Human RPE cell medicines and their uses

Abstract

To provide compositions for treating Stargardt's Macular Dystrophy (SMD) and Dry Age-Related Macular Degeneration (AMD).SOLUTION: A pharmaceutical composition comprises a plurality of retinal pigment epithelium (RPE) cells, and a pharmaceutically acceptable carrier, wherein the plurality of RPE cells have an average melanin content of less than 8 pg / cell.SELECTED DRAWING: Figure 1A-B

Description

Technical Field

[0001] Cross - reference to Related Applications This patent application claims the benefit of U.S. Provisional Application No. 61 / 559,521, filed Nov. 14, 2011; U.S. Provisional Application No. 61 / 724,047, filed Nov. 8, 2012; and U.S. Provisional Application No. 61 / 589,741, filed Jan. 23, 2012, each of which is hereby incorporated by reference in its entirety.

[0002] Human embryonic stem cells (hESCs) are considered a promising source of alternative cells for regenerative medicine (1). Despite significant scientific progress, hESCs are one of the most complex biological therapeutic entities ever proposed for clinical use to date (2). In addition to their biological dynamic complexity, numerous regulatory concerns, including the risk of teratoma formation and issues related to tissue incompatibility, have hindered clinical translation. Until cell reprogramming technologies such as somatic cell nuclear transfer (3) or induced pluripotent stem cells (4, 5) further develop, diseases affecting the eye and other immune - privileged sites are likely to be the first pluripotent stem cell - based therapies in patients. It is well - established that the subretinal space is protected by the blood - eye barrier and is characterized by antigen - specific inhibition of both cellular and humoral immune responses (6).

[0003] Within the retina, degeneration of the retinal pigment epithelium (RPE) leads to photoreceptor loss in diseases threatening diverse vision, including dry age-related macular degeneration (AMD) and Stargardt macular dystrophy (SMD), which are the two leading causes of blindness worldwide in adults and juveniles, respectively. Although both are currently incurable, there is evidence that transplantation of hESC-derived RPE can rescue photoreceptors and prevent vision loss in preclinical models of macular degeneration (7, 8). As its functions, the RPE maintains photoreceptor health by recycling photopigments, delivering, metabolizing, and storing vitamin A, phagocytosing the outer segments of photoreceptor cells, transporting iron and small molecules between the retina and choroid, and absorbing stray light to enable better image resolution (9, 10). In Royal College of Surgeons (RCS) rats, an animal model of vision deterioration caused by RPE dysfunction, subretinal transplantation of hESC-derived RPE led to large-scale photoreceptor rescue and improved vision (more than 100% above untreated controls) without signs of harmful lesions (7). The retinal pigment epithelium (RPE) is a layer of pigmented cells outside the retinal neurosensory epithelium, between the underlying choroid (the vascular layer behind the retina) and the overlying retinal photoreceptors (e.g., the rods and cones of the photoreceptors). The RPE is important for the function and health of the photoreceptors and the retina. The RPE recycles photopigments, delivers and metabolizes and stores vitamin A, phagocytoses the outer segments of rod photoreceptors, transports iron and small molecules between the retina and the choroid, maintains Bruch's membrane, and absorbs stray light to enable better image resolution, thereby maintaining photoreceptor function. See also Engelmann and Valtink (2004) "RPE Cell Cultivation". Graefe's Archive for Clinical and Experimental Ophthalmology 242(1):65-67 (Non-Patent Document 1); Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, in STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009) (Non-Patent Document 2). Degeneration of the RPE is associated with several visual degenerative diseases that result in photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt disease (macular dystrophy), and can cause retinal detachment, retinal dysplasia, or retinal atrophy. See, for example, WO 2009 / 051671 pamphlet for reference. Please refer to.

Background Art

[0004] The RPE cells, methods of making RPE cell compositions, and their uses, among other specific subjects, are incorporated herein by reference in their entireties to the following: U.S. Patent No. 7,736,896, which was filed as U.S. Patent Application No. 11 / 186,720 on July 20, 2005; U.S. Patent No. 7,795,025, which was filed as U.S. Patent Application No. 11 / 490,953 on July 21, 2006; U.S. Patent No. 7,794,704, which was filed as U.S. Patent Application No. 11 / 041,382 on January 24, 2005; U.S. Provisional Patent Application No. 60 / 538,964, which was filed on January 23, 2004; U.S. Provisional Patent Application No. 12 / 682,712, which was filed on October 10, 2010; U.S. Provisional Patent Application No. 60 / 998,668, which was filed on October 12, 2007; U.S. Provisional Patent Application No. 60 / 998,766, which was filed on October 12, 2007; U.S. Provisional Patent Application No. 61 / 009,908, which was filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 009,911, which was filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 367,038, which was filed on July 23, 2010; U.S. Provisional Patent Application No. 61 / 414,770, which was filed on November 17, 2010; International Patent Application No. PCT / US11 / 45232, which was filed on July 25, 2011; U.S. Provisional Patent Application No. 12 / 682,712, which was filed on December 14, 2010; International Patent Application No. PCT / US05 / 02273, which was filed on January 24, 2005; International Patent Application No. PCT / US2010 / 57056, which was filed on November 17, 2010 (published as WO 2011 / 063005 pamphlet); and U.S. Provisional Patent Application No. 61 / 262,002, which was filed on November 17, 2009, among other commonly-owned U.S. patent applications and patents. [Prior Art Documents] [Non-Patent Documents]

[0005] [Non-Patent Document 1] Engelmann and Valtink(2004)”RPE Cell Cultivation”. Graefe’s Archive for Clinical and Experimental Ophthalmology 242(1):65-67 [Non-Patent Document 2] Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, in STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009) [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] Transplantation of intact sheets and suspensions of primary RPE cells has been previously attempted in human subjects, but the results have been mixed with respect to both graft survival and visual improvement. (11-19). To date, no consistently effective human therapeutic agent using primary RPE cells has been reported. [Means for Solving the Problems]

[0007] This disclosure reports 1 / 2 phase clinical data that helps demonstrate the safety of human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells for treating Stargardt macular dystrophy (SMD) and dry age-related macular degeneration (dry AMD). Results are reported for two patients, the first cases in each of the 1 / 2 phase clinical trials. In addition to showing no adverse safety issues, structural evidence demonstrated that the hESC-derived cells survived and continued to persist throughout the reported study period. Both patients had measurable improvements in vision and it persisted for at least one year.

[0008] At any time after treatment, no overgrowth, tumorigenicity, ectopic tissue formation, or overt rejection was observed in either patient. Detailed clinical and diagnostic test evaluations were performed at multiple time points after transplantation. In stem cell-based therapies, particularly those based on hESC-derived stem cells, abnormal growth (or tumor formation) is considered a major safety concern due to their pluripotency; therefore, controlling the differentiation of hESCs is important. The reported results indicate that stem cell differentiation was well controlled in these patients. No adverse safety signals were detected.

[0009] Anatomical evidence of successful stem cell-derived RPE transplantation was observed clinically and by high-resolution imaging techniques in SMD patients. This evidence included increased pigmentation at the RPE level within the transplant area, starting at 1 week after transplantation and throughout the follow-up period. The transplanted stem cell-derived RPE appeared to engraft in the appropriate location and assume a normal RPE morphology. In dry AMD patients, no engraftment or increased pigmentation was detected. However, both patients showed some visual improvement during the 4-month follow-up period, which persisted for at least 1 year.

[0010] As detailed below, the visual acuity of the Stargardt disease patient improved from only light perception to 20 / 800 vision. Before treatment, the patient could not read any letters on the ETDRS visual acuity chart. However, 2 weeks after transplantation, she was able to start reading letters with the treated eye, which improved to 5 letters at 1 - 3 months and 15 letters at 1 year (20 / 500 vision).

[0011] There are several new drugs available for treating wet AMD, but there are currently no proven treatments for either dry AMD or Stargardt's disease. Despite the progressive nature of these conditions, the vision of both groups of patients appeared to improve after cell transplantation, even at the lowest doses. The applicants expect even more significant improvements if patients are treated earlier in the disease course, when more significant results might potentially be expected. Increasing the cell dose might also result in more significant improvements.

[0012] Human embryonic stem cells can potentially provide an excellent source of replacement tissue by generating unlimited numbers of healthy "young" cells that are potentially less immunogenic. The eye is a site with immune privilege due to protection of the subretinal space by a barrier between the blood and the eye, and as a result, only low transient doses of immunosuppression were used. No signs of rejection or inflammation were observed in any of the patients, and the physicians continue to monitor both groups of patients.

[0013] The results presented herein highlight the promise of stem cell therapy regenerative medicine for the realization of the potential to repair or replace tissue damaged by disease.

[0014] Extensive safety testing was performed on hESC-derived RPE cells prior to transplantation. The cells were confirmed to be free of animal and human pathogens, and sensitive assays were performed to eliminate the presence of any undifferentiated hESCs, which are risk factors for tumor formation, from the final product. Controlled hESC differentiation resulted in nearly 100 percent pure RPE. A central feature of hESCs is the ability to control the in vitro differentiation stage to maximize survival and functionality. The data herein indicate that the degree of RPE maturity and pigmentation may dramatically affect the subsequent attachment and growth of the cells after transplantation.

[0015] Both trials were prospective, non-blinded studies, and following subretinal transplantation into SMD and dry AMD patients, at month 12, which is the primary endpoint of the study, of hESC-derived RPE cells It is designed to determine safety and tolerance. In each clinical trial, 12 patients are enrolled in each cohort of 3 patients in an escalating dose format. Patients with SMD and dry AMD both received subretinal transplantation of fully differentiated RPE cells derived from hESCs at the lowest dose (50,000 cells).

[0016] In one aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of retinal pigment epithelial (RPE) cells and a pharmaceutically acceptable carrier, wherein the average melanin content of the plurality of RPE cells is less than 8 pg / cell. The RPE cells may be contained in a suspension, gel, colloid, substrate, matrix, scaffold, or implant.

[0017] The pharmaceutically acceptable carrier may comprise a sterile solution having an osmotic pressure of about 290 mOsm / kg to about 320 mOsm / kg, or about 300 mOsm / kg to 310 mOsm / kg or about 305 mOsm / kg. The pharmaceutically acceptable carrier may comprise a balanced salt solution. The balanced salt solution may comprise, consist of, or consist essentially of 7.14 mg of sodium chloride, 0.38 mg of potassium chloride, 0.154 mg of calcium chloride dihydrate, 0.2 mg of magnesium chloride hexahydrate, 0.42 mg of dibasic sodium phosphate, 2.1 mg of sodium bicarbonate, 0.92 mg of dextrose, 0.184 mg of glutathione disulfide (oxidized glutathione) per 1 mL of water, and hydrochloric acid and / or sodium hydroxide (to adjust the pH to about 7.4).

[0018] The volume of the pharmaceutical composition may be about 100 μL to 1000 μL, or at least about 150 μL. The pharmaceutical composition may be about 1,000 to about 1×10 9It may comprise individual viable RPE cells. The pharmaceutical composition may comprise from about 333 viable RPE cells / μL to about 2,000 viable RPE cells / μL, from about 444 viable RPE cells / μL to about 1766 viable RPE cells / μL, about 333 viable RPE cells / μL, about 444 viable RPE cells / μL, about 666 viable RPE cells / μL, about 888 viable RPE cells / μL, about 999 viable RPE cells / μL, or about 1,333 viable RPE cells / μL.

[0019] The concentration of RPE cells in the pharmaceutical composition may be sufficiently high such that about 30% or less of the RPE cells lose viability within 60 minutes, and optionally about 10% or less of the RPE cells lose viability within 4 hours. The concentration of the RPE cells may be at least about 1,000 cells / μL, at least about 2,000 cells / μL, about 1,000 - 10,000 cells / μL, or about 2,000 - 5,000 cells / μL.

[0020] The pharmaceutical may comprise cells that are not RPE cells at about 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or less than 0.0001%.

[0021] The average melanin content of the RPE cells is 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; 0.1 - 8 pg / cell, 0.1 - 7 pg / cell, 0.1 - 6 pg / cell, 0.1 - 5 pg / cell, 0.1 - 4 pg / cell, 0.1 - 3 pg / cell, 0.1 - 2 pg / cell, 0.1 - 1 pg / cell, 1 - 7 pg / cell, 0.5 - 6 pg / cell, or 1 - 5 pg / cell.

[0022] At least 50%, at least 60%, at least 70%, or at least 80% of the cells in the pharmaceutical composition may be bestrophin +. The cells in the pharmaceutical composition At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% may be PAX6+ and / or MITF+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be PAX6+ and / or bestrophin+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be ZO-1+. At least 50%, at least 60%, or at least 70% of the cells in the pharmaceutical composition may be PAX6+ and bestrophin+. At least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be PAX6+.

[0023] In an exemplary embodiment, about 1 or fewer cells per 1 million cells and optionally 2 or fewer cells per 9 million cells in the pharmaceutical composition may be positive for both OCT-4 and alkaline phosphatase (AP) expression.

[0024] The needle or injection cannula may contain at least a portion of the RPE cells. The concentration of the RPE cells may be from about 444 viable cells / μL to about 1,766 viable cells / μL upon loading into the needle or injection cannula. The concentration of viable RPE cells delivered from the needle or injection cannula may be from about 333 viable cells / μL to about 1,333 viable cells / μL. The needle or injection cannula diameter may be from about 0.3 mm to about 0.9. The needle or injection cannula diameter may be from about 0.5 to about 0.6 mm. The needle or injection cannula may comprise a tip having a diameter of from about 0.09 mm to about 0.15 mm. The cannula may be a MEDONE POLYTIP® cannula 25 / 38g (0.12 mm (38g) × 5 mm tip attached to 0.50 mm (25g) × 28 mm cannula) or a Synergetics Angled 39g injection cannula.

[0025] The RPE cells may comprise RPE cells that have been cryopreserved and thawed.

[0026] The RPE cells may be human cells.

[0027] The pharmaceutical composition may further comprise at least one angiogenesis inhibitor that is administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells. Exemplary angiogenesis inhibitors include pegaptanib sodium; aflibercept; bevacizumab; rapamycin; AGN-745; vitalanib; pazopanib; NT-502; NT-503; PLG101; CPD791; an anti-VEGF antibody or a functional fragment thereof; bevacizumab; ranibizumab; an anti-VEGFR1 antibody; an anti-VEGFR2 antibody; an anti-VEGFR3 antibody; IMC-1121(B); IMC-18F1; a fragment or domain of VEGF; a fragment or domain of a VEGFR receptor; VEGF-Trap (aflibercept); AZD-2171 (cediranib); a tyrosine kinase inhibitor (TKI); a TKI that inhibits VEGFR-1 and / or VEGFR-2; sorafenib (nexavar); SU5416 (semaxinib); SU11248 / sunitinib (sutent); vandetanib (ZD6474); Ly317615 (enzastaurin); an anti-α5β1 integrin antibody or a functional fragment thereof; brolucizumab; 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-1-(alkyl-amino)-propionic acid; (S)-2-[(2,4,6-trimethylphenyl)sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]carbonylaminopropionic acid; EMD478761; or RC * D(ThioP)C * (Arg-Cys-Asp-thioproline-Cys (the asterisk indicates cyclization by a disulfide bond through the cysteine residues) taste; 2-Methoxyestradiol; αVβ3 inhibitor; Angiopoietin 2; anti-angiogenic steroid and heparin; Angiostatin; Angiostatin-related molecule; anti-Cathepsin S antibody; antithrombin III fragment; Calreticulin; Canstatin; Carboxyamidotriazole; cartilage-derived angiogenesis inhibitor; CDAI; CM101; CXCL10; Endostatin; IFN-α; IFN-β; IFN-γ; IL-12; IL-18; IL-4; Linomide; Maspin; matrix metalloproteinase inhibitor; Meth-1; Meth-2; Osteopontin; Pegaptanib; Platelet factor-4; Prolactin; Proliferin-related protein; Prothrombin (kringle domain-2); Restin; Soluble NRP-1; Soluble VEGFR-1; SPARC; SU5416; Suramin; Tecogalan; Tetrathiomolybdate; Thalidomide; Lenalidomide; Thrombospondin; TIMP; TNP-470; TSP-1; TSP-2; Vasostatin; VEGFR antagonist; VEGI; Brolocizumab (M200); Fibronectin fragment or domain; Anastrozole; Lenvatinib (E7080); Motesanib (AMG706); Pazopanib (Votrient); inhibitor of VEGF; inhibitor of VEGFR1; inhibitor of VEGFR2; inhibitor of VEGFR2; inhibitor of α5β1 integrin; peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitor of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; IL-6 antagonist; anti-IL-6 antibody; and any combination thereof may be optionally selected in an amount sufficient to prevent or treat a proliferative (angiogenic) eye disease.

[0028] The RPE cells may be genetically modified. For example, the RPE cells may be generated from genetically modified pluripotent cells. The genetic manipulation may result in the production of one or more factors by the RPE cells that inhibit angiogenesis. Exemplary factors that inhibit angiogenesis include fibronectin fragments or domains; anastellin; specific anti-VEGF antibodies or functional fragments or domains thereof; specific anti-VEGF receptor antibodies or functional fragments or domains thereof; specific anti-α5β1 integrin antibodies or functional fragments or domains thereof; VEGF fragments or domains; VEGFR receptor fragments or domains; VEGF-Trap; and at least one factor selected from the group consisting of any combination thereof.

[0029] The production of the factors that inhibit angiogenesis may be regulated by an RPE-specific promoter. The RPE-specific promoter may be selected from the group consisting of the RPE65 promoter, the cathepsin D proximal promoter, and the VMD2 promoter.

[0030] The RPE cells may be generated from pluripotent cells. The pluripotent stem cells may be positive for the expression of one or more markers, which may comprise OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80. The pluripotent cells may be human pluripotent cells that may be cultured in a multilayer population or embryoid body for a time sufficient for pigmented epithelial cells to appear during the culture. The time sufficient for pigmented epithelial cells to appear during the culture may comprise at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, or at least about seven weeks, at least about eight weeks. The multilayer population or embryoid body may be cultured in a medium that may comprise DMEM. The medium may comprise, consist essentially of, or consist of EB-DM. The pigmented epithelial cells may be isolated and cultured to thereby generate a population of RPE cells. The isolation may comprise separating cells or cell clusters enzymatically, chemically, or physically from the culture, and selecting pigmented epithelial cells, or cell clusters that may comprise pigmented epithelial cells. The embryoid body may be cultured in suspension and / or as an adherent culture (e.g., suspension followed by adherent culture). The embryoid body cultured as an adherent culture may give rise to one or more growths comprising pigmented epithelial cells. The pluripotent stem cells have a reduced HLA antigen complexity. Prior to RPE formation, the pluripotent cells may be cultured on a substrate selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The substrate may comprise Matrigel™ (soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). The pluripotent stem cells have a reduced HLA antigen complexity. Prior to RPE formation, the pluripotent cells may be cultured on a substrate selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The substrate may comprise Matrigel™ (soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

[0031] A pharmaceutical composition that may comprise cells lacking substantial expression of one or more embryonic stem cell markers. The one or more embryonic stem cell markers may comprise OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

[0032] The RPE cells may be positive for the expression of one or more RPE cell markers. The one or more RPE cell markers may comprise RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and / or tyrosinase.

