Methods for producing retinal pigment epithelium cells

Abstract

The present invention provides an improved method of producing highly pure retinal pigment epithelial (RPE) cells by differentiation of pluripotent stem cells. The present invention also provides compositions of RPE cells and methods of use thereof in treating retinal diseases.

Description

[0001] 127206-12220

[0002] METHODS FOR PRODUCING RETINAL PIGMENT EPITHELIUM CELLS

[0003] RELATED APPLICATIONS

[0004] [1] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 790,895, filed on April 18, 2025, and U.S. Provisional Application No. 63 / 931,007, filed on December 4, 2025. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] [2] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2026 is named 127206-12220. xml and is 46,367 bytes in size.

[0007] BACKGROUND

[0008] [3] The retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina. This layer of cells nourishes retinal visual cells, and is positioned between the underlying choroid (the layer of blood vessels behind the retina) and overlying retinal visual cells. The RPE transports nutrients from the retina to the choroid. Additionally, the RPE provides a barrier between the retina and the choroid. Breakdown of the RPE interferes with the metabolism of the retina, causing thinning of the retina. Thinning of the retina can have serious consequences that can have an impact on vision. For example, thinning of the retina may cause "dry" macular degeneration (in particular geographic atrophy) and may also lead to the inappropriate blood vessel formation that can cause "wet" macular degeneration.

[0009] [4] Given the importance of the RPE in maintaining visual and retinal health, there have been significant efforts in studying the RPE and in developing methodologies for producing RPE cells in vitro. RPE cells produced in vitro can be used to study the developments of the RPE, to identify factors that cause the RPE to breakdown, or to identify agents that can be used to stimulate repair of endogenous RPE cells. Additionally, RPE cells produced in vitro can themselves be used as a therapy for replacing or restoring all or a portion of a patient's damaged RPE cells. When used in this manner, RPE cells may provide an approach to treat127206-12220

[0010] macular degeneration, as well as other diseases and conditions caused, in whole or in part, by damage to the RPE.

[0011] [5] In vitro methods for producing retinal pigment epithelial (RPE) cells by inducing differentiation of pluripotent stem cells in the presence of a differentiation-inducing factor in a culture medium are known (see, e.g., Kuroda et al., PLoS One. 2012; 7(5): e37342.).

[0012] However, these methods require multiple steps combining adhesion culture and floating culture in order to obtain a highly concentrated RPE cell population. These known methods also require a purification step.

[0013] [6] Furthermore, using conventionally known methods, when RPE cells are obtained from pluripotent stem cells, cells other than the target cells are generally obtained simultaneously. Consequently, these methods can obtain only a portion of the RPE cells induced in a culture container. Moreover, the purity of the obtained RPE cells is largely influenced 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.

[0014] [7] Macular dystrophies with a Stargardt-type clinical presentation represent a group of rare, inherited, progressive, serious, bilateral conditions with a high unmet medical need. Such macular dystrophies include Stargardt’s disease (STGD) and STGD-like macular dystrophies which are associated with considerable genetic heterogeneity. Macular dystrophies are chronically debilitating conditions which pose significant public health challenges by impairing central vision, hindering daily activities and diminishing the quality of life of patients. Currently, there are no approved treatments for either STGD or any of the STGD-like macular dystrophies. In addition, there are no gene-specific, cell-based, or small molecule based treatments in active development for most types of STGD-like macular dystrophies.

[0015] [8] Accordingly, there is a need in the art for a simple and efficient method for producing highly pure RPE cells from pluripotent stem cells, as well as a high unmet medical need for RPE cells produced in vitro which can be used for the treatment of macular dystrophies, such as macular dystrophies with a Stargardt type clinical presentation.

[0016] SUMMARY

[0017] [9] 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, the127206-12220

[0018] invention is based, in part, on the discovery of stages during differentiation of pluripotent stem cells to RPE cells when RPE cells can be isolated at an intermediate stage to generate a cell stock, cryopreserved, and / or further expanded to mature RPE cells without manual picking of the cells. The methods of the invention are both simple and efficient, and result in a high yield of RPE cells that are substantially pure.

[0019]

[0010] The present disclosure also provides compositions of RPE cells which have a long shelf life (e.g., at least 18-24 months and up to 3, 4, or 5 years), improved long-term stability, improved commercial viability, and improved safety profile as compared to other compositions of RPE cells. Compositions of RPE cells provided herein can be stored at a central storage depot or a clinical site and then thawed and reconstituted at the clinical site.

[0020]

[0011] The present disclosure also provides RPE cells for use in the treatment of retinal diseases. For example the present disclosure provides RPE cells for use as a gene agnostic cell therapy in a broad spectrum of patients with macular dystrophies with a Stargardt-type clinical presentation. RPE cells of the disclosure can augment and / or replace the function of the RPE cells that are dysfunctional in the patient, leading to the improvement of retinal biology and function and vision. The improvement of retinal function and vision after administration of RPE cells of the disclosure may result from:

[0021] - the anatomical replacement of RPE in areas of cell loss and functional replacement of essential RPE support functions;

[0022] supplementation of the treated area with RPE expressing physiological levels of proteins necessary for maintenance of retinal structure and function;

[0023] - preservation of retinal cell function and potential improvement of visual function in affected areas through improved homeostasis and photoreceptor outer segment phagocytosis, along with the removal of accumulated cellular and extracellular subretinal debris;

[0024] - re-establishment of RPE-mediated metabolic exchange and transport functions, including nutrient delivery, ion regulation and maintenance of outer blood-retinal barrier integrity;

[0025] - restoration of retinoid recycling and chromophore regeneration capacity required for sustained phototransduction; and

[0026] - release of diffusible tropic factors within the microenvironment to support photoreceptor and outer retinal health.127206-12220

[0027]

[0012] The present disclosure also provides RPE cells for use in the treatment of dry age-related macular degeneration (AMD). The present disclosure also provides RPE cells for use in the treatment of geographic atrophy such as geographic atrophy secondary to AMD.

[0028]

[0013] Upon administration to the eyes, the RPE cells of the disclosure display exclusive localization to the eye in which they are administered, durable persistence within the subretinal space, maintenance of RPE phenotype, no evidence of systemic distribution (e.g., to the gonads), no systemic toxicity and no tumorigenicity.

[0029]

[0014] Accordingly, in one aspect, the present invention provides a method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising: (i) culturing pluripotent stem cells in a first differentiation medium to generate RPE progenitor cells, (ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells, (iii) expanding the RPE cells produced in step (ii), (iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock, (v) expanding the RPE cells of the cell stock, and (vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

[0030]

[0015] In another aspect, the present invention provides a method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising: (i) culturing pluripotent stem cells in a first differentiation medium to generate RPE progenitor cells, (ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells, (iii) expanding the RPE cells produced in step (ii) by passaging one time, (iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock, (v) expanding the RPE cells of the cell stock by passaging at least one time, and (vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

[0031]

[0016] In some embodiments, the pluripotent stem cells are human embryonic stem cells. In some embodiments, the human embryonic stem cells are genetically engineered to reduce their immunogenicity.

[0032]

[0017] In some embodiments, the pluripotent stem cells are human induced pluripotent stem cells. In some embodiments, the human induced pluripotent stem cells are genetically engineered to reduce their immunogenicity. In some embodiments, the human induced pluripotent stem cells comprise a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene. In some embodiments, the human induced pluripotent127206-12220

[0033] stem cells lack substantial endogenous expression of class I and class II HLA genes. In some embodiments, the human induced pluripotent stem cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G. In some embodiments, the human induced pluripotent stem cells comprise one or more safety switch genes. In some embodiments, the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch.

[0034]

[0018] In some embodiments, step (i) of any of the methods described further comprises dissociating the RPE progenitor cells, fractionating the RPE progenitor cells to collect cell clusters, and dissociating the cell clusters of RPE progenitor cells into single cells.

[0035]

[0019] In some embodiments, step (ii) of any of the methods described further comprises dissociating the RPE cells, fractionating the RPE cells to collect cell clusters, and dissociating the cell clusters of RPE cells into single cells.

[0036]

[0020] In some embodiments, the dissociation step is carried out by treating the cells with a dissociation reagent. In some embodiments, the dissociation reagent is selected from the group consisting of collagenase, accutase, chelator, trypsin, TrypLE, dispase, and combinations thereof. In some embodiments, the collagenase is collagenase IV. In some embodiments, the collagenase is animal origin free.

[0037]

[0021] In some embodiments, prior to step (i) of the methods described herein, the pluripotent stem cells are cultured on feeder cells in a medium that supports pluripotency. In some embodiments, prior to step (i) of the methods described herein, the pluripotent stem cells are cultured under feeder-free conditions in a medium that supports pluripotency.

[0038]

[0022] In some embodiments, step (i), step (ii), step (iii), and / or step (v) of the methods described herein is performed in a non-adherent culture. In some embodiments, step (i), step (ii), step (iii), and / or step (v) of the methods described herein is performed in an adherent culture.

[0039]

[0023] In some embodiments, in step (ii), step (iii), and / or step (v) of the methods described herein, the RPE cells are cultured on an extracellular matrix selected from the group consisting of gelatin, laminin, fibronectin, vitronectin, Matrigel, CellStart, and collagen.

[0040]

[0024] In some embodiments, the first differentiation medium and the second differentiation medium are the same. In some embodiments, the first differentiation medium and the second differentiation medium are different. In some embodiments, the first differentiation medium127206-12220

[0041] and the second differentiation medium is Embryoid Body Differentiation Medium (EBDM). In some embodiments, the first differentiation medium and / or the second differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, a transforming factor-P (TGFP) superfamily member (e.g., activin A, activin B, and activin AB), nodal, anti-mullerian hormone (AMH), bone morphogenetic proteins (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth and differentiation factors (GDF)), WNT pathway inhibitor (e.g., CKI-7, DKK1), a TGF pathway inhibitor (e.g., LDN193189, Noggin), a BMP pathway inhibitor (e.g., SB431542), a sonic hedgehog signal inhibitor, a bFGF inhibitor, and an MEK inhibitor (e.g., PD0325901).

[0042]

[0025] In some embodiments, wherein the RPE cells are expanded in step (iii) and / or step (v) of the methods described herein, by culturing the cells in a maintenance media. In some embodiments, the maintenance media is RPE-Growth Medium / Maintenance Medium (RPE-GM / MM). In some embodiments, the RPE-GM / MM is supplemented with FGF.

[0043]

[0026] In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose. In some embodiments, the medium for cryopreservation does not comprise DMSO.

[0044]

[0027] In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising albumin, glucose, buffered saline, and one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, and ethylene glycol, and wherein the medium does not comprise DMSO. In some embodiments, the albumin is present in a range of about 2% to about 8% (w / v); the glucose is present in a range of about 0.01% to about 1.5% (w / v); and / or the buffered saline is phosphate buffered saline.

[0045]

[0028] In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising about 4-10% (v / v) cryoprotectant, about 2-8% (w / v) albumin, about 0.1-1.5% glucose or 0.1-1.5% dextrose, and a buffer. In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising a) about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer; or b) about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer. In some embodiments, in step (iv) and / or step (vi)127206-12220

[0046] of the methods described herein, the RPE cells are cryopreserved in a medium comprising about 4-6% (v / v) cryoprotectant. In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising fetal bovine serum (FBS). In some embodiments the RPE cells are cryopreserved in a medium comprising 1-20% DMSO in FBS, 5-20% DMSO in FBS or 5-10% DMSO in FBS. In some embodiments, in step (iv) and / or step (vi) of the methods described herein, the RPE cells are cryopreserved in a medium comprising about 0.08-0.10% (w / v) glucose, or about 0.6% (w / v) glucose.

[0047]

[0029] In some embodiments, the methods described herein further comprise a step of thawing and formulating the RPE cells in a medium comprising sodium hyaluronate, a tonicity agent, and a buffer. In some embodiments, the tonicity agent is selected from the group consisting of D(+)-glucose anhydrous, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. In some embodiments, the buffer is selected from the group consisting of sodium acetate, trisodium citrate, and monosodium phosphate monobasic.

[0048]

[0030] In some embodiments, the RPE cells are formulated in a medium comprising sodium hyaluronate, D(+)-glucose anhydrous, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, trisodium citrate, monosodium phosphate monobasic dehydrate and / or sodium hydroxide.

[0049]

[0031] In some embodiments, the methods described herein do not comprise a step of formulating or diluting the RPE cells in a medium.

[0050]

[0032] In some embodiments, in step (iii) of the methods described herein, the RPE cells are expanded by passaging up to one time, two times, five times, or ten times. In some embodiments, in step (v) of the methods described herein, the RPE cells are expanded by passaging up to two times, five times, or ten times.

[0051]

[0033] In some embodiments, the duration of culturing in step (i) of the methods described herein is about 1 week to about 12 weeks. In some embodiments, the duration of culturing in step (ii) of the methods described herein is about 1 week to about 8 weeks.

[0052]

[0034] In some embodiments, the cell clusters of RPE progenitor cells are about 40 pm to about 200 pm, or about 40 pm to about 100 pm in size. In some embodiments, the cell clusters of RPE cells are about 40 pm to 200 pm, or about 40 pm to about 100 pm in size.127206-12220

[0053]

[0035] In some embodiments, 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.

[0054]

[0036] In some embodiments, the RPE cells are human RPE cells.

[0055]

[0037] In some embodiments, the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1. In some embodiments, the RPE cells express gp-100. In some embodiments the RPE cells express gp-100 and MITF. In some embodiments the RPE cells express gp-100 and CRALBP. In some embodiments, the RPE cells express gp-100, MITF and CRALBP.

[0056]

[0038] In some embodiments, 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)-l-60, and TRA-1-80.

[0057]

[0039] The present invention also provides a pharmaceutical composition comprising a population of RPE cells produced by any one of the methods described herein.

[0058]

[0040] The present invention also provides a composition of RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1, wherein the RPE cells comprise an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene. In some embodiments, the RPE cells lack substantial endogenous expression of class I and class II HLA genes. In some embodiments, the RPE cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G. In some embodiments, the RPE cells comprise one or more safety switch genes. In some embodiments, the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch.

[0059]

[0041] In some embodiments, the composition of RPE cells further comprises a cryoprotectant solution. In some embodiments, the cryoprotectant solution comprises one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, dimethyl sulfoxide (DMSO), ethylene glycol,127206-12220

[0060] glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose. In some embodiments, the cryoprotectant solution comprises 1-20% DMSO in fetal bovine serum; 5-20% DMSO in fetal bovine serum or 5-10% DMSO in fetal bovine serum. In some embodiments, the cryoprotectant solution does not comprise DMSO. In some embodiments, the cryoprotectant solution comprises albumin, glucose, buffered saline, and one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, and ethylene glycol, and wherein the medium does not comprise DMSO.

[0061]

[0042] In some embodiments, the cryoprotectant solution comprises albumin at a range of about 2% to about 8% (w / v), glucose at a range of about 0.01% to about 1.5% (w / v); and / or phosphate buffered saline. In some embodiments, the cryoprotectant solution further comprises about 4-10% (v / v) of DMSO. In some embodiments, the cryoprotectant solution further comprises about 4-10% (v / v) of one or more of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose.

[0062]

[0043] In some embodiments, The composition of RPE cells further comprises sodium hyaluronate, a tonicity agent, and a buffer. In some embodiments, the tonicity agent is selected from the group consisting of glucose, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. In some embodiments, the buffer is selected from the group consisting of sodium acetate, trisodium citrate, and monosodium phosphate monobasic. In some embodiments, the composition comprises sodium hyaluronate, glucose, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, trisodium citrate, monosodium phosphate monobasic and / or sodium hydroxide.

[0063]

[0044] In some embodiments, the composition comprises about 1 x 105- 1 x 107RPE cells.

[0064]

[0045] In another aspect, the present invention provides a composition comprising: a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1, and comprising a genetically engineered disruption of an endogenous [32 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene; about 2-8% (v / v) albumin; about 0.01% to about 1.5% (w / v) glucose; about 3-7% (v / v) DMSO; and phosphate buffered saline.127206-12220

[0065]

[0046] In another aspect, the present invention provides a composition comprising: a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1, and comprising a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene; sodium hyaluronate; glucose; sodium chloride; potassium chloride; calcium chloride; magnesium chloride; sodium acetate; trisodium citrate; and monosodium phosphate monobasic.

[0066]

[0047] In another aspect, the present invention provides a composition comprising: a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1; about 1-4% (v / v) albumin; about 0.01% to about 1.5% (w / v) glucose; about 4-10% (v / v) of one or more of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose; and phosphate buffered saline.

[0067]

[0048] In some embodiments, the RPE cells comprise a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene. In some embodiments, the RPE cells lack substantial endogenous expression of class I and class II HLA genes. In some embodiments, the RPE cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G. In some embodiments, the RPE cells comprise one or more safety switch genes. In some embodiments, the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch

[0068]

[0049] In another aspect, the present invention further provides a method of treating a subject with a retinal disease, or at risk of developing, a retinal disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition comprising a population of RPE cells produced by any one of the methods described herein, thereby treating the subject .127206-12220

[0069]

[0050] In some embodiments, the population of RPE cells is administered at a dose of about 10,000 cell / dose to about 500,000 cells / dose. In some embodiments, the population of RPE cells is administered at a dose of about 50, 000, 100,000, 133,000, 167,000, 200,000, 233,000, 267,000, 300,000, 333,333, 367,000, or 400,000 cells / dose.

[0070]

[0051] In some embodiments, the population of RPE cells is administered via injection. In some embodiments, the population of RPE cells is administered via an injection into the sub-retinal space of the eye. In some embodiments, the population of RPE cells is administered via a sub-retinal non-fovea involving injection. In some embodiments, the population of RPE cells is administered via a pars plana vitrectomy.

[0071]

[0052] In some embodiments, the retinal disease is selected from the group consisting of retinal degeneration, retinal damage, retinal detachment, dry age-related macular degeneration (AMD), wet AMD, geographic atrophy secondary to AMD, macular dystrophies with a Stargardt-type clinical presentation, choroideremia, retinitis pigmentosa, Leber congenital amaurosis, Best vitelliform macular dystrophy, Best vitelliform macular dystrophy, autosomal recessive bestrophinopathies (ARBs), Autosomal dominant vitreoretinochoroidopathy (ADViRC), Usher syndrome IB, Angioid streaks, myopic macular degeneration, and glaucoma.

[0072]

[0053] In some embodiments, the subject has severe visual impairment. In some embodiments, the subject has a baseline best corrected visual acuity (BCVA) ranging from about 20 / 80 to about 20 / 500. In some embodiments, the subject has a BCVA between light perception and 37 early treatment diabetic retinopathy study (ETDRS) letters. In some embodiments, the subject has between 9 - 12 disc areas of geographic atrophy.

[0073]

[0054] In some embodiments, the patient has moderate visual impairment. In some embodiments, the subject has a BCVA ranging from about 20 / 40 to about 20 / 200. In some embodiments, the subject has a BCVA ranging from 38 to 65 ETDRS letters. In some embodiments, the subject has between 1 - 8 disc areas of geographic atrophy.

[0074]

[0055] In some embodiments, the subject has geographic atrophy. In some embodiments, administration of the composition or the pharmaceutical composition to a subject’s eye results in one or more of the following as compared to the untreated eye of the subject: improvement in visual acuity; improvement in retinal function; stabilization of geographic atrophy; stabilization of ellipsoid zone loss; increase in treatment site cellularity; improvement in outer nuclear layer (ONL) structure; and / or photoreceptor preservation.127206-12220

[0075]

[0056] In some embodiments, the subject is suffering, or prone to suffering, from a macular dystrophy with a Stargardt-type clinical presentation. In some embodiments, the macular dystrophy with a Stargardt-type clinical presentation is Stargardt’s disease (STGD) or a STGD-like macular dystrophy. In some embodiments, the macular dystrophy with Stargardt-type clinical presentation is Stargardt’s disease (STGD) or a STGD-like macular dystrophy, for example often caused by mutations in genes such as PRPH2, ELOVL4, PR0M1, MT-TL1, CDHR1 and CTNNA1. In some embodiments, the subject has one or more symptoms selected from the group consisting of macular RPE loss and atrophy, macular photoreceptor loss and atrophy, accumulation of drusen-like macular deposits, decline of BCVA, and central vision loss. In some embodiments, administration of the composition or the pharmaceutical composition to the subject’s eye results in one or more of the following as compared to a control: improvement in visual acuity; improvement in retinal function; increase in treatment site cellularity; improvement in outer nuclear layer (ONL) structure; and / or photoreceptor preservation.

[0076]

[0057] In some embodiments, the control is measured in the subject prior to administration of the composition or the pharmaceutical composition; or the control is measured in a subject to whom the composition or the pharmaceutical composition have not been administered; or the control is measured in an untreated eye of the subject.

[0077]

[0058] In some embodiments, the subject is a human subject. In some embodiments, the subject is 6 years of age or older or 12 years of age or older. In some embodiments, the subject is 18 years of age or older. In some embodiments, the subject is 30 years of age or older, 40 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, 80 years of age or older, 85 years of age or older or 90 years of age or older.

[0078] BRIEF DESCRIPTION OF DRAWINGS

[0079]

[0059] Figure 1 shows representative microscopy images of the hiPSC cell culture on days 1 and 4 post thaw (top row), and days 1 and 6 post-passage (bottom row) under phase contrast at lOx magnification. Scale bar represents 100 pm.

[0080]

[0060] Figure 2 shows representative microscopy images of the cell culture on days 29 (top row) and 55 under Hoffman Modulation Contrast (HMC) (bottom row) at lOx magnification. Scale bar represents 100 pm.127206-12220

[0081]

[0061] Figure 3 shows representative microscopy images of the cell culture at days 1, 8 under phase contrast (top row), and days 20 and 27 under HMC (bottom row), at lOx magnification. Scale bar represents 100 pm.

[0082]

[0062] Figure 4 shows representative microscopy images of the P0 cell culture at days 2 and 3 under phase contrast (top row), and days 9 and 14 under HMC (bottom row), at lOx magnification. Scale bar represents 100 pm.

[0083]

[0063] Figure 5 shows representative microscopy images of the Pl cell culture at days 2 and 3 under phase contrast (top row), and days 9 and 12 under HMC (bottom row), at lOx magnification. Scale bar represents 100 pm.

[0084]

[0064] Figure 6 is a plot showing changes in mean best corrected visual acuity (BCVA) from baseline for cohort 1 in the clinical study, over a time period of 52 weeks.

[0085]

[0065] Figure 7 is a plot showing changes in mean BCVA from baseline for cohort 2 in the clinical study, over a time period of 52 weeks. The solid line represents BCVA for subject eyes (SE), and dashed line represents BCVA for fellow eyes (FE).

[0086]

[0066] Figure 8 shows plots of natural killer (NK)-mediated cytotoxicity in myeloid cells expressing HLA-E either through endogenous expression in unedited control iPSC or through gene editing in hiPSC cells in coculture assays with NK cells from 3 different donors. NIH2 C.2B9 cells do not express HLA-E and serve as positive control. Lines represent mean ± SD from triplicate wells from a single experiment. Statistical Test (hiPSC vs. NIH2 c.2B9): P > 0.05 (not significant), *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 (significant; multiple unpaired t-test); n.s.: not significant.

[0087]

[0067] Figure 9 shows plots of allogeneic CD4+ T cell response, as measured by T cell proliferation, in 3 different donors. NIH2 WT c.H12 is an unedited control. Asterisk indicates P< 0.05 in comparison to WT of the same parental line. Bar graphs represent mean ± SD from triplicate wells from a single experiment. Statistical Test: *P< 0.05 vs WT line (unpaired t-test with two- stage step-up; Benjamini, Krieger and Yekutieli correction); RLU: relative light unit.

[0088]

[0068] Figure 10 shows plots of allogeneic CD4+ T cell response, as measured by IL-2 secretion, in 3 different donors. Dotted line indicates the lower level of quantification of the assay; some values fall below this threshold. Bar graphs represent mean ± SD from triplicate127206-12220

[0089] wells from a single experiment. Statistical Test: *P < 0.05 (multiple unpaired T-tests with Holm-Sidak correction for adjusted P-values vs T cells and WT target cells).

[0090]

[0069] Figure 11 shows plots of myeloid cell death after exposure to CD8+ T cells from 3 different donors. Cell death was measured by flow cytometry gating on CD1 lb+Live / Dead+ cells. Bar graphs represent mean ± SD from triplicate wells from a single experiment.

[0091] Statistical Test: * P< 0.05, ** P< 0.01, *** P< 0.001 vs T cell + NIH2 WT c.H12 coculture (unpaired t-test).

[0092]

[0070] Figure 12 shows histograms of extracellular expressions of HLA-I, HLA-II, and HLA-E in IFNy untreated (-IFNy) and pre-treated (+IFNy) unedited iPSC-derived RPE cells (top panel) and hiPSC-derived RPE cells (bottom panel). The x-axis represents fluorescence intensity, while the y-axis, normalized to mode, represents the percentage of events relative to the highest peak which is set to 100%.

[0093]

[0071] Figure 13 shows plots of CD8+ T Cell activation, as measured by the expression of the late activation marker CD25, from 3 individual donors. The % CD25+ cells was calculated within the gated CD8+ T cell population. CD8+ T cells activated with anti-CD3 / CD28 Dynabeads or cocultured with irradiated Raji cells (irrRaji) serve as positive controls. CD8+ T cells alone were used as baseline (dotted line). Bars represent mean ± SD. **P< 0.01, ***P< 0.001, **** P< 0.0001 unedited iPSC-derived RPE cell cocultures vs hiPSC-derived RPE cell cocultures (unpaired t-test).

[0094]

[0072] Figure 14 shows plots of CD8 activation, as measured by IFNy secretion in the culture supernatants from 3 independent CD8+ T cell donors, analyzed by ELISA. CD8+ T cells activated with anti-CD3 / CD28 Dynabeads as well as coculture with irradiated Raji cells (irrRaji) served as positive controls. CD8+ T cells alone were used as baseline. Bars represent mean ± SD. ** P< 0.01, ***P< 0.001, **** P< 0.0001 unedited iPSC-derived RPE cell cocultures vs hiPSC-derived RPE cell cocultures (t-test); MDD: minimal detectable dose; STD OD: optical density for standard concentrations of IFNy.

[0095]

[0073] Figure 15 shows plots of CD8 proliferation, as measured by CD8+ T cells expressing the nuclear marker of cell proliferation Ki67 detected by flow cytometry, from 3 different donors. The % Ki67+ cells was calculated within the gated CD8+ T cell population. CD8+ T cells activated with anti-CD3 / CD28 Dynabeads or cocultured with irradiated Raji cells (irrRaji) served as positive controls. CD8+ T cells alone were used as baseline (dotted line).127206-12220

[0096] Bars represent mean ± SD. ** P< 0.01, **** P< 0.0001 unedited iPSC-derived RPE cell cocultures vs hiPSC-derived RPE cell cocultures (t-test); Ki67: antigen Kiel 67.

[0097]

[0074] Figure 16 shows plots of CD8+ T cell induced cell death, as measured by the viability of cocultured RPE cells assessed by flow cytometry, from 3 different donors. Target RPE cell death was quantified by gating on the CD45- RPE cell population and determining the proportion of viability dye-positive cells (CD45- Live / Dead+ cells). hiPSC-derived RPE cells and unedited iPSC-derived RPE cells alone were used to detect background lysis (0:1 samples, target alone). Cell death was measured by flow cytometry gating on CD45-Live / Dead+ cells. Bars represent mean ± SD from 3 independent experiments. * P< 0.05, ** P< 0.01, *** P< 0.001 for unedited iPSC-derived RPE cell cocultures vs hiPSC-derived RPE cell cocultures for each ratio (t-test).

[0098]

[0075] Figure 17 is a plot showing the confluence of cells in an in vitro GCV cytotoxicity assay to assess TK safety switch function. There are 8 replicates of each concentration and error is standard error of the mean; TK: thymidine kinase.

[0099]

[0076] Figure 18 shows plots of absolute teratoma size (top panel) and normalized teratoma size (bottom panel) against days-post GCV treatment, analyzed by simple linear regression (SLR) according to treatment group (vehicle control, 30 mg / kg GCV, and 100 mg / kg GCV) for all iPSC clones. Mice that had teratomas exceeding the threshold specified under the study parameters were not included. The 95% CI are shown in dashed lines. **P> 0.01, 0.0001, slopes analyzed via ordinary One-Way ANOVA compared to the saline treated group. SLR used for data analysis.

[0100]

[0077] Figure 19 shows representative histopathology images from rat studies. Figure 19A shows H&E images for injected (left panel) and noninjected (right panel) eyes from Group 2 animals (hiPSC-derived RPE cells supplemented With 1% iPSC). Black arrows show a thickening of the RPE layer in the temporal retina. Figure 19B shows IHC images for injected (left panel) and noninjected (right panel) eyes from Group 2 animals (hiPSC-derived RPE cells supplemented With 1% iPSC). *: background stain; Blue: Nuclear Hoescht; Red: Ku80; Green: PMEL17.

[0101]

[0078] Figure 20 shows dose-specific improvement in spatial frequency threshold in the OMR assay in RCS rats following hiPSC-derived subretinal administration. Two-Way ANOVA Mixed-effects model, p< 0.01. Tukey’s multiple comparisons test (p-values127206-12220

[0102] indicated in graph above: *p< 0.05, **p< 0.01, ***p< 0.001, 0.0001). Error bars are SEM; eye: cycle; deg: degree; P60: postnatal day 60; P90: postnatal day 90.

[0103]

[0079] Figure 21 shows plots of Electroretinogram (ERG) signals in RCS rats following hiPSC-derived subretinal administration. Figure 21A is a plot showing ERG b-wave amplitudes. Figure 21B is a plot showing OP1 amplitudes. Figure 21C is a plot showing OP2 amplitudes. Figure 21D is a plot showing OP3 amplitudes. Two-Way ANOVA Mixed-effects model, p< 0.0X; Tukey’s multiple comparisons test (p-values indicated in graph above: *p<0.05, **p< 0.01, ***p< 0.001,

[0104]

[0105] 0.0001). Error bars are SEM.

[0106]

[0080] Figure 22 shows plots of histopathology studied in RCS rats following hiPSC-derived subretinal administration. Figure 22A is a plot showing treatment site cellularity evaluated using a semi quantitative scoring scale ranging from 0 (no increase in cells observed) to 5 (greater than five cell layers or widespread multifocal clusters of cells and extending around a large portion of the globe periphery). Figure 22B is a plot showing ONL cellularity evaluated using a semi-quantitative scale from 0 (absent or nearly absent ONL) through increasing scores which indicate progressively thicker nuclear layering. Figure 22C is a plot showing the number of labeled cells in Ku80 IF-stained sections using a semi-quantitative scoring scale ranging from 0 (no labeled cells) to 5 ( > 100 labeled cells). Figure 22D is a plot showing Ku80 / PMEL17 co-labeling evaluated using a semi-quantitative scoring scale ranging from 0 (no labeled cells) to 5 ( > 75% co-expression. Ku80: X-ray repair cross complementing 5 (also known as XRCC5); PMEL17: premelanosome protein 17. Error bars are SEM.

[0107]

[0081] Figure 23 is a graph showing improvement in BCVA in eyes treated with RPE cells at 26 weeks. Figure 23A is a graph showing improvement in BCVA for low dose RPE cell cohort. Figure 23B is a graph showing improvement in BCVA for medium dose RPE cell cohorts. Figure 23C is a graph showing improvement in BCVA for high dose RPE cell cohorts.

[0108]

[0082] Figure 24 is a graph showing improvement in BCVA in eyes treated with RPE cells up to 52 weeks in low dose and medium dose RPE cell cohorts, and up to 26 weeks in high dose RPE cell cohort. Figure 24A is a graph showing improvement in BCVA in eyes treated with low dose RPE cells. Figure 24B is a graph showing improvement in BCVA in eyes treated with medium dose RPE cells. Figure 24C is a graph showing improvement in BCVA in eyes treated with high dose RPE cells).127206-12220

[0109]

[0083] Figure 25 is a graph showing improvement in GA area size in eyes treated with RPE cells. Figure 25A is a graph showing improvement in GA area size for medium dose cohorts up to week 52. Figure 25B is a graph showing improvement in GA area size for high dose RPE cells up to week 26.

[0110]

[0084] Figure 26 is a set of spectral domain optical coherence tomography (SD-OCT) images from baseline, week 26 and week 52 of the study eye treated with RPE cells and fellow untreated eye from a study participant. The images show stabilization of the RPE-bruch’s membrane layer in the supero-temporal macula of the treated eye, stabilization of region of ellipsoid zone (EZ) loss in treated eye (shown in pink) and slow progression of RPE-Bruch’s membrane loss and EZ loss in the fellow eye, from baseline to week 52.

