19 21 2 10 IGFBP—4 9 11 IGF-I 9 13 IL-10 59 59 IL- 1 3 81 77 IL-1 5 56 55 62 IL- 1 6 3 3 1 IL- 1 alpha 77 76 63 72 78 IL- 1 beta 8 12 16 12 8 n- IL- 1ra 65 58 68 58 60 IL-2 54 53 62 51 54 190 IL-3 56 49 52 50 52 177 IL-4 7 6 7 —n- IL-5 81 79 82 67 87 -m IL-6 280 1,053 386 377 IL-7 64 56 62 59 63 Leptin 15 19 14 17 15 LIGHT 8 12 MCP- l 67 1,460 4,269 3,963 5,061 MCP-2 16 19 22 22 22 MCP-3 8 10 MCP-4 9 11 M-CSF 19 18 MDC 9 8 2015/051269 MIP- l -delta MIPa1 ha NAP-2 NT-3 PARC PDGF-BB RANTES SDF-l TARC TGF-beta 1 TGF-beta 3 TNF-alpha TNF-beta RPE secreted proteins can be divided into 3 functional groups: 1) Angiogenic ns such as VEGF and Angiogenin, 2) Extracellular matrix regulators such as TIMP—1 and TIMP-2, and 3) Immunomodulatory proteins such as IL—6, MIF, sgpl30, sTNF—Rl, —R3, MCP—l, and Osteoprotegerin. The or tyrosine kinase Axl was also found to be secreted by the RPE cells. 6 ns that demonstrated high levels of secretion and/or demonstrated a polarized secretion l/basal) pattern were selected for validation by ELISA (angiogenin, TlMP—2, MIF, sgpl30, sTNF—Rl and -R3). The array data also demonstrated secretion of VEGF as seen in the polarization assay.

Angiogenin: Protein array data demonstrated increased secretion of angiogenin along the production process (Tables 9 and 10). These results were confirmed by ELISA demonstrating that the level of angiogenin secreted by differentiating cells that were treated with nicotinamide prior to the addition of Activin A was 0.52 ng/mL, whereas after the 2 weeks treatment with nicotinamide and n A, agiogenin secretion level increased to 0.91 ng/mL (Figure 32A). RPE cells which were cultured for 2 weeks in a 12 well plate 106 cells/well; Passage 3) post thawing secreted angiogenin (Figure 32B). Polarized RPE cells (week 3 on transwell; TEER > 35052, PEDF apical/basal and VEGF basal/apical ratios >1) secreted angiogenin in a polarized manner to the basal side with low to no secretion to the apical side (basal angiogenin levels were in the range of 01—025 ng/mL and apical angiogenin levels in the range of 0.05—0.12 ng/mL; Figure 32B). RPE 7 cells generated ing to Idelson et al., 2009 were unable to te barrier function in the transwell system (TEER below 1009) gh could secrete VEGF and PEDF. The ability of RPE7 cells to secrete angiogenin was tested when plated in a 12 well plate for 14 days. RPE7 secreted angiogenin on day 14 of culture in a level that is within the range of the RPE cells generated as bed herein (Figure 32C).

TIMP-1 and TIMP-2 Secretion: Protein array screen demonstrated secretion of TIMP—1 and TIMP-2 from polarized and non—polarized RPE cells e 33A—E).

Interestingly, the array data showed polarized secretion of TlMP—2 to the apical side and TIMP—l to the basal side (Figure 33A). ELISA data confirmed that TIMP—2 is secreted mainly to the apical side by all RPE batches tested so far (Figures 33C—D apical range of 69.9 — ll3.3 ng/mL and basal range of 11.9 — 43.7 ng/mL). TIMP—2 was also ed by non-polarized OpRegen® cells in levels similar to the levels secreted by normal human fetal RPE cells (HuRPE, ScienCell) (Figures 33C—E). RPE 7 cells also secreted TIMP—2 in levels similar to the OpRegen® cells (Figures 33C-E). Interestingly, very low levels of TIMP—2 were detected along the production process at QC3 and QC4 oints (Figure 33B).