[0033] The RPE cells may be generated by a method comprising maintaining the RPE cells as quiescent cells for a time sufficient to obtain the average melanin content. The RPE cells may be generated by a method comprising maintaining the RPE cells as quiescent cells for a time sufficient to establish bestrophin expression in at least 50% of the RPE cells.

[0034] The pharmaceutical composition may be substantially free of mouse embryonic fibroblast (MEF) and human embryonic stem cells (hES).

[0035] The RPE may be generated by a method comprising culturing the RPE cells under conditions that increase the expression of one or more of the α-integrin subunits, such as α-integrin subunit 1, α-integrin subunit 2, α-integrin subunit 3, α-integrin subunit 4, α-integrin subunit 5, α-integrin subunit 6, or α-integrin subunit 9. The conditions may include exposure to manganese, exposure to an anti-CD29 antibody, exposure to the monoclonal antibody HUTS-21, exposure to the monoclonal antibody mAb TS2 / 16, and / or at least about 4 passages of the RPE cells.

[0036] The RPE cells meet at least one of the criteria listed in Table 5 and / or are manufactured in accordance with Good Manufacturing Practice (GMP).

[0037] The pharmaceutical composition may further comprise at least one immunosuppressive or immunomodulatory agent that is administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells. The immunosuppressive or immunomodulatory agent comprises one or more of mesenchymal stem cells, anti-lymphocyte globulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab (registered trademark) (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), daclizumab (registered trademark) (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab (registered trademark) (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil. It may be.

[0038] In one aspect, the present disclosure provides a kit comprising a pharmaceutical composition as described above and another container comprising a pharmaceutically acceptable diluent in a volume sufficient to dilute the plurality of RPE cells to a desired target concentration. The volume of the pharmaceutically acceptable diluent may be such that when the total volume of the pharmaceutically acceptable diluent is combined with the entirety of the plurality of RPE cells, it results in the plurality of RPE cells having the desired target concentration. The temperature of the pharmaceutically acceptable diluent may be from about 0 to 10 °C, optionally from about 2 to 8 °C. The temperature of the plurality of RPE cells, or of the pharmaceutically acceptable carrier containing the plurality of RPE cells, may be from about 0 to 10 °C, optionally from about 2 to 8 °C.

[0039] The kit may further comprise at least one immunosuppressive or immunomodulatory agent that may be administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells, and examples of the immunosuppressive or immunomodulatory agent include one or more of those listed above.

[0040] The kit may further comprise one or more angiogenesis inhibitors, such as one or more of the angiogenesis inhibitors listed above, that may be administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells.

[0041] In one aspect, the present disclosure provides a cryopreservation composition comprising a plurality of cryopreserved retinal pigment epithelial (RPE) cells having an average maturity level at the time of freezing such that the RPE cells, which may be recovered subsequent to thawing, have a seeding efficiency of at least about 60%. The seeding efficiency may be at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. The average maturity level may be determined by measuring the average melanin content of a cell population that is typical of the plurality of cryopreserved RPE cells. The average melanin content of the plurality of cryopreserved RPE cells may be less than 8 pg / cell.

[0042] In one aspect, the present disclosure provides a cryopreserved composition comprising a plurality of cryopreserved retinal pigment epithelial (RPE) cells, wherein the average melanin content of the plurality of cryopreserved RPE cells may be less than 8 pg / cell.

[0043] The cells may be contained in a cryopreservation medium. The cryopreservation medium may comprise one or more of DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), propylene glycol, and sucrose, such as, for example, about 5% to about 50% DMSO and about 30% to about 95% serum, and the serum may optionally be fetal bovine serum (FBS). The cryopreservation medium may comprise about 90% FBS and about 10% DMSO.

[0044] The RPE cells recovered subsequent to thawing may have a seeding efficiency of at least about 60%, such as at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0045] The cryopreserved composition may comprise from about 5,000 to about 1×10 8 viable RPE cells at the time of freezing, such as from about 200,000 to about 10,000,000, from about 20,000 to about 50,000,000, from about 250,000 to about 5,000,000, from about 500,000 to about 4,000,000, or from about 1,000,000 to about 4,000,000 live RPE cells at the time of freezing.

[0046] The RPE cells recovered subsequent to thawing may have a seeding efficiency of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% at a time point at least about 3, 6, 9, or 12 months after freezing.

[0047] At least 85% of the cells with viability during thawing may maintain viability when stored at 2-8°C for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, or up to 6 hours after thawing.

[0048] The cryopreservation composition may comprise less than about 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001% of cells that are not RPE cells.

[0049] The average melanin content of the RPE cells is 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; 0.1-8 pg / cell, 0.1-7 pg / cell, 0.1-6 pg / cell, 0.1-5 pg / cell, 0.1-4 pg / cell, 0.1-3 pg / cell, 0.1-2 pg / cell, 0.1-1 pg / cell, 1-7 pg / cell, 0.5-6 pg / cell, or 1-5 pg / cell.

[0050] In one embodiment, the average melanin content of the RPE cells may be less than 10 pg / cell. In one embodiment, the average melanin content of the RPE cells may be less than 9 pg / cell. In one embodiment, the average melanin content of the RPE cells may be less than 8 pg / cell. In one embodiment, the average melanin content of the RPE cells may be less than 7 pg / cell. In one embodiment, the average melanin content of the RPE cells may be less than 6 pg / cell. In one embodiment, the average melanin content of the RPE cells may be less than 5 pg / cell.

[0051] At least 50%, at least 60%, at least 70%, or at least 80% of the cells in the cryopreservation composition may be Bestrophin+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition may be PAX6+ and / or MITF+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition may be PAX6+ and / or Bestrophin+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition may be ZO-1+. At least 50%, at least 60%, or at least 70% of the cells in the cryopreservation composition may be PAX6+ and Bestrophin+. At least 95% or at least 99% of the cells in the cryopreservation composition may be PAX6+.

[0052] In the cryopreservation composition, optionally, there may be about 1 or fewer cells per 1 million cells in the cryopreservation composition, and optionally, 2 or fewer cells per 9 million cells may be positive for both OCT-4 and alkaline phosphatase (AP) expression.

[0053] The cryopreservation composition may further comprise at least one angiogenesis inhibitor, such as one or more of the angiogenesis inhibitors listed above, which may be administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells.

[0054] The RPE cells may be genetically modified. The RPE cells may be generated from pluripotent cells. The RPE cells may be generated from pluripotent cells that may be genetically modified. The genetic manipulation results in the production of one or more factors by the RPE cells that inhibit angiogenesis. The one or more factors that inhibit angiogenesis include at least one factor selected from the group consisting of fibronectin fragments or domains, anastellin, specific anti-VEGF antibodies or functional fragments or domains thereof, specific anti-VEGF receptor antibodies or functional fragments or domains thereof, specific anti-α5β1 integrin antibodies or functional fragments or domains thereof, VEGF fragments or domains, VEGFR receptor fragments or domains, VEGF-Trap, and any combination thereof, which are regulated by RPE-specific promoters such as the RPE65 promoter, the cathepsin D proximal promoter, and the VMD2 promoter.

[0055] The pluripotent stem cells may be positive for the expression of one or more markers comprising OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

[0056] The pluripotent cells may be human pluripotent cells that may be cultured in a multilayer population or embryoid body for a time sufficient for pigmented epithelial cells to appear during the culture.

[0057] The time sufficient for pigmented epithelial cells to appear during the culture may comprise at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, or at least about 7 weeks, at least about 8 weeks.

[0058] In one aspect, the present disclosure provides a method for generating retinal pigment epithelial (RPE) cells for use in a pharmaceutical, comprising: (a) culturing RPE cells under adherent conditions to form a substantially monolayer culture of pigmented RPE cells having a cobblestone morphology; and (b) harvesting the RPE cells from the culture for cryopreservation or formulation, wherein at the time of harvest, the population of harvested pigmented RPE cells has an average melanin content of less than 8 pg / cell.

[0059] At least 10 6 RPE cells may be harvested for cryopreservation or formulation. The RPE cells may be generated from pluripotent stem cells, which may optionally be human embryonic stem cells or human iPS cells.

[0060] The average melanin content may be determined for a cell population excluding the 5% of harvested RPE cells with the darkest pigment and the 5% of harvested RPE cells with the lightest pigment. The average melanin content 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; 0.1 - 8 pg / cell, 0.1 - 7 pg / cell, 0.1 - 6 pg / cell, 0.1 - 5 pg / cell, 0.1 - 4 pg / cell, 0.1 - 3 pg / cell, 0.1 - 2 pg / cell, 0.1 - 1 pg / cell, 1 - 7 pg / cell, 0.5 - 6 pg / cell, or 1 - 5 pg / cell.

[0061] In one aspect, the present disclosure provides: (a) culturing RPE cells under adherent conditions to form a substantially monolayer culture of pigmented RPE cells having a cobblestone morphology; (b) passaging the RPE cells at least once before the RPE cells reach an average melanin content of more than 8 pg / cell; and (c) optionally, after one or more passages, cryopreservation Provided is a method for generating retinal pigment epithelium (RPE) cells for use in a pharmaceutical, comprising the step of harvesting RPE cells for a pharmaceutical formulation, wherein the RPE cells have an average melanin content of less than 8 pg / cell at the time of harvest.

[0062] In one aspect, the present disclosure provides a method for generating retinal pigment epithelial (RPE) cells, comprising: (a) culturing pluripotent stem cells, which may optionally be human embryonic stem cells or human iPS cells, to form embryoid bodies (EBs), or culturing pluripotent stem cells to form a multilayered population; (b) culturing the multilayered cell population or EBs for a time sufficient for the appearance of pigmented cells, which may comprise brown pigment dispersed in the cytoplasm; and (c) isolating and culturing the pigmented cells of (b) to generate a cultured population containing RPE cells having an average pigment level. Step (b) may comprise culturing the embryoid bodies to form an adherent culture. Step (a) may comprise overgrowing the culture of pluripotent cells to thereby form a multilayered population. Step (a) may comprise culturing the pluripotent cells on a low-attachment substrate or culturing the pluripotent cells using the hanging drop method to thereby form embryoid bodies from the pluripotent cells. The pluripotent stem cells may be induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells, fetal stem cells, mesenchymal stem cells, postpartum stem cells, multipotent stem cells, or embryonic germ cells. The pluripotent stem cells may be human ES cells or human iPS cells. The pluripotent stem cells may be genetically modified. The genetic manipulation results in the production of factors that inhibit angiogenesis, such as those identified above, by the RPE cells. The medium in which embryoid bodies may be formed in step (a) and / or the medium in which pigmented cells may be cultured in step (c) may comprise DMEM. The embryoid bodies may be formed in step (a) and / or the pigmented cells may be cultured in step (c) and may comprise, consist essentially of, or consist of EB-DM. The medium in which the pigmented cells may be cultured in step (c) may comprise EB-DM. The pigmented epithelial cells may be cultured in step (c) and may comprise, consist essentially of, or consist of RPE-GM / MM. The culture period of step (b) may be at least about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or at least about 1, 2, 3, 4, 5, or 6 months.The medium used in step (a), (b), or (c) may be EB-DM, RPE-GM / MM, MDBK-GM, OptiPro SFM, VP-SFM, EGM-2, or MDBK-MM. Step (c) may comprise contacting the culture with an enzyme selected from the group consisting of trypsin, collagenase, dispase, papain, a mixture of collagenase and dispase, and a mixture of collagenase and trypsin, or may comprise mechanically disrupting or isolating the culture, or may comprise contacting the culture with EDTA or EGTA, thereby interfering with the attachment of said pigmented cells to the culture substrate. The pluripotent stem cells have a reduced HLA antigen complexity. The RPE cells may lack substantial expression of one or more embryonic stem cell markers. The one or more embryonic stem cell markers may be Oct-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, DPPA-2, and / or DPPA-4.

[0063] For example, to allow the outgrowths to grow, the embryoid bodies may subsequently be cultured as adherent cultures following their formation. The RPE cells may be positive for at least one RPE cell marker. The at least one RPE cell marker may be one or more of RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, or tyrosinase, or optionally may include PAX6 and bestrophin.

[0064] The method may further comprise culturing the RPE cells, for example as described above, under conditions that increase α integrin subunit expression thereof.

[0065] The EB may be formed in the presence of a rho-associated protein kinase (ROCK) inhibitor such as Y-27632. Prior to the formation of the RPE, the pluripotent cells may be cultured on Matrigel (trademark) (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

[0066] In one aspect, the present disclosure provides a pharmaceutical composition comprising RPE cells suitable for the treatment of retinal degeneration, which have an average melanin content of less than 8 pg / cell, maintain their phenotype in culture for at least about one month, maintain their phenotype for at least about one month after transplantation, are incorporated into the host after transplantation, do not substantially proliferate after transplantation, may be phagocytotic, deliver, metabolize or store vitamin A, transport iron between the retina and choroid after transplantation, adhere to Bruch's membrane after transplantation, absorb stray light after transplantation, have an increased expression of an α-integrin subunit, have a greater average telomere length compared to RPE cells derived from the provided human tissue, have a longer replicative lifespan in culture compared to RPE cells derived from the provided human tissue, have a greater expression of one or more α-integrin subunits compared to RPE cells derived from the provided human tissue, have a lower A2E content compared to RPE cells derived from the provided human tissue, have a lower lipofuscin content compared to RPE cells derived from the provided human tissue, exhibit less ultraviolet damage accumulation compared to RPE cells derived from the provided human tissue, or have at least one of the characteristics of containing a greater number of phagosomes compared to RPE cells derived from the provided human tissue. In one aspect, the present disclosure provides a pharmaceutical composition comprising RPE cells suitable for the treatment of retinal degeneration, which may have an average melanin content of less than 8 pg / cell, adhere to Bruch's membrane after transplantation, absorb stray light after transplantation, have a greater average telomere length compared to RPE cells derived from the provided human tissue, have a longer replicative lifespan in culture compared to RPE cells derived from the provided human tissue, have a lower A2E content compared to RPE cells derived from the provided human tissue, have a lower lipofuscin content compared to RPE cells derived from the provided human tissue, exhibit less ultraviolet damage accumulation compared to RPE cells derived from the provided human tissue, or have at least one of the characteristics of containing a greater number of phagosomes compared to RPE cells derived from the provided human tissue.

[0067] In one aspect, the present disclosure provides a method for treating a retinal degenerative condition, comprising administering to the eye of a subject in need thereof a pharmaceutical composition or kit comprising RPE cells, or a pharmaceutical manufactured according to the method described above, in an amount effective to treat the retinal degenerative condition.

[0068] The retinal degenerative condition may comprise choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt disease, pigmentary striations, or myopic macular degeneration. The administering step may comprise intravitreally administering the RPE cells to the eye in need thereof. The intravitreal administration may comprise injecting the RPE cells into the subretinal space. The intravitreal administration may optionally comprise injecting an aqueous solution, which is an isotonic solution and / or a saline solution, into the subretinal cavity to form a pre-bleb thereby, and removing the aqueous solution before administering the RPE cells into the same subretinal cavity. The injection may be through a needle or an injection cannula. The needle or injection cannula diameter may be from about 0.3 mm to 0.9 mm or about 0.5 to about 0.6 mm. The needle or injection cannula may comprise a tip having a diameter of about 0.09 mm to about 0.15 mm. The cannula may be a MEDONE POLYTIP® cannula 25 / 38g (0.12 mm (38g) × 5 mm tip with 0.50 mm (25g) × 28 mm cannula). The effectiveness of the treatment may be evaluated by determining one or more visual acuity results of slit-lamp biomicroscopy, fundus photography, IVFA, and SD-OCT, and best corrected visual acuity (BCVA). The method may improve the corrected visual acuity (BCVA) and / or increase the number of readable characters in the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart. The retinal degenerative condition may be dry AMD or Stargardt disease.

[0069] An effective amount for treating the retinal degenerative condition may be about 20,000 to 200,000 RPE cells, about 20,000 to 500,000 RPE cells, about 20,000 to 2,000,000 RPE cells, or at least about 20,000 RPE cells, or at least about 20,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, 200,000, or 500,000 RPE cells.

[0070] The subject may not be administered a corticosteroid prior to or simultaneously with the administration of the RPE cells, such as prednisone or methylprednisolone. The subject may not be administered a corticosteroid within at least 3, 6, 12, 24, 48, 72, or 96 hours prior to or simultaneously with the administration of the RPE cells. The subject may not be administered a corticosteroid within at least 1 hour prior to or immediately before or simultaneously with the administration of the RPE cells. The subject may not be administered a corticosteroid within at least 12, 24, 48, 72, or 96 hours subsequent to the administration of the RPE cells. The subject may not be administered a corticosteroid within at least 48 hours subsequent to the administration of the RPE cells.

[0071] The RPE cells may be administered to a patient in combination with one or more agents selected from the group consisting of an angiogenesis inhibitor, an antioxidant, an antioxidant cofactor, other factors contributing to increased antioxidant activity, macular xanthophyll, long-chain ω-3 fatty acids, an amyloid inhibitor, a CNTF agonist, an inhibitor of RPE65, a factor targeting A2E and / or lipofuscin accumulation, a downregulator or inhibitor of photoreceptor function and / or metabolism, an α2-adrenergic receptor agonist, a selective serotonin 1A agonist, a factor targeting C-5, a membrane attack complex (C5b-9) and any other optional drusen components, an immunosuppressant, and an agent for preventing or treating lipofuscin accumulation.

[0072] The one or more agents may be administered to the patient simultaneously with, prior to, and / or subsequent to the RPE cell preparation.

[0073] The composition, kit, or pharmaceutical may be used in the manufacture of a medicament for treating retinal pathologies such as choroideremia, diabetic retinopathy, dry age-related macular degeneration, wet age-related macular degeneration, retinal detachment, retinitis pigmentosa, Stargardt's disease, pigmentary striation, or myopic macular degeneration.