[0111] DETAILED DESCRIPTION

[0112]

[0085] The present invention provides improved methods 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 invention is based on the discovery of stages during differentiation of pluripotent stem cells to RPE cells when RPE cells can be isolated at an intermediate stage to generate a cell stock, cryopreserved, and / or further expanded to mature RPE cells without manual picking of the cells. The methods of the invention are both simple and efficient, and result in a high yield of RPE cells that are substantially pure.

[0113]

[0086] The current invention provides several advantages over methods known in the art for producing RPE cells, including, for example, greatly enhanced RPE cell yields, greatly enhanced shelf-life, greatly enhanced commercial viability, greatly enhanced safety profile, greatly enhanced RPE cell purity, improved ease of manual RPE cell isolation, the ability for automated RPE cell selection, the absence of the requirement for any further purification by manual or automated selection, and the use of simple constituents, which enables commercial large-scale manufacturing. In some embodiments, the methods of the invention increase the yield of RPE, e.g., up to more than 50-90 times greater, as compared to cells produced by the conventional manufacturing method, e.g., involving manual picking, and produces RPE cells with high consistency of purity over 98% to 99%. In some embodiments, the RPE cell drug products produced by the methods of the invention have a stability of at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 48 months,127206-12220

[0114] or at least 120 months. The RPE cell drug products produced by the methods of the invention have greatly improved long-term stability, e.g., at least 50-500 times greater, as compared to RPE cell drug products produced by other methods. In some embodiments, the RPE cell preparation is suitable for transplantation into a subject once thawed and diluted with a diluent. In some embodiments, the diluent is GS2, GS2 Plus, described hereinbelow, BSS, BSS Plus®, a dextran and HSA solution, PBS, DMEM, MEM, albumin, or any other appropriate diluent. In some embodiments, the diluent is GS2 or GS2 Plus or any other appropriate diluent. In some embodiments, the yield of RPE cells produced by the methods of the invention is sufficient to treat at least 500 patients, at least 1,000 patients, at least 5,000 patients, at least 10,000 patients, or at least 20,000 patients. The yield of RPE cells produced by the methods of the invention is sufficient to treat a greater number of patients, e.g., 100-400 times greater, as compared to the yield of RPE cells produced by other methods. The methods of the invention are both simple and efficient.

[0115]

[0087] In some embodiments, the methods of the invention result in cultures of RPE cells that are substantially pure. A substantially purified population of RPE cells is one in which the RPE cells comprise at least about 65% of the cells in the population. In other embodiments, a substantially purified population of RPE cells is one in which the RPE cells comprise at least about 65%, 70%, 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.

[0116]

[0088] In order to make the invention described herein fully understood, the following detailed description and Examples are provided. Various embodiments of the invention have been described in detail, and may be further illustrated by the examples provided herein. All technical and scientific terms used herein unless otherwise defined, have the same meaning as those skilled in the art to which the invention pertains generally understood.

[0117] Definitions

[0118]

[0089] Unless otherwise specified, each of the following terms have the meaning set forth in this section.

[0119]

[0090] 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.”

[0120]

[0091] The conjunctions “or” and “and / or” are used interchangeably as non-exclusive disjunctions.127206-12220

[0121]

[0092] As used herein, the terms “retinal pigment epithelial cell” and “RPE cell” and “retinal pigment epithelium cell” are used interchangeably herein to refer to an epithelial cell constituting the retinal pigment epithelium. The term is used generically to refer to differentiated RPE cells, regardless of the level of maturity of the cells, and thus may encompass RPE cells of various levels of maturity. RPE cells can be visually recognized by their cobblestone morphology and the initial appearance of pigment. RPE cells can also be identified molecularly based on substantial lack of expression of embryonic stem cell markers such as OCT4 and NANOG, as well as based on the expression of one or more RPE markers such as RPE65, PEDF, CRALBP (also known as RLBP1), premelanosome protein (PMEL), gp-100 (also known as gplOO or PMEL17), bestrophin (BEST1), MITF and TRYP1. In one embodiment, RPE cells are identified by the presence of glycoprotein gp-100 (PMEL17). In one embodiment, the RPE cells lack substantial expression of one or more of embryonic stem cell markers including but not limited to OCT4, NANOG, REXI, alkaline phosphatase, SOX2, TDGF- 1, DPPA-2, DPPA-4, stage specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-l -60 and / or TRA-1-80. In another embodiment, the RPE cells express one or more RPE cell markers including but not limited to RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, premelanosome protein (PMEL), gp-100, and / or tyrosinase. In one embodiment, RPE cells express glycoprotein gp-100 (PMEL17). In another embodiment, the RPE cells express ZO1. In an embodiment, the RPE cells express MITF and at least one marker selected from Bestrophin and PAX6. In another embodiment, the RPE cells express CRALBP and premelanosome protein (PMEL). In another embodiment the RPE cells express gp-100 and MITF. In another embodiment the RPE cells express gplOO and CRALBP. In another embodiment, the RPE cells express gp-100, MITF and CRALBP. Note that when other RPE-like cells are referred to, they are generally referred to as adult RPEs, fetal RPEs, primary cultures of adult or fetal RPEs, and immortalized RPE cell lines such as ARPE19 cells. Thus, unless otherwise specified, RPE cells, as used herein, refers to RPE cells obtained from pluripotent stem cells (PSC-RPE) and may refer to RPE cells obtained from human pluripotent stem cells (hRPE). Marker expression can be determined by methods known in the art including Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and flow cytometry.

[0122]

[0093] Pigmentation of the RPE cells may vary with cell density in the culture and the maturity of the RPE cells. However, when cells are referred to as pigmented, the term is understood to refer to any and all levels of pigmentation. Thus, the present invention provides127206-12220

[0123] RPE cells with varying degrees of pigmentation. In certain embodiments, the pigmentation of a RPE is the same as the average pigmentation as other RPE-like cells, such as adult RPEs, fetal RPEs, primary cultures of adult or fetal RPEs, or immortalized RPE cell lines such as ARPE19. In certain embodiments, the degree of pigmentation of a RPE is higher than the average pigmentation of other RPE-like cells, such as adult RPEs, fetal RPEs, primary cultures of adult or fetal RPEs, or immortalized RPE cell lines such as ARPE19. In certain other embodiments, the degree of pigmentation of a RPE is lower than of the average pigmentation of other RPE-like cells, such as adult RPEs, fetal RPEs, primary cultures of adult or fetal RPEs, or immortalized RPE cell lines such as ARPE19. The level of pigmentation may be measured as an average melanin content per cell for a population, e.g., expressed as picograms per cell (pg / cell), as it will be appreciated that in general there may be some variation in the level of melanin in cells in a population. In certain embodiments, the average melanin content is between 1-5 pg / cell, for example, between 1-4 pg / cell, between 1- 3 pg / cell, between 1-2 pg / cell, between 3-5 pg / cell, or between 4-5 pg / cell, such as 4.2-4.8 pg / cell. In a further example, the average melanin content may be 1-5 pg / cell, 2-5 pg / cell, 3-5 pg / cell, 4-5 pg / cell, or 4.5-5 pg / cell. The average melanin content of said RPE cells may be less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, less than 5 pg / cell, less than 4 pg / cell, less than 3 pg / cell, less than 2 pg / cell and at least 0.1 pg / cell and optionally at least 0.5 pg / cell or 1 pg / cell; between 0.1-8 pg / cell, between 0.1-7 pg / cell, between 0.1-6 pg / cell, between 0.1-5 pg / cell, between 0.1-4 pg / cell. between 0.1-3 pg / cell, between 0.1-2 pg / cell, between 0.1-1 pg / cell, between 1-7 pg / cell, between 0.5-6 pg-cell, or between 1-5 pg / cell.

[0124]

[0094] Functional evaluation of RPE cells can be confirmed using, for example, phagocytic capacity, polarized secretion of cytokines (VEGF or PEDF, etc.), phagocytosis of shed rod and cone outer segments (or phagocytosis of another substrate, such as polystyrene beads), absorption of stray light, vitamin A metabolism, regeneration of retinoids, trans-epithelial resistance, cell polarity, polarized secretion of PEDF and / or VEGF, and tissue repair.

[0125] Evaluation may also be performed by testing in vivo function after RPE cell implantation into a suitable host animal (such as a human or non-human animal suffering from a naturally occurring or induced condition of retinal degeneration), e.g., using behavioral tests, fluorescent angiography, histology, tight junctions conductivity, or evaluation using electron microscopy. These functional evaluation and confirmation operations can be performed by those of ordinary skill in the art. RPE cells, as used herein, include human RPE (hRPE) cells.127206-12220

[0126]

[0095] As used herein, the terms “progenitor cell of an RPE cell” and “RPE progenitor cell” are used interchangeably herein to refer to a cell directed to differentiate into a retinal cell. In an embodiment, the term RPE progenitor cell may be used to refer to any cell directed to differentiate into a retinal cell up to the time of harvesting the RPE cell (e.g., for plating at PO as described herein). It will be appreciated that in the latter stages of differentiation, the differentiation culture may comprise a mixture of RPE progenitor cells and RPE cells. In an embodiment, a progenitor cell expresses MITF (pigment epithelial cell, progenitor cell), PAX6 (progenitor cell), Rx (retinal progenitor cell), Crx (photoreceptor precursor cell), and / or ChxlO (bipolar cell) etc., and the like. In an embodiment, the RPE progenitor cell expresses PAX6 and MITF.

[0127]

[0096] The terms "mature RPE cell'' and "mature differentiated RPE cell'" are used interchangeably throughout to refer to changes that occur following initial differentiation of RPE cells. Specifically, although RPE cells may be recognized, in part, based on initial appearance of pigment, after differentiation mature RPE cells may be recognized based on enhanced pigmentation. Pigmentation post-differentiation may not be indicative of a change in the RPE state of the cells (e.g., the cells are still differentiated RPE cells). The changes in pigment post-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 RPEs stop proliferating, for example, due to high cell density within the culture dish. Mature RPE cells may be subcultured at a lower density such that it allows proliferation of the mature RPE cells. Proliferation of the mature RPEs in culture may be accompanied by dedifferentiation - loss of pigment and epithelial morphology, both of which are restored after the cells form a monolayer and become quiescent. In this context, mature RPE cells may be cultured to produce RPE cells. Such RPE cells are still differentiated RPE cells that express markers of RPE. Thus, in contrast to the initial appearance of pigmentation that occurs when RPE cells begin to appear, pigmentation changes post-differentiation are phenomenological and do not reflect dedifferentiation of the cells away from an RPE fate. Changes in pigmentation post-differentiation may also correlate with changes in one or more of PAX2, PAX6, tyrosinase, neural markers such as tubulin beta III, bestrophin, RPE65, and CRALBP expression. In an embodiment, changes in pigmentation post-differentiation shows a reverse correlation with one or more of PAX6 and127206-12220

[0128] neural markers (such as tubulin beta III). In another embodiment, changes in pigmentation post-differentiation shows a direct correlation with RPE65 and CRALBP.

[0129]

[0097] As used herein, the terms “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 derived. Pluripotent stem cells are defined functionally as stem cells that: (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); (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, REXI, etc); and d) are capable of self-renewal. The term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper). For example, embryonic stem cells and induced pluripotent stem cells are types of pluripotent stem cells that are able to form cells from each of the three germs layers: the ectoderm, the mesoderm, and the endoderm. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell). Exemplary pluripotent stem cells can be generated using, for example, methods known in the art.

[0130] Exemplary pluripotent stem cells include, but are not limited to, embryonic stem cells derived from the ICM of blastocyst stage embryos, embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo), induced pluripotent stem cells produced by reprogramming of somatic cells into a pluripotent state, and pluripotent cells produced 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 produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis.

[0131]

[0098] In an embodiment, pluripotent stem cells may be genetically engineered or otherwise modified, for example, to increase longevity, potency, homing, to prevent or reduce immune responses, or to deliver a desired factor in cells that are obtained from such pluripotent cells (for example, RPEs). For example, the pluripotent stem cell and therefore, the resulting differentiated cell, can be engineered or otherwise modified to lack or be devoid of or substantially lack or have reduced endogenous expression of beta 2 microglobulin, HLA-A,127206-12220

[0132] HLA-B, HLA-C, TAPI, TAP2, Tapasin, CTIIA, RFX5, TRAC, or TRAB genes. The pluripotent stem cell and the resulting differentiated cell may be engineered or otherwise modified to increase expression of a gene. There are a variety of techniques for engineering cells to modulate the 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, as well as the use of transcriptional and translational inhibitors such as antisense and RNA interference (which can be achieved using stably integrated vectors and episomal vectors).

[0133]

[0099] The term "embryo" or "embryonic" is meant a developing cell mass that has not been implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained in an embryo. This also includes blastomeres, obtained as early as the two-cell stage, or aggregated blastomeres after extraction.

[0134]

[0100] The term “embryo-derived cells” (EDC), as used herein, refers broadly to morula-derived cells, blastocyst-derived cells including those of the inner cell mass, embryonic shield, or epiblast, or other pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm and their derivatives. “EDC” also includes blastomeres and cell masses from aggregated single blastomeres or embryos from varying stages of development, but excludes human embryonic stem cells that have been passaged as cell lines.

[0135]

[0101] The term "embryonic stem cells", “ES cells,” or “ESCs” as used herein, refers broadly to cells isolated from the inner cell mass of blastocysts or morulae that have been serially passaged as cell lines. The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo (see, e.g., Chung et al., Cell Stem Cell. 2008 Feb 7;2(2): 1 13-7; U.S. Pub No. 20060206953; U.S. Pub No. 2008 / 0057041, each of which is hereby incorporated by reference in its entirety). The ES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by any means to generate ES cells with homozygosity in the HLA region. ES cells may also refer to cells derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce a cell. In an embodiment, the embryonic stem cell may be a human embryonic stem cell (or “hES cells”). In an embodiment, human embryonic stem cells are not derived from embryos over 14 days from fertilization. In another embodiment, human embryonic stem cells are not derived from127206-12220

[0136] embryos that have been developed in vivo. In another embodiment, human embryonic stem cells are derived from preimplantation embryos produced by in vitro fertilization.

[0137]

[0102] “Induced pluripotent stem cells” or “iPS cells,” as used herein, generally refer to pluripotent stem cells obtained by reprogramming a somatic cell. An iPS cell may be generated by expressing or inducing expression of a combination of factors ("reprogramming factors"), for example, OCT4 (sometimes referred to as OCT 3 / 4), SOX2, MYC (e.g., c-MYC or any MYC variant), NANOG, LIN28, and KLF4, in a somatic cell. In an embodiment, the reprogramming factors comprise OCT4, SOX2, c-MYC, and KLF4. In another embodiment, reprogramming factors comprise OCT4, SOX2, NANOG, and LIN28. In certain embodiments, at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least four reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In another embodiment, at least five reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In yet another embodiment, at least six reprogramming factors are expressed in the somatic cell, for example, OCT4, SOX2, c-MYC, NANOG, LIN28, and KLF4. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram a somatic cell to a pluripotent stem cell.

[0138]

[0103] iPS cells may be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. Somatic cells may 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 an embodiment, the somatic cells are fibroblast cells, such as a dermal fibroblast, synovial fibroblast, or lung fibroblast, or a non-fibroblastic somatic cell.

[0139]

[0104] iPS cells may be obtained from a cell bank. Alternatively, iPS cells may be newly generated by methods known in the art. iPS cells may be specifically generated using material from a particular patient or matched donor with the goal of generating tissue-matched cells. In an embodiment, iPS cells may be universal donor cells that are not substantially immunogenic.

[0140]

[0105] The induced pluripotent stem cell may be produced by expressing or inducing the expression of one or more reprogramming factors in a somatic cell. Reprogramming factors may be expressed in the somatic cell by infection using a viral vector, such as a retroviral127206-12220

[0141] vector or other gene editing technologies, such as CRISPR, Talen, zinc-finger nucleases (ZFNs). Also, reprogramming factors may be expressed in the somatic cell using a non-integrative vector, such as an episomal plasmid, or RNA, such as synthetic mRNA or via an RNA virus such as Sendai virus. When reprogramming factors are expressed using non-integrative vectors, the factors may be expressed in the cells using electroporation, transfection, or transformation of the somatic cells with the vectors. For example, in mouse cells, expression of four factors (OCT3 / 4, SOX2, c-MYC, and KLF4) using integrative viral vectors is sufficient to reprogram a somatic cell. In human cells, expression of four factors (OCT3 / 4, SOX2, NANOG, and LIN28) using integrative viral vectors is sufficient to reprogram a somatic cell.

[0142]

[0106] Expression of the reprogramming factors may be induced by contacting the somatic cells with at least one agent, such as a small organic molecule agents, that induce expression of reprogramming factors.

[0143]

[0107] The somatic cell may also be reprogrammed using a combinatorial approach wherein the reprogramming factor is expressed (e.g., using a viral vector, plasmid, and the like) and the expression of the reprogramming factor is induced (e.g., using a small organic molecule).

[0144]

[0108] Once the reprogramming factors are expressed or induced in the cells, the cells may be cultured. Over time, cells with ES characteristics appear in the culture dish. The cells may be chosen and subcultured based on, for example, ES cell morphology, or based on expression of a selectable or detectable marker. The cells may be cultured to produce a culture of cells that resemble ES cells.

[0145]

[0109] To confirm the pluripotency of the iPS cells, the cells may be tested in one or more assays of pluripotency. For examples, the cells may be tested for expression of ES cell markers; the cells may be evaluated for ability to produce teratomas when transplanted into SCID mice; the cells may be evaluated for ability to differentiate to produce cell types of all three germ layers.

[0146]

[0110] iPS cells may 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, newborn, and adult tissue. Accordingly, one may readily generate iPS cells using a donor cell from any species. Thus, one may generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions,127206-12220

[0147] tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0148]

[0111] As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, for example, an RPE cell. A differentiated cell is one that has taken on a more specialized position within the lineage of a cell. For example, an hES cell can be differentiated into various more differentiated cell types, including an RPE cell.

[0149]

[0112] As used herein, the term "cultured" or "culturing" refers to the placing of cells in a medium containing, among other things nutrients needed to sustain the life of the cultured cells, any specified added substances. Cells are cultured "in the presence of" a specified substance when the medium in which such cells are maintained contains such specified substance. Culturing can take place in any vessel or apparatus in which the cells can be maintained exposed to the medium, including without limitation petri dishes, culture dishes, blood collection bags, roller bottles, flasks, test tubes, microtiter wells, hollow fiber cartridges or any other apparatus known in the art.

[0150]

[0113] As used herein, the term "subculturing” or “passaging,” refers to transferring some or all cells from a previous culture to fresh growth medium and / or plating onto a new culture dish and further culturing the cells. Subculturing may be done, e.g., to prolong the life, enrich for a desired cell population, and / or expand the number of cells in the culture. For example, the term includes transferring, culturing, or plating some or all cells to a new culture vessel at a lower cell density to allow proliferation of the cells.

[0151]

[0114] As used herein, the term “selectively picking” or “selective picking” refers to mechanically picking or separating a subset of cells from a larger population based on visual or other phenotypic characteristic. Selective picking may be performed manually or by an automated system, and may be performed with the aid of a microscope, computer imaging system, or other means for identifying the cells to be picked.

[0152]

[0115] As used herein, the term “dissociation reagent” refers to an enzymatic or non-enzymatic reagent that promotes cell dissociation or detachment into cell aggregates or into single cells. Examples of dissociation reagents include, but are not limited to, collagenase (such as collagenase I or collagenase IV), accutase, chelator e.g., EDTA-based dissociation solution), trypsin, dispase, or any combinations thereof.

[0153]

[0116] As used herein, the term “extracellular matrix” refers to any substance to which cells can adhere in culture and typically contains extracellular components to which the cells127206-12220

[0154] can attach and thus it provides a suitable culture substrate. Suitable for use with the present invention are extracellular matrix components derived from basement membrane

[0155] or extracellular matrix components that form part of adhesion molecule receptor-ligand couplings. Examples of an extracellular matrix includes, but is not limited to, laminin or a fragment thereof, e.g., laminin 521, laminin 511, or iMatrix511, fibronectin, vitronectin, Matrigel, CellStart, collagen, gelatin, proteoglycan, entactin, heparin sulfate, and the like, alone or in various combinations.

[0156]

[0117] As used herein, the term “laminin” refers to a heterotrimer molecule consisting of a, P, y chains, or fragments thereof, which is an extracellular matrix protein containing isoforms having different subunit chain compositions. Specifically, laminin has about 15 kinds of isoforms including heterotrimers of combinations of 5 kinds of a chain, 4 kinds of P chain and 3 kinds of y chain. The number of each of a chain (al-a5), P chain (P1-P4) and y chain (yl-y3) is combined to determine the name of a laminin. For example, a laminin composed of a combination of al chain, pi chain, yl chain is named laminin-111, a laminin composed of a combination of a5 chain, pi chain, yl chain is named laminin-511, and a laminin composed of a combination of a5 chain, P2 chain, yl chain is named laminin-521. A laminin derived from a mammal can be used in the methods of the invention. Examples of mammals include mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, swine, bovine, horse, goat, monkey and human. Human laminin is preferably used when RPE cells are produced. In an embodiment, the laminin is a recombinant laminin. Currently, human laminin is known to include 15 kinds of isoforms.

[0157]

[0118] Any laminin fragment may be used in the present invention as long as it retains the function of each corresponding laminin. That is, a “laminin fragment” used in the present invention is not limited as to the length of each chain as long as it is a molecule having laminin a chain, p chain and y chain constituting a heterotrimer, retaining binding activity to integrin, and maintaining cell adhesion activity. A laminin fragment shows integrin binding specificity that varies for the original laminin isoform, and can exert an adhesion activity to a cell that expresses the corresponding integrin. In an embodiment, the laminin is a recombinant laminin-511 E8 fragment e.g., iMatrix-511 (Takara Bio)).

[0158]

[0119] As used herein, “administration”, “administering” and variants thereof refers to introducing a composition or agent into a subject and includes concurrent and sequential introduction of a composition or agent. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration”127206-12220

[0159] also encompasses in vitro and ex vivo treatments. Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the composition or the 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 a vein of the subject.

[0160]

[0120] As used herein, the term “retinal disease” refers to a disease, disorder or condition that affects the retina of the eye. A retinal disease may affect the structure and / or function of the retina. Retinal diseases include, but are not limited to, rod or cone dystrophy, retinal degeneration, retinal detachment, retinal atrophy, retinal damage, retinal dysplasia, retinal pigment epithelium tears, retinitis pigmentosa, diabetic retinopathy, macular degeneration, for example wet or dry age-related macular degeneration (AMD), geographic atrophy such as geographic atrophy secondary to AMD, macular dystrophies with a Stargardt-type clinical presentation (e.g., but not limited to, Stargardt's Disease (STGD) with or without fundus flavimaculatus, and STGD-like macular dystrophies) or any other type of macular dystrophy caused by a gene defect expressed directly and / or primarily in the retinal pigment epithelium (e.g., but not limited to, CRB1- and EFEMP1- related macular dystrophies), Angioid streaks, myopic macular degeneration, Leber congenital amaurosis, Best vitelliform macular dystrophy, and BEST1 -related retinal dystrophies including autosomal recessive bestrophinopathies (ARBs), and Autosomal dominant vitreoretinochoroidopathy (ADViRC), Usher syndrome IB, or diseases associated with a number of vision altering ailments that result in photoreceptor damage and blindness, choroideremia and glaucoma

[0161]

[0121] As used herein, the terms “subject”, “individual”, “host”, and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. 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 a human.

[0162]

[0122] As used herein, the terms “therapeutic amount”, "therapeutically effective amount ", an "amount effective ", or “pharmaceutically effective amount” of an active agent (e.g., an RPE cell) are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, anticipated cell engraftment, long term survival, and / or the particular active agent employed. Thus the dosage regimen may vary127206-12220

[0163] widely, but can be determined routinely by a physician using standard methods.

[0164] Additionally, the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.

[0165]

[0123] As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

[0166]

[0124] For the therapeutic agents described herein (e.g., RPE cells), a therapeutically effective amount may be initially determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.

[0167]

[0125] Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the agent's plasma concentration can be measured and related to therapeutic window, additional guidance for dosage modification can be obtained.

[0168]

[0126] As used herein, the terms “treat”, “treating”, and / or “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing127206-12220

[0169] the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0170]

[0127] Beneficial or desired clinical results, such as pharmacologic and / or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (z.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0171] I. METHODS OF THE INVENTION

[0172]

[0128] The present invention is based on the discovery of stages during differentiation of pluripotent stem cells to RPE cells when RPE cells can be isolated at an intermediate stage to generate a cell stock, cryopreserved, and / or further expanded to mature RPE cells without manual picking of the cells. The methods of the invention are both simple and efficient, and result in a high yield of RPE cells that are substantially pure.

[0173]

[0129] Any method for differentiating pluripotent cells into RPE cells may be used. For example, RPE cells may be obtained by differentiating pluripotent stem cells through a monolayer method as described herein and also described in WO 2021 / 086911 and WO 2005 / 070011 and, each of which is incorporated herein by reference in its entirety. Other methods include obtaining embryoid bodies from pluripotent stem cells and differentiating the embryoid bodies into RPE cells, also described in WO 2005 / 070011 as well as in WO 2014 / 121077, each of which is incorporated by reference in its entirety. In another example, pluripotent stem cells may be differentiated towards the RPE cell lineage using a first differentiating agent and then further differentiated towards RPE cells using a member of the transforming factor-P (TGFP) superfamily, as well as homologous ligands including activin (e.g., activin A, activin B, and activin AB), nodal, anti-mullerian hormone (AMH), bone127206-12220

[0174] morphogenetic proteins (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth and differentiation factors (GDF)), as described in, for example, WO 2019 / 130061, which is incorporated herein by reference in its entirety.

[0175]

[0130] In an embodiment, RPE cells may be obtained by (a) culturing pluripotent stem cells in a medium comprising a first differentiating agent e.g., nicotinamide) and (b) culturing the cells obtained in step (a) in a medium comprising a member of the TGFp superfamily (e.g., activin A) and the first differentiating agent (e.g., nicotinamide), as described in WO 2019 / 130061. In yet another example, a single cell suspension of pluripotent stem cells may be used to differentiate into RPEs as described in WO 2017 / 044488, which is incorporated herein by reference in its entirety. Accordingly, the RPEs may be obtained from pluripotent stem cells in which the pluripotent stem cells are differentiated in one or more steps in one or more differentiation media that may comprise differentiation factors, such as one or more of a WNT pathway inhibitor (e.g., CKI-7, DKK1), a TGF pathway inhibitor (e.g., LDN193189), a BMP pathway inhibitor (e.g., SB431542), a MEK inhibitor (e.g., PD0325901), a member of the transforming factor-P (TGFP) superfamily, and homologous ligands such as activin.

[0176] Additionally, the RPE cells may be obtained from non-adherent or adherent cultures and from feeder or feeder-free cultures. During the differentiation process when there is a sufficient number of clusters of RPE progenitor cells (e.g., identified as PAX6 / MITF positive cells) that stay together, the clusters of RPE progenitor cells may be treated with a dissociation reagent, followed by size fractionation of the clusters and subsequent subculture of the RPE progenitor cells as single cells or cell clusters to produce RPE cells. The methods of the invention are both simple and efficient, and result in cultures of RPE cells that are substantially pure.

[0177]

[0131] In one aspect, the present invention provides a method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising: (i) culturing pluripotent stem cells in a first differentiation medium to generate RPE progenitor cells, (ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells, (iii) expanding the RPE cells produced in step (ii), (iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock, (v) expanding the RPE cells of the cell stock, and (vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

[0178]

[0132] In another aspect, the present invention provides a method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising: (i) culturing pluripotent127206-12220

[0179] stem cells in a first differentiation medium to generate RPE progenitor cells, (ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells, (iii) expanding the RPE cells produced in step (ii) by passaging one time, (iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock, (v) expanding the RPE cells of the cell stock by passaging at least one time, and (vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

[0180]

[0133] In an 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 medium that supports pluripotency. In a further embodiment, prior to step (i), the pluripotent stem cells are cultured feeder-free in a medium that supports pluripotency. In an embodiment, the medium that supports pluripotency is supplemented with bFGF.

[0181]

[0134] The methods may further comprise harvesting 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.

[0182]

[0135] In an embodiment, pluripotent stem cells are human pluripotent stem cells and the RPE cells are human RPE cells. Any of these steps may be performed in non-adherent or adherent cultures, and under feeder or feeder-free conditions.

[0183]

[0136] In an embodiment, the RPE progenitor cell clusters and / or the RPE cell clusters have a size of between about 20 to about 200 pm, about 30 to about 200 pm , about 40 to about 200 pm, about 20 to about 100 pm, about 30 to about 100 pm, about 40 to about 100 pm, about 40pm to about 70pm, about 70pm to about 100 pm, about 70pm to about 200 pm, or about 100 pm to about 200 pm.

[0184]

[0137] 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 following harvesting. In some embodiments, the methods of the invention result in cultures of RPE cells that are substantially pure.

[0075]

[0185]

[0138] In any of the embodiments of the present invention, the RPE cells express one or more of markers selected from the group RPE65, CRALBP, PEDF, Bestrophin (BEST1), MITF, OTX2, PAX2, PAX6, premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1. In an 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 an embodiment, the RPE cells express MITF and at least one marker selected from127206-12220

[0186] Bestrophin and PAX6. In an embodiment the RPE cells express PMEL or gp-100 and CRALBP. In an embodiment the RPE cells express gp-100.

[0187] Engineered Non- Immunogenic Cells

[0188]

[0139] In some embodiments, a cell population provided herein is a population of engineered cells wherein the cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The population of cells also comprise a genetically engineered disruption of one or more copies (e.g., all copies) of endogenous P-2 microglobulin (B2M) and / or a genetically engineered disruption of one or more copies (e.g., all copies) of a human leukocyte antigen (HLA) class Il-related gene selected from the group consisting of regulatory factor X associated ankyrin containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X associated protein (RFXAP), and class II transactivator (CIITA) (UC-RPE). For example, the cells comprise a genetically engineered disruption of endogenous B2M and RFXANK, and express HLA-E. In another example, the cells comprise a genetically engineered disruption of all copies of endogenous B2M and all copies of RFXANK, and express HLA-E. In various aspects, the cells further comprise a nucleic acid molecule encoding a suicide gene product (e.g., a herpes simplex virus thymidine kinase (TK) suicide gene). The disruptions allow the cells to, e.g., reduce or evade immune detection, and allow production of a cell population which does not require HLA matching for therapeutic applications and provides for a homogenous population of cells with reduced occurrence of undesired cells, improved safety profiles, strong persistence in vivo, enhanced ability to engraft in a subject, and / or enhanced ability to reduce tumor burden (see for example WO2012145384 and WO2013158292, which are herein incorporated by reference in their entireties). In some embodiments, the cells, for example RPE cells, comprise a safety switch. Safety switches comprise genetic elements expressed within cells, which cause cell death only in combination with a drug / inducer (for example a suicide gene). Examples of safety switches include, but are not limited to, thymidine kinase (e.g., Herpes simplex virus thymidine kinase), inducible caspases (e.g., caspase 9) and human FK506 binding protein.

[0189]

[0140] The cells, for example RPE cells (or a PSC from which the cell, for example RPE cell, is derived or an intermediate thereof) are genetically engineered to disrupt one or more copies (e.g., all copies) of the cells’ endogenous B2M gene. B2M is a critical component of the HLA class I (HLA-I) complex. HLA is a cell surface complex that mediates leukocyte127206-12220

[0190] interactions or interactions of leukocytes with other cells. HLA-I molecules are cell surface complexes which present antigens to CD8+ cytotoxic T cells, thereby mediating cellular immunity. HLA-I molecules comprise an HLA-I heavy chain and B2M. Genetic disruption of one or more (e.g., all) copies of the B2M gene reduces or abrogates production of the B2M protein (SEQ ID NO: 2), thereby reducing or abrogating display of HLA-I complexes on the cell. A representative nucleic acid sequence encoding B2M is provided in SEQ ID NO: 1 (Table 1).

[0191]

[0141] Also contemplated are cells, for example an RPE cell (or a PSC from which the cell is derived or an intermediate thereof) genetically engineered to disrupt one or more copies (e.g., all copies) of a subset of HLA-I. Six HLA class I alpha (a) chains have been identified, including three classical (HLA-A, HLA-B and HLA-C) and three non-classical (HLA-E, HLA-F, and HLA-G), which are responsible for specificity of peptide binding on the HLA-I binding cleft. In this regard, the disclosure contemplates cells genetically engineered to disrupt one or more copies (e.g., all copies) of an HLA-I gene selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, as well as combinations thereof. For example, the cells (or a PSC from which the cell is derived or an intermediate thereof) may be genetically engineered to disrupt one or more copies (e.g., all copies) of HLA-A, HLA-B, and / or HLA-C. HLA molecules are further described in, e.g., Choo, Yonsei Med J., 48(1): 11-23 (2007); www.ebi.ac.uk / ipd / imgt / hla / ; and GenBank Accession Nos. NG_029217 (HLA-A), XP_041536701 (HLA-A), 9 NG_023187 (HLA-B), NP_005505 (HLA-B), NG_029422 (HLA-C), NP_001229971 (HLA-C), NM_005516 (HLA-E), NP_005507 (HLA-E), NG_012009 (HLA-F), NP_001091949 (HLA-F), NG_029039 (HLA-G), and NP_001371219 (HLA-G).

