RPE cell populations and methods of generating the same

Abstract

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

Description

Technical Field

[0001] In some aspects thereof, the present invention relates to retinal pigment epithelial cells, and more particularly to evaluating such cells as therapeutic agents, but is not limited thereto. The present invention also relates to the production of retinal pigment epithelial cells from embryonic stem cells.

Background Art

[0002] The retinal pigment epithelium (RPE) is a single layer of pigment cells between the neural retina and the choroidal capillary lamina. RPE cells play a crucial role in the maintenance and function of the retina and its photoreceptors. These maintenance and functions include the formation of the blood-retinal barrier, the absorption of stray light, the supply of nutrients to the neural retina, the regeneration of visual substances, and the uptake and reuse of shed photoreceptor outer segments.

[0003] Retinal tissue can degenerate for many reasons. Among the reasons are arterial or venous occlusion, diabetic retinopathy, and retinopathy of prematurity, which are usually genetic. Diseases such as retinitis pigmentosa, retinal detachment, lattice degeneration of the retina, Best disease, and age-related macular degeneration (AMD) are characterized by progressive retinal degeneration.

[0004] RPE cells have the potential to be used in cell replacement therapy for degenerated RPE in the above-mentioned retinal diseases. It may also be used as a vehicle for introducing genes to treat retinal degenerative diseases. These cells can also be used as a tool for high-throughput screening to determine whether low-molecular-weight substances have a therapeutic effect in an in vitro model of retinal degenerative diseases, and for discovering and testing new drugs for retinal degenerative diseases. RPE cells can also potentially be used in basic research on the development, maturation, characteristics, properties, metabolism, immunogenicity, function, and interaction with other cell types of RPE.

[0005] Human fetal RPE and adult RPE have been used as alternative donor sources for allotransplantation. However, due to practical problems in obtaining sufficient tissue supply and ethical issues regarding the use of tissues from aborted fetuses, the popularity of these donor sources is limited. Considering these constraints in the supply of adult RPE grafts and fetal RPE grafts, the possibility of alternative donor sources has been studied. Human pluripotent stem cells have great advantages as a source of RPE cells for transplantation. Because human pluripotent stem cells have pluripotent developmental potential, they have the potential to differentiate into authentic functional RPE cells, and considering their unlimited self-renewal ability, they may serve as an unlimited source of RPE cell donors. In fact, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPS) have been shown to differentiate into RPE cells in vitro and, after subretinal transplantation into the Royal College of Surgeons (RCS) rat retinal degeneration model induced by RPE dysfunction, attenuate retinal degeneration and preserve visual function. Therefore, pluripotent stem cells may be an unlimited source for producing RPE cells.

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

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

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

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

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

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

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

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

[0014] According to an embodiment of the present invention, the ratio of the apical secretion of sgp130 to the basolateral secretion of sgp130 is greater than 1.

[0015] According to an aspect of the present invention, the ratio of the apical secretion of sTNF-R1 to the basolateral secretion of sTNF-R1 is greater than 1.

[0016] According to an aspect of the present invention, the ratio of the basolateral secretion of angiogenin to the apical secretion of angiogenin is greater than 1.

[0017] According to an aspect of the present invention, the ratio of the apical secretion of TIMP2 to the basolateral secretion of TIMP2 is greater than 1.

[0018] According to an aspect of the present invention, Oct4 + TRA-1-60 + The number of cells in the population is less than 1:250,000.

[0019] According to an aspect of the present invention, when measured by immunostaining, at least 80% of the cells express bestrophin 1.

[0020] According to an aspect of the present invention, when measured by immunostaining, at least 80% of the cells express microphthalmia-associated transcription factor (MITF).

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

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

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

[0024] According to an aspect of the present invention, the ratio of the apical secretion of PEDF to the basolateral secretion of PEDF is greater than 1.

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

[0026] According to an aspect of the present invention, the transepithelial electrical resistance of a cell population is greater than 100 ohms.

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

[0028] According to an aspect of the present invention, the ratio of the basolateral secretion of VEGF to the apical secretion of VEGF is greater than 1.

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

[0030] According to an aspect of the present invention, a cell population can rescue vision in RCS rats after subretinal administration.

[0031] According to an aspect of the present invention, a cell population can rescue photoreceptors in RCS rats for at least 180 days after subretinal administration.

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

[0033] According to an aspect of the present invention, a cell population (a) culturing human embryonic stem cells in a medium containing nicotinamide and lacking activin A so as to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A so as to generate cells that are further differentiated into the RPE lineage; and (c) culturing the cells that are further differentiated into the RPE lineage in a medium containing nicotinamide and lacking activin A is prepared by.

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

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

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

[0037] According to an aspect of the present invention, the method further includes, after step (c), culturing the differentiated cells in a medium under conditions where the atmospheric oxygen level exceeds about 10% in the presence of nicotinamide.

[0038] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the cell population described herein as an active substance and a pharmaceutically acceptable carrier.

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

[0040] According to one aspect of some embodiments of the present invention, (a) culturing pluripotent stem cells in a medium containing a differentiation substance and lacking a member of the transforming growth factor β (TGFβ) superfamily so as to generate differentiated cells; (b) culturing the differentiated cells in a medium containing a member of the transforming growth factor β (TGFβ) superfamily and a differentiation substance so as to generate cells that are further differentiated into the RPE lineage; (c) culturing the cells that are further differentiated into the RPE lineage in a medium containing a differentiation substance and lacking a member of the transforming growth factor β (TGFβ) superfamily so as to generate RPE cells A method for producing RPE cells, comprising steps (a) to (c), wherein steps (a) to (c) are performed under conditions where the atmospheric oxygen level is less than about 10%, is provided.

[0041] According to an aspect of the present invention, step (a) is performed under non-adherent conditions.

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

[0043] According to an aspect of the present invention, step (a) is (i) culturing a population of human pluripotent stem cells to be cultured in a medium containing nicotinamide under non-adherent conditions in the absence of activin A so as to generate a cluster of cells containing differentiated cells, and then, (ii) culturing the differentiated cells of (i) in a medium containing nicotinamide under adherent conditions in the absence of activin A including.

[0044] According to an aspect of the present invention, the method further includes dissociating the cell cluster before step (ii) to prepare a cell aggregate or a single cell suspension of the cells.

[0045] According to an aspect of the present invention, the method further includes culturing the differentiated cells in a medium under conditions where the atmospheric oxygen level exceeds about 10% in the presence of a differentiation substance after step (c).

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

[0047] According to an aspect of the present invention, the differentiation substance in step (a) and the differentiation substance in step (c) are the same.

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

[0049] According to an aspect of the present invention, the method further includes, after step (c), a step of selecting a polygonal cell.

[0050] According to an aspect of the present invention, the method further includes a step of growing the polygonal cells.

[0051] According to an aspect of the present invention, the growing step is performed on an adherent surface or on an extracellular matrix.

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

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

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

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

Brief Description of the Drawings

[0056] Some aspects of the present invention, by way of example only, are described herein in connection with the accompanying drawings. Now, the drawings will be specifically described in detail. It is emphasized that the matters shown are by way of example and for purposes of illustrative discussion of aspects of the present invention. In this regard, the description employed with the drawings will make apparent to those skilled in the art how aspects of the present invention can be implemented.

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BRIEF DESCRIPTION OF THE INVENTION

[0057] Description of Specific Embodiments of the Present Invention In some aspects, the present invention relates to retinal pigment epithelial cells, and more particularly, to the evaluation of such cells as therapeutic agents, although not limited thereto. The present invention also relates to the production of retinal pigment epithelial cells from human embryonic stem cells.

[0058] Before explaining at least one aspect of the present invention in detail, it is to be understood that the present invention is not necessarily limited to the details shown in the following description or exemplified by the examples. The present invention may have other aspects or may be implemented or carried out in various ways.

[0059] The neural retina initiates vision and is supported by the underlying retinal pigment epithelium (RPE). Dysfunction, degeneration, and loss of RPE cells are prominent features of Best disease, subtypes of retinitis pigmentosa (RP), and age-related macular degeneration (AMD), the leading cause of visual impairment in the Western world. In these situations, vision is gradually lost and blindness often ensues.

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

[0061] Human embryonic stem cells (hESCs) may serve as an unlimited donor source of RPE cells for transplantation. The ability of mouse ESCs, primate ESCs, and human ESCs to differentiate into RPE-like cells, attenuate retinal degeneration after subretinal transplantation, and preserve visual function has been demonstrated.

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

[0063] The inventors have now discovered a unique and simple method for determining the eligibility of a cell population successfully differentiated into RPE cells based on the expression of specific polypeptides. The inventors have discovered that the combination of two specific markers can be used to demonstrate successful differentiation from among the numerous potential polypeptides expressed on the surface of these differentiated cells.

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

[0065] While further narrowing the present invention for implementation, the inventors have identified additional proteins secreted by RPE cells that may be used as a signature defining RPE cells in some embodiments.

[0066] Accordingly, one aspect of the present invention provides a method for determining the eligibility of a cell population as an appropriate therapeutic agent for treating an eye disorder, the method comprising analyzing the co-expression of premelanosome protein (PMEL17) and at least one polypeptide selected from the group consisting of cellular retinaldehyde-binding protein (CRALBP), lecithin retinol acyltransferase (LRAT), and sex-determining region Y-box 9 (SOX9) in the cell population, and determining that the cell population is eligible as an appropriate therapeutic agent for treating a retinal disorder when the number of cells co-expressing PMEL17 and the at least one polypeptide exceeds a predetermined level.

[0067] According to another aspect, there is provided a method for determining the eligibility of a cell population as a suitable therapeutic substance for treating eye abnormalities, the method comprising analyzing the co-expression of cellular retinaldehyde-binding protein (CRALBP) and at least one polypeptide selected from the group consisting of premelanosome protein (PMEL17), lecithin retinol acyltransferase (LRAT), and sex-determining region Y-box 9 (SOX9) in the cell population, and determining that the cell population is eligible as a suitable therapeutic substance for treating eye abnormalities when the number of cells co-expressing CRALBP and the at least one polypeptide exceeds a predetermined level.

[0068] As used herein, the phrase "suitable therapeutic substance" refers to a cell population being suitable for treating eye abnormalities. Cells that are therapeutic substances can exert their effects by any one of a plurality of mechanisms. One exemplary mechanism is a trophic effect that promotes the survival of degenerating photoreceptors or other cells within the retina. Therapeutic RPE cells may also exert their effects via a regenerative mechanism that replenishes dysfunctional and / or degenerating host RPE cells. According to one aspect, the RPE cells are mature and have the functional ability to phagocytose the shed outer segments of photoreceptors containing rhodopsin. According to another aspect, the RPE cells are not fully mature.