[0074] The pluripotent stem cells express one or more markers selected from the group consisting of OCT-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-80.

[0075] The RPE cells may have a replication lifespan that is longer compared to the replication lifespan of RPE cells obtained from other sources; an average telomere length that is at least 30 percent of the telomere length of hESC and / or human iPS cells (or the average of an hESC and / or human iPS cell population), or at least 40, 50, 60, 70, 80, or 90 percent of the telomere length of hESC and / or human iPS cells; longer than 4 kb, or 5, 6 An average terminal restriction fragment length (TRF) that is longer than 7, 8, 9, 10, 11, 12 or 13 kb, or may be 10 kb or more; an average lipofuscin content that is less than 50 percent of the average lipofuscin content of an equivalent number of RPE cells isolated from an adult eye, or may be less than 40, 30, 20 or 10 percent of the average lipofuscin content of an equivalent number of RPE cells isolated from an adult eye; an average N-retinylidene-N-retinylethanolamine (A2E) content that may be less than 50 percent of the average A2E content of an equivalent number of RPE cells isolated from isolated adult eyes, or may be less than 40, 30, 20 or 10 percent of the average A2E content of an equivalent number of RPE cells isolated from an adult eye; 10 5(100,000) cells may have an average N-retinylidene-N-retinylethanolamine (A2E) content of less than 50 ng per cell; at least 50 percent greater than the phagocytosis rate of the outer segments of photoreceptor cells (POS) of an equivalent number of RPE cells isolated from isolated adult eyes, or at least 75, 100, 150 or 200 percent greater than the phagocytosis rate of the POS of an equivalent number of RPE cells isolated from isolated adult eyes, the phagocytosis rate of POS; at least 20 percent of the total POS concentration after 24 hours, or may be at least 25, 30, 25, 40 or 50 percent of the total POS concentration after 24 hours, the phagocytosis rate of the outer segments of photoreceptor cells (POS); a reduced level of oxidative stress and / or DNA damage accumulation compared to RPE cells isolated from an adult host; may be at least 50 percent greater than the average proteasome activity of an equivalent number of RPE cells isolated from isolated adult eyes, or at least 60, 70, 80, 90 or 100 percent greater than the average proteasome activity of an equivalent number of RPE cells isolated from isolated adult eyes, the average proteasome activity; may be less than 50 percent of the average accumulation of ubiquitin conjugates of an equivalent number of RPE cells isolated from isolated adult eyes, or may be less than 40, 30, 20 or 10 percent of the average accumulation of ubiquitin conjugates of an equivalent number of RPE cells isolated from isolated adult eyes, showing one or more of the characteristics of the average accumulation of ubiquitin conjugates.

Brief Description of the Drawings

[0076]

Figure 1A - B

Figure 1C - D

Figure 1E - F

Figure 1G - H

Figure 1I

Figure 1J

Figure 1K

Figure 2A - C

Figure 2D - F

Figure 3A - B

Figure 3C - D

Figure 3E - F

Figure 3G

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13A - C

Figure 13D - G

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Mode for Carrying Out the Invention

[0077] This disclosure describes the first results for two patients in a prospective clinical trial exploring the safety and tolerability of these hESC-derived RPE in patients with dry AMD and Stargardt disease. The hESC-derived RPE cells did not show signs of rejection or tumorigenicity at the time of this report. Visual measurements suggest improvement in both patients. These results suggest that hESCs can serve as a potentially safe and inexhaustible source of RPE for effectively treating a range of retinal degenerative diseases.

[0078] Also described is a method for generating a population of hESC-derived RPE cells with advantageous properties. By controlling the differentiation pathway including the degree of gene and pigment expression, it has been demonstrated that cell survival, attachment, and growth after injection are significantly improved. Specifically, the data presented here show that the degree of RPE maturation and pigmentation dramatically affects the continued attachment and growth of cells in vitro. These results illustrate the advantages that may be obtained using cells differentiated from therapeutic hESCs compared to the use of primary cells. These results show that, in addition to being able to generate an unlimited number of healthy “young” cells with potentially reduced immunogenicity (20, 21), the in vitro differentiation stage can be controlled at the cellular and molecular levels to ensure safety, identity, purity, and efficacy prior to transplantation into patients.

[0079] We initiated two prospective clinical trials to determine the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt macular dystrophy (SMD) and dry age-related macular degeneration (AMD), leading causes of blindness in developed countries. Pre- and post-operative ophthalmic examinations including visual acuity, fluorescein angiography, optical coherence tomography (OCT), and visual field testing were performed on the first patient in each trial.

[0080] Controlled hESC differentiation resulted in a nearly 100% pure RPE population. Immediately after surgery, hyperpigmentation was visible at the transplant sites in both patients, and there was continued evidence that the cells had attached to and integrated into the native RPE layer. Signs of inflammation or hyperproliferation were not observed. Visual acuity measurements showed signs of improvement during the first two months. By two weeks, in the study eyes of the AMD patients, the best-corrected visual acuity (BCVA) improved from 20 / 500 before treatment to 20 / 200, with continued improvement on the Early Treatment Diabetic Retinopathy Study (ETDRS) acuity chart (20 / 200 - 20 / 320), and an increase in the number of letters read. The SMD patient improved from hand motion to counting fingers during the same period; at one and two months, the BCVA improved to 20 / 800. Before RPE transplantation, the patient could not read any letters on the ETDRS chart, but began reading letters at two weeks, which continued to improve during the study period (five letters at one and two months).

[0081] hESC-derived RPE cells did not show signs of rejection or tumorigenicity at the time of this report. Visual measurements demonstrate improvement in both patients.

[0082] Definitions For a complete understanding of the invention described herein, the following detailed description is provided. Various embodiments of the invention are described in detail and may also be further illustrated by the examples provided.

[0083] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but the appropriate methods and materials are as described below. The materials, methods, and examples are intended only to illustrate and are not intended to be limiting. The following terms and definitions are provided herein.

[0084] Throughout the description of this specification and the following claims, the meanings of "a", "an", and "the" include references to the plural, unless the context clearly dictates otherwise. As used in the description of this specification, unless the context clearly dictates otherwise, the meaning of "in" includes "in" and "on".

[0085] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" imply the inclusion of the recited integer or group of integers, but do not imply the exclusion of any other integer or group of integers.

[0086] "Effective amount", as used in this specification, broadly refers to an amount of a compound or cell that, when administered to a patient for treating a disease, is sufficient to effect such treatment. An effective amount may be an amount effective for a prophylactic method and / or an amount effective for prevention. An effective amount may be an amount effective to reduce, prevent the occurrence of signs / symptoms, reduce the severity of the occurrence of signs / symptoms, eliminate the occurrence of signs / symptoms, delay the progression of the occurrence of signs / symptoms, prevent the progression of the occurrence of signs / symptoms, and / or effect a prophylactic method for the occurrence of signs / symptoms. An "effective amount" may vary depending on the disease and its severity, and the age, weight, medical history, susceptibility, and existing medical condition of the patient being treated. For the purposes of this disclosure, the term "effective amount" is synonymous with "therapeutically effective amount".

[0087] "Embryo" or "embryonic", as used in this specification, broadly refers to a developing cell population that has not implanted in the uterine lining of the maternal host. "Embryonic cells" are cells isolated from or contained within an embryo. This also includes blastomeres obtained as early as the two-cell stage and aggregated blastomeres.

[0088] "Embryonic stem cells" (ES cells), as used herein, broadly refers to cells derived from the inner cell mass of a blastocyst or morula that are continuously passaged as a cell line. ES cells may be derived from fertilization of an egg by sperm or DNA, nuclear transfer, parthenogenesis, or by means of creating ES cells that are homozygous in the HLA region. ES cells may also refer to mammalian embryos at the zygote, blastomere, or blastocyst stage that result from the fusion of sperm and egg; nuclear transfer; parthenogenesis; or cells derived from the incorporation of reprogrammed chromatin into the plasma membrane for subsequent cell creation following chromatin reprogramming. Embryonic stem cells can be identified based on (i) their ability to differentiate cells into the three germinal layers, (ii) the expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to form teratomas when transplanted into immunodeficient animals, regardless of their origin or the specific method used to create them. The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remaining embryo (see, e.g., Chung et al., Cell Stem Cell. 2008 Feb 7;2(2):113-7; US Patent Application Publication No. 20060206953; US Patent Application Publication No. 2008 / 0057041, each of which is incorporated herein by reference in its entirety). The term also includes cells generated by somatic cell nuclear transfer, even when non-embryonic cells are used in the process. ES cells may be derived from fertilization of an egg by sperm or DNA, nuclear transfer, parthenogenesis, or by means of creating ES cells that are homozygous in the HLA region. ES cells are also cells derived from the fusion of sperm and egg, resulting in mammalian embryos at the zygote, blastomere, or blastocyst stage; nuclear transfer; parthenogenesis; or the incorporation of reprogrammed chromatin into the plasma membrane for subsequent cell creation following chromatin reprogramming. Examples of human embryonic stem cells of the present disclosure include, but are not limited to, MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. In certain embodiments, the human ES cells used to create RPE cells are derived and maintained in accordance with GMP standards.

[0089] As used herein, "embryo-derived cells" (EDC) broadly refers to cells derived from a morula, blastocyst-derived cells including those of the inner cell mass, the embryonic shield, or the epiblast, or the primitive endoderm, ectoderm, and mesoderm, and other pluripotent stem cells of the early embryo, and their derivatives. "EDC" also includes blastomeres, and cell populations from aggregated single blastomeres or embryos at various stages of development, but excludes human embryonic stem cells that have been passaged as a cell line.

[0090] As used herein, "macular degeneration" broadly refers to diseases characterized by progressive loss of central vision associated with abnormalities of Bruch's membrane, the neural retina, and the retinal pigment epithelium. Macular degeneration diseases include, but are not limited to, age-related macular degeneration, North Carolina macular dystrophy, Stargardt's fundus dystrophy, Stargardt disease, pattern dystrophy, Best disease, Malattia Leventinese, Doyne honeycomb choroidopathy, dominant drusen, and radial drusen.

[0091] As used herein, "pluripotent stem cells" broadly refers to cells that can proliferate in vitro for a long term or substantially indefinitely while maintaining their undifferentiated state, exhibit a stable (preferably normal) karyotype, and have the ability to differentiate into the three germ layers (i.e., ectoderm, mesoderm, and endoderm) under appropriate conditions.

[0092] As used herein, "pluripotent embryonic stem cells" (a) can induce teratomas when transplanted into immunodeficient (SCID) mice; (b) can differentiate into cell types of the three germ layers (e.g., ectoderm, mesoderm, and endoderm cell types); (c) express at least one molecular embryonic stem cell marker (e.g., Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA blastomere, and cell populations from aggregated single blastomeres or embryos at various stages of development, but excludes human embryonic stem cells that have been passaged as a cell line. Cells that express 4 surface antigens, NANOG, TRA 1 60, TRA 1 81, SOX2, and REX1 are broadly referred to. As additional examples, pluripotent cells may express OCT-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80. Exemplary pluripotent stem cells can be generated, for example, using methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst-stage embryos, as well as embryonic stem cells derived from one or more blastomeres of cleavage-stage or morula-stage embryos (optionally without destruction of the remaining embryo). Such embryonic stem cells can be generated from embryonic materials produced by fertilization or by asexual reproductive means including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In another embodiment, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct-4, Sox2, Nanog, and Lin28. In another embodiment, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In another embodiment, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. iPS cells can typically be identified by the expression of the same markers as embryonic stem cells, although the expression profile of certain iPS cell lines may vary.

[0093] "RPE cells", "differentiated RPE cells", "ES-derived RPE cells", and the like are used synonymously throughout the present specification and may broadly refer to RPE cells differentiated from pluripotent stem cells using, for example, the methods disclosed herein. The terms are used inclusively to refer to differentiated RPE cells regardless of the level of cell maturity and thus may encompass RPE cells at various maturity levels. RPE cells can be visually recognized by their cobblestone morphology and initial pigment appearance. RPE cells can also be molecularly identified based on the substantial absence of expression of embryonic stem cell markers such as Oct-4 and NANOG, as well as based on the expression of RPE markers such as RPE 65, PEDF, CRALBP, and bestrophin. For example, a cell may be considered positive for a given marker if predicted staining patterns such as nuclear localization of PAX6, plasma membrane localization of bestrophin in a polygonal pattern (indicating localization of bestrophin staining by a distinct line around the cell perimeter), ZO-1 staining present in tight junctions outlining the cell in a polygonal pattern, and MITF staining detected in nuclear confinement are observed. Unless otherwise specified, in the usage of the present specification, RPE cells refer to RPE cells differentiated in vitro from pluripotent stem cells.

[0094] "Mature RPE cells" and "mature differentiated RPE cells" are used synonymously throughout the present specification and may broadly refer to changes that occur subsequent to the initial differentiation of RPE cells. Specifically, RPE cells can be recognized to some extent based on the initial appearance of pigment, but after differentiation, mature RPE cells can be recognized based on high levels of pigmentation.

[0095] "Seeding efficiency", in the usage of the present specification, refers to the recovered cell fraction that can maintain viability upon thawing and attach to the culture substrate. For example, seeding efficiency is determined by thawing, washing, and (preferably) seeding the cells (onto gelatin); measuring the total cell number before seeding and the number of live cells after seeding It can be determined by measurement, and then as a fraction of all cells before seeding, the seeding efficiency can be calculated for those that are viable and attach to the substrate after seeding. As a more specific example, the seeding efficiency is determined by thawing the cells in a 37°C water bath with constant stirring (such as for 1 - 2 minutes or for a time sufficient for the cells to thaw), then washing the cells 3 times with phosphate - buffered saline (or another suitable washing solution), measuring the total number of cells including live and non - live cells (using a hemocytometer, etc.), seeding the cells onto gelatin supplemented with growth medium (such as RPE - GM), culturing the cells (preferably at 37°C), allowing the cells to attach to the gelatin for about 24 hours, and then measuring the live cell count (for example, using a hemocytometer where trypan blue exclusion is used to measure viability); then after seeding, dividing the number of live cells by the total number of cells before seeding to determine the seeding efficiency.

[0096] "Pigmentation" as used herein refers broadly to any level of pigment formation, such as the initial pigment formation that occurs when, for example, RPE cells differentiate from ES cells. Pigment formation may vary depending on the cell density and maturity of the differentiated RPE cells. The pigment formation of RPE cells may be the same as that of average RPE cells after the final differentiation of RPE cells. The pigment formation of RPE cells may be more abundant than that of average RPE cells after the final differentiation of RPE cells. The pigment formation of RPE cells may be less abundant than that of average RPE cells after the final differentiation.

[0097] A "sign" of a disease, as used herein, refers broadly to any abnormality detectable in a patient's examination that indicates the disease; an objective indicator of the disease as contrasted with a symptom, which is a subjective indicator of the disease.

[0098] A "symptom" of a disease, as used herein, refers broadly to any pathological phenomenon experienced by a patient that suggests the disease, or any deviation from normal in structure, function, or perception.

[0099] The terms "therapy", "therapeutic", "treating", "treat", or "treatment" as used herein in their broadest sense refer to the treatment of a disease, the inhibition or reduction of the progression of a disease or its clinical symptoms, and / or the regression of a disease or its clinical symptoms. Therapy includes disease prevention methods, prevention, treatment, cure, correction, reduction, alleviation, and / or the provision of remission of a disease, a sign, and / or a symptom. Therapy includes the alleviation of signs and / or symptoms in a patient having ongoing disease signs and / or symptoms (e.g., blindness, retinal degeneration). Therapy also includes "prevention methods" and "prevention". Prevention methods include preventing the occurrence of a disease in a patient subsequent to treatment of the disease in the patient, or reducing the incidence or severity of a disease in the patient. The term "reduced" in the context of therapy refers in its broadest sense to a clinically significant reduction of signs and / or symptoms. Therapy includes treating recurrence or recurrent signs and / or symptoms (e.g., retinal degeneration, vision loss). Therapy always includes, but is not limited to, preventing the appearance of signs and / or symptoms, reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Therapy includes the treatment ("maintenance") of chronic diseases and the treatment of acute diseases. For example, treatment includes treating or preventing recurrence or relapse of signs and / or symptoms (e.g., blindness, retinal degeneration).

[0100] As used herein, the term "corticosteroid" refers to a class of steroid hormones that bind to glucocorticoid receptors, including natural and synthetic corticosteroids, analogs, etc. Exemplary corticosteroids include prednisone, hydrocortisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, fluticasone (including fluticasone propionate (FP)), budesonide, ciclesonide, mometasone , and include, but are not limited to, flunisolide.

[0101] RPE Cell Preparations and Combination Therapies The present disclosure provides RPE cell preparations, including RPE cells, substantially purified RPE cell populations, pharmaceuticals comprising RPE cells, and cryopreserved preparations of RPE cells. The RPE cells described herein may be substantially free of at least one protein, molecule, or other impurity found in their native environment (e.g., "isolated"). The RPE cells may be mammalian cells, including human RPE cells. The present disclosure also provides human RPE cells, substantially purified human RPE cell populations, pharmaceuticals comprising human RPE cells, and cryopreserved preparations of human RPE cells. The preparations may be preparations comprising RPE cells derived from human embryonic stem cells, human iPS cell-derived RPE cells, and substantially purified (with respect to non-RPE cells) preparations comprising differentiated ES-derived RPE cells.

[0102] The RPE cells of the preparation may have a longer replicative lifespan compared to the replicative lifespan of RPE cells obtained from other sources (e.g., cultures derived from provided human tissues such as fetal, infant, pediatric, adolescent, or adult tissues). The replicative lifespan may be evaluated by measuring the population doubling number during culture before senescence of replication. For example, the RPE cells of the preparation may be at least 10 percent longer, preferably at least 20, 30, 40, 50, 60, 70, 80, 90, 100 percent or more longer, compared to the RPE population derived from the provided human tissue.

[0103] The RPE cells of the preparation may have an average telomere length that is at least 30 percent of the telomere length of hESCs and / or human iPS cells (or the average of the hESC and / or human iPS cell population), preferably reaching at least 40, 50, 60, 70, 80, or even 90 percent of the telomere length of hESCs and / or human iPS cells (or the average of the hESC and / or human iPS cell population). For example, the hESCs and / or human iPS cells (or the hESC and / or human iPS cell population) may be cells or a cell population from which the RPE cells were differentiated.

[0104] The RPE of the preparation may have a restriction enzyme terminal fragment length (TRF) that is longer than 4 kb, preferably longer than 5, 6, 7, 8, 9, 10, 11, 12, or even reaching 13 kb. In an exemplary embodiment, the RPE cells of the preparation may have a TRF of 10 kb or more.