[0192]

[0142] The cells, for example RPE cells, of the disclosure (or a PSC from which the cell is derived or an intermediate thereof) are genetically engineered to disrupt one or more (e.g., all) copies of an HLA class II (HLA-II)-related gene selected from RFXANK, RFX5, RFXAP, and CIITA. HLA-II molecules are transmembrane proteins found on antigen-presenting cells (APCs) and, at times, solid organs. HLA-II molecules comprise two homologous subunits, the alpha (a) subunit and the beta (0) subunit. HLA-II related genes encode HLA-II regulatory proteins that regulate the expression of HLA class II molecules. Genetic disruption of one or more copies (e.g., all copies) of one or more of the HLA-II related genes RFXANK, RFX5, RFXAP, and / or CIITA reduces or abrogates production of the encoded proteins, thereby reducing or abrogating display of HLA-II complexes on the127206-12220

[0193] cell. The cell (or a PSC from which the cell is derived or an intermediate thereof) may comprise disruptions of one or more copies (e.g., all copies) of the genes encoding one of RFXANK, RFX5, RFXAP, and / or CIITA (e.g., the cell is engineered to disrupt one or more copies (e.g., all copies) of the RFXANK gene); one or more copies (e.g., all copies) of the genes encoding any combination of two of RFXANK, RFX5, RFXAP, and / or CIITA; one or more copies (e.g., all copies) of the genes encoding any combination of three of RFXANK, RFX5, RFXAP, and / or CIITA; or one or more copies (e.g., all copies) of the genes encoding all of RFXANK, RFX5, RFXAP, and CIITA). RFXANK is encoded by, e.g., the sequence of SEQ ID NOs: 3 and 5; RFX5 is encoded by, e.g., the sequence of SEQ ID NOs: 7 and 9; RFXAP is encoded by, e.g., SEQ ID NO: 11; and CIITA is encoded by, e.g., SEQ ID NO: 13. In various aspects of the disclosure, the cells are engineered to disrupt all copies of B2M and RFXANK.

[0194]

[0143] Any suitable technique for introducing a disruption in a target gene (e.g., an HLA-II related gene, a B2M gene, or any other gene of interest) may be used. Many techniques for disrupting endogenous coding sequences are known in the art, including use of gene editing systems such as CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases, as well as targeting vectors that cleave and / or insert nucleic acid sequences into a target site in the cellular genome. An exemplary technique employs adeno- associated virus (AAV) vector-based editing, which optionally involves targeted insertion of an edited sequence (potentially a transgene) into the cellular genome so as to interrupt an endogenous coding sequence, thereby resulting in reduction or avoidance of production of the endogenous protein. In various aspects, serotype 3B AAV vectors are employed. Use of AAV vectors to engineer stem cells is further described in, e.g., Riolobos et al., Molecular Therapy, 21(6), 1232-1241 (2013), hereby incorporated by reference in its entirety and particularly with respect to the disclosure of use of AAV vectors and disruption of HLA complex production. Methods of confirming disruption of a target gene are known in the art and include, but are not limited to, PCR detection methods (e.g., real time polymerase chain reaction (qRT-PCR)), RNA sequencing, next generation sequencing methods, fluorescence-activated cell sorting (FACS), and immunostaining.

[0195]

[0144] In various aspects, the engineered cells, for example RPE cells, express at least one polypeptide selected from the group consisting of HLA-E, HLA-F, and HLA-G. In the context of the disclosure, the cell is engineered to express HLA-E, HLA-F, and / or HLA-G,127206-12220

[0196] i.e., a nucleic acid encoding HLA-E, HLA-F, and / or HLA-G is introduced into the cell (or a PSC from which the cell is derived or an intermediate thereof) and the encoded protein is produced and displayed on the cell surface. Optionally, the engineered cells express HLA-E. Optionally, the engineered cell expresses a single chain fusion HLA-E comprising at least of portion of B2M linked, either directly or via a linker sequence, to at least of portion of the HLA-I a-chain, e.g., HLA-E, HLA-F and HLA-G. In some embodiments, the engineered cells express an HLA-E- B2M fusion protein. Optionally, the engineered cell expresses a single chain fusion HLA-F or HLA-G comprising at least a portion of B2M linked, either directly or via a linker sequence, to at least of portion of the HLA-I alpha-chain, e.g., HLA-F or HLA-G. The engineered cell may overexpress a protein which is naturally produced by the cell, may express a protein encoded by an exogenous nucleic acid following knock-out or disruption of the corresponding endogenous coding sequence for the protein, or may express a protein which is not naturally encoded by the wild- type cell genome. Representative DNA and protein sequences for HLA-E are provided in SEQ ID NOs: 15 and 16. Representative DNA and protein sequences for HLA-F are provided in SEQ ID NOs: 17 and 18.

[0197] Representative DNA and protein sequences for HLA-G are provided in SEQ ID NOs: 19 and 20. It will be appreciated that variants of the sequences provided herein may be used and, as such, the disclosure contemplates use of a nucleic acid comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequences disclosed herein. The disclosure further contemplates use of a nucleic acid which encodes a peptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the amino acid sequences described herein. Preferably, a variant retains HLA-I functions such as, e.g., forming a functional peptide binding cleft to display peptides and / or engaging inhibitory receptors on NK cells. Cells which express HLA-E, HLA-F, and / or HLA-G minimize the risk of rejection when used as an adoptive cell therapy. In various aspects of the disclosure, the cells are engineered to disrupt all copies of B2M and RFXANK and engineered to express HLA-E, e.g., single chain fusion HLA-E.

[0198]

[0145] Optionally, the cells, for example RPE cells, further comprise a nucleic acid molecule encoding a suicide gene product. Combinations of nucleic acid molecules encoding different suicide gene products also are contemplated. Suicide genes are useful in selectively killing cells by inducing apoptosis or converting a nontoxic compound to a toxic compound, resulting in death of unwanted cells. Examples of suicide gene products include, but are not127206-12220

[0199] limited to, caspases (e.g., caspase 9), thymidine kinases, cytosine deaminases, cytochrome P450, telomerases, and DNases. See, e.g., U.S. Patent Publication No. 2022 / 0025001, hereby incorporated by reference. Optionally, the nucleic acid encoding the suicide gene product is operably linked to an inducible promoter. See, e.g., Straathof et al. Blood, 105(11), 4247-4254 (2005), hereby incorporated by reference. Optionally, the engineered cells comprise a herpes simplex virus thymidine kinase (TK) suicide gene. See, e.g., Bonini, et al., Science, 276, 1719-1724 (1997), hereby incorporated by reference. In various aspects, the cell comprises a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. For example, cells may comprise a nucleic acid encoding HSV TK and a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. In some embodiments, the cells, for example RPE cells, comprise a safety switch. Safety switches comprise genetic elements (for example suicide genes) expressed within cells, which cause cell death only in combination with a drug / inducer. Examples of safety switches include, but are not limited to, thymidine kinase (e.g., Herpes simplex virus thymidine kinase), inducible caspases (e.g., caspase 9) and human FK506 binding protein. In some embodiments, the engineered cells comprise 2 copies of a Herpes Simplex Virus type 1-thymidine kinase (HSV-TK) safety switch. In some embodiments, the safety switch confers to the cell sensitivity to ganciclovir (GCV). In some embodiments, administration of GCV to cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) allows for selective ablation of proliferating cells. In some embodiments the cells of the disclosure comprise induced pluripotent stem cells, RPE cells or intermediate cells comprising a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene. In some embodiments, the genetically engineered cells of the disclosure lack substantial expression of class I and class II HLA genes. In some embodiments, the cells of the disclosure comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G.

[0200]

[0146] In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are genetically engineered to be devoid of endogenous HLAs. In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are genetically127206-12220

[0201] engineered to be devoid of Class I and Class II HLAs. In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are genetically engineered to incorporate a nonclassical HLA Class I gene, HLA-E, into one allele of the B2M gene, leading to the expression of an HLA-E-B2M fusion protein. In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are genetically engineered to incorporate one or more safety switch genes. In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are genetically engineered to incorporate two HSV-TK safety switch genes. In some embodiments, the cells of the disclosure, for example RPE cells (or a PSC from which the cell is derived or an intermediate thereof) are sensitive to GCV. In some embodiments, administration of GCV to RPE cells of the disclosure results in the selective ablation of proliferating cells.

[0202]

[0147] Methods of genetically engineering a host cell to stably produce one or more gene products of interest are known in the art. Exemplary methods involve the use of viral vectors. Viral vectors may include any suitable viral vectors including, for example, retrovirus, adenovirus, parvovirus (for example, adeno-associated viruses), coronavirus, orthomyxovirus (for example, influenza virus), rhabdovirus (for example, rabies and vesicular stomatitis virus), paramyxovirus (for example, measles and Sendai), picomavirus, alphavirus, herpesvirus (for example, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (for example, vaccinia, fowlpox, and canarypox). Exogenous nucleic acids also may be introduced into a host cell via gene editing techniques using, e.g., the systems described herein. Preferably, the nucleic acid encoding HLA-E, HLA-F, or HLA-G are integrated into cellular genome, such as into the B2M or the HLA-II loci. Thus, in various aspects, the B2M loci are disrupted by inserting in the B2M loci a nucleic acid encoding a different gene product to replace the expression of the endogenous wild type B2M protein. Alternatively or in addition, certain HLA-II loci are optionally disrupted by inserting in the HLA-II loci a nucleic acid encoding a different gene product to replace the expression of the endogenous wild type HLA-II protein.

[0203]

[0148] In various aspects of the disclosure, cells of a cell population, for example, RPE cells, are derived from pluripotent stem cells. The term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are typically127206-12220

[0204] defined functionally as stem cells that are: (a) capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REXI, etc.). Pluripotent stem cells can be generated using, for example, methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells. "Embryonic stem cells" (ESC) include, e.g., cells derived from the inner cell mass of human blastocysts or morulae, including those that have been serially passaged as cell lines.

[0205] Embryonic stem cells, regardless of their source or the particular method used to produce them, can be identified based on, for instance, (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) ability to produce teratomas when transplanted into immunodeficient animals.

[0206]

[0149] Another exemplary pluripotent stem cell is an induced pluripotent stem cell (iPSC). iPSCs are generated by, e.g., reprogramming a somatic cell by expressing a combination of factors (“reprogramming factors”) such as, but not limited to, Oct4, Sox2, c-Myc, Nanog, Lin28, and / or Klf4. Induced pluripotent stem cells can be generated using, as a starting point, virtually any somatic cell of any developmental stage. For example, the cell can be from an embryo, fetus, neonate, juvenile, or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by a skin sample or biopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. In certain embodiments, the somatic cell is not a fibroblast.

[0207]

[0150] The pluripotent stem cells can be from any species. Embryonic stem cells have been successfully derived from, for example, mice, multiple species of non-human primates, and humans. Thus, one of skill in the art can generate embryonic stem cells from any species, including but not limited to, human, non-human primates, rodents (mice and rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, and cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. Similarly, iPSCs can be from any species. iPSCs have been successfully generated using, for instance, mouse and human cells. Accordingly, one can readily generate an iPSC using a donor cell from any species, such as any species described herein.127206-12220

[0208] Table 1

[0209] Name Genbank Sequence SEQ ID NO

[0210] B2M NA GenBank aatataagtggaggcgtcgcgctggcgggcattcctgaagctgacagcattcgggccgagatgtctcgct 1 seq Accession ccgtggccttagctgtgctcgcgctactctctctttctggcctggaggctatccagcgtactccaaagattca Number ggtttactcacgtcatccagcagagaatggaaagtcaaatttcctgaattgctatgtgtctgggtttcatccat NM_ ccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcagacttgtctttca 004048) gcaaggactggtctttctatctcttgtactacactgaattcacccccactgaaaaagatgagtatgcctgccg tgtgaaccatgtgactttgtcacagcccaagatagttaagtgggatcgagacatgtaagcagcatcatgga ggtttgaagatgccgcatttggattggatgaattccaaattctgcttgcttgctttttaatattgatatgcttatac acttacactttatgcacaaaatgtagggttataataatgttaacatggacatgatcttctttataattctactttga gtgctgtctccatgtttgatgtatctgagcaggttgctccacaggtagctctaggagggctggcaacttaga ggtggggagcagagaattctcttatccaacatcaacatcttggtcagatttgaactcttcaatctcttgcactc aaagcttgttaagatagttaagcgtgcataagttaacttccaatttacatactctgcttagaatttgggggaaa atttagaaatataattgacaggattattggaaatttgttataatgaatgaaacattttgtcatataagattcatattt acttcttatacatttgataaagtaaggcatggttgtggttaatctggtttatttttgttccacaagttaaataaatc ataaaacttgatgtgttatctctta

[0211] B2M AA MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCY 2 seq VSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPT EKDEYACRVNHVTLSQPKIVKWDRDM RFXANK GenBank ccggacgccgcacggctcctgttccggtgtcagagggcccgccctccccgctcctcagtctttgcggac 3

[0212] Accession aagaaaggggctgtgtgagacgcagggaaggaggcacacccgggggtggcgcagtgaggaggggg Numbers cgcgacggccaggaggctggtggagcgacacccaggcaggagagggggaagaactctctccctttct NM_ gaacccccttttccttgagagacgagttgggggagtcctccacgcattacccactcgggccgcaaaaact 134440.1 cccttctttagccctctgcccccgcccttgcttataagcctttgagaccgcagaagggaccttgttgtggaa cgggacggccaagaggaagccagatcgctgagggtccggtctccagtttgcctcctgctatatccattgg aagagaaaagtttgtgacttgggcccccaagttttgagagaactgggctttcggcgcggggggacagag gaggctcgtggggagctttccccatggagcttacccagcctgcagaagacctcatccagacccagcaga cccctgcctcagaacttggggaccctgaagaccccggagaggaggctgcagatggctcagacactgtg gtcctcagtctctttccctgcacccctgagcctgtgaatcctgaaccggatgccagtgtttcctctccacagg gcagctccctgaagcactccaccactctcaccaaccggcagcgagggaacgaggtgtcagctctgccg gccaccctagactgtgacaacctcgtcaacaagccagacgagcgcggcttcacccccctcatctgggcc tccgcctttggagagattgagaccgttcgcttcctgctggagtggggtgccgacccccacatcctggcaa aagagcgagagagcgccctgtcgctggccagcacaggcggctacacagacattgtggggctgctgctg gagcgtgacgtggacatcaacatctatgattggaatggagggacgccactgctgtacgctgtgcgcggg aaccacgtgaaatgcgttgaggccttgctggcccgaggcgctgacctcaccaccgaagccgactctggc tacaccccgatggaccttgccgtggccctgggataccggaaagtgcaacaggtgatcgagaaccacatc ctcaagctcttccagagcaacctggtgcccgctgaccctgagtgaaggccgcctgccggggactcagac actcagggaacaaaatggtcagccagagctggggaaacccagaactgacttcaaaggcagcttctgga caggtggtgggaggggacccttcccaagaggaaccaataaaccttctgtgcagaatga

[0213] RFXANK GenBank MELTQPAEDLIQTQQTPASELGDPEDPGEEAADGSDTVVLSLFPCT 4

[0214] Accession PEPVNPEPDASVSSPQGSSLKHSTTLTNRQRGNEVSALPATLDCDN Numbers LVNKPDERGFTPLIWASAFGEIETVRFLLEWGADPHILAKERESAL NM_ SLASTGGYTDIVGLLLERDVDINIYDWNGGTPLLYAVRGNHVKCV 134440.1 EALLARGADLTTEADSGYTPMDLAVALGYRKVQQVIENHILKLFQ SNLVPADPE RFXANK GenBank ccggacgccgcacggctcctgttccggtgtcagagggcccgccctccccgctcctcagtctttgcggac 5

[0215] Accession aagaaaggggctgtgtgagacgcagggaaggaggcacacccgggggtggcgcagtgaggaggggg Numbers cgcgacggccaggaggctggtggagcgacacccaggcaggagagggggaagaactctctccctttct NM_ gaacccccttttccttgagagacgagttgggggagtcctccacgcattacccactcgggccgcaaaaact 003721.2 cccttctttagccctctgcccccgcccttgcttataagcctttgagaccgcagaagggaccttgttgtggaa cgggacggccaagaggaagccagatcgctgagggtccggtctccagtttgcctcctgctatatccattgg aagagaaaagtttgtgacttgggcccccaagttttgagagaactgggctttcggcgcggggggacagag gaggctcgtggggagctttccccatggagcttacccagcctgcagaagacctcatccagacccagcaga cccctgcctcagaacttggggaccctgaagaccccggagaggaggctgcagatggctcagacactgtg gtcctcagtctctttccctgcacccctgagcctgtgaatcctgaaccggatgccagtgtttcctctccacagg caggcagctccctgaagcactccaccactctcaccaaccggcagcgagggaacgaggtgtcagctctg

[0216]

[0217] ccggccaccctagactccctgtccatccaccagctcgcagcacagggggagctggaccagctgaagga127206-12220

[0218] gcatttgcggaaaggtgacaacctcgtcaacaagccagacgagcgcggcttcacccccctcatctgggc ctccgcctttggagagattgagaccgttcgcttcctgctggagtggggtgccgacccccacatcctggca aaagagcgagagagcgccctgtcgctggccagcacaggcggctacacagacattgtggggctgctgct ggagcgtgacgtggacatcaacatctatgattggaatggagggacgccactgctgtacgctgtgcgcgg gaaccacgtgaaatgcgttgaggccttgctggcccgaggcgctgacctcaccaccgaagccgactctg gctacaccccgatggaccttgccgtggccctgggataccggaaagtgcaacaggtgatcgagaaccac atcctcaagctcttccagagcaacctggtgcccgctgaccctgagtgaaggccgcctgccggggactca gacactcagggaacaaaatggtcagccagagctggggaaacccagaactgacttcaaaggcagcttct ggacaggtggtgggaggggacccttcccaagaggaaccaataaaccttctgtgcagaatga RFXANK GenBank MELTQPAEDLIQTQQTPASELGDPEDPGEEAADGSDTVVLSLFPCT 6

[0219] Accession PEPVNPEPDASVSSPQAGSSLKHSTTLTNRQRGNEVSALPATLDSL Numbers SIHQLAAQGELDQLKEHLRKGDNLVNKPDERGFTPLIWASAFGEIE NM_0037 TVRFLLEWGADPHILAKERESALSLASTGGYTDIVGLLLERDVDINI 21.2 YDWNGGTPLLYAVRGNHVKCVEALLARGADLTTEADSGYTPMD LAVALGYRKVQQVIENHILKLFQSNLVPADPE RFX5 GenBank attcagaaaaaggaaaataaagcaacgcacgagccggctgggccaggaccttggggctgggctggag 7

[0220] Accession tgaaggtggctacgagttttccagatttaggagacttcagaaaggtggggcagatagaatggagatggca Number aagatctctttgggcatatatgggcctggcgaagtaatggaataatttctaattttcggagaaggcaagtgc NM_ cctcatgccgggatggcagaagatgagcctgatgctaagagccccaagactgggggaagggcccccc 000449.3 caggtggtgctgaggctggggaacctaccacccttcttcagaggctccgaggtaccatttccaaggccgt gcagaacaaagtagaggggatcctgcaagatgtacagaaattttctgacaatgacaagctgtatctctacc ttcagctcccctcaggacccaccactggagacaaaagctcagagccaagtacactgagcaatgaggagt acatgtatgcctataggtggatccgcaaccacctggaagagcacactgacacctgtctgccaaagcaaa gtgtttatgatgcctatcggaagtactgtgagagtcttgcctgttgccgcccactcagcacagccaactttg gcaagatcatcagagagatcttccctgacatcaaagctcgaaggcttggtggccggggccagtccaaat attgctacagtggcataaggaggaagaccttggtgtctatgccacccctgcctggacttgacctaaagggt tctgagagtccagaaatgggcccagaagtaaccccagcacctcgagatgaactggtggaggcagcgtg tgccctgacctgtgactgggcagagcggatcctgaaacggtccttcagttccatcgttgaggtcgcccgct tcctgctacagcagcatctcatctctgcccgatctgcacatgcccatgtgcttaaggccatggggcttgctg aagaggacgaacatgcacctcgggaacggtcatctaaaccaaagaatggtttagagaacccagagggt ggagcccacaagaagccagagagactggcccagcctcctaaggatctggaagcccgaactggggcc ggtcctctcgcacgtggagagcggaagaagagtgtagttgagagctcggccccaggagccaataacct gcaggttaatgccctagtggctcggctgcctctgctccttccccgggcccctcgctcactaattccgccaat cccagtctctccacctattctggcccccaggctttcttcaggtgccctgaaagtggctacactgcctctgtct agtagggccggggcacccccagcagctgtgcccatcattaacatgatcttaccaactgttcctgctttgcct ggacctggacctgggcctgggcgagctccacctgggggactcactcagccccggggcacagagaaca gagaggtaggcataggtggtgaccaaggaccacatgacaagggtgtcaagaggacagctgaagtacct gtgagtgaggccagtgggcaggctccaccagctaaagcagcaaagcaggatatagaggatacagcaa gtgatgccaaaaggaaacgggggcgccctcgaaaaaagtcaggtggaagtggggaaaggaattctac ccctctcaagtcagcagctgccatggaatctgcccagtcctcaaggttaccatgggagacatggggctca ggaggggaaggcaactcagctggaggggcagagaggccagggccaatgggagaggctgaaaagg gggcagtacttgcccagggtcagggagatggtactgtttccaaaggaggaaggggccccggttcccag cataccaaagaagcagaagataaaattcccttggtcccctcaaaagtgagtgtcatcaagggcagcagaa gccaaaaggaggcttttcctttggcaaagggagaggtagacactgcaccacagggtaataaagacttaa aggagcatgtgcttcaaagttccttatcccaggagcataaagacccaaaagcaacacccccatgatacag gtctgtggggaagagtgtttatatccctacgttaactttgcctagtagaggcccttctttgcacttgcttctcatt tggctattcttttcctaaggaagtccattctcctctgtacagacagctgagtcacccagtctacttagtacctg gttgctgcctctgaccttttcagcttgataccctgggctttagtgtaaccaataaatctgtagtgaccttacctg tattccctgtgctatcctgtgggaaggtaggaatgggctaagtatgatgaatatataggttagggatcttttg gttttaaatcacagaaaacctaattcaaactggcttaaaataaaaaggatttattggttcatgtaactagaaag tccataggtagtgctggctccaggtgaagacttgacccagtagttcagtatgtctctaaataccggactgac ttttttctcactgttgcatcttctgtaggaccatttaagtctgggccacttaatggctgccagcattcctaagatt acacttttccccatttatgtccaatcagaaaaagaaggcatctttgtaccagaaatctcagcaaaagccctaa tattcacactgattaggcctgggtcacatgtccaccctgaccaatcactgtggccaggaggatgatacatg ctaatttgcttattctatatcatggacaacacctttggggaaaagggtgggggtcagcctccccaaaatcac atggattccccaagtggaaactaggagcagggagttgcttgggtggccgctaacaccaggctactcttatt ttagcttgctaagttgagatcagctagacctgctttcttttctcctcagtcttgcatttccctcaatacaagctgt agcctctttcctcgtttctagtctcagaaggaaggagagggaagccattctcctctagggactcttcagtctc

[0221]

[0222] atttagatgatagtccctttttttctacctccatattagagatggagctccttccttttcctgtttcttaatttttgtctt127206-12220

[0223] ctcattcctgcttccctctcaccctattgccagttccaccaactagagtgaaagacttcctagccatttcattaa atctattctgtatccaccaggtggcagcatcttgtcatacgtgtcaggacttaggactgcggggtttaggtta gatgtcacggaaaaagctagttctgtggtcaggcggcaccaatgagaaaggaatgcagaccctccagat gtatccttgggaaaagcagtaaaccaactaatatttattgaagacctactttgtcctctacatagggtagcttc tgtcagggaatcttggttcttcccaagaaacactgattttctttcagggagacttcatgtgttcatttatttccac cacagcagattttaagaaattataatatgtaatatttgatatctataaagagtatatctaacgtgaataaattatg aagcatactaatgagtacctatgacccataacacatatacattaaaacattttaaatacca

[0224] RFX5 GenBank MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNK 8

[0225] Accession VEGILQDVQKFSDNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMY Number AYRWIRNHLEEHTDTCLPKQSVYDAYRKYCESLACCRPLSTANFG NM_ KIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSMPPLPGLDLKGS 000449.3 ESPEMGPEVTPAPRDELVEAACALTCDWAERILKRSFSSIVEVARF LLQQHLISARSAHAHVLKAMGLAEEDEHAPRERSSKPKNGLENPE GGAHKKPERLAQPPKDLEARTGAGPLARGERKKSVVESSAPGAN NLQVNALVARLPLLLPRAPRSLIPPIPVSPPILAPRLSSGALKVATLP LSSRAGAPPAAVPIINMILPTVPALPGPGPGPGRAPPGGLTQPRGTE NREVGIGGDQGPHDKGVKRTAEVPVSEASGQAPPAKAAKQDIEDT ASDAKRKRGRPRKKSGGSGERNSTPLKSAAAMESAQSSRLPWET WGSGGEGNSAGGAERPGPMGEAEKGAVLAQGQGDGTVSKGGRG PGSQHTKEAEDKIPLVPSKVSVIKGSRSQKEAFPLAKGEVDTAPQG NKDLKEHVLQSSLSQEHKDPKATPP RFX5 GenBank attcagaaaaaggaaaataaagcaacgcacgagccggctgggccaggaccttggggctgggctggag 9

[0226] Accession tgaaggtggctacgagttttccagatttaggagacttcagaaaggtggggcagatagaatggagatggca Number aagatctctttgggcatatatgggcctggcgaagtaatggaataatttctaattttcggagaagccctcatgc NM_ cgggatggcagaagatgagcctgatgctaagagccccaagactgggggaagggcccccccaggtggt 00102560 gctgaggctggggaacctaccacccttcttcagaggctccgaggtaccatttccaaggccgtgcagaac 3.1 aaagtagaggggatcctgcaagatgtacagaaattttctgacaatgacaagctgtatctctaccttcagctc ccctcaggacccaccactggagacaaaagctcagagccaagtacactgagcaatgaggagtacatgtat gcctataggtggatccgcaaccacctggaagagcacactgacacctgtctgccaaagcaaagtgtttatg atgcctatcggaagtactgtgagagtcttgcctgttgccgcccactcagcacagccaactttggcaagatc atcagagagatcttccctgacatcaaagctcgaaggcttggtggccggggccagtccaaatattgctaca gtggcataaggaggaagaccttggtgtctatgccacccctgcctggacttgacctaaagggttctgagagt ccagaaatgggcccagaagtaaccccagcacctcgagatgaactggtggaggcagcgtgtgccctgac ctgtgactgggcagagcggatcctgaaacggtccttcagttccatcgttgaggtcgcccgcttcctgctac agcagcatctcatctctgcccgatctgcacatgcccatgtgcttaaggccatggggcttgctgaagaggac gaacatgcacctcgggaacggtcatctaaaccaaagaatggtttagagaacccagagggtggagccca caagaagccagagagactggcccagcctcctaaggatctggaagcccgaactggggccggtcctctcg cacgtggagagcggaagaagagtgtagttgagagctcggccccaggagccaataacctgcaggttaat gccctagtggctcggctgcctctgctccttccccgggcccctcgctcactaattccgccaatcccagtctct ccacctattctggcccccaggctttcttcaggtgccctgaaagtggctacactgcctctgtctagtagggcc ggggcacccccagcagctgtgcccatcattaacatgatcttaccaactgttcctgctttgcctggacctgga cctgggcctgggcgagctccacctgggggactcactcagccccggggcacagagaacagagaggta ggcataggtggtgaccaaggaccacatgacaagggtgtcaagaggacagctgaagtacctgtgagtga ggccagtgggcaggctccaccagctaaagcagcaaagcaggatatagaggatacagcaagtgatgcc aaaaggaaacgggggcgccctcgaaaaaagtcaggtggaagtggggaaaggaattctacccctctcaa gtcagcagctgccatggaatctgcccagtcctcaaggttaccatgggagacatggggctcaggagggg aaggcaactcagctggaggggcagagaggccagggccaatgggagaggctgaaaagggggcagta cttgcccagggtcagggagatggtactgtttccaaaggaggaaggggccccggttcccagcataccaaa gaagcagaagataaaattcccttggtcccctcaaaagtgagtgtcatcaagggcagcagaagccaaaag gaggcttttcctttggcaaagggagaggtagacactgcaccacagggtaataaagacttaaaggagcatg tgcttcaaagttccttatcccaggagcataaagacccaaaagcaacacccccatgatacaggtctgtggg gaagagtgtttatatccctacgttaactttgcctagtagaggcccttctttgcacttgcttctcatttggctattct tttcctaaggaagtccattctcctctgtacagacagctgagtcacccagtctacttagtacctggttgctgcct ctgaccttttcagcttgataccctgggctttagtgtaaccaataaatctgtagtgaccttacctgtattccctgt gctatcctgtgggaaggtaggaatgggctaagtatgatgaatatataggttagggatcttttggttttaaatca cagaaaacctaattcaaactggcttaaaataaaaaggatttattggttcatgtaactagaaagtccataggta gtgctggctccaggtgaagacttgacccagtagttcagtatgtctctaaataccggactgacttttttctcact gttgcatcttctgtaggaccatttaagtctgggccacttaatggctgccagcattcctaagattacacttttccc

[0227]

[0228] catttatgtccaatcagaaaaagaaggcatctttgtaccagaaatctcagcaaaagccctaatattcacact127206-12220

[0229] gattaggcctgggtcacatgtccaccctgaccaatcactgtggccaggaggatgatacatgctaatttgctt attctatatcatggacaacacctttggggaaaagggtgggggtcagcctccccaaaatcacatggattccc caagtggaaactaggagcagggagttgcttgggtggccgctaacaccaggctactcttattttagcttgct aagttgagatcagctagacctgctttcttttctcctcagtcttgcatttccctcaatacaagctgtagcctctttc ctcgtttctagtctcagaaggaaggagagggaagccattctcctctagggactcttcagtctcatttagatga tagtccctttttttctacctccatattagagatggagctccttccttttcctgtttcttaatttttgtcttctcattcctg cttccctctcaccctattgccagttccaccaactagagtgaaagacttcctagccatttcattaaatctattctg tatccaccaggtggcagcatcttgtcatacgtgtcaggacttaggactgcggggtttaggttagatgtcacg gaaaaagctagttctgtggtcaggcggcaccaatgagaaaggaatgcagaccctccagatgtatccttgg gaaaagcagtaaaccaactaatatttattgaagacctactttgtcctctacatagggtagcttctgtcaggga atcttggttcttcccaagaaacactgattttctttcagggagacttcatgtgttcatttatttccaccacagcaga ttttaagaaattataatatgtaatatttgatatctataaagagtatatctaacgtgaataaattatgaagcatacta atgagtacctatgacccataacacatatacattaaaacattttaaatacca

[0230] RFX5 GenBank MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNK 10

[0231] Accession VEGILQDVQKFSDNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMY Number AYRWIRNHLEEHTDTCLPKQSVYDAYRKYCESLACCRPLSTANFG NM_0010 KIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSMPPLPGLDLKGS 25603.1 ESPEMGPEVTPAPRDELVEAACALTCDWAERILKRSFSSIVEVARF LLQQHLISARSAHAHVLKAMGLAEEDEHAPRERSSKPKNGLENPE GGAHKKPERLAQPPKDLEARTGAGPLARGERKKSVVESSAPGAN NLQVNALVARLPLLLPRAPRSLIPPIPVSPPILAPRLSSGALKVATLP LSSRAGAPPAAVPIINMILPTVPALPGPGPGPGRAPPGGLTQPRGTE NREVGIGGDQGPHDKGVKRTAEVPVSEASGQAPPAKAAKQDIEDT ASDAKRKRGRPRKKSGGSGERNSTPLKSAAAMESAQSSRLPWET WGSGGEGNSAGGAERPGPMGEAEKGAVLAQGQGDGTVSKGGRG PGSQHTKEAEDKIPLVPSKVSVIKGSRSQKEAFPLAKGEVDTAPQG NKDLKEHVLQSSLSQEHKDPKATPP RFXAP GenBank cgcgcaggctgcggtcgcgcaggcgcagtcggggcgccttcccggtataggcgccttttaccccagcg 11