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

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

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

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

[0073] It will be understood that more than two of the polypeptides mentioned herein, for example, three of the polypeptides described above, and even all four of the polypeptides described above, can be measured.

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

[0075] In the case of flow cytometry, the antibody may be attached to a fluorescent moiety and analyzed using a fluorescence-activated cell sorter (FACS). Alternatively, the use of a secondary antibody with a fluorescent moiety is also contemplated.

[0076] Since the polypeptide to be analyzed is an intracellular polypeptide, it will be understood that the cells are typically permeabilized so that the antibody can bind to the target. First, the cells may be fixed to ensure the stability of soluble antigens or antigens with a short half-life. Thereby, the target protein should be maintained in its original cellular location. To ensure that the permeability of the cells is maintained, the antibody may be prepared in a permeabilization buffer. It will be understood that when gating the cell population, the light scattering profile of the cells in the flow cytometer will change significantly after permeabilization and fixation.

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

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

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

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

[0081] In order to determine the suitability of the cell as a therapeutic substance, the amounts of at least two of the polypeptides co-expressed in the cell must be increased at a statistically significant level compared to non-RPE cells (e.g., undifferentiated embryonic stem cells).

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

[0083] According to another aspect, in order to determine the eligibility of the cells being useful as a therapeutic substance, the level of co-expression of CRALBP and one of the polypeptides described above (e.g., PMEL17) (when measured by, for example, mean fluorescence intensity) should be increased by at least 2-fold, more preferably at least 3-fold, more preferably at least 4-fold, even more preferably at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold compared to undifferentiated ESCs.

[0084] According to a particular aspect, in order to determine the eligibility of the cells being useful as a therapeutic substance, when assayed by a method known to those skilled in the art (e.g., FACS), at least 80% of the cells within the population should express a detectable level of CRALBP and one of the polypeptides described above (e.g., PMEL17), more preferably at least 85% of the cells within the population should express a detectable level of CRALBP and one of the polypeptides described above (e.g., PMEL17), more preferably at least 90% of the cells within the population should express a detectable level of CRALBP and one of the polypeptides described above (e.g., PMEL17), more preferably at least 95% of the cells within the population should express a detectable level of CRALBP and one of the polypeptides described above (e.g., PMEL17), more preferably 100% of the cells within the population should express a detectable level of CRALBP and one of the polypeptides described above (e.g., PMEL17).

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

[0086] The cells may be qualified or characterized by additional techniques including, for example, karyotype analysis, morphology, cell number, as well as viability, efficacy (barrier function and polarized secretion of PEDF and VEGF), level of residual hESCs, Gram staining, and sterility. Exemplary assay methods that may be performed are described in Example 4.

[0087] Furthermore, the cells may be analyzed for barrier function and level of secretion in a polarized pattern of growth factors (such as pigment epithelium-derived factor (PEDF) or VEGF, cytokines, interleukins, and / or chemokines).

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

[0089] Furthermore, the direction of secretion of PEDF and VEGF in the cells may be analyzed. This may be performed using a transwell assay method as shown in FIG. 28. Before or after qualification determination, the cells may be stored (such as frozen or cryopreserved) according to methods known in the art and administered directly to a subject.

[0090] The present invention is intended to analyze a cell population containing retinal pigment epithelial (RPE) cells derived from any source. Thus, this cell population may include RPE cells obtained from a donor (i.e., native RPE cells of the pigment layer of the retina), and may also include RPE cells differentiated ex vivo from a population of stem cells (hSC-derived RPE cells, for example, pluripotent stem cells, such as human embryonic stem cells). According to another aspect, the RPE cells are obtained by differentiation conversion. See, for example, Zhang et al., Protein Cell 2014, 5(1): 48-58. This content is incorporated herein by reference.

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

[0092] According to another aspect, the RPE cells to be analyzed express Pax6.

[0093] "Retinal pigment epithelial cells", "RPE cells", "RPE" may be used synonymously when the context permits, and refer to cells of a cell type that is functionally similar to native RPE cells forming the pigment epithelial cell layer of the retina (for example, when transplanted into the eye, it exhibits functional activities similar to native RPE cells).

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

[0095] According to yet another aspect, the RPE cells are RPE cells differentiated from embryonic stem cells according to the methods described in the Examples section below of this specification. The content of the Examples is the content as included in this specification itself.

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

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

[0098] According to another aspect, the RPE cells have a morphology similar to that of native RPE cells that form the pigment epithelial cell layer of the retina, i.e., are pigment cells and / or have a characteristic polygonal shape.

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

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

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

[0102] According to certain aspects, the RPE cells are obtained by the directed differentiation of hSCs in the presence of one or more members of the TGFβ superfamily and exhibit at least one of the following characteristics: - During differentiation, the cultured cells respond to TGFβ signaling; - The RPE cells express markers indicative of terminal differentiation, such as bestrophin 1, CRALBP, and / or RPE65; - After transplantation (i.e., in situ), the RPE cells exhibit trophic effects that support photoreceptors adjacent to the RPE cells; - Further, in situ, the RPE cells can function with phagocytosis of shed photoreceptor outer segments as part of the normal regeneration process of these photoreceptors; - Further, in situ, the RPE cells can form the retinal pigment epithelium and function in the visual cycle.

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

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

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

[0106] According to some aspects of the present invention, it will be understood that commercially available stem cells can also be used. Human ES cells can be purchased from the NIH Human Embryonic Stem Cells Registry [Hypertext Transfer Protocol: / / grants(dot)nih(dot)gov / stem_cells / registry / current(dot)htm] and other European registries. Non-limiting examples of commercially available embryonic stem cell lines include HAD-C 102, ESI, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, HUES27, HUES28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES45, Shef3, Shef6, BJNhem19, BJNhem20, SA001, SA001.

[0107] Furthermore, ES cells can be obtained from other species, 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: 288-92], 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 (rhesus 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].

[0108] [[ID=D4]]Expanded blastocyst cells (EBC) can be obtained from blastocysts at least 9 days after fertilization, which is a pre-gastrulation stage. Before culturing the blastocysts, the zona pellucida is digested [e.g., by Tyrode's acidic solution (Sigma Aldrich, St Louis, MO, USA)] to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos using standard embryonic stem cell culture methods for at least 9 days and up to 14 days after fertilization (i.e., before gastrulation events).

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

[0110] Yet another method for preparing ES cells is by parthenogenesis. The embryo is not destroyed in this process either.

[0111] Current ES culture methods are mainly based on the use of feeder cell layers that secrete factors necessary for stem cell proliferation while simultaneously inhibiting stem cell differentiation. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), mouse fetal fibroblasts (MFF), human embryonic fibroblasts (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 placenta, and human bone marrow stromal cells (hMSC). Growth factors may be added to the medium to maintain ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGFβ. In another aspect, agents may be added to the medium to maintain hESCs in a naive undifferentiated state. See, for example, Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

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

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

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

[0115] In one exemplary differentiation protocol, embryonic stem cells are differentiated into the RPE cell lineage using a first differentiating agent and then further differentiated into RPE cells using a member of the transforming growth factor-β (TGFβ) superfamily (e.g., TGFβ1, TGFβ2, and TGFβ3 subtypes, as well as activin (e.g., activin A, activin B, and activin AB), nodal, anti-Müllerian hormone (AMH), some bone morphogenetic proteins (BMPs), e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth differentiation factor (GDF)-containing homolog ligands).

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

[0117] According to certain aspects, the member of the transforming growth factor-β (TGFβ) superfamily is activin A, e.g., 20 - 200 ng / ml, e.g., 100 - 180 ng / ml of activin A.

[0118] The first differentiating agent promotes differentiation into the RPE lineage. For example, the first differentiating agent may promote differentiation of pluripotent stem cells into neural precursors. Such cells may express neural precursor markers such as PAX6.

[0119] According to certain aspects, the first differentiating agent is nicotinamide (NA), e.g., 1 - 100 mM, 5 - 50 mM, 5 - 20 mM, e.g., 10 mM of nicotinamide (NA).

[0120] NA is also known as "nicotinamide" and is an amide derivative form of vitamin B3 (niacin) that is thought to protect and improve β-cell function. The chemical formula of NA is C6H6N2O. NA is essential for growth and the conversion of food into energy and has been used in the treatment of arthritis and the treatment and prevention of diabetes. TIFF2025111765000002.tif43128

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

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

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

[0124] According to certain embodiments, the differentiation is carried out as follows: (a) culturing ESCs in a medium containing a first differentiation substance (e.g., nicotinamide); and (b) culturing the cells obtained from step (a) in a medium containing a member of the TGFβ superfamily (e.g., activin A) and the first differentiation substance (e.g., nicotinamide).

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

[0126] 6]The protocol may be continued by culturing the cells obtained in step (b) in a medium containing the first differentiation substance (e.g., nicotinamide) but no member of the TGFβ superfamily (e.g., activin A). This step is referred to herein as step (c).

[0127] Now, the protocol will be described in more detail using additional embodiments.

[0128] Once a sufficient amount of ESCs is obtained, the differentiation process is initiated. ESCs are typically removed from adherent cell cultures (e.g., by using collagenase A, dispase, TrypLE select, EDTA) and plated on a non-adherent substrate (e.g., Hydrocell non-adherent cell culture plate) in the presence of nicotinamide (and in the absence of activin A). Exemplary nicotinamide concentrations are 1 - 100 mM, 5 - 50 mM, 5 - 20 mM, e.g., 10 mM. Once the cells are plated on the non-adherent substrate, the cell culture is sometimes referred to as a cell suspension, preferably a floating cluster in suspension culture, i.e., an aggregate of cells derived from human embryonic stem cells (hESCs). This cell cluster does not attach to any substrate (e.g., culture plate, carrier). The source of floating stem cells has been previously described in WO06 / 070370, which is hereby incorporated by reference in its entirety. This stage may be carried out for at least 1 day, more preferably 2 days, 3 days, 1 week, or even 10 days. Preferably, the cells are not cultured for more than 2 weeks in suspension with nicotinamide (and in the absence of TGFβ superfamily members, e.g., activin A).

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

[0130] Examples of non-adherent cell culture plates include non-adherent cell culture plates manufactured by Hydrocell (e.g., catalog number 174912), Nunc, etc.