[0105] The RPE cells of the preparation have an average lipofuscin content that is less than 50 percent of the average lipofuscin content of an equal number of RPE cells isolated from the eyes of an adult (e.g., a human adult patient aged 25 to 80 years old, more preferably an adult aged 50 to 80 years old), and more preferably less than 40, 30, 20, or 10 percent of the average lipofuscin content of an equal number of RPE cells isolated from the eyes of an adult.

[0106] The RPE cells of the preparation have an average N-retinylidene-N-retinylethanolamine (A2E) content that is less than 50 percent of the average A2E content of an equal number of RPE cells isolated from the eyes of an adult (e.g., a human adult patient aged 25 to 80 years old, more preferably an adult aged 50 to 80 years old), and more preferably less than 40, 30, 20, or 10 percent of the average A2E content of an equal number of RPE cells isolated from the eyes of an adult.

[0107] The RPE cells of the preparation may be judged from the integrated peak intensity (as described in Sparrow et al., Invest. Ophthalmol. Vis. Sci. November 1999 vol.40 no.12, pg.2988-2995) and have an average N-retinylidene-N-retinylethanolamine (A2E) content of less than 50 ng per (100,000) cells, more preferably less than 40 ng, 30 ng, 20 ng, 10 ng, and may be 5 ng per 10 cells. 5 (100,000) cells, more preferably less than 40 ng, 30 ng, 20 ng, 10 ng, and may be 5 ng per 10 cells. 5

[0108] The RPE cells of the preparation may have a phagocytosis rate of photoreceptor outer segments (POS) that is at least 50 percent greater than the phagocytosis rate of an equivalent number of RPE cells isolated from the eyes of adults (i.e., human adult patients aged 25 to 80 years, more preferably adults aged 50 to 80 years), more preferably at least 75, 100, 150, 200 percent greater. POS phagocytosis can be evaluated using the protocol described in Bergmann et al. FASEB Journal March 2004 vol.18 pages 562-564 as an exemplary and non-limiting example.

[0109] The RPE cells of the preparation may have a phagocytosis rate of rod photoreceptor outer segments (POS) that is at least 20 percent of the total POS concentration after 24 hours, more preferably at least (at least than) 25, 30, 25, 40, or even up to 50 percent of the total POS concentration after 24 hours. POS phagocytosis can be evaluated using the protocol described in Bergmann et al. FASEB Journal March 2004 vol.18 pages 562-564 as an exemplary and non-limiting example.

[0110] The RPE cells may exhibit reduced levels of oxidative stress and / or accumulation of DNA damage compared to RPE cells isolated from an adult host.​​

[0111] The RPE cells of the formulation have an average proteasome activity that is at least 50 percent greater than the average proteasome activity of an equivalent number of RPE cells isolated from the eyes of adults (i.e., human adult patients aged 25 to 80 years, more preferably adults aged 50 to 80 years), and more preferably at least 60, 70, 80, 90, or 100 percent of the average proteasome activity of an equivalent number of RPE cells isolated from the eyes of adults. The proteasome activity can be measured using, by way of illustrative and non-limiting example, succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVY-AMC) for chymotrypsin-like activity, N-t-butyloxycarbonyl-Leu-Ser-Thr-Arg-amidomethylcoumarin (LSTR-AMC) for trypsin-like activity, and benzyloxycarbonyl-Leu-Leu-Glu-amidomethylcoumarin (LLE-AMC) for peptidylglutamyl-peptide hydrolase activity.

[0112] The RPE cells of the formulation have an average accumulation of ubiquitin conjugates that is less than 50 percent of the average accumulation of ubiquitin conjugates of an equivalent number of RPE cells isolated from the eyes of adults (e.g., human adult patients aged 25 to 80 years, more preferably adults aged 50 to 80 years), and more preferably less than 40, 30, 20, or 10 percent of the average accumulation of ubiquitin conjugates of an equivalent number of RPE cells isolated from the eyes of adults. The accumulation of ubiquitin conjugates can be evaluated using the protocol described in Zhang et al. Invest. Ophthalmol. Vis. Sci. August 2008 vol.49 no.8 3622-3630.

[0113] Preferably, one or more angiogenesis inhibitors may be administered in a therapeutically effective amount for preventing or treating an ocular disease such as an angiogenesis-related ocular disease, in combination with an RPE cell preparation. Exemplary ocular diseases include macular degeneration (e.g., wet AMD or dry AMD), diabetic retinopathy, and choroidal neovascularization. Exemplary angiogenesis inhibitors include, for example, pegaptanib sodium, aflibercept, bevacizumab, rapamycin, AGN-745, batranib (vitalanib), pazopanib, NT-502, NT-503, or PLG101, CPD791 (a di-Fab’ polyethylene glycol (PEG) conjugate that inhibits VEGFR-2), an anti-VEGF antibody or a functional fragment thereof (such as bevacizumab (Avastin®) or ranibizumab (Lucentis®), etc.), or an anti-VEGF receptor antibody (such as IMC-1121(B) (a monoclonal antibody against VEGFR-2), or IMC-18F1 (an antibody against the extracellular binding domain of VEGFR-1), etc.), and VEGF antagonists such as peptides, peptidomimetics, small molecules, chemicals, or nucleic acids that inhibit VEGF and / or VEGF receptors (VEGFRs, e.g., VEGFR1 (FLT1, FLT), VEGFR2 (KDR, FLK1, VEGFR, CD309), VEGFR3 (FLT4, PCL)). Additional exemplary VEGF activity inhibitors include fragments or domains of VEGFR receptors, an example of which is VEGF-Trap (aflibercept), which is a fusion protein of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 with the Fc fragment of IgG1. Another exemplary VEGFR inhibitor is AZD-2171 (cediranib), which inhibits VEGFR1 and 2. Additional exemplary VEGF antagonists include tyrosine kinase inhibitors (TKIs) such as sorafenib (Nexavar), SU5416 (Semaxinib), SU11248 / sunitinib (Sutent), and vandetanib (ZD6474), which have been reported to inhibit VEGFR-1 and / or VEGFR-2. Additional exemplary VEGF antagonists include Ly317615 (enzastaurin), which is thought to target downstream kinases involved in VEGFR signaling (protein kinase C).Additional exemplary angiogenesis inhibitors include anti-α5β1 integrin antibodies or functional fragments thereof (such as volociximab), peptides, peptidomimetics, small molecules, 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkyl-amino)-propionic acid, (S)-2-[(2,4,6-trimethylphenyl)sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]carbonylaminopropionic acid, EMD478761, or RC. * D(ThioP)C *Examples of such chemical substances or nucleic acids include (including and) α5β1 integrin activity inhibitors such as (Arg-Cys-Asp-thioproline-Cys; the asterisk indicates cyclization by a disulfide bond through cysteine residues). Additional exemplary angiogenesis inhibitors include 2-methoxyestradiol, αVβ3 inhibitors, angiopoietin 2, anti-angiogenic steroids and heparin, angiostatin, angiostatin-related molecules, anti-α5β1 integrin antibodies, anti-cathepsin S antibodies, anti-thrombin III fragments, bevacizumab, calreticulin, canstatin, carboxyamidotriazole, cartilage-derived angiogenesis inhibitor, CDAI, CM101, CXCL10, endostatin, IFN-α, IFN-β, IFN-γ, IL-12, IL-18, IL-4, linomide, maspin, matrix metalloproteinase inhibitors, Meth-1, Meth-2, osteopontin, pegaptanib, platelet factor-4, prolactin, proliferin-related protein, prothrombin (kringle domain-2), ranibizumab, restin, soluble NRP-1, soluble VEGFR-1, SPARC, SU5416, slamin, tecogalan, tetrathiomolybdate, thalidomide, lenalidomide, thrombospondin, TIMP, TNP-470, TSP-1, TSP-2, vasostatin, VEGFR antagonists, VEGI, volociximab (also known as M200), anastellin such as fibronectin fragments (see Yi and Ruoslahti, Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):620-4) or any combination thereof. Said angiogenesis inhibition The agent is preferably in an amount sufficient to prevent or treat proliferative (neovascular) eye diseases such as choroidal neovascular membrane (CNV) associated with wet AMD and other retinal diseases. Additional exemplary angiogenesis inhibitors include lenvatinib (E7080), motesanib (AMG 706), pazopanib (Votrient), and IL-6 antagonists such as anti-IL-6 antibodies. Additional exemplary angiogenesis inhibitors include any of the foregoing fragments, mimetics, chimeras, fusions, analogs, and / or domains. Additional exemplary angiogenesis inhibitors include any combination of the foregoing. In an exemplary embodiment, the RPE cell preparation comprises an anti-VEGF antibody such as bevacizumab at about 0.1 mg to about 6.0 mg, such as about 1.25 mg and about 2.5 mg of bevacizumab per intravitreal injection. In a further exemplary embodiment, the RPE cell preparation comprises one or more VEGF activity inhibitors and one or more α5β1 integrin activity inhibitors.

[0114] In combination with the RPE cell preparation, one or more anti-inflammatory agents may be administered. Exemplary anti-inflammatory agents include glucocorticoids, non-steroidal anti-inflammatory drugs, aspirin, ibuprofen, naproxen, cyclooxygenase (COX) enzyme inhibitors, aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate ester, fluocinolone acetonide (e.g., ILUVIEN (registered trademark)), glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Optionally, the anti-inflammatory agent may not be a corticosteroid. For example, the anti-inflammatory agent may be a non-steroidal anti-inflammatory drug.

[0115] Furthermore, the patient may not be administered corticosteroids before, simultaneously with, and / or subsequent to administration of the RPE cell preparation. Without wishing to be bound by theory, the applicants hypothesize that corticosteroid administration interferes with RPE cell sedimentation and / or engraftment. In certain preferred embodiments, the patient is not treated with prednisone or methylprednisolone before, simultaneously with, and / or subsequent to administration of the RPE cell preparation. In a more preferred embodiment, the patient is not treated with prednisone before, simultaneously with, and / or subsequent to administration of the RPE cell preparation. For example, the patient may not be administered prednisone or methylprednisolone or another corticosteroid within at least 3, 6, 12, 24, 48, 72, 96, or 120 hours or more before administration of the RPE cell preparation. Furthermore, the patient may not be administered prednisone or methylprednisolone or another corticosteroid within at least 3, 6, 12, 24, 48, 72, 96, or 120 hours or more subsequent to administration of the RPE cell preparation.

[0116] The patient may be administered a non-corticosteroid immunosuppressant before and / or subsequent to administration of the RPE cell preparation. Exemplary non-corticosteroid immunosuppressants include tacrolimus (FK-506 macrolide) and MMF (mycophenolic acid prodrug).

[0117] In combination with the RPE cell preparation, one or more antioxidants, antioxidant cofactors, and / or other factors contributing to increased antioxidant activity may be administered, examples of which include OT-551 (Othera), vitamin C, vitamin E, beta-carotene, zinc (e.g., zinc oxide), and / or copper (e.g., copper oxide).

[0118] In combination with the RPE cell preparation, one or more macular xanthophylls (such as lutein and / or zeaxanthin) may be administered.

[0119] In combination with the RPE cell preparation, one or more long-chain omega-3 fatty acids such as docosahexaenoic acid (DHA) and / or eicosapentaenoic acid (EPA) may be administered.

[0120] In combination with the RPE cell preparation, one or more amyloid inhibitors such as fenretinide, Arc-1905, Copaxone (glatiramer acetate, Teva), RN6G (PF-4382923, Pfizer) (humanized monoclonal antibody against ABeta40 and ABeta42), GSK933776 (GlaxoSmithKline) (anti-amyloid antibody) may be administered.

[0121] In combination with the RPE cell preparation, one or more ciliary neurotrophic factor (CNTF) agonists (e.g., CNTF that may be delivered by an intraocular device such as NT-501 (Neurotech)) may be administered.

[0122] In combination with the RPE cell preparation, one or more RPE65 inhibitors such as ACU-4429 (Aculea, Inc.) may be administered.

[0123] In combination with the RPE cell preparation, one or more factors targeting A2E and / or lipofuscin accumulation such as fenretinide and ACU-4429 may be administered.

[0124] In combination with the RPE cell preparation, one or more photoreceptor function and / or metabolic downregulators or inhibitors such as fenretinide and ACU-4429 may be administered.

[0125] In combination with the RPE cell preparation, one or more alpha2 adrenergic receptor agonists such as brimonidine tartrate may be administered.

[0126] In combination with the RPE cell preparation, one or more selective serotonin 1A agonists such as tandospirone (AL-8309B) may be administered.

[0127] In combination with the RPE cell preparation, one or more factors targeting C-5, the membrane attack complex (C5b-9) and / or any other optional drusen component may be administered, examples of which include ARC1905 (Ophthotec) (an anti-C5 aptamer that selectively inhibits C5), POT-4 (Potentia) (a compstatin derivative that inhibits C3), complement factor H, eculizumab (Soliris, Alexion) (a humanized IgG antibody that inhibits C5), and / or FCFD4514S (Genentech, San Francisco) (a monoclonal antibody against complement factor D), such as complement factor D, C-3, C-3a, C5, and C5a inhibitors, and / or factor H agonists.

[0128] In combination with the RPE cell preparation, one or more immunosuppressive agents such as sirolimus (rapamycin) may be administered.

[0129] In combination with the RPE cell preparation, one or more agents that prevent or treat lipofuscin accumulation, such as piracetam, centrophenoxine, acetyl-L-carnitine, Ginkgo biloba (Ginko Biloba) or its extract or preparation, and / or DMAE (dimethyl ethanolamine), may be administered.

[0130] When administered in combination with an RPE cell preparation, one or more agents (angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors contributing to increased antioxidant activity, macular xanthophylls, long-chain ω-3 fatty acids, amyloid inhibitors, CNTF agonists, RPE65 inhibitors, factors targeting A2E and / or lipofuscin accumulation, downregulators or inhibitors of photoreceptor function and / or metabolism, α2-adrenergic receptor agonists, selective serotonin 1A agonists, factors targeting C-5, membrane attack complex (C5b-9) and / or optional other drusen components, immunosuppressants, agents for preventing or treating lipofuscin accumulation, etc.) may be administered simultaneously with, prior to, and / or subsequent to the RPE cell preparation. For example, the agent may be administered to the patient's eye in the procedure in which the RPE cell preparation is implanted into the patient's eye. Administration of the agent may begin before and / or continue after administration of the RPE cells to the patient's eye. For example, the agent may be provided in solution, suspension, in a sustained release form, and / or within a sustained release system (e.g., Allergan Novadur™ delivery system, NT-501, or another intraocular device or sustained release system).

[0131] The RPE cell population may include differentiated RPE cells at various levels of maturity, or may be substantially pure with respect to differentiated RPE cells at a particular level of maturity. The RPE cells may be a substantially purified preparation comprising RPE cells at various levels of maturity / pigmentation. For example, a substantially purified RPE cell culture may contain both differentiated RPE cells and mature differentiated RPE cells. Among mature RPE cells, the pigment level may vary. However, mature RPE cells may be visually distinguishable from RPE cells based on an increase in the level of pigmentation and a more columnar shape. A substantially purified RPE cell preparation comprises RPE cells at different levels of maturity (e.g., differentiated RPE cells and mature differentiated RPE cells). In such cases, there may be variation throughout the preparation with respect to the expression of markers indicative of pigmentation. Pigmentation of RPE cells in cell culture may be homogeneous. Additionally, pigmentation of RPE cells in cell culture may be heterogeneous, and the RPE cell culture may comprise both differentiated RPE cells and mature RPE cells. Preparations comprising RPE cells include those that are substantially pure with respect to non-RPE cell types but contain a mixture of differentiated RPE cells and mature differentiated RPE cells. Preparations comprising RPE cells also include those that are substantially pure with respect to non-RPE cell types as well as with respect to RPE cells at other levels of maturity.

[0132] The percentage of mature differentiated RPE cells in the culture may be decreased by lowering the culture density. Thus, the methods described herein may further comprise the step of passaging a population of mature RPE cells to produce a culture containing a lower percentage of mature RPE cells. The number of RPE cells in the formulation includes differentiated RPE cells, regardless of the level of maturity and regardless of the relative percentages of differentiated RPE cells and mature differentiated RPE cells. The number of RPE cells in the formulation refers to the number of either differentiated RPE cells or mature RPE cells. The formulation may comprise at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% differentiated RPE cells. The formulation may comprise at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mature RPE cells. The RPE cell formulation may comprise a mixed population of differentiated RPE cells and mature RPE cells.

[0133] The present disclosure provides a cell culture comprising human RPE cells that express at least one gene that is pigmented and not expressed in non-human RPE cells. For example, such RPE cells may have expression of RPE65, PEDF, CRALBP, and bestrophin that is substantially the same as native human RPE cells. The RPE cells may vary in terms of the level of maturity with respect to the expression of one or more of PAX2, Pax6, MITF, and / or tyrosinase. Note that the change in pigmentation after differentiation also correlates with the change in PAX2 expression. Mature RPE cells may be distinguished from RPE cells by pigmentation level, PAX2, Pax6, and / or tyrosinase expression level. For example, mature RPE cells may have a higher pigmentation level or a higher expression level of PAX2, Pax6, and / or tyrosinase compared to RPE cells. It may vary according to the level of maturity.

[0134] The preparation is substantially purified with respect to non-RPE cells and may comprise at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells. The RPE cell preparation may essentially contain no non-RPE cells or may consist of RPE cells. For example, a substantially purified RPE cell preparation may comprise less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% non-RPE cell types. For example, the RPE cell preparation may comprise less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% non-RPE cells.

[0135] The RPE cell preparation may be substantially pure with respect to non-RPE cells as well as with respect to RPE cells at other levels of maturity. The preparation may be substantially purified with respect to non-RPE cells and may be enriched in mature RPE cells. For example, in an RPE cell preparation enriched in mature RPE cells, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% of the RPE cells are mature RPE cells. The preparation may be substantially purified with respect to non-RPE cells and may be enriched in differentiated RPE cells rather than mature RPE cells. For example, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the RPE cells may be differentiated RPE cells rather than mature RPE cells.

[0136] The RPE cell preparation contains at least about 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×106 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10It may comprise a plurality of RPE cells. The RPE cell preparation may comprise at least about 5,000 - 10,000, 50,000 - 100,000, 100,000 - 200,000, 200,000 - 500,000, 300,000 - 500,000, or 400,000 - 500,000 RPE cells. The RPE cell preparation may comprise at least about 20,000 - 50,000 RPE cells. Also, the RPE cell preparation may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 100,000, or 500,000 RPE cells.

