Methods for generating retinal pigment epithelial cells

Abstract

To provide an improved method for generating highly pure retinal pigment epithelial (RPE) cells by differentiation of pluripotent stem cells.SOLUTION: A method for generating a population of retinal epithelial (RPE) cells comprising: (I) obtaining clusters of cells of PAX6 + / MITF + RPE progenitor cells and dissociating the clusters of cells into single cells; (ii) culturing the single cells in a differentiating medium such that the cells differentiate into RPE cells; and (iii) collecting the RPE cells generated in step (ii); thereby generating a population of RPE cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 928,125, filed October 30, 2019, the entire contents of which are expressly incorporated herein by reference. [Background technology]

[0002] background The retinal pigment epithelium (RPE) is a layer of pigmented cells immediately outside the neurosensory retina. This layer of cells nourishes retinal photoreceptors and is attached to the underlying choroid (a layer of blood vessels behind the retina), covering them. The RPE acts as a filter to determine which nutrients reach the retina from the choroid. Additionally, the RPE provides insulation between the retina and choroid. Breakdown of the RPE disrupts retinal metabolism, causing retinal thinning. Retinal thinning can have serious consequences. For example, retinal thinning can lead to "dry" macular degeneration and can also lead to improper angiogenesis, which can cause "wet" macular degeneration.

[0003] Given the importance of the RPE in maintaining vision and retinal health, there has been significant effort in studying the RPE and developing methodologies for generating RPE cells in vitro. In vitro-generated RPE cells can be used to study RPE development, identify factors that disrupt the RPE, or identify agents that can be used to stimulate repair of endogenous RPE cells. Furthermore, in vitro-generated RPE cells can also be used as a therapy to replace or restore all or part of a patient's damaged RPE cells. Used in this way, RPE cells may provide an approach for treating macular degeneration and other diseases and conditions caused, in whole or in part, by damage to the RPE.

[0004] In vitro methods for generating retinal pigment epithelial (RPE) cells by inducing differentiation of pluripotent stem cells in the presence of differentiation-inducing factors in culture medium are known (see, e.g., Kuroda et al., PLoS One. 2012; 7(5): e37342). However, these methods require multiple steps that combine adherent and suspension culture to obtain highly enriched RPE cell populations. These known methods also require purification steps.

[0005] In addition, when RPE cells are obtained from pluripotent stem cells using conventional known methods, cells other than target cells are generally obtained at the same time.As a result, these methods can only obtain a portion of the RPE cells induced in culture vessels.In addition, the purity of the obtained RPE cells is greatly affected by the technique of the experimenter, which makes these methods unsuitable for obtaining a pure population of RPE cells in a short period of time.

[0006] Therefore, there is a need in the art for a simple and efficient method for generating highly pure RPE cells from pluripotent stem cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kuroda et al., PLoS One. 2012; 7(5): e37342 Summary of the Invention

[0008] overview The present invention provides an improved method for obtaining retinal pigment epithelium (RPE) from pluripotent stem cells, such as human embryonic stem (hES) cells. In particular, the present invention is based on the discovery of a stage during the differentiation of pluripotent stem cells into RPE cells, at which RPE precursors can be isolated, partially purified, and further differentiated into mature RPE cells with minimal or no manual collection of cells. As described herein, following the initiation of differentiation of pluripotent cells, the inventors have identified a time point during the culture process at which a high percentage of clusters of RPE precursor cells (e.g., identified as PAX6 / MITF-positive cells) remain together. Thus, the method described herein includes treating clusters of RPE precursor cells with a dissociation reagent, such as collagenase or dispase, that detaches the cells in the clusters, followed by size fractionation of the clusters, and then subculturing the cells to generate RPE cells. The method of the present invention is simple and efficient, and in some embodiments, results in a culture of substantially pure RPE cells.

[0009] In one aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising the steps of: (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells and dissociating the cell clusters into single cells; (ii) culturing the single cells in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells.

[0010] In another aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising the steps of: (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells; (ii) culturing the cell clusters in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In any of the embodiments of the present invention, the PAX6+ / MITF+ RPE progenitor cells can be obtained from a population of pluripotent stem cells.

[0011] In one aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect RPE progenitor cell clusters, dissociating the RPE progenitor cell clusters into single cells, and subculturing the single cells in a second differentiation medium so that the single cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In another aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, the method comprising the steps of: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect RPE progenitor cell clusters, and subculturing the collected RPE progenitor cell clusters in a second differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In one embodiment of the present invention, the RPE progenitor cells are positive for PAX6 / MITF. In another embodiment, prior to step (i), the pluripotent stem cells are cultured on feeder cells in a pluripotency-supporting medium. In a further embodiment, prior to step (i), the pluripotent stem cells are cultured feeder-free in a pluripotency-supporting medium. In one embodiment, the pluripotency-supporting medium is supplemented with bFGF.

[0012] The method can further comprise recovering the RPE cells produced in step (ii) of any of the described methods by dissociating the RPE cells, fractionating the RPE cells to collect RPE cell clusters, dissociating the RPE cell clusters into single RPE cells, and culturing the single RPE cells. In another embodiment, the method can further comprise recovering the RPE cells produced in step (ii) of any of the described methods by dissociating the RPE cells, collecting RPE cell clusters, and selectively collecting the RPE cell clusters. The method can further comprise dissociating the selectively collected RPE cell clusters into single RPE cells and culturing the single RPE cells.

[0013] In any of the embodiments of the present invention, the method can further comprise the step of expanding RPE cells. RPE cells can be expanded by culturing them in a maintenance medium supplemented with FGF. In one embodiment, RPE cells are cultured in a maintenance medium containing FGF for the first 1, 2, or 3 days of RPE growth at each passage, and then the RPE cells are cultured in a maintenance medium lacking FGF. In one embodiment, FGF is added before the RPE cells reach confluence. In another embodiment, RPE cells are passaged up to two times.

[0014] In any of the embodiments of the present invention, any one of the dissociation steps is performed by treating the cells with a dissociation reagent, hi one embodiment, the dissociation reagent is selected from the group of collagenase (e.g., collagenase I or collagenase IV), accutase, chelating agent (e.g., EDTA-based dissociation solution), trypsin, dispase, or any combination thereof.

[0015] In any of the embodiments, the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells. In any of the embodiments of the invention, the population of pluripotent stem cells is embryoid bodies. In any of the embodiments of the invention, the cells are cultured on feeder cells. In yet another embodiment, the cells are cultured under feeder-free conditions. In a further embodiment, the cells are cultured in non-adherent culture. In another embodiment, the cells are cultured in adherent culture.

[0016] In one embodiment of the present invention, the differentiation medium is EBDM. In another embodiment, the differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), bone morphogenetic protein (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factor (GDF)), WNT pathway inhibitor (e.g., CKI-7, DKK1), TGF pathway inhibitor (e.g., LDN193189, noggin), BMP pathway inhibitor (e.g., SB431542), sonic hedgehog signal inhibitor, bFGF inhibitor, and MEK inhibitor (e.g., PD0325901). In a further embodiment, the differentiation medium comprises nicotinamide. In yet another embodiment, the differentiation medium comprises activin. In one embodiment, the first differentiation medium and the second differentiation medium are the same. In another embodiment, the first differentiation medium and the second differentiation medium are different. In yet another embodiment, the first differentiation medium and the second differentiation medium are EBDM. In one embodiment, the first differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, Noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and MEK inhibitors (e.g., PD0325901).In one embodiment, the second differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and MEK inhibitors (e.g., PD0325901). In another embodiment, the first differentiation medium comprises nicotinamide. In another embodiment, the second differentiation medium comprises activin. In any of the embodiments of the present invention, the differentiation medium may further comprise heparin and / or a ROCK inhibitor.

[0017] In one embodiment of the invention, the cell clusters of RPE progenitor cells are between about 40 μm and about 200 μm in size, and in another embodiment, the cell clusters of RPE progenitor cells are between about 40 μm and about 100 μm in size.

[0018] In any of the embodiments of the present invention, in step (ii), the cells are cultured on an extracellular matrix selected from the group consisting of laminin or a fragment thereof, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin. In one embodiment, the extracellular matrix is ​​laminin or a fragment thereof. In another embodiment, the laminin is selected from laminin-521 and laminin-511. In a further embodiment, the laminin is iMatrix511.

[0019] In any of the aspects of the present invention, the duration of the step of culturing the population of pluripotent stem cells in the first differentiation medium is about 1 week to about 12 weeks. In another aspect, the duration of the step of culturing the population of pluripotent stem cells in the first differentiation medium is at least about 3 weeks. In another aspect, the duration of the step of culturing the population of pluripotent stem cells in the first differentiation medium is about 6 weeks to about 10 weeks. In any of the aspects of the present invention, the duration of the culture in step (ii) is about 1 week to about 8 weeks. In another aspect, the duration of the culture in step (ii) is at least about 3 weeks. In yet another aspect, the duration of the culture in step (ii) is about 6 weeks.

[0020] In some embodiments of the present invention, RPE progenitor cell clusters or RPE progenitor single cells are passaged on an extracellular matrix selected from the group consisting of laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin. In one embodiment, the extracellular matrix comprises laminin or a fragment thereof. In one embodiment, the laminin or a fragment thereof is selected from laminin-521 and laminin-511.

[0021] In one embodiment of the present invention, single RPE cells are cultured in a medium that supports the proliferation or differentiation of RPE.In another embodiment, single RPE cells are cultured on an extracellular matrix selected from the group consisting of laminin or its fragment, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin.In one embodiment, the extracellular matrix is ​​gelatin.In yet another embodiment, the extracellular matrix is ​​laminin or its fragment.

[0022] In certain embodiments, the composition of RPE cells comprises a substantially purified population of RPE cells. For example, the composition of RPE cells may comprise less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of cells other than RPE cells. In some embodiments, a substantially purified population of RPE cells is one in which RPE cells comprise at least about 75% of the cells in the population. In other embodiments, a substantially purified population of RPE cells is one in which RPE cells comprise at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, or even more than 99% of the cells in the population. In some embodiments, the pigmentation level of RPE cells in the cell culture is uniform. In other embodiments, the pigmentation of RPE cells in the cell culture is heterogeneous. The cell culture of the present invention comprises at least about 10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or at least about 10 10 In any of the embodiments of the present invention, the RPE cells are human RPE cells.

[0023] In one embodiment of the invention, the RPE cell clusters are between about 40 μm and 200 μm in size, hi another embodiment, the RPE cell clusters are between about 40 μm and 100 μm in size.

[0024] In any of the embodiments of the present invention, RPE cells express one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of the following genes (at the mRNA and / or protein level): RPE65, CRALBP, PEDF, bestrophin (BEST1), MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1. In one embodiment, RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1. In a further embodiment, RPE cells express bestrophin, PAX6, MITF, and RPE65. In another embodiment, RPE cells express at least one gene selected from MITF, bestrophin, and PAX6. In certain embodiments, gene expression is measured by mRNA expression. In other embodiments, gene expression is measured by protein expression.

[0025] In one embodiment of the present invention, RPE cells lack substantial expression of one or more stem cell markers.The stem cell markers can be selected from the group consisting of OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80.In one embodiment, RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase.In another embodiment, RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

[0026] In any of the embodiments of the present invention, RPE cells are cryopreserved after collection. In certain embodiments of any of the aforementioned aspects, RPE cells are frozen for storage. Cells can be frozen by any suitable method known in the art, for example, cryogenic freezing, and can be frozen at any temperature suitable for cell preservation. In one embodiment, the cryopreserved composition comprises RPE cells and a cryopreservative. Any cryopreservative known in the art can be used, and the cryopreservative can include one or more of DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, and sucrose. In one embodiment, the cryopreservative comprises about 5% to about 50% DMSO and about 30% to about 95% serum, which may optionally be fetal bovine serum (FBS). In certain embodiments, the cryopreservative comprises about 90% FBS and about 10% DMSO. In another embodiment, the cryopreservative comprises about 2% to about 5% DMSO. In one embodiment, cells can be frozen at approximately -20°C to -196°C, or any other temperature suitable for cell storage. In one embodiment, cells are frozen at about -80°C or about -196°C. In another embodiment, cells are frozen at about -135°C to about -196°C. In a particular embodiment, cells are frozen at about -135°C. In a further embodiment, cells can be frozen using an automated slow-freezing protocol, whereby cells are cooled to a specified temperature in a computer-controlled process. Cryogenically frozen cells are stored in an appropriate container and prepared for storage to reduce the risk of cell damage and maximize the likelihood that the cells will survive thawing. In other embodiments, RPE cells are maintained or transported at about 2°C to about 37°C. In one embodiment, RPE cells are maintained or transported at room temperature, about 2°C to about 8°C, about 4°C, or about 37°C.

[0027] In certain embodiments of any of the foregoing, the methods are performed in accordance with current good manufacturing practices (cGMP). In certain embodiments of any of the foregoing, the pluripotent stem cells from which the RPE cells differentiate are obtained in accordance with current good manufacturing practices (cGMP).

[0028] The present invention also provides a composition comprising a population of RPE cells produced by any one of the methods described herein. In certain embodiments of any of the foregoing, the method comprises producing at least 10 RPE cells, at least 100 RPE cells, at least 1000 RPE cells, at least 1 x 10 RPE cells, or at least 1 x 10 RPE cells. 4 RPE cells, at least 1 x 10 5 RPE cells, at least 5 x 10 5 RPE cells, at least 1 x 10 6 RPE cells, at least 5 x 10 6 RPE cells, at least 1 x 10 7 RPE cells, at least 2 x 10 7 RPE cells, at least 3 x 10 7 RPE cells, at least 4 x 10 7 RPE cells, at least 5 x 10 7 RPE cells, at least 6 x 10 7 RPE cells, at least 7 x 10 7 RPE cells, at least 8 x 10 7 RPE cells, at least 9 x 10 7 RPE cells, at least 1 x 10 8 RPE cells, at least 2 x 10 8 RPE cells, at least 5 x 10 8 RPE cells, at least 7 x 10 8 RPE cells, at least 1 x 10 9 RPE cells, at least 1 x 10 10 RPE cells, at least 1 x 10 11 RPE cells, or at least 1 x 10 12 In one embodiment, the composition comprises about 1 x 10 RPE cells. 8 ~1×10 12 RPE cells, approximately 1 x 10 9 ~1×10 11 RPE cells, or approximately 5 x 10 9 ~1×10 10In certain embodiments, the number of RPE cells in the composition includes RPE cells of different maturity levels. In other embodiments, the number of RPE cells in the composition refers to the number of mature RPE cells.

[0029] The present invention further provides a method for treating a patient with or at risk of a retinal disease, comprising administering an effective amount of a composition comprising a population of RPE cells produced by any one of the methods described herein, or a pharmaceutical composition comprising a population of RPE cells produced by any of the methods described herein and a pharmaceutically acceptable carrier. In one embodiment, the retinal disease is selected from the group consisting of retinal degeneration, choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt's disease, angioid streaks, myopic macular degeneration, and glaucoma. In certain embodiments, the method further comprises formulating the RPE cells to produce a composition of RPE cells suitable for transplantation.

[0030] In another aspect, the present invention provides a method for treating or preventing a condition characterized by retinal degeneration, comprising administering an effective amount of a composition comprising RPE cells to a subject in need thereof, wherein the RPE cells are derived from human embryonic stem cells or other pluripotent stem cells.Conditions characterized by retinal degeneration include, for example, Stargardt's macular dystrophy, age-related macular degeneration (dry or wet), diabetic retinopathy, and retinitis pigmentosa.In certain embodiments, the RPE cells are obtained from human pluripotent stem cells using one or more of the methods described herein.

[0031] In certain embodiments, the preparation is previously cryopreserved and thawed prior to implantation.