[0232] Accession tgtcctgagtctttggttcgcgaagtgccgttaggccaagcaggtgctaaaagcccggggtcgtggaccc Number cggccaggtcttagcagcatggaggcgcagggtgtagcggagggcgcggggccgggcgccgccag NM_0005 cggcgtgccccaccccgcggccctagccccggctgcggctcccaccttggcgccagcctcggtggcg 38.3 gccgcggcctctcaattcaccctgctagtgatgcaaccctgtgctgggcaggacgaggctgcggccccc gggggcagcgttggggcgggcaagcccgttaggtacctgtgcgaaggggccggggatggcgaagag gaggctggggaggacgaggcggacctgttagacacttcggaccctccggggggaggcgagagcgcg gctagtttggaggatctagaggacgaggagactcactcggggggcgagggcagcagcgggggcgcc cggaggcggggcagcggtgggggcagcatgagcaagacctgcacctacgaaggctgcagcgagac cacgagccaggtggccaagcagcgcaaaccgtggatgtgcaagaaacaccgcaacaagatgtacaag gacaagtataaaaagaagaagagcgaccaggccctgaactgcggtgggactgcctcgactggcagcg cgggaaacgtcaaactcgaggaaagtgcagataacatactctccattgttaaacaaagaacaggatctttt ggggatcgtcctgcaagacctactcttttagaacaagtgttaaatcaaaaaagactgtcgttactaagaagt ccagaagtagtgcaatttttacagaaacagcaacagctattaaatcagcaagttttggagcaaagacaaca gcagtttccaggaacatcaatgtgagggaacttaccaagaacatctacatggttttttatcttattgtaatagat gagcatatttttttaccagacataaatggggtaataatctatgcctgtagaacataaacattttcctgtaaatgt atgtgtgcatttggggataagtaagtattgcactttgtgcatctaatctttcagattactgtgagtttgaagaag tcagcttatctttccaaataacatttaattataatgttttttaaaaaatatattcctcttcagtcattgttactgaagg taatgaagcagttactttctgtggaagtcataaagttaatagatattaatcttgactcatctagctcagtggttct catcaagggtcaatttgattgtcatagtgaccttgaaaaccactggcttttagtgagtggccaggaatgctaa atgttctgcagtgtcaggggtagtcccacatactaaagattgtctcacccgcagtgccaataacactcctaa gaaatgttgatggctattttgtggtgctaacatgtagttggggcacctataattgggttctcttaataacctttct ttgcagttaagactgaagctgtcaaagaggtaagcacattttatatagacgtaaggaaagtgattattgttta atatctgtgaatttaggatgtgcatctcttttcagaggtgtgttagtaaaacctgacggattaactaagcacac tgggatgtgtctcctacagttggcttctctctttgatgttacctgttagtgctgatctcttaaagcagacatttctt gtttgttgaatttgtgaacagtatagatctcagcccaccaatgccaagacaaaattatttttcttatacttatttttt attaaacaaaatgaaaaagatccttttcaaaaaggtgatcctgaaaataaaactaacactccagtattttgtc attgtttttcgcaattgagctatctgaaaactgttattcctaagtaatgttcaaaaatgataagtaatctggatac ctttttcttatactttctcctaggaaaactttaaaactttaaaaaggcaaacctaccaataggaataacaaatta aatgtcaagagagtatatccaatattaggatataaatgtatgtgtctcaagtttaactctacaaaaatttgttact tgttttttaaactctatatataaagttcgacttaatcatggctgttctaagaagtacttatggagagcaagaacg

[0233]

[0234] tttttgttcatttcttaatgtgtgtgtttttacttgcatatctgttcaaaacacttttaacaaaattaattcattaaagtc127206-12220

[0235] cagttgttgacctttgagttagctgatttctttattctgttctttagtttattcttactagatgcagaggaattcatct actgtctgttattaactgttagtttattctcatacttacgatgttgagagtttttttgaagcttaagttaccctttatg gtggaaaacattagcttatgcttctttagatggaataatgggaaaggagggaaatgggaaatggatggaa atgggaaaggagggaaaataatagcccagtgagagctgaatgaaaagggactgaatttaaatatttgtaa gaactttgtgatgatgagtaattgtcagacgtgggatagataactgagaggctcagaatctttaccaaggat attttttaggataaggtagctgcctgttcatgaatttggataagaatagtaggacaatattcaacacaatttaat ttttgtctgccacattagacatttttttaccttataaaatgatcaataaagcaataaggtttattttgggt RFXAP GenBank MEAQGVAEGAGPGAASGVPHPAALAPAAAPTLAPASVAAAASQF 12

[0236] Accession TLLVMQPCAGQDEAAAPGGSVGAGKPVRYLCEGAGDGEEEAGE Number DEADLLDTSDPPGGGESAASLEDLEDEETHSGGEGSSGGARRRGS NM_0005 GGGSMSKTCTYEGCSETTSQVAKQRKPWMCKKHRNKMYKDKY 38.3 KKKKSDQALNCGGTASTGSAGNVKLEESADNILSIVKQRTGSFGD RPARPTLLEQVLNQKRLSLLRSPEVVQFLQKQQQLLNQQVLEQRQ QQFPGTSM CIITA GenBank ggttagtgatgaggctagtgatgaggctgtgtgcttctgagctgggcatccgaaggcatccttggggaag 13

[0237] Accession ctgagggcacgaggaggggctgccagactccgggagctgctgcctggctgggattcctacacaatgcg Number ttgcctggctccacgccctgctgggtcctacctgtcagagccccaaggcagctcacagtgtgccaccatg NM_0002 gagttggggcccctagaaggtggctacctggagcttcttaacagcgatgctgaccccctgtgcctctacc 46.3 acttctatgaccagatggacctggctggagaagaagagattgagctctactcagaacccgacacagaca ccatcaactgcgaccagttcagcaggctgttgtgtgacatggaaggtgatgaagagaccagggaggctt atgccaatatcgcggaactggaccagtatgtcttccaggactcccagctggagggcctgagcaaggaca ttttcaagcacataggaccagatgaagtgatcggtgagagtatggagatgccagcagaagttgggcaga aaagtcagaaaagacccttcccagaggagcttccggcagacctgaagcactggaagccagctgagccc cccactgtggtgactggcagtctcctagtgggaccagtgagcgactgctccaccctgccctgcctgccac tgcctgcgctgttcaaccaggagccagcctccggccagatgcgcctggagaaaaccgaccagattccc atgcctttctccagttcctcgttgagctgcctgaatctccctgagggacccatccagtttgtccccaccatctc cactctgccccatgggctctggcaaatctctgaggctggaacaggggtctccagtatattcatctaccatg gtgaggtgccccaggccagccaagtaccccctcccagtggattcactgtccacggcctcccaacatctcc agaccggccaggctccaccagccccttcgctccatcagccactgacctgcccagcatgcctgaacctgc cctgacctcccgagcaaacatgacagagcacaagacgtcccccacccaatgcccggcagctggagag gtctccaacaagcttccaaaatggcctgagccggtggagcagttctaccgctcactgcaggacacgtatg gtgccgagcccgcaggcccggatggcatcctagtggaggtggatctggtgcaggccaggctggagag gagcagcagcaagagcctggagcgggaactggccaccccggactgggcagaacggcagctggccc aaggaggcctggctgaggtgctgttggctgccaaggagcaccggcggccgcgtgagacacgagtgatt gctgtgctgggcaaagctggtcagggcaagagctattgggctggggcagtgagccgggcctgggcttg tggccggcttccccagtacgactttgtcttctctgtcccctgccattgcttgaaccgtccgggggatgcctat ggcctgcaggatctgctcttctccctgggcccacagccactcgtggcggccgatgaggttttcagccaca tcttgaagagacctgaccgcgttctgctcatcctagacggcttcgaggagctggaagcgcaagatggctt cctgcacagcacgtgcggaccggcaccggcggagccctgctccctccgggggctgctggccggccttt tccagaagaagctgctccgaggttgcaccctcctcctcacagcccggccccggggccgcctggtccag agcctgagcaaggccgacgccctatttgagctgtccggcttctccatggagcaggcccaggcatacgtg atgcgctactttgagagctcagggatgacagagcaccaagacagagccctgacgctcctccgggaccg gccacttcttctcagtcacagccacagccctactttgtgccgggcagtgtgccagctctcagaggccctgc tggagcttggggaggacgccaagctgccctccacgctcacgggactctatgtcggcctgctgggccgtg cagccctcgacagcccccccggggccctggcagagctggccaagctggcctgggagctgggccgca gacatcaaagtaccctacaggaggaccagttcccatccgcagacgtgaggacctgggcgatggccaaa ggcttagtccaacacccaccgcgggccgcagagtccgagctggccttccccagcttcctcctgcaatgct tcctgggggccctgtggctggctctgagtggcgaaatcaaggacaaggagctcccgcagtacctagcat tgaccccaaggaagaagaggccctatgacaactggctggagggcgtgccacgctttctggctgggctg atcttccagcctcccgcccgctgcctgggagccctactcgggccatcggcggctgcctcggtggacagg aagcagaaggtgcttgcgaggtacctgaagcggctgcagccggggacactgcgggcgcggcagctg ctggagctgctgcactgcgcccacgaggccgaggaggctggaatttggcagcacgtggtacaggagct ccccggccgcctctcttttctgggcacccgcctcacgcctcctgatgcacatgtactgggcaaggccttgg aggcggcgggccaagacttctccctggacctccgcagcactggcatttgcccctctggattggggagcc tcgtgggactcagctgtgtcacccgtttcagggctgccttgagcgacacggtggcgctgtgggagtccct gcagcagcatggggagaccaagctacttcaggcagcagaggagaagttcaccatcgagcctttcaaag ccaagtccctgaaggatgtggaagacctgggaaagcttgtgcagactcagaggacgagaagttcctcgg aagacacagctggggagctccctgctgttcgggacctaaagaaactggagtttgcgctgggccctgtctc

[0238]

[0239] aggcccccaggctttccccaaactggtgcggatcctcacggccttttcctccctgcagcatctggacctgg127206-12220

[0240] atgcgctgagtgagaacaagatcggggacgagggtgtctcgcagctctcagccaccttcccccagctga agtccttggaaaccctcaatctgtcccagaacaacatcactgacctgggtgcctacaaactcgccgaggc cctgccttcgctcgctgcatccctgctcaggctaagcttgtacaataactgcatctgcgacgtgggagccg agagcttggctcgtgtgcttccggacatggtgtccctccgggtgatggacgtccagtacaacaagttcacg gctgccggggcccagcagctcgctgccagccttcggaggtgtcctcatgtggagacgctggcgatgtg gacgcccaccatcccattcagtgtccaggaacacctgcaacaacaggattcacggatcagcctgagatg atcccagctgtgctctggacaggcatgttctctgaggacactaaccacgctggaccttgaactgggtactt gtggacacagctcttctccaggctgtatcccatgagcctcagcatcctggcacccggcccctgctggttca gggttggcccctgcccggctgcggaatgaaccacatcttgctctgctgacagacacaggcccggctcca ggctcctttagcgcccagttgggtggatgcctggtggcagctgcggtccacccaggagccccgaggcct tctctgaaggacattgcggacagccacggccaggccagagggagtgacagaggcagccccattctgcc tgcccaggcccctgccaccctggggagaaagtacttctttttttttatttttagacagagtctcactgttgccc aggctggcgtgcagtggtgcgatctgggttcactgcaacctccgcctcttgggttcaagcgattcttctgct tcagcctcccgagtagctgggactacaggcacccaccatcatgtctggctaatttttcatttttagtagagac agggttttgccatgttggccaggctggtctcaaactcttgacctcaggtgatccacccacctcagcctccca aagtgctgggattacaagcgtgagccactgcaccgggccacagagaaagtacttctccaccctgctctcc gaccagacaccttgacagggcacaccgggcactcagaagacactgatgggcaacccccagcctgcta attccccagattgcaacaggctgggcttcagtggcagctgcttttgtctatgggactcaatgcactgacatt gttggccaaagccaaagctaggcctggccagatgcaccagcccttagcagggaaacagctaatgggac actaatggggcggtgagaggggaacagactggaagcacagcttcatttcctgtgtcttttttcactacattat aaatgtctctttaatgtcacaggcaggtccagggtttgagttcataccctgttaccattttggggtacccactg ctctggttatctaatatgtaacaagccaccccaaatcatagtggcttaaaacaacactcacattta

[0241] CIITA GenBank MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPL 14

[0242] Accession CLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEET Number REAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAE NM_0002 VGQKSQKRPFPEELPADLKHWKPAEPPTVVTGSLLVGPVSDCSTLP 46.3 CLPLPALFNQEPASGQMRLEKTDQIPMPFSSSSLSCLNLPEGPIQFV PTISTLPHGLWQISEAGTGVSSIFIYHGEVPQASQVPPPSGFTVHGLP TSPDRPGSTSPFAPSATDLPSMPEPALTSRANMTEHKTSPTQCPAA GEVSNKLPKWPEPVEQFYRSLQDTYGAEPAGPDGILVEVDLVQAR LERSSSKSLERELATPDWAERQLAQGGLAEVLLAAKEHRRPRETR VIAVLGKAGQGKSYWAGAVSRAWACGRLPQYDFVFSVPCHCLN RPGDAYGLQDLLFSLGPQPLVAADEVFSHILKRPDRVLLILDGFEE LEAQDGFLHSTCGPAPAEPCSLRGLLAGLFQKKLLRGCTLLLTARP RGRLVQSLSKADALFELSGFSMEQAQAYVMRYFESSGMTEHQDR ALTLLRDRPLLLSHSHSPTLCRAVCQLSEALLELGEDAKLPSTLTG LYVGLLGRAALDSPPGALAELAKLAWELGRRHQSTLQEDQFPSAD VRTWAMAKGLVQHPPRAAESELAFPSFLLQCFLGALWLALSGEIK DKELPQYLALTPRKKRPYDNWLEGVPRFLAGLIFQPPARCLGALL GPSAAASVDRKQKVLARYLKRLQPGTLRARQLLELLHCAHEAEE AGIWQHVVQELPGRLSFLGTRLTPPDAHVLGKALEAAGQDFSLDL RSTGICPSGLGSLVGLSCVTRFRAALSDTVALWESLQQHGETKLLQ AAEEKFTIEPFKAKSLKDVEDLGKLVQTQRTRSSSEDTAGELPAVR DLKKLEFALGPVSGPQAFPKLVRILTAFSSLQHLDLDALSENKIGD EGVSQLSATFPQLKSLETLNLSQNNITDLGAYKLAEALPSLAASLL RLSLYNNCICDVGAESLARVLPDMVSLRVMDVQYNKFTAAGAQQ LAASLRRCPHVETLAMWTPTIPFSVQEHLQQQDSRISLR HLA-E GenBank gcagactcagttctcattcccaatgggtgtcgggtttctagagaagccaatcagcgtcgccacgactcccg 15

[0243] Accession actataaagtccccatccggactcaagaagttctcaggactcagaggctgggatcatggtagatggaacc Number ctccttttactcctctcggaggccctggcccttacccagacctgggcgggctcccactccttgaagtatttcc NM_0055 acacttccgtgtcccggcccggccgcggggagccccgcttcatctctgtgggctacgtggacgacaccc 16 agttcgtgcgcttcgacaacgacgccgcgagtccgaggatggtgccgcgggcgccgtggatggagca ggaggggtcagagtattgggaccgggagacacggagcgccagggacaccgcacagattttccgagtg aatctgcggacgctgcgcggctactacaatcagagcgaggccgggtctcacaccctgcagtggatgcat ggctgcgagctggggcccgacgggcgcttcctccgcgggtatgaacagttcgcctacgacggcaagg attatctcaccctgaatgaggacctgcgctcctggaccgcggtggacacggcggctcagatctccgagc aaaagtcaaatgatgcctctgaggcggagcaccagagagcctacctggaagacacatgcgtggagtgg ctccacaaatacctggagaaggggaaggagacgctgcttcacctggagcccccaaagacacacgtgac

[0244]

[0245] tcaccaccccatctctgaccatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatc127206-12220

[0246] acactgacctggcagcaggatggggagggccatacccaggacacggagctcgtggagaccaggcct gcaggggatggaaccttccagaagtgggcagctgtggtggtgccttctggagaggagcagagatacac gtgccatgtgcagcatgaggggctacccgagcccgtcaccctgagatggaagccggcttcccagccca ccatccccatcgtgggcatcattgctggcctggttctccttggatctgtggtctctggagctgtggttgctgct gtgatatggaggaagaagagctcaggtggaaaaggagggagctactctaaggctgagtggagcgaca gtgcccaggggtctgagtctcacagcttgtaaagcctgagacagctgccttgtgtgcgactgagatgcac agctgccttgtgtgcgactgagatgcaggatttcctcacgcctcccctatgtgtcttaggggactctggcttc tctttttgcaagggcctctgaatctgtctgtgtccctgttagcacaatgtgaggaggtagagaaacagtcca cctctgtgtctaccatgacccccttcctcacactgacctgtgttccttccctgttctcttttctattaaaaataag aacctgggcagagtgcggcagctcatgcctgtaatcccagcacttagggaggccgaggagggcagatc acgaggtcaggagatcgaaaccatcctggctaacacggtgaaaccccgtctctactaaaaaatacaaaa aattagctgggcgcagaggcacgggcctgtagtcccagctactcaggaggcggaggcaggagaatgg cgtcaacccgggaggcggaggttgcagtgagccaggattgtgcgactgcactccagcctgggtgacag ggtgaaacgccatctcaaaaaataaaaattgaaaaataaaaaaagaacctggatctcaatttaatttttcata ttcttgcaatgaaatggacttgaggaagctaagatcatagctagaaatacagataattccacagcacatctc tagcaaatttagcctattcctattctctagcctattccttaccacctgtaatcttgaccatataccttggagttga atattgttttcatactgctgtggtttgaatgttccctccaacactcatgttgagacttaatccctaatgtggcaat actgaaaggtggggcctttgagatgtgattggatcgtaaggctgtgccttcattcatgggttaatggattaat gggttatcacaggaatgggactggtggctttataagaagaggaaaagagaactgagctagcatgcccag cccacagagagcctccactagagtgatgctaagtggaaatgtgaggtgcagctgccacagagggcccc caccagggaaatgtctagtgtctagtggatccaggccacaggagagagtgccttgtggagcgctgggag caggacctgaccaccaccaggaccccagaactgtggagtcagtggcagcatgcagcgcccccttggg aaagctttaggcaccagcctgcaacccattcgagcagccacgtaggctgcacccagcaaagccacagg cacggggctacctgaggccttgggggcccaatccctgctccagtgtgtccgtgaggcagcacacgaagt caaaagagattattctcttcccacagataccttttctctcccatgaccctttaacagcatctgcttcattcccctc accttcccaggctgatctgaggtaaactttgaagtaaaataaaagctgtgtttgagcatcatttgtatttcaaa aaaaaaaaaaaaaaa

[0247] HLA-E GenBank MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISV 16

[0248] Accession GYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSAR Number DTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRG NM_ YEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQ 005507 RAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATL RCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWA AVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGL VLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESH SL HLA-F GenBank tttctcactcccattgggcgtcgcgtttctagagaagccaatcagtgtcgccgcagttcccaggttctaaagt 17

[0249] Accession cccacgcaccccgcgggactcatatttttcccagacgcggaggttggggtcatggcgccccgaagcctc Number ctcctgctgctctcaggggccctggccctgaccgatacttgggcgggctcccactccttgaggtatttcag NM_0189 caccgctgtgtcgcggcccggccgcggggagccccgctacatcgccgtggagtacgtagacgacacg 50 caattcctgcggttcgacagcgacgccgcgattccgaggatggagccgcgggagccgtgggtggagc aagaggggccgcagtattgggagtggaccacagggtacgccaaggccaacgcacagactgaccgagt ggccctgaggaacctgctccgccgctacaaccagagcgaggctgggtctcacaccctccagggaatga atggctgcgacatggggcccgacggacgcctcctccgcgggtatcaccagcacgcgtacgacggcaa ggattacatctccctgaacgaggacctgcgctcctggaccgcggcggacaccgtggctcagatcaccca gcgcttctatgaggcagaggaatatgcagaggagttcaggacctacctggagggcgagtgcctggagtt gctccgcagatacttggagaatgggaaggagacgctacagcgcgcagatcctccaaaggcacacgttg cccaccaccccatctctgaccatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagat cacgctgacctggcagcgggatggggaggaacagacccaggacacagagcttgtggagaccaggcc tgcaggggatggaaccttccagaagtgggccgctgtggtggtgcctcctggagaggaacagagataca catgccatgtgcagcacgaggggctgccccagcccctcatcctgagatgggagcagtctccccagccc accatccccatcgtgggcatcgttgctggccttgttgtccttggagctgtggtcactggagctgtggtcgct gctgtgatgtggaggaagaagagctcagatagaaacagagggagctactctcaggctgcagtgtgaga cagcttccttgtgtgggactgagaagcaagatatcaatgtagcagaattgcacttgtgcctcacgaacatac ataaattttaaaaataaagaataaaaatatatctttttatagataaaaaaaaaaaaaaaaaa

[0250] HLA-F GenBank MAPRSLLLLLSGALALTDTWAGSHSLRYFSTAVSRPGRGEPRYIAV 18

[0251] Accession EYVDDTQFLRFDSDAAIPRMEPREPWVEQEGPQYWEWTTGYAKA Number NAQTDRVALRNLLRRYNQSEAGSHTLQGMNGCDMGPDGRLLRG

[0252]

[0253] YHQHAYDGKDYISLNEDLRSWTAADTVAQITQRFYEAEEYAEEFR127206-12220

[0254] NP_06182 TYLEGECLELLRRYLENGKETLQRADPPKAHVAHHPISDHEATLR

[0255] 3 CWALGFYPAEITLTWQRDGEEQTQDTELVETRPAGDGTFQKWAA VVVPPGEEQRYTCHVQHEGLPQPLILRWEQSPQPTIPIVGIVAGLV VLGAVVTGAVVAAVMWRKKSSDRNRGSYSQAAV HLA-G GenBank agtgtggtactttgtcttgaggagatgtcctggactcacacggaaacttagggctacggaatgaagttctca 19

[0256] Accession ctcccattaggtgacaggtttttagagaagccaatcagcgtcgccgcggtcctggttctaaagtcctcgctc Number acccacccggactcattctccccagacgccaaggatggtggtcatggcgccccgaaccctcttcctgctg NM_0021 ctctcgggggccctgaccctgaccgagacctgggcgggctcccactccatgaggtatttcagcgccgcc 27 gtgtcccggcccggccgcggggagccccgcttcatcgccatgggctacgtggacgacacgcagttcgt gcggttcgacagcgactcggcgtgtccgaggatggagccgcgggcgccgtgggtggagcaggaggg gccggagtattgggaagaggagacacggaacaccaaggcccacgcacagactgacagaatgaacctg cagaccctgcgcggctactacaaccagagcgaggccagttctcacaccctccagtggatgattggctgc gacctggggtccgacggacgcctcctccgcgggtatgaacagtatgcctacgatggcaaggattacctc gccctgaacgaggacctgcgctcctggaccgcagcggacactgcggctcagatctccaagcgcaagtg tgaggcggccaatgtggctgaacaaaggagagcctacctggagggcacgtgcgtggagtggctccac agatacctggagaacgggaaggagatgctgcagcgcgcggacccccccaagacacacgtgacccac caccctgtctttgactatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatcatact gacctggcagcgggatggggaggaccagacccaggacgtggagctcgtggagaccaggcctgcagg ggatggaaccttccagaagtgggcagctgtggtggtgccttctggagaggagcagagatacacgtgcca tgtgcagcatgaggggctgccggagcccctcatgctgagatggaagcagtcttccctgcccaccatccc catcatgggtatcgttgctggcctggttgtccttgcagctgtagtcactggagctgcggtcgctgctgtgct gtggagaaagaagagctcagattgaaaaggagggagctactctcaggctgcaatgtgaaacagctgcc ctgtgtgggactgagtggcaagtccctttgtgacttcaagaaccctgactcctctttgtgcagagaccagcc cacccctgtgcccaccatgaccctcttcctcatgctgaactgcattccttccccaatcacctttcctgttccag aaaaggggctgggatgtctccgtctctgtctcaaatttgtggtccactgagctataacttacttctgtattaaa attagaatctgagtataaatttactttttcaaattatttccaagagagattgatgggttaattaaaggagaagatt cctgaaatttgagagacaaaataaatggaagacatgagaacttt

[0257] HLA-G GenBank MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPR 20

[0258] Accession FIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETR Number NTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRL NP_ LRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANV 002118 AEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFD YEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTF QKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMG

[0259]

[0260] I V AGL V VL A A V VTG A A V A A VLWRKKS SD

[0261] a. Culturing Pluripotent Stem Cells

[0262]

[0151] Pluripotent stem cells, e.g., embryonic stem (ES) cells or iPS cells, may be the starting material of the disclosed method. In any of the embodiments herein, the pluripotent stem cell may be human pluripotent stem cells (hPSCs). In some embodiments, the pluripotent stem cells e.g., embryonic stem (ES) cells or iPS cells) may be genetically engineered to be hypoimmune PSCs or non-immunogenic PSCs prior to differentiation. Pluripotent stem cells (PSCs) may be cultured in any way known in the art, such as in the presence or absence of feeder cells. Additionally, PSCs produced using any method can be used as the starting material to produce RPE cells. For example, the hES cells may be derived from blastocyst stage embryos that were the product of in vitro fertilization of egg and sperm. Alternatively, the hES cells may be derived from one or more blastomeres removed from an early cleavage stage embryo, without destroying the remainder of the127206-12220

[0263] embryo. In still other embodiments, the hES cells may be produced using nuclear transfer. In some embodiments, commercially available cell lines of embryonic stem cells may be used. The human embryo used to make the embryonic stem cells is one that has not undergone in vivo development and that is within 14 days of fertilization. Further, the embryonic stem cells are not germ cells or fertilized eggs. In a further embodiment, iPSCs may be used. As a starting material, previously cryopreserved PSCs may be used. In another embodiment, PSCs that have never been cryopreserved may be used.

[0264]

[0152] In one aspect of the present invention, PSCs are plated onto 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, laminin may be selected from the group comprising laminin 521, laminin 511, or iMatrix511. In some embodiments, the feeder cells are human dermal fibroblasts (HDF). In other embodiments, the feeder cells are mouse embryo fibroblasts (MEF).

[0265]

[0153] In certain embodiments, the media used when culturing the PSCs may be selected from any media appropriate for culturing PSCs. In some embodiments, any media that is capable of supporting PSC cultures may be used. For example, one of skill in the art may select amongst commercially available or proprietary media. In further embodiments, the 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.

[0266]

[0154] The medium that supports pluripotency may be any such medium known in the art. In some embodiments, the medium that supports pluripotency is Nutristem™. In some embodiments, the medium that supports pluripotency is TeSR™. In some embodiments, the medium that supports pluripotency is StemFit™. In other embodiments, the medium that supports pluripotency is Knockout™ DMEM (Gibco), which may be supplemented with Knockout™ Serum Replacement (Gibco), FBS (e.g., gamma-irradiated FBS (g-Irr FBS)), EIF, GlutaMAX™, bFGF, or any other factors. Each of these exemplary media is known in the art and commercially available. In further embodiments, the medium that supports pluripotency may be supplemented with bFGF or any other factors. In an embodiment, bFGF may be supplemented at a low concentration (e.g., 4ng / mE). In another embodiment, bFGF may be supplemented at a higher concentration (e.g., 100 ng / mE), which may prime the PSCs for differentiation.127206-12220

[0267]

[0155] The concentration of PSCs to be used in the production method of the present invention is not particularly limited. For example, when a 10 cm dish is used, lxlO4-lxlO8cells per dish, preferably 5xl04-5xl06cells per dish, more preferably 1X105-1X107cells, per dish are used.

[0268]

[0156] In some embodiments, the PSCs are plated with a cell density of about 1,000-100,000 cells / cm2. In some embodiments, the PSCs are plated with a cell density of about 5000 -100,000 cells / cm2, about 5000 - 50,000 cells / cm2, or about 5000 - 15,000 cells / cm2. In other embodiments, the PSCs are plated at a density of about 10,000 cells / cm2.

[0269]

[0157] In some embodiments, the medium that supports pluripotency, e.g., StemFit™ or other similar medium, is replaced with a differentiation medium e.g., a medium without pluripotency- supporting factors such as bFGF) to differentiate the cells into RPE cells. In an embodiment, embryoid bodies (EBs) are formed from the PSCs and the EBs are further differentiated into RPE cells.

[0270]

[0158] In some embodiments, replacement of the media from the medium that supports pluripotency to a differentiation medium may be performed at different time points during the cell culture of PSCs and may also depend on the initial plating density of the PSCs. In some embodiments, replacement of the media can be performed after 3-14 days of culture of the PSCs in the pluripotency medium. In some embodiments, replacement of the media may be performed at day 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0271] b. Differentiation of Pluripotent Stem Cells

[0272]

[0159] Differentiation of pluripotent stem cells to RPE cells is initiated following replacement of the medium that supports pluripotency with one or more differentiation medium, e.g., EBDM. In some embodiments, the pluripotent stem cells are spontaneously differentiated into RPE cells in the absence of differentiation-inducing factors. In other embodiments, differentiation-inducing factors such as activin, a nodal signal inhibitor, a Wnt signal inhibitor, or a sonic hedgehog signal inhibitor may be used to differentiate pluripotent stem cells into RPE cells.

[0273]

[0160] In some embodiments, the differentiation medium is EB differentiation medium (EBDM). EBDM comprises Knockout™ DMEM (Gibco) with Xeno-free KnockOut™ Serum Replacement (XF-KSR) (Gibco), beta-mercaptoethanol, Minimum Essential Medium (MEM) non-essential amino acids (NEAA), and / or GlutaMAX. Any other differentiation medium known in the art may be used. In another embodiment, the differentiation medium127206-12220

[0274] may comprise one or more differentiation agents, such as nicotinamide, a member of the transforming factor-P (TGFP) superfamily (e.g., activin A, activin B, and activin AB), nodal, anti-mullerian hormone (AMH), bone morphogenetic proteins (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth and differentiation factors (GDF)), WNT pathway inhibitor (e.g., CKI-7, DKK1), a TGF pathway inhibitor (e.g., LDN193189, Noggin), a BMP pathway inhibitor (e.g., SB431542), a sonic hedgehog signal inhibitor, a bFGF inhibitor, and / or a MEK inhibitor (e.g., PD0325901). In an embodiment, the pluripotent stem cells may be differentiated towards the RPE cell lineage in a first differentiation medium comprising a first differentiation agent and then further differentiated towards RPE cells in a second differentiation medium comprising a second differentiation agent. In an embodiment, the first differentiation medium comprises nicotinamide and the second differentiation medium comprises activin (e.g., activin A). Additionally, the RPE cells may be obtained from nonadherent or adherent cultures, and under feeder or feeder-free conditions.

[0275]

[0161] In an embodiment, the differentiation media may be changed every day during differentiation. In some embodiments, the differentiation media is subsequently changed every 2-3 days during differentiation. In some embodiments, the cells are cultured in differentiation media for about 3-12 weeks, e.g., 6-10 weeks, 2-8 weeks, 7-9 weeks or 3-6 weeks. In some embodiments, the cells are cultured in differentiation media for about 8 weeks.

[0276]

[0162] In an embodiment, following replacement of the medium that supports pluripotency with a differentiation medium, molecular markers and morphological features may be detected in order to determine differentiation of pluripotent cells and identify RPE progenitor cells in culture. Whether or not a cell is an RPE cell or an RPE progenitor may be judged by changes in cell morphology (e.g., intracellular melanin pigment deposition, polygonal and flat cell morphology, formation of polygonal actin bundle, etc.) as an index by using an optical or electron microscope. Detection of molecular, morphological, and other features of RPEs are described, for example, in U.S. Pat. No. 7,794,704; U.S. Pat. No. 7,736,896; WO 2009 / 051671; WO 2012 / 012803; WO 2013 / 074681; WO 2011 / 063005; and WO 2016 / 154357, incorporated in their entireties herein by reference. Accordingly, in some optional embodiments, after the medium that supports pluripotency is replaced with a differentiation medium, the differentiation of pluripotent cells may be observed by the identification of morphological features of the RPE progenitor cells in culture.127206-12220

[0277]

[0163] In further embodiments, after the medium that supports pluripotency is replaced with a differentiation medium, the differentiation of pluripotent cells is identified by observing the changes in gene expression of the 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, the changes in gene expression of the molecular markers of differentiated cells can be confirmed by qPCR / scorecard and / or by immuno staining. In some embodiments, the changes in gene expression of the molecular markers of differentiated cells are observed after about three weeks of differentiation.