Sgp130 Secretion by ® 0pRegen Cells: Protein array data demonstrated increased secretion of sgpl30 along OpRegen® production process as seen in the IPC/QC check points 3 and 4 (Tables 9 and 10). ELISA data med higher levels of sgpl30 secretion following 2 weeks Activin A treatment C4; 1.64 ng/mL) as compared to the levels secreted by the cells following nicotinamide treatment prior to the addition of n A (IPC/QC3; 0.68 ng/mL) (Figure 34A). OpRegen® cells which were cultured for 2 weeks in a 12 well plate (0.5){106 cells/well; Passage 3) post thawing secreted sgpl30 (Figures 34B—C). RPE 7 cells cultured under similar conditions secreted sgp130 in levels that were within the range of OpRegen® cells (1.0 ng/mL at day 14; Figure 34D). Fetal HuRPE cells secreted low sgpl30 levels both on day 4 and on day 14.

Polarized OpRegen® cells secreted sgpl30 in a polarized manner to the apical side with low to no secretion to the basal side (apical sgpl30 secretion levels were n .06 ng/mL and basal sgp130 levels were in the range of 0—0.2 ng/mL; Figures 34B—C).

Shed sTNF-RI: Very low levels of shed sTNF-Rl were detected by ELISA in the supernatant of differentiating cells prior (IPC/QC3 0.01ng/mL) and post two weeks treatment with nicotinamide and Activin A (IPC/QC4 0.02 ng/mL) e 35A).

OpRegen® cells which were cultured for 2 weeks in a 12 well plate 106 cells/well; Passage 3) post thawing contained sTNF—Rl in the atant of culture day 14 (Figures 35B—C). HuRPE cells cultured under similar ions had similar levels of sTNF—Rl in their culture supernatant while RPE 7 cells demonstrated relatively low sTNF—Rl levels (Figure 35D).

Polarized OpRegen® cells secreted shed sTNF—Rl in higher levels to the apical side (apical and basal sTNF—Rl levels were in the range of 0.22—1.83 ng/mL and 0.01— 0.11 ng/mL, respectively; Figures 35C—D). sTRAIL-R3: Protein array data detected sTRAIL-R3 in the atant of OpRegen® cells (Tables 9 and 10). ELISA confirmed the presence of sTRAIL—R3 along OpRegen® production process (493 pg/mL in QC3 and 238 pg/mL in QC4). In fetal HuRPE culture there was no sTRAIL—R3 and in RPE 7 culture, very low levels of sTRAIL—R3 (4 pg/mL).

Detection of MIF: Protein array data detected MIF in the supernatant of OpRegen® cells s 9 and 10). ELISA confirmed the presence of MIF along n® production process (100.3 ng/mL in QC3 and 44.7 ng/mL in QC4).

Polarized OpRegen® cells demonstrated higher levels of MIF in the apical side (apical MIF levels in the range of 266—1383 ng/mL and basal in the range of 19—305 ng/mL).

EXAMPLE 10 Comparison of OpRegen® to RPE] & RPE7 Objective: To compare OpRegen® (RPE cells) with RPE cells generated according to the protocol of Idelson et al, 2009.

MATERIALS AND METHODS OpRegen® (RPE cells) were generated as bed in Example 3.

RPE cells were generated according to the protocol of Idelson et a1, 2009 and named RPEl and RPE7.

WO 08239 A transwell system (as rated in Figure 28) was used to enable the development of a polarized RPE monolayer with stable r properties and polarized PEDF and VEGF secretion. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of the RPE monolayer, and Enzyme—Linked Immunosorbent Assay (ELISA) was used to assess polarized PEDF and VEGF secretion. Cells were thawed and cultured for 14 days in the presence of Nicotinamide.

PEDF secretion was tested on days 7 and 14. Then cells were transferred to a transwell (Costar 3460, 0.4nm) for additional 4 weeks during which TEER was measured and medium was collected (for assessment of cytokine ion) from the upper and lower transwell chambers on a weekly basis up to 4 weeks. When the cells are polarized, TEER should be above 100 Q and the ratio between the apical to basal PEDF secretion and the basal to apical VEGF secretion should be above 1.

All OpRegen® batches that were tested demonstrated the ability to te barrier function (TEER range of 368-688 9) and secrete PEDF and VEGF in a polarized manner (Apical/Basal PEDF ratio ranged from 3.47—8.75 and Basal/Apical VEGF ratio of .74) (see Table 11).