[0032] In certain embodiments, the method of treatment further comprises administering one or more immunosuppressants. In one embodiment, the immunosuppressant can comprise one or more of the following: antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX1MAB® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil (MMF). When an immunosuppressant is used, it can be administered systemically or locally and can be administered prior to, simultaneously with, or after administration of RPE cells. In certain embodiments, immunosuppressive therapy continues for weeks, months, years, or indefinitely following administration of RPE cells. In other embodiments, the method of treatment does not require the administration of immunosuppressants. In certain embodiments, the method of treatment comprises the administration of a single dose of RPE cells. In other embodiments, the method of treatment comprises a course of therapy in which RPE cells are administered multiple times over a period of time. Exemplary courses of treatment can include weekly, biweekly, monthly, quarterly, twice-yearly, or annually. Alternatively, treatment can proceed stepwise, requiring multiple administrations initially (e.g., daily administration during the first week), followed by fewer and less frequent administrations. Multiple treatment regimens are contemplated.

[0033] In certain embodiments, the composition comprising RPE cells is implanted in a suspension, matrix, or substrate. In certain embodiments, the composition is administered to the subretinal space of the eye by injection. In certain embodiments, the composition is administered in an amount of about 10 4 ~about 10 6RPE cells are administered to the subject. In certain embodiments, the method further comprises monitoring the effectiveness of the treatment or prevention by measuring the electroretinogram response, optomotor acuity threshold, or luminance threshold in the subject. The method can also comprise monitoring the effectiveness of the treatment or prevention by monitoring the immunogenicity of the cells or the migration of the cells in the eye. In other embodiments, the efficacy of the treatment can be evaluated by determining visual outcomes by one or more of the following: slit-lamp biomicroscopy, fundus photography, 1VFA, and SD-OCT, and best-corrected visual acuity (BCVA). The method can result in an improvement in best-corrected visual acuity (BCVA) and / or an increase in the number of letters that can be read on an eye chart, for example, the Early Treatment Diabetic Retinopathy Study (ETDRS).

[0034] In certain aspects, the present invention provides a pharmaceutical composition for treating or preventing a condition characterized by retinal degeneration, comprising an effective amount of RPE cells, wherein the RPE cells are derived from human embryonic stem cells or other pluripotent stem cells. The pharmaceutical composition can be formulated in a pharmaceutically acceptable carrier according to the administration route. For example, the preparation can be formulated for administration into the subretinal space of the eye. The composition can be administered in a concentration of at least 10 ... 3 , 10 4 , 10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , or 10 7 In certain embodiments, the composition may comprise at least 1 x 10 RPE cells. 4 , 5×10 4 , 1×10 5, 1.5×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 The RPE cells may be included.

[0035] In certain embodiments, RPE cells are formulated in a pharmaceutical composition comprising RPE cells and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the present invention provides pharmaceutical preparations comprising human RPE cells derived from human embryonic stem cells or other pluripotent stem cells. The pharmaceutical preparations comprise at least about 10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 1.5×10 5 , 2 × 10 5 , 5×10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or about 10 10 The cells may contain hRPE cells.

[0036] In another aspect, the present invention provides a screening method for identifying the agent that modulates the survival of RPE cells.For example, RPE cells obtained from human embryonic stem cells can be used to screen for the agent that promotes the survival of RPE.The identified agent can be used alone or in combination with RPE cells as part of a treatment regimen.Alternatively, the identified agent can be used as part of a culture method to improve the survival of RPE cells differentiated in vitro.

[0037] In another aspect, the present invention provides a method for screening to identify agents that modulate the maturation of RPE cells. For example, RPE cells derived from human ES cells can be used to screen for agents that promote RPE maturation. [Brief explanation of the drawings]

[0038] [Figure 1] 1 shows the time course of PAX6 and MITF mRNA expression by qPCR in RPE progenitor cells compared to normalized GAPDH mRNA expression. [Figure 2] 1 shows the time course of PAX6 and MITF expression by immunofluorescence assay (IFA) of various cell fractions obtained after the initiation of differentiation into RPE cells. [Figure 3A] 1 shows a schematic diagram of a single RPE progenitor cell subculture method. [Figure 3B] 1 shows a schematic diagram of a method for subculturing RPE progenitor cell clusters. [Figure 4-1] 1 shows exemplary workflows for single RPE progenitor cell subculture and RPE progenitor cell cluster subculture methods. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] 1 shows the characteristics of RPE cells obtained by single RPE progenitor cell subculture and RPE progenitor cell cluster subculture methods according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description The present invention provides an improved method for obtaining retinal pigment epithelial (RPE) cells from pluripotent stem cells, such as human embryonic stem (hES) cells, embryo-derived cells, and induced pluripotent stem cells (iPS cells). In particular, the present invention is based on the discovery of a stage during pluripotent stem cell differentiation at which RPE precursors can be isolated, partially purified, and further differentiated into mature RPE cells with minimal or no selective collection of cells. In particular, as described herein, following the initiation of pluripotent cell differentiation, the inventors identified a time point during the culture process at which there are sufficient clusters of RPE precursor cells (identified as PAX6 / MITF-positive cells) that remain together when the culture is dissociated with dissociation reagents, such as collagenase and dispase. The culture is not too mature, so that the majority of non-RPE cells in the culture or attached to such RPE precursor cell clusters can be eliminated as single cells. Furthermore, large clusters of non-RPE cells and clusters containing a mixture of RPE and non-RPE can be eliminated by size fractionation, thereby increasing purity.Therefore, the method described herein includes treating clusters of RPE progenitor cells with a dissociation reagent, such as collagenase or dispase, followed by size fractionation to isolate RPE progenitor cell clusters of a specific size, and subculturing the RPE progenitor cells as single cells or cell clusters to generate RPE cells.

[0040] In one embodiment, the methods of the present invention involve isolating RPE progenitor cell clusters between about 40 and about 200 μm in size, or between about 40 and about 100 μm in size. In one embodiment, the RPE progenitor cell clusters are collected using a cell strainer or a series of cell strainers to collect cell clusters of the desired size. For example, to obtain cell clusters between about 40 and about 200 μm or between about 40 and about 100 μm, cell strainers of 40 μm, 70 μm, 100 μm, 200 μm, or any other filter size that will allow for obtaining the desired cell cluster size can be used. The methods of the present invention are simple and efficient. In some embodiments, the methods of the present invention result in a culture of substantially pure RPE cells. A substantially purified population of RPE cells is one in which RPE cells constitute at least about 75% of the cells in the population. In other embodiments, a substantially purified population of RPE cells is one in which RPE cells constitute at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5, 99%, or even greater than 99% of the cells in the population.

[0041] The present invention offers several advantages over methods known in the art for producing RPE cells, including, for example, greatly enhanced RPE cell yield, greatly enhanced RPE cell purity, improved ease of manual RPE cell isolation, the ability for automated RPE cell selection, the elimination of the need for any further purification by manual or automated selection, and the use of simple components that enable commercial large-scale production. In some embodiments, the methods of the present invention increase RPE yield, e.g., by more than 50-90 fold, and produce RPE cells with a high consistency of greater than 98%-99% purity, compared to cells produced by conventional production methods involving manual collection.

[0042] In order to fully understand the invention described herein, the following detailed description is provided. Various aspects of the invention are described in detail and can be further illustrated by the examples provided herein. All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs, unless otherwise defined.

[0043] definition Unless otherwise specified, each of the following terms has the meaning set forth in this section.

[0044] The indefinite articles "a" and "an" refer to at least one of the associated noun and are used interchangeably with the terms "at least one" and "one or more."

[0045] The conjunctions "or" and "and / or" are used interchangeably as non-exclusive disjunctions.

[0046] As used herein, the term "retinal pigment epithelial cell" or "RPE cell" is used interchangeably herein to refer to the epithelial cells that constitute the retinal pigment epithelium.This term is generally used to refer to differentiated RPE cells regardless of the maturity level of cells, and therefore can encompass RPE cells of various maturity levels.RPE cells can be visually recognized by their cobblestone morphology and early appearance of pigment.RPE cells can also be molecularly identified based on the substantial absence of the expression of embryonic stem cell markers such as OCT4 and NANOG, and based on the expression of RPE markers such as RPE65, PEDF, CRALBP, and / or bestrophin (BEST1). In one embodiment, RPE cells lack substantial expression of one or more embryonic stem cell markers, including but not limited to, OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and / or TRA-1-80. In another embodiment, RPE cells express one or more RPE cell markers, including but not limited to, RPE65, CRALBP, PEDF, bestrophin, MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), and / or tyrosinase. In another embodiment, RPE cells express ZO1. In one embodiment, RPE cells express at least one marker selected from MITF, bestrophin, and PAX6. Please note that when other RPE-like cells are mentioned, they are generally referred to as adult RPE, fetal RPE, primary cultures of adult or fetal RPE, and immortalized RPE cell lines, such as APRE19 cells.Therefore, unless otherwise specified, as used herein, RPE cells refer to RPE cells obtained from pluripotent stem cells (PSC-RPE), and may also refer to RPE cells obtained from human pluripotent stem cells (hRPE).

[0047] The pigmentation of RPE cells can vary according to the cell density of culture and the maturity of RPE cells.However, when cells are referred to as pigmented, this term is understood to refer to any and all levels of pigmentation.Therefore, the present invention provides RPE cells with various levels of pigmentation.In certain embodiments, the pigmentation of RPE is the same as the average pigmentation of other RPE-like cells, such as adult RPE, fetal RPE, the primary culture of adult or fetal RPE, or immortalized RPE cell line, such as ARPE19.In certain embodiments, the pigmentation of RPE is higher than the average pigmentation of other RPE-like cells, such as adult RPE, fetal RPE, the primary culture of adult or fetal RPE, or immortalized RPE cell line, such as ARPE19. In certain other embodiments, the degree of pigmentation of the RPE is lower than the average pigmentation of other RPE-like cells, such as adult RPE, fetal RPE, primary cultures of adult or fetal RPE, or immortalized RPE cell lines, such as ARPE19.

[0048] Functional evaluation of RPE cells can be confirmed, for example, by secreting cytokines (such as VEGF or PEDF), phagocytic ability, such as phagocytosis of shed outer segments of rods and cones (or phagocytosis of other substrates such as polystyrene beads), absorption of stray light, metabolism of vitamin A, retinoid regeneration, transepithelial resistance, cell polarity, and tissue repair. Evaluation can also be performed by testing in vivo function after implantation of RPE cells into a suitable host animal (e.g., a human or non-human animal suffering from a natural or induced retinal degeneration condition) using, for example, behavioral tests, fluorescence angiography, histological examination, tight junction conductivity, or electron microscopy. These functional evaluations and confirmation procedures can be performed by those skilled in the art. As used herein, RPE cells include human RPE (hRPE) cells.

[0049] As used herein, the terms "RPE cell precursor cells" or "RPE precursor cells" are used interchangeably herein to refer to cells that are instructed to differentiate into retinal cells. In one embodiment, the term RPE precursor cells can be used to refer to any cells that are instructed to differentiate into retinal cells by the time RPE cells are harvested (e.g., for plating at P0 as described herein). It will be recognized that in later stages of differentiation, the differentiation culture may contain a mixture of RPE precursor cells and RPE cells. In one embodiment, the precursor cells express, for example, MITF (pigment epithelial cells, precursor cells), PAX6 (precursor cells), Rx (retinal precursor cells), Crx (photoreceptor precursor cells), and / or Chx10 (bipolar cells). In one embodiment, the RPE precursor cells express PAX6 and MITF.

[0050] The terms "mature RPE cells" and "mature differentiated RPE cells" are used interchangeably throughout to refer to changes that occur following the initial differentiation of RPE cells. In particular, RPE cells can be recognized to some extent based on the initial appearance of pigment, but after differentiation, mature RPE cells can be recognized based on enhanced pigmentation. Pigmentation after differentiation may not indicate a change in the RPE state of the cells (e.g., the cells are still differentiated RPE cells). The change in pigment after differentiation may correspond to the density at which the RPE cells are cultured and maintained. Mature RPE cells may have increased pigmentation that accumulates after initial differentiation. Mature RPE cells may be more pigmented than immature RPE cells and may appear after the RPE has stopped proliferating, for example, due to high cell density in a culture dish. Mature RPE cells can be subcultured at lower densities to allow for the expansion of mature RPE cells. The growth of mature RPE cells in culture may be accompanied by dedifferentiation—the loss of pigment and epithelial morphology, which are both restored after the cells form a monolayer and become quiescent.In this regard, mature RPE cells can be cultured to generate RPE cells.Such RPE cells are still differentiated RPE cells that express RPE markers.Therefore, in contrast to the initial appearance of pigmentation that occurs when RPE cells begin to appear, the changes in pigmentation after differentiation are phenotypic and do not reflect the dedifferentiation of cells away from the fate of RPE.The changes in pigmentation after differentiation can also be correlated with changes in one or more of the expression of PAX2, PAX6, tyrosinase, neural markers such as tubulin beta III, bestrophin, RPE65, and CRALBP.In one embodiment, the changes in pigmentation after differentiation show an inverse correlation with one or more of PAX6 and neural markers (e.g., tubulin beta III).In another embodiment, the changes in pigmentation after differentiation show a direct correlation with RPE65 and CRALBP.

[0051] As used herein, the term "pluripotent stem cells," "PS cells," or "PSCs" includes embryonic stem cells, induced pluripotent stem cells, and embryo-derived pluripotent stem cells, regardless of the method by which the pluripotent stem cells are obtained. Pluripotent stem cells are functionally defined as stem cells that (a) can induce teratomas when transplanted into immunodeficient (SCID) mice; (b) can differentiate into all three germ layers of cell types (e.g., ectoderm, mesoderm, and endoderm); (c) express one or more markers of embryonic stem cells (e.g., express OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, NANOG, TRA-1-60, TRA-1-81, SOX2, REX1, etc.); and d) can self-renew. The term "pluripotency" refers to the ability of a cell to form all lineages of the body or cell body (i.e., the embryonic body). For example, embryonic stem cells and induced pluripotent stem cells are types of pluripotent stem cells that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism. Exemplary pluripotent stem cells can be generated, for example, using methods known in the art. Exemplary pluripotent stem cells include, but are not limited to, embryonic stem cells derived from the ICM of a blastocyst-stage embryo, embryonic stem cells derived from one or more blastomeres of a cleavage-stage or morula-stage embryo (optionally without destroying the rest of the embryo), induced pluripotent stem cells generated by reprogramming somatic cells to a pluripotent state, and pluripotent cells generated from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF). Such embryonic stem cells can be generated from embryonic material by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis.

[0052] In one aspect, pluripotent stem cells are modified to, for example, enhance longevity, efficacy, homing, etc. , to prevent or reduce immune responses, or to deliver desirable factors obtained from such pluripotent cells (e.g., RPE) to cells. For example, pluripotent stem cells, and thus the resulting differentiated cells, can be engineered or otherwise modified to lack or have reduced expression of beta2 microglobulin, HLA-A, HLA-B, HLA-C, TAP1, TAP2, tapasin, CTIIA, RFX5, TRAC, or TRAB genes. Pluripotent stem cells and the resulting differentiated cells can be engineered or otherwise modified to increase gene expression. A variety of techniques exist for engineering modulated expression of one or more genes (or proteins), including the use of viral vectors such as AAV vectors, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas-based methods for genome engineering, and the use of transcription and translation inhibitors, e.g., antisense and RNA interference, which can be achieved using stably integrating and episomal vectors.

[0053] The term "embryonic" refers to a developing mass of cells that has not implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained within an embryo. It also includes blastomeres obtained as early as the two-cell stage or aggregated blastomeres after extraction.

[0054] As used herein, the term "embryonic-derived cells" (EDCs) broadly refers to blastocyst-derived cells, including those from the morula, inner cell mass, embryonic shield, or epiblast, or other pluripotent stem cells of early embryos, including primitive endoderm, ectoderm, and mesoderm, and their derivatives. "EDCs" also includes cell masses from blastomeres and aggregated single blastomeres or embryos from various stages of development, but excludes human embryonic stem cells that have been passaged as cell lines.