[0278]

[0164] In some embodiments, a molecular marker of retinal lineage is PAX6, and a marker of pigmented cells is MITF. Therefore, a population of cells expressing PAX6 and / or MITF indicate that the progenitors of retinal lineage / RPE are present and can be isolated from the culture.

[0279]

[0165] In other embodiments, it may not be necessary to determine differentiation of pluripotent cells and identify RPE progenitors as long as the culture conditions are known to produce RPE progenitor cells. Thus, PAX6 and MITF-positive clusters may be isolated without having to test for PAX6 / MITF.

[0280] c. Isolation and Subculture of RPE Progenitor Cells

[0281]

[0166] The cells of epithelial morphology are held together in culture by formation of tight junctions and generate clusters of similar type of cells during differentiation. Thus, in some embodiments, for isolation of the desired RPE progenitor cell population, the differentiating culture is digested or dissociated, e.g., with an enzymatic or non-enzymatic dissociation reagent, e.g., a collagenase or dispase, to form a suspension containing cellular clusters comprising RPE progenitor cells and single cells. Single cells and non-epithelial cells may be separated and discarded as described below. Additionally, large clusters of non-RPE cells as well as clusters containing a mixture of RPEs and non-RPEs may be eliminated by size fractionation as described below, allowing for increased purity.

[0282]

[0167] In some embodiments, to isolate the desired RPE progenitor cell population, the differentiating culture can be digested with a dissociation reagent and allow for 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, the dissociation with the dissociation reagent is carried out overnight. In some embodiments, the127206-12220

[0283] dissociation with the dissociation reagent is carried out for about 2-30 hours. In an embodiment, the dissociation with the dissociation reagent is carried out for about 3-10 hrs or about 3-6 hrs. In an embodiment, the dissociation with the dissociation reagent is carried out for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours.

[0284]

[0168] In some embodiments, dissociation is performed at about 2 to 12 weeks after onset of differentiation. In some embodiments, dissociation is performed at about 2 weeks, about 3 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks or about 12 weeks after onset of differentiation. In further embodiments, dissociation is performed on clusters of epithelial morphology positive for PAX6 and MITF.

[0285]

[0169] In another aspect of the methods disclosed herein, in order to isolate RPE progenitor cell clusters, the suspension containing cellular clusters and single cells are fractionated. Any method for collecting the desired RPE progenitor cell clusters may be used. In an embodiment, single cells and other undesirable cells may be passed through a cell strainer or a series of cell strainers and the desired cell cluster populations may be collected by harvesting the cells remaining on the cell strainer. In some embodiments, the cell clusters collected for further processing comprise cell clusters of between about 20 pm and about 100 pm in size , about 20 pm and about 100 pm in size , about 30 pm and about 100 pm in size, about 30 and about 200 pm in size, or about 40 pm and about 100 pm in size. In other embodiments, the collected cell clusters comprise cell clusters of between about 20 pm and about 100 pm in size, 30 pm and about 100 pm in size or 40 pm and about 200 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 20 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 30 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 40 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 50 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 60 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 70 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 80 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 90 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 100 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 110 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 120 pm in size. In some embodiments, the127206-12220

[0286] collected cell clusters comprise cell clusters of about 130 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 140 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 150 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 160 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 170 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 180 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 190 pm in size. In some embodiments, the collected cell clusters comprise cell clusters of about 200 pm in size.

[0287]

[0170] In some embodiments, single cells and cell cultures that do not meet the desired size requirement are discarded. In some embodiments, a cell strainer or a series of cell strainers may be used to collect cell clusters having the desired size requirements. For instance, the first cell strainer may have a low mesh size (e.g., 40pm) and the cell cluster population that remains on the first cell strainer are collected. The collected cell cluster population may then be placed on a second cell strainer having a higher mesh size (e.g., 200pm, 100pm), and the cell cluster population that pass through the second cell strainer may be collected to obtain the desired size requirement (e.g., 40pm - 200pm or 40pm - 100pm). Alternatively, the first cell strainer may be a first cell strainer with a higher mesh size (e.g., 200pm, 100pm) such that the cell cluster population that passes through the cell strainer is collected and larger cell clusters remaining on the first cell strainer are discarded. The pass-through cells may then be placed on a second cell strainer having a smaller mesh size (e.g., 40pm) such that the cell clusters remaining on the second cell strainer are collected and have the desired size requirement (e.g., 40pm - 200pm or 40pm - 100pm).

[0288]

[0171] The collected RPE progenitor cells may be subcultured as clusters or as single cells to obtain proliferating and mature RPE cells according to the methods described below.

[0289] d. Single RPE progenitor cell subculture method for obtaining RPE cells

[0290]

[0172] In the single RPE progenitor cell subculture method, the RPE progenitor cell clusters obtained as described above may be dissociated with a dissociation reagent to obtain single cells, and the population of RPE progenitor single cells are subcultured in a differentiation medium until RPE cells are obtained. In an embodiment, the cells are subcultured on laminin, e.g., laminin 521, laminin 511, or iMatrix511, or other extracellular matrix, such as, fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the127206-12220

[0291] cells are subcultured for about 1 to 8 weeks, e.g., about 4-5 weeks. In some embodiments, the cells are subcultured for about 2 weeks, 3, weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In other embodiments, the cells are subcultured for at least 8 weeks. In an embodiment, the cells may be subcultured under adherent conditions, such as on an adherent culture dish. In another embodiment, the cells may be subcultured under non-adherent conditions, and under feeder or feeder-free conditions.

[0292]

[0173] The RPE cells may then be harvested, for example, with a dissociation reagent in order to obtain RPE cell clusters. RPE cell clusters may be obtained by harvesting the RPE cells and removing single cells by any method known in the art. In an embodiment, the RPE cells may be harvested and passed through a strainer or a series of strainers as described above, to obtain RPE cell clusters. Any cell strainer size may be used, for example, 20 pm, 30 pm, 40 pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 110pm, 120pm, 130pm, 140pm, 150pm, 160pm, 170pm, 180pm, 190pm, or 200pm in size, or a combination thereof. The RPE cell clusters obtained may be at least 20 pm, 30 pm, 40 pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 110pm, 120pm, 130pm, 140pm, 150pm, 160pm, 170pm, 180pm, 190pm, or 200pm in size. In some embodiments, the RPE cell clusters collected for further processing comprise cell clusters of about 40 pm and about 100 pm in size. In other embodiments, the collected RPE cell clusters comprise cell clusters of about 40 pm and about 200 pm in size. In some embodiments, the collected RPE cell clusters comprise cell clusters of about 40 pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 110pm, 120pm, 130pm, 140pm, 150pm, 160pm, 170pm, 180pm, 190pm, or 200pm in size. For example, to obtain a cell cluster between about 40 to about 200 pm or between about 40 to about 100 pm, cell strainers of 40 pm, 70 pm, 100 pm, 200 pm or any other filter size that would allow obtaining the desired cell cluster size may be used.

[0293]

[0174] In an embodiment, the RPE cell clusters obtained may be dissociated into single cells with an enzymatic or non-enzymatic dissociation reagent and cultured to expand the RPE cells, further described below.

[0294]

[0175] In any of the embodiments of the present invention, the RPE cells express one or more of markers selected from the group RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1. In an 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 an127206-12220

[0295] embodiment, the RPE cells express MITF and at least one marker selected from Bestrophin and PAX6. In an embodiment, the RPE cells express gp-100. In any of the embodiments of the present invention, the RPE cells lack substantial expression of one or more stem cell markers selected from the group OCT4, NANOG, REXI, alkaline phosphatase, SOX2, TDGF- 1, DPPA-2, DPPA-4, stage specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-l -60 and TRA-1-80. In any of the embodiments of the present invention, the expression in the RPE cells of one or more stem cell markers selected from the group OCT4, NANOG, REXI, alkaline phosphatase, SOX2, TDGF- 1, DPPA-2, DPPA-4, stage specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-l -60 and TRA-1-80 is down-regulated compared to their expression in pluripotent stem cells. In any of the embodiments of the present invention, the expression in the RPE cells of one or more stem cell markers selected from the group OCT4, NANOG, REXI, alkaline phosphatase, SOX2, TDGF- 1, DPPA-2, DPPA-4, stage specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-l -60 and TRA-1-80 is down-regulated at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 100-fold compared to their expression in pluripotent stem cells. In an embodiment, the RPE cells lack substantial expression of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase. In an embodiment, the expression in the RPE cells of OCT4, SSEA4, TRA-1-81, and alkaline phosphatase is down-regulated at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 100-fold compared to their expression in pluripotent stem cells. In another embodiment, the RPE cells lack substantial expression of OCT4, NANOG, and SOX2. In an embodiment, the expression in the RPE cells of OCT4, NANOG, and SOX2 is down-regulated at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 100-fold compared to their expression in pluripotent stem cells.

[0296]

[0176] In some embodiments, a sample of the RPE cells produced may be tested for the desired molecular marker profile and then harvested. In other embodiments, it may not be necessary to test the RPE cells for molecular markers before harvesting as long as the culture conditions are known to produce RPE cells. Thus, RPE cells may be harvested without having to test for molecular markers.127206-12220

[0297] e. RPE progenitor cell cluster subculturing method for obtaining RPE cells

[0298]

[0177] In the RPE progenitor cell cluster subculturing method, the RPE progenitor cell clusters obtained after size fractionation as described above are subcultured in differentiation medium as cell clusters until RPE cells are obtained. In an embodiment, the RPE progenitor cell clusters are subcultured onto laminin, e.g., laminin 521, laminin 511, or iMatrix511, or other extracellular matrix, such as fibronectin, vitronectin, Matrigel, CellStart, collagen, or gelatin. In some embodiments, the cell clusters are subcultured for about 1 to 8 weeks, e.g., about 4-5 weeks. In some embodiments, the cell clusters are subcultured for about 2 weeks, 3, weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In other embodiments, the cell clusters are subcultured for at least 8 weeks. In an embodiment, the cell clusters may be subcultured under non-adherent conditions. In another embodiment, the cell clusters may be subcultured under adherent conditions. In another embodiment, the cell clusters may be cultured under feeder or feeder-free conditions.

[0299]

[0178] The RPE cells may then be harvested, for example, with a dissociation reagent to obtain RPE cell clusters. RPE cell clusters may be obtained by harvesting the RPE cells and removing single cells by any method known in the art. In an embodiment, the RPE cells may be harvested and passed through a strainer or a series of strainers as described above, to obtain RPE cell clusters as described above.

[0300]

[0179] In an embodiment, the RPE cell clusters obtained may be dissociated into single cells with an enzymatic or non-enzymatic dissociation reagent and cultured to expand the RPE cells, further described below.

[0301]

[0180] In some embodiments, a sample of the RPE cells produced may be tested for the desired molecular marker profile and then harvested. In other embodiments, it may not be necessary to test the RPE cells for molecular markers before harvesting as long as the culture conditions are known to produce RPE cells. Thus, RPE cells may be harvested without having to test for molecular markers.

[0302] f. Expansion of RPE cells

[0303]

[0181] In some embodiments, the RPE cells obtained from the single RPE progenitor cell subculture or RPE progenitor cell cluster subculture method may be cultured onto 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.127206-12220

[0304]

[0182] The RPE cell population first cultured in this step is referred to herein as “PO.” In an 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 an embodiment, the laminin is selected from laminin 521, laminin 511, or iMatrix511. In further embodiments, laminin comprises e-cadherin. In another embodiment, the extracellular matrix is gelatin. In some embodiments, the medium is RPE-MM (also referred to as RPE-GM / MM, MM or maintenance medium and comprising DMEM / KO-DMEM with KSR and FBS, beta-mercaptoethanol, NEAA, and glutamine), StemFit, EGM2, or EBDM. In some embodiments, the RPE-MM is supplemented with FGF (MM / FGF), and / or FBS (e.g., g-Irr FBS). In other embodiments, other medium known in the art that supports RPE growth and expansion may be used. Any such medium may be supplemented with or without 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 e.g., WO2013 / 074681A, which is incorporated herein by reference in its entirety.

[0305]

[0183] In an embodiment, the RPE cells may be passaged and cultured until adequate numbers of RPE cells are obtained. In an embodiment, the RPE cells are passaged indefinitely. In another embodiment, the RPE cells are passaged at least one time (“Pl”) up to 30 times (“P30”).

[0306]

[0184] In an embodiment, the RPE cells are harvested and cryopreserved after passaging one time (“Pl”) to generate a cell stock.

[0307]

[0185] In an embodiment, the RPE cells e.g., the RPE cells of the cell stock) are passaged at least two times (“P2”) up to 10 times (“PIO”).

[0308]

[0186] In a further embodiment, the RPE cells are passaged two times (“P2”), three times (“P3”), four times, (“P4”), five times (“P5”), six times (“P6”), seven times (“P7”), eight times (“P8”), nine times (“P9”), or ten times (“PIO”). The RPE cells may be cryopreserved until further use.

[0309]

[0187] In an embodiment, the duration of each expansion phase may vary from days, weeks, to months. In an embodiment, the duration of the expansion phase is between about 2-90 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, or 2-14 days, or 2-10 days. During the expansion phase, fresh medium may be added at intervals, such as every 1-2 days. In an embodiment, bFGF is added at a concentration of about l-100ng / ml to the127206-12220

[0310] RPE cell culture medium during the first 1-5 days, 1-4 days, 1-3 days, 1-2 days, 1 day, 2 days, 3 days, 4 days, or 5 days of RPE expansion at each passage (e.g., PO, Pl, P2) and then removed until further passaged. In an embodiment, the bFGF concentration is about 1 - 50 ng / ml, about 2-40 ng / ml, about 3-30 ng / ml, about 4-20 ng / ml, or about 4-10ng / ml. In a specific embodiment, the bFGF concentration is about 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml.

[0311]

[0188] In some embodiments, a sample of the RPE cells produced may be tested for the desired molecular marker profile and then harvested. In other embodiments, it may not be necessary to test the RPE cells for molecular markers before harvesting as long as the culture conditions are known to produce RPE cells. Thus, RPE cells may be harvested without having to test for molecular markers.

[0312] g. Feeder and Feeder-Free Based Cultures

[0313] Mouse Feeder Layers

[0314]

[0189] The PSCs, as disclosed herein, may be cultured on mouse embryonic fibroblasts (MEF) as a feeder cell (see, e.g., Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel J J, Marshall V S, Jones J M. (1998); Science 282: 1145-7; Reubinoff B E, Pera M F, Fong C, Trounson A, Bongso A. (2000); Reubinoff et al., 2000, Nat. Biotechnol. 18: 399-404). MEF cells may be derived from day 12-13 mouse embryos in medium supplemented with fetal bovine serum.

[0315]

[0190] PSCs may be cultured on MEF under serum-free conditions using serum replacement supplemented with basic fibroblast growth factor (bFGF) (see, e.g., Amit M, Carpenter M K, Inokuma M S, Chiu C P, Harris C P, Waknitz M A, Itskovitz-Eldor J, Thomson J A. (2000)). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture (see, e.g., Dev. Biol. 227: 271-8). In addition, following 6 months of culturing under serum replacement the PSCs may still maintain their pluripotency when cultured under conditions that promote maintenance of the pluripotent state. The pluripotency of PSCs may be indicated by their ability to form teratomas which contain all three embryonic germ layers. Additionally, the differentiation of PSCs to RPEs may be performed in the presence of mouse feeder cells. Accordingly, the PSCs used in the methods described herein may be cultured on mouse feeder cells.127206-12220

[0316] Human Feeder Cells

[0317]

[0191] PSCs may be cultured, maintained, or differentiated on human feeder cells, as described in, for example, PCT publication No. W02009 / 048675, the entire contents of which are expressly incorporated herein by reference. PSCs may be maintained in the undifferentiated state by multiple sequential passages of the PSCs on human feeder cells (see, e.g., Richards et al., 2002, Nat. Biotechnol. 20: 933-6). PSCs may also be differentiated to RPEs in the presence of human feeder cells. Accordingly, the PSCs used in the methods described herein can be cultured on human feeder cells.

[0318] Feeder-Free Cultures

[0319]

[0192] PSCs may be cultured in a feeder-free system on a solid surface such as an extracellular matrix e.g., Matrigel® or laminin) in the presence of a culture medium. Various methods are known in the art to differentiate PSCs ex vivo into RPE cells, as summarized in Rowland et al., Journal Cell Physiology, 227:457-466, 2012, incorporated herein by reference. Accordingly, the PSCs used in the methods described herein may be cultured on feeder-free cultures.

[0320] f. Use of FGF / bFGF and ROCK inhibitors

[0321]

[0193] In mammalian development, RPE shares the same progenitor with neural retina, the neuroepithelium of the optic vesicle. Under certain conditions, RPE can transdifferentiate into neuronal progenitors (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 which can stimulate the change of RPE 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 the eye development, including rx / rax, chxlO / 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 margins of the chick retina contain neural stem cells (Fischer and Reh, 2000; Dev Biol. 15;220(2): 197-210) and that the pigmented cells in that area, which express PAX6 / mitf, can form neuronal cells in response to FGF (Fischer and Reh, 2001, Dev Neurosci. 23(4-5):268-76).

[0322]

[0194] In some embodiments, the PSCs of the invention may be maintained in a pluripotent state in a culture medium that includes 1-200 ng / ml bFGF. In an embodiment, the bFGF concentration is about 1-100 ng / ml, about 2-100ng / ml, about 3-100 ng / ml, or about 4-127206-12220

[0323] lOOng / ml. In a specific embodiment, the bFGF concentration is about 100 ng / ml. In some embodiments, PSCs may be differentiated into RPE cells in the presence of bFGF. In other embodiments, as discussed above and herein, RPE cells may be expanded in the presence of bFGF.

[0324]

[0195] During RPE formation, the pluripotent cells may be cultured in the presence of an inhibitor of rho-associated protein kinase (ROCK). ROCK inhibitors refer to any substance that inhibits or reduces the function of Rho-associated kinase or its signaling pathway in a cell, such as a small molecule, an siRNA, a miRNA, an antisense RNA, or the like. "ROCK signaling pathway," as used herein, may include any signal processors involved in the ROCK-related signaling pathway, such as the Rho-ROCK-Myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway in a cell. An exemplary ROCK inhibitor that may be used is Stemgent’s Stemolecule Y-27632 (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):68 1 -6). Other ROCK inhibitors include, e.g., H- 1 1 52, Y-3014 1, 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., 52:3 1 9-324, 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 may be utilized with concentrations and / or culture conditions as known in the art, for example as described in US Pub. No. 2012 / 0276063 which is hereby incorporated by reference in its entirety. For example, the ROCK inhibitor may have a concentration of about 0.05 to about 50 pM, for example, 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 pM, including any range derivable therein, or any concentration effective for promoting cell growth or survival. In further embodiments, the RPE expansion culture may be further supplemented with ROCK inhibitors and / or bFGF as described by PCT publication No. WO2013074681A1; incorporated in its entirety herein by reference.

[0325] i. Adherent and non-adherent culture

[0326]

[0196] The “adherent culture” as used in the present disclosure means culture in a state where the cells of interest are adhered to a tissue culture vessel via a cell culture substrate, e.g., laminin. Cells may also adhere to plastic that has been treated for cell adhesion (“tissue culture treated”) without any additional substrate coating.127206-12220

[0327]

[0197] In some embodiments, the differentiation from pluripotent stem cells to RPE cells is performed by adherent culture. Adherent culture can be performed by using a cell-adhesive culture vessel. While the cell-adhesive culture vessel is not particularly limited as long as the surface of the culture vessel is treated to improve adhesiveness to the cell, for example, a culture vessel having a coated layer containing an extracellular matrix, a synthetic polymer and the like can be used. The coated layer may be constituted with one or more kinds of components, or may be formed by a single layer or multiple layers. While the extracellular matrix is not particularly limited as long as it can form a coated layer showing adhesiveness to a pluripotent stem cell, for example, collagen, gelatin, laminin, fibronectin and the like, which can be used alone or in combination. As a commercially available product containing multiple kinds of extracellular matrices, Matrigel (BD), CELLStart (Invitrogen) and the like are available. As the synthetic polymer, biologically or chemically produced polymers can be used. For example, cationic polymers such as polylysine (poly-D-lysine, poly-L-lysine), poly omithinepoly ethyleneimine (PEI), poly-N-propylacrylamide (PIPAAm) and the like are preferably used. The extracellular matrix or synthetic polymer may be biologically produced by using bacterium, cells and the like and introducing genetic modification as necessary, or chemically synthesized. In other embodiments, cells may bind to the extracellular matrix via RGD peptides, which are bound by integrin adhesion receptors found on many extracellular matrices.

[0328]

[0198] In some embodiments, adherent culture may be performed on a tissue culture vessel that has not been treated with any cell culture substrate or for cell adhesion. For example, media components such as FBS, fibronectin, or vitronectin may be absorbed by the tissue culture vessel and serve as cell adhesion substrates. In other embodiments, the cells in the tissue culture vessels may secrete extracellular matrices that may also serve as cell adhesion substrates.

[0329]

[0199] The “non-adherent culture” as used in the present disclosure means culture in a state where the cells of interest do not adhere or substantially do not adhere to a tissue culture vessel. Accordingly, single cells or clusters of cells in a non-adherent culture may float in culture and may be in suspension. Single cells in a non-adherent culture may form clusters or aggregates under appropriate conditions. In an embodiment, the culture vessel surface may be coated with a hydrophilic, neutrally charged coating that is covalently bound to the polystyrene vessel surface, such as the Corning® Ultra- Low Attachment Surface. The nonbinding surface inhibits specific and nonspecific immobilization, forcing cells into a127206-12220

[0330] suspended state. The cells may also be cultured in a spinner flask (Coming) to culture cells in suspension. Other methods of culturing cells in non-adherent culture are known to those skilled in the art and may be used in the methods of the present invention.

[0331] II. CRYOPRESERVATION FORMULATIONS

[0332]

[0200] Cell-based therapies including those involving transplantation of cells, e.g., in the context of a regenerative medicine approach, often require formulating, storing, transporting, and / or injecting delicate or fragile cells that can be damaged or lose repopulation capacity upon inappropriate handling, extensive manipulation and / or processing steps or exposure to non-physiological conditions.

[0333]

[0201] The present disclosure also provides formulations and methods for cell cryopreservation, cell storage, cell transport, and ultimately cell administration to a subject with minimal processing and manipulation of the cells, and increased viability post-thaw. The formulations provided herein have several advantages over currently available formulations. For example, in contrast to currently available cryopreservative formulations, the formulations provided herein require minimal post-thaw processing and can be administered to a subject along with the cell preparation with little, if any, adverse effect such as toxicity. In some embodiments, the formulations provided herein allow for cryopreservation at a high density in a low volume which offers a number of advantages including a thawing regimen that requires only a single step of adding a diluent. Since there is no need to wash the formulation away post-thaw, there is less chance of cell loss and cell damage, thereby increasing the therapeutic efficacy of the cryopreserved cellular preparation. Currently available cryoprotectants and cryoprotective formulations require extensive cell processing procedures that may be costly, time-consuming, prone to human error, and ultimately may diminish cell survival, re-plating efficiency, and repopulation capacity of the cells contained therein. Also, notably, increased viability has been observed for cell populations that are cryopreserved using the formulations provided herein, compared to prior art cryopreservative formulations and methods. Improved cell growth post-thaw has also been observed when using the formulations provided herein. The formulations provided herein may provide for improved functionality, survival and stability of cell populations.

[0334]

[0202] In addition to the aforementioned advantages, the cryopreservative formulations of this disclosure are also surprisingly and unexpectedly associated with reduced frequency (or prevalence) of multi-nucleated cells in culture, particularly in RPE cell cultures. Multi-127206-12220

[0335] nucleated cells, such as multi-nucleated RPE cells, have been observed using other formulations. However, surprisingly, the cryopreservative formulations provided herein appear to reduce the frequency of such cells.

[0336] a. RPE Cells

[0337]

[0203] The present invention provides a composition comprising a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1. In some embodiments, the RPE cells express the markers RPE65, CRALBP, PEDF, Bestrophin, MITF, PAX6, PAX6 and ZO-1. In some embodiments, the RPE cells express the markers gp-100 and bestrophin. In some embodiments, the RPE cells express the markers gp-100 and MITF. In some embodiments, the RPE cells express the markers gp-100 and CRALBP. In some embodiments, the RPE cells express gp-100 (PMEL17).

[0338]

[0204] In some embodiments, at least 60% of RPE cells in an RPE cell population of the disclosure express BEST1. In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express BEST1. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express BEST1. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express BEST1.

[0339]

[0205] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express MITF and / or PAX6. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express MITF and / or PAX6. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express MITF and / or PAX6. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express MITF and / or PAX6.

[0340]

[0206] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express BEST1 and / or PAX6. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express BEST1 and / or PAX6. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express BEST1 and / or PAX6. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express BEST1 and / or PAX6.

[0341]

[0207] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express ZO-1. In some embodiments, at least 80% of RPE cells in an RPE cell127206-12220

[0342] population of the disclosure express ZO-1. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express ZO-1. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express ZO-1.

[0343]

[0208] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express gp-100.

[0344]

[0209] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or Bestrophin. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or Bestrophin. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or Bestrophin. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or Bestrophin.

[0345]

[0210] In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or MITF. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or MITF. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or MITF. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or MITF.

[0346]

[0211] e embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or CRALBP. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or CRALBP. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or CRALBP. In some embodiments, at least 95% of RPE cells in an RPE cell population of the disclosure express gp-100 and / or CRALBP.

[0347]

[0212] In some embodiments, at least 30% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 35% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 40% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 45% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 50% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 55% of RPE cells in an RPE127206-12220

[0348] cell population of the disclosure express bestrophin. In some embodiments, at least 60% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 70% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express bestrophin. In some embodiments, at least 90% or more RPE cells in an RPE cell population of the disclosure express bestrophin. .

[0349]

[0213] In some embodiments, at least 75% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 80% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 85% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 90% of RPE cells in an RPE cell population of the disclosure express gp-100. In some embodiments, at least 90% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the RPE cells in an RPE cell population of the disclosure express gp-100.

[0350]

[0214] In certain embodiments, the purity of an RPE cell population and / or the maturity of RPE cells, for example in an RPE cell population, is assessed by measuring or determining expression of one or more markers, for example gplOO, bestrophin, MITF, CRALBP and TRYP1.

[0351]

[0215] In some embodiments, the ratio of apical to basal PEDF secretion is at least 1.0. In some embodiments, the ratio of apical to basal PEDF secretion is at least 1.5. In some embodiments, the ratio of apical to basal PEDF secretion is at least 2.0. In some embodiments, the ratio of apical to basal PEDF secretion is at least 3. In some embodiments, the ratio of apical to basal PEDF secretion is greater than 3. In some embodiments, the ratio of basil to apical VEGF secretion is at least 1.0. In some embodiments, the ratio of basil to apical VEGF secretion is at least 1.5. In some embodiments, the ratio of basal to apical VEGF secretion is at least 2.0. In some embodiments, the ratio of basal to apical VEGF secretion is at least 3.0. In some embodiments, the ratio of apical to basal PEDF secretion is greater than 3.

[0352]

[0216] In some embodiments, the expression in the RPE cells 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)-l-60, and TRA-1-80 is down-regulated compared to their expression in stem cells. In any of the embodiments of the present invention, the expression127206-12220

[0353] in the RPE cells expression of one or more stem cell markers selected from the group OCT4, NANOG, REXI, alkaline phosphatase, SOX2, TDGF- 1, DPPA-2, DPPA-4, stage specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection antigen (TRA)-l -60 and TRA-1-80 is down-regulated at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 100-fold compared to their expression in pluripotent stem cells.

[0354]

[0217] In some embodiments, RPE cells of the disclosure lack one or more endogenous human leukocyte antigen (HLA) genes (e.g., class I and class II HLAs). In some embodiments, RPE cells of the disclosure lack substantial endogenous expression of human leukocyte antigen (HLA) genes (e.g., class I and class II HLAs). In some embodiments, the expression in the RPE cells of endogenous HLA genes (e.g., class I and class II HLAs) is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold lower than in RPE cells which have one or more HLA genes. In some embodiments, RPE cells of the disclosure express an HLA-E-beta2-microglobulin (HLA-E-B2M) fusion protein. In some embodiments, RPE cells of the disclosure comprise one or more safety switch or suicide genes (e.g., herpes simplex virus thymidine kinase (TK) safety switch). In some embodiments, RPE cells of the disclosure have GCV sensitivity. In some embodiments, administration of GCV to RPE cells of the disclosure results in the selective ablation of proliferating cells.

[0355]

[0218] In some embodiments, RPE cells of the disclosure evade immune detection by allogeneic NK cells. In some embodiments, RPE cells of the disclosure do not induce NK-mediated cytotoxicity. In some embodiments, RPE cells of the disclosure evade immune detection by allogeneic CD4+ T cells. In some embodiments, RPE cells of the disclosure do not elicit an allogeneic CD4+ T cell response. In some embodiments, RPE cells of the disclosure evade immune detection by allogeneic CD8+ T cell. In some embodiments, RPE cells of the disclosure do not elicit an allogeneic CD8+ T cell response.

[0356]

[0219] In some embodiments, when administered to a subject, RPE cells of the disclosure display exclusive localization to the eye in which they are administered. In some embodiments, RPE cells of the disclosure show no systemic distribution after administration to a subject. In some embodiments, RPE cells of the disclosure show no systemic distribution to the gonads after administration to a subject.

[0357]

[0220] In some embodiments, RPE cells of the disclosure display a durable persistence within the subretinal space when administered to a subject’s eye. In some embodiments, RPE127206-12220

[0358] cells of the disclosure show a cell persistence of at least 2 months within the subretinal space. In some embodiments, RPE cells of the disclosure show a cell persistence of at least 3 months within the subretinal space. In some embodiments, RPE cells of the disclosure show a cell persistence of at least 4 months within the subretinal space. In some embodiments, RPE cells of the disclosure show a cell persistence of at least 5 months within the subretinal space. In some embodiments, RPE cells of the disclosure show a cell persistence of at least 6 months within the subretinal space.

[0359]

[0221] In some embodiments, RPE cells of the disclosure maintain RPE phenotype for at least 6 months after administration to a subject. In some embodiments, RPE cells of the disclosure maintain RPE phenotype for at least 8 months after administration to a subject. In some embodiments, RPE cells of the disclosure maintain RPE phenotype for at least 10 months or more after administration to a subject.

[0360]

[0222] In some embodiments, RPE cells of the disclosure show no systemic toxicity for at least 8 months after administration to a subject. In some embodiments, RPE cells of the disclosure show no systemic toxicity for at least 9 months after administration to a subject. In some embodiments, RPE cells of the disclosure show no systemic toxicity for at least 10 months or more after administration to a subject.

[0361]

[0223] In some embodiments, RPE cells of the disclosure have low risk of tumorigenicity. In some embodiments, RPE cells of the disclosure do not induce tumor formation (e.g., in the subretinal space).

[0362]

[0224] The RPE cells may be cryopreserved at higher concentration (or density) compared to standard prior art techniques. For example, the cells may be cryopreserved at a concentration (or density) of about 3 x 102cells per pL or higher, including about 103cells per pL, about 2 x 103cells per pL, about 3 x 103cells per pL, 104cells per pL or higher, including about 2 x 104cells per pL, about 3 x 104cells per pL, about 4 x 104cells per pL, about 5 x 104cells per pL, about 6 x 104cells per pL, about 7 x 104cells per pL, about 8 x 104cells per pL, about 9 x 104cells per pL, or about 105 cells per pL, or higher. In some embodiments, the cell density is about 3 x 102cells per pL. In some embodiments, the cell density is about 3.3 x 103cells per pL. In some embodiments, the cell density is about 5 x 104cells per pL.

[0363]

[0225] In some embodiments, the cell density is in the range of about 103cells per pL to about 6 x 104cells per pL, including about 3 x 103cells per pL to about 5 x 104cells per pL. In some embodiments, the cell density is in the range of about 104cells per pL to about 6 x 104cells per pL, including 1.5 x 104cell per pL to about 5 x 104cells per pL, including about127206-12220

[0364] 2 x 104cells per pL to about 5 x 104cells per pL, and about 3 x 104cells per pL to about 5 x 104cells per pL. In some embodiments, the cell density is about 3 x 102cells per pL. In some embodiments, the cell density is about 3.3 x 103cells per pL. In some embodiments, the cell density is about 5 x 104cells per pL.