[0137] The RPE cell preparation may comprise at least about 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×106 , 9 × 10 6 , 1 × 10 7 , 2 × 10 7 , 3 × 10 7 , 4 × 10 7 , 5 × 10 7 , 6 × 10 7 , 7 × 10 7 , 8 × 10 7 , 9 × 10 7 , 1 × 10 8 , 2 × 10 8 , 3 × 10 8 , 4 × 10 8 , 5 × 10 8 , 6 × 10 8 , 7 × 10 8 , 8 × 10 8 , 9 × 10 8 , 1 × 10 9 , 2 × 10 9 , 3 × 10 9 , 4 × 10 9 , 5 × 10 9 , 6 × 10 9 , 7 × 10 9 , 8 × 10 9 , 9 × 10 9 , 1 × 10 10 , 2 × 10 10 , 3 × 10 10 , 4 × 10 10 , 5 × 10 10 , 6 × 10 10 , 7 × 10 10 , 8 × 10 10 , or 9 × 10 10 It may contain 9 × 10 cells / mL of RPE cells. The RPE cell preparation may contain at least about 5,000 - 10,000, 50,000 - 100,000, 100,000 - 200,000, 200,000 - 500,000, 300,000 - 500,000, or 400,000 - 500,000 cells / mL of RPE cells. The RPE cell preparation may contain at least about 20,000 - 50,000 cells / mL of RPE cells. Also, the RPE cell preparation may contain at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 cells / mL of RPE cells.

[0138] The formulations described herein may be substantially free of bacterial, viral, or fungal contamination or infection, including but not limited to the presence of HIV1, HIV2, HBV, HCV, CMV, HTLV1, HTLV2, parvovirus B19, Epstein - Barr virus, or herpesvirus 6. The formulations described herein may be substantially free of mycoplasma contamination or infection.

[0139] The RPE cells described herein may also act as functional RPE cells after transplantation, and the RPE cells form a monolayer between the neurosensory retina and the choroid of the patient who received the cell transplantation. The RPE cells may also supply nutrients to adjacent photoreceptors and may process shed rod outer segments by phagocytosis. Further, the RPE cells described herein may have a greater proliferative potential than cells derived from the eye donor (e.g., the RPE cells are "younger" than those of the donor). This allows the RPE cells described herein to have a longer useful life than cells derived from the eye donor.

[0140] Formulations comprising RPE cells may be prepared in accordance with Good Manufacturing Practice (GMP) (e.g., the formulation is GMP compliant) and / or current Good Tissue Practice (GTP) (e.g., the formulation may be GTP compliant).

[0141] RPE cell culture The present disclosure also provides substantially purified RPE cell cultures, including human RPE cells. The RPE cultures described herein may comprise at least about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; or 9,000 RPE cells. The culture may be for at least about 1×10 4 、2×10 4 、3×10 4 、4×10 4 、5×10 4 、6×10 4、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6× 10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10, 8 × 10 10 , or 9 × 10 10 and may comprise 8 × 10 10 , or 9 × 10 10 RPE cells.

[0142] The RPE cells may be further cultured to produce a mature RPE cell culture. The RPE cells may be matured. The RPE cells may be further cultured, for example, in RPE-GM / MM or MDBK MM medium until the desired level of maturity is obtained. This may be determined by monitoring the increase in the level of pigment formation during maturation. As an alternative to RPE-GM / MM or MDBK MM medium, a functionally equivalent or similar medium may be used. Regardless of the specific medium used to mature the RPE cells, the medium may optionally be supplemented with growth factors or agents. Both RPE cells and mature RPE cells are differentiated RPE cells. However, mature RPE cells are characterized by an increase in pigment level compared to differentiated RPE cells. The level of maturity and pigment formation may be adjusted by increasing or decreasing the culture density of the differentiated RPE cells. Thus, the RPE cell culture may be further cultured to produce mature RPE cells. As an alternative, the density of the culture containing mature RPE cells may be decreased to reduce the percentage of mature differentiated RPE cells and increase the percentage of differentiated RPE cells.

[0143] The RPE cells may be identified by comparing messenger RNA transcripts between such cells and in vivo-derived cells. Cell aliquots are collected at various intervals during the differentiation of embryonic stem cells into RPE cells and assayed for the expression of any of the markers described above. These characteristics distinguish the differentiated RPE cells.

[0144] The RPE cell culture may be a substantially purified culture comprising at least about 30%, 35%, 40%, or 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% differentiated RPE cells. A substantially purified culture may comprise at least about 30%, 35%, 40%, or 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mature differentiated RPE cells.

[0145] The RPE cell cultures may be prepared in accordance with Good Manufacturing Practice (GMP) (e.g., the cultures are GMP compliant) and / or current Good Tissue Practice (GTP) (e.g., the cultures may be GTP compliant).

[0146] Cryopreserved preparations of RPE cells RPE cells may be preserved by any suitable method known in the art (e.g., deep-freezing), and freezing may be at any temperature suitable for preserving cells. For example, cells may be frozen at approximately −20° C., −80° C., −120° C., −130° C., −135° C., −140° C., −150° C., −160° C., −170° C., −180° C., −190° C., −196° C., and any other temperature suitable for preserving cells. Deep-frozen cells may be stored in an appropriate container and prepared for storage to reduce the risk of cell damage and maximize the likelihood that the cells will survive thawing. RPE cells may be differentiated and immediately subjected to in vitro cell culture. Following maturation, or after some period in culture, the RPE cells may be cryopreserved. RPE cells may also be maintained at room temperature or refrigerated, for example at about 4°C.

[0147] Similarly, a method for cryopreserving RPE cells is also provided. The RPE cells are harvested, washed in a buffer or medium, counted, concentrated (by centrifugation), formulated in a cryopreservation medium (e.g., 90% FBS / 10% DMSO), or any combination of these procedures may be used. For example, the RPE cells may be seeded in several culture vessels and grown continuously. The RPE cells are harvested and maintained in FBS at about 4°C, while combining the RPE cells from several flasks into a single lot. The RPE cells may also be washed at least 1, 2, 3, 4, or 5 times with a saline solution (e.g., DPBS). Further, the RPE cells may be cryopreserved after dystrophin is organized in the cell membrane and PAX6 expression is decreased. Additionally, the vials may be labeled with primary and / or secondary labels. Information on the label may include cell type (e.g., hRPE cells), lot number and date, cell count (e.g., 1×10 6 cells / mL), expiration date (e.g., the recommended date by which the vial should be used), manufacturing information (e.g., name and address), warnings, and storage means (e.g., storage in liquid nitrogen).

[0148] The cryopreserved RPE cell preparation described herein may comprise at least about 50,000 to 100,000 RPE cells. The cryopreserved RPE cell preparation may also comprise at least about 20,000 to 500,000 RPE cells. Also, the cryopreserved RPE cell preparation may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, or 100,000 RPE cells. The cryopreserved RPE cell preparation may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 RPE cells. The cryopreserved RPE cell preparation may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1×10 4、2×10 4 、3×10 4 、4×10 4 、5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×1010 or 4×10 10 or 5×10 10 or 6×10 10 or 7×10 10 or 8×10 10 or 9×10 10 and may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 RPE cells. The RPE cells of the cryopreserved RPE cell preparation may be mammalian RPE cells including human RPE cells.

[0149] Further, the cryopreserved RPE cell preparation described herein may comprise at least about 50,000 to 100,000 RPE cells / mL. The cryopreserved RPE cell preparation may also comprise at least about 20,000 to 500,000 RPE cells / mL. Also, the cryopreserved RPE cell preparation may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, and 100,000 RPE cells / mL. The cryopreserved RPE cell preparation may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,00 0 RPE cells / mL. The cryopreserved RPE cell preparation may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1×10 4 or 2×10 4 or 3×10 4 or 4×10 4 or 5×10 4 or 6×10 4 or 7×10 4 or 8×10 4 or 9×10 4 or 1×10 5 or 2×10 5 or 3×10 5 or 4×10 5 or 5×10 5 or 6×10 5 or 7×10 5 or 8×10 5, 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 may also contain 9×10 cells / mL. The RPE cells of the cryopreserved RPE cell preparation may be mammalian RPE cells including human RPE cells.

[0150] The RPE cells of the present disclosure may be recovered from storage following cryopreservation. RPE cells recovered from cryopreservation also maintain their viability and differentiation state. For example, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the RPE cells may retain viability and differentiation following cryopreservation. Further, the RPE cells of the present disclosure may be cryopreserved and maintain their viability after storage for at least about 1, 2, 3, 4, 5, 6, or 7 days. The RPE cells of the present disclosure may also be cryopreserved and maintain their viability after storage for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The RPE cells of the present disclosure may be cryopreserved and maintain their viability after storage for at least about 1, 2, 3, 4, 5, 6, or 7 years. For example, the RPE cells of the present disclosure may be cryopreserved for at least about 4 years and exhibit at least about 80% viability. A cryopreservation formulation comprising RPE cells may be substantially free of DMSO.

[0151] Method for generating RPE cells The cell population analyzed by the subject method may be generated from pluripotent stem cells. Cell types that may be generated include, but are not limited to, RPE cells, RPE progenitor cells, iris pigmented epithelial (IPE) cells, and other vision-related nerve cells such as interneurons and amacrine cells (such as the "relay" neurons of the inner nuclear layer (INL)). Further, retinal cells, rods, cones, and corneal cells may be generated. Cells providing the vasculature of the eye may also be generated by the methods described herein.

[0152] Without being bound by a particular theory, the inventors have discovered that the methods described herein may act through FGF, EGF, WNT4, TGF-β, and / or oxidative stress, signal through the MAP-kinase and possibly the c-Jun N-terminal kinase pathways, and induce the expression of the Paired-box 6 (PAX6) transcription factor. PAX6 acts synergistically with PAX2 to ultimately differentiate mature RPE through the cooperation of MITF and Otx2, and transcribe RPE-specific genes such as tyrosinase (Tyr) and downstream targets such as RPE65, bestrophin, CRALBP, and PEDF. See Figure 1 of WO 2009 / 051671 pamphlet.

[0153] The RPE cells described herein may differentiate from pluripotent stem cells such as human embryonic stem cells and may be molecularly distinct from embryonic stem cells, adult-derived RPE cells, and fetal-derived RPE cells. For example, the manufacturing process steps described herein may endow the final RPE cell product with unique structural and functional properties such that these cells closely resemble native RPE cells and are different from fetal-derived RPE cells or RPE cell lines (e.g., ARPE19).

[0154] Applicants have previously disclosed methods for generating RPE from pluripotent cells. See U.S. Patent No. 7,736,896, U.S. Patent No. 7,795,025, and U.S. Patent No. 7,794,704, and International Publication No. WO / 2012 / 012803 and International Publication No. WO 2011 / 063005, each of which is incorporated herein by reference in its entirety. RPE may be generated from pluripotent cells cultured as a multilayer population or embryoid bodies. For example, embryoid bodies may be formed by culturing pluripotent cells under non-adherent conditions, such as on a low-adhesion substrate or in a "hanging drop". During these cultures, ES cells can form cell masses or clusters called embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb;6(2):88-95, which is incorporated herein by reference in its entirety. Typically, embryoid bodies first form as solid masses or clusters of pluripotent cells and, over time, some embryoid bodies come to contain cavities filled with liquid, with the former being referred to as "simple" EBs and the latter as "cystic" embryoid bodies in the same reference. As applicants have previously reported, cells in these EBs (both solid and cystic forms) can differentiate and increase the number of RPE cells over time. Optionally, the EBs may then be cultured as an adherent culture to form a growth. Similarly, applicants have previously reported that pluripotent cells overgrown to form a multilayer cell population can differentiate over time to form RPE cells. Once RPE is formed, they can be easily identified based on their morphological characteristics, including pigment formation and a cobblestone appearance, and isolated for further use.

[0155] Pluripotent cells may be expanded and maintained prior to RPE cell formation using any culture method known in the art. For example, pluripotent cells may be cultured in the presence of feeder cells such as mouse cells (e.g., mouse embryonic fibroblasts (MEF)), human feeder cells (e.g., human adult skin cells, neonatal dermal fibroblasts (HNDF), etc.). Pluripotent cells may be cultured in a culture free of xenogeneic components and / or under conditions free of feeder cells. See Klimanskaya et al., Lancet. 2005 May 7-13;365(9471):1636-41; Richards et al., Stem Cells. 2003;21(5):546-56; U.S. Pat. No. 7,410,798; Ilic et al., Stem Cells Dev. 2009 Nov;18(9):1343-5; Xu et al. Nat Biotechnol. 2001 Oct;19(10):971-4, the entire contents of each of which are incorporated herein by reference. For example, pluripotent cells may be cultured on a substrate. The substrate may be selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel (trademark) (soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The substrate may be of human origin or of non-human animal origin such as bovine, mouse or rat. Pluripotent cells may be cultured in conditioned medium. For example, the conditioned medium may be conditioned by pluripotent cells such as ES cells, iPS cells, feeder cells, fetal cells, etc., whether or not any of them are human cells.

[0156] During RPE production, pluripotent cells may be cultured in the presence of a rho-associated protein kinase (ROCK) inhibitor. ROCK inhibitor refers to any substance such as a small molecule, siRNA, miRNA, antisense RNA, etc. that inhibits or reduces the rho-associated kinase function or its signaling pathway in cells. The "ROCK signaling pathway" is described in this specification In the method of the book, it may include any signal processor involved in the ROCK-related signal transduction pathway, such as the Rho-ROCK-myosin II signal transduction pathway in cells, the signal transduction pathway upstream thereof, or the signal transduction pathway downstream thereof. An exemplary ROCK inhibitor that may be used is Stemgent’s Stemolecule Y-27632 (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):681-6), and other ROCK inhibitors include, for example, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, and SB-772077-B. Each of them is incorporated herein by reference as if fully described, Doe et al., J. Pharmacol. Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. Pharmacol., 57:976-983, 2000; Nakajima et al., Cancer Chemother. Pharmacol., 52:319-324, 2003; and Sasaki et al., Pharmacol. Ther., 93:225-232, 2002. The ROCK inhibitor may be utilized at known concentrations and / or culture conditions in the art, as described in U.S. Patent Application Publication No. 2012 / 0276063 (published prior to grant), which is incorporated herein by reference in its entirety. For example, the ROCK inhibitor may have a concentration of about 0.05 to about 50 μM, such as at least, or about 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μM, including any range that can be inferred therefrom, or any target concentration effective to promote cell growth or survival.

[0157] For example, pluripotent cell viability may be improved by the inclusion of a ROCK inhibitor. In an exemplary embodiment, pluripotent cells may be maintained under feeder cell-free conditions such as Matrigel™ or another substrate. Thereafter, embryoid bodies may be generated from the isolated pluripotent cells using EDTA, collagenase, etc. without using trypsin, or mechanically. The embryoid bodies may be formed in a medium comprising Y-27632 or another ROCK inhibitor. For example, the ROCK inhibitor may enhance cell viability in embryoid bodies generated from pluripotent cells cultured in Matrigel™ or another substrate. Thereby, the RPE cell yield may be improved.

[0158] An exemplary method of generating RPE cells comprises: (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells as embryoid bodies in a nutrient-rich, low-protein medium optionally comprising a serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium optionally comprising a serum-free B27 supplement; (d) culturing the adherent cultures of (c) in a nutrient-rich, low-protein medium without a serum-free B27 supplement; (e) culturing the cells of (d) in a medium capable of supporting the growth of a high-density somatic cell culture, whereby RPE cells appear in the cell culture; (f) separating the cells or cell aggregates from the culture of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme, or another separation medium); (g) selecting the RPE cells from the culture and transferring the RPE cells to a culture containing another medium supplemented with a growth factor to generate an enriched culture of RPE cells; and (g) expanding the enriched culture of RPE cells to generate RPE cells. These method steps may be performed at least once to generate a substantially purified RPE cell culture. Further, these method steps may be repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times to generate more RPE cells.

[0159] Furthermore, the present disclosure also provides a method for generating mature retinal pigment epithelium (RPE) cells, comprising: (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells as embryoid bodies in a nutrient-rich, low-protein medium optionally comprising a serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium optionally comprising a serum-free B27 supplement; (d) culturing the adherent-cultured cells of step (c) in a nutrient-rich, low-protein medium without a serum-free B27 supplement; (e) culturing the cells of (d) in a medium capable of supporting the growth of a high-density somatic cell culture, whereby RPE cells appear in the cell culture; (f) separating the cells or cell aggregates from the culture of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme, or another separation medium); (g) selecting the RPE cells from the culture and transferring the RPE cells to a culture containing another medium supplemented with growth factors to produce an enriched culture of RPE cells; (h) expanding the enriched culture of RPE cells; and (i) culturing the enriched culture of RPE cells to produce mature RPE cells. These method steps may be performed at least once to produce a substantially purified mature RPE cell culture. Furthermore, these method steps may be repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times to produce more mature RPE cells.

[0160] ​In any of the linked procedures, the cells may be cultured for at least about 1 to 10 weeks. For example, the cells may be cultured for at least about 3 to 6 weeks. In any of the linked procedures, the cells may be cultured for about 1 day to 50 days, such as, for example, at least about 1 to 3, 3 to 4, 7, 4 to 9, 7 to 10, 7 to 12, 8 to 11, 9 to 12, 7 to 14, 14 to 21, and 3 to 45 days. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. For example, the cells may be cultured for 2 to 4 and 3 to 6 hours. In each of the above-linked method procedures, the cells may be cultured for the same period at each step, or for different periods in one or more of the procedures. Further, any of the above-linked method procedures may be repeated to generate more RPE cells (e.g., scale up to generate a large number of RPE cells).

[0161] In the methods described herein, the RPE cells may begin to differentiate among the cells during adherent culture of the EBs. The RPE cells may be visually recognized based on their cobblestone morphology and the initial appearance of pigment formation. As the RPE cells continue to differentiate, clusters of RPE cells may be observed.

[0162] Mechanical or enzymatic methods may be used to select RPE cells among non-RPE cell clusters during embryoid body culture or to facilitate the subculture of adherent cells. Exemplary mechanical methods include, but are not limited to, tituration with a pipette or cutting with a pulled needle. Exemplary enzymatic methods include, but are not limited to, any enzyme suitable for cell dissociation (e.g., trypsin (e.g., trypsin / EDTA), collagenase (e.g., collagenase B, collagenase IV), dispase, papain, a collagenase and dispase mixture, a collagenase and trypsin mixture). Non-enzymatic solutions such as solutions with a high EDTA content, such as Hank's-based cell dissociation buffer, may be used to separate cells.