[0055] As used herein, the term "embryonic stem cell," "ES cell," or "ESC" broadly refers to cells that are isolated from the inner cell mass of a blastocyst or morula and are continuously subcultured as a cell line. This term also includes cells that are preferably isolated from one or more blastomeres of an embryo without destroying the rest of the embryo (see, for example, Chung et al., Cell Stem Cell. 2008 Feb 7;2(2):113-7; US Patent Application No. 20060206953; US Patent Application No. 2008 / 0057041, each of which is incorporated herein by reference in its entirety). ES cells can be obtained by fertilizing egg cells with sperm or DNA, nuclear transfer, parthenogenesis, or by any means that can generate ES cells with homozygosity in the HLA region. ES cells can also refer to cells obtained from a zygote, blastomere, or blastocyst stage mammalian embryo produced by the fusion of a sperm and an egg cell, nuclear transfer, parthenogenesis, or the generation of cells by chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the plasma membrane. In one embodiment, the embryonic stem cells can be human embryonic stem cells (or "hES cells"). In one embodiment, the human embryonic stem cells are not derived from an embryo greater than 14 days post-fertilization. In another embodiment, the human embryonic stem cells are not derived from an embryo developed in vivo. In another embodiment, the human embryonic stem cells are derived from a pre-implantation embryo generated by in vitro fertilization.

[0056] As used herein, "induced pluripotent stem cells" or "iPS cells" generally refer to pluripotent stem cells obtained by reprogramming somatic cells. iPS cells can be generated by expressing or inducing the expression of a combination of factors ("reprogramming factors") in somatic cells, such as OCT4 (sometimes referred to as OCT3 / 4), SOX2, MYC (e.g., c-MYC or any MYC variant), NANOG, LIN28, and KLF4. In one embodiment, the reprogramming factors include OCT4, SOX2, c-MYC, and KLF4. In another embodiment, the reprogramming factors include OCT4, SOX2, NANOG, and LIN28. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram them. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram them. In another embodiment, at least four reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In another embodiment, at least five reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In yet another embodiment, at least six reprogramming factors, such as OCT4, SOX2, c-MYC, NANOG, LIN28, and KLF4, are expressed in somatic cells. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0057] iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. Somatic cells can include, but are not limited to, fibroblasts, keratinocytes, adipocytes, muscle cells, organ and tissue cells, and various blood cells, including, but not limited to, hematopoietic cells (e.g., hematopoietic stem cells). In one embodiment, the somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or pulmonary fibroblasts, or non-fibroblastic cells.

[0058] iPS cells can be obtained from cell banks. Alternatively, iPS cells can be generated de novo by methods known in the art. iPS cells can be specifically generated using material from a particular patient or matched donor with the goal of generating histocompatible cells. In one embodiment, iPS cells can be universal donor cells that are substantially non-immunogenic.

[0059] Induced pluripotent stem cells can be generated by expressing one or more reprogramming factors in somatic cells or inducing the expression of said reprogramming factors.Reprogramming factors can be expressed in somatic cells by infection with viral vectors such as retroviral vectors, or other gene editing techniques such as CRISPR, Talen, zinc finger nuclease (ZFN).Reprogramming factors can also be expressed in somatic cells by using non-integrated vectors, such as episomal plasmids, or RNA, such as synthetic mRNA, or via RNA viruses, such as Sendai virus.When using non-integrated vectors to express reprogramming factors, the factors can be expressed in cells by using electroporation, transfection, or transformation of somatic cells with vectors.For example, in mouse cells, the expression of four factors (OCT3 / 4, SOX2, c-MYC, and KLF4) using integrating viral vectors is sufficient to reprogram somatic cells. In human cells, expression of four factors (OCT3 / 4, SOX2, NANOG, and LIN28) using integrating viral vectors is sufficient to reprogram somatic cells.

[0060] Expression of the reprogramming factors can be induced by contacting the somatic cells with at least one agent, such as a small organic molecule agent, that induces expression of the reprogramming factors.

[0061] Somatic cells can also be reprogrammed using combinatorial approaches in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules).

[0062] Once reprogramming factor is expressed or induced in cell, cell can be cultured.With time, cells with ES characteristics appear in culture dish.For example, based on the morphology of ES cell or based on the expression of selectable or detectable marker, cell can be selected and subcultured.Cell can be cultured to produce the culture of cells that resemble ES cell.

[0063] To confirm the pluripotency of iPS cells, the cells can be tested in one or more assays for pluripotency. For example, the cells can be tested for the expression of ES cell markers; the cells can be evaluated for their ability to generate teratomas when transplanted into SCID mice; and the cells can be evaluated for their ability to generate cell types of all three germ layers.

[0064] iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, neonatal, and adult tissues. Therefore, donor cells from any species can be used to easily generate iPS cells. Therefore, iPS cells can be generated from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, e.g., lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc.

[0065] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as an RPE cell. A differentiated cell is one that has taken a more specialized position within the lineage of a cell. For example, hES cells can differentiate into a variety of more differentiated cell types, including RPE cells.

[0066] As used herein, the terms "cultured" or "culturing" refer to placing cells in a medium containing, among other things, nutrients, any particular added substances required to sustain the survival of the cultured cells. Cells are cultured "in the presence of" a particular substance if the medium in which such cells are maintained contains such a particular substance. Culturing can be performed in any vessel or device capable of maintaining cells exposed to the medium, including, but not limited to, petri dishes, culture dishes, blood collection bags, roller bottles, flasks, test tubes, microtiter wells, hollow fiber cartridges, or any other device known in the art.

[0067] As used herein, the term "subculture" or "passage" refers to transferring some or all of the cells from a previous culture into fresh growth medium and / or plating them onto a new culture dish, and further culturing the cells. Subculture is performed, for example, to extend survival, enrich for a desired cell population, and / or expand the number of cells in culture. For example, this term includes transferring, culturing, or plating some or all of the cells into a new culture vessel at a lower cell density to allow cell growth.

[0068] As used herein, the terms "selectively harvest" or "selective collection" refer to the mechanical collection or separation of a subset of cells from a larger population based on appearance or other phenotypic characteristics. Selective collection can be performed manually or by an automated system, and can be performed using a microscope, computer imaging system, or other means to identify the cells to be harvested.

[0069] As used herein, the term "dissociation reagent" refers to an enzymatic or non-enzymatic reagent that promotes dissociation or detachment of cells into cell aggregates or single cells. Examples of dissociation reagents include, but are not limited to, collagenase (e.g., collagenase I or collagenase IV), activase, chelating agents (e.g., EDTA-based dissociation solutions), trypsin, dispase, or any combination thereof.

[0070] As used herein, the term "extracellular matrix" refers to any material to which cells can adhere in culture, typically containing extracellular components to which cells can attach, thereby providing a suitable culture substrate. Suitable for use in the present invention are extracellular matrix components derived from basement membranes or that form part of adhesion molecule receptor-ligand coupling. Examples of extracellular matrix include, but are not limited to, laminin or fragments thereof, such as laminin 521, laminin 511, or iMatrix 511, fibronectin, vitronectin, Matrigel, CellStart, collagen, gelatin, proteoglycans, entactin, heparin sulfate, and the like, alone or in various combinations.

[0071] As used herein, the term "laminin" refers to a heterotrimeric molecule consisting of α, β, and γ chains, or fragments thereof, which are extracellular matrix proteins containing isoforms with different subunit chain compositions. In particular, laminin has approximately 15 isoforms, including a heterotrimer of five α chains, four β chains, and three γ chains. The number of α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ3) are combined to determine the name of a laminin. For example, a laminin consisting of an α1, β1, and γ1 chain is designated laminin-111; an α5, β1, and γ1 chain is designated laminin-511; and an α5, β2, and γ1 chain is designated laminin-521. Mammalian-derived laminins can be used in the methods of the present invention. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. When RPE cells are produced, human laminin is preferably used. In one embodiment, the laminin is recombinant laminin. Currently, human laminin is known to contain 15 isoforms.

[0072] Any laminin fragment can be used in the present invention as long as it retains the function of the corresponding laminin. That is, the "laminin fragment" used in the present invention is not limited by the length of each chain, as long as it is a molecule that has laminin α, β, and γ chains that form a heterotrimer, retains integrin-binding activity, and maintains cell adhesive activity. Laminin fragments exhibit different integrin-binding specificities depending on the original laminin isoform and can exert adhesive activity toward cells expressing the corresponding integrin. In one embodiment, the laminin is a recombinant laminin-511 E8 fragment (e.g., iMatrix-511 (Takara Bio)).

[0073] As used herein, "administration," "administering," and variations thereof refer to the introduction of a composition or agent into a subject, including simultaneous and sequential introduction of compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. Administration includes self-administration and administration by another person. Administration can be carried out by any suitable route. A suitable administration route allows the composition or agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0074] As used herein, the terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in particular embodiments, the subject is human.

[0075] As used herein, the terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., RPE cells) are used interchangeably to refer to an amount sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, expected cell engraftment, long-term survival, and / or the specific active agent used. Thus, dosage regimens can vary widely but can be routinely determined by a physician using standard methods. Furthermore, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the compositions of the present invention described. In the preventive or preventive application of the present invention described, the pharmaceutical composition or medicament is administered to a patient who is susceptible to or otherwise at risk of a disease, disorder, or condition in an amount sufficient to eliminate or reduce the risk of the disease, disorder, or condition, reduce the severity of the disease, disorder, or condition, or delay the onset of the disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes that appear during the development of the disease, disorder, or condition. Generally, it is preferable to use the maximum dose, i.e., the safest dose according to certain medical judgment. The terms "dose" and "dosage" are used interchangeably herein.

[0076] As used herein, the term "therapeutic effect" refers to the outcome of treatment, which is deemed to be desirable and beneficial. Therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of disease manifestations. Therapeutic effect can also include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease manifestations.

[0077] For the therapeutic agent (e.g., RPE cells) described herein, therapeutically effective amount can be determined first from preliminary in vitro studies and / or animal models.Therapeutically effective amount can also be determined from human data.Dosage can be adjusted based on the relative bioavailability and efficacy of administered compound.It is within the ability of those skilled in the art to adjust dosage to achieve maximum efficacy based on the above method and other well-known methods.

[0078] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve the desired degree of therapeutic efficacy while minimizing unacceptable adverse effects. In situations where the plasma concentration of an agent can be measured and correlated with a therapeutic window, further guidance for dosage modifications can be obtained.

[0079] As used herein, the terms "treat," "treating," and / or "treatment" include preventing, substantially inhibiting, slowing, or reversing the progression of a condition, substantially ameliorating the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, and obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the occurrence of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0080] Beneficial or desirable clinical results, e.g., pharmacological and / or physiological effects, include, but are not limited to, preventing a disorder or condition from occurring in a subject who may be predisposed to the disease, disorder, or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of a disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, improvement or remission of the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0081] I. Methods of the Invention The present invention is based on the discovery of a stage during the differentiation of pluripotent stem cells into RPE cells, which allows RPE progenitor cells to be isolated, partially purified, and further differentiated into mature RPE cells, with or without the need for manual collection of minimal RPE cells.Any method for differentiating pluripotent cells into RPE cells can be used.For example, RPE cells can be obtained by differentiating pluripotent stem cells through the monolayer method described herein and also described in WO 2005 / 070011, the entire contents of which are incorporated herein by reference.Other methods include obtaining embryoid bodies from pluripotent stem cells, and differentiating embryoid bodies into RPE cells, and are also described in WO 2005 / 070011 and WO 2014 / 121077, the entire contents of which are incorporated herein by reference. In another example, as described in, for example, WO 2019130061, which is incorporated herein by reference in its entirety, pluripotent stem cells can be differentiated toward an RPE cell lineage using a first differentiation agent, and then further differentiated toward RPE cells using members of the transforming factor-β (TGFβ) superfamily and homologous ligands including activins (e.g., activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth differentiation factors (GDFs)). In one embodiment, RPE cells can be obtained by (a) culturing pluripotent stem cells in a medium containing a first differentiation agent (e.g., nicotinamide), and (b) culturing the cells obtained in step (a) in a medium containing a member of the TGFβ superfamily (e.g., activin A) and the first differentiation agent (e.g., nicotinamide), as described in WO 2019130061. In yet another example, single-cell suspensions of pluripotent stem cells can be used to differentiate into RPE, as described in WO 2017 / 044488, the entire contents of which are incorporated herein by reference.Thus, RPE cells can be obtained from pluripotent stem cells, which can be differentiated in one or more steps in one or more differentiation media that can contain differentiation factors such as WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189), BMP pathway inhibitors (e.g., SB431542), MEK inhibitors (e.g., PD0325901), members of the transforming factor-β (TGFβ) superfamily and homologous ligands, such as activin. Furthermore, RPE cells can be obtained from non-adherent or adherent cultures, and from feeder or feeder-free cultures.

[0082] During the differentiation process, when there are clusters of sufficient numbers of RPE progenitor cells (e.g., identified as PAX6 / MITF-positive cells) that remain together, the clusters of RPE progenitor cells can be treated with a dissociation reagent, followed by size fractionation of the clusters, and then subculture of the RPE progenitor cells as single cells or cell clusters to generate RPE cells. The methods of the present invention are simple and efficient, and in some embodiments, result in cultures of substantially pure RPE cells.

[0083] In one aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising the steps of (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells and dissociating the cell clusters into single cells; (ii) culturing the single cells in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In another aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising the steps of (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells, (ii) culturing the cell clusters in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In any of the embodiments of the present invention, PAX6+ / MITF+ RPE progenitor cells can be obtained from a population of pluripotent stem cells.

[0084] In one aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, comprising: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect RPE progenitor cell clusters, dissociating the RPE progenitor cell clusters into single cells, and subculturing the single cells in a second differentiation medium so that the single cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby providing a method for generating a population of RPE cells. In another aspect, the present invention provides a method for generating a population of retinal epithelial (RPE) cells, the method comprising the steps of: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect RPE progenitor cell clusters, and subculturing the collected RPE progenitor cell clusters in a second differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells generated in step (ii), thereby generating a population of RPE cells. In one embodiment of the present invention, the RPE progenitor cells are positive for PAX6 / MITF. In another embodiment, prior to step (i), the pluripotent stem cells are cultured on feeder cells in a pluripotency-supporting medium. In a further embodiment, prior to step (i), the pluripotent stem cells are cultured feeder-free in a pluripotency-supporting medium. In one embodiment, the pluripotency-supporting medium is supplemented with bFGF.

[0085] The method can further comprise recovering the RPE cells produced in step (ii) by dissociating the RPE cells, fractionating the RPE cells to collect RPE cell clusters, dissociating the RPE cell clusters into single RPE cells, and culturing the single RPE cells. In another embodiment, the method can further comprise recovering the RPE cells produced in step (ii) by dissociating the RPE cells, collecting RPE cell clusters, and selectively collecting the RPE cell clusters. The method can further comprise dissociating the selectively collected RPE cell clusters into single RPE cells and culturing the single RPE cells.

[0086] In one embodiment, the pluripotent stem cells are human pluripotent stem cells and the RPE cells are human RPE cells. Any of these steps can be performed in non-adherent or adherent culture, under feeder or feeder-free conditions.

[0087] In one embodiment, the RPE progenitor cell clusters and / or RPE cell clusters have a size between about 40 and about 200 μm, about 40 and about 100 μm, about 40 μm and about 70 μm, about 70 μm and about 100 μm, about 70 μm and about 200 μm, or about 100 μm and about 200 μm.

[0088] In some embodiments, the pluripotent stem cells are human embryonic stem cells. In other embodiments, the pluripotent stem cells are human iPS cells. In some embodiments, the RPE cells are further expanded after collection. In some embodiments, the method of the present invention results in a culture of substantially pure RPE cells. A substantially purified population of RPE cells is one in which RPE cells constitute at least about 75% of the cells in the population. In other embodiments, a substantially purified population of RPE cells is one in which RPE cells constitute at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, or even more than 99% of the cells in the population. In any of the embodiments, the RPE cells are human RPE cells.