[0131] Typically, the clusters contain at least 50 to 500,000, 50 to 100,000, 50 to 50,000, 50 to 10,000, 50 to 5,000, or 50 to 1,000 cells. According to one aspect, the cells in the cluster are not organized into layers and form an irregular shape. In one aspect, the cluster does not contain pluripotent embryonic stem cells. In another aspect, the cluster contains a small number of pluripotent embryonic stem cells (e.g., 5% or less, or 3% or less (e.g., 0.01 to 2.7%) of the cells that co-express OCT4 and TRA1-60 at the protein level). Typically, the cluster contains cells that are partially differentiated under the influence of nicotinamide. Such cells may express neural precursor markers such as PAX6. The cells may also express precursor markers of other lineages, such as, for example, alpha-fetoprotein, MIXL1, and Brachyuri.

[0132] The clusters may be dissociated using enzymatic or non-enzymatic methods (e.g., mechanical methods) known in the art. According to one aspect, the cells are dissociated such that they no longer form aggregates or clumps in the form of clusters, e.g., aggregates or clumps of 2 to 100,000 cells, 2 to 50,000 cells, 2 to 10,000 cells, 2 to 5,000 cells, 2 to 1,000 cells, 2 to 500 cells, 2 to 100 cells, or 2 to 50 cells. According to a particular aspect, the cells are present in a single cell suspension.

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

[0134] Generally speaking, the cells are typically exposed to nicotinamide (at a concentration of 1-100 mM, 5-50 mM, 5-20 mM, for example, 10 mM) for about 2-3 weeks, preferably 4 weeks or less, before the addition of the second differentiation factor (e.g., activin A).

[0135] Examples of the adherent substrate include, but are not limited to, collagen, fibronectin, laminin (e.g., laminin 521).

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

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

[0138] According to certain embodiments, when the atmospheric oxygen conditions are returned to normal atmospheric conditions, the cells are cultured for at least another day (e.g., up to 2 weeks) in the presence of nicotinamide (e.g., 10 mM) and in the absence of activin A.

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

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

[0141] According to certain embodiments, the differentiation of ESCs is performed under xenofree conditions.

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

[0143] Another method for culturing ESCs under xenofree conditions is shown in U.S. Patent Application Publication No. 20130196369, the contents of which are incorporated herein by reference in their entirety.

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

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

[0146] Thus, according to another aspect of the invention, (a) culturing pluripotent stem cells in a medium containing a differentiation agent and lacking a member of the transforming growth factor β (TGFβ) superfamily to generate differentiated cells; (b) culturing the differentiated cells in a medium containing a member of the transforming growth factor β (TGFβ) superfamily and a differentiation agent to generate cells further differentiated into the RPE lineage; (c) Analyzing the secretion of pigment epithelium-derived factor (PEDF) from cells that are further differentiated into the RPE series; and (d) Culturing cells that are further differentiated into the RPE series in a medium containing differentiating substances and lacking members of the transforming growth factor β (TGFβ) superfamily so as to generate RPE cells comprising step (d) is performed when the amount of PEDF exceeds a predetermined level, A method for producing retinal epithelial cells is provided.

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

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

[0149] The cells may be selected / or increased once promoted to RPE cells.

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

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

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

[0153] The increase of RPE cells may be carried out on an extracellular matrix, for example, gelatin, collagen, or poly-D-lysine, and laminin. For the increase, the cells may be cultured in serum-free KOM, a medium containing serum (e.g., DMEM + 20%), or Nuristem medium (06-5102-01-1A Biological Industries). Optionally, the cells may be exposed to nicotinamide at a concentration of 1 to 100 mM, 5 to 50 mM, 5 to 20 mM, for example, 10 mM during the increase period. Under these culture conditions, the pigment cells reduce pigmentation and acquire a morphology similar to uterine fibroids. After further culturing for a long time and growing to a high-density culture, the cells reacquire a characteristic polygonal morphology, and preferably, the pigmentation of RPE cells is also reacquired.

[0154] RPE cells may be increased in suspension or increased in a monolayer state. The increase of RPE cells in monolayer culture may be changed to large-scale increase in a bioreactor by methods well known to those skilled in the art.

[0155] A population of RPE cells produced according to the methods described herein may be characterized according to a number of different parameters.

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

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

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

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

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

[0161] According to a particular aspect, the TEER is from 100 to 1000 ohms, more preferably from 100 to 900 ohms, for example, from 200 to 900 ohms, from 300 to 800 ohms, from 300 to 700 ohms, from 400 to 800 ohms, or from 400 to 700 ohms.

[0162] Devices for measuring transepithelial electrical resistance (TEER) are known in the art. An exemplary configuration for measuring TEER is shown in Figure 28.

[0163] It will be understood that the cell populations disclosed herein do not contain undifferentiated human embryonic stem cells. According to one aspect, for example, when measured by FACS, fewer than 1:250,000 cells are Oct4 + TRA-1-60 + It is. Said cells also do not express GDF3 or TDGF related to hESC or downregulate GDF3 or TDGF related to hESC when measured by PCR.

[0164] Another method of characterizing the cell populations disclosed herein is by marker expression. Thus, for example, when measured by immunostaining, at least 80%, 85%, or 90% of said cells express Bestrophin 1. According to one aspect, 90-95% of said cells express Bestrophin.

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

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

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

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

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

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

[0171] In some cases, it will be understood that all cells that co-express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) also secrete / releases angiogenin, tissue metalloprotease inhibitor 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor α ubiquitous membrane receptor 1 (sTNF-R1).

[0172] In other cases, the majority (more than 50%, 60%, 70%, 80, 90%) of cells that co-express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) secrete / releases angiogenin, tissue metalloprotease inhibitor 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor α ubiquitous membrane receptor 1 (sTNF-R1).

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

[0174] According to certain aspects, the ratio of apical secretion of sgp130 to basolateral secretion of sgp130 is greater than 1. According to certain aspects, the ratio of apical secretion of sgp130 to basolateral secretion of sgp130 is greater than 2. According to certain aspects, the ratio of apical secretion of sgp130 to basolateral secretion of sgp130 is greater than 3.

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

[0176] Furthermore, the ratio of basal secretion of angiogenin to apical secretion of angiogenin is greater than 1. According to certain embodiments, the ratio of basal secretion of angiogenin to apical secretion of angiogenin is greater than 1.5, 2, 2.5, or 3.

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

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

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

[0180] Those skilled in the art will well understand that it is highly beneficial to obtain RPE cells. RPE cells may be used as an in vitro model for developing new drugs that promote the survival, regeneration, and function of RPE cells. RPE cells may be useful for high-throughput screening of compounds that have a toxic or regenerative effect on RPE cells. RPE cells may be used to clarify important mechanisms, new genes, soluble factors, or membrane-bound factors for the generation, differentiation, maintenance, survival, and function of photoreceptor cells.

[0181] RPE cells may also serve as an unlimited source of RPE cells for transplanting, replenishing, and supporting dysfunctional or degenerated RPE cells in retinal degeneration. Furthermore, genetically modified RPE cells may serve as vectors for carrying and expressing genes in the eye and retina after transplantation.

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

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

[0184] The RPE cells prepared as described herein can be transplanted into various target sites within the eye of a subject. According to one aspect, transplantation of the RPE cells is into the subretinal space of the eye, which is the normal anatomical location of the RPE (between the photoreceptor outer segments and the choroid). Further, depending on the migratory ability and / or positive paracrine effects of the cells, transplantation into additional eye compartments can be considered, including the inner or outer retina, the retinal periphery, and within the choroid.

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

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

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

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

[0189] Accordingly, the present invention also relates to a pharmaceutical composition of the RPE cells described herein. This composition is preferably suitable for transplantation into the eye. Thus, for example, RPE cells may be formulated in an intraocular irrigation solution such as BSS plus (trademark).

[0190] During the period of the patent that is being established from this application, it is expected that many related technologies for producing RPE cells will be developed. The term RPE cells is intended to include a priori all such new technologies.

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

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

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

[0194] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or parts, so long as such additional components, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, and structure.

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

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

[0197] As used herein, the term "method" refers to a way, means, technique, and procedure for accomplishing a given task, including, but not limited to, those known to, or readily developed from those known to, persons of ordinary skill in the fields of chemistry, pharmacology, biology, biochemistry, and medicine.

[0198] As used herein, the term "treating" includes preventing the progression of an abnormality, substantially inhibiting, delaying or reversing it, or substantially alleviating the clinical or aesthetic symptoms associated with the abnormality, or substantially preventing the appearance of the clinical or aesthetic symptoms associated with the abnormality.

[0199] It is understood that certain features of the invention, which are described in separate contexts for ease of understanding, may be provided in combination in one context. Conversely, various features of the invention, which are described in the context of one aspect for brevity, may be provided separately, or in any suitable sub-combination, or as appropriate in any other described aspect of the invention. Certain features described in the context of various aspects should not be regarded as essential features of these aspects, except where the aspect would not function without these elements.

[0200] The various aspects and aspects of the invention described above and claimed in the following claims sections are experimentally verified in the following examples.

Examples

[0201] Reference is now made to the following examples. The following examples illustrate, non-limitingly, some aspects of the invention together with the above description.

[0202] Generally, the nomenclature used herein and the experimental procedures used in the present invention include molecular techniques, biochemical techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are described in the literature in fine detail. 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, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vol. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods shown in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volume I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volume I-III Coligan J. E., ed.See (1994); Stites et al. (eds), 「Basic and Clinical Immunology」 (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), 「Selected Methods in Cellular Immunology」, W. H. Freeman and Co., New York (1980). Immunassay methods available are described extensively in patents and scientific literature. See, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771, and 5,281,521. 「Oligonucleotide Synthesis」 Gait, 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. I., ed. (1986); 「Immobilized Cells and Enzymes」 IRL Press, (1986); 「A Practical Guide to Molecular Cloning」 Perbal, B., (1984), and 「Methods in Enzymology」 Vol.See, e.g., Sambrook et al., 「Molecular Cloning: A Laboratory Manual」, 2nd Edition, Cold Spring Harbor Laboratory Press (1989); Ausubel et al., 「Current Protocols in Molecular Biology」, John Wiley & Sons, Inc., New York (1995); Harlow and Lane, 「Antibodies: A Laboratory Manual」, Cold Spring Harbor Laboratory Press (1988); Colligan et al., 「Current Protocols in Immunology」, John Wiley & Sons, Inc., New York (1994); 「PCR Protocols: A Guide To Methods And Applications」, Academic Press, San Diego, CA (1990); Marshak et al., 「Strategies for Protein Purification and Characterization - A Laboratory Course Manual」 CSHL Press (1996). All of these are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. Some of the procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All of the information therein is incorporated herein by reference.