[0365]

[0226] In some embodiments, the number of cells per vial is about 105to 107cells per vial. In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells per vial. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells per vial. In some embodiments the cells are formulated such that the number of cells per vial is about 450,000 to about 1,500,000 in a volume of a diluent or cryopreservative as disclosed herein of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 125 pL, about 150 pL, about 175 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL or about 500 pL. In some embodiments the cells are formulated such that the number of cells per vial is about 50,000 to about 500,000 in a volume of a diluent or cryopreservative as disclosed herein of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 125 pL, about 150 pL, about 175 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL or about 500 pL. In some embodiments the cells are formulated such that the number of cells per vial is about 50,000 to about 500,000 in a volume of a diluent or cryopreservative as disclosed herein of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 125 pL, about 150 pL, about 175 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL or about 500 pL. In some embodiments the cells are formulated such that the number of cells per vial is about 133,000 cells / dose, about 143,000 cells / dose, about 150,000 cells / dose, about 167,000 cells / dose, about 200,000 cells / dose, about 225,000 cells / dose, about 233,000 cells / dose, about 243,000 cells / dose, about 250,000 cells / dose, about 260,000 cells / dose, about 265,000 cells / dose, about 263,000 cells / dose, about 267,000 cells / dose, about 275,000 cells / dose, about 300,000 cells / dose, about 350,000 cells / dose, about 333,333 cell / dose, about 343,333 cell / dose, about 367,000 cells / dose, about 400,000 cells / dose and about 443,333 cells in a volume of a diluent or cryopreservative as disclosed herein of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100127206-12220

[0366] pL, about 125 pL, about 150 |aL, about 175 |aL, about 200 |aL, about 250 |aL, about 300 |aL, about 350 |aL, about 400 |aL, about 450 |aL or about 500 |aL.

[0367]

[0227] Additionally, the volume of the cryopreserved cell preparation may be reduced compared to standard prior art techniques. In some embodiments, the cells may be cryopreserved in volumes ranging from about 10 pL to about 500 pL. Since the cells can be frozen in large numbers yet in low volumes, the frozen preparation can then be diluted with a suitable diluent to arrive at a desired volume and concentration, and then directly administered to a subject without the need for performing a washing step. Standard prior art techniques typically freeze lower concentrations of cells in larger volumes (e.g., on the order of 1 mL), and then require that the thawed preparation be washed, sometimes multiple times, in order to remove the cryoprotectant before administration to a subject.

[0368]

[0228] The volume and the number of cells in the cell preparations to be administered will depend on the specific application. Typically, for cell transplantation applications, it is desirable to reduce the volume administered as much as possible. Accordingly, the cell preparations may be formulated so that minimized volumes may be delivered. Cell concentrations for injection may be at any concentration that is effective and non-toxic. In some embodiments, the volume of the cell preparation to be administered is between 1 to 1000 pL. In some embodiments, the volume of the RPE cell preparation to be administered is between 1 to 50 pl, or between 10 to 50 pl or between 10 to 100 pl or between 25 to 50 pl or between 50 to 100 pl or between 50 to 200 pl or between 50 to 300 pl or between 50 to 400 pl or between 50 to 500 pl, or between 100 to 500 pl, or between 100 to 400 pl or between 100 to 300 pl, or between 100 to 300 pl or about 200 pl or about 150 pl. In some embodiments the volume of the RPE cell preparation to be administered is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950 or 1000 pl.

[0369]

[0229] In some embodiments, the formulations or preparations provided herein exhibit a physiological pH and a physiological osmotic pressure, also referred to as a physiological osmolarity. A physiological pH refers to a pH that is not cytotoxic and resembles the pH of the cell or tissue in its natural environment. For most cells and tissues, a physiological pH is a pH of about 6.8 to about 7.8, for example, a pH of about 7 to about 7.7, a pH of about 7.2 to about 7.6, a pH of about 7.2 to about 7.4, or a pH of about 7.4 to about 7.5. Accordingly, in some embodiments, the formulations or preparations provided herein exhibit a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. A127206-12220

[0370] physiological osmotic pressure refers to an osmotic pressure that is not cytotoxic and resembles the osmotic pressure of the cell or tissue in its natural environment. For some cells and tissues, a physiological osmotic pressure is about 270 to about 345 mOsm / 1, for example, about 280 to about 330 mOsm / 1, about 290 to about 325 mOsm / 1, about 300 to about 315 mOsm / 1. In some embodiments, a physiological osmotic pressure is about 300, about 305, about 310, about 315, about 320, or about 325 mOsm / 1. In some embodiments, the formulations or preparations provided herein exhibit a physiological pH and an osmotic pressure that is greater than physiological osmotic pressure, for example, about 350 to about 450 mOsm / 1, for example, about 360 to about 440 mOsm / 1, about 370 to about 430 mOsm / 1, about 380 to about 420 mOsm / 1, or about 390 to about 410 mOsm / 1. In some embodiments, an osmotic pressure that is greater than physiological osmotic pressure is about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445 or about 450 mOsm / 1. In some embodiments, the formulations or preparations provided herein have an osmolality of about 1250 mOsm / kg, for example about 1250 mOsm / kg, about 1000 mOsm / kg, about 900 mOsm / kg, about 800 mOsm / kg, about 700 mOsm / kg, about 600 mOsm / kg, about 500 mOsm / kg, about 400 mOsm / kg or about 300 mOsm / kg. In some embodiments, the formulations or preparation provided herein have an osmolality about 300-450 mOsm / kg, about 300-400 mOsm / kg, about 300-450 mOsm / kg, about 350-450 mOsm / kg or about 350-400 mOsm / kg.

[0371]

[0230] Some aspects of this disclosure provide RPE cell preparations comprising a population of cells or a tissue in a cryopreservative formulation as provided herein. In some embodiments, the RPE cell preparation is cryopreserved. The RPE cell preparation provided herein may be cryopreserved by being cryogenically frozen at any appropriate temperature known in the art. For example, in some embodiments, the RPE cell preparations provided herein are cryopreserved at temperatures between -100 °C and -200 °C. In some embodiments the RPE cell preparations provided herein are cryopreserved at temperatures below -125 °C. In some embodiments the RPE cell preparations provided herein are cryopreserved at temperatures at or below -135 °C. In some embodiments, the RPE cell preparations provided herein are cryopreserved at temperatures below -150 °C. The pharmaceutical compositions may be cryopreserved in liquid nitrogen or in the gas phase of liquid nitrogen.

[0372]

[0231] In some embodiments, the RPE cell preparation is suitable for transplantation into a subject once thawed, optionally without being diluted. In some embodiments, the RPE cell127206-12220

[0373] preparation is suitable for transplantation into a subject once thawed and diluted with a diluent. In some embodiments, the diluent is GS2, GS2 Plus, described hereinbelow, BSS, BSS Plus®, a dextran and HSA solution, PBS, DMEM, MEM, albumin, or any other appropriate diluent. In some embodiments, the diluent is GS2 or GS2 Plus or any other appropriate diluent. In some embodiments, the diluent is a solution comprising (a) a buffer that maintains the solution at a physiological pH; (b) at least 2 mM glucose; and (c) an osmotically active agent maintaining the solution at a physiological osmolarity. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is a solution comprising (a) a buffer that maintains the solution at a physiological pH; (b) at least 2 mM glucose; and (c) an osmotically active agent maintaining the solution at an osmolarity that is higher than physiological osmolarity, for example in the range of 350-450 mOsm / kg or an osmolality in the range of 300-1250 mOsm / kg. In some embodiments, the diluent further comprises divalent cations. In some embodiments, divalent cations comprise calcium and / or magnesium. In some embodiments, the diluent comprises calcium. In some embodiments, the diluent further comprises magnesium. In some embodiments, the diluent comprises an acetate buffer and / or a citrate buffer. In some embodiments, the diluent is a solution which combines two or more or any number of criteria (e.g., pH, osmolarity, solutes (buffer, glucose, osmotically active agent, magnesium, calcium, potassium, polymer) concentrations, etc.). In some embodiments, the diluent is a solution comprising a buffering agent, glucose, and an osmotically active agent with or without added polymer, a solution comprising potassium and a solution not comprising potassium, as well as a solution comprising any combination of solutes at any concentration provided for the respective solute. It will also be understood that the disclosure embraces a solution comprising the listed solutes as well as a solution essentially consisting of or consisting of the listed solutes and a solvent, e.g., water. These alternatives are not spelled out here for purposes of brevity.

[0374]

[0232] For example, in some embodiments, the diluent is a solution comprising or consisting essentially of calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose, e.g., D-glucose, in water. In some embodiments, the diluent is a solution comprising or consisting essentially of calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose, e.g., D-glucose, and potassium chloride, in water. In some embodiments, the diluent is GS2 or GS2 Plus, as described herein below and in W02017 / 031312A1, the contents of which are incorporated by reference herein.127206-12220

[0375]

[0233] In some embodiments, the diluent is a solution comprising about 0.1 to about 1.2 mM CaCh, about 0.05 to about 5 mM MgCh, about 1 to about 2.5 mM KC1, about 0.5 to about 2 mM sodium citrate, about 14 to about 17 mM dextrose, and about 125 to about 175 mM NaCl. In some embodiments, the diluent is a solution comprising about 0.9 mM CaCh, about 0.3 mM MgCh, about 2 mM KC1, about 1.2 mM sodium citrate, about 15 mM dextrose, and about 145 mM NaCl. The diluent may further comprise sodium acetate. The diluent may further comprise a polymer such as hyaluronic acid or a solvate thereof such as sodium hyaluronate, optionally at a concentration of about 0.01 to about 0.05% (w / v), including about 0.05% (w / v). It may further comprise buffering components such as sodium phosphate dibasic heptahydrate and / or sodium phosphate monobasic monohydrate and / or sodium phosphate monobasic dihydrate.

[0376]

[0234] In some embodiments, the diluent is a solution comprising about 0.008% to about 0.012% CaC12 dihydrate, about 0.0048% to about 0.0072% MgCh hexahydrate, about 0.012% to about 0.018% KC1, about 0.028% to about 0.042% sodium citrate dihydrate, about 0.23% to about 0.35% dextrose, and about 0.68% to about 1.02% NaCl. In some embodiments, the diluent is a solution that comprises about 0.01% CaCh dihydrate, about 0.006% MgCh hexahydrate, about 0.015% KC1, about 0.035% sodium citrate dihydrate, at least 0.25% dextrose, and about 0.85% NaCl. The diluent may further comprise sodium acetate. The diluent may further comprise a polymer such as hyaluronic acid or a solvate thereof such as sodium hyaluronate, optionally at a concentration of about 0.01 to about 0.05% (w / v), including about 0.05% (w / v). It may further comprise buffering components such as sodium phosphate dibasic heptahydrate and / or sodium phosphate monobasic monohydrate and / or sodium phosphate monobasic dihydrate.

[0377]

[0235] In some embodiments, the diluent comprises 5% dextrose, 0.9% NaCl, BSS® sterile irrigating solution, 0.1N NaOH, and 0.9% NaCl irrigation. In some embodiments, the diluent comprises 3% sodium hyaluronate, 5% dextrose anhydrous, sodium phosphate dibasic dodecahydrate, BSS® sterile irrigating solution (e.g., 0.64% NaCl, 0.075% KC1, 0.048% CaCh dihydrate, 0,03% MgCh hexahydrate, 0.39% sodium acetate trihydrate, and 0.17% sodium citrate dihydrate).

[0378]

[0236] In some embodiments, the diluent comprises about 0.27% glucose, about 0.84% sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate, (e.g., at about 0.08% ), optionally sodium hyaluronate (e.g., at about 0.049%), and optionally127206-12220

[0379] sodium phosphate dibasic heptahydrate (e.g., at about 0.0007%) and sodium phosphate monobasic monohydrate e.g., at about 0.0001%). In some embodiments, the diluent comprises about 15 mM glucose, about 144 mM sodium chloride, about 2.1 mM potassium chloride, about 0.9 mM calcium chloride, about 0.3 mM magnesium chloride, about 1.2 mM sodium citrate, optionally sodium acetate (e.g., at about 6 mM), optionally sodium hyaluronate, and optionally sodium phosphate dibasic heptahydrate (e.g., at about 0.027 mM) and sodium phosphate monobasic monohydrate (e.g., at about 0.007 mM). Such diluents include GS2.

[0380]

[0237] In some embodiments, the diluent comprises about 0.27% glucose, about 0.84% sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate (e.g., at about 0.08%), optionally sodium hyaluronate (e.g., at about 0.049%), and optionally sodium phosphate monobasic dihydrate (e.g., at about 0.047%). In some embodiments, the diluent comprises about 15 mM glucose, about 144 mM sodium chloride, about 2.1 mM potassium chloride, about 0.9 mM calcium chloride, about 0.3 mM magnesium chloride, about 1.2 mM sodium citrate, optionally sodium acetate (e.g., at about 6 mM), optionally sodium hyaluronate, and optionally sodium phosphate monobasic dihydrate (e.g., at about 3 mM). Such diluents include GS2 Plus.

[0381]

[0238] In some embodiments, the RPE cell preparations provided herein are formulated for administration to a subject, for example, for administration via injection, once thawed and diluted.

[0382]

[0239] Exemplary cell or tissue preparations, post-thaw may be formulated to be suitable for use in treating a human patient, e.g., pyrogen-free or essentially pyrogen-free, pathogen-free, sterile, and at physiological pH and osmolarity. In some embodiments, the preparations provided herein are formulated for injection into a specific site, e.g., in the case of ophthalmologic preparations for treating retinal diseases or disorders, into the vitreous humor for delivery to the site of retinal or choroidal damage or to an area of geographic atrophy. In some embodiments, the RPE cell preparation is within a storage container. In some embodiments, the storage container is a vial, ampule, bottle, syringe, a cooler package, or other suitable storage container. In some embodiments, the RPE cell preparation is within a vial. In some embodiments, the RPE cell preparation is within a syringe.127206-12220

[0383]

[0240] RPE Cell preparations provided by the present disclosure may include additionally therapeutic agents, for example, an immunosuppressant, a pro-angiogenic agent, or nutrients or growth factors supporting survival and / or implantation of the cells in the preparation.

[0384]

[0241] In some embodiments, the number of cells and / or the concentration of cells in a RPE cell preparation provided herein may be determined by counting viable cells and excluding non-viable cells. For example, non-viable cells may be detected by failure to exclude a vital dye (such as Trypan Blue), or using a functional assay (such as the ability to adhere to a culture substrate, phagocytosis, etc.). Additionally, the number of cells or the concentration of cells of a desired cell type may be determined by counting cells that express one or more cell markers characteristic of that cell type and / or excluding cells that express one or more markers indicative of a cell type other than the desired cell type.

[0385]

[0242] In some embodiments, a RPE cell preparation to be administered comprises about or at least IxlO3, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, IxlO4, 2xl04, 3xl04, 4xl04, 5xl04, 6xl04, 7xl04, 8xl04, 9xl04, IxlO5, 2xl05, 3xl05, 4xl05, 5xl05, 6xl05, 7xl05, 8xl05, 9xl05, IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, IxlO7, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xlO10, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xlO10, or 9xlO10cells, or more cells.

[0386]

[0243] In some embodiments, the number of RPE cells per vial is about 105to 107cells per vial prior to cryopreservation. In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells per vial prior to cryopreservation. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells per vial prior to cryopreservation. In some embodiments, the number of cells per vial is about 105to 107cells per vial following cryopreservation. In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells per vial following cryopreservation, thawing and dilution. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells per vial prior following cryopreservation, thawing and diluting.

[0387]

[0244] The aforementioned numbers of cells may be present in the cryopreserved RPE cell preparation in a single cryovial. The cells may be present in about 10 to about 500 pL of cryoprotective formulation, including in about 100 to about 500 pL of cryopreservative formulation, or in about 200 to about 500 pL of cryoprotective formulation, or in about 300 to about 500 pL of cryopreservative formulation, or in about 400 to about 500 pL of cryopreservative formulation, or in about 10 to about 200 pL of cryoprotective formulation,127206-12220

[0388] or in about 10 to about 100 |aL of cryoprotective formulation, or about 10 to about 50 pL of cryoprotective formulation, or about 20 to about 50 L of cryoprotective formulation. In some embodiments, the cells are present in about 450 pL of cryopreservative formulation.

[0389]

[0245] In some embodiments, the RPE cell preparations exhibit cell viability of at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%). In some embodiments, the RPE cell preparations exhibit robust cell growth index. In some embodiments, the RPE cell preparations exhibit a time to reach a cell growth index of 3 of at least 110 hours, at least 109 hours, at least 108 hours, at least 107 hours, at least 106 hours, at least 105 hours, at least 104 hours, at least 103 hours, at least 102 hours, at least 101 hours, at least 100 hours, at least 99 hours, at least 98 hours, at least 97 hours, at least 96 hours, at least 95 hours, at least 94 hours, at least 93 hours, at least 92 hours, at least 91 hours, at least 90 hours, at least 89 hours, at least 88 hours, at least 87 hours, at least 86 hours, at least 85 hours, at least 84 hours, at least 83 hours, at least 82 hours, at least 81 hours, or at least 80 hours. In some embodiments, the RPE cell preparations are resistant to variations in storage temperature while maintaining robust viability and cell growth index.

[0390]

[0246] Provided herein is an RPE cell preparation comprising a population of cells in a 1 : 1 (v / v) mixture of (i) the cryopreservative formulation provided herein and (ii) a suspension solution comprising albumin, glucose and buffered saline, each at the same concentration as in the formulation, wherein the suspension solution does not contain a (non-albumin) cryoprotectant. In some embodiments, the RPE cell preparation is free of DMSO (DMSO-free). If DMSO is present in an RPE cell preparation provided herein, it is present at less than 10% (v / v), less than 5% (v / v), less than 1% (v / v), less than 0.1% (v / v), or less than 0.01% (v / v).

[0391]

[0247] In some embodiments, the population of cells is suitable for transplantation into the eye of a subject. The cells so formulated may be generated by directed differentiation of pluripotent or multipotent stem cells, including induced human pluripotent stem cells, human embryonic stem cells (hESC) and somatic cells (including transdifferentiated cells and stem cells).

[0392]

[0248] In some embodiments, the RPE cell preparation comprising a population of cells suitable for transplantation into the eye of a subject is suitable for injection into the eye of the127206-12220

[0393] subject. In some embodiments, such an RPE cell preparation may be used for treating retinal disease as defined herein.

[0394]

[0249] The RPE cells may be stable, terminally differentiated RPE cells that do not dedifferentiate to a non-RPE cell type. The RPE cells described herein may be functional RPE cells, characterized by the ability to integrate into the retina upon corneal, sub retinal, or other administration into a human or a non-human animal.

[0395]

[0250] The level of maturity of the RPE cells may be assessed by measuring expression of at least one of gp-100, PAX2, PAX6, and tyrosinase, or their respective expression levels.

[0396]

[0251] In some embodiments, a preparation comprising RPE cells in a cell cryopreservative formulation described herein, is provided, wherein the RPE cells maintain their phenotype following transplantation of the RPE cells to a subject, e.g., following injection of the preparation into the eye of the subject. The RPE cells may maintain their phenotype for the lifespan of the recipient after transplantation. For example, the RPE cells may maintain their phenotype following transplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Further, the RPE cells may maintain their phenotype following transplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. The RPE cells may maintain their phenotype following transplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The RPE cells may maintain their phenotype following transplantation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more years.

[0397]

[0252] Cells may be formulated in a preparation as provided herein for delivery in a pharmaceutically acceptable ophthalmic vehicle, such that the preparation is maintained in contact with the ocular surface for a sufficient time period to allow the cells to penetrate the affected regions of the eye, as for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary, lens, choroid, retina, sclera, suprachoridal space, conjunctiva, subconjunctival space, episcleral space, intracorneal space, epicomeal space, pars plana, surgically-induced avascular regions, the macula, the fovea, or an area of geographic atrophy.

[0398]

[0253] In some embodiments, a preparation is provided herein, in which cells are individual cells, a dissociated cell population, a cell monolayer, in clusters, spheres, or aggregates, sheets or any combination thereof which may be contained in an aqueous vehicle, gel, matrix, polymer or the like. In some embodiments, the cell population is a human population. In some embodiments, cells provided herein are administered in combination with a biomaterial,127206-12220

[0399] for example a biomaterial that can be polymerized. In some embodiments, the cell population is in combination with a biomaterial and / or is part of a 3D organoid.

[0400]

[0254] In some embodiments, a preparation is provided herein, in which cells are contained in a sheet of cells. For example, RPE cells may be contained in a sheet. For example, a sheet of cells comprising RPE cells may be prepared by culturing RPE cells on a substrate from which an intact sheet of cells can be released, e.g., a thermoresponsive polymer such as a thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surface, upon which cells adhere and proliferate at the culture temperature, and then upon a temperature shift, the surface characteristics are altered causing release of the cultured sheet of cells e.g., by cooling to below the lower critical solution temperature (LCST) (see da Silva et al., Trends Biotechnol. 2007 Dec;25(12):577-83; Hsiue et al., Transplantation. 2006 Feb 15;81 (3):473-6; Ide, T. et al. (2006); Biomaterials 27, 607-614, Sumide, T. et al. (2005), FASEB J. 20, 392-394; Nishida, K. et al. (2004), Transplantation 77, 379-385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187-1196 each of which is incorporated by reference herein in its entirety). The sheet of cells may be adherent to a substrate suitable for transplantation, such as a substrate that may dissolve in vivo when the sheet is transplanted into a host organism, e.g., prepared by culturing the cells on a substrate suitable for transplantation, or releasing the cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. An exemplary substrate potentially suitable for transplantation may comprise gelatin (see Hsiue et al., supra). Alternative substrates that may be suitable for transplantation include fibrin-based matrixes and others, he sheet of cells may be used in the manufacture of a medicament for the prevention or treatment of a disease of retinal degeneration.

[0401]

[0255] In some embodiments, cells provided herein (e.g., RPE cells) are administered as an implant or device or organoid. In certain embodiments, the device is a bioerodible implant for treating a medical condition of the eye comprising an active agent dispersed within a biodegradable polymer matrix, wherein at least about 75% of the particles of the active agent have a diameter of less than about 10 pm. The bioerodible implant is sized for implantation in an ocular region. The ocular region can be any one or more of the anterior chamber, the posterior chamber, the vitreous cavity, the choroid, the suprachoroidal space, the conjunctiva, the subconjunctival space, the episcleral space, the intracorneal space, the epicomeal space, the sclera, the pars plana, surgically-induced avascular regions, the macula, the retina and an area of geographic atrophy. The biodegradable polymer can be, for example, a poly(lactic-co-127206-12220

[0402] glycolic)acid (PLGA) copolymer. In certain embodiments, the ratio of lactic to glycolic acid monomers in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, 75 / 25 weight percentage, more preferably about 50 / 50. Additionally, the PLGA copolymer can be about 20, 30, 40, 50, 60, 70, 80 to about 90 percent by weight of the bioerodible implant. In certain preferred embodiments, the PLGA copolymer can be from about 30 to about 50 percent by weight, preferably about 40 percent by weight of the bioerodible implant. Cells can be transplanted in conjunction with a biocompatible polymer such as polylactic acid, poly (lactic-glycolic acid), PDLGA 50:50, PdLg A 85:15, and biodegradable membrane INION GTR® (mixture of biocompatible polymers). See U.S. Patent Nos. 6,331,313, 7,462,471 and 7,625,582. See also Hutala, et al. (2007) "In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: Direct contact studies". Journal of Biomedical Materials Research 83A (2): 407-413; Lu et al. (1998) J Biomater Sci Polym Ed 9: 1187205; and Tomita et al. (2005) Stem Cells 23: 1579-88. In some instances the biodegradable material can be polymerized.

[0403]

[0256] The volume of a pharmaceutical composition provided by some embodiments of this disclosure depends on factors such as the mode of administration, number of cells to be delivered, age and weight of the patient, and type and severity of the disease being treated. For example, if administered by injection, the volume of a pharmaceutical composition of cells of the disclosure may be about 1-1000 pL. In some embodiments, the volume may be about 1-200 pL. For example, the volume of a composition of the disclosure may be about 10-50, 20-50, 25-50, or 1-200 pL. The volume of a composition of the disclosure may be about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 450 pL, or higher.

[0404]

[0257] In some embodiments, the concentration of RPE cells in the cryopreservative formulation (or the cryopreserved RPE cell preparation) is about 300, 500, 1 x 103, 1.5 x 103, 2 x 103, 2.5 x 103, 3 x 103, 3.333 x 103, 3.5 x 103, 4 x 103, 4.4 x 103, 5 x 103, 5.5 x 103, 7.5 x 103, 1 x 104, 1.5 x 104, 2 x 104, 2.5 x 104, 3 x 104, 3.5 x 104, 4 x 104, 4.5 x 104, 5 x 104, 5.5 x 104, 6 x 104, 6.5 x 104, 7 x 104, 7.5 x 104, 8 x 104, 8.5 x 104, 9 x 104, 9.5 x 104, 1 x 105, 1.05 x 105, 1.1 x 105, 1.15 x 105, 1.2 x 105, 1.25 x 105, 1.3 x 105, 1.35 x 105, 1.4 x 105, 1.45 x 105, or 1.5 x 105cells / pL. In some embodiments, the concentration of cells in the cryopreservative formulation (or the cryopreserved cell preparation) is about 2,000-80,000 cells / pL, 2,000-70,000 cells / pL, 3,000-70,000 cells / pL, 3,000-60,000 cells / pL, 3,000-50,000 cells / pL, 3,000-10,000 cells / pL, 3,000-5,000 cells / pL, 3,000-4,000 cells / pL, 3,500 cells / pL, 10,000-100,000127206-12220

[0405] cells / pL, 10,000-90,000 cells / pL, 10,000-80,000 cells / pL, 10,000-70,000 cells / pL, 10,000-60,000 cells / pL, 10,000-50,000 cells / pL, 10,000-40,000 cells / pL, 10,000-30,000 cells / pL, 10,000-20,000 cells / pL, 20,000-100,000 cells / pL, 20,000-90,000 cells / pL, 20,000-80,000 cells / pL, 20,000-70,000 cells / pL, 20,000-60,000 cells / pL, 20,000-50,000 cells / pL, 20,000-40,000 cells / pL, 20,000-30,000 cells / pL, 30,000-100,000 cells / pL, 30,000-90,000 cells / pL, 30,000-80,000 cells / pL, 30,000-70,000 cells / pL, 30,000-60,000 cells / pL, 30,000-50,000 cells / pL, 30,000-40,000 cells / pL, 40,000-100,000 cells / pL, 40,000-90,000 cells / pL, 40,000-80,000 cells / pL, 40,000-70,000 cells / pL, 40,000-60,000 cells / pL, 40,000-50,000 cells / pL, 50,000-100,000 cells / pL, 50,000-90,000 cells / pL, 50,000-80,000 cells / pL, 50,000-70,000 cells / pL, 50,000-60,000 cells / pL, 60,000-100,000 cells / pL, 60,000-90,000 cells / pL, 60,000-80,000 cells / pL, 60,000-70,000 cells / pL, 70,000-100,000 cells / pL, 70,000-90,000 cells / pL, 70,000-80,000 cells / pL, 80,000-100,000 cells / pL, 80,000-90,000 cells / pL, 90,000-100,000 cells / pL, 10,000-150,000 cells / pL, 20,000-150,000 cells / pL, 30,000-150,000 cells / pL, 40,000-150,000 cells / pL, 50,000-150,000 cells / pL, 60,000-150,000 cells / pL, 70,000-150,000 cells / pL, 80,000-150,000 cells / pL, 90,000-140,000 cells / pL, 100,000-150,000 cells / pL, 100,000-130,000 cells / pL, 120,000-150,000 cells / pL, or 120,000-125,000 cells / pL.

[0406]

[0258] In some embodiments, the preparation supports survival of the RPE cells in the population of cells during storage of the preparation. In some embodiments, the preparation supports survival of the cells during storage for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years at least 5 years or more. In some embodiments, the preparation supports survival of the cells during storage for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 1 week to about 8 weeks, about 1 week to about 4 weeks, about 1 week to about 2 weeks. In some embodiments, the preparation supports survival of the cells during storage for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, or about 1 day to about 7 days, about 1 day to about 5 days, about 1 day to about 3 days or about 1 day to about 2 days. The preparation may be stored at temperatures ranging from about -100 to about -200 °C, or about -135 °C to about -200 °C, without limitation.127206-12220

[0407]

[0259] In some embodiments, at least 30% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 40% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 50% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 55% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 60% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 65% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 70% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 80% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C. In some embodiments, at least 90% of the cells in the cell population are viable after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C . In some embodiments, the preparation supports maintenance of the plating efficiency of127206-12220

[0408] the population of cells during storage of the preparation. In some embodiments, after about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years or about 1-2 years or less of storage of the preparation at -100 to -200 °C, preferably at or less than -135 °C, the population of cells exhibits at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of its original plating efficiency, wherein the original plating efficiency refers to the plating efficiency of the population of cells at the beginning of the storage period. In some embodiments, the preparation is within a storage container. In some embodiments, the storage container is a vial, ampule, bottle, syringe, a cooler package, or other suitable storage container. In some embodiments, the preparation is within a vial. In some embodiments, the preparation is within a syringe.

[0409]

[0260] The RPE cell preparations provided herein, post-thaw, are suitable for administration to a subject following minimal processing (e.g. dilution). In some embodiments, the preparation consists essentially of cells, a cell population, or a tissue and a cryopreservative formulation as provided herein. In some embodiments, the preparation comprises one or more pharmaceutically active ingredients, for example, a preservative, an antioxidant, a radical scavenger, an immunosuppressant, a pro-angiogenic factor, an anti-angiogenic factor, a hormone (e.g., a growth hormone), or a cell nutrient or substrate supporting cell growth, survival, and implantation.

[0410]

[0261] Preservatives may include but are not limited to benzalkonium chloride (BAK), benzethonium chloride, chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, polyquatemium- 1 (POLYQUAD®), stabilized oxychloro complex (PURITE®) or methyl or propylparabens. Antioxidants and / or radical scavengers may include but are not limited to vitamin C, vitamin E, P-carotene, conenzyme Q-10, selenium, thioredoxin reductase, glutathione, a-tocopherol, L-cysteine and N-acetylcysteine (NAC), Ascorbic acid-2-phosphate (AAP), zinc (e.g., zinc oxide), copper (e.g., copper oxide), dimethyl sulfoxide (DMSO), glycerol, mannitol, and polyphenolic compounds. Immunosuppressants may include but are not limited to anti-lymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB® (anti-IL-2Ra receptor antibody), cyclosporin (cyclosporin A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, and tacrolimus, mesenchymal stem cells, and mycophenolate mofetil. Pro-127206-12220

[0411] angiogenic factors may include but are not limited to VEGF, HGF, KGF, bFGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF, TGF- 1, MMP-9, and MMP-2. Anti-angiogenic factors may include but are not limited to somatostatin, angiostatin, and endostatin, thrombospondin (TSP), pigment epithelium derived factor (PEDF), transforming growth factor beta (TGF-beta), ranibizurnab (LUCENTIS®) or bevacizumab (AVASTIN®), tissue inhibitors of metalloproteinases (TiMPs), Plasminogen Activator Inhibitor (PAI), prolactin, interferons, and angiopoietin 2. Hormones and hormone antagonists may include 17B -estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte stimulating hormone, chorionic gonadotropin, corticosteroid-binding globulin, corticosterone, dexamethasone, estriol, follicle stimulating hormone, gastrin 1, glucagon, gonadotropin, hydrocortisone, insulin, insulin-like growth factor binding protein. L-3,3',5'-triiodothyronine, L-3,3',5'-triiodothyronine, leptin, leutinizing hormone, L-thyroxine, melatonin, MZ4, oxytocin, parathyroid hormone. PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone binding globulin, thyroid stimulating hormone, thyrotropin releasing factor, thyroxine-binding globulin, and vasopressin. Cell nutrients may include but are not limited to albumin, B-27 supplement, fetal bovine serum (FBS), ethanolamine, fetuin, glutamine, insulin, peptone, purified lipoprotein material, sodium selenite, transferrin, vitamin A, vitamin B9, vitamin B 12, vitamin C, or vitamin E, glucose, galactose, riboflavin, thiamine, biotin, cholesterol, iron zinc, copper, selenium, magnesium, and tricarboxylic acid. Substrates supporting cell growth, survival, and implantation may include but are not limited to extracellular matrix from bovine corneal epithelium, Matrigel ™ (basement membrane substrate), or gelatin.

[0412]

[0262] Some aspects of this disclosure provide methods that comprise contacting a population of cells with a cell cryopreservation formulation provided herein. The population of cells may be a population of RPE cells. The population of cells may be generated by in vitro differentiation of pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells. The population of cells may be human cells or non-human cells.

[0413]

[0263] Some aspects of this disclosure provide methods comprising contacting a population of cells with a cell cryopreservation formulation provided herein to form a pharmaceutical composition, and then cryopreserving the pharmaceutical composition. The pharmaceutical compositions may be cryopreserved using any suitable process, including at any appropriate temperature. For example, the pharmaceutical compositions may be cryopreserved at temperatures between -100 °C and -200 °C. In some embodiments the pharmaceutical127206-12220

[0414] compositions are cryopreserved at temperatures below -125°C. In some embodiments the pharmaceutical compositions are cryopreserved at temperatures at or below 135°C. In some embodiments the pharmaceutical compositions are cryopreserved at temperatures below -150°C. The pharmaceutical compositions may be cryopreserved in liquid nitrogen or in the gas phase of liquid nitrogen.