[0089] In any of the embodiments of the present invention, the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, bestrophin (BEST1), MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1. In one embodiment, the RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1. In a further embodiment, the RPE cells express bestrophin, PAX6, MITF, and RPE65. In one embodiment, the RPE cells express at least one marker selected from MITF, bestrophin, and PAX6.

[0090] In one embodiment of the present invention, the RPE cells lack substantial expression of one or more stem cell markers selected from the group consisting of OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80. In one embodiment, the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase. In another embodiment, the RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

[0091] Pluripotent stem cell culture Pluripotent stem cells, such as embryonic stem (ES) cells or iPS cells, can be the starting material for the disclosed methods. In any of the embodiments herein, the pluripotent stem cells can be human pluripotent stem cells (hPSCs). Pluripotent stem cells (PSCs) can be cultured by any method known in the art, for example, with or without feeder cells. Furthermore, PSCs generated using any method can be used as the starting material for generating RPE cells. For example, hES cells can be derived from a blastocyst-stage embryo that was the product of in vitro fertilization of an egg and sperm. Alternatively, hES cells can be derived from one or more blastomeres removed from an early cleavage-stage embryo, optionally without destruction of the remainder of the embryo. In yet another embodiment, hES cells can be generated using nuclear transfer. In a further embodiment, iPSCs can be used. Previously cryopreserved PSCs can be used as the starting material. In another embodiment, PSCs that have never been cryopreserved can be used.

[0092] In one aspect of the present invention, PSCs are plated on an extracellular matrix under feeder or feeder-free conditions. In some embodiments, the extracellular matrix is ​​laminin with or without e-cadherin. In some embodiments, the laminin can be selected from the group including laminin 521, laminin 511, or iMatrix 511. In some embodiments, the feeder cells are human dermal fibroblasts (HDFs). In other embodiments, the feeder cells are mouse embryonic fibroblasts (MEFs).

[0093] In certain embodiments, the medium used when culturing PSCs can be selected from any medium suitable for culturing PSCs. In some embodiments, any medium that can support PSC culture can be used. For example, those skilled in the art can select from commercially available or proprietary media. In further embodiments, PSCs can be cultured on an extracellular matrix, including but not limited to laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin, in a medium that supports pluripotency.

[0094] The medium that supports pluripotency can be any such medium known in the art. In some embodiments, the medium that supports pluripotency is Nutristem (商標) In some embodiments, the pluripotency-supporting medium is TeSR. (商標) In some embodiments, the pluripotency-supporting medium is StemFit (商標) In another embodiment, the medium that supports pluripotency is Knockout (商標) DMEM (Gibco), which is a Knockout (商標) The medium may be supplemented with Serum Replacement (Gibco), LIF, bFGF, or any other factor. Each of these exemplary media is known in the art and commercially available. In a further embodiment, the medium supporting pluripotency may be supplemented with bFGF or any other factor. In one embodiment, bFGF may be supplemented at a low concentration (e.g., 4 ng / mL). In another embodiment, bFGF may be supplemented at a higher concentration (e.g., 100 ng / mL), which can stimulate PSCs to differentiate.

[0095] The concentration of PSCs used in the production method of the present invention is not particularly limited. For example, when a 10 cm dish is used, 1 × 10 4 ~1×10 8 Cells, preferably 5 x 10 per dish 4 ~5×10 6 cells, more preferably 1 x 10 per dish 5 ~1×107 Cells are used.

[0096] In some embodiments, the PSCs are cultured at about 1,000-100,000 cells / cm. 2 In some embodiments, PSCs are plated at a cell density of about 5,000-100,000 cells / cm. 2 , about 5000~50,000 cells / cm 2 , or approximately 5,000–15,000 cells / cm 2 In other embodiments, the PSCs are plated at a cell density of about 10,000 cells / cm. 2 The cells are plated at a density of 1000 μg / ml.

[0097] In some embodiments, a medium that supports pluripotency, e.g., StemFit (商標) Or other similar medium is exchanged with a differentiation medium (e.g., a medium that does not contain pluripotency support factors such as bFGF) to differentiate the cells into RPE cells. In one embodiment, embryoid bodies (EBs) are formed from PSCs, and the EBs are further differentiated into RPE cells.

[0098] In some embodiments, the change of medium from pluripotency-supporting medium to differentiation medium can be performed at different time points during cell culture of PSCs and may also depend on the initial plating density of the PSCs. In some embodiments, the change of medium can be performed after culturing PSCs in pluripotency medium for 3 to 14 days. In some embodiments, the change of medium can be performed on days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0099] Pluripotent stem cell differentiation The differentiation of pluripotent stem cells into RPE cells is initiated after replacing the pluripotency-supporting medium with one or more differentiation mediums, such as EBDM.In some embodiments, pluripotent stem cells spontaneously differentiate into RPE cells in the absence of differentiation inducer.In other embodiments, differentiation inducer, such as activin, Nodal signal inhibitor, Wnt signal inhibitor or sonic hedgehog signal inhibitor, can be used to make pluripotent stem cells differentiate into RPE cells.

[0100] In some embodiments, the differentiation medium is EB differentiation medium (EBDM). EBDM is a Xeno-free Knockout (商標) Knockout with Serum Replacement (XF-KSR) (Gibco) (商標)DMEM (Gibco), beta-mercaptoethanol, NEAA, and glutamine. Any other differentiation medium known in the art can be used. In another embodiment, the differentiation medium can include one or more differentiation agents, such as nicotinamide, members of the transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, Noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and / or MEK inhibitors (e.g., PD0325901). In one embodiment, pluripotent stem cells can be differentiated into RPE cell lineage in a first differentiation medium containing a first differentiation agent, and then further differentiated into RPE cells in a second differentiation medium containing a second differentiation agent. In one embodiment, the first differentiation medium contains nicotinamide, and the second differentiation medium contains activin (e.g., activin A). Furthermore, RPE cells can be obtained from non-adherent or adherent culture under feeder or feeder-free conditions.

[0101] In one embodiment, the differentiation medium can be changed daily during differentiation. In some embodiments, the differentiation medium is then changed every 2-3 days during differentiation. In some embodiments, the cells are cultured in the differentiation medium for about 3-12 weeks, e.g., 6-10 weeks, 2-8 weeks, or 3-6 weeks.

[0102] In one embodiment, after replacing the medium supporting pluripotency with differentiation medium, molecular markers and morphological characteristics can be detected to determine the differentiation of pluripotent cells in culture and identify RPE progenitor cells.Whether cells are RPE cells or RPE precursors can be determined by using light microscope or electron microscope and by the change of cell morphology (for example, intracellular melanin pigmentation, polygonal and flat cell morphology, formation of polygonal actin bundles, etc.) as indicators.Detection of molecular characteristics, morphological characteristics and other characteristics of RPE is described in, for example, United States Patent No. 7,794,704; United States Patent No. 7,736,896; WO 2009 / 051671; WO 2012 / 012803; WO 2013 / 074681; WO 2011 / 063005; and WO 2016 / 154357, which are incorporated herein by reference in their entirety. Thus, in some embodiments, after the pluripotency-supporting medium is replaced with a differentiation medium, differentiation of the pluripotent cells is monitored by identifying morphological characteristics of the RPE progenitor cells in culture.

[0103] In further embodiments, after the pluripotency-supporting medium is replaced with a differentiation medium, differentiation of pluripotent cells is identified by observing changes in gene expression of molecular markers of differentiated cells. In some embodiments, the molecular markers of differentiated cells are upregulated. In further embodiments, the molecular markers of pluripotency are downregulated. In some embodiments, changes in gene expression of molecular markers of differentiated cells can be confirmed by qPCR / scorecard and / or immunostaining. In some embodiments, changes in gene expression of molecular markers of differentiated cells are observed after about 3 weeks of differentiation.

[0104] In some embodiments, the molecular marker for the retinal lineage is PAX6 and the marker for pigmented cells is MITF. Thus, a population of cells expressing PAX6 and / or MITF indicates that retinal lineage / RPE precursors are present and can be isolated from the culture.

[0105] In other embodiments, it may not be necessary to determine the differentiation of pluripotent cells and identify RPE precursors, as long as the culture conditions are known to generate RPE precursor cells. Thus, PAX6 and MITF positive clusters can be isolated without the need to test for PAX6 / MITF.

[0106] Isolation and subculture of RPE progenitor cells Epithelial morphology cells are held together in culture by the formation of tight junctions, generating clusters of similar cell types during differentiation. Thus, in some embodiments, for isolation of the desired RPE progenitor cell population, the differentiating culture is digested or dissociated, for example, with an enzymatic or non-enzymatic dissociation reagent, such as collagenase or dispase, to form a suspension containing RPE progenitor cells and cell clusters containing single cells. As described below, single cells and non-epithelial cells can be separated and discarded. Furthermore, as described below, large clusters of non-RPE cells and clusters containing a mixture of RPE and non-RPE can be eliminated by size fractionation, thereby increasing purity.

[0107] In some embodiments, to isolate the desired RPE progenitor cell population, the differentiating culture can be digested with a dissociation reagent, allowing for the isolation of free-floating clusters of cells. In some embodiments, the dissociation reagent is collagenase. In other embodiments, the dissociation reagent is dispase. In some embodiments, dissociation with the dissociation reagent is performed overnight. In some embodiments, dissociation with the dissociation reagent is performed for about 2-30 hours. In one embodiment, dissociation with the dissociation reagent is performed for about 3-10 hours or about 3-6 hours. In one embodiment, dissociation with the dissociation reagent is performed for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours.

[0108] In some embodiments, dissociation is performed about 2-12 weeks after initiation of differentiation. In some embodiments, dissociation is performed about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after initiation of differentiation. In further embodiments, dissociation is performed on clusters of epithelial morphology positive for PAX6 and MITF.

[0109] In another aspect of the methods disclosed herein, a suspension containing cell clusters and single cells is fractionated to isolate RPE progenitor cell clusters. Any method for collecting the desired RPE progenitor cell clusters can be used. In one embodiment, single cells and other unwanted particles can be passed through a cell strainer or a series of cell strainers, and the desired cell cluster population can be collected by collecting the cells remaining on the cell strainer. In some embodiments, the cell clusters collected for further processing comprise cell clusters between about 40 μm and about 100 μm in size. In other embodiments, the collected cell clusters comprise cell clusters between about 40 μm and about 200 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 40 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 50 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 60 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 70 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 80 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 90 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 100 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 110 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 120 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 130 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 140 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 150 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 160 μm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 170 μm in size.In some embodiments, the collected cell clusters comprise cell clusters about 180 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 190 μm in size. In some embodiments, the collected cell clusters comprise cell clusters about 200 μm in size.

[0110] In some embodiments, single cells and cell cultures that do not meet the desired size requirements are discarded. In some embodiments, a series of cell strainers can be used to collect cell clusters that meet the desired size requirements. For example, a first cell strainer can have a small mesh size (e.g., 40 μm), and the cell cluster population remaining on the first cell strainer is collected. The collected cell cluster population can then be placed on a second cell strainer with a larger mesh size (e.g., 200 μm, 100 μm), and the cell cluster population that passes through the second cell strainer can be collected to achieve the desired size requirements (e.g., 40 μm to 200 μm, or 40 μm to 100 μm). Alternatively, the first cell strainer can be a first cell strainer with a larger mesh size (e.g., 200 μm, 100 μm), such that the cell cluster population that passes through the cell strainer is collected and the larger cell clusters remaining on the first cell strainer are discarded. The passaged cells can then be placed on a second cell strainer with a smaller mesh size (e.g., 40 µm), resulting in collection of cell clusters remaining on the second cell strainer, which have the desired size requirements (e.g., 40 µm to 200 µm, or 40 µm to 100 µm).

[0111] The collected RPE progenitor cells can be subcultured as clusters or as single cells to obtain proliferative, mature RPE cells according to the methods described below.

[0112] A method for subculturing single RPE progenitor cells to obtain RPE cells In the single RPE progenitor cell subculture method, the RPE progenitor cell clusters obtained as described above can be dissociated with a dissociation reagent to obtain single cells, and the population of RPE progenitor single cells is subcultured in a differentiation medium until RPE cells are obtained. In one embodiment, the cells are subcultured on laminin, such as laminin 521, laminin 511, or iMatrix 511, or other extracellular matrices, such as fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the cells are subcultured for approximately 1 to 8 weeks. In some embodiments, the cells are subcultured for approximately 2, 3, 4, 5, 6, 7, or 8 weeks. In other embodiments, the cells are subcultured for at least 8 weeks. In one embodiment, the cells can be subcultured under adherent conditions, such as on an adherent culture dish. In another embodiment, the cells can be subcultured under non-adherent conditions, and under feeder or feeder-free conditions.

[0113] Then, for example, by using dissociation reagent, RPE cells are collected, and RPE cell clusters can be obtained.RPE cell clusters can be obtained by collecting RPE cells and removing single cells by any method known in the art.In one embodiment, as described above, RPE cells are collected, and passed through a strainer or a series of strainers to obtain RPE cell clusters.Can use any cell strainer size, for example, 40 μ m, 50 μ m, 60 μ m, 70 μ m, 80 μ m, 90 μ m, 100 μ m, 110 μ m, 120 μ m, 130 μ m, 140 μ m, 150 μ m, 160 μ m, 170 μ m, 180 μ m, 190 μ m or 200 μ m size, or combinations thereof. The resulting RPE cell clusters can be at least 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm in size. In some embodiments, the RPE cell clusters collected for further processing comprise cell clusters between about 40 μm and about 100 μm in size. In other embodiments, the collected RPE cell clusters comprise cell clusters between about 40 μm and about 200 μm in size. In some embodiments, the collected RPE cell clusters comprise cell clusters of approximately 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm in size.

[0114] In one embodiment, the resulting RPE cell clusters can be dissociated into single cells with enzymatic or non-enzymatic dissociation reagents and cultured to expand the RPE cells, as further described below.

[0115] In an alternative embodiment, islands of pigmented cells can be selectively harvested from the resulting RPE cell clusters. This selective / minimal harvesting process is substantially simpler and results in highly purified RPE cells, since the desired cell population has been enriched in the previous subculture step. RPE can be selectively harvested manually, for example, mechanically using glass capillaries, by using an optical microscope, or by an automated system that can distinguish RPE cells from other types of cells. The selected RPE clusters can then be dissociated to generate single RPE cells. The single RPE cells can be cultured and expanded as further described below.

[0116] In any of the embodiments of the present invention, the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, bestrophin, MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1. In one embodiment, the RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1. In a further embodiment, the RPE cells express bestrophin, PAX6, MITF, and RPE65. In one embodiment, the RPE cells express at least one marker selected from MITF and bestrophin and PAX6. In one embodiment of the present invention, the RPE cells lack substantial expression of one or more stem cell markers selected from the group consisting of OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80. In one embodiment, the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase. In another embodiment, the RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

[0117] In some embodiments, the sample of RPE cells produced can be tested for desired molecular marker profile, and then collected.In other embodiments, as long as the culture conditions are known to produce RPE cells, it may not be necessary to test RPE cells for molecular marker before collecting.Therefore, RPE cells can be collected without the need to test for molecular marker.

[0118] Method for subculturing RPE progenitor cell clusters to obtain RPE cells In the RPE progenitor cell cluster subculture method, the RPE progenitor cell clusters obtained after the size fractionation described above are subcultured as cell clusters in a differentiation medium until RPE cells are obtained. In one embodiment, the RPE progenitor cell clusters are subcultured on laminin, such as laminin 521, laminin 511, or iMatrix 511, or other extracellular matrices, such as fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the cell clusters are subcultured for approximately 1 to 8 weeks. In some embodiments, the cell clusters are subcultured for approximately 2, 3, 4, 5, 6, 7, or 8 weeks. In other embodiments, the cell clusters are subcultured for at least 8 weeks. In one embodiment, the cell clusters can be subcultured under non-adherent conditions. In another embodiment, the cell clusters can be subcultured under adherent conditions. In another embodiment, the cell clusters can be cultured under feeder or feeder-free conditions.