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

[0204] Staining protocol: Negative control hESC cells obtained from a cryopreserved hESC bank (HAD-C 102 p48 4.5.2014) were thawed in Nutristem (containing HSA) according to the sponsor protocol. Positive control RPE cell stock: OpRegen® batch 5C cells (reference strain) were thawed in 20% HS-DMEM according to the sponsor protocol. The thawed OpRegen® 5C and HAD-C 102 hESCs were centrifuged, resuspended in 1 ml of PBS(-), filtered through a 35 μM cell strainer, and counted using trypan blue. The cell concentration was adjusted to 0.73×10 6 ~10 6 cells per ml in PBS(-). After adding 1 μl / ml of FVS450 to each cell suspension, it was vortexed and incubated at 37°C for 6 minutes. FVS450 was washed with 0.1% BSA and resuspended in 0.1% BSA-Fc-block (for 5 minutes at RT) to block all Fc epitopes on the cells. The cells were then washed with PBS(-) and fixed with 80% methanol (for 5 minutes at 4°C). The fixed cells were washed once with PBS(-) and once with 0.1% PBS-T, and permeabilized with 0.1% PBS-T (for 20 minutes at RT). The permeabilization solution was replaced with 10% normal goat serum (NGS) blocking solution (200,000 cells / 50 μl) for at least 30 minutes (maximum 1 hour) at RT. Quality sample tubes (QS) were prepared during the incubation time, and the cells were separated and immunostained at the end of blocking. The cells were incubated with the primary antibody for 30 minutes, then washed 3 times with 0.1% PBS-T, incubated with the secondary antibody for 30 minutes, and washed 3 times with 0.1% PBS-T.

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

[0206] Results Accuracy: The accuracy of the assay was determined from the test results of four levels of spiked RPE (25%, 50%, 75%, and 95%). The accuracy of the RPE stock (OpRegen® 5C) was determined relative to potentially 100% RPE cells. Each level value was analyzed by six independent runs / measurements.

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

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

[0209] (Table 1) TIFF2025111765000003.tif232111

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

[0211] (Table 2) TIFF2025111765000004.tif230105

[0212] Repeatability: The repeatability of the samples was tested in three runs (#2, #3, and #4), and in the three runs (#2, #3, and #4), two identical OpRegen® SC samples were stained and obtained in parallel. From the results, it was confirmed that the sample identity obtained within the experiment was reproducible and consistent across the entire sample.

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

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

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

[0216] Stability: From the results, the stained samples were stable at 4°C even after 1 day and 4 days, and the accuracy was maintained within the expected acceptance criteria. Thus, it can be seen that the samples can be prepared and data can be obtained within 96 hours.

[0217] Conclusion From the results shown herein, the disclosed method was determined to be qualified and suitable for the intended use of determining in vitro the purity of RPE in the OpRegen® final product and the purity of RPE at different stages along the production process of OpRegen®. In RPE cells in the range of 50% to 99.5%, the accuracy of relative bias was <25% and the precision of %CV was <20%.

[0218] Example 2 Evaluation of the level of OpRegen® purity A FACS-based method was developed to evaluate the purity of human retinal pigment epithelial cells (RPE) and the level of non-RPE cell impurities in RPE cells. Cellular retinaldehyde-binding protein (CRALBP), one of the visual cycle components, was identified using bioinformatics as a unique marker for mature RPE cells. Preliminary studies using a CRALBP-specific monoclonal antibody have shown a purity exceeding 98% in RPE cells prepared according to the method described herein. Furthermore, these results were supported by immunostaining of PMEL17, a melanosome marker found in RPE. Additionally, unlike some RPE-specific markers, CRALBP is not expressed in melanocytes, which are potential neural crest cell contaminants.

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

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

[0221] FACS data acquisition was performed using a validated Navios flow cytometer (Beckman Coulter), and analysis was performed using FlowJo7.6.

[0222] Results From the initial FACS data using the anti - CRALBP monoclonal antibody, the purity level of OpRegen® was found to be above 98%. Melanocytes, potential neural crest cell contaminants, were found to be negative for CRALBP, a unique RPE - specific marker (1.7%). The parental strain HADC102 - hESC was CRALBP - negative as expected (0.2%).

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

[0224] Example 3 Description of the manufacturing process and process control OpRegen (registered trademark) is manufactured from a xeno-free GMP-grade HAD-C 102 hESC line that was grown on irradiated, xeno-free GMP-grade human umbilical cord fibroblast feeders. Clinical-grade human fibroblast feeder cell lines (CRD008; MCB) and working cell banks (WCB) were produced, appropriately tested, characterized, and banked under the manufacturing and quality control regulations for pharmaceuticals and quasi-drugs (Good Manufacturing Practice) (GMP) and under xeno-free conditions. These were then used to obtain the clinical-grade hESC HAD-C 102 line from surplus human blastocysts under GMP and under xeno-free conditions.

[0225] In the initial stage of production, hESCs are increased as colonies on irradiated feeders. Then, to initiate directed differentiation, the hESCs are transferred to suspension culture. Spheroids (SB) are formed and then plated as adherent cultures under continuous directed differentiation conditions towards a neural fate and then towards RPE cells. At the end of the differentiation stage, the non-pigmented area is physically excised, and the pigment cells are collected enzymatically, seeded, and increased. Purified hESC-derived RPE cells (DS) are collected at passage 2 and immediately processed into DP. The duration of the manufacturing process depends on the growth rate of the hESCs (about 2 months from thawing) and usually lasts for a total of 4 to 5 months or more.

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

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

[0228] Production Stages 1 - 3 are illustrated in Figure 12.

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

[0230] Stages 6 - 13: Differentiation into RPE cells. The increased HAD-C 102-hESC were enzymatically treated with collagenase (4152, Worthington) in a 6 cm cell culture plate for further expansion (Figure 14, Stage 6). Then, the increased HAD-C 102-hESC were used in obtaining OpRegen® DS.

[0231] The HAD-C 102-hESC clusters collected from the stage 6 cultures with collagenase A were mechanically transferred into feeder-free non-adherent 6 cm Hydrocell culture dishes in the presence of Nutristem "Minus" medium (containing no growth factors bFGF and TGF-β; 06-5102-01-1A Biological Industries, special order) supplemented with 10 mM nicotinamide (N-5535, Sigma) to initiate the differentiation of each OpRegen® batch (Figure 14, stage 7). The plates were then cultured for up to 1 week under hypoxic atmosphere (5%) conditions (37 °C, 5% CO2) to allow spheroid formation. Then, spheroids of a certain age were collected into suspension, gently dissociated by pipetting, and transferred to 6-well plates coated with human laminin (511, Biolamina) for further growth for 1 week in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide under hypoxic (5%) atmosphere (Figure 14, stage 8). The cells were grown for up to 4 weeks under hypoxic (5%) atmosphere; for 2 weeks in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide and 140 ng / ml activin A (G-120-14E, Peprotech) (Figure 14, stage 9), followed by up to 2 weeks in the presence of Nutristem "Minus" medium supplemented with only 10 mM nicotinamide (Figure 14, stage 10). When distinct areas of faint pigmentation were visibly present in the polygonal cell patches, the plates were returned to normoxic (20%) atmosphere (37 °C, 5% CO2) and grown for up to 2 weeks in the presence of Nutristem "Minus" medium containing 10 mM nicotinamide (Figure 14, stage 11). By 2 weeks later, enlarged polygonal patches with distinct pigmentation were visibly present within the areas of non-pigmented cells (Figure 14, stage 12). The remaining pigmented cells were removed and manually collected after treatment with TrypLE Select (12563-011, Invitrogen) at 37 °C for 15 minutes (Figure 14, stage 13). Production stages 6 - 13 are illustrated in Figure 14.

[0232] Stages 14 - 17: Increase in OpRegen (registered trademark) cells. Next, the pigment cells were transferred to a 6 - well gelatin - coated plate (0.5 - 1×10 6 cells / plate; P0) to proliferate for 2 - 3 days in the presence of DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (14 - 498E, Lonza) (Figure 15, stage 14). Next, the DMEM was replaced with Nutristem "Minus" medium, and the cells were grown for 2 - 3 weeks until the plate was covered with slightly pigmented polygonal cells (Figure 15, stage 14). Next, these P0 cells were placed in a flask coated with gelatin and increased during two additional passages (P1, P2). Cells at P0 and P1 were collected after treatment with TrypLE Select at 37°C, washed, placed on a gelatin - coated flask, and cultured for 2 - 3 days in the presence of DMEM supplemented with 20% human serum. The DMEM was replaced with Nutristem "Minus" medium, and the cells were grown for 2 - 3 weeks until the plate was covered with thinly pigmented polygonal cells (Figure 15, stages 15 - 16). Next, the cells at P2 grown in a T175 flask were collected after treatment with TrypLE Select at 37°C, resuspended in DMEM supplemented with 20% human serum, pooled, and counted.

[0233] For sterility, mycoplasma, and LAL tests, growth medium samples were taken from each batch. The cell morphology was observed and recorded (Figure 15, stage 17). Production stages 14 - 17 are illustrated in Figure 15.

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

[0235] IPC Point 1: Mechanically expanded HAD-C 102 hESCs with a normal karyotype before differentiation. This is the starting material from which the highest levels of hESCs are expected. This point was added for the purpose of assessing the maximum hESC levels before differentiation.

[0236] IPC Point 2: HAD-C 102 hESCs expanded by collagenase before differentiation. At this stage, some degree of differentiation is expected, which would result in a decrease in the levels of cells expressing Oct4 and TRA-1-60 and a decrease in the expression levels of GDF3 and TDGF. This point was added for the purpose of assessing hESC impurities during the non-directed differentiation stage.

[0237] IPC Point 3: Spheroids produced after 1 week of inducing hESC differentiation in the presence of nicotinamide under feeder-free conditions. At this early stage of differentiation, it is expected that the hESC impurities during differentiation are at their maximum levels, and thus this assessment is expected to indicate the highest levels of safety concerns.

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

[0239] IPC Points 5 - 7: Cells at the end of the differentiation process before and after separating the non-pigmented area (IPC Point 6) from the pigmented area (IPC Point 7). IPC Points 5 and 6 contain cell impurities, while sample 7 corresponds to the product at the end of the differentiation process before expansion. The cell contaminants observed in sample 6 are present in small amounts in sample 7 and even less in the product.