[0163] RPE cells may differentiate from embryoid bodies. Isolating RPE cells from EBs allows RPE cells to proliferate in in vitro enrichment culture. In human cells, RPE cells may be obtained from EBs grown for less than 9 0 days. Furthermore, RPE cells may occur in human EBs grown for at least about 7-14 days, 14-28 days, 28-45 days, or 45-90 days. The medium used to culture pluripotent stem cells, embryoid bodies, and RPE cells may be removed at optional intervals and / or replaced with the same or a different medium. For example, the medium may be removed and / or replaced at least about 0-7 days, 7-10 days, 10-14 days, 14-28 days, or 28-90 days later. Furthermore, the medium may be replaced at least daily, every other day, or at least every 3 days.

[0164] To enrich RPE cells and establish a substantially purified RPE cell culture, mechanical and / or chemical (including enzymatic) methods may be used to separate RPE cells from each other and from non-RPE cells. The RPE cell suspension may then be transferred to fresh medium and a fresh culture vessel to provide an enriched population of RPE cells.

[0165] RPE cells may be selected from dissociated cells and cultured separately to generate a substantially purified RPE cell culture. RPE cells are selected based on characteristics associated with RPE cells. For example, RPE cells can be recognized by their cobblestone cell morphology and pigmentation. In addition to this, cellular retinaldehyde-binding protein (CRALBP), a cytoplasmic protein also found in apical microvilli; RPE65, a cytoplasmic protein involved in retinoid metabolism; bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2); and pigment epithelium-derived factor (PEDF), a 48 kD secreted protein with anti-angiogenic properties, among others, are some known RPE markers. Messenger RNA transcripts of these markers may be assayed using PCR (e.g., RT-PCR) or Northern blot. Also, immunoblot techniques or Western blot may be used to assay the protein levels of these markers.

[0166] RPE cells may also be selected based on cell function, such as phagocytosis of shed rod and cone outer segments (or phagocytosis of another substrate such as polystyrene beads), stray light absorption, vitamin A metabolism, retinoid regeneration, and tissue repair. The evaluation may also be performed by examining in vivo function, for example, using behavioral tests, fluorescence angiography, histology, tight junction conductance, or electron microscopy, after transplantation of RPE cells into a suitable host animal (such as a human or non-human animal suffering from a naturally occurring or induced retinal pathological condition).

[0167] Enriched cultures of RPE cells may be cultured in a suitable medium, such as EGM2 medium. This medium, or a functionally equivalent or similar medium, may be supplemented with growth factors or agents (such as bFGF, heparin, hydrocortisone, vascular endothelial growth factor, recombinant insulin-like growth factor, ascorbic acid, or human epidermal growth factor). RPE cells may have a stable phenotype over a long period during culture (e.g., >6 weeks).

[0168] Optionally, the RPE may be cultured in the presence of a rho-associated protein kinase (ROCK) inhibitor such as Stemgent's Stemolecule Y-27632. For example, the RPE may be cultured in the presence of a ROCK inhibitor prior to cryopreservation.

[0169] Pluripotent stem cells The methods described herein may be used for differentiated cells (such as RPE cells) generated from pluripotent stem cells. Suitable pluripotent stem cells include, but are not limited to, embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are induced. Pluripotent stem cells may be prepared, for example, using methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst-stage embryo, as well as embryonic stem cells derived from one or more blastomeres of a cleavage-stage or morula-stage embryo (optionally without destruction of the remaining embryo). Such embryonic stem cells can be prepared from embryonic material generated by fertilization or by asexual means including somatic cell nuclear transfer (SCNT), parthenogenesis, cellular reprogramming, and androgenesis. Further, suitable pluripotent stem cells include, but are not limited to, human embryonic stem cells, human embryo-derived stem cells, and human induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are induced. Pluripotent stem cells (e.g., hES cells) may be cultured as a suspension culture to generate embryoid bodies (EBs). The EBs may be cultured in suspension for about 7 to 14 days. However, in certain embodiments, the EBs may be cultured in suspension for less than 7 days (less than 7, 6, 5, 4, 3, 2 days, or less than 1 day) or for more than 14 days. The EBs may be cultured in a medium supplemented with B27 supplement.

[0170] Pluripotent stem cells (e.g., hES cells) may be cultured as a suspension culture to generate embryoid bodies (EBs). The EBs may be cultured in suspension for about 7 to 14 days. However, in certain embodiments, the EBs may be cultured in suspension for less than 7 days (less than 7, 6, 5, 4, 3, 2 days, or less than 1 day) or for more than 14 days. The EBs may be cultured in a medium supplemented with B27 supplement.

[0171] After culturing the EBs in suspension culture, the EBs may be transferred to generate adherent culture. For example, the EBs may be seeded onto a gelatin-coated plate in the medium. When culturing as adherent culture, the EBs may be cultured in the same type of medium as when growing in suspension. When culturing the cells as adherent culture, a B27 supplement may be added to the medium. Also, the medium is initially supplemented with B27 (e.g., for about 7 days or less), and then for the remaining period, it is continuously cultured in the absence of B27 as adherent culture. The EBs may be cultured as adherent culture for at least about 14 - 28 days. However, in certain embodiments, the EBs may be cultured as adherent culture for less than about 14 days (less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or less than 1 day) or for more than about 28 days.

[0172] Human embryonic stem cells In the methods described herein, human embryonic stem (hES) cells may be used as pluripotent stem cells. Human embryonic stem cells (hES) include descendants of the inner cell mass (ICM) of a blastocyst, or cells derived from another origin, and may remain pluripotent substantially indefinitely. hES cells may be derived from one or more blastomeres of an early cleavage stage embryo, optionally without destruction or damage to the embryo. hES cells may be generated using nuclear transfer. hES cells may also be induced pluripotent stem cells (iPS cells), which are described in more detail below. Cryopreserved hES cells may also be used. hES cells may be cultured by any method known in the art, such as in the presence or absence of feeder cells. For example, hES cells may be cultured in EB-DM, MDBK GM, hESC medium, INVITROGEN® stem cell medium, OptiPro SFM, VP SFM, EGM 2, or MDBK MM. See Stem Cell Information (Culture of Human Embryonic Stem Cells (hESC)) [NIH website, 2010]. hES cells may be used and maintained in accordance with GMP standards.

[0173] When cultured on a feeder cell layer under defined conditions, hES cells maintain a specific morphology and form flat colonies that consist of small, tightly packed cells with a high nucleus-to-cytoplasm ratio, clear cell-cell borders, and distinct refractive colony boundaries. hES cells express a set of molecular markers such as octamer-binding protein 4 (Oct-4, also known as Pou5f1), stage-specific embryonic antigen (SSEA) 3 and SSEA 4, tumor rejection antigen (TRA) 1 60, TRA 1 80, alkaline phosphatase, NANOG, and Rex1. Similar to ICM cells that differentiate into a given lineage, hES cells in culture may be induced to differentiate. For example, hES cells may be differentiated into human RPE under the defined conditions described herein.

[0174] Human embryonic stem cells that may be used include, but are not limited to, MA01, MA04, MA09, ACT 4, MA03, H1, H7, H9, and H14. Additional exemplary cell lines include NED1, NED2, NED3, NED4, and NED5. See also the NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that may be used is the MA09 cell. The isolation and preparation of MA09 cells have been previously described in Klimanskaya, et al. (2006) “Human Embryonic Stem Cell lines Derived from Single Blastomeres.” Nature 444:481-485. are not.

[0175] hES cells may initially be co-cultured with mouse embryonic fibroblast (MEF) cells. The MEF cells may be mitotically inactivated by exposure to mitomycin C prior to seeding of the hES cells into the co-culture, and thus the MEF do not proliferate during culture. Further, the hES cell cultures may be examined microscopically and, for example, colonies containing non-hES cell morphology may be selected and discarded using a stem cell cutting tool, laser ablation, or other means. Typically, no additional MEF cells are used in the process after harvesting of the hES cells for seeding for embryoid body formation. The time between MEF removal and RPE cell harvesting as described herein may be at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days in at least 1, 2, 3, 4, or 5 passages in MEF-free cell culture. The time between MEF removal and RPE cell harvesting may also be at least about 80 - 90 days in at least about 3 passages in MEF-free cell culture. For the production methods described herein, the RPE cell cultures and the formulations described herein may be substantially free of mouse embryonic fibroblast (MEF) and human embryonic stem (hES) cells.

[0176] Induced pluripotent stem cells (iPS cells) Additional exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) created by reprogramming somatic cells by expression or induction of expression of a factor combination (a "reprogramming factor"). The iPS cells may be created using fetal, postnatal, neonatal, juvenile, or adult somatic cells. The iPS cells may be obtained from a cell bank. Alternatively, the iPS cells may be newly created (by methods known in the art) prior to initiating differentiation into RPE cells or another cell type. Generation of the iPS cells may be the first step in generating differentiated cells. The iPS cells may be specifically created for the purpose of creating histocompatible RPE cells using materials from a particular patient or compatible donor. The iPS cells can be generated from cells that are substantially non-immunogenic in the intended recipient, for example, from autologous cells or from cells histocompatible with the intended recipient.

[0177] Induced pluripotent stem cells may be generated by expressing or inducing the expression of one or more reprogramming factors in somatic cells. The somatic cells are fibroblasts such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or are non-fibroblast somatic cells. The somatic cells are reprogrammed by expressing at least 1, 2, 3, 4, 5. The reprogramming factors may be selected from Oct 3 / 4, Sox2, NANOG, Lin28, cMyc, and Klf4. Expression of the reprogramming factors may be induced by contacting the somatic cells with at least one agent such as a small organic molecule agent that induces expression of the reprogramming factors.

[0178] The somatic cells may also be reprogrammed using a combinatorial approach, expressing the reprogramming factors (e.g., using a viral vector, plasmid, etc.) and inducing reprogramming factor expression (e.g., using small organic molecules). For example, the reprogramming factors may be expressed in somatic cells by infection using a viral vector such as a retroviral vector or a lentiviral vector. Also, episomal pla Reprogramming factors may be expressed in somatic cells using non-integrating vectors such as Sendai. See, for example, Yu et al., Science. 2009 May 8;324(5928):797-801, which is incorporated by reference in its entirety. When using a non-integrating vector to express reprogramming factors, the factors may be expressed in the cells using electroporation, transfection, or vector-mediated conversion of somatic cells. For example, in mouse cells, expression of four factors (Oct3 / 4, Sox2, c-myc, and Klf4) using an integrating viral vector is sufficient to reprogram somatic cells. In human cells, expression of four factors (Oct3 / 4, Sox2, NANOG, and Lin28) using an integrating viral vector is sufficient to reprogram somatic cells.

[0179] Once the reprogramming factors have been expressed in the cells, the cells may be cultured. Over time, cells with ES-like characteristics will appear in the culture dish. The cells may be selected and passaged, for example, based on ES morphology or based on the expression of a selectable or detectable marker. The cells may be cultured to generate a cell culture similar to ES cells, which are presumably iPS cells.

[0180] To confirm the pluripotency of the iPS cells, the cells may be tested in one or more pluripotency assays. For example, the cells may be tested for the expression of ES cell markers; the cells may be evaluated for the ability to form teratomas when transplanted into SCID mice; the cells may be evaluated for the ability to differentiate into cell types of the three germ layers. Once pluripotent iPS cells are obtained, they may be used to generate RPE cells.

[0181] Retinal pigment epithelial (RPE) cells The present disclosure provides RPE cells that may differentiate from pluripotent stem cells such as human embryonic stem cells and that may be molecularly different from embryonic stem cells, adult-derived RPE cells, and fetal-derived RPE cells. The RPE generated according to exemplary embodiments of the methods disclosed herein and above may be different from what was achievable by previous methods and from those from other sources of RPE cells. For example, the manufacturing process steps described herein may, like cells from these isolated RPE cells obtained from other sources such as fetal-derived RPE cells or RPE cell lines (e.g., ARPE19), impart unique structural and functional properties to the final RPE cell product.

[0182] Furthermore, exemplary embodiments of the methods of generating RPE cells described herein do not permit ES cells, do not allow ES cells to persist, and do not pose a risk of contamination that is unacceptable for RPE cell cultures and formulations.

[0183] Cells provided by the present disclosure include, but are not limited to, RPE, RPE progenitor cells, iris pigmented epithelial (IPE) cells, and other vision-related nerve cells such as interneurons and amacrine cells (such as the “relay” neurons of the inner nuclear layer (INL)). Embodiments of the present disclosure may also provide retinal cells, rods, cones, and corneal cells, as well as cells that provide the vasculature of the eye.

[0184] RPE cells may be used to treat retinal degenerative diseases associated with several visual degenerative diseases that result in photoreceptor damage and blindness, such as retinoschisis, retinal dysplasia, pigmentary streaks, myopic macular degeneration, or retinal atrophy, or that result from choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt disease (macular dystrophy).

[0185] The RPE cells may be stable terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. The RPE cells described herein may be functional RPE cells characterized by the ability to be incorporated into the retina upon corneal, subretinal, or other administration to a human or non-human animal.

[0186] The RPE cells may express RPE cell markers. For example, the expression levels of markers such as RPE65, PAX2, PAX6, tyrosinase, bestrophin, PEDF, CRALBP, Otx2, and MITF may be equivalent to those of native RPE cells. The level of maturity of the RPE cells may be evaluated by measuring the expression of at least one of PAX2, PAX6, and tyrosinase, or their respective expression levels.

[0187] In contrast, the RPE cells may express ES cell markers. For example, the expression levels of the ES cell genes Oct-4, NANOG, and / or Rex-1 may be about 100 to 1000 times lower in RPE cells than in ES cells. For example, the RPE cells may substantially lack the expression of ES cell markers including, but not limited to, octamer-binding protein 4 (Oct-4, also known as Pou5f1), stage-specific embryonic antigen (SSEA)-3 and SSEA-4, tumor rejection reaction antigen (TRA)-1-60, TRA-1-80, alkaline phosphatase, NANOG, Rex-1, Sox2, TDGF-1, DPPA2, DPPA3 (STELLA), DPPA4, and / or DPPA5. Thus, the RPE cells preferably substantially lack the expression of Oct-4, NANOG, and / or Rex-1 compared to ES cells.

[0188] The RPE cells described herein may also show an increased expression level of α-integrin subunits 1-6 or 9, as compared to non-cultured RPE cells or other RPE cell preparations. The RPE cells described herein may also show an increased expression level of α-integrin subunits 1, 2, 3, 4, 5, or 9. The RPE cells described herein may be cultured under conditions that promote the expression of α-integrin subunits 1-6. For example, the RPE cells may be cultured with an integrin activator, including but not limited to manganese and activating monoclonal antibody (mAb) TS2 / 16. See Afshari, et al. Brain (2010) 133(2):448-464. The RPE cells are seeded on laminin (1 μg / mL) and may be exposed to Mn 2+ (500 μM) for at least about 8, 12, 24, 36, or 48 hours. The RPE cells may also be cultured for several passages (e.g., at least about 4, 5, 6, 7, or 8 passages), which may increase the expression of α-integrin subunits.

[0189] The RPE cells may express RPE markers and not express hES markers and may show a normal karyotype.

[0190] The RPE cells described herein may also be identified and characterized based on the degree of cell pigmentation. The change in pigment can be controlled by the density at which the RPE cells are cultured and maintained, and the time during which the RPE is maintained in culture. Differentiated RPE cells that are dividing rapidly have less pigmentation. In contrast, RPEs that divide more slowly or do not divide take on their characteristic polygonal or hexagonal shape and increase their pigmentation levels by accumulating melanin and lipofuscin. For example, (e.g., due to confluence) quiescent RPE cultures typically increase their pigmentation levels over time. Thus, the accumulation of pigmentation serves as an indicator of RPE differentiation and increased pigmentation, which is associated with cell density that serves as an indicator of RPE maturity. For example, mature RPE cells may be passaged at a lower density to reduce pigmentation. In this context, mature RPE cells may be cultured to generate more immature RPE cells. Such RPE cells are still differentiated RPE cells that express RPE differentiation markers.

[0191] The RPE cells described herein may maintain their phenotype in vitro over a long period of time. For example, RPE cells may maintain their phenotype over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 passages. RPE cells may maintain their phenotype over 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. RPE cells may maintain their phenotype over at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0192] Furthermore, the RPE cells described herein may maintain their phenotype following transplantation. The RPE cells may maintain their phenotype following transplantation for the lifetime of the recipient. For example, the RPE cells may maintain their phenotype 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 following transplantation. Furthermore, the RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks following transplantation. Furthermore, the RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following transplantation. The RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 years or more following transplantation.

[0193] Melanin content of the RPE cell population Exemplary embodiments of the present disclosure provide an RPE cell population having a low or moderate average pigmentation level and a pharmaceutical composition comprising RPE cells having a low or moderate average pigmentation level. As detailed in the examples below, Applicants have shown that RPE cells having a relatively lower pigmentation level functioned better in an assay evaluating the ability of the cells to attach and survive. Without wishing to be bound by theory, it is believed that as RPE cells mature, they may have a reduced ability to form cell attachments and survive and proliferate after cryopreservation, which may be due to an increase in the level of pigmentation (melanin) contained in the more mature RPE cells and / or an increase in other phenotypes of mature RPE that generally correlate with increased pigmentation (such as changes in the cytoskeleton, membrane composition, cell surface receptor expression, adhesion strength, nuclear structure, gene expression, or other phenotypes, or combinations of phenotypes). Also without wishing to be bound by theory, it is believed that as RPE cells become more mature, they may have a reduced ability to form cell attachments and survive and proliferate even when passaged and / or maintained without cryopreservation; this may also be due to an increase in the level of pigmentation (melanin) contained in the more mature RPE cells and / or in other phenotypes of mature RPE that generally correlate with increased pigmentation.