[0119] Then, for example, by using dissociation reagent, RPE cells can be collected to obtain RPE cell clusters.RPE cell clusters can be obtained by collecting RPE cells and removing single cells by any method known in the art.In one embodiment, as described above, RPE cells can be collected and passed through a strainer or a series of strainers to obtain RPE cell clusters.Can use any cell strainer size, for example, 40 μ m, 50 μ m, 60 μ m, 70 μ m, 80 μ m, 90 μ m, 100 μ m, 110 μ m, 120 μ m, 130 μ m, 140 μ m, 150 μ m, 160 μ m, 170 μ m, 180 μ m, 190 μ m or 200 μ m size, or combinations thereof. The resulting RPE cell clusters can be at least 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm in size. In some embodiments, the RPE cell clusters collected for further processing comprise cell clusters about 40 μm to about 100 μm in size. In other embodiments, the collected RPE cell clusters comprise cell clusters about 40 μm to about 200 μm in size. In some embodiments, the collected RPE cell clusters comprise cell clusters of approximately 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm in size.

[0120] In one embodiment, the resulting RPE cell clusters can be dissociated into single cells with enzymatic or non-enzymatic dissociation reagents and cultured to expand the RPE cells, as further described below.

[0121] In an alternative embodiment, islands of pigmented cells can then be selectively harvested from the resulting RPE cell clusters. This selective / minimal harvesting process is substantially simpler and results in highly purified RPE cells, since the desired cell population has been enriched in the previous subculture step. RPE can be selectively harvested manually, for example, mechanically using glass capillaries, by using an optical microscope, or by an automated system that can distinguish RPE cells from other types of cells. The selected RPE clusters can then be dissociated to generate single RPE cells. The single RPE cells can be cultured and expanded as further described below.

[0122] In any of the embodiments of the present invention, the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, bestrophin, MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1. In one embodiment, the RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1. In a further embodiment, the RPE cells express bestrophin, PAX6, MITF, and RPE65. In one embodiment, the RPE cells express at least one marker selected from MITF and bestrophin and PAX6. In one embodiment of the present invention, the RPE cells lack substantial expression of one or more stem cell markers selected from the group consisting of OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80. In one embodiment, the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase. In another embodiment, the RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

[0123] In some embodiments, the sample of RPE cells produced can be tested for desired molecular marker profile, and then collected.In other embodiments, as long as the culture conditions are known to produce RPE cells, it may not be necessary to test RPE cells for molecular marker before collecting.Therefore, RPE cells can be collected without the need to test for molecular marker.

[0124] Expansion of RPE cells In some embodiments, RPE cells obtained from the single RPE progenitor cell subculture or RPE progenitor cell cluster subculture methods can be cultured on an extracellular matrix, such as laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin, in a medium that supports RPE growth or proliferation to expand the RPE cell population.

[0125] The RPE cell population initially cultured in this step is referred to herein as "P0." In one embodiment, the extracellular matrix is ​​selected from the group consisting of laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin. In some embodiments, the extracellular matrix is ​​laminin. In one embodiment, the laminin is selected from laminin-521, laminin-511, or iMatrix-511. In a further embodiment, the laminin comprises e-cadherin. In another embodiment, the extracellular matrix is ​​gelatin. In some embodiments, the culture medium is RPE-MM (also referred to as RPEGMMM, MM, or maintenance medium, which contains DMEM / KO-DMEM containing KSR and FBS, beta-mercaptoethanol, NEAA, and glutamine), StemFit, EGM2, or EBDM. In some embodiments, the RPE-MM is supplemented with FGF (MM / FGF). In other embodiments, other media known in the art that support the growth and expansion of RPE can be used.Any such media may or may not be supplemented with FBS and / or bFGF, or any other factors, such as heparin, hydrocortisone, vascular endothelial growth factor, recombinant insulin-like growth factor, ascorbic acid, or human epidermal growth factor.See, for example, WO2013074681A, the entire contents of which are incorporated herein by reference.

[0126] In one embodiment, RPE cells can be passaged and cultured until a sufficient number of RPE cells are obtained. In one embodiment, RPE cells are passaged indefinitely. In another embodiment, RPE cells are passaged at least once ("P1") and up to 20 times ("P20"). In one embodiment, RPE cells are passaged at least twice ("P2") and up to 8 times ("P8"). In a further embodiment, RPE cells are passaged two ("P2"), three ("P3"), four ("P4"), five ("P5"), six ("P6"), seven ("P7"), or eight ("P8") times. RPE cells can be cryopreserved until further use. In one embodiment, the duration of each expansion phase can vary from several days, weeks, to several months. In one embodiment, the duration of an expansion phase is between about 2 and 90 days. In another embodiment, the duration of the expansion phase is between about 2-60 days, 3-50 days, 3-40 days, 3-30 days, 3-25 days, 8-25 days, 10-25 days, 2-14 days, or 2-10 days. Fresh medium can be added at intervals, such as every 1-2 days, during the expansion phase. In one embodiment, bFGF is added to the RPE cell culture medium at a concentration of about 1-100 ng / ml for the first 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, or 5 days of RPE expansion at each passage (e.g., P0, P1, P2), and then removed until further passage. In one embodiment, the concentration of bFGF is about 1-50 ng / ml, about 2-40 ng / ml, about 3-30 ng / ml, about 4-20 ng / ml, or about 4-10 ng / ml. In certain embodiments, the concentration of bFGF is about 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml.

[0127] In any of the embodiments of the present invention, the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, bestrophin, MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1. In one embodiment, the RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1. In a further embodiment, the RPE cells express bestrophin, PAX6, MITF, and RPE65. In one embodiment, the RPE cells express at least one marker selected from MITF and bestrophin and PAX6. In one embodiment of the present invention, the RPE cells lack substantial expression of one or more stem cell markers selected from the group consisting of OCT4, NANOG, REX1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80. In one embodiment, the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase. In another embodiment, the RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

[0128] In some embodiments, the sample of RPE cells produced can be tested for desired molecular marker profile, and then collected.In other embodiments, as long as the culture conditions are known to produce RPE cells, it may not be necessary to test RPE cells for molecular marker before collecting.Therefore, RPE cells can be collected without the need to test for molecular marker.

[0129] Feeder and feeder-free based culture Mouse feeder layer As disclosed herein, PSCs can be cultured on mouse embryonic fibroblasts (MEFs) as feeder cells (see, for example, Thomson JA, Itskovitz-Eldor J, Shapiro S Xenon, Waknitz MA, Swiergiel J Siegel, Marshall VS, Jones J M. (1998); Science 282: 1145-7; Reubinoff BE, Pera MF, Fong C, Trounson A, Bongso A. (2000); Reubinoff et al., 2000, Nat. Biotechnol. 18: 399-404). MEF cells can be derived from 12-13 day old mouse embryos in a medium supplemented with fetal bovine serum.

[0130] PSCs can be cultured on MEFs under serum-free conditions using serum replacement supplemented with basic fibroblast growth factor (bFGF) (see, e.g., Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA. (2000)). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferation over long periods of culture (see, e.g., Dev. Biol. 227: 271-8). Furthermore, when cultured under conditions that promote the maintenance of the pluripotent state, PSCs can still maintain their pluripotency after six months of culture under serum replacement. Pluripotency of PSCs can be demonstrated by their ability to form teratomas containing all three embryonic germ layers. Furthermore, differentiation of PSCs into RPE can be performed in the presence of mouse feeder cells. Thus, the PSCs used in the methods described herein can be cultured on mouse feeder cells.

[0131] Human feeder cells PSCs can be cultured, maintained or differentiated on human feeder cells, as described in, for example, PCT Publication No. WO2009048675.PSCs can be maintained in undifferentiated state by multiple continuous passages of PSCs on human feeder cells (see, for example, Richards et al., 2002, Nat. Biotechnol. 20: 933-6).PSCs can also be differentiated into RPE in the presence of human feeder cells.Therefore, the PSCs used in the methods described herein can be cultured on human feeder cells.

[0132] Feeder-free culture PSCs can be cultured in feeder-free system on solid surface such as extracellular matrix (for example, Matrigel® or laminin) in the presence of culture medium.As outlined in Rowland et al., Journal Cell Physiology, 227:457-466, 2012, which is incorporated herein by reference, various methods for differentiating PSCs into RPE cells ex vivo are known in the art.Therefore, the PSCs used in the methods described herein can be cultured in feeder-free culture.

[0133] Use of FGF / bFGF and ROCK inhibitors During mammalian development, the RPE shares the same precursor as the neural retina, i.e., the neuroepithelium of the optic vesicle. Under certain conditions, the RPE can transdifferentiate into neuronal precursors (Opas and Dziak, 1994, Dev Biol. 161(2):440-54), neurons (Chen et al., 2003, J Neurochem. 84(5):972-81; Vinores et al., 1995, Exp Eye Res. 60(6):607-19), and lens epithelium (Eguchi, 1986). One of the factors that can stimulate RPE transformation into neurons is bFGF (Opas and Dziak, 1994, Dev Biol. 161(2):440-54), a process associated with the expression of transcriptional activators normally required for eye development, such as rx / rax, chx10 / vsx-2 / alx, ots-1, otx-2, six3 / optx, six6 / optx2, mitf, and PAX6 / pax2 (Fischer and Reh, 2001, Dev Neurosci. 23(4-5):268-76; Baumer et al., 2003, Development;130(13):2903-15). It has been shown that the edge of the chick retina contains neural stem cells (Fischer and Reh, 2000; Dev Biol. 15;220(2):197-210), and that pigmented cells in that region that express PAX6 / mitf can form neurons in response to FGF (Fischer and Reh, 2001, Dev Neurosci. 23(4-5):268-76).

[0134] In some embodiments, the PSCs of the present invention can be maintained in a pluripotent state in a culture medium containing 1 to 200 ng / ml bFGF. In one embodiment, the concentration of bFGF is about 1 to 100 ng / ml, about 2 to 100 ng / ml, about 3 to 100 ng / ml, or about 4 to 100 ng / ml. In certain embodiments, the concentration of bFGF is about 100 ng / ml. In some embodiments, the PSCs can be differentiated into RPE cells in the presence of bFGF. In other embodiments, the RPE cells can be expanded in the presence of bFGF, as described above and herein.

[0135] During RPE formation, pluripotent cells can be cultured in the presence of an inhibitor of rho-associated protein kinase (ROCK).ROCK inhibitor refers to any substance, such as small molecule, siRNA, miRNA, antisense RNA, etc., that inhibits or reduces the function of Rho-associated kinase or its signaling pathway in cells.As used herein, "ROCK signaling pathway" can include any signal processor involved in ROCK-related signaling pathways, such as the Rho-ROCK-myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway in cells.An exemplary ROCK inhibitor that can be used is Stemolecule Y-27632 from Stemgent (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):68 1-6). Other ROCK inhibitors include, for example, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, and SB-772077-B.Doe et al., J. Pharmacol. Exp. Ther., 32:89-98, 2007;Ishizaki et al., Mol. Pharmacol., 57:976-983, 2000;Nakajima et al., Cancer Chemother. Pharmacol., 52:319-324, 2003;And Sasaki et al., Pharmacol. Ther., 93:225-232, 2002, each of which is incorporated herein by reference as if set forth in its entirety. ROCK inhibitors can be utilized at concentrations and / or culture conditions known in the art, for example, as described in U.S. Patent Application Publication No. 2012 / 0276063, which is incorporated herein by reference in its entirety.For example, the ROCK inhibitor can have a concentration of about 0.05 to about 50 microM, e.g., at least or about 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 microM, including any range derivable therein, or any concentration effective to promote cell proliferation or survival. In a further embodiment, the expansion culture of RPE may be further supplemented with a ROCK inhibitor and / or bFGF, as described by PCT Publication No. WO2013074681A1, which is incorporated herein by reference in its entirety.

[0136] Adherent and non-adherent cultures As used in this disclosure, "adherent culture" refers to culture in which the cells of interest are attached to a cell culture substrate, e.g., tissue culture vessel, via laminin. Cells can also adhere to plastic that has been treated for cell attachment ("tissue culture treated") without any additional substrate coating.

[0137] In some embodiments, differentiation of pluripotent stem cells into RPE cells is carried out by adherent culture. Adherent culture can be carried out using a cell-adhesive culture vessel. The cell-adhesive culture vessel is not particularly limited as long as the surface of the culture vessel is treated to improve cell adhesion. For example, a culture vessel having a coated layer containing an extracellular matrix, a synthetic polymer, or the like can be used. The coated layer may be composed of one or more components, or may be formed as a single layer or multiple layers. The extracellular matrix is ​​not particularly limited as long as it can form a coated layer that exhibits adhesive properties for pluripotent stem cells. For example, collagen, gelatin, laminin, fibronectin, and the like can be used alone or in combination. Commercially available products containing multiple types of extracellular matrix include Matrigel (BD) and CellStart (Invitrogen). Biologically or chemically produced polymers can be used as synthetic polymers. For example, cationic polymers such as polylysine (poly-D-lysine, poly-L-lysine), polyornithine polyethyleneimine (PEI), poly-N-propylacrylamide (PIPAAm), etc. are preferably used. The extracellular matrix or synthetic polymer can be produced biologically using bacteria, cells, etc., and optionally by introducing genetic modifications, or can be chemically synthesized. In another embodiment, cells can be bound to the extracellular matrix via RGD peptides, which bind to integrin adhesion receptors found in the extracellular matrix.

[0138] In some embodiments, adherent culture can be performed in tissue culture vessels that are not treated with any cell culture substrate or for cell adhesion. For example, media components such as FBS, fibronectin, or vitronectin can be absorbed into the tissue culture vessel and serve as a cell adhesion substrate. In other embodiments, cells in the tissue culture vessel can secrete an extracellular matrix that can also serve as a cell adhesion substrate.

[0139] As used herein, "non-adherent culture" refers to culture in which the cells of interest do not or substantially do not adhere to the tissue culture vessel. Thus, single cells or clusters of cells in non-adherent culture can float in culture and remain in suspension. Single cells in non-adherent culture can form clusters or aggregates under appropriate conditions. In one embodiment, the surface of the culture vessel can be coated with a hydrophilic, neutrally charged coating, such as Corning® Ultra-Low Attachment Surface, that is covalently attached to the surface of the polystyrene vessel. The non-attaching surface inhibits specific and non-specific immobilization, placing the cells in suspension. To culture the cells in suspension, cells can also be cultured in spinner flasks (Corning). Other methods for culturing cells in non-adherent culture are known to those skilled in the art and can be used in the methods of the present invention.

[0140] II. Methods of Using Retinal Pigment Epithelial Cells RPE cells and pharmaceutical compositions comprising RPE cells produced by the methods described herein can be used in cell-based therapies where RPE cells are required or would improve treatment. Methods using RPE cells provided by the present invention to treat various conditions that may benefit from RPE cell-based therapy are described herein and, for example, in U.S. Patent No. 10,077,424, the contents of which are incorporated herein by reference. Specific treatment regimens, administration routes, and optional adjunctive therapies will be tailored based on the specific condition, the severity of the condition, and the patient's overall health. Furthermore, in certain embodiments, administration of RPE cells may be effective in fully restoring any vision loss or other symptoms. In other embodiments, administration of RPE cells may be effective in reducing the severity of symptoms and / or preventing further deterioration of the patient's condition. The present invention contemplates that administration of compositions comprising RPE cells can be used to treat any of the conditions described herein (including reducing the severity of symptoms in whole or in part). Additionally, administration of RPE cells can be used to help treat the symptoms of any injury to the endogenous RPE layer.

[0141] The present invention contemplates that RPE cells obtained using any of the methods described herein, including compositions comprising RPE cells, can be used in the treatment of any of the indications described herein.Furthermore, the present invention contemplates that any of the compositions comprising RPE cells described herein can be used in the treatment of any of the indications described herein.In another embodiment, the RPE cells of the present invention can be administered together with other therapeutic cells or agents.RPE cells can be administered simultaneously or sequentially in combined or separate formulations.