[0240] IPC Point 8: Pigmented cells at P0. Pigmented cells at the end of the differentiation process expanded for 2 - 3 weeks. These cells correspond to the product two steps before the end of the production process.

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

[0242] IPC point 10: Pigment cells in P2 before cryopreservation. P1 cells expanded for 2-3 weeks are collected and pooled. These cells represent the pre-cryopreservation bulk material (DS).

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

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

[0245] Relative expression of pluripotent hESC markers GDF3 and TDGF: The relative expression of the pluripotent genes GDF3 and TDGF was analyzed at various IPC points along the production process. The expression levels of GDF3 and TDGF gradually decreased. This was correlated with the gradual decrease in the number of TRA-1-60+Oct4 + cells. At the end of P0, P1, and P2 before cryopreservation (original) and P2 after cryopreservation (formulation), two steps before the end of the production process, the expression levels of GDF3 and TDGF were similar to those observed in the negative control OpRegen® 5C cells.

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

[0247] (Table 3) TIFF2025111765000005.tif25128DP, Drug Product.* The IPC point 8 was tested after cryopreservation. The accuracy error from the internal standard of RPE cells spiked into hESC (HAD-C 102, negative control) and OpRegen® 5C (positive control) was proven to be ≤25%.

[0248] Confocal imaging of cells immunostained with Bestrophin 1, MITF, and CRALBP along simulated test production runs 4 and 5: At the end of the differentiation stage (IPC point 7), at the end of the expansion stage (IPC point 10, DS), and after cryopreservation (IPC point 11, DP), cells were immunostained for the RPE markers Bestrophin 1, MITF, ZO-1, and CRALBP. For immunostaining, non-pigmented cells manually isolated (IPC point 6) were plated but detached from the plate during fixation and thus could not be stained. Selected pigmented cells (IPC point 7) plated for 12 days (simulated test 5 only. In parallel with cells at P0 from ongoing production) and 28 days were positively stained for all RPE markers tested, and the percentages of cells expressing Bestrophin 1 and MITF were 93% and 93.3 - 96.5%, respectively. Similar levels of Bestrophin 1-positive and MITF-positive cells were detected at P0 (94.9% and 95.9% respectively; tested in simulated test 4 only), P2 before cryopreservation, the original body (92.2 - 92.75% and 93.7 - 95.5% respectively), and P2 after cryopreservation, the formulation (91.1 - 95.7% and 83.8 - 94.9% respectively; outliers in randomly selected areas for analysis were proven from the decrease in MITF immunostaining in simulated test 5). CRALBP (and ZO-1) expression was detected in all IPC7, 10, and 11 samples (Figure 18).

[0249] Relative expression of bestrophin 1, CRALBP, and RPE65, RPE markers along simulated production 2, 4, and 5: At various IPC points along the production process, the relative expression of the RPE genes bestrophin 1, CRALBP, and RPE65 was measured. Along the production process, the relative expression levels of bestrophin 1, CRALBP, and RPE65 gradually increased. At the end of activin A treatment directed towards RPE cell differentiation (IPC point 4), the relative levels of bestrophin 1, CRALBP, and RPE65 were 685-fold, 36-fold, and 325-fold, respectively, of their respective relative levels in mechanically passaged hESCs before differentiation (IPC point 1; simulated trial 4). The relative expression levels of bestrophin 1, CRALBP, and RPE65 reached a peak from the end of the differentiation stage (IPC point 5) to the P1 stage (IPC point 9). At these stages, the respective expression levels were 5,838 - 11,841-fold, 211 - 299-fold, and 5,708 - 8,687-fold of the levels in mechanically passaged hESCs before differentiation (IPC point 1).

[0250] Morphological evaluation along simulated production 4 and 5: At the end of the differentiation stage (IPC point 5), to estimate the relative area of pigmented cells and during the expansion phase P0 - P2 (IPC points 8 - 10), to verify a confluent polygonal morphology, cell morphology was analyzed. At the end of the differentiation stage (IPC point 5), the relative area of pigmented cells was estimated before excision of the non-pigmented area. The relative area of pigmented cells was 32.5% ± 13.5% (mean ± SD, n = 7 wells of a 6-well plate) in simulated trial 4 and 60% ± 13% in simulated trial 5 (mean ± SD, n = 7 wells of a 6-well plate) (see representative images in Figure 11). The pigmented cell area was selected and magnified. From the morphology at the end of expansion phase P0 (IPC point 8), P1 (IPC point 9), and P2 (IPC point 10), cultures densely packed with a typical polygonal epithelial monolayer morphology were demonstrated (Figure 11).

[0251] Measurement of PEDF secretion and efficacy according to simulated test productions 4 and 5: At various IPC points according to simulated test productions 4 and 5, pigment epithelium-derived factor (PEDF), which is known to be secreted from RPE cells, was measured in the cell culture medium. As can be seen from Table 4, very low levels of PEDF in the range of 4 to 79 ng / mL / day were secreted by hESCs (IPC points 1 and 2) as well as spheroids (IPC point 3; end of the first week using nicotinamide). At the end of activin A treatment directed towards RPE cell differentiation (IPC point 4), the levels of secreted PEDF were in the range of 682 to 1,038 ng / mL / day, which was 31 to 37 times higher compared to the levels secreted by spheroids. After incubating cells under normal oxygen conditions with nicotinamide (IPC point 5), it was observed that PEDF secretion further increased (2.2 to 4.6 times) to 1,482 to 4,746 ng / mL / day. During the increasing phase (P0 to P2, IPC 8 to 10 respectively), the PEDF secretion levels were in the range of 2,187 to 8,681 ng / mL / day and reached a peak at P0 to P1.

[0252] (Table 4) PEDF secretion according to simulated test productions 4 and 5 TIFF2025111765000006.tif61154ND, not performed; NA, not applicable; DS, prototype; DP, formulation.

[0253] The tight junctions formed between RPE cells enable the formation of the blood-retinal barrier and the polarized secretion of PEDF and VEGF. PEDF is secreted on the apical side and acts as an anti-angiogenic and neurotropic growth factor on the apical side. VEGF is mainly secreted on the basal side and acts as an angiogenesis-promoting growth factor on the basal side for choroid plexus endothelium. At the end of P0 (IPC point 8), at the end of P2 before cryopreservation (IPC point 10), and at the end of P2 after cryopreservation (IPC point 11), RPE polarization (barrier function as well as polarized secretion of PEDF and VEGF) was measured in a transwell system. As can be seen from Table 5, at all IPC points, barrier function / trans-epithelial electrical resistance (TEER) as well as polarized secretion of PEDF and VEGF were demonstrated.

[0254] (Table 5) TIFF2025111765000007.tif 233166 ND, not performed; DS, original body; DP, preparation. PEDF and VEGF were measured by ELISA. During culture in 12-well plates, PEDF on day 14 was collected from the cells. Subsequently, the cells were subcultured onto transwells and cultured for 6 weeks. During this period, TEER and the secretion of VEGF and PEDF from the basolateral and apical sides of the transwell were measured.

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

[0256] Conclusion Three simulation test production runs (simulation runs 2, 4, and 5) were conducted under research-grade conditions using the same GMP production method, xeno-free GMP-grade cells (HAD-C 102 hESCs grown on irradiated CRD008 feeders), xeno-free GMP-grade reagents, and GMP-grade experimental equipment used for GMP production of clinical batches. Simulation test productions 2, 4, and 5 were aimed at evaluating the level of hESC impurities along the production process, and simulation test productions 4 and 5 were also aimed at identifying what is important in process quality control.

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

[0258] Identity tests performed three steps before the end of production (isolation of pigment cells) demonstrated that Bestrophin 1 and MITF were expressed in 93% and 96.5% of immunostained cells, respectively, and that CRALBP and ZO-1 were expressed (not quantified). One step further (i.e., P0, which is two steps before the end of the production process), after one cycle of the increased cycle of negatively selected pigment cells was completed, an RPE purity test showed that >98.5% of the cells were + PMEL17 + double positive by FACS. Similar levels of RPE purity (i.e., >99.6%) were also detected in the formulation. These results were supported by morphological tests demonstrating a typical polygonal epithelial monolayer morphology and quantitative RT-PCR analysis demonstrating that the expression of the RPE genes Bestrophin 1, CRALBP, and RPE65 was upregulated to levels similar to those of the positive control (OpRegen® 5C cells).

[0259] PEDF, which is known to be secreted from RPE cells, was measured in the cell culture medium at various stages along the production processes of simulation runs 4 and 5. At the end of activin A treatment (IPC point 4), which had been previously shown by Idelson et al., 2009 to direct differentiation towards RPE cells, the level of secreted PEDF increased significantly compared to the previous production stage (induction of spheroids) (31-fold in simulation run 4 and 37-fold in simulation run 5). The PEDF secretion level continued to increase and reached a peak between P0 and P1 (1.7 - 5.8-fold increase compared to the level after activin A). Evaluation of the relative area of melanocytes at the end of the differentiation process (IPC point 5) was confirmed to be another important quality control measure for assessing RPE differentiation. Using this measure, a two-fold difference in melanocyte yield was observed between simulation runs 4 and 5 (32.5% in simulation run 4 and 60% in simulation run 5). This correlated with a similar difference observed in PEDF secretion at this stage (1,482 ng / ml / day in simulation run 4 and 4,746 ng / ml / day in simulation run 5).

[0260] As a conclusion, TRA-1-60 + Oct4 + hESC impurities were not observed. This was correlated with low expression levels of GDF3 and TDGF, high expression levels of bestrophin 1, CRALBP, and RPE65, as well as high levels of bestrophin 1 single-positive cells and MITF single-positive cells, and a large amount of CRALBP + PMEL17 + double-positive cells (tested one stage too late). Important safety IPCs and efficacy IPCs were identified at critical production stages.

[0261] Example 5 Efficacy evaluation Experimental setup: The inventors investigated whether the progression of RDD in a Royal College of Surgeons (RCS) rat model could be retarded by subretinal transplantation of RPE cells prepared as described in Example 4.

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

[0263] Local and full - field ERGs were evaluated in all study groups at P60 and P100. At the assigned sacrifice day of each animal, both eyes were enucleated, fixed with 4% paraformaldehyde, cryopreserved, embedded in Optimum Cutting Temperature compound (OCT), and sectioned. Cresyl violet staining was used to identify and count photoreceptor structural rescue. Immunofluorescent staining (IF) was used to identify transplanted cells and evaluate their fate, proliferation state, and ability to phagocytose photoreceptor outer segments. Additionally, immunofluorescence was used when measuring host cone rescue.