Table 11 Non-GMP Mock OpRegen® Clinical- OpRegen® GMP Produced RPE Criteri Grade Batches Research-Grade Batches Productlog According to a for OpRegen Idelson et 21., release Batches 2009 RPE Purity ELI 7+ Polarizati on 7 TEER at Week 3 PEDF Apical/Ba sal Ratio at Week 3 informa VEGF tion Basal/Api only cal Ratio PEDF secretion day 14 ND: Not determined since TEER was below 100 S2 and big holes were seen in the culture 2015/051269 RPEl and RP7, that were produced under GMP conditions according to Idelson et al (2009) were unable to generate barrier function (TEER < 100 Q) in 3 independent studies. Cells seeded on the transwell were unable to generate a homogeneous closed polygonal monolayer and big holes were seen (Figure 36). Although the cells could not te barrier function, RPEl and RPE7 could secrete PEDF (see Table 11) and VEGF (not shown) in levels r to OpRegen® and their level of CRALBPJ'PMEL17+ purity was 99.91% and 96.29%, respectively, similar to n® (Figure 37).

Based on these data, it may be concluded that RPEl and RPE7 are defective in their ability to generate tight on. gh the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and ions 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.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, 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 that section headings are used, they should not be construed as necessarily limiting.

Claims (46)

WHAT IS CLAIMED IS:

1. A population of human polygonal RPE cells, wherein at least 95 % of the cells thereof co—express premelanosome protein (PMEL17) and cellular retinaldehyde binding n P), wherein the trans—epithelial electrical resistance of the population of cells is greater than 100 ohms.

2. A population of human RPE cells, wherein at least 80 % of the cells f co—express premelanosome protein (PMEL17) and cellular ldehyde binding protein (CRALBP) and wherein cells of the population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), e glycoprotein 130 (sgpl30) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor—0t (sTNF— R1).

3. The cell population of claim 1, wherein cells of the population secrete each of angiogenin, tissue tor of metalloproteinase 2 (TIMP 2), e glycoprotein 130 0) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor—0c (sTNF—Rl).

4. The cell population of claims 2 or 3, wherein the cells secrete said angiogenin, said TIMP2, said sgp130 or said sTNF—Rl in a polarized manner.

5. The cell population of claims 2 or 3, wherein the cells secrete each of said angiogenin, said TIMP2, said sgpl30 and said sTNF—Rl in a polarized manner.

6. The cell population of claims 4 or 5, wherein the ratio of apical secretion of sgpl30: basal secretion of sgpl30 is greater than 1.

7. The cell population of claims 4 or 5, wherein the ratio of apical secretion of l: basal secretion of sTNF—Rl is greater than 1.

8. The cell population of claims 4 or 5, wherein the ratio of basal secretion of angiogenin: apical secretion of angiogenin is greater than 1.

9. The cell population of claims 4 or 5, wherein the ratio of apical secretion of TIMPZ: basal ion of TIMP2 is greater than 1.

10. The cell tion of claims 1 or 2, wherein the number of Oct4+TRA—1— 60+ cells in the population is below 1:250,000.

11. The cell tion of any one of claims 1—10, wherein at least 80 % of the cells express Bestrophin 1, as measured by staining.

12. The cell population of any one of claims 1—11, wherein at least 80 % of the cells express Microphthalmia—associated transcription factor {MITF), as measured by immunostaining.

13. The cell population of any one of claims 1—12, wherein more than 50 % of the cells express paired box gene 6 (FAX—6) as measured by FACS.

14. The cell population of any one of claim 1—13, wherein the cells secrete greater than 750 ng of Pigment epithelium—derived factor (PEDF) per ml per day.

15. The cell population of any one of claim 1—14, wherein the cells secrete PEDF and ar endothelial growth factor (VEGF) in a polarized manner.

16. The cell population of claim 15, wherein the ratio of apical secretion of PEDF: basal secretion of PEDF is greater than 1.

17. The cell population of claim 16, n said ratio remains greater than 1 following incubation for 8 hours at 2—8 ° C.

18. The cell population of claim 2, wherein the trans—epithelial electrical resistance of the population of cells is greater than 100 ohms.

19. The cell population of claim 1 or 18, wherein said trans—epithelial electrical resistance of the cells remains greater than 100 ohms following incubation for 8 hours at 2—8 ° C.