[0142] In one aspect, the present invention provides a method for treating retinal disease or disorder.In one aspect, retinal disease or disorder includes, for example, retinal degeneration, such as choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt's disease, angioid streaks or myopic macular degeneration, or glaucoma.In a specific aspect, the RPE cells of the present invention can be used to treat central nervous system disorders, such as Parkinson's disease.

[0143] Retinitis pigmentosa is an inherited condition in which visual receptors are gradually destroyed through aberrant genetic programming. Some forms cause total blindness at a relatively young age, while others present with characteristic "bone spicule" retinal changes with little or no visual disruption. The disease affects approximately 1.5 million people worldwide. Several genetic defects causing autosomal recessive retinitis pigmentosa have been found in genes expressed exclusively in the RPE. One involves an RPE protein (cis-retinalaldehyde-binding protein (CRLBP)) involved in vitamin A metabolism. Another involves a protein unique to the RPE, RPE65. Mutations in the MER proto-oncogene, i.e., tyrosine kinase (MERTK) gene, have also been associated with disruption of the RPE phagocytic pathway and the development of autosomal recessive retinitis pigmentosa. Other genetic defects and forms of RPE-associated retinitis pigmentosa are known. See, for example, Verbakel et al., Progress in Retinal and Eye Research 66:157-186 (2018). The present invention provides methods and compositions for treating any or all forms of RPE-associated retinitis pigmentosa by administration of RPE cells.

[0144] Animal models of retinitis pigmentosa that can be treated or used to test the efficacy of RPE cells generated using the methods described herein include rodents (rd mice, RPE-65 knockout mice, tabby-like mice, LRAT mice, RCS rats), cats (Abyssinian cats), and dogs (cone degeneration "cd" dogs, progressive rod-cone degeneration "prcd" dogs, early retinal degeneration "erd" dogs, rod-cone dysplasia 1, 2 & 3 "rcd1, rcd2 & rcd3" dogs, photoreceptor dysplasia "pd" dogs, and Briard "RPE-65" (dog)).

[0145] In another aspect, the present invention provides methods and compositions for treating disorders associated with retinal degeneration, including macular degeneration.

[0146] A further aspect of the present invention is the use of RPE cells for the therapy of ocular diseases, including inherited and acquired ocular diseases, such as age-related macular degeneration, glaucoma, and diabetic retinopathy.

[0147] Age-related macular degeneration (AMD) is the most common cause of legal blindness in Western countries. Submacular atrophy of the retinal pigment epithelium and the development of choroidal neovascularization (CNV) secondary to central vision loss. For the majority of patients with subfoveal CNV and geographic atrophy, no treatment is currently available to prevent central vision loss. The earliest sign of AMD is deposits (drusen) between the retinal pigment epithelium and Bruch's membrane. During the disease, there is sprouting of choroidal blood vessels into the subretinal space of the macula. This leads to loss of central vision and reading ability.

[0148] Glaucoma is the name given to a group of disorders in which pressure inside the eye increases abnormally. This leads to a limited field of vision and an overall decrease in the ability to see. The most common form is primary glaucoma; two types are distinguished: chronic open-angle glaucoma and acute angle closure glaucoma. Secondary glaucoma can be caused by infection, tumor, or injury. The third type, hereditary glaucoma, usually results from developmental disorders during pregnancy. Aqueous humor in the eye is under a certain pressure, necessary for the eye's optical properties. This intraocular pressure, usually 15–20 mm of mercury, is controlled by the balance between aqueous humor production and aqueous humor outflow. In glaucoma, aqueous humor outflow at the angle of the anterior chamber is blocked, resulting in increased pressure inside the eye. Glaucoma usually occurs in middle age or older adults, but hereditary forms and disorders are not uncommon in children and adolescents. Although intraocular pressure is only slightly elevated and there are no obvious symptoms, slow damage, particularly limited visual field, occurs. In contrast, acute angle-closure glaucoma causes pain, redness, dilated pupils, and severe visual impairment. The cornea becomes cloudy, and intraocular pressure increases significantly. As the disease progresses, the visual field becomes increasingly narrower, which can be easily detected using an ophthalmic instrument called a perimeter. Chronic glaucoma generally responds well to locally administered medications that enhance aqueous humor outflow. Systemic active substances may also be given to reduce aqueous humor production. However, drug treatment is not always successful. If drug therapy fails, laser therapy or conventional surgery is used to create new outflow of aqueous humor. Acute glaucoma is a medical emergency. If intraocular pressure is not reduced within 24 hours, permanent damage occurs.

[0149] Diabetic retinopathy occurs in patients with diabetes mellitus. This can lead to thickening of the basement membrane of vascular endothelial cells as a result of protein glycosylation. This is the cause of early vascular sclerosis and the formation of microaneurysms. These vascular changes lead to diabetic retinopathy over the course of several years. These vascular changes cause hypoperfusion in the capillary area, leading to lipid deposits (hard exudates) and vascular proliferation. The clinical course varies among patients with diabetes mellitus. In age-related diabetes (type 2 diabetes), microaneurysms appear first. Subsequently, hard and soft exudates and punctate hemorrhages appear in the retinal stroma due to impaired capillary perfusion. In later stages of diabetic retinopathy, fatty deposits form in a coronal pattern around the macula (retinitis circumflexis). These changes are often accompanied by edema in the posterior pole of the eye. If the edema involves the macula, there is acute and severe visual deterioration. The main problem in type 1 diabetes is vascular proliferation in the fundus area. The standard treatment is laser coagulation of the affected area of ​​the fundus. Laser coagulation is initially performed locally in the affected area of ​​the retina. If vitiligo persists, the area of ​​laser coagulation is expanded. The center of the retina, which contains the clearest vision, i.e., the macula and papillomacular bundle, cannot be coagulated because the procedure would destroy the part of the retina most important for vision. If proliferation has already occurred, it is often necessary to compress the lesion very tightly based on the proliferation. This inevitably involves the destruction of areas of the retina. The result is a corresponding loss of visual field. In type 1 diabetes, timely laser coagulation is often the only chance to save the patient from blindness.

[0150] Another aspect of the present invention is a method for inducing RPE cells or precursors to RPE cells with increased ability to prevent angiogenesis. Alternatively, such cells can be genetically modified with exogenous genes that inhibit angiogenesis.

[0151] The present invention contemplates that compositions of RPE cells derived from human pluripotent stem cells (e.g., human embryonic stem cells or other pluripotent stem cells) can be used to treat any of the aforementioned diseases or conditions, as well as injuries to the endogenous RPE layer. These diseases can be treated with compositions of RPE cells containing RPE cells at various levels of maturity, and with compositions of RPE cells enriched in mature RPE cells.

[0152] III. Methods for administering retinal pigment epithelial cells The RPE cells of the present invention can be administered by any administration route that is suitable for the disease or disorder to be treated.In one embodiment, the RPE cells of the present invention can be administered locally, systemically, or locally, for example, by injection (e.g., subretinal injection) or as part of a device or implant (e.g., sustained-release implant).For example, when treating patients with retinal disorders or diseases, such as macular degeneration, Stargardt's disease, and retinitis pigmentosa, the RPE cells of the present invention can be transplanted into the subretinal space by using vitrectomy surgery.In another example, when treating patients with CNS disorders, such as Parkinson's disease, the RPE cells of the present invention can be transplanted systemically or locally.Those skilled in the art will be able to determine the administration route for the disease or disorder to be treated.

[0153] The RPE cells of the present invention can be delivered by intraocular injection, more specifically subretinal, in a pharmaceutically acceptable ophthalmic formulation. The concentration for injection can be any amount that is effective and non-toxic, depending on the factors described herein. In some embodiments, the RPE cells for treatment of a patient can be about 5 cells / 150 μl to 1×10 cells / 150 μl. 7 cells / 150μl, 50 cells / 150μl~1×10 6 cells / 150 μl, or 50 cells / 150 μl ~ 5 × 10 5 In other embodiments, RPE cells for treatment of patients are formulated at a dose of about 10, 50, 100, 500, 5000, 1 x 10 cells / 150 μl. 4 , 5×10 4 , 1×10 5, 5×10 5 , or 1 × 10 6 The RPE cells are formulated at a dose of 150 μl. In one embodiment, approximately 50,000 to 500,000 cells can be administered to a patient. In certain embodiments, approximately 50,000, 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, or 500,000 RPE cells can be administered to a patient.

[0154] The RPE cells can be formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle so that the composition is maintained in contact with the ocular surface for a period of time sufficient to allow the cells to penetrate into the affected area of ​​the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, epicorneal space, pars plana, surgically induced avascular area, or macula. Products and systems comprising the agents of the present invention, such as delivery vehicles, particularly those formulated as pharmaceutical compositions, and kits comprising such delivery vehicles and / or systems are also contemplated as part of the present invention.

[0155] In certain embodiments, the therapeutic method of the present invention comprises administering the RPE cells of the present invention in an implant or device.In certain embodiments, the device is a bioerodible implant for treating ocular medical conditions, which comprises an active agent dispersed in a biodegradable polymer matrix, and at least about 75% of the particles of the active agent have a diameter of less than about 10 um.The bioerodible implant is sized for implantation in an ocular region.The ocular region can be any one or more of the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, extracorneal space, sclera, pars plana, surgically induced avascular region, macula, and retina.The biodegradable polymer can be, for example, poly(lactic-co-glycolic) acid (PLGA) copolymer. In certain embodiments, the ratio of lactic acid to glycolic acid monomers in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, or 75 / 25 weight percent, more preferably about 50 / 50. Additionally, the PLGA copolymer can be about 20, 30, 40, 50, 60, 70, 80, to about 90 weight percent of the bioerodible implant. In certain preferred embodiments, the PLGA copolymer can be about 30 to about 50 weight percent, preferably about 40 weight percent, of the bioerodible implant.

[0156] The volume of the composition administered according to the methods described herein also depends on factors such as the mode of administration, the number of RPE cells, the age of the patient, and the type and severity of the disease being treated. When administered by injection, the volume of liquid containing the composition of the present invention can be about 5.0 microliters to about 50 microliters, about 50 microliters to about 250 microliters, or about 250 microliters to about 1 milliliter. In one embodiment, the volume for injection can be about 150 microliters.

[0157] When administered via intraocular injection, RPE cells can be delivered periodically, one or more times throughout the patient's life. For example, RPE cells can be delivered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. When administered via implant or device, RPE cells can be administered once, one or more times periodically throughout the patient's life, depending on the needs of the particular patient and disorder or condition being treated. Similarly, treatment regimens that vary over time are contemplated. In certain embodiments, the patient is also administered immunosuppressive therapy either prior to, concurrently with, or after administration of RPE cells. Immunosuppressive therapy may be required throughout the patient's life or for a shorter period of time. Examples of immunosuppressive therapies include, but are not limited to, one or more of the following: antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX1MAB® (anti-CD20 antibody), sirolimus, tacrolimus (Prograf (商標) ), and mycophenolate mofetil (MMF).

[0158] In certain embodiments, the RPE cells of the present invention are formulated with a pharmaceutically acceptable carrier. For example, the RPE cells can be administered alone or as a component of a pharmaceutical preparation. The subject compounds can be formulated for administration in any convenient manner for use in human medicine. In certain embodiments, pharmaceutical compositions suitable for parenteral administration can include RPE cells in combination with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which can include antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0159] In one aspect, the RPE cells of the present invention are formulated in GS2, which is described in WO 2017 / 031312, which is incorporated herein by reference in its entirety.

[0160] The contents of any publications cited herein, including patents and patent applications, are hereby incorporated by reference in their entirety to the extent that they are disclosed herein. [Example]

[0161] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way.

[0162] Example 1: Time course of PAX6 / MITF expression in RPE progenitor cells J1 cell differentiation was initiated by plating J1 hES cells onto laminin-521 / e-cadherin-coated plates containing mitomycin C-inactivated HDF in EBDM. Approximately 1, 2, 3, 4, 6, and 8 weeks after initiation of culture in EBDM, cells were harvested and assessed for PAX6 and MITF expression by qPCR. As shown in Figure 1, PAX6+ / MITF+ RPE progenitor cells began to appear at approximately 3-4 weeks of culture, and PAX6 and MITF mRNA expression in culture increased over time (e.g., see weeks 6-8).

[0163] In another experiment, J1 hES cells were plated on laminin-521 / e-cadherin-coated plates containing mitomycin C-inactivated HDFs in Nutristem (Stemgent) for 4 days, followed by TeSR2 (STEMCELL Technologies) for 8 days. The medium was then switched to EBDM to initiate J1 cell differentiation. Approximately 5.5, 9, and 10 weeks after initiation of culture in EBDM, the cells were treated with collagenase, and the released, digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. We collected cells that passed through the 40-micron strainer (cells <40 μm), cells retained on the 100-micron strainer (cells >100 μm), and clusters retained on the 40-micron strainer (cells approximately 40-100 μm). We plated each fraction onto LN521-coated wells in EBDM for 3 days, fixed the cells, and stained them for PAX6 / MITF. As shown in Figure 2, cells <40 μm showed little or no PAX6 / MITF staining even at 5.5, 9, and 10 weeks after the start of differentiation. By 9-10 weeks after the start of differentiation, cells obtained from the 40-100 μm fraction showed stronger PAX6 / MITF staining compared to the >100 μm fraction.

[0164] Based on these results, the timing of harvesting PAX6+ / MITF+ RPE progenitor cells for subculture was identified. An exemplary process for generating RPE cells according to some embodiments of the present invention is outlined in Figure 3. Detailed steps of these exemplary method embodiments are described as follows.

[0165] Example 2: Generation of retinal pigment epithelial (RPE) cells by a single RPE progenitor cell subculture method In the first experiment, mitomycin-C-treated HDF cells were plated onto laminin-521 / E-cadherin-coated wells. J1 hESCs were seeded into the wells and cultured in Nutristem (Stemgent) for approximately 4 days, followed by TeSR2 (STEMCELL Technologies) for 4 days. The medium was then switched to EBDM to promote RPE generation, and the EBDM was changed daily for 7 days, then every 2–3 days.

[0166] After 83 days (approximately 12 weeks) in EBDM, cells were treated with collagenase overnight. The released and digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. Clusters retained on the 40-micron strainer were collected and dissociated into single cells by trypsinization for 15 minutes. Single cells were plated onto LN521-coated wells in EBDM, and the EBDM was changed every 2–3 days. After 30 days (approximately 4 weeks) in EBDM after plating, cells were treated with collagenase for approximately 6 hours. The released and digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. Clusters retained on the 40-micron strainer were collected and dissociated into single cells by trypsinization for 15 minutes in MM / FGF medium (DMEM; GlutaMAX (商標)Single cells were plated as passage 0 RPE cells ("P0") onto gelatin-coated wells in MM / FGF medium (100x TrypLE supplement, liquid, 200 mM; FBS; Knockout DMEM; non-essential amino acids; 2-mercaptoethanol; Knockout Serum Replacement [KSR] + bFGF). MM / FGF medium was changed daily until approximately >90% confluent, then replaced with MM medium [MM / FGF medium described above without bFGF] and fed every 2 days until harvest. P0 RPE cells were cultured for 16 days. P0 cells were harvested by 15 minutes of 10x TrypLE treatment and replated as single cells onto gelatin-coated wells in MM / FGF medium as passage 1 RPE cells ("P1"). The culture method was repeated as described above for P0 RPE cells, by first culturing in MM / FGF and then switching to MM medium. P1 RPE cells were cultured for 14 days. P1 RPE cells were harvested and initially cultured in MM / FGF, then re-plated as passage 2 RPE cells ("P2") as described above by switching to MM medium. P2 RPE cells were cultured for 14 days, harvested by 15 minutes of 10x TrypLE treatment, and then cryopreserved. The cells were then thawed, formulated in GS2, and subjected to quality testing. The results are shown in Table 1.

[0167] In the second experiment, mitomycin-C-inactivated HDF cells were plated onto iMatrix511 (Takara Bio)-coated wells. J1 hES cells were then plated onto iMatrix511-HDF wells and cultured in StemFit medium (Ajinomoto) for 8 days. The medium was then switched to EBDM to promote RPE generation. EBDM was changed daily for 7 days, then every 2–3 days.