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

[0265] (Table 6) TIFF2025111765000008.tif67152

[0266] Materials and Methods Cell number: After counting the cells, aliquots were made at appropriate dosing concentrations. The mean pre - injection cell viability was 94.0% ± 0.03 for all injection time points. The mean post - injection cell viability was 92.4% ± 0.02.

[0267] Surgical procedure: Using needles with gradually decreasing gauges: 18, 22, 25, and 30, small incisions were made through the conjunctiva and sclera. To reduce the emergence of injected cells, intraocular pressure was lowered using a puncture at the outer peripheral part of the cornea. Subsequently, a glass pipette was inserted into the subretinal space and 2 μl of the suspension was injected. The scleral incision was then closed with sutures. The success of the injection of cells or buffer only (BSS+) was initially confirmed by manually visualizing the subretinal bleb and then by photographing it using a fundus camera (Micron III).

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

[0269] Electroretinagram (ERG): Two types of ERG were measured: a diagnostic form of local ERG that stimulates local parts of the retina using small light spots and a standard form of full-field ERG that stimulates the entire visual field.

[0270] Histology and immunohistochemistry: Both eyes were collected from each animal, fixed, cryoprotected, embedded, and frozen. The frozen blocks were made into 12-μm frozen sections. Approximately 60 slides were obtained, each containing 4 sections per slide.

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

[0272] Immunofluorescence (IF): For IF, slides of eyes treated with RPE cells were selected from cresyl violet-stained sections containing cells in the subretinal space that matched the size and morphology of transplanted human cells. Additionally, protection of the host ONL was used as a secondary criterion. All IF staining was performed as double staining. DAPI served as background nuclear staining. At least one slide from each animal treated with cells was used for each run.

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

[0274] Run #2 was performed using rabbit monoclonal anti-Ki67 (Ki67; clone EPR3610, human-specific, Abcam, catalog number ab92742) and an anti-nuclear marker to detect human proliferating cells.

[0275] Run #3 was performed using rabbit polyclonal anti-rat cone arrestin (Millipore catalog number ab15282) to evaluate cone counts in the sections (see Section 6.8.3). Additionally, selected slides were stained using a combination of mouse monoclonal anti-rhodopsin (clone Rho1D4, Millipore, MAB5356) and PMEL17 to identify transplanted human cells containing host rhodopsin / outer segments as a measure of phagocytic activity.

[0276] Cone counting: Confocal z-stack images were obtained from retinal sections from all eyes transplanted with cells and from age-matched controls injected with saline. Sections from eyes injected with cells were selected in the photoreceptor rescue area revealed using previously evaluated cresyl violet-stained sections. Three observers counted cones in a blinded fashion. The three counts were then averaged and the counts were compared between treatment groups and ages.

[0277] Rhodopsin uptake: A potential rescue mechanism utilized by transplanted cells is the mechanism of taking up photoreceptor outer segments and shed debris. Removal of debris compartments reduces the toxic stress on photoreceptors and thus helps to sustain photoreceptor survival. Here, the inventors selected specific animals to evaluate rhodopsin uptake by RPE cells based on cell viability and photoreceptor protection index. This evaluation was performed using immunofluorescence.

[0278] Results Fundus imaging: From fundus images collected at autopsy of eyes treated with cells, the locations where subretinal blebs were formed during surgery; hyperpigmented and hypopigmented areas of the retina corresponding to the locations where cells were deposited into the subretinal space were revealed (Figs. 19A - C). These mottled areas were not clearly distinguishable in eyes injected with BSS+ or in non-injected eyes.

[0279] Visual motor tracking threshold: The OKT threshold was rescued in all age groups and in all cell treatment groups (Figure 20). The cell treatment groups were superior in performance to the non-operated or saline-injected eyes at all ages. There was a significant dose-dependent effect particularly in older animals between the low dose (25K) and two higher doses (100K (p<0.0001) and 200K (p<0.0001)), but no clear benefit for OKT was observed from the higher dose (200K) beyond the middle dose (100K) (p = 0.5646). While the OKT threshold was rescued in all cell treatment groups, the absolute visual acuity decreased slowly over time. The OKT thresholds of untreated animals and saline-injected animals continued to decrease during the study. The eyes injected with BSS+ did not differ from the naive untreated group (p = 0.6068) and the other untreated eyes.

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

[0281] Full-field ERG: Full-field ERG was measured from 125 RCS rats at P60 and 63 RCS rats at P100. As shown in Figure 21B, individual animals treated with RPE cells functioned well and had significantly better performance than the controls.

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

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

[0284] Immunofluorescence: Transplanted RPE cells were identified as positive by immunofluorescence in animals of each age at survival (Figure 24). However, as the age advanced, the number of animals with identified cells decreased. Repeated staining of additional slides from animals that initially did not show transplanted cells identified positive cells in additional animals, but not in all cases.

[0285] By IF analysis, transplanted cells were not found in all animals, but from the measurement results of ONL thickness, significant photoreceptor rescue confirmed by OKT rescue was present in 70 - 90% of the animals treated with cells. This suggested that transplanted cells were present at some point in most of the treated eyes. The proliferation marker Ki67 was used to identify proliferating human cells. Ki67-positive human cells were not observed (Figure 24).

[0286] Cone counting: The number of cones in animals that received cell transplantation was significantly higher than that in the control eyes (Figure 25; p = <0.0001 for each comparison). Generally, there was no difference in the number of cones across the low, medium, and high doses of cells overall. Representative images from each age are shown in Figure 24.

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

[0288] Conclusion When transplanted into the subretinal space of RCS rats, RPE cells rescued the vision of RCS rats above that of the controls at all ages tested. The ERG responses were protected when the grafts were large enough to evaluate or in retinal areas accessible for evaluation. By 180 days after transplantation, rod and cone photoreceptors were rescued in the transplanted area. In summary, this data demonstrates that OpRegen® maintains the functional and structural integrity of the host retina over the long term. Therefore, OpRegen® holds great potential for the treatment of human RPE cell disorders such as RP and AMD.

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

[0290] The viability and cell concentration of RPE cells were stable at the time point of incubation for 8 hours for all dose formulations; percent average viability (±SD) for the following concentrations. · Low concentration (70×10 3 cells per 100 μl of BSS plus) changed from 93% ± 5 at time point 0 hour to 91% ± 1 at time point 8 hours, with no significant decrease. · High concentration (70×10 3 cells per 100 μl of BSS plus) changed from 92% ± 3 at time point 0 hour to 91% ± 2 at time point 8 hours, with no significant decrease.

[0291] For the medium concentration tested (250×10 3 cells per 100 μl of BSS plus), there was no significant change throughout the time points.

[0292] For all time points and prescribed dosages, the overall range was 88% - 97% from time point 0 hours to 8 hours. When averaging all the results at time point 0 hours (93% ± 3) and time point 8 hours (91% ± 1), a 2% decrease was found.

[0293] Neither at the time points nor at the prescribed dosages was a significant change in cell concentration observed. The cell concentration did not change in all three studies except for a slight decrease (2%) in one batch at high dosage.

[0294] The appearance of the various dosage forms did not change throughout the time points tested. The cell suspension contains no foreign particles and non - dissociative aggregates.

[0295] At all time points tested, the identity and purity of each prescribed RPE cell dosage were stable up to 24 hours and within the batch release criteria. At 8 hours (for all prescribed RPE cell dosages), the levels of MITF - positive cells and bestrophin - positive cells were 86 - 97% and 90 - 94% respectively, and the levels of + PMEL17 + double - positive cells were in the range of 98.35 - 99.64%.

[0296] The prescribed RPE cell dosages maintained efficacy at all time points tested (4 hours, 8 hours, 24 hours), both secreted high levels of PEDF, and formed a polarized RPE monolayer due to the mainly apical - side secretion of PEDF and basal - side secretion of VEGF. Results at the 8 - hour time point tested: TEER was in the range of 376 - 724 ohms, the apical - side ratio of PEDF to the basal - side was in the range of 2.77 - 5.70, and the basal - side ratio of VEGF to the apical - side was in the range of 2.04 - 3.88.

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

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

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

[0300] (Table 7) TIFF2025111765000009.tif96151

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

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

[0303] (Table 8) TIFF2025111765000010.tif90151

[0304] Results Viability, total cell number / vial, and RPE identity were maintained consistently over 3 years. Furthermore, as shown, efficacy and purity were demonstrated at levels similar to those collected prior to storage.

[0305] After cryopreservation, a normal karyotype was observed four years later. From this, it can be seen that long-term storage in the gas phase has not, so far, had a harmful effect on the genomic stability of RPE.

[0306] Sterility of the sample was demonstrated by testing for the absence of bacterial / fungal growth in all clinical batches at three months. Another batch showed negative test results four years after cryopreservation. Based on these uniformly acceptable stability results covering up to three years of stability testing, it can be concluded that RPE cell products are stable for at least three years when stored at temperatures below -180 °C in the gas phase of liquid nitrogen.

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

[0308] At the time of injection, NOD-SCID mice (NOD.CB17-Prkdcscid) aged 5 - 6 weeks were injected with BSS Plus (vehicle control), or two doses of RPE cells suspended in 1 μL of BSS Plus: 50×10 3 cells or 100×10 3 cells (the maximum dose feasible). RPE was administered subretinally via a transvitreal route (the proposed clinical route of administration) using a 33G Hamilton needle. A single dose of 50×10 3 cells or 100×10 3Individual cells were injected into one eye, while the other eye served as an internal standard. Each dosing session included mice (male and female) from each group. Mice included in the study after the preliminary test were randomly assigned to various test groups. Randomization was performed twice. A weight-based measurement randomization procedure was used to place the treatment groups before administering the vehicle / test article. After administration, sequential randomization was used to place the final treatment groups, and animals suitable for use in the study were transferred to the target study. Mice with eye abnormalities, abnormal clinical observations, or a body weight of less than 16 grams, and mice in which subretinal RPE injection was unsuccessful were excluded from the study in the preliminary test.

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

[0310] Results In the survival tests, which included detailed clinical observations, body weight, ophthalmic examinations, and clinical pathology consisting of hematology and serum clinical chemistry, there were no toxicological findings related to the RPE. In the detailed clinical observations and ophthalmic examinations, it was found that in mice treated with RPE pigment cells at both dose levels, "the eyes changed to a darker color" was observed in the left eye with an albino background. From the ophthalmic examinations of the surviving animals, this observation was found to consist of foci with dark pigment deposits in the mid-vitreal. The foci with pigment deposits were randomly distributed along a line extending from the temporal posterior lens capsule to the nasal retinal surface. These foci were interpreted to be RPE cells that leaked from the injection cannula when removing the injection cannula from the eye after injection, or RPE cells that leaked into the vitreous humor after subretinal transplantation, as supported by the backflow of the vitreous humor seen during injection.