[0415]

[0264] In some embodiments, the RPE cell preparation is in a cryopreserved form. The RPE cell preparation provided herein may be cryopreserved by being cryogenically frozen at any appropriate temperature known in the art. For example, in some embodiments, the RPE cell preparations provided herein are cryopreserved at temperatures between -100°C and -200°C. In some embodiments the RPE cell preparations provided herein are cryopreserved at temperatures below -125°C. In some embodiments the cell preparations provided herein are cryopreserved at temperatures at or below -135°C. In some embodiments the RPE cell preparations provided herein are cryopreserved at temperatures below -150°C. In some embodiments, the cryopreserved RPE cell preparations provided herein are stored in liquid nitrogen or in the vapor phase of liquid nitrogen.

[0416]

[0265] The cryopreserved RPE cell preparations are produced by combining a cell population, with the cryopreservative formulation provided herein, optionally dispersing the cell population in the formulation, and then slow-freezing (e.g., controlled-rate freezing) the mixture. Slow-freezing can be accomplished manually or by device (e.g., a controlled-rate freezer, as described in the Examples). In one embodiment, the preparation is frozen at a rate of -2 °C per minute until a desired, optionally intermediate, temperature is reached, such as for example -80 °C. Alternatively, the rate of controlled-rate freezing may be -1 °C per minute or -1.5 °C per minute, etc. The cryopreserved RPE cell preparation may be kept at the intermediate temperature for hours, days, or weeks, or longer. It may then be kept at a lower temperature. As an example, the preparation may be brought to -80 °C using a slow-freeze process, after which it may be stored at -80 °C overnight (or for up to about 12 or about 24 hours), after which it may be stored in the vapor phase of a liquid nitrogen tank (e.g., at about -125 °C). Once at the lower temperature, it may be stored there for days, weeks, months, or even years. It may even be transported, for example to a clinical site, while in the cryopreserved form at the lower temperature, if so desired. The temperature of the cryopreservative formulation can be, for example, between 0 °C and 25 °C, for example, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C. In some embodiments, the RPE cell preparation has been cryopreserved and thawed.127206-12220

[0417] Once an RPE cell preparation has been cryopreserved and thawed, it is considered a “postthaw” cell preparation. The RPE cell preparations provided herein may be thawed without the need for diluent, wash, or other manipulation.

[0418]

[0266] The following is a non-limiting process for preparing cells for cryopreservation using any one of the cryopreservation formulas provided herein.

[0419]

[0267] After RPE cells are harvested from in vitro cultures they may be washed one or more times. A suitable washing buffer comprises albumin (e.g., an about 5% (w / v) albumin buffered solution). After washing, the cell pellet may be resuspended in a suitable resuspension buffer that minimally comprises albumin and glucose, and may also comprise PBS such as DPBS. Typically, the resuspension buffer will comprise the same concentration of albumin and glucose as the cryopreservative formulation. As an example, the resuspension buffer and the cryopreservative formulation may both comprise about 2.5% (w / v) albumin, about 0.09% (w / v) glucose in a buffered saline. The resuspended cells may have a cell density in the range of for example about 200 cells per microliter to about 20,000 cells per microliter, including about 1000 cells per microliter to about 10,000 cells per microliter, and about 3000 cells per microliter to about 10,000 cells per microliter. In some specific examples, the cell density may be about 6000 cells per microliter to about 7000 cells per microliter, including about 6500 cells per microliter, about 6600 cells per microliter, or about 6700 cells per microliter.

[0420]

[0268] The resuspended cell mixture is then mixed 1 : 1 with one of the cryopreservation buffers provided herein. Such 1 : 1 dilution serves to half the cell density, and half the concentration of each of the cryoprotectants in the cryopreservative formulation.

[0421] Accordingly, the cell density for example may be in the range of about 100 cells per microliter to about 10,000 cells per microliter, including about 500 cells per microliter to about 5,000 cells per microliter, and about 1500 cells per microliter to about 5,000 cells per microliter. In some specific examples, the cell density may be about 2000 cells per microliter to about 4000 cells per microliter, including about 3000 cells per microliter. This RPE cell preparation may then be placed into freezing vials such as for example 2 mL CZ vials. Each vial may be filled with for example about 450 microliters of the cell preparation comprising about 1.5 million cells.

[0422]

[0269] Those vials may then be subjected to cryopreservation, for example using a controlled-rate freezer and program. One non-limiting program may comprise the following steps: (a) introduce the vial to a 4 °C environment, (b) hold the vial at 4 °C for 10 minutes,127206-12220

[0423] (c) ramp the temperature downwards at a rate of 2 °C per minute until -40 °C is reached, (d) ramp the temperature downwards at a rate of 10 °C per minute until -80 °C is reached, and optionally placement in liquid nitrogen (LN2) (-180 °C) thereafter.

[0424]

[0270] It is to be understood that the cell pellet may be directly resuspended in a cryopreservative formulation that has been diluted 1 : 1 with another solution provided the formulation and such solution comprise the same concentrations of albumin, glucose and buffering agent. Thus, in some instances, the resuspension buffer or the dilution solution are identical to the cryopreservative formulation except that the cryopreservative formulation comprises one or two, or optionally more, cryopreservatives and the resuspension buffer and dilution solution do not.

[0425]

[0271] When ready to be thawed, a cryopreserved vial may be placed in a 37 °C water bath for about 2-3 minutes. Thereafter, the thawed RPE cell preparation may be gently pipetted several times (e.g., 7-10 times) in the vial. The RPE cell preparation may be used directly and immediately, or it may be kept at 4 °C or at room temperature for 0-4 hours. In certain embodiments a cryopreserved RPE preparation is thawed at room temperature.

[0426]

[0272] The RPE cell preparation may also be evaluated for cell viability and cell growth potential (referred to herein as cell growth index). Viability may be measured from several aliquots of the thawed RPE cell preparation using any standard technique or instrumentation known in the art. Typically, viability may be measured within about 5 to about 30 minutes after thawing.

[0427]

[0273] Cell growth potential may be measured in a number of ways. In one approach, the cell density of the thawed RPE cell preparation may be adjusted to about 100-200 cells per microliter (e.g., about 160 cells per microliter), and about 80 cells per microliter may be seeded per well in a 96 well plate (e.g., an e-plate). These plates may then be incubated for 7 days at 37 °C and evaluation of cell growth may be monitored during that time. One metric that may be used is the time it takes the cells to triple in number. Cells are typically seeded within about 2 hours after thawing.

[0428]

[0274] In some instances, suitable RPE cell preparations are those demonstrating equal to or greater than about 75%, or equal to or greater than about 80%, or equal to or greater than about 85%, or equal to or greater than about 90%, or equal to or greater than about 95% viability. In some instances, suitable RPE cell preparations are those demonstrating a time to reach a cell growth index of 3 of less than or equal to about 100 hours, less than or equal to about 90 hours, less than or equal to about 80 hours, or less than or equal to about 70 hours.127206-12220

[0429] In some instances, suitable RPE cell preparations are those demonstrating equal to or greater than about 80% viability and a time to reach a cell growth index of 3 of less than or equal to about 100 hours. Other suitable RPE cell preparations include those demonstrating equal to or greater than about 90% viability and a time to reach a cell growth index of 3 of less than or equal to about 80 hours.

[0430]

[0275] In some embodiments, the RPE cell preparation is kept at room temperature (about 20 °C to about 25 °C). In some embodiments, the cell preparation is kept at about 4 °C. In some embodiments, the RPE cell preparation is kept at about -20 °C. In some embodiments, the RPE cell preparation is kept at about -70 °C. In some embodiments, the RPE cell preparation is kept at about -180 °C.

[0431]

[0276] In some embodiments, the RPE cell preparation has been cryopreserved, thawed, and is kept at room temperature (about 20 °C to about 25 °C). In some embodiments, the RPE cell preparation has been cryopreserved, thawed, and is kept at about 4 °C. In some embodiments, RPE cell preparation has been kept at room temperature (about 20 °C to about 25 °C) for about 1 to about 2 hours prior to cryopreservation at -180 °C. In some embodiments, the RPE cell preparation has been kept at about 4 °C for about 4 to about 7 hours, prior to cryopreservation at -180 °C. In some embodiments, RPE cell preparation has been kept at room temperature (about 20 °C to about 25 °C) for about 1 to about 2 hours, and at about 4 °C for about 4 to about 7 hours, prior to cryopreservation at -180 °C.

[0432]

[0277] In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 50%. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 60%. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70%. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 80%. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 90%. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 60% upon thawing after storage at a temperature lower than about -135°C. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% upon thawing after storage at a temperature lower than about -135°C. In some embodiments, cell viability is measured by an automated cell counter.

[0433]

[0278] In some embodiments, RPE cell populations of the disclosure have a residual pluripotent stem cell content of less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02% or less than 0.01%. In some embodiments,127206-12220

[0434] RPE cell populations of the disclosure have a residual pluripotent stem cell content of less than 0.01%. In some embodiments, residual pluripotent stem cell content is determined by ddPCR.

[0435]

[0279] In some embodiments, RPE cell populations of the disclosure have a residual human dermal fibroblast (HDF) content of less than 1%, less than 0.5%, less than 0.25%, less than 0.1%. In some embodiments, RPE cell populations of the disclosure have an HDF content of less than 0.25%. In some embodiments, residual pluripotent stem cell content is determined by ddPCR.

[0436]

[0280] In some embodiments, RPE cells of the disclosure show functional activity through phagocytosis of bioparticles. In some embodiments, the ratio of phagocytosis by RPE cells at 37 °C compared to 12-15°C is greater than 40%. In some embodiments, the ratio of phagocytosis by RPE cells at 37 °C compared to 12-15°C is greater than 50%. In some embodiments, the ratio of phagocytosis by RPE cells at 37 °C compared to 12-15°C is greater than 60%. In some embodiments, the ratio of phagocytosis by RPE cells at 37 °C compared to 12-15°C is greater than 70%. In some embodiments, the ratio of phagocytosis by RPE cells at 37 °C compared to 12-15°C is greater than 80%.

[0437]

[0281] In some embodiments, RPE cells of the disclosure reach a cell index of 3 in less than about 150 hours. In some embodiments, RPE cells of the disclosure reach a cell index of 3 in less than about 125 hours. In some embodiments, RPE cells of the disclosure reach a cell index of 3 in less than about 100 hours. In some embodiments, RPE cells of the disclosure reach a cell index of 3 in less than about 75 hours.

[0438]

[0282] In some embodiments, at least 50% of RPE cells within an RPE cell population of the disclosure express the marker gp-100. In some embodiments, at least 60% of RPE cells within an RPE cell population of the disclosure express the marker gp-100. In some embodiments, at least 70% of RPE cells within an RPE cell population of the disclosure express the marker gp-100. In some embodiments, at least 80% of RPE cells within an RPE cell population of the disclosure express the marker gp-100. In some embodiments, at least 90% of RPE cells within an RPE cell population of the disclosure express the marker gp-100. In some embodiments, expression of the gp-100 marker is determined by flow cytometry.

[0439]

[0283] The stability of an RPE cell population may be determined by RPE cell viability (e.g., using a cell counter). In some embodiments, RPE cell populations of the disclosure are stable after storage at a temperature lower than about -135°C for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least127206-12220

[0440] 36 months, at least 48 months, or at least 60 months. In some embodiments, RPE cell populations are stable after storage at a temperature lower than about -135°C for at least 60 months. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% after storage at a temperature lower than about -135°C for at least 12 months. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% after storage at a temperature lower than about -135°C for at least 24 months. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% after storage at a temperature lower than about -135°C for at least 36 months. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% after storage at a temperature lower than about -135°C for at least 48 months. In some embodiments, RPE cell populations of the disclosure have a cell viability of at least 70% after storage at a temperature lower than about -135°C for at least 60 months.

[0441]

[0284] The stability of RPE cells may also be determined by phagocytosis potency, or time taken to reach an appropriate cell index, for example between 1 and 15 or between 1 and 10, or between 1 and 5 or between 1 and 4, or between 1 and 3 or between 1 and 2, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0442]

[0285] Also provided herein is an RPE cell preparation comprising a population of cells in a formulation comprising albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants, and having equal to or greater than 80% cell viability and / or equal to or less than 110 hours to reach a cell growth index of 3 after storage at room temperature (about 20 °C to about 25 °C) for about 1 to about 2 hours and then at about 4 °C for about 4 to about 7 hours, optionally at room temperature for about 1.5 hours and then at 4 °C for about 4.5 hours or 6.5 hours; cryopreservation at -180 °C for about 1 to about 2 years, optionally for about 1 month to about 1 year; cryopreservation at -180 °C and then post-thaw at about 4 °C for about 3 to about 6 hours, optionally for about 4 hours; cryopreservation at -180 °C and then post- thaw at room temperature (about 20 °C to about 25 °C) for about 3 to about 6 hours, optionally for about 4 hours; cryopreservation at -180 °C, then at about - 20 °C for about 0.5 to about 1 hour, then cryopreservation at -180 °C, optionally then at about - 20 °C for about 0.5 to about 1 hour, then cryopreservation at -180 °C, or cryopreservation at -180 °C, then at about - 70 °C for about 2 to about 4 weeks, then cryopreservation at -180 °C.

[0443]

[0286] Some aspects of this disclosure provide methods for treating a disease or condition comprising (a) thawing a cryopreserved RPE cell preparation provided herein by diluting the RPE cell preparation with a diluent at a ratio in the range of about 1:1 to about 1:20, or about127206-12220

[0444] 1:2 to about 1:20, or about 1:3 to about 1:20, or about 1:4 to about 1:20, or about 1:5 to about 1:20, or about 1:4 to about 1:19, or about 1:4 to about 1:18, or about 1:4 to about 1:17, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 and 1:20 or more, for example 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100 to form a diluted RPE cell preparation, and (b) administering the diluted RPE cell preparation to the subject. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the RPE cell preparation is prepared by diluting the cryopreserved RPE cells with GS2 or GS2 plus diluent at a ratio of about 1:14. In some embodiments, the cryopreserved formulations may be thawed through simple addition of a diluent such as GS2 or GS2 Plus, following which they may be administered to a subject without the need for wash or other manipulation. In some embodiments, the RPE cell preparation is diluted with warmed diluent. In some embodiments, the RPE cell preparation is diluted with cold diluent. In some embodiments, the RPE cell preparation is pipetted gently after dilution. In some embodiments, the RPE cell preparation is pipetted vigorously after dilution. There were no differences between embodiments in which the RPE cell preparation was diluted with a warmed diluent and embodiments in which the cell preparation was diluted with cold diluent. There were also no differences in the viability for cells between embodiments in which the RPE cell preparations were pipetted gently after dilution and embodiments in which the RPE cell preparations were pipetted vigorously after dilution, for example into the storage container or any container into which the RPE cell preparations are transferred to for dilution. In some embodiments, the cryopreserved formulations may be thawed without addition of a diluent, following which they may be administered to a subject without the need for wash or other manipulation.

[0445] Exemplary Uses and Methods

[0446]

[0287] In some embodiments, the cell population so formulated is intended for clinical use in a regenerative medicine approach. For example, the cell population may be a cell population that can replace cells lost or degenerated in a subject, or repair or replace a tissue that has been damaged or is dysfunctional in a subject. For example, in some embodiments, the cell population may comprise a pluripotent stem cell, for example an iPS cell or an ES cell, a multipotent stem or progenitor cell, or a functional differentiated cell, or a population or tissue comprising such cells or a combination of such cells.127206-12220

[0447]

[0288] In some embodiments, the cryopreserved RPE cell preparation can be stored indefinitely or it can be stored for defined periods of time, and optionally it can be transported to a clinic or other end user location.

[0448]

[0289] The cryopreserved formulations may be stored in a frozen state, such as in the vapor phase of a liquid nitrogen tank, for an extended period of time, such as those recited herein and including but not limited to at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years at least 5 years or more with minimal impact on cell viability or cellular function, as measured for example by dye exclusion, re-plating efficiency, or repopulating capacity. Cells or cell populations cryopreserved and thawed according to the present disclosure may have a cell viability, re-plating efficiency, and / or repopulation capacity of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the viability, re-plating efficiency, and / or repopulation capacity prior to cryopreservation.

[0449]

[0290] In some embodiments, an RPE cell preparation to be administered comprises about 100 cells per microliter to about 10,000 cells per microliter.

[0450]

[0291] In some embodiments, the number of cells per vial is about 45,000 to about 4,500,000 cells per vial. In some embodiments, the number of cells per vial is about 150,000 cells per vial. In some embodiments, the number of cells per vial is about 500,000 cells per vial. In some embodiments, the number of cells per vial is about 750,000 cells per vial. In some embodiments, the number of cells per vial is about 1,000,000 cells per vial. In some embodiments, the number of cells per vial is about 1,250,000 cells per vial. In some embodiments, the number of cells per vial is about 1,500,000 cells per vial. In some embodiments, the number of cells per vial is about 1,750,000 cells per vial.

[0451]

[0292] Some aspects of this disclosure provide methods for treating a subject having a disorder or condition. In some embodiments, the subject has a disorder or condition that would benefit from a cell transplant. In some embodiments, the method comprises127206-12220

[0452] administering to a subject in need thereof an effective amount of the RPE cell preparation provided herein without washing or dilution post-thaw. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of the RPE cell preparation prepared according to a method provided herein without washing or dilution postthaw. In some embodiments, the RPE cell preparation is kept at about 4 °C for about 4 hours before administration to the subject. In some embodiments, the RPE cell preparation is kept at room temperature (about 20 °C to about 25 °C) for about 4 hours before administration to the subject. In some embodiments, the RPE cell preparation is administered in a cell dose effective to treat the disorder or condition in the subject. In some embodiments, the disorder or condition includes, but is not limited to a retinal disease as defined herein. In some embodiments, the method further comprises monitoring at least one symptom of the disease or disorder or condition in the subject, optionally in order to determine if such symptom is lessened in severity or frequency post-administration.

[0453]

[0293] Some aspects of this disclosure provide methods for treating a retinal disease, wherein the methods comprise administering an effective amount of an RPE cell preparation provided herein to the eye of a subject having a retinal disease as defined herein. In some embodiments, the subject has or is diagnosed with the retinal disease.

[0454]

[0294] The cryopreservative formulations provided herein are compatible with various cell types, cell populations, and tissues, including, but not limited to, adult stem and progenitor cells, differentiated cells, and populations and tissues comprising such cells.

[0455]

[0295] Significantly, it can be thawed through simple addition of a diluent, and then administered to a subject without further manipulation. The cryopreserved formulation is typically a low volume formulation. For example, it may have a volume of less than 1 mL, less than 500 p.L, less than 250 p.L, less than 100 p.L, or less than or about 50 pL or any volume disclosed herein. It may be diluted at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more, in order to arrive at a volume and cell concentration suitable for the intended clinical use. The ratio of cryoprotective formulation volume to diluent volume may be127206-12220

[0456] expressed as a ratio and may include but is not limited to ratios of about 1:1 to about 1:20, or about 1:2 to about 1:20, or about 1:3 to about 1:20, or about 1:4 to about 1:20, or about 1:5 to about 1:20, or about 1:4 to about 1:19, or about 1:4 to about 1:18, or about 1:4 to about 1:17, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9. 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1: 80, 1:85, 1:90, 1:95, 1:100 or more.

[0457]

[0296] The cryopreserved RPE cell preparation typically comprises cells at a high density such as but not limited to at least or about 3xl02per pL, or at least or about 103per pL, at least or about 2 x 103per pL, at least or about 3 x 103per pL, at least or about 3.5 x 103per pL, at least or about 5 x 103per pL, at least or about 1.5 x 104per pL, at least or about 5 x 104per pL, at least or about 105per pL, or at least or about 5 x 105per pL, or higher. As will be understood by the skilled person, the cell concentration and the intended use will guide the degree of dilution that is required prior to administration to a subject.

[0458]

[0297] The cryopreserved RPE cell preparations are produced by combining the cells, cell population, or tissue of interest in the cryoprotective formulation provided herein, optionally dispersing the cells, cell population, tissue in the formulation, and then slow-freezing the mixture. Slow-freezing can be accomplished manually or by device, as described in the Examples. In one embodiment, the preparation is frozen at a rate of -1 °C per minute until a desired, optionally intermediate, temperature is reached, such as for example -80 °C.

[0459] Alternatively, the rate of freezing may be -1.5 °C per minute or -2 °C per minute, etc. The cryopreserved RPE cell preparation may be kept at the intermediate temperature for hours, days, or weeks, or longer. It may then be kept at a lower temperature. As an example, the preparation may be brought to -80°C using a slow-freeze process, after which it may be stored at -80°C overnight (or for up to about 12 or about 24 hours), after which it may be stored in the vapor phase of a liquid nitrogen tank (e.g., at about -125°C). Once at the lower temperature, it may be stored there for days, weeks, months, or even years. It may even be transported, for example to a clinical site, while in the cryopreserved form at the lower temperature, if so desired. The temperature of the cryoprotective formulation can, for example, between 0 °C and 45 °C, for example, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C and about 40 °C.

[0460]

[0298] Some aspects of this disclosure provide methods for treating a subject having a retinal disease, disorder or condition as described herein, that would benefit from a cell transplant.127206-12220

[0461] The methods may include thawing a cryopreserved RPE cell preparation comprising a cryopreservative formulation as provided herein, and then administering the thawed RPE cell preparation to a subject in need thereof, optionally without dilution with a diluent. In some embodiments, the methods may include thawing a cryopreserved RPE cell preparation comprising a cryopreservative formulation as provided herein, by addition of a diluent, and then administering the thawed and diluted RPE cell preparation to a subject in need thereof. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Plus®, a dextran and HSA solution, PBS, DMEM, MEM, or albumin. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is GS2 or GS2 Plus. The subject may have or may have been diagnosed with a disease or disorder that can be treated by administering a cell, cell population, or tissue. The RPE cell preparation is administered in a cell dose effective to treat the disorder or condition in the subject. In some embodiments, the method further comprises monitoring at least one symptom of the disorder or condition in the subject, optionally in order to determine if such symptom is lessened in severity or frequency postadministration.

[0462]

[0299] The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a cell preparation may refer to the amount of the preparation that comprises a number of cells or amount of tissue that is sufficient to provide a therapeutic benefit to a subject having a disorder or condition, e.g., sufficient to improve vision in a subject with a retinal disease or disorder. As will be appreciated by the skilled artisan, the effective amount may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific disorder being treated, a specific symptom to be alleviated, or the cell or tissue being targeted, and on the subject’s age, gender, and general health status.

[0463] b. Kits

[0464]

[0300] Some aspects of this disclosure provide pharmaceutical packs and / or kits comprising (a) a cryopreserved pharmaceutical composition comprising cells of interest as provided herein; (b) a diluent such as but not limited to GS2 Diluent for Subretinal Injection or GS2 Plus Diluent for Subretinal Injection, and (c) instructions for adding the diluent to the composition, thereby thawing and diluting the composition, and rendering it suitable for administration to a subject. Those instructions may also provide that no washing or other127206-12220

[0465] manipulation of the cryopreserved composition is necessary post-thaw. The kit may further comprise a device for administering the composition such as a syringe and needle.

[0466]

[0301] Some aspects of this disclosure provide kits comprising (a) a cryopreserved pharmaceutical composition comprising cells of interest as provided herein; and (b) instructions for thawing the composition, and rendering it suitable for administration to a subject. Those instructions may provide that no diluting, washing, or other manipulation of the cryopreserved composition is necessary. The kit may further comprise a device for administering the composition such as a syringe, canula, and needle.

[0467]

[0302] Some aspects of this disclosure provide kits comprising (a) a diluent or clinical solution as provided hereinbelow; and (b) instructions for contacting an RPE cell population with the diluent or clinical solution of (a) to generate an RPE cell preparation; and (c) a container for the contacting of (b) and / or for storing the RPE cell preparation of (b). In some embodiments, the solution of (a) and the container of (c) are suitable for use of the RPE cell preparation of (b) for transplantation to a subject.

[0468] Also embraced by the present disclosure are pharmaceutical packs and / or kits.

[0469] Pharmaceutical packs and / or kits provided may comprise a pharmaceutical composition provided herein, a diluent, and instructions for thawing the pharmaceutical composition by diluting the pharmaceutical composition prior to administration to a subject, preferably without washing the cells prior to addition of the diluent. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Plus®, a dextran and HSA solution, PBS, DMEM, MEM, albumin, or any other appropriate diluent. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the pharmaceutical composition is cryopreserved. In some embodiments, the instructions further comprise instructions for thawing the cryopreserved pharmaceutical composition, and optionally for combining the composition with the diluent and then administering the resultant composition to a subject. In some embodiments, the pharmaceutical composition is not cryopreserved. In some embodiments, the instructions further comprise instructions for combining the composition with the diluent and then administering the resultant composition to a subject.

[0470] c. DMSO Cryopreservatives

[0471]

[0303] This disclosure provides novel cryoprotective formulations that minimally include a cryoprotectant and albumin. In certain embodiments the cryoprotective formulations minimally include a cryoprotectant, albumin and optionally a sugar. The cryoprotectant may127206-12220

[0472] be DMSO, glycerol, ethylene glycol, trehalose, or taurine. In some embodiments, the cryoprotectant is DMSO. The albumin may be human albumin. The sugar may be glucose. The formulation may further comprise a buffer or a buffered saline such as but not limited to phosphate-buffered saline (PBS). The formulation may have a pH that is about a physiological pH (e.g., in the range of 6-8). The cryoprotectant may be present in a range of about 1% to about 10% volume / volume (v / v). The cryoprotectant may be present in a range of about 2% to about 10% volume / volume (v / v). The cryoprotectant may be present in a range of about 3% to about 10% volume / volume (v / v). The cryoprotectant may be present in a range of about 4% to about 10% volume / volume (v / v), for example 4%, 5%, 6%, 7%, 8%, 9% or 10%. The albumin may be present in a range of about 2% to about 3% (w / v). The albumin may be present in a range of about 2% to about 10% (w / v). The albumin may be present in a range of about 2% to about 8% (w / v). The albumin may be present in a range of about 2% to about 7.5% (w / v). The albumin may be present in a range of about 3.5% to about 7.5% (w / v). The albumin may be present in a range of about 4% to about 7.5% (w / v). The albumin may be present in a range of about 4% to about 8% (w / v). The albumin may be present in a range of about 4% to about 8.5% (w / v). The albumin may be present in a range of about 4% to about 9% (w / v). The albumin may be present in a range of about 4% to about 10% (w / v). The sugar may be present in a range of about 0 - about 1.5% (w / v).

[0473]

[0304] In some embodiments, the cryopreservative formulation comprises about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The formulation may consist essentially of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The formulation may consist of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

[0474]

[0305] In some embodiments, the cryopreservative formulation comprises about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and buffer or buffered saline. In some embodiments, the cryopreservative formulation consists essentially of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and buffer or buffered saline. In some embodiments, the cryopreservative formulation consists of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and127206-12220

[0475] buffer or buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

[0476]

[0306] In some embodiments, the cryopreservative formulation comprises about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The formulation may consist essentially of about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The formulation may consist of about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and buffer or buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

[0477]

[0307] In some embodiments, the cryopreservative formulation comprises about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and buffer or buffered saline. In some embodiments, the cryopreservative formulation consists essentially of about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and buffer or buffered saline. In some embodiments, the cryopreservative formulation consists of about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v) and buffer or buffered saline.

[0478]

[0308] Formulations comprising about 2.5% (v / v) DMSO, about 50 mM trehalose, about 7.5% (w / v) dextran-40, about 2.5% albumin (w / v) and buffered saline were associated with a reduced incidence of multinucleated RPE cells having more than 4 nuclei per cell, following freezing and thawing. DMSO at the 2.5% v / v level was more effective at reducing the incidence of multinucleated RPE cells having more than 4 nuclei per cell, as compared to DMSO at the 1% v / v level, following freezing and thawing. Formulations comprising even higher levels of DMSO (e.g., 5%) and optionally also containing ethylene glycol (e.g., 5%) were associated with reduced incidence of multinucleated cells having more than 3 nuclei per cell. Formulations comprising 5% glycerol did not give rise to cells having more than 4 nuclei per cell.

[0479]

[0309] In some embodiments, the cryoprotectant is a cell-penetrating cryoprotectant such as DMSO, glycerol or ethylene glycol, all of which were found to be associated with multinucleation of RPE cells.127206-12220

[0480]

[0310] In some embodiments, the cryoprotectant is a cell-penetrating cryoprotectant such as DMSO, glycerol or ethylene glycol, all of which were found to be associated with reduced incidence of multinucleated RPE cells.

[0481]

[0311] Still other formulations comprise about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.09% glucose (w / v) and buffer or buffered saline. Other formulations comprise about 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and buffer or buffered saline. Other formulation comprise 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and buffer or buffered saline.

[0482]

[0312] Still other formulations comprise about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.6% glucose (w / v) and buffer or buffered saline. Other formulations comprise about 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and buffer or buffered saline. Other formulation comprise 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and buffer or buffered saline.

[0483]

[0313] Still other formulations comprise about 5% DMSO (v / v), about 7.5% albumin (w / v), about 0.6% glucose (w / v) and buffer or buffered saline. Other formulations comprise about 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and buffer or buffered saline. Other formulation comprise 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and buffer or buffered saline.

[0484]

[0314] The disclosure further contemplates that in any of the embodiments provided herein, “glycerol” could also encompass “glycerin” and conversely, in any of the embodiments provided herein, “glycerin” could also encompass “glycerol”.

[0485]

[0315] The cryopreserved cell preparation comprises RPE cells, 5% or 10% DMSO, 27 pg glucose, 750 pg recombinant human albumin, 110 pg sodium chloride, 26.6 pg sodium phosphate, 2.10 pg calcium chloride, 4.19 pg potassium chloride, 4.19 pg potassium phosphate monobasic, 2.10 pg magnesium chloride-6H20, 168 pg sodium chloride, and 24.1 pg sodium phosphate dibasic. The cryopreserved cell preparation may comprise, in some embodiments, the cells of interest (e.g., RPE cells), 5% or 10% DMSO. 0.9 pg / ml Glucose, 259.5 pg / ml Recombumin Elite (recombinant human albumin) and Dulbecco’s phosphate buffered saline as a buffering agent.127206-12220

[0486]

[0316] Upon thaw and dilution in a diluent as provided herein, the final cell preparation to be administered to a subject may comprise, in some embodiments, the cells of interest (e.g., RPE cells), sodium hyaluronate, sodium chloride, potassium chloride, sodium phosphate dibasic dodecahydrate, dextrose (anhydrous), calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium citrate dihydrate.

[0487]

[0317] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (w / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline, and is used to cryopreserve RPE cells. This formulation results in viability of the RPE cells, post thaw, of at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95%. It also results in a lower frequency of multi-nucleated cells. In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 10% DMSO (v / v), about 2.4% albumin (w / v), 0-1.5% glucose (w / v) and buffer.

[0488]

[0318] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer, and is used to cryopreserve photoreceptor rescue cells, photoreceptor cells or photoreceptor progenitor cells. The DMSO may be used at about 5% or about 10%. The cells may be frozen at a concentration of about 50 x 103cells per pL. The formulation results in improved viability of the cells, post-thaw, compared to a commercially available cryoprotective formulation, CS10.

[0489]

[0319] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline, and is used to cryopreserve corneal endothelial progenitor cells or corneal endothelial cells. The DMSO may be present at about 5% or about 10% (v / v). The cells may be frozen at a concentration of about 30 x 103cells per p L. The formulation results in improved viability of the cells, post-thaw, compared to the conventional cryoprotective formulation of 90% FBS (v / v) and 10% DMSO (v / v).

[0490]

[0320] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline. The albumin may be present at about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). The glucose may be present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.10% (w / v).127206-12220

[0491]

[0321] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline. The DMSO may be present at about 4% (v / v), about 5% (v / v), and about 6% (v / v). The albumin may be human albumin, including recombinant human albumin, and may be present at about 2% (w / v), or about 2.5% (w / v), or about 3% (w / v), or about 4% (w / v), or about 5% (w / v), or about 6% (w / v), or about 7% (w / v), or about 7.5% (w / v), or about 8% (w / v), or about 9% (w / v) or about 10% w / v). The glucose may be absent, or it may be present at about 0.08% (w / v) or about 0.09% (w / v), or about 0.1% (w / v). The buffered saline may be phosphate buffered saline.

[0492]

[0322] In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline. The albumin may be present at about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline. The albumin may be present at about 2% (w / v), about 5% (w / v), or about 7.5% (w / v). The glucose may be present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.10% (w / v).