[0168] After 47 days (approximately 7 weeks) in EBDM, cells were treated with collagenase for 6 hours. The released and digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. Clusters retained on the 40-micron strainer were collected and dissociated into single cells by treatment with 10x TrypLE for 15 minutes. Single cells were plated onto iMatrix511-coated wells in EBDM, and the EBDM was changed every 2–3 days. After 39 days (approximately 5 weeks) in EBDM after plating, cells were treated with collagenase overnight. The released and digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. Clusters retained on the 40-micron strainer were collected and dissociated into single cells by treatment with 10x TrypLE (Thermo Fisher Scientific) for 15 minutes. Single cells were plated onto gelatin-coated wells in MM / FGF medium as passage 0 RPE cells ("P0"). MM / FGF medium was changed daily until approximately >90% confluent, then changed to MM medium every two days until harvest. P0 RPE cells were cultured for 16 days. P0 cells were harvested by 15 minutes of 10x TrypLE treatment and replated onto gelatin-coated wells in MM / FGF medium as passage 1 RPE cells ("P1"). The culture method was repeated as described above for P0 RPE cells by first culturing in MM / FGF and then switching to MM medium. P1 RPE cells were cultured for 14 days. P1 RPE cells were harvested and replated as passage 2 RPE cells ("P2") as described above by first culturing in MM / FGF and then switching to MM medium. P2 RPE cells were cultured for 14 days, harvested by 10x TrypLE treatment for 15 minutes, and then cryopreserved. The cells were then thawed, formulated in GS2, cultured on gelatin (for certain tests), and subjected to quality testing. The results are shown in Table 2.

[0169] Quality testing is generally performed as described in US Patent Application Publication No. 2015 / 0366915, the entire contents of which are incorporated herein by reference. For example, purity (MITF / PAX6), bestrophin, and ZO1 levels are determined by immunofluorescence assay (IFA). Phagocytosis / efficacy assay is performed as described in WO 2016 / 154357, the entire contents of which are incorporated herein by reference.

[0170] (Table 1) TIFF2026032026000001.tif89164

[0171] (Table 2) TIFF2026032026000002.tif129164

[0172] Example 3: RPE cells generated by the single RPE progenitor cell subculture method and the RPE progenitor cell cluster subculture method In the first experiment, RPE cells were generated by single RPE progenitor cell subculture and cluster RPE progenitor cell subculture methods, as shown in Figure 4. Briefly, mitomycin C-inactivated HDF cells were plated onto iMatrix511-coated wells. J1 hESCs were then plated onto iMatrix511-HDF wells and cultured in StemFit medium for 8 days. The medium was then changed to EBDM to promote RPE generation. After 69 days (approximately 10 weeks) in EBDM, the cells were treated with collagenase overnight. The released, digested material was passed through a column of strainers consisting of a 100-micron strainer resting on top of a 40-micron strainer placed on a collection tube. Clusters retained on the 40-micron strainer were collected. For the single RPE progenitor cell subculture procedure, clusters were dissociated into single cells with 10x TrypLE and cultured in EBDM on iMatrix511. For the RPE progenitor cluster subculture procedure, clusters obtained after collagenase and strainer fractionation were seeded intact in EBDM on iMatrix 511. All seeded wells had the EBDM medium changed every other or every two days.

[0173] Approximately 24 days (approximately 4 weeks) after replating in EBDM, wells from the single RPE progenitor cell subculture process were subjected to the same collagenase treatment and strainer fractionation described above to dissociate the clusters into single RPE cells. Wells from the RPE progenitor cell cluster subculture process were treated with collagenase, strained to remove single cells, and then subjected to negative and positive selection by inspection and manual manipulation. Isolated patches were dissociated into single RPE cells with 10x TrypLE. Single RPE cells obtained from the single RPE progenitor cell subculture and RPE progenitor cell cluster process were separately seeded as P0 RPE cells onto gelatin- or iMatrix511-coated wells in MM / FGF. MM / FGF medium was changed daily until approximately >90% confluence (approximately 3 days), and then changed to MM medium every 2 days until harvest. This process was repeated until P2 RPE cells were obtained and cryopreserved. The cells were then thawed, formulated into GS2, cultured on gelatin (if necessary), and subjected to quality testing. Quality testing was generally performed as described in U.S. Patent Application Publication No. 2015 / 0366915, the entire contents of which are incorporated herein by reference. For example, purity (MITF / PAX6), bestrophin, and ZO1 levels were determined by immunofluorescence assay (IFA). Phagocytosis / efficacy assays were performed as described in WO 2016 / 154357, the entire contents of which are incorporated herein by reference. The results are shown in Figure 5.

[0174] Example 4: Evaluation of two immunosuppressive therapy regimens as a method to prevent graft rejection after subretinal transplantation of RPE cells and determination of proof of concept for RPE cells as a treatment for atrophy secondary to age-related macular degeneration in patients with moderate to severe visual impairment The disclosed human pluripotent stem cell-derived retinal pigment epithelial (hPSC-RPE) cells can be used for subretinal transplantation as a treatment for atrophy secondary to age-related macular degeneration in patients with moderate to severe visual impairment. This study evaluates the efficacy, safety, and tolerability of two regimens of short-term, low-dose, systemic immunosuppressive therapy (IMT) as a method to prevent graft rejection after administration of hPSC-RPE cells (Part 1). This study also demonstrates the efficacy of hPSC-RPE cells for atrophy secondary to age-related macular degeneration in patients with moderate to severe visual impairment (Part 2).

[0175] Part 1 of the study involves serial evaluation of hPSC-RPE cells with one of two immunosuppressive therapy regimens in up to 15 subjects for each regimen. The occurrence of graft failure or rejection in Part 1 will determine the immunosuppressive therapy regimen used for the next subject treated in Part 2 of the study. Part 2 of the study is a proof-of-concept study, which includes subjects treated with the selected immunosuppressive therapy or a longer immunosuppressive therapy regimen from Part 1.

[0176] Dosage and Administration A single dose of hPSC-RPE cells and optional GS diluent will be administered to the study eye via subretinal injection. The dose of hPSC-RPE cells will be determined prior to treatment of the first subject in this study based on the results of separate dose-escalation studies in which subjects will be treated with 50,000; 150,000; and 500,000 hPSC-RPE cells.

[0177] The immunosuppressive therapy regimen includes Prograf® 0.5 mg capsules, Prograf® 1 mg capsules, and mycophenolate mofetil (MMF) 500 mg tablets, all of which are administered orally. Prograf® is administered at an initial dose of 0.05 mg / kg per day divided into two doses daily and titrated to achieve a target trough level of 3-5 ng / mL. For subjects taking CYP3A4 inhibitors (other than protease inhibitors, direct factor Xa inhibitors, direct thrombin inhibitors, or erythromycin), such as azole antifungals (e.g., voriconazole, ketoconazole), or antibiotics (e.g., clarithromycin, chloramphenicol), the initial dose of Prograf® may need to be adjusted. MMF is administered orally at a dose of 1.0 g twice daily. There are two IMT regimens: during regimen 1, Prograf® and MMF are initiated one week prior to the day of hPSC-RPE cell transplant. Both IMT drugs are continued for six weeks after transplant. During regimen 2, Prograf® and MMF are taken one week prior to the day of transplant and then discontinued.

[0178] hPSC-RPE cells will be administered to the study eye via subretinal injection after a standard three-port pars plana vitrectomy. Subjects will remain supine for at least 6 hours after transplantation. SSC will recommend the location for cell transplant injection. The dose of hPSC-RPE cells will be determined through separate dose-escalation studies in which subjects will be treated with 50,000; 150,000; and 500,000 hPSC-RPE cells.

[0179] After transplantation, all subjects treated with hPSC-RPE cells will be evaluated for safety and efficacy in the study eye on Day 1, weekly through Weeks 1-4 (no Week 3 visit for the 1-week immunosuppressive therapy regimen), every 2 weeks through Weeks 6-14, at Weeks 20, 26, 52, and 78, and annually thereafter until the end of Year 5. Untreated controls will be evaluated for efficacy in the study eye on Study Entry Criteria Day 0, and at Weeks 4, 8, 12, 20, 26, and 52. Week 52 is the end of study (EoS) for the control group.

[0180] All adverse events (AEs) will be captured from the screening visit through Week 52. Thereafter, only AEs of particular interest will be captured, including all ocular and immune-mediated events.

[0181] The image reading center will evaluate fundus photography, fundus autofluorescence, spectral-domain optical coherence tomography (SD-OCT), optical coherence tomography-angiography (OCT-A), adaptive optics (AO), and fluorescein angiography (FA) results. A central microperimetry data collection center and central laboratory will also be utilized. Whenever possible, visual function testers and reading centers will be masked to treatment groups.

[0182] Evaluation of immunosuppressive therapy Subjects initially enrolled in the study and randomized to the hPSC RPE cell therapy arm will be sequentially assigned to one of two regimens of low-dose combination immunosuppressive therapy (Prograf® and mycophenolate mofetil) and infection prophylaxis:

[0183] Cohort 1 / Immunosuppressive Therapy Regimen 1: 7 weeks of immunosuppressive therapy and prophylactic medication starting 1 week prior to transplantation.

[0184] Cohort 2 / immunosuppressive therapy regimen 2: One week of immunosuppressive therapy and prophylactic medication starting one week prior to transplantation.

[0185] While the subject is receiving immunosuppressive therapy, the subject will be monitored for safety by the immunosuppressive therapy physician.

[0186] Each cohort will consist of up to 15 subjects treated with hPSC-RPE cells. If there is one or no incidence of graft failure or rejection in Cohort 1, randomization to the treatment arm of Cohort 2 will begin once Cohort 1 is fully enrolled and the last treated subject completes the 14-week visit.

[0187] If more than one subject in a cohort or across cohorts has evidence of transplant failure or rejection, the immunosuppressive therapy regimen for treated and as yet untreated subjects is altered.

[0188] Graft failure or rejection, unless attributable to another cause, consists of: · Evidence of unexpected, persistent, or increasing non-infectious ocular inflammation (e.g., vasculitis, retinitis, choroiditis, vitritis, pars planitis, or anterior segment inflammation / uveitis). · Appearance and subsequent disappearance after implantation of pigmented spots on fundus photography or hyperreflective material on Bruch's membrane on SD-OCT. If a gain of ≥10 letters is confirmed by repeat measurements or at the next scheduled visit within the first 52 weeks of the study, a subsequent confirmed loss of ≥10 letters that cannot be attributed to another cause can be considered evidence of graft failure or rejection. Any other ocular signs or symptoms that, in the opinion of the investigator and / or the Data Safety Monitoring Board (DSMB), may be due to graft failure or rejection. The final determination of whether a report of "other ocular signs or symptoms" constitutes graft failure or graft rejection will be made by the sponsor based on guidance from the DSMB.

[0189] Effectiveness The primary analysis set is the full analysis set, which includes all randomized treated subjects from the hPSC-RPE group who received the selected IMT regimen or the longer IMT regimen, and randomized subjects from the untreated control group (from both parts of the study) who reached day 0. A two-sided 5% significance level will be used to assess statistical significance for all analyses.

[0190] The primary endpoint is the change from baseline in total area of ​​atrophy at week 52. Analysis of the primary endpoint is estimated from a mixed model repeated measures (MMRM) analysis of the change from baseline to each week (weeks 4, 8, 12, 20, 26, and 52). The model includes the following fixed effects: study group (hPSC-RPE or untreated), stratification group for baseline area of ​​DDAF (two levels) and hyperAF (two levels) around the area of ​​DDAF in the study eye, site (pooled as necessary), time (study week), and treatment x time interaction, as well as baseline covariates. Parameters are estimated using restricted maximum likelihood, and degrees of freedom are estimated using the Kenward-Roger approximation. An unstructured variance-covariance structure is used to estimate the within-subject error of the model. If the fit of the unstructured covariance structure does not converge, other variance-covariance structures are used until convergence. Missing data are not imputed in this analysis.

[0191] Least squares means (with standard errors) for both study groups and the difference between hPSC-RPE versus untreated control study groups (with 95% confidence intervals) are shown for weeks 4, 8, 12, 20, 26, and 52.

[0192] Analysis of the secondary endpoint "Subject visual function response, defined as a confirmed gain of ≥ 15 letters in the study eye (within the visit acceptance period)" (change from baseline to week 52) will use a chi-square test for comparison of study groups. If there are fewer than five subjects in any cell of the 2x2 table, Fisher's exact test will be used instead. The proportion of subjects who gained ≥ 15 letters in the study eye for study group and study group difference (with 95% confidence interval) will be shown. In addition to observed data analysis, subjects with missing values ​​will be evaluated using non-response for missing data.

[0193] The secondary endpoints of "change from baseline in area of ​​atrophy in the index quadrant," "change from baseline in mean microperimetry sensitivity of perilesional test sites at week 52," "change from baseline in log contrast sensitivity at week 52," and "change from baseline in BCVA at week 52" will be analyzed using the same MMRM model described for the primary endpoint. Time points included for area of ​​atrophy are weeks 4, 8, 12, 20, 26, and 52; for BCVA, weeks 4, 8, 12, 20, 26, and 52 (time points common to both the RPE cell and untreated groups); for microperimetry sensitivity, weeks 4, 12, 20, 26, and 52; and for contrast sensitivity, weeks 4, 12, 26, and 52.

[0194] Analysis of the "change from baseline" summary score representing all items on the Impact of Vision Impairment questionnaire (IVI) at week 52 used an analysis of covariance (ANCOVA) model, which included terms for study group (ASP7317 or untreated), stratification group for baseline area of ​​DDAF (2 levels) and for hyper AF (2 levels) surrounding the area of ​​DDAF in the study eye, and site (pooled as appropriate).

[0195] The primary and secondary endpoints will also be analyzed separately for severe (baseline BCVA 20 / 320 to <20 / 200) and moderate (baseline BCVA 20 / 200 to 20 / 80) visual impairment groups (provided there are sufficient numbers of subjects in each subgroup analysis).

[0196] The Week 52 / ET time point will be analyzed for all endpoints above using ANCOVA as above, except for "subject response, defined as a confirmed ≥ 15 letter improvement in the study eye," which will use the chi-square test as above.

[0197] Example 5: Comparison of RPE cell generation from a conventional selective collection method without subculture, an RPE progenitor cell cluster subculture method with selective collection, and a single RPE progenitor cell subculture method without selective collection A comparison of RPE cell generation was performed between 1) a conventional RPE cell generation method involving labor-intensive selective collection without subculture, 2) the RPE progenitor cell cluster subculture method using selective collection described herein, and 3) the single RPE progenitor cell subculture method without selective collection described herein. The conventional RPE cell generation method was performed using an adherent hESC monolayer method, generally as described in WO 2005 / 070011. Briefly, J1 hESCs were differentiated on HDFs in EBDM for 90-100 days until pigmented spots with polygonal cobblestone morphology and brown pigment in the cytoplasm formed. These pigmented polygonal cells were digested, and pigmented islands were manually selectively collected. The collected pigmented clusters were dissociated into single cells, counted, and seeded as P0 RPE cells. RPE cells obtained from the RPE progenitor cluster subculture method with selective collection and the single RPE progenitor cell subculture method without selective collection were similarly counted and then seeded as P0 RPE cells.

[0198] Table 3 shows the RPE cells generated from methods involving selective collection: a conventional selective collection method without subculturing, and an RPE progenitor cell cluster subculture method using selective collection according to the present invention. Table 3 shows that the RPE progenitor cell cluster subculture method using selective collection can produce a greater number of cells per lot than the conventional method, but more importantly, it shows that the RPE progenitor cell cluster subculture method using selective collection produced a greater average number of cells per hour of manual labor required to selectively collect RPE cells than the conventional method. Furthermore, because the conventional method did not include a subculture step that enriches RPE precursors, selective collection from a population with low purity using the conventional method resulted in fewer cells being obtained, greater variability in morphology, and longer labor times for selectively collecting RPE.