[0311] All the eye lesions observed in this study were considered to be secondary to anesthesia and surgical injection procedures, or incidentally occurred as age-related changes. The discovery of multiple foci with pigment deposits in the vitreous humor suggests that RPE cells may be able to survive in the vitreous. The presence of pigment cells in the vitreous was confirmed at the microscopic level in some of the animals treated with RPE.

[0312] In terms of biodistribution when evaluated by qPCR using a set of human β-globin gene probes / primers at intervals of 2 weeks, 2 months, and 6 months, 100×10 3The left eyes treated with individual OpRegen® cells were RPE DNA positive in 8 / 12, 11 / 12, and 16 / 16 animals, and the group mean levels were 38 copies, 47 copies, and 249 copies per μg of total DNA of the eye, respectively. This indicates a tendency to increase over time. There was no significant difference between males and females. In these animals, with the exception of the spinal cord from one 2-week-old male animal (27 copies / μg DNA) and skeletal muscle (16 copies / μg DNA) and spinal cord from one 2-week-old female animal (below the level of qualification) (presumably due to inadvertent contamination by exogenous human DNA during DNA extraction from these tissues), RPE DNA was not detected in the untreated right eyes and all non-eye tissues, including blood, femoral marrow, brain, heart, kidney, liver, lung, submandibular lymph nodes, ovaries, skeletal biceps femoris, spinal cord, spleen, testes, and thymus.

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

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

[0315] Figure 27B is a photograph showing cells concentrated at the location of the bleb after injection. Figure 27C is a photograph showing that the cells then spread and formed a monolayer after injection.

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

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

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

[0319] There was no RPE-dependent effect on animal mortality and survival.

[0320] Conclusion After a single injection of RPE at a dose level up to 100,000 cells / μl / eye, no local or systemic toxicological effects, lethal effects, or tumorigenic effects were observed in the NOD / SCID animal model during the 6-month study period. The in vivo distribution of RPE cells was limited to the treated left eye, and the subretinal cells spread locally from the subretinal injection site as a function of time. At intervals of 2 weeks, 2 months, and 6 months, in most of the animals examined in the high-dose group, RPE cells were mainly present in the subretinal space, followed by the vitreous, and the positivity of immunostaining with antibodies against human nuclear-specific biomarkers and / or human RPE-specific biomarkers was not consistent. The persistence of RPE cells in the eye was estimated to be at least 6 months, and cell proliferation was very limited. The limited proliferation occurred mainly in the vitreous and had no harmful effects. There was evidence that the number of RPE cells increased over time in the treated eyes, but this was accompanied by a decrease in the occurrence of proliferation in the examined subretinal population. The RPE-specific markers RPE65 and PMEL17 were expressed mainly in RPE cells within the subretinal space, in contrast to the vitreous. Most of the Ki67-positive cell occurrence was found in the vitreous. The latter suggests that the increase in RPE cells over time is limited to the vitreous space and that the expression of the specific RPE65 and PMEL17 RPE markers may be regulated by the microenvironment. In conclusion, based on the data presented above, there were no significant safety issues associated with the injection of RPE cells described herein when compared to the vehicle control group.

[0321] Example 8 Pax-6 Expression in RPE Cells Objective: Development of a FACS-based method to evaluate PAX-6 levels in human retinal pigment epithelium (RPE) cells.

[0322] Materials and Methods (Frozen RPE cells (prepared as described in Example 4) were thawed, centrifuged, resuspended in 1 ml of PBS minus, filtered through a 35 μM cell strainer, and counted using an NC-200 cell counter. The cell concentration was adjusted to approximately 1×10 per 1 ml in PBS minus6 Adjusted to individual cells. After adding 1 μl / ml of FVS450 per 1 ml of cell suspension, it was vortexed and incubated at 37°C for 6 minutes. FVS450 was quenched with 0.1% BSA(-Ig)-PBS minus and resuspended in 0.1% BSA(-Ig)-Fc-block (for 5 minutes at RT) to block all Fc epitopes on the cells. Then the cells were fixed and stained with an anti-Pax-6 antibody (AF647 catalog number 562249).

[0323] Results As can be seen from Figure 29, the cells at P0 and P2 are PAX6 positive (81.5% - 82.5% at P0 and 91.3% - 96.1% at P2). P2 is the subculture at the end of the production process, and P0 is before the two-increase stage. As shown in Figures 29 and 30, the data were shown to be consistent throughout the batch. Furthermore, the inventors showed by FACS analysis that RPE cells are double-stained for PAX-6 and CRALBP (Figure 31).

[0324] Example 9 Identification of Proteins Secreted by RPE Cells Objective: To identify the signatures of proteins (known and novel) secreted by OpRegen® (RPE cells) that can be used as batch release validity assay methods and process control assay methods.

[0325] Supernatants were collected from RPE cells cultured under various culture conditions shown below (prepared as described in Example 3). Then, using G6 and G7 RayBiotech arrays and following the manufacturer's instructions, the relevant arrays and supernatants were screened overnight, and then the supernatants were screened. 1. RPE formulation cells after thawing (0.5×10 6 cells / well) (referred to as OpRegen® in this specification) cultured on a 12-well plate for 4 days and 14 days. 2. RPE preparation cells after thawing, which were cultured on a 12-well plate for 14 days and then on Transwell for 3 weeks (according to AM-RPE-15) and showed a TEER of more than 500 Ω. Supernatants were collected from the apical and basal chambers. 3. Cells prepared according to the protocol described in Example 3 before (QC3) and after (QC4) activin A treatment. 4. Nutristem medium without TGFβ and FGF (Nut-).

[0326] Supernatants were also collected from the following cell cultures and tested by ELISA. 1. OpRegen® preparation cells after thawing, which were cultured on a 12-well plate for 14 days and then on Transwell for 3 weeks (according to AM-RPE-15) and showed TEERs of 355 Ω and 505 Ω, respectively. Supernatants were collected from day 14 (passage 3) and from the apical and basal chambers. 2. RPE7 cells after thawing, cultured on a 12-well plate for 14 days (0.5×10 6 cells / well at passage 3). 3. Mock test VI cells at the end of passage 1 of the production process, which were isolated by enzyme or mechanical means (as described in Example 3) and then grown on laminin 521. These cells were tested for efficacy according to AM-RPE-15, and supernatants were collected from the cells on day 14 (passage 2) on a 12-well plate and from the cells in the apical and basal chambers after 3 weeks on Transwell. 4. Fetal HuRPE cells at passage 3 on day 4 and day 14 (0.5×10 6 cells / well).

[0327] The ELISA tests were validated according to the manufacturer's instructions for each ELISA kit. Incubation with the supernatants was overnight in each protocol.

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

[0329] Results The G7 array results are shown in Table 9 below in this specification.

[0330] (Table 9) TIFF2025111765000011.tif212147TIFF2025111765000012.tif163153

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

[0332] (Table 10) TIFF2025111765000013.tif51145TIFF2025111765000014.tif235145TIFF2025111765000015.tif91145

[0333] RPE-secreted proteins can be divided into three functional groups: (1) angiogenesis proteins, 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, sgp130, sTNF-R1, sTRAIL-R3, MCP-1, and osteoprotegerin. Receptor tyrosine kinase Ax1 was also found to be secreted by RPE cells. To verify by ELISA, six proteins (angiogenin, TIMP-2, MIF, sgp130, sTNF-R1, and sTRAIL-R3) that showed high levels of secretion and / or a polarized secretion (apical / basolateral) pattern were selected. VEGF secretion as seen in the polarization assay was also demonstrated from the array data.

[0334] Angiogenin: It was demonstrated from the protein array data that angiogenin secretion increased along the production process (Tables 9 and 10). These results were confirmed by ELISA, and from this ELISA, the level of angiogenin secreted by differentiated cells treated with nicotinamide before adding activin A was 0.52 ng / mL, whereas it was demonstrated that the angiogenin secretion level increased to 0.91 ng / mL after treatment with nicotinamide and activin A for 2 weeks (Figure 32A). After thawing, RPE cells (0.5×10 6 cells / well; passage 3) placed in a 12-well plate and cultured for 2 weeks secreted angiogenin (Figure 32B). Polarized RPE cells (week 3 on transwell; TEER > 350 Ω, ratio of PEDF apical / basal and VEGF basal / apical > 1) secreted angiogenin in a polarized manner to the basal side, and secretion to the apical side was low or absent (the basal angiogenin level was in the range of 0.1 - 0.25 ng / mL, and the apical angiogenin level was in the range of 0.05 - 0.12 ng / mL; Figure 32B). RPE7 cells prepared according to Idelson et al., 2009 were unable to generate a barrier function in the transwell system (TEER was less than 100 Ω), but were able to secrete VEGF and PEDF. The ability of RPE7 cells to secrete angiogenin was tested when plated in a 12-well plate for 14 days. On the 14th day of culture, RPE7 secreted angiogenin at levels within the range of RPE cells prepared as described herein (Figure 32C).

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

[0336] sgp130 secretion by OpRegen® cells: Protein array data also demonstrated an increase in sgp130 secretion along the OpRegen® production process as seen at IPC / QC checkpoints 3 and 4 (Tables 9 and 10). ELISA data confirmed high levels of sgp130 secretion after 2 weeks of treatment with activin A compared to levels secreted by cells before addition of activin A after treatment with nicotinamide (IPC / QC3; 0.68 ng / mL) (IPC / QC4; 1.64 ng / mL) (Fig. 34A). OpRegen® cells (0.5×10 6 cells / well; passage 3) placed in 12-well plates and cultured for 2 weeks after thawing secreted sgp130 (Figs. 34B-C). RPE7 cells cultured under similar conditions secreted sgp130 at levels within the range of OpRegen® cells (1.0 ng / mL on day 14; Fig. 34D). Fetal HuRPE cells secreted low sgp130 levels on days 4 and 14.

[0337] Polarized OpRegen® cells secrete sgp130 in a polarized manner with the apical side as the top, and a small amount is secreted or not secreted on the basal side (the apical sgp130 secretion level was 0.93 - 2.06 ng / mL, and the basal sgp130 level was in the range of 0 - 0.2 ng / mL; Figures 34B - C).