[0493]

[0323] In some embodiments, the formulations comprise a cryoprotectant. Cryoprotectants are used to protect cells from damage during the freezing process. In some embodiments, the formulations comprise a cryoprotectant selected from dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the formulations comprise about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (volume / volume, v / v) DMSO. In some embodiments, the solution comprises about 4%-10%, about 4%-9.5%, about 4%-9%, about 4%-8.5%, about 4%-8%, about 4%-7.5%, about 4%-7%, about 4%-6.5%, about 4%-6%, about 4%-5.5%, about 4%-5%, about 4%-4.5%, about 4.5%-10%, about 4.5%-9.5%, about 4.5%-9%, about 4.5%-8.5%, about 4.5%-8%, about 4.5%-7.5%, about 4.5%-7%, about 4.5%-6.5%, about 4.5%-6%, about 4.5%-5.5%, about 4.5%-5%, about 5%- 10%, about 5%-9.5%, about 5%-9%, about 5%-8.5%, about 5%-8%, about 5%-7.5%, about 5%-7%, about 5%-6.5%, about 5%-6%, about 5%-5.5%, about 5.5%-10%, about 5.5%-9.5%, about 5.5%-9%, about 5.5%-8.5%, about 5.5%-8%, about 5.5%-7.5%, about 5.5%-7%, about 5.5%-6.5%, about 5.5%-6%, about 6%- 10%, about127206-12220

[0494] 6%-9.5%, about 6%-9%, about 6%-8.5%, about 6%-8%, about 6%-7.5%, about 6%-7%, about 6%-6.5%, about 6.5%- 10%, about 6.5%-9.5%, about 6.5%-9%, about 6.5%-8.5%, about 6.5%-8%, about 6.5%-7.5%, about 6.5%-7%, about 7%-10%, about 7%-9.5%, about 7%-9%, about 7%-8.5%, about 7%-8%, about 7%-7.5%, about 7.5%-10%, about 7.5%-9.5%, about 7.5%-9%, about 7.5%-8.5%, about 7.5%-8%, about 8%-10%, about 8%-9.5%, about 8%-9%, about 8%-8.5%, about 8.5%-10%, about 8.5%-9.5%, about 8.5%-9%, about 9%-10%, about 9%-9.5%, or about 9.5%- 10% (v / v) DMSO. In some embodiments, the formulations comprise about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9% or about 10% (volume / volume, v / v) DMSO.

[0495]

[0324] In some embodiments, the formulations comprise albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is recombinant human (rh) albumin. The term “recombinant albumin” is interchangeably used with the term “rA” or “rAlbumin”. The term “recombinant human albumin” is interchangeably used with the term “rHA”, “rHSA”, “rhAlbumin”. In some embodiments, the cryopreservative formulations comprises about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0% about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, or about 10.0% (w / v) albumin.127206-12220

[0496]

[0325] In some embodiments, the cryopreservative formulations comprise about 2.0%-10.0%, about 2.0%-9.5%, about 2.0%-9.0%, about 2.0%-8.5%, about 2.0%-8.0%, about 2.0%-7.5%, about 2.0%-7.0%, about 2.0%-6.5%, about 2.0%-6.0%, about 2.0%-5.5%, about 2.0%-5.0%, about 2.0%-4.5%, about 2.0%-4.0%, about 2.0%-3.5%, about 2.0%-3.0%, about 2.0%-2.5%, about 2.5%-10.0%, about 2.5%-9.5%, about 2.5%-9.0%, about 2.5%-8.5%, about 2.5%-8.0%, about 2.5%-7.5%, about 2.5%-7.0%, about 2.5%-6.5%, about 2.5%-6.0%, about 2.5%-5.5%, about 2.5%-5.0%, about 2.5%-4.5%, about 2.5%-4.0%, about 2.5%-3.5%, about 2.5%-3.0%, about 3.0%-10.0%, about 3.0%-9.5%, about 3.0%-9.0%, about 3.0%-8.5%, about 3.0%-8.0%, about 3.0%-7.5%, about 3.0%-7.0%, about 3.0%-6.5%, about 3.0%-6.0%, about 3.0%-5.5%, about 3.0%-5.0%, about 3.0%-4.5%, about 3.0%-4.0%, about 3.0%-3.5%, about 3.5%-10.0%, about 3.5%-9.5%, about 3.5%-9.0%, about 3.5%-8.5%, about 3.5%-8.0%, about 3.5%-7.5%, about 3.5%-7.0%, about 3.5%-6.5%, about 3.5%-6.0%, about 3.5%-5.5%, about 3.5%-5.0%, about 3.5%-4.5%, about 3.5%-4.0%, about 4.0%-10.0%, about 4.0%-9.5%, about 4.0%-9.0%, about 4.0%-8.5%, about 4.0%-8.0%, about 4.0%-7.5%, about 4.0%-7.0%, about 4.0%-6.5%, about 4.0%-6.0%, about 4.0%-5.5%, about 4.0%-5.0%, about 4.0%-4.5%, about 4.5%-10.0%, about 4.5%-9.5%, about 4.5%-9.0%, about 4.5%-8.5%, about 4.5%-8.0%, about 4.5%-7.5%, about 4.5%-7.0%, about 4.5%-6.5%, about 4.5%-6.0%, about 4.5%-5.5%, about 4.5%-5.0%, about 5.0%-10.0%, about 5.0%-9.5%, about 5.0%-9.0%, about 5.0%-8.5%, about 5.0%-8.0%, about 5.0%-7.5%, about 5.0%-7.0%, about 5.0%-6.5%, about 5.0%-6.0%, about 5.0%-5.5%, about 5.5%-10.0%, about 5.5%-9.5%, about 5.5%-9.0%, about 5.5%-8.5%, about 5.5%-8.0%, about 5.5%-7.5%, about 5.5%-7.0%, about 5.5%-6.5%, about 5.5%-6.0%, about 6.0%-10.0%, about 6.0%-9.5%, about 6.0%-9.0%, about 6.0%-8.5%, about 6.0%-8.0%, about 6.0%-7.5%, about 6.0%-7.0%, about 6.0%-6.5%, about 6.5%-10.0%, about 6.5%-9.5%, about 6.5%-9.0%, about 6.5%-8.5%, about 6.5%-8.0%, about 6.5%-7.5%, about 6.5%-7.0%, about 7.0%-10.0%, about 7.0%-9.5%, about 7.0%-9.0%, about 7.0%-8.5%, about 7.0%-8.0%, about 7.0%-7.5%, about 8.0%-10.0%, about 8.0%-9.5%, about 8.0%-9.0%, about 8.0%-8.5%, about 8.5%-10.0%, about 8.5%-9.5%, about 8.5%-9.0%, about 9.0%-10.0%, or about 9.5%-10.0% (w / v) albumin.

[0497]

[0326] In some embodiments, the cryopreservative formulations comprise about 2.0%-3.0%, about 2.0%-2.9%, about 2.0%-2.8%, about 2.0%-2.7%, about 2.0%-2.6%, about 2.0%-2.5%, about 2.0%-2.4%, about 2.0%-2.3%, about 2.0%-2.2%, about 2.0%-2.1%, about 2.1%-3.0%, about 2.1%-2.9%, about 2. l%-2.8%, about 2.1%-2.7%, about 2.1%-2.6%, about 2. l%-2.5%, about 2.1%-2.4%, about 2. l%-2.3%, about 2.1%-2.2%, about 2.2%-3.0%, about 2.2%-2.9%,127206-12220

[0498] about 2.2%-2.8%, about 2.2%-2.7%, about 2.2%-2.6%, about 2.2%-2.5%, about 2.2%-2.4%, about 2.2%-2.3%, about 2.3%-3.0%, about 2.3%-2.9%, about 2.3%-2.8%, about 2.3%-2.7%, about 2.3%-2.6%, about 2.3%-2.5%, about 2.3%-2.4%, about 2.4%-3.0%, about 2.4%-2.9%, about 2.4%-2.8%, about 2.4%-2.7%, about 2.4%-2.6%, about 2.4%-2.5%, about 2.5%-3.0%, about 2.5%-2.9%, about 2.5%-2.8%, about 2.5%-2.7%, about 2.5%-2.6%, about 2.6%-3.0%, about 2.6%-2.9%, about 2.6%-2.8%, about 2.6%-2.7%, about 2.7%-3.0%, about 2.7%-2.9%, about 2.7%-2.8%, about 2.8%-3.0%, about 2.8%-2.9%, or about 2.9%-3.0% (w / v) albumin.

[0499]

[0327] In some embodiments, the cryopreservative formulations comprise a buffer. A buffer refers to an agent that can maintain the pH of a solution, preparation or formulation relatively stable by neutralizing added acid or base. Typically, a buffer comprises a weak conjugate acid-base pair, i.e., either a weak acid and its conjugate base, or a weak base and its conjugate acid. In some embodiments, the buffer comprised in the cryopreservative formulations provided herein is a water-based salt solution comprising disodium hydrogen phosphate (Na2HPO4) and sodium chloride (NaCl). In some embodiments, the buffer comprised in the formulations provided herein further comprises potassium chloride (KC1) and potassium dihydrogen phosphate (KH2PO4). In some embodiments, the formulations comprise a buffered saline such as phosphate-buffered saline (PBS). In some embodiments, the buffer is Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, the buffer is 4-(2-hy droxy ethyl) -1 -piperazineethanesulfonic acid (HEPES). In some embodiments, the buffer is 3-(N-morpholino) propanesulfonic acid (MOPS). In some embodiments, the buffer is N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). In some embodiments, the buffer is 3-morpholino-2-hydroxypropanesulfonic acid sodium salt (MOPSO). In some embodiments, the buffer is N-tris(Hydroxymethyl) Methyl Glycine (Tricine). In some embodiments, the buffer is 2- [Tris (hydroxymethyl)methylamino]-l -ethanesulfonic acid (TES). In some embodiments, the buffer is 4- (2-Hy droxy ethyl)- 1 -piperazinepropanesulfonic acid (EPPS or HEPPS).

[0500]

[0328] In some embodiments, the buffer comprises divalent cations. Suitable divalent cations include, without limitation, e.g., Ca2+, Mg2+, Zn2+, Fe2+, Mn2+, Cr2+, Cu2+, Ba2+, and Sr2+. In some embodiments, the divalent cations comprise calcium. In some embodiments, the divalent cations comprise magnesium. In some embodiments, the divalent cations comprise a two or more different divalent cations, e.g., calcium and magnesium. In some embodiments, the buffer comprises calcium. In some embodiments, the buffer comprises a pharmaceutically acceptable calcium salt. In some embodiments, the buffer127206-12220

[0501] comprises magnesium. In some embodiments, the buffer comprises a pharmaceutically acceptable magnesium salt. In some embodiments of the solutions provided herein, the buffer comprises calcium chloride. In some embodiments, the buffer comprises calcium chloride dihydrate. In some embodiments, the buffer comprises magnesium chloride. In some embodiments, the buffer comprises magnesium chloride hexahydrate.

[0502]

[0329] In some embodiments, the cryopreservative formulations provided herein comprise glucose. In some embodiments, the solution comprises about 0%-5.0% (w / v) glucose. For example, in some embodiments, the cryopreservative formulation comprises about 0%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9% or about 5% (w / v) glucose. The amount of glucose can be expressed as a range. The disclosure contemplates and herein provides any range of glucose selected from a lower limit of any one of the following and a upper limit of any one of the following. Any combination of these is disclosed herein. The lower limit of the range includes any one of the following 0%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, and 4.9% and the range includes an upper limit of any of the following 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5% (w / v) glucose. This disclosure contemplates any range of glucose having any combination of lower limit as disclosed and upper limit as disclosed. For example, provided herein are ranges such as about 0-.5%, about 0-1%, about 0-1.5%, about 0-2%, about 0-2.5%, about 0-3%, about 0-3.5%, about 0-4%, about 0-4.5%, about 0-5%, about 0.5-1%, about 0.5-1.5%, about 0.5-2%, about 0.5-2.5%, about 0.5-3%, about 0.5-3.5%, about 0.5-4%, about 0.5-4.5%, about 0.5-5%,127206-12220

[0503] about 1-1.5%, about 1-2%, about 1-2.5%, about 1-3%, about 1-3.5%, about 1-4%, about 1-4.5%, about 1-5%, about 1.5-2%, about 1.5-2.5%, about 1.5-3%, about 1.5-3.5%, about 1.5-4%, about 1.5-4.5%, about 1.5-5%, about 2-2.5%, about 2-3%, about 2-3.5%, about 2-4%, about 2-4.5%, about 2-5%, about 2.5-3%, about 2.5-3.5%, about 2.5-4%, about 2.5-4.5%, about 2.5-5%, about 3-3.5%, about 3-4%, about 3-4.5%, about 3-5%, about 3.5-4%, about 3.5-4.5%, about 3.5-5%, about 4-4.5%, about 4-5%, and about 4.5-5% (w / v) glucose. In some embodiments, the solution comprises about 0-1.5% (w / v) glucose. It is to be understood that a range of about 1-1.5%, as an example, means about 1% to about 1.5%. A similar meaning is imparted to each and every range recited herein.

[0504]

[0330] In some embodiments, the formulation comprises about 4-10% (v / v) cell cryoprotectant, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer, optionally as a buffered saline. In some embodiments, the formulation comprises about 4-10% (v / v) cell cryoprotectant, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer, optionally as a buffered saline. In some embodiments, the formulation comprises about 4-6% (v / v) cell cryoprotectant, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer, optionally as a buffered saline.

[0505]

[0331] In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-6% (v / v) DMSO, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-6% (v / v) DMSO, about 2-8% (w / v) albumin, about 0 -1.5% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 7.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer.127206-12220

[0506]

[0332] This disclosure further contemplates any of the foregoing formulations comprising about 0% - 5% (w / v) glucose or comprising any one of the ranges specified herein, such as in the preceding paragraphs.

[0507]

[0333] In some embodiments, the formulations provided herein further comprise at least one further excipient. In some embodiments, the formulation comprises about lmM-500mM. about 5mM-400mM, about 10mM-300mM, about 25mM-250mM, about 50mM-200mM of the at least one further excipient. In some embodiments, the formulation comprises about 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM, 82mM, 83mM, 84mM, 85mM, 86mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, lOOmM, lOlmM, 102mM, 103mM, 104mM, 105mM, 106mM, 107mM, 108mM, 109mM, llOmM, lllmM, 112mM, 113mM, 114mM, 115mM, 116mM, 117mM, 118mM, 119mM, 120mM, 121mM, 122mM, 123mM, 124mM, 125mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM, 136mM, 137mM, 138mM, 139mM, 140mM, 141mM, 142mM, 143mM, 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 164mM, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171mM, 172mM, 173mM, 174mM, 175mM, 176mM, 177mM, 178mM, 179mM, 180mM, 181mM, 182mM, 183mM, 184mM, 185mM, 186mM, 187mM, 188mM, 189mM, 190mM, 191mM, 192mM, 193mM, 194mM, 195mM, 196mM, 197mM, 198mM, 199mM, 200mM, 201mM, 202mM, 203mM, 204mM, 205mM, 206mM, 207mM, 208mM, 209mM, 210mM, 211mM, 212mM, 213mM, 214mM, 215mM, 216mM, 217mM, 218mM, 219mM, 220mM, 221mM, 222mM, 223mM, 224mM, 225mM, 226mM, 227mM, 228mM, 229mM, 230mM, 231mM, 232mM, 233mM, 234mM, 235mM, 236mM, 237mM, 238mM, 239mM, 240mM, 241mM, 242mM, 243mM, 244mM, 245mM, 246mM, 247mM, 248mM, 249mM, or 250mM of the at least one further excipient. In some embodiments, the at least one further excipient is selected from the group consisting of taurine, trehalose, sucrose, lactose, propanediol, mannitol, sorbitol, proline, glutathione, glycine, alanine, or lysine. In some embodiments, the at least one further excipient is taurine. In some embodiments, the at least one further excipient is127206-12220

[0508] trehalose. In some embodiments, the at least one further excipient is 1,2 propanediol or 1,3 propanediol. In some embodiments, the at least one further excipient is propylene glycol.

[0509]

[0334] In some embodiments, the formulations comprise a polymeric excipient. In some embodiments, the formulations comprise about l%-20% of the polymeric excipient. In some embodiments, the formulations comprise about 3%-18%, about 5%- 15%, or about 7.5%-12.5%, of a polymeric excipient. In some embodiments, the formulations comprise about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, or about 20% of a polymeric excipient. In some embodiments, the polymeric excipient is selected from the g...

Claims

127206-12220CLAIMSWe Claim:

1. A method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising:(i) culturing pluripotent stem cells in a first differentiation medium to generate RPE progenitor cells,(ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells,(iii) expanding the RPE cells produced in step (ii),(iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock,(v) expanding the RPE cells of the cell stock, and(vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

2. A method for producing a population of retinal pigment epithelium (RPE) cells, the method comprising:(i) culturing pluripotent stem cells in a first differentiation medium to generate RPE progenitor cells,(ii) culturing the RPE progenitor cells in a second differentiation medium to generate RPE cells,(iii) expanding the RPE cells produced in step (ii) by passaging one time, (iv) harvesting and cryopreserving the RPE cells produced in step (iii) to generate a cell stock,(v) expanding the RPE cells of the cell stock by passaging at least one time, and(vi) harvesting and cryopreserving the RPE cells produced in step (v); thereby producing a population of RPE cells.

3. The method of any one of claims 1-2, wherein the pluripotent stem cells are human embryonic stem cells.127206-122204. The method of claim 3, wherein the human embryonic stem cells are genetically engineered to reduce their immunogenicity.

5. The method of any one of claims 1-2, wherein the pluripotent stem cells are human induced pluripotent stem cells.

6. The method of claim 4, wherein the human induced pluripotent stem cells are genetically engineered to reduce their immunogenicity.

7. The method of claim 6, wherein the human induced pluripotent stem cells comprise a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene.

8. The method of claim 7, wherein the human induced pluripotent stem cells lack substantial endogenous expression of class I and class II HLA genes9. The method of claim 6, wherein the human induced pluripotent stem cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G.

10. The method of claim 6, wherein the human induced pluripotent stem cells comprise one or more safety switch genes.

11. The method of claim 10, wherein the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch.

12. The method of any one of claims 1-11, wherein step (i) further comprises dissociating the RPE progenitor cells, fractionating the RPE progenitor cells to collect cell clusters, and dissociating the cell clusters of RPE progenitor cells into single cells.127206-1222013. The method of any one of claims 1-12, wherein step (ii) further comprises dissociating the RPE cells, fractionating the RPE cells to collect cell clusters, and dissociating the cell clusters of RPE cells into single cells.

14. The method of claim 12 or 13, wherein the dissociation step is carried out by treating the cells with a dissociation reagent.

15. The method of claim 14, wherein the dissociation reagent is selected from the group consisting of collagenase, accutase, chelator, trypsin, TrypLE, dispase, and combinations thereof.

16. The method of claim 15, wherein the collagenase is collagenase IV.

17. The method of claim 15 or 16, wherein the collagenase is animal origin free.

18. The method of any one of claims 1-17, wherein prior to step (i), the pluripotent stem cells are cultured on feeder cells in a medium that supports pluripotency.

19. The method of any one of claims 1-17, wherein prior to step (i), the pluripotent stem cells are cultured under feeder-free conditions in a medium that supports pluripotency.

20. The method of any one of claims 1-19, wherein step (i), step (ii), step (iii), and / or step (v) is performed in a non-adherent culture.

21. The method of any one of claims 1-19, wherein step (i), step (ii), step (iii), and / or step (v) is performed in an adherent culture.

22. The method of any one of claims 1-21, wherein in step (ii), step (iii), and / or step (v) the RPE cells are cultured on an extracellular matrix selected from the group consisting of gelatin, laminin, fibronectin, vitronectin, Matrigel, CellStart, and collagen.

23. The method of any one of claims 1-22, wherein the first differentiation medium and the second differentiation medium are the same media.127206-1222024. The method of any one of claims 1-22, wherein the first differentiation medium and the second differentiation medium are different media.

25. The method of any one of claims 1-23, wherein the first differentiation medium and the second differentiation medium is Embryoid Body Differentiation Medium (EBDM).

26. The method of any one of claims 1-25, wherein the first differentiation medium and / or the second differentiation medium comprises one or more differentiation agents selected from the group consisting of nicotinamide, a transforming factor-P (TGFP) superfamily member (e.g., activin A, activin B, and activin AB), nodal, anti-mullerian hormone (AMH), bone morphogenetic proteins (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth and differentiation factors (GDF)), WNT pathway inhibitor (e.g., CKI-7, DKK1), a TGF pathway inhibitor (e.g., LDN193189, Noggin), a BMP pathway inhibitor (e.g., SB431542), a sonic hedgehog signal inhibitor, a bFGF inhibitor, and an MEK inhibitor (e.g., PD0325901).

27. The method of any one of claims 1-26, wherein the RPE cells are expanded in step (iii) and / or step (v) by culturing the cells in a maintenance media.

28. The method of claim 27, wherein the maintenance media is RPE-Growth Medium / Maintenance Medium (RPE-GM / MM).

29. The method of claim 28, wherein the RPE-GM / MM is supplemented with FGF.

30. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose.

31. The method of claim 30, wherein the medium for cryopreservation does not comprise DMSO.127206-1222032. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising albumin, glucose, buffered saline, and one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, and ethylene glycol, and wherein the medium does not comprise DMSO.

33. The method of claim 32, wherein:(a) the albumin is present in a range of about 2% to about 8% (w / v);(b) the glucose is present in a range of about 0.01% to about 1.5% (w / v); and / or (c) the buffered saline is phosphate buffered saline.

34. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising about 4-10% (v / v) cryoprotectant, about 2-8% (w / v) albumin, about 0.1-1.5% glucose or 0.1-1.5% dextrose, and a buffer.

35. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprisinga) about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer; orb) about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

36. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising about 4-6% (v / v) cryoprotectant.

37. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising fetal bovine serum (FBS).

38. The method of claim 37, wherein the RPE cells are cryopreserved in a medium comprising 1-20% DMSO in FBS, 5-20% DMSO in FBS or 5-10% DMSO in FBS.127206-1222039. The method of any one of claims 1-29, wherein in step (iv) and / or step (vi) the RPE cells are cryopreserved in a medium comprising about 0.08-0.10% (w / v) glucose, or about 0.6% (w / v) glucose.

40. The method of any one of claims 1-39, further comprising a step of thawing the RPE cells and formulating the RPE cells in a medium comprising sodium hyaluronate, a tonicity agent, and a buffer.

41. The method of claim 40, wherein the tonicity agent is selected from the group consisting of D(+)-glucose anhydrous, sodium chloride, potassium chloride, calcium chloride dehydrate, and magnesium chloride hexahydrate.

42. The method of claim 40 or 41, wherein the buffer is selected from the group consisting of sodium acetate, trisodium citrate, and monosodium phosphate monobasic.

43. The method of any one of claims 40-42, wherein the RPE cells are formulated in a medium comprising sodium hyaluronate, D(+)-glucose anhydrous, sodium chloride, potassium chloride, calcium chloride dehydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, trisodium citrate dehydrate, monosodium phosphate monobasic dehydrate and / or sodium hydroxide.

44. The method of any one of claim 1-39, wherein said method does not comprise a step of formulating or diluting the RPE cells in a medium.

45. The method of any one of claims 1 and 3-44, wherein in step (iii) the RPE cells are expanded by passaging up to one time, two times, five times, or ten times.

46. The method of any one of claims 1-45, wherein in step (v) the RPE cells are expanded by passaging up to two times, five times, or ten times.

47. The method of any one claims 1-46, wherein the duration of culturing in step (i) is about 1 week to about 12 weeks.

48. The method of any one of claims 1-47, wherein the duration of culturing in step (ii) is about 1 week to about 8 weeks.127206-1222049. The method of any one of claims 12-48, wherein the cell clusters of RPE progenitor cells are about 40 pm to about 200 pm, or about 40 pm to about 100 pm in size.

50. The method of any one of claims 13-49, wherein the cell clusters of RPE cells are about 40 pm to 200 pm, or about 40 pm to about 100 pm in size.

51. The method of any one of claims 1-50, 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.

52. The method of any one of claims 1-51, wherein the RPE cells are human RPE cells.

53. The method of any one of claims 1-52, wherein the RPE cells express one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, TRYP1, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1.

54. The method of any one of claims 1-53, wherein the RPE cells express gp-10055. The method of any one of claims 1-54, 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)-l-60, and TRA-1-80.

56. A pharmaceutical composition comprising the population of RPE cells produced by the method of any one of claims 1-55.

57. A composition comprising a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO I ^wherein the RPE cells comprise an endogenous P2 microglobulin127206-12220(B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene.

58. The composition of claim 57, 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)-l-60, and TRA-1-80.

59. The composition of claim 57 or 58, wherein the RPE cells lack substantial endogenous expression of class I and class II HLA genes.

60. The composition of claim 59, wherein the RPE cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G61. The composition of claim 57 or 58, wherein the RPE cells comprise one or more safety switch genes.

62. The composition of claim 61, wherein the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch.

63. The composition of claim 57 or 58, further comprising a cryoprotectant solution.

64. The composition of claim 63, wherein the cryoprotectant solution comprises one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose.127206-1222065. The composition of any one of claims 57-64, wherein the cryoprotectant solution comprises 1-20% DMSO in fetal bovine serum; 5-20% DMSO in fetal bovine serum or 5-10% DMSO in fetal bovine serum66. The composition of claim 63, wherein the cryoprotectant solution does not comprise DMSO.

67. The composition of claim 63, wherein the cryoprotectant solution comprises albumin, glucose, buffered saline, and one or more cryopreservatives selected from the group consisting of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, and ethylene glycol, and wherein the medium does not comprise DMSO.

68. The composition of claim 63 wherein the cryoprotectant solution comprises albumin at a range of about 2% to about 8% (w / v), glucose at a range of about 0.01% to about 1.5% (w / v); and / or phosphate buffered saline.

69. The composition of claim 68, wherein the cryoprotectant solution further comprises about 4-10% (v / v) of DMSO.

70. The composition of claim 68, wherein the cryoprotectant solution further comprises about 4-10% (v / v) of one or more of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose.

71. The composition of claim 63, further comprising sodium hyaluronate, a tonicity agent, and a buffer.

72. The composition of claim 71, wherein the tonicity agent is selected from the group consisting of glucose, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride.

73. The composition of claim 71, wherein the buffer is selected from the group consisting of sodium acetate, trisodium citrate, and monosodium phosphate monobasic.

74. The composition of any one of claims 71-73, wherein the composition comprises sodium hyaluronate, glucose, sodium chloride, potassium chloride, calcium chloride,127206-12220magnesium chloride, sodium acetate, trisodium citrate, monosodium phosphate monobasic and / or sodium hydroxide.

75. The composition of any one of claims 57-74, wherein the composition comprises about 1 x 105- 1 x 107RPE cells.

76. A composition comprising:a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1, and comprising a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class II-related gene;about 2-8% (v / v) albumin;about 0.01% to about 1.5% (w / v) glucose;about 3-7% (v / v) DMSO; andphosphate buffered saline.

77. A composition comprisinga population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1, and comprising a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class II-related gene;sodium hyaluronate;glucose;sodium chloride;potassium chloride;calcium chloride;magnesium chloride;sodium acetate;127206-12220trisodium citrate; andmonosodium phosphate monobasic.

78. A composition comprising:a population of human stem cell-derived RPE cells expressing one or more markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, OTX2, PAX2, PAX6, a premelanosome protein (PMEL), gp-100, tyrosinase, and ZO1;about 1-4% (v / v) albumin;about 0.01% to about 1.5% (w / v) glucose;about 4-10% (v / v) of one or more of propanediol, dextran-40, proline, trehalose, glycerin, pentastarch, ethylene glycol, glycerol, 2-methyl-2-4-pentanediol (MPD), propylene glycol, hydroxyethyl starch, and sucrose; andphosphate buffered saline.

79. The composition of claim 78, wherein the RPE cells comprise a genetically engineered disruption of an endogenous P2 microglobulin (B2M) gene and / or a genetically engineered disruption in a human leukocyte antigen (HLA) class Il-related gene80. The composition of any one of claims 76-79, wherein the RPE cells lack substantial endogenous expression of class I and class II HLA genes.

81. The composition of any one of claims 76-80, wherein the RPE cells comprise one or more polynucleotides capable of encoding a single chain fusion HLA class I protein, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M linked to at least a portion of an HLA class I a chain selected from the group consisting of HLA-E , HLA-F and HLA-G.

82. The composition of any one of claims 76-81, wherein the RPE cells comprise one or more safety switch genes.

83. The composition of claim 82, wherein the one or more safety switch genes comprise a herpes simplex virus thymidine kinase (TK) safety switch.127206-1222084. A method of treating a subject with a retinal disease, or at risk of developing a retinal disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 56, or the composition of any one of claims 57-83, thereby treating the subject.

85. The method of claim 84, wherein the population of RPE cells is administered at a dose of about 10,000 cells / dose to about 500,000 cells / dose.

86. The method of claim 85, wherein the population of RPE cells is administered at a dose of about 50, 000, 100,000, 133,000, 167,000, 200,000, 233,000, 267,000, 300,000, 333,333, 367,000, or 400,000 cells / dose.

87. The method of any one of claims 84-86, wherein the population of RPE cells is administered via injection.

88. The method of claim 87, wherein the population of RPE cells is administered via an injection into the sub-retinal space of the eye.

89. The method of claim 88, wherein the population of RPE cells is administered via a sub-retinal non-fovea involving injection.

90. The method of claim 88, wherein the population of RPE cells is administered via a pars plana vitrectomy.

91. The method of any one of claims 84-90, wherein the retinal disease is selected from the group consisting of retinal degeneration, retinal damage, retinal detachment, dry age-related macular degeneration (AMD), wet AMD, geographic atrophy secondary to AMD, macular dystrophies with a Stargardt-type clinical presentation, choroideremia, retinitis pigmentosa, Leber congenital amaurosis, Best vitelliform macular dystrophy, Best vitelliform macular dystrophy, autosomal recessive bestrophinopathies (ARBs), Autosomal dominant vitreoretinochoroidopathy (ADViRC), Usher syndrome IB, Angioid streaks, myopic macular degeneration, and glaucoma.127206-1222092. The method of any one of claims 84-90, wherein the subject has severe visual impairment.

93. The method of claim 92, wherein the subject has a baseline best corrected visual acuity (BCVA) ranging from about 20 / 80 to about 20 / 500.

94. The method of claim 92, wherein the subject has a BCVA between light perception and 37 early treatment diabetic retinopathy study (ETDRS) letters.

95. The method of claim 92, wherein the subject has between 9 - 12 disc areas of geographic atrophy.

96. The method of any one of claims 84-95, wherein the subject has moderate visual impairment.

97. The method of claim 96, wherein the subject has a BCVA ranging from about 20 / 40 to about 20 / 200.

98. The method of claim 96, wherein the subject has a BCVA ranging from 38 to 65 ETDRS letters.

99. The method of claim 97, wherein the subject has between 1 - 8 disc areas of geographic atrophy.

100. The method of any one of claims 84-99, wherein the subject has geographic atrophy.

101. The method of claim 100, wherein administration of the composition or the pharmaceutical composition to a subject’s eye results in one or more of the following as compared to the untreated eye of the subject:improvement in visual acuity;improvement in retinal function;stabilization of geographic atrophy;stabilization of ellipsoid zone loss;127206-12220increase in treatment site cellularity;improvement in outer nuclear layer (ONL) structure; and / orphotoreceptor preservation.

102. The method of claim 91 or 92, wherein the subject is suffering, or prone to suffering, from a macular dystrophy with a Stargardt-type clinical presentation103. The method of claim 102, wherein the macular dystrophy with a Stargardt-type clinical presentation is Stargardt’s disease (STGD) or a STGD-like macular dystrophy.

104. The method of claim 102 or 103, wherein the subject has one or more symptoms selected from the group consisting of macular RPE loss and atrophy, macular photoreceptor loss and atrophy, accumulation of drusen-like macular deposits, decline of BCVA, and central vision loss.

105. The method of any one of claims 102-104, wherein administration of the composition or the pharmaceutical composition to the subject’s eye results in one or more of the following as compared to a control:improvement in visual acuity;improvement in retinal function;increase in treatment site cellularity;improvement in outer nuclear layer (ONL) structure; and / orphotoreceptor preservation.

106. The method of claim 105,wherein the control is measured in the subject prior to administration of the composition or the pharmaceutical composition;wherein the control is measured in a subject to whom the composition or the pharmaceutical composition have not been administered; and / orwherein the control is measured in an untreated eye of the subject.

107. The method of any one of claims 84-106, wherein the subject is a human subject.127206-12220108. The method of claim 107, wherein the subject is 6 years of age or older or 12 years of age or older.

109. The method of claim 107, wherein the subject is 18 years of age or older.

110. The method of claim 107, wherein the subject is 30 years of age or older, 40 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, 80 years of age or older, 85 years of age or older or 90 years of age or older.

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