[0194] The level of pigment formation may be measured as average melanin per cell of the population, for example expressed as picograms per cell (pg / cell), and it will generally be understood that there may be some variation in the melanin levels among the cells within the population. For example, the average melanin content may be less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, or less than 5 pg / cell, such as 0.1 - pg / cell, 0.1 - 7 pg / cell, 0.1 - 6 pg / cell, 0.1 - 5 pg / cell, 0.1 - 4 pg / cell, 0.1 - 3 pg / cell, 0.1 - 2 pg / cell, 0.1 - 1 pg / cell, 1 - 8 pg / cell, 1 - 7 pg / cell, 1 - 6 pg / cell, 1 - 5 pg / cell, 1 - 4 pg / cell, 1 - 3 pg / cell, 1 - 2 pg / cell, 2 - 6 pg / cell, 3 - 5 pg / cell, or 4.2 - 4.8 pg / cell, etc., 4 - 5 pg / cell, or it may be 0.1 - 5 pg / cell. In further embodiments, the average melanin content may be less than 5 pg / cell, such as 0.1 - 5 pg / cell, 0.2 - 5 pg / cell, 0.5 - 5 pg / cell, 1 - 5 pg / cell, 2 - 5 pg / cell, 3 - 5 pg / cell, 4 - 5 pg / cell, or 4.5 - 5 pg / cell.

[0195] The melanin content may be measured using a variety of methods including methods that utilize cell extracts, FACS-based methods, etc. For example, Boissy et al., Cytometry. 1989 Nov;10(6):779 - 87; Swope et al., J Invest Dermatol. 1997 Sep;109(3):289 - 95; Watts et al., Cancer Res 1981;41:467 - 472; Rosenthal, each of which is incorporated herein by reference in its entirety. See, e.g., et al., Anal Biochem. 1973 Nov;56(1):91-9. For example, to determine the average melanin content, one may measure the number of cells in a representative sample, lyse the cells in the representative sample, measure the total melanin content of the cell lysate (e.g., from a NaOH-extracted cell pellet) (e.g., by spectrophotometry), and divide by the number of cells in the representative sample to obtain the average melanin content per cell. Optionally, one may ignore cells other than RPE in culture, measure the number of cells in a representative sample (e.g., count cells that are positive for one or more markers of RPE and / or exhibit the morphological characteristics of RPE cells), thereby obtaining the average melanin content per RPE cell in the representative sample.

[0196] The average melanin content may be determined for a cell population excluding the harvested RPE cells that are in the 5 percent with the darkest pigment and the 5 percent with the lightest pigment.

[0197] An RPE population having a desired average melanin content can be readily obtained. For example, while the melanin content of non-dividing metabolically active RPE (e.g., in confluent cultures) tends to increase over time due to the accumulation of synthesized melanin, the accumulated melanin is diluted by cell division such that the melanin content is relatively decreased in dividing cells. See, e.g., Dunn et al., Exp Eye Res. 1996 Feb;62(2):155-69. Thus, an RPE population having a desired average melanin content can be obtained by selecting an appropriate growth history, such as maintenance as a quiescent population for a duration that results in the desired average melanin content, e.g., for 1, 2, 3, 4, 5, or 6 days, or for 1, 2, 3, 4, 5, 6, 7, 8 weeks or more. Additional growth histories, such as maintaining as a quiescent population for a while followed by re-dividing the cells at a defined time or number of divisions (thereby decreasing the average melanin content achieved in the quiescent population), can also be used to adjust the average melanin content. The melanin content can also be adjusted by the use of various media and / or medium supplements. For example, melanin accumulation in cultured RPE is decreased in the presence of protein kinase inhibitors (e.g., H-7, W-7, H-8, and staurosporine) (Kishi et al., Cell Biol Int. 2000;24(2):79-83) and increased in the presence of all-trans retinoic acid (10(-5) to 10(-7) M) or TGF-β1 (1 to 100 U / ml) (Kishi et al., Curr Eye Res. 1998 May;17(5):483-6). The melanin content can also be increased by treatment of cells with zinc α-2-glycoprotein (see U.S. Patent No. 7,803,750) and / or by adenosine-1 receptor antagonists, adenosine-2 receptor agonists, adenosine-1 receptor agonists, adenosine-2 receptor antagonists, and combinations of adenosine-1 receptor antagonists and adenosine-2 receptor agonists, or combinations thereof (see U.S. Patent No. 5,998,423).Each of the foregoing documents is incorporated herein by reference in its entirety.

[0198] As an alternative to, or in addition to, the foregoing methods, RPE cells having a desired average melanin content may be obtained through cell sorting, for example using a flow cytometer. For example, melanin-containing cells are detectable by their light scattering properties, including increased side scatter and decreased forward scatter; these properties may be used to sort the population by pigmentation level, thereby purifying a population having the desired average melanin content. Boissy et al., Cytometry. 1989 Nov;10(6):779-87; Swope et al., J Invest Dermatol. 1997 Sep;109(3):289-95, each of which is incorporated herein by reference in its entirety.

[0199] Modifying MHC genes in human embryonic stem cells to obtain RPE cells with reduced complexity (For example, as described herein, from which RPE may also be derived) Human embryonic stem (hES) cells may be derived from a human embryonic stem cell library. The human embryonic stem cell library may comprise stem cells, each of which is hemizygous, homozygous, or null-deficient for at least one MHC allele present within the human population, and each member of the stem cell library is hemizygous, homozygous, or null-deficient for a different set of MHC alleles relative to the remaining members of the library. The human embryonic stem cell library may comprise stem cells that are hemizygous, homozygous, or null-deficient for all MHC alleles present within the human population. In the context of the present disclosure, stem cells that are homozygous for one or more histocompatibility antigen genes include cells that are null-deficient for one or more (and in some embodiments all) such genes. A null-deficiency at a locus means that the gene is deleted at that position (i.e., both alleles of the gene are deleted or inactivated).

[0200] The hES cells may comprise a modification in one of the alleles of the sister chromosomes in the MHC complex of the cell. A variety of methods for creating genetic modifications such as gene targeting may be used to modify the genes in the MHC complex. Further, the modified allele of the MHC complex in the cell may be subsequently modified so that the same allele exists on the sister chromosomes to make it homozygous. Methods such as loss of heterozygosity (LOH) may be utilized to genetically engineer the cells to have homozygous alleles in the MHC complex. For example, one or more genes of a set of MHC genes from the parental alleles may be targeted to create hemizygous cells. The other set of MHC genes may be removed by gene targeting or LOH to create a null line. This null line may be further utilized as a germ cell line into which an HLA gene array or individual genes are inserted to create a hemizygous or homozygous bank with a genetically uniform background in other respects. Stem cells that are null-deficient for all MHC genes may be generated by standard methods known in the art such as, for example, gene targeting and / or loss of heterozygosity (LOH). See, for example, U.S. Patent Application Publication No. 2004 / 0091936, U.S. Patent Application Publication No. 2003 / 0217374, and U.S. Patent Application Publication No. 2003 / 0232430, and U.S. Provisional Patent Application No. 60 / 729,173.

[0201] Accordingly, the present disclosure relates to methods of obtaining RPE cells, including a library of RPE cells with reduced MHC complexity. Using RPE cells with reduced MHC complexity may increase the supply of cells available for therapeutic use, as it may eliminate the difficulties associated with patient matching. Such cells may be derived from stem cells modified to be hemizygous or homozygous for the genes of the MHC complex.

[0202] The present disclosure relates to RPE cells ( Also provided is a library of RPE cells and / or RPE lineage cells). These RPE cells and / or RPE lineage cells may be used for patients in need of cell-based therapies. The present disclosure also provides a library of RPE cells, each of which is hemizygous, homozygous, or null-deficient for at least one MHC allele present within the human population, wherein each member of the RPE cell library is hemizygous, homozygous, or null-deficient for a different set of MHC alleles relative to the remaining members of the library. The present disclosure provides a library of human RPE cells that are hemizygous, homozygous, or null-deficient for all MHC alleles within the human population.

[0203] Medium In the methods described herein, any medium capable of supporting cell culture, such as a medium for viral, bacterial, or eukaryotic cell culture, may be used. For example, the medium may be EB-DM or RPE-GM / MM. As a further example, the medium may be a high-nutrient protein-free medium or a high-nutrient low-protein medium. Additionally, the medium may also contain nutrient components such as albumin, B-27 supplement, ethanolamine, fetuin, glutamine, insulin, peptone, purified lipoproteins, sodium selenite, transferrin, vitamin A, vitamin C, or vitamin E. For example, a nutrient-rich low-protein medium may be any medium that supports cell growth during culture and has a low protein content. Examples of nutrient-rich low-protein media include, but are not limited to, MDBK-GM, OptiPro SFM, VP-SFM, DMEM, RPMI Medium 1640, IDMEM, MEM, F-12 Nutrient Mixture, F-10 Nutrient Mixture EGM-2, DMEM / F-12 Medium, Medium 1999, or MDBK-MM. See also Table 1. Additionally, the nutrient-rich low-protein medium may be a medium that does not support the growth or maintenance of embryonic stem cells.

[0204] When a low-protein medium is used, the medium may contain components that contain at least about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.20%, 0.10%, 0.05%, 0.02%, 0.016%, 0.015%, or 0.010% animal-derived protein (e.g., 10% FBS). Note that reference to the percentage of protein present in the low-protein medium refers only to the medium and does not take into account proteins present in, for example, the B-27 supplement. Thus, it is understood that when cells are cultured in a low-protein medium and a B-27 supplement, the percentage of protein present in the medium may be higher.

[0205] A serum-free B-27 supplement is added to the low-protein or protein-free medium. The nutritional components of the B27 supplement may comprise biotin, L-carnitine, corticosterone, ethanolamine, D+ galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, triodo-1-thyronine (T3), DL-α tocopherol (vitamin E), DL-α tocopheryl acetate, bovine serum albumin, catalase, insulin, superoxide dismutase, and transferrin. When cells are cultured in a protein-free medium supplemented with B-27, protein-free refers to the medium prior to the addition of B-27.

[0206] The growth factors, agents, and other supplements described herein may be used alone or in combination with other factors, agents, or supplements for inclusion in the medium. The factors, agents, and supplements may be added to the medium immediately or at any point during or after cell culture.

[0207] The medium may also contain supplements such as heparin, hydrocortisone, ascorbic acid, serum (e.g., fetal bovine serum); or growth substrates (e.g., extracellular matrix from bovine corneal epithelium, Matrigel (trademark) (basement membrane substrate), or gelatin); fibronectin; proteolytic fragments of fibronectin, laminin, thrombospondin, aggrecan, and syndecan (syndezan).

[0208] One or more factors or agents may be supplemented to the medium.

[0209] Growth factors that may be used include, for example, EGF, FGF, VEGF, and recombinant insulin-like growth factor. Growth factors that may be used in the present disclosure also include 6Ckine (recombinant), activin A, alpha interferon, alpha interferon, amphiregulin, angiogenin, beta endothelial cell growth factor, betacellulin, beta interferon, brain-derived neurotrophic factor, cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemoattractant-1, endothelial cell growth supplement, eotaxin, epidermal growth factor, epithelial neutrophil activating peptide-78, erythropoiten, estrogen receptor alpha, estrogen receptor beta, fibroblast growth factor (acidic / basic heparin-stabilized recombinant), FLT-3 / FLK-2 ligand (FLT-3 ligand), gamma interferon, glial cell line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, GRO-alpha / MGSA, GRO-B, GRO-gamma, HCC-1, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, heregulin-alpha (EGF domain), insulin growth factor binding protein-1, insulin-like growth factor binding protein-1 / IGF-1 complex, insulin-like growth factor, insulin-like growth factor II, 2.5S nerve growth factor (NGF), 7S-NGF, macrophage inflammatory protein 1 beta, macrophage inflammatory protein 2, macrophage inflammatory protein 3 alpha, macrophage inflammatory protein 3 beta, monocyte chemoattractant protein 1, monocyte chemoattractant protein 2, monocyte chemoattractant protein 3, neurotrophin 3, neurotrophin 4, NGF-beta (human or rat recombinant), oncostatin M (human or mouse recombinant), pituitary extract, placental growth factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, pleiotrophin, rantes, stem cell factor, stromal cell-derived factor 1B / pre-B cell growth-stimulating factor, thrombopoetin, transforming growth factor alpha, transforming growth factor beta1, transforming growth factor beta2, transforming growth factor beta3, transforming growth factor beta5, tumor necrosis factor (alpha and beta), and vascular endothelial growth factor.

[0210] Agents that may be used in accordance with the present disclosure include cytokines such as interferon α, interferon α A / D, interferon β, interferon γ, interferon γ - inducible protein 10, interleukin - 1, interleukin - 2, interleukin - 3, interleukin - 4, interleukin - 5, interleukin - 6, interleukin - 7, interleukin - 8, interleukin - 9, interleukin - 10, interleukin - 11, interleukin - 12, interleukin - 13, interleukin - 15, interleukin - 17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony - stimulating factor, and macrophage inflammatory protein 1α.

[0211] The medium contains 17β - estradiol, adrenocorticotropic hormone, adrenomedullin, α - 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, luteinizing hormone, L - thy Hormones and hormone antagonists, including but not limited to Roxine, Melatonin, MZ-4, Oxytocin, Parathyroid hormone, PEC-60, Pituitary growth hormone, Progesterone, Prolactin, Secretin, Sex hormone-binding globulin, Thyroid-stimulating hormone, Thyroid-stimulating hormone releasing factor, Thyroxine-binding globulin, and Vasopressin, may be supplemented. The medium may be supplemented with antibodies against various factors, including but not limited to anti-low density lipoprotein receptor antibody, anti-progesterone receptor, internal antibody, anti-alpha interferon receptor chain 2 antibody, anti-c-c chemokine receptor 1 antibody, anti-CD 118 antibody, anti-CD 119 antibody, anti-colony stimulating factor-1 antibody, anti-CSF-1 receptor / c-fins antibody, anti-epidermal growth factor (AB-3) antibody, anti-epidermal growth factor receptor antibody, anti-epidermal growth factor receptor, phospho-specific antibody, anti-epidermal growth factor (AB-1) antibody, anti-erythropoietin receptor antibody, anti-estrogen receptor antibody, anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor B antibody, anti-fibroblast growth factor receptor antibody, anti-fibroblast growth factor, basic antibody, anti-gamma interferon receptor chain antibody, anti-gamma interferon human recombinant antibody, anti-GFR α-1 C-terminal antibody, anti-GFR α-2 C-terminal antibody, anti-granulocyte colony stimulating factor (AB-1) antibody, anti-granulocyte colony stimulating factor receptor antibody, anti-insulin receptor antibody, anti-insulin-like growth factor-1 receptor antibody, anti-interleukin-6 human recombinant antibody, anti-interleukin-1 human recombinant antibody, anti-interleukin-2 human recombinant antibody, anti-leptin mouse recombinant antibody, anti-nerve growth factor receptor antibody, anti-p60, chicken antibody, anti-parathyroid hormone-like protein antibody, anti-platelet-derived growth factor receptor antibody, anti-platelet-derived growth factor receptor B antibody, anti-platelet-derived growth factor alpha antibody, anti-progesterone receptor antibody, anti-retinoic acid receptor alpha antibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroid hormone nuclear receptor alpha 1 / Bi antibody, anti-transferrin (transfesferin) receptor / CD71 antibody, anti-transforming growth factor alpha antibody, anti-transforming growth factor B3 antibody, anti-tumor necrosis factor alpha antibody, and anti-vascular endothelial growth factor antibody.

[0212] Exemplary growth media potentially suitable for use in the methods described herein are listed in Table 1.

[0213] [Table 1]

[0214] treatment method RPE cells and RPE cell-containing materials produced by the methods described herein. Pharmaceutical preparations containing RPE cells may be used for cell-based therapy. The present disclosure provides a method for treating a condition involving retinal degeneration, comprising administering an effective amount of a pharmaceutical preparation comprising RPE cells, wherein the RPE cells are derived from ex vivo pluripotent stem cells. Conditions involving retinal degeneration include, for example, choroideremia, diabetic retinopathy, retinal atrophy, retinal detachment, retinal dysplasia, retinitis pigmentosa, angioid striae (also known as Knapp's striae or Knapp's lines, characterized by small breaks in Bruch's membrane that calcify and crack), and myopic macular degeneration (also known as degenerative myopia). The RPE cells described herein may also be used in methods of treating macular degeneration, including, but not limited to, age-related macular degeneration (dry or wet), North Carolina macular dystrophy, Thorsby fundus dystrophy, Stargardt disease, pattern dystrophy, Best disease, Malachialeventinis, Doyne honeycomb choroidopathy, dominant drusen, and radial drusen. The RPE cells described herein may also be used in methods of treating Parkinson's disease (PD).

[0215] A common feature of cell transplantation described in the prior art is the low survival rate of the graft. For example, in many cell transplantation studies, cells tend to be lost immediately (e.g., within the first week) following transplantation. This cell loss does not seem to be due to rejection of the transplanted cells, but rather to the inability of a certain percentage of cells to be retained at the transplantation site. This lack of cell retention is due to several factors, such as failure of cell adhesion to the underlying structure, lack of sufficient nutrients, or physical stress at the transplantation site. Following this initial decrease in cell number, cell survival at various time points after transplantation can vary significantly from study to study. Thus, some studies show a steady decrease in number, while others show results where the transplanted cells can reach a stable number. However, an important factor in considering transplantation success is the percentage of recipients with surviving grafts following cell transplantation.

[0216] In contrast to prior formulations, the RPE cells in the formulations described herein may survive for extended periods following transplantation. For example, the RPE cells may survive for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Additionally, the RPE cells may survive for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks; at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Further, the RPE cells may survive for the entire lifespan of the transplant recipient. Additionally, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the recipients of the RPE cells described herein may show survival of the transplanted RPE cells. Further, the RPE cells described herein may be successfully incorporated into the RPE layer of the transplant recipient, form a semi - continuous cell line, and maintain the expression of key RPE molecular markers (such as RPE65 and bestrophin). The RPE cells described herein may also adhere to Bruch's membrane and form a stable RPE layer in the transplant recipient. Also, the RPE cells described herein substantially do not contain ES cells, and the transplant recipient does not show abnormal proliferation or tumor formation at the transplant site.

[0217] A method of treating a patient suffering from a condition associated with retinal degeneration may comprise the step of locally administering a composition of the present disclosure (such as by intravitreal injection or insertion of a substrate comprising a pharmaceutical of the present disclosure). Examples of intravitreal administration of the pharmaceuticals of the present disclosure include delivery into the vitreous, trans - corneal, sub - conjunctival, sub - retinal, sub - macular (such as by sub - foveal macular injection), near the sclera, posterior sclera, and sub - Tenon's capsule portion of the eye. See, for example, U.S. Patent No. 7,794,704; U.S. Patent No. 7,795,025; 6,943,145; and U.S. Patent No. 6,943,153.