[0338] Released sTNF - R1: In the supernatant of differentiated cells, a very low level of released sTNF - R1 was detected by ELISA before treatment with nicotinamide and activin A for 2 weeks (IPC / QC3 0.01 ng / mL) and after 2 weeks of treatment (IPC / QC4 0.02 ng / mL) (Figure 35A). After thawing, OpRegen® cells (0.5×10 6 cells / well; passage 3) placed in a 12 - well plate and cultured for 2 weeks contained sTNF - R1 in the supernatant on day 14 of culture (Figures 35B - C). There was a similar level of sTNF - R1 in the culture supernatant of HuRPE cells cultured under the same conditions, while RPE7 cells showed a relatively low sTNF - R1 level (Figure 35D).

[0339] Polarized OpRegen® cells secreted a high level of released sTNF - R1 to the apical side (the apical sTNF - R1 level was in the range of 0.22 - 1.83 ng / mL, and the basal sTNF - R1 level was in the range of 0.01 - 0.11 ng / mL; Figures 35C - D).

[0340] sTRAIL - R3: According to protein array data, sTRAIL - R3 was detected in the supernatant of OpRegen® cells (Tables 9 and 10). From ELISA, it was confirmed that sTRAIL - R3 was present along the OpRegen® production process (493 pg / mL in QC3 and 238 pg / mL in QC4). There was no sTRAIL - R3 in fetal HuRPE cultures, and there was a very low level of sTRAIL - R3 (4 pg / mL) in RPE7 cultures.

[0341] Detection of MIF: MIF was detected in the supernatant of OpRegen® cells by protein array data (Tables 9 and 10). By ELISA, the presence of MIF was confirmed along the OpRegen® production process (100.3 ng / mL in QC3 and 44.7 ng / mL in QC4). It was demonstrated that there was a high level of MIF on the apical side from polarized OpRegen® cells (the MIF level on the apical side ranged from 26.6 to 138.3 ng / mL, and the MIF level on the basal side ranged from 1.9 to 30.5 ng / mL).

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

[0343] Materials and Methods OpRegen® (RPE cells) were prepared as described in Example 3.

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

[0345] Using a Transwell system (as shown in Fig. 28), a polarized RPE monolayer with stable barrier properties and polarized secretion of PEDF and VEGF could be developed. The barrier function of the RPE monolayer was evaluated using transepithelial electrical resistance (TEER) measurements, and the polarized secretion of PEDF and VEGF was evaluated using enzyme-linked immunosorbent assay (ELISA). Cells were thawed and cultured for 14 days in the presence of nicotinamide. PEDF secretion was tested on days 7 and 14. The cells were then transferred to Transwells (Costar 3460, 0.4 μm) for an additional 4 weeks. During this time, TEER was measured and media was collected weekly from the upper and lower Transwell chambers for up to 4 weeks (to evaluate cytokine secretion). When the cells are polarized, the TEER should exceed 100 Ω, and the ratio of apical-side PEDF secretion to basal-side PEDF secretion and the ratio of basal-side VEGF secretion to apical-side VEGF secretion should exceed 1.

[0346] All tested OpRegen® batches demonstrated the ability to generate a barrier function (TEER in the range of 368 - 688 Ω) and the ability to secrete PEDF and VEGF in a polarized manner (the ratio of apical-side PEDF / basal-side PEDF was 3.47 - 8.75 and the ratio of basal-side VEGF / apical-side VEGF was 1.39 - 2.74) (see Table 11).

[0347] (Table 11) TIFF2025111765000016.tif90154ND: TEER was not measured because it was less than 100 Ω. Large holes were observed in the culture.

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

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

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

[0351] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Further, any reference or identification of a 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 shall not necessarily be construed as limiting.

Claims

1. A population of human polygonal RPE cells, wherein at least 95% of those cells co-express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP), and the transepithelial electrical resistance of the population of cells is greater than 100 ohms.

2. A population of human RPE cells, wherein at least 80% of those cells co-express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP), and the cells within the population secrete angiogenin, tissue metalloprotease inhibitor 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor α ubiquitous membrane receptor 1 (sTNF-R1) respectively.

3. The cell population according to claim 1, wherein the cells within the population secrete angiogenin, tissue metalloprotease inhibitor 2 (TIMP2), soluble glycoprotein 130 (sgp130), and soluble tumor necrosis factor α ubiquitous membrane receptor 1 (sTNF-R1) respectively.

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

5. The cell population according to claim 2 or 3, wherein the cells secrete the angiogenin, the TIMP2, the sgp130, and the sTNF-R1 in a polarized manner respectively.

6. The cell population according to claim 4 or 5, wherein the ratio of apical secretion of sgp130 to basolateral secretion of sgp130 is greater than 1.

7. The cell population according to claim 4 or 5, wherein the ratio of apical secretion of sTNF-R1 to basolateral secretion of sTNF-R1 is greater than 1.

8. The cell population according to claim 4 or 5, wherein the ratio of basolateral secretion of angiogenin to apical secretion of angiogenin is greater than 1.

9. The cell population according to claim 4 or 5, wherein the ratio of apical secretion of TIMP2 to basolateral secretion of TIMP2 is greater than 1.

10. Oct4 in the said population + TRA-1-60 + The cell population according to claim 1 or 2, wherein the number of cells is less than 1:250,000.

11. The cell population according to any one of claims 1 to 10, wherein at least 80% of the cells express bestrophin 1 (Bestrophin 1) when measured by immunostaining.

12. The cell population according to any one of claims 1 to 11, wherein when measured by immunostaining, at least 80% of said cells express microphthalmia-associated transcription factor (MITF).

13. The cell population according to any one of claims 1 to 12, wherein when measured by FACS, more than 50% of said cells express paired box gene 6 (PAX-6).

14. The cell population according to any one of claims 1 to 13, wherein said cells secrete more than 750 ng of pigment epithelium-derived factor (PEDF) per ml per day.

15. The cell population according to any one of claims 1 to 14, wherein said cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner.

16. The cell population according to claim 15, wherein the ratio of apical secretion of PEDF to basolateral secretion of PEDF is greater than 1.

17. The cell population according to claim 16, wherein after incubation at 2 - 8°C for 8 hours, the ratio is still greater than 1.

18. The cell population according to claim 2, wherein the transepithelial electrical resistance of said cell population is greater than 100 ohms.

19. The cell population according to claim 1 or 18, wherein after incubation at 2 - 8°C for 8 hours, the transepithelial electrical resistance of said cells is still greater than 100 ohms.

20. The cell population according to claim 15 or 16, wherein the ratio of basolateral secretion of VEGF to apical secretion of VEGF is greater than 1.

21. The cell population according to claim 20, wherein after incubation at 2 - 8°C for 8 hours, the ratio is still greater than 1.

22. The cell population according to any one of claims 1 to 21, which can rescue vision in RCS rats after subretinal administration.

23. The cell population according to any one of claims 1 to 21, which can rescue photoreceptors in RCS rats for at least 180 days after subretinal administration.

24. The cell population according to any one of claims 1 to 23, produced by ex vivo differentiation of human embryonic stem cells.

25. (a) culturing human embryonic stem cells in a medium containing nicotinamide and lacking activin A to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A so as to generate cells that are further differentiated into the RPE lineage; and (c) culturing the cells that are further differentiated into the RPE lineage in a medium containing nicotinamide and lacking activin A A cell population according to any one of claims 1 to 24, produced by the above. **Claim 26** The cell population according to claim 25, wherein the embryonic stem cells are proliferated in a medium containing bFGF and TGFβ. **Claim 27** The cell population according to claim 25, wherein the embryonic stem cells are cultured on human dermal fibroblasts. **Claim 28** The cell population according to any one of claims 25 to 27, wherein steps (a) to (c) are performed under conditions where the atmospheric oxygen level is less than about 10%. **Claim 29** The cell population according to claim 28, further comprising, after step (c), culturing the differentiated cells in a medium under conditions where the atmospheric oxygen level exceeds about 10% in the presence of nicotinamide. **Claim 30** A pharmaceutical composition comprising a cell population according to any one of claims 1 to 29 as an active substance and a pharmaceutically acceptable carrier. **Claim 31** Use of a cell population according to any one of claims 1 to 30 for treating retinal degeneration. **Claim 32** (a) culturing pluripotent stem cells in a medium containing a differentiating substance and lacking a member of the transforming growth factor β (TGFβ) superfamily so as to generate differentiated cells; (b) culturing the differentiated cells in a medium containing a member of the transforming growth factor β (TGFβ) superfamily and the differentiating substance so as to generate cells that are further differentiated into the RPE lineage; (c) culturing the cells that are further differentiated into the RPE lineage in a medium containing a differentiating substance and lacking a member of the transforming growth factor β (TGFβ) superfamily so as to generate RPE cells A method for producing RPE cells, comprising: The method, wherein steps (a) to (c) are performed under conditions where the atmospheric oxygen level is less than about 10%. **Claim 33** The method according to claim 32, wherein step (a) is performed under non-adherent conditions. **Claim 34** The method according to claim 33, wherein the non-adherent conditions include a non-adherent culture plate. **Claim 35** Step (a) is (i) Culturing a population of human pluripotent stem cells to be cultured in a medium containing nicotinamide under non-adherent conditions in the absence of activin A to generate a cluster of cells containing differentiated cells, and then, (ii) culturing the differentiated cells of (i) in a medium containing nicotinamide under adherent conditions in the absence of activin A The method according to claim 32, comprising:

36. The method according to claim 35, further comprising dissociating the cell cluster before step (ii) to produce a cell aggregate or a single cell suspension of the cells.

37. The method according to claim 32, further comprising culturing the differentiated cells in a medium under conditions where the atmospheric oxygen level exceeds about 10% in the presence of a differentiating substance after step (c).

38. The method according to claim 32, wherein the member of the transforming growth factor β (TGFβ) superfamily is selected from the group consisting of TGFβ1, TGFβ3, and activin A.

39. The method according to claim 32, wherein the differentiating substance in step (a) and the differentiating substance in step (c) are the same.

40. The method according to claim 32, wherein the differentiating substance in step (a) is nicotinamide (NA) or 3-aminobenzamide.

41. The method according to claim 32, further comprising selecting polygonal cells after step (c).

42. The method according to claim 41, further comprising proliferating the polygonal cells.

43. The method according to claim 42, wherein the proliferating step is performed on an adherent surface.

44. The method according to claim 32, wherein the pluripotent stem cells include embryonic stem cells.

45. The method according to claim 44, wherein the embryonic stem cells are proliferated in a medium containing bFGF and TGFβ.

46. The method according to claim 44, wherein the embryonic stem cells are cultured on human foreskin fibroblasts.

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