20. The cell population of claims 15 or 16, wherein the ratio of basal ion of VEGF: apical secretion of VEGF is greater than 1.

21. The cell population of claim 20, wherein said ratio remains greater than 1 following incubation for 8 hours at 2—8 0 C.

22. The cell tion of any one of claims 1—21, being capable of rescuing visual acuity in the RC5 rat following subretinal administration.

23. The cell tion of any one of claims 1—21, being capable of rescuing photoreceptors for at least 180 days post—subretinal administration in the RC8 rat.

24. The cell population of any one of claims 1—23, being generated by ex— Vivo entiation of human embryonic stem cells.

25. The cell population of any one of claims 1—24, being generated by: (a) culturing human embryonic stem cells in a medium comprising nicotinamide so as to te differentiating cells, wherein said medium is devoid of activin A; (b) culturing said differentiating cells in a medium comprising nicotinamide and n A to generate cells which are further differentiated towards the RPE lineage; (c) culturing said cells which are further differentiated towards the RPE lineage in a medium comprising nicotinamide, wherein said medium is devoid of activin

26. The cell population of claim 25. wherein said embryonic: stem. cells, are propagated in a medium comprising bFGF and ’IGFB.

27. The cell population of claim 25, wherein said embryonic stem cells are cultured on human cord fibroblasts.

28. The cell population of claims , wherein steps (a)—(c) are effected under ions wherein the heric oxygen level is less than about 10 %.

29. The cell population of claim 28, wherein the method further comprises culturing said differentiated cells in a medium under conditions wherein the atmospheric oxygen level is greater than about 10 % in the presence of nicotinamide ing step (c).

30. A pharmaceutical composition comprising the cell population of any one of claims 1—29, as the active agent and a pharmaceutically acceptable carrier.

31. Use of the cell population of any one of claims 1—30, for treating a retinal degeneration.

32. A method of generating RPE cells comprising: (a) culturing otent stem cells in a medium comprising a entiating agent so as to generate differentiating cells, wherein said medium is devoid of a member of the transforming growth factor B (TGF [3) superfamily; (b) culturing said differentiating cells in a medium comprising said member of the transforming growth factor B (TGF [3) superfamily and said differentiating agent to generate cells which are r differentiated towards the RPE lineage; (c) culturing said cells which are further differentiated towards the RPE lineage in a medium comprising a differentiating agent so as to generate RPE cells, wherein said medium is devoid of a member of the transforming growth factor B (TGF [3) superfamily, n steps (a)-(c) are effected under conditions wherein the atmospheric oxygen level is less than about 10 %.

33. The method of claim 32, wherein step (a) is effected under non—adherent conditions.

34. The method of claim 33, wherein said non—adherent conditions comprise a non—adherent culture plate.

35. The method of claim 32, wherein step (a) comprises: i) culturing said cultured population of human otent stem cells in a medium comprising nicotinamide, in the absence of activin A; under non—adherent conditions to generate a r of cells comprising differentiating cells; and subsequently ii) culturing said differentiating cells of (i) in a medium comprising nicotinamide, in the absence of activin A under adherent conditions.

36. The method of claim 35, further comprising dissociating said cluster of cells prior to step (ii) to generate clumps of cells or a single cell suspension of cells.

37. The method of claim 32, further comprising culturing said differentiated cells in a medium under conditions wherein the atmospheric oxygen level is greater than about 10 % in the presence of a entiating agent following step (c).

38. The method of claim 32, wherein said member of the transforming growth factor B (TGF B) superfamily is selected from the group consisting of TGFBl, TGFB3 and activin A.

39. The method of claim 32, wherein said differentiating agent of step (a) and said differentiating agent of step (c) are identical.

40. The method of claim 32, wherein said differentiating agent of step (a) is namide (NA) or 3— aminobenzamide.

41. The method of claim 32, further comprising selecting polygonal cells following step (c).

42. The method of claim 41, further comprising propagating said polygonal cells.

43. The method of claim 42, n said propagating is effected on an adherent surface.

44-. The method of claim 32, n said pluripotent stem cells comprise. embryonic stem cells.

45. The method of Claim 44, wherein said embryonic stem cells are propagated in a medium comprising bFGF and, TGFB.

46. The method of claim 44, wherein said embryonic stem cells are cultured on human cord fibroblasts.