[0199] Table 4 shows the RPE cells generated from the single RPE progenitor cell subculture method, which did not involve manual selective collection of RPE cells. The single RPE progenitor cell subculture method produced significantly more RPE cells than the conventional method or the RPE progenitor cell cluster subculture method using selective collection. Furthermore, the total number of cells obtained per unit time required to isolate P0 RPE cells was also significantly higher.

[0200] The method of the present invention offers a significant improvement over conventional methods, which require manual selective collection of RPE cells from a low-purity population. Manual collection is physically and mentally demanding, requiring several hours of continuous, highly precise, and focused work over several days to produce a single, properly sized lot. Training new operators to conventional methods is also difficult because it requires both precise mechanical manipulation under a microscope and experience with cell morphology, as a small number of contaminating cells, if mistakenly accepted, can overgrow the RPE and result in lot failure. Each collected cluster must be evaluated for morphology by the operator before it is accepted or rejected. Some clusters may have less than ideal RPE morphology, requiring the operator to subjectively decide whether to accept or reject the cluster. Once each cluster is evaluated, it must be moved quickly. This procedure is repeated two to three times to eliminate single cells and ensure the quality of the collected clusters. Slow operator speed can result in very low yields, and poor decision-making can lead to low purity and lot failure. Therefore, a skilled operator must have experience with aseptic procedures, skill in micromanipulation under a microscope in a sterile environment, experience that allows for relatively fast movement of selected and withdrawn clusters, and experience with cell morphology that allows for fast decision-making about each cluster evaluated. The method of the present invention allows for the use of standard cell culture methods that can be used by operators with minimal cell culture experience, and the cell yield is significantly higher.

[0201] (Table 3) TIFF2026032026000003.tif55164

[0202] (Table 4) TIFF2026032026000004.tif42128*IFA was not performed on P0 RPE cells, however all 4 lots passed QC testing with purity >95% at P1.

Claims

1. 1. A method for generating a population of retinal epithelial (RPE) cells, comprising: (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells and dissociating the cell clusters into single cells; (ii) culturing the single cells in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells produced in step (ii). Including, The method thereby generates a population of RPE cells.

2. 1. A method for generating a population of retinal epithelial (RPE) cells, comprising: (i) obtaining cell clusters of PAX6+ / MITF+ RPE progenitor cells; (ii) culturing the cell clusters in a differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells produced in step (ii). Including, The method thereby generates a population of RPE cells.

3. recovering the RPE cells produced in step (ii) by dissociating the RPE cells, fractionating the RPE cells, collecting RPE cell clusters, dissociating the RPE cell clusters into single RPE cells, and culturing the single RPE cells.

3. The method of claim 1 or 2, further comprising:

4. recovering the RPE cells produced in step (ii) by dissociating the RPE cells, collecting RPE cell clusters, and selectively harvesting the RPE cell clusters.

3. The method of claim 1 or 2, further comprising:

5. dissociating the selectively collected RPE cell clusters into single RPE cells and culturing the single RPE cells.

5. The method of claim 4, further comprising:

6. The method of any one of the preceding claims, wherein the PAX6+ / MITF+ RPE progenitor cells are obtained from a population of pluripotent stem cells.

7. The method of claim 6, wherein the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

8. The method of any one of the preceding claims, further comprising expanding the RPE cells.

9. 9. The method of claim 8, wherein the RPE cells are expanded by culturing the cells in a maintenance medium supplemented with FGF.

10. 10. The method of claim 9, wherein the maintenance medium contains FGF for the first 1, 2, or 3 days of RPE growth at each passage, followed by culturing the RPE cells in maintenance medium lacking FGF.

11. 11. The method of claim 9 or 10, wherein FGF is added prior to confluence.

12. The method according to any one of the preceding claims, wherein the differentiation medium further comprises heparin and / or a ROCK inhibitor.

13. 2. The method of claim 1, wherein the RPE cells are passaged up to two times.

14. 14. The method of any one of claims 1 and 3 to 13, wherein any one of the dissociation steps is carried out by treating the cells with a dissociation reagent.

15. 15. The method of claim 14, wherein the dissociation reagent is selected from the group of collagenase (e.g., collagenase I or collagenase IV), accutase, a chelating agent (e.g., an EDTA-based dissociation solution), trypsin, dispase, or any combination thereof.

16. The method of any one of the preceding claims, wherein the RPE cells are cryopreserved after collection.

17. 17. The method of claim 16, wherein the cells are cryopreserved in a medium comprising one or more cryopreservatives selected from the group consisting of DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, and sucrose.

18. The method of any one of claims 6 to 17, wherein the population of pluripotent stem cells is an embryoid body.

19. 10. The method of any one of the preceding claims, wherein the cells are cultured on feeder cells.

20. The method of any one of claims 1 to 18, wherein the cells are cultured under feeder-free conditions.

21. The method of any one of the preceding claims, wherein the cells are cultured in non-adherent culture.

22. The method of any one of claims 1 to 20, wherein the cells are cultured in adherent culture.

23. The method of any one of the preceding claims, wherein the differentiation medium is EBDM.

24. 23. The method of any one of claims 1 to 22, wherein the differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and MEK inhibitors (e.g., PD0325901).

25. 25. The method of claim 24, wherein the differentiation medium comprises nicotinamide.

26. 26. The method of claim 24 or 25, wherein the differentiation medium comprises activin.

27. 2. The method of claim 1, wherein the cell clusters of the PAX6+ / MITF+ RPE progenitor cells are between about 40 μm and about 200 μm in size.

28. 2. The method of claim 1, wherein the cell clusters of the PAX6+ / MITF+ RPE progenitor cells are between about 40 μm and about 100 μm in size.

29. The method of any one of the preceding claims, wherein in step (ii), the cells are cultured on an extracellular matrix selected from the group consisting of laminin or a fragment thereof, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin.

30. 30. The method of claim 29, wherein the extracellular matrix is ​​laminin or a fragment thereof.

31. 31. The method of claim 30, wherein the laminin is selected from laminin-521 and laminin-511.

32. 32. The method of claim 31, wherein the laminin is iMatrix511.

33. The method according to any one of the preceding claims, wherein the culture period in step (ii) is from about 1 week to about 8 weeks.

34. 2. The method according to claim 1, wherein the culturing period in step (ii) is at least about 3 weeks.

35. The method according to any one of the preceding claims, wherein the culture period in step (ii) is about 6 weeks.

36. The method of any one of claims 3 to 35, wherein the RPE cell clusters are between about 40 μm and 200 μm in size.

37. 37. The method of claim 36, wherein the RPE cell clusters are between about 40 μm and 100 μm in size.

38. The method of any one of claims 3 to 37, wherein the single RPE cell is cultured in a medium that supports proliferation or differentiation of RPE.

39. 39. The method of claim 38, wherein the single RPE cells are cultured on an extracellular matrix selected from the group consisting of laminin or a fragment thereof, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin.

40. 40. The method of claim 39, wherein the extracellular matrix is ​​gelatin.

41. 40. The method of claim 39, wherein the extracellular matrix is ​​laminin or a fragment thereof.

42. 2. The method of any one of the preceding claims, wherein the population of RPE cells is at least 75% pure, at least 80% pure, at least 90% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, or at least 99% pure.

43. The method of any one of the preceding claims, wherein the RPE cells are human RPE cells.

44. 1. A method for generating a population of retinal epithelial (RPE) cells, comprising: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect cell clusters, dissociating the cell clusters into single cells, and subculturing the single cells in a second differentiation medium so that the single cells differentiate into RPE cells; and (iii) recovering the RPE cells produced in step (ii). Including, The method thereby generates a population of RPE cells.

45. 1. A method for generating a population of retinal epithelial (RPE) cells, comprising: (i) culturing a population of pluripotent stem cells in a first differentiation medium so that the pluripotent stem cells differentiate into RPE progenitor cells; (ii) dissociating the RPE progenitor cells, fractionating the cells to collect cell clusters, and subculturing the collected cell clusters in a second differentiation medium so that the cells differentiate into RPE cells; and (iii) recovering the RPE cells produced in step (ii). Including, The method thereby generates a population of RPE cells.

46. recovering the RPE cells produced in step (ii) by dissociating the RPE cells, fractionating the RPE cells to collect RPE cell clusters, dissociating the RPE cell clusters into single RPE cells, and culturing the single RPE cells.

46. ​​The method of claim 44 or 45, further comprising:

47. recovering the RPE cells produced in step (ii) by dissociating the RPE cells, collecting RPE cell clusters, and selectively harvesting the RPE cell clusters.

46. ​​The method of claim 44 or 45, further comprising:

48. 48. The method of claim 47, further comprising dissociating the selectively collected RPE cell clusters into single RPE cells and culturing the single RPE cells.

49. The method of any one of claims 44 to 48, wherein the RPE progenitor cells are positive for PAX6 / MITF.

50. 50. The method of any one of claims 44 to 49, further comprising expanding the RPE cells.

51. 51. The method of claim 50, wherein the RPE cells are expanded by culturing the cells in a maintenance medium supplemented with FGF.

52. 52. The method of claim 51, wherein the maintenance medium contains FGF for the first 1, 2 or 3 days of RPE growth at each passage, followed by culturing the RPE cells in maintenance medium lacking FGF.

53. 53. The method of claim 51 or 52, wherein FGF is added prior to confluence.

54. The method of any one of claims 44 to 53, wherein the first differentiation medium and / or the second differentiation medium further comprises heparin and / or a ROCK inhibitor.

55. The method of any one of claims 44 to 54, wherein the RPE cells are passaged up to two times.

56. 56. The method of any one of claims 44 to 55, wherein any one of said dissociation steps is carried out by treating the cells with a dissociation reagent.

57. 57. The method of claim 56, wherein the dissociation reagent is selected from the group of collagenase (e.g., collagenase I or collagenase IV), accutase, a chelating agent (e.g., an EDTA-based dissociation solution), trypsin, dispase, or any combination thereof.

58. The method of any one of claims 44 to 57, wherein the RPE cells are cryopreserved after collection.

59. 59. The method of claim 58, wherein the cells are cryopreserved in a medium comprising one or more cryopreservatives selected from the group consisting of DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, and sucrose.

60. The method of any one of claims 44 to 59, wherein the pluripotent stem cells are human embryonic stem cells.

61. The method of any one of claims 44 to 59, wherein the pluripotent stem cells are human induced pluripotent stem cells.

62. The method of any one of claims 44 to 61, wherein the population of pluripotent stem cells is an embryoid body.

63. 63. The method of any one of claims 44 to 62, wherein prior to step (i), the pluripotent stem cells are cultured on feeder cells in a medium that supports pluripotency.

64. 63. The method of any one of claims 44 to 62, wherein prior to step (i), the pluripotent stem cells are cultured feeder-free in a medium that supports pluripotency.

65. 65. The method of claim 63 or 64, wherein the pluripotency-supporting medium is supplemented with bFGF.

66. 66. The method of any one of claims 44 to 65, wherein steps (i), (ii), and / or (iii) are performed in non-adherent culture.

67. 66. The method of any one of claims 44 to 65, wherein steps (i), (ii), and / or (iii) are performed in adherent culture.

68. 68. The method of any one of claims 44 to 67, wherein the first differentiation medium and the second differentiation medium are the same.

69. 68. The method of any one of claims 44 to 67, wherein said first differentiation medium and said second differentiation medium are different.

70. 69. The method of any one of claims 44 to 68, wherein the first differentiation medium and the second differentiation medium are EBDM.

71. 70. The method of any one of claims 44-69, wherein the first differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, Noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and MEK inhibitors (e.g., PD0325901).

72. 70. The method of any one of claims 44-69, wherein the second differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth differentiation factors (GDFs)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, Noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signal inhibitors, bFGF inhibitors, and MEK inhibitors (e.g., PD0325901).

73. 73. The method of claim 71 or 72, wherein said first differentiation medium comprises nicotinamide.

74. 74. The method of any one of claims 71 to 73, wherein said second differentiation medium comprises activin.

75. The method of any one of claims 44 to 74, wherein the culturing period in step (i) is from about 1 week to about 12 weeks.

76. The method of any one of claims 44 to 75, wherein the culturing period in step (i) is at least about 3 weeks.

77. The method of any one of claims 44 to 76, wherein the culturing period in step (i) is from about 6 weeks to about 10 weeks.

78. 78. The method of any one of claims 44 to 77, wherein the cell clusters collected in step (ii) are between about 40 μm and about 200 μm in size.

79. 79. The method of any one of claims 44 to 78, wherein the cell clusters collected in step (ii) are between about 40 μm and about 100 μm in size.

80. 80. The method of any one of claims 44 to 79, wherein in step (ii), the cells are passaged on an extracellular matrix selected from the group consisting of laminin, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin.

81. 81. The method of claim 80, wherein the extracellular matrix comprises laminin or a fragment thereof.

82. 82. The method of claim 81, wherein the laminin or fragment thereof is selected from laminin-521 and laminin-511.

83. The method of any one of claims 44 to 82, wherein the period of subculturing in step (ii) is from about 1 week to about 8 weeks.

84. The method of any one of claims 44 to 83, wherein the period of subculturing in step (ii) is at least about 3 weeks.

85. The method of any one of claims 44 to 84, wherein the period of subculturing in step (ii) is about 6 weeks.

86. 86. The method of any one of claims 46 and 48-85, wherein the RPE cell clusters are between about 40 μm and 200 μm in size.

87. 87. The method of claim 86, wherein the RPE cell clusters are between about 40 μm and 100 μm in size.

88. 88. The method of any one of claims 46 and 48-87, wherein the single RPE cell is cultured in a medium that supports RPE proliferation or differentiation.

89. 89. The method of claim 88, wherein the single RPE cells are cultured on an extracellular matrix selected from the group consisting of laminin or a fragment thereof, fibronectin, vitronectin, Matrigel, CellStart, collagen, and gelatin.

90. 90. The method of claim 89, wherein the extracellular matrix is ​​gelatin.

91. 90. The method of claim 89, wherein the extracellular matrix is ​​laminin or a fragment thereof.

92. 92. The method of any one of claims 44-91, wherein the population of RPE cells is at least 75% pure, at least 80% pure, at least 90% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, or at least 99% pure.

93. The method of any one of claims 44 to 92, wherein the RPE cells are human RPE cells.

94. The method of any one of the preceding claims, wherein the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, bestrophin, MITF, OTX2, PAX2, PAX6, pre-melanosome protein (PMEL or gp-100), tyrosinase, and ZO1.

95. The method of any one of the preceding claims, wherein the RPE cells express bestrophin, PMEL, CRALBP, MITF, PAX6, and ZO1.

96. The method of any one of claims 1 to 94, wherein the RPE cells express bestrophin, PAX6, MITF, and RPE65.

97. The method of any one of claims 1 to 94, wherein the RPE cells express at least one marker selected from MITF and bestrophin, and PAX6.

98. 2. The method of any one of the preceding claims, wherein the RPE cells lack substantial expression of one or more stem cell markers selected from the group consisting of OCT4, NANOG, Rex-1, alkaline phosphatase, SOX2, TDGF-1, DPPA-2, DPPA-4, stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-1-60 and TRA-1-80.

99. 2. The method of claim 1, wherein the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase.

100. 99. The method of any one of claims 1-98, wherein the RPE cells lack substantial expression of OCT4, NANOG, and SOX2.

101. A composition comprising a population of RPE cells produced by the method of any one of the preceding claims.

102. 101. A pharmaceutical composition comprising a population of RPE cells produced by the method of any one of claims 1 to 100 and a pharmaceutically acceptable carrier.

103. 104. A method of treating a patient having or at risk of having a retinal disease, comprising administering an effective amount of the composition of claim 101 or the pharmaceutical composition of claim 102.

104. 104. The method of claim 103, wherein the retinal disease is selected from the group of retinal degeneration, choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt's disease, angioid streaks, myopic macular degeneration, and glaucoma.

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