[0218] The present disclosure also provides a method of administering to a patient human RPE cells derived from embryonic stem cells with reduced complexity. The method may comprise: (a) identifying a patient in need of treatment involving administration of human RPE cells; (b) identifying MHC proteins expressed on the cell surface of the patient; (c) providing a library of human RPE cells with reduced MHC complexity generated by the method of generating RPE cells of the present disclosure; (d) selecting from the library an RPE cell that matches the MHC protein on the cells of this patient; and (e) administering to the patient any cell of step (d). The method may be performed at a regional center, such as a hospital, clinic, dispensary, and other medical facilities. Further, RPE cells selected as matching the patient may be expanded prior to the patient's treatment if the number of stored cells is low.

[0219] Prior to transplantation, the RPE cells may be cultured under conditions that increase the expression of alpha integrin subunits 1-6 or 9, as compared to uncultured RPE cells or other RPE cell preparations. The RPE cells described herein may be cultured to enhance the expression level of alpha integrin subunits 1, 2, 3, 4, 5, 6, or 9. The RPE cells described herein may be cultured under conditions that promote the expression of alpha integrin subunits 1-6. For example, the RPE cells may be cultured with an integrin activator, including but not limited to manganese and activating monoclonal antibody (mAb) TS2 / 16. See Afshari, et al. Brain (2010) 133(2):448-464.

[0220] Based on a specific medical condition, the severity of the medical condition, and the overall health of the patient, a specific treatment plan, route of administration, and combination therapy may be adjusted. Administration of a formulation comprising RPE cells may be effective in reducing the severity of symptoms and / or preventing further degeneration of the patient's medical condition. For example, administration of a pharmaceutical comprising RPE cells may improve the patient's vision. Further, in certain embodiments, administration of RPE cells may be effective in completely restoring any blindness or other symptoms. Additionally, administration of RPE cells may treat symptoms of endogenous RPE layer damage.

[0221] RPE cell pharmaceutical RPE cells may be formulated with a pharmaceutically acceptable carrier. For example, RPE cells may be administered alone or as a formulation ingredient. The test compound may be formulated for administration by any convenient method for use in medicine. Suitable pharmaceuticals for administration may be one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (such as balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be combined with a sterile injectable solution or dispersion which may contain, immediately prior to use, antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents. Exemplary pharmaceuticals may comprise RPE cells combined with ALCON® BSS PLUS (aqueous balanced salt solution containing 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg dibasic sodium phosphate, 2.1 mg sodium bicarbonate, 0.92 mg dextrose, 0.184 mg glutathione disulfide (oxidized glutathione), hydrochloric acid and / or sodium hydroxide (to adjust the pH to approximately 7.4)).

[0222] Exemplary compositions of the present disclosure may be formulations suitable for use in the treatment of human patients, such as without or essentially without pyrogenic substances and sterile. At the time of administration, the pharmaceuticals used in the present disclosure may be in a sterile, physiologically acceptable form without pyrogenic substances. A formulation comprising RPE cells used in the methods described herein may be transplanted in a suspension, gel, colloid, slurry, or mixture. Further, the formulation may desirably be encapsulated or injected into the vitreous humor in a viscous form for delivery to the site of retinal or choroidal damage. Also at the time of injection, cryopreserved RPE cells may be resuspended in a commercially available balanced salt solution to achieve the osmotic pressure and concentration desired for administration by subretinal injection. The formulation may be administered to the area surrounding the fovea of the retina that is not completely lost due to the disease, which may promote the attachment and / or survival of the administered cells.

[0223] The compositions of the present disclosure may include a rho-associated protein kinase (ROCK) inhibitor such as Stemgent’s Stemolecule Y-27632. For example, exemplary compositions may include RPE and a ROCK inhibitor present in an amount sufficient to promote RPE survival and / or engraftment after administration to a patient.

[0224] The RPE cells of the present disclosure may be delivered by intracameral injection in a pharmaceutically acceptable ophthalmic solution. For example, when administering the formulation by intravitreal injection, the solution may be concentrated so that a minimized volume may be delivered. The concentration for injection may be any optional amount that is effective and non-toxic depending on the factors described herein. An RPE cell pharmaceutical for treating a patient may be formulated at a dose of at least about 10 4 cells / mL. An RPE cell formulation for treating a patient may be at least about 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、or 10 10They are formulated at a dose of individual RPE cells / mL. For example, the RPE cells may be formulated in a pharmaceutically acceptable carrier or excipient.

[0225] The RPE cell pharmaceutical described herein may comprise at least about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; or 9,000 RPE cells. The RPE cell pharmaceutical is at least about 1×10 4 、2×10 4 、3×10 4 、4×10 4 、5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×108 , 9 × 10 8 , 1 × 10 9 , 2 × 10 9 , 3 × 10 9 , 4 × 10 9 , 5 × 10 9 , 6 × 10 9 , 7 × 10 9 , 8 × 10 9 , 9 × 10 9 , 1 × 10 10 , 2 × 10 10 , 3 × 10 10 , 4 × 10 10 , 5 × 10 10 , 6 × 10 10 , 7 × 10 10 , 8 × 10 10 , or 9 × 10 10 may also comprise 9 × 10 RPE cells. The RPE cell pharmaceutical may comprise at least about 1 × 10 2 to 1 × 10 3 , 1 × 10 2 to 1 × 10 4 , 1 × 10 4 to 1 × 10 5 , or 1 × 10 3 to 1 × 10 6 RPE cells. The RPE cell pharmaceutical may comprise at least about 10,000, 20,000, 25,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, or 200,000 RPE cells. For example, the RPE cell pharmaceutical may comprise at least about 20,000 to 200,000 RPE cells in a volume of at least about 50 - 200 μL. Further, the RPE cell pharmaceutical may comprise about 50,000 RPE cells in a volume of 150 μL, about 200,000 RPE cells in a volume of 150 μL, or at least about 180,000 RPE cells in a volume of at least about 150 μL.

[0226] In the aforementioned pharmaceuticals and compositions, the number or concentration of RPE cells may be determined by counting viable cells and excluding non-viable cells. For example, non-viable RPE may be detected by its inability to exclude vital dyes (such as trypan blue) or by using functional assays (such as the ability to adhere to a culture substrate, phagocytosis, etc.). Furthermore, the number or concentration of RPE cells may be determined by counting cells expressing one or more RPE cell markers and / or excluding cells expressing one or more markers indicative of cell types other than RPE.

[0227] RPE may be formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle such that the formulation maintains contact with the ocular surface for a time sufficient for the cells to penetrate into an affected area of the eye, such as the anterior chamber, posterior chamber, vitreous, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoridal space, conjunctiva, subconjunctival space, episcleral space, intrastromal space of the cornea, supraepithelial space of the cornea, pars plana of the ciliary body, surgically induced avascular area, or macula of the retina.

[0228] RPE cells may be contained in the cell sheet. For example, a cell sheet comprising RPE cells may be prepared by culturing RPE cells on a substrate from which a cell sheet without damage can be detached, such as a thermoresponsive polymer surface of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) grafted surface, which changes surface properties during temperature transition, for example, by cooling below the lower critical solution temperature (LCST), causing detachment of the cultured cell sheet, and allowing the cells to adhere and grow thereon at the culture temperature. (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, the entire contents of which are incorporated herein by reference respectively). The cell sheet may be attached to a substrate suitable for transplantation, such as a substrate that may dissolve in vivo when the sheet is transplanted into the host organism, and may be prepared, for example, by culturing the cells on a substrate suitable for transplantation or by detaching the cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. Exemplary substrates potentially suitable for transplantation may comprise gelatin (see Hsiue et al., supra). Alternative substrates that may be suitable for transplantation include cellulose-based substrates and the like. The cell sheet may be used in the manufacture of a medicament for preventing or treating retinal degenerative diseases. The RPE cell sheet may be formulated for implantation into the eye of a subject in need thereof. For example, the cell sheet may be implanted into the eye in need thereof by subfoveal membrane resection for transplanting the RPE cell sheet, or may be used for manufacturing a medicament for transplantation after subfoveal membrane resection.

[0229] The volume of the formulation administered by the methods described herein may depend on factors such as the mode of administration, the number of RPE cells, the age and weight of the patient, and the type and severity of the disease being treated. When administered by injection, the volume of the pharmaceutical of the RPE cells of the present disclosure may be from at least about 1, 1.5, 2, 2.5, 3, 4, or 5 mL. The volume may be at least about 1-2 mL. For example, when administered by injection, the volume of the RPE cell pharmaceutical of the present disclosure may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 μL (microliters) may also be used. For example, the volume of the formulation of the present disclosure may be at least about 10 - 50, 20 - 50, 25 - 50, or 1 - 200 μL. The volume of the formulation of the present disclosure may be at least about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL or more.

[0230] For example, the formulation may contain at least about 1×10 per μL 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , or 9×10 4It may comprise a plurality of RPE cells. The preparation may comprise 2000 RPE cells per μL, such as, for example, 100,000 RPE cells per 50 μL or 180,000 RPE cells per 90 μL.

[0231] The method for treating retinal degeneration may further comprise the step of administering an immunosuppressant. Immunosuppressants that may be used include, but are not limited to, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB (registered trademark) (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB (registered trademark) (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab (registered trademark) (anti-CD20 antibody), sirolimus, and tacrolimus. The immunosuppressant may be administered at at least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg / kg. When using immunosuppressants, they may be administered systemically or locally, and they may be administered before, simultaneously with, or following the administration of RPE cells. The immunosuppressive therapy may continue for several weeks, months, years, or indefinitely following the administration of RPE cells. For example, 5 mg / kg of cyclosporine may be administered to a patient for 6 weeks following the administration of RPE cells.

[0232] The treatment method for retinal degeneration may comprise the administration of a single dose of RPE cells. Also, the treatment methods described herein may comprise a treatment process in which RPE cells are administered multiple times over a certain period. Exemplary treatment processes may comprise treatment weekly, biweekly, monthly, four times a year, twice a year, or annually. Alternatively, the treatment may proceed stepwise, whereby multiple administrations are first performed (e.g., daily administration for the first week), followed by less frequent administrations at lower doses.

[0233] When administered by intraocular injection, RPE cells may be delivered periodically one or more times throughout the patient's lifetime. For example, RPE cells may be delivered once a year, once every 6 - 12 months, once every 3 - 6 months, once every 1 - 3 months, or once every 1 - 4 weeks. Alternatively, in certain medical conditions or disorders, more frequent administration may be desirable. When administered by graft or device, depending on the specific patient being treated and the needs of the disease or medical condition, RPE cells may be administered once, or periodically one or more times throughout the patient's lifetime. A drug administration schedule that changes over time may also be considered. For example, initially, more frequent treatment may be required (e.g., daily or weekly treatment). As time passes and the patient's medical condition improves, less frequent treatment may be needed, or in some cases, further treatment may not be required at all.

[0234] The methods described herein may further comprise monitoring the efficacy of treatment or prevention by measuring electroretinogram responses, optomotor acuity thresholds, or luminance thresholds in a subject. The methods may also comprise monitoring the efficacy of treatment or prevention by monitoring the immunogenicity or cell migration of cells in the eye.

[0235] RPE cells may be used in the manufacture of agents for treating retinal degeneration. The present disclosure also encompasses the use of a formulation comprising RPE cells in the treatment of blindness. For example, using a formulation comprising human RPE cells, retinal degeneration associated with several visual degenerative diseases such as diabetic retinopathy, which causes photoreceptor damage and blindness, (including age-related macular degeneration such as wet age-related macular degeneration and dry age-related macular degeneration), age-related macular degeneration, retinitis pigmentosa, and Stargardt's disease (macular fundus) may be treated. The formulation may comprise at least about 5,000 to 500,000 RPE cells (e.g., 100,000 RPE cells), and administering it to the retina may treat retinal degeneration associated with several visual degenerative diseases such as diabetic retinopathy, (including age-related macular degeneration), age-related macular degeneration, retinitis pigmentosa, and Stargardt's disease (macular fundus) that cause photoreceptor damage and blindness.

[0236] The RPE cells provided herein may be human RPE cells. However, it should be noted that human cells may also be used in animal models or animal patients, as well as in human patients. For example, human cells may be tested in mouse, rat, cat, dog, or non-human primate models of retinal degeneration. Additionally, in veterinary medicine and the like, human cells may be used therapeutically to treat animals in need thereof.

[0237] Mode of Administration The pharmaceutical may be formulated in a pharmaceutically acceptable carrier depending on the route of administration. For example, the formulation may be formulated to be administered into the subretinal space of the eye. A formulation comprising RPE cells may be administered to one or both eyes of the same patient. Administration to both eyes may be sequential or simultaneous. For example, a formulation comprising RPE cells may be formulated as a suspension, solution, slurry, gel, or colloid.

[0238] The RPE cells of the present disclosure may be administered locally by injection (e.g., intravitreal injection), or as part of a device or implant (e.g., an implant) or a part thereof. As described above, the RPE cells may have various possible configurations such as individual cells, masses, clusters, sheets, or any combination thereof, and may be contained in an aqueous carrier, gel, substrate, polymer, etc. For example, the formulation may be administered by injection into the subretinal space of the eye. The formulation may also be administered transcorneally. For example, the cells of the present disclosure may be transplanted into the subretinal space using vitrectomy. Further, at the time of injection, the RPE cells may be resuspended in a commercially available balanced salt solution (e.g., Alcon BSS Plus (registered trademark)) to achieve the desired osmotic pressure and concentration for administration by subretinal injection.

[0239] Optionally, the RPE cells may be administered by a method comprising a pars plana vitrectomy such as a three-port pars plana vitrectomy. Examples of the method include a small retinal incision. Before cell administration, a subretinal bleb may be formed (a "pre-bleb") by injection of, for example, saline or another suitable fluid, and then removed before cell administration. However, the cells may be administered without pre-bleb formation. The cells may be administered in a bleb at the temporal position. For example, the bleb may optionally be expanded within the arcade vessels. The bleb may be positioned so as not to detach the fovea centralis of the retina.

[0240] Depending on the administration method, the RPE cells may be added to a buffered electrolyte balanced aqueous solution; a buffered electrolyte balanced aqueous solution added with a lubricating polymer, mineral oil or petrolatum-based ointment, other oils, liposomes, cyclodextrins, sustained release polymers or gels.

[0241] Substrates used for RPE cells The methods described herein may comprise administering the RPE cells of the present disclosure as a graft or device. In certain embodiments, the device is an in vivo biodegradable implant for the treatment of eye diseases, comprising an active agent dispersed in a biodegradable polymer substrate, wherein at least about 75% of the particles of the active agent have a diameter of less than about 10 μm. The in vivo biodegradable implant may be sized for implantation into the eye region. The eye region may be any one or more of the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, episcleral space, intrastromal corneal space, supraepithelial corneal space, sclera, pars plana, surgically induced avascular area, macula of the retina, and the retina. The biodegradable polymer may be, for example, a poly(lactic-co-glycolic) acid (PLGA) copolymer, a biodegradable poly(DL-lactic-co-glycolic acid) film, or a PLLA / PLGA polymer matrix. The ratio of glycolic acid monomer in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, 75 / 25, more preferably about 50 / 50 weight percent. The PLGA copolymer may be about 20, 30, 40, 50, 60, 70, 80 to about 90 weight percent of the in vivo biodegradable implant. The PLGA copolymer may be about 30 to about 50 weight percent, preferably about 40 weight percent of the in vivo biodegradable implant. The RPE cells may be transplanted in combination with a biocompatible polymer such as polylactic acid, poly(lactic-co-glycolic acid), 50:50 PDLGA, 85:15 PDLGA, and INION GTR® biodegradable membrane (a biocompatible polymer mixture). See U.S. Patent No. 6,331,313; U.S. Patent No. 7,462,471; and U.S. Patent No. 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 9:1187-205; and Tomita, et al. (2005) Stem Cells 23:1579-88.

[0242] In another aspect, the present disclosure provides a composition comprising RPE located on a membrane, and a method of using the composition for the prevention or treatment of retinal diseases, disorders, or conditions. For example, the membrane may be the membrane described in U.S. Patent Application Publication No. 20110236464 (published before grant), the entire contents of which are incorporated herein by reference. The membrane is substantially non-biodegradable and porous, and the pores may have a diameter of approximately 0.2 μm to 0.5 μm. For example, the pore diameter may be 0.3 μm to 0.45 μm. The use of a non-biodegradable membrane may ensure that it continues to support cells for, for example, at least 5 years, at least 10 years, or at least 15 years following insertion into the body once it has been implanted into the eye.

[0243] The pore density may be approximately 1×10^7 to 3×10^8 pores per square centimeter, such as 5×10^7 and 1×10^8 pores per square centimeter. This density may allow for the desired level of permeability and may also allow for angiogenesis. In particular, the pore size and density may allow for the movement of nutrients from one side of the membrane to the other, for example, after implantation, and may also allow for angiogenesis through the membrane. The polymer body may receive angiogenesis from the rich choroid bed. This is shown by the rich vascular bed outside the eye (Cassell et al, 2002; Patrick et al, 1999; Saxena et al 1999, Peter et al 1998), and occurs only when the porosity is sufficient (Menger et al, 1990).

[0244] For example, the water permeability of the membrane may exceed 50×10^-10 msec^-1 Pa^-1. Specifically, the water permeability of the membrane may be approximately 33 mL / min / cm^2. This is equal to 801.21×10^-10 msec^-1 Pa^-1 and is 8 times the water permeability of the Bruch's membrane of the macula of young people. Since the artificial membrane may depend entirely on a passive process, this excess permeability is potentially useful. From the perspective of nutrient diffusion, it can not only meet the requirements of the overlying cells, but preferably does not impede fluid transport from the bottom side of the RPE layer; otherwise, the RPE may peel off from the polymer surface. Consistent with this prediction, the hypothesis has been proposed that the decrease in the water permeability of the Bruch's membrane in the elderly causes pigment epithelial detachment in AMD (Bird & Marshall, 1986).

[0245] Preferably, the membrane may be sterilized by gamma irradiation, ethylene oxide, autoclaving or UV sterilization without decomposition.

[0246] Preferably, the membrane may be sealed by ultrasonic sealing, high-frequency sealing or insert molding. This allows other layers to be attached to the membrane, for example, attaching a formulation or a coating layer to the membrane. For example, it may be desirable to attach a more rigid biodegradable layer such as PLGA to provide rigidity to the membrane and assist in delivery. As an alternative, a layer containing a pharmacological agent or a biological agent, or a layer supporting other cells may be attached.

[0247] The membrane preferably has a maximum thickness of approximately 11 μm. More preferably, the thickness of the membrane is 9 μm to 11 μm. The thickness of the membrane can be selected so as to allow diffusion of nutrients to induce angiogenesis and to enable easy insertion of the membrane into the eye.

[0248] Thus, the RPE may be provided on or cultured on a membrane that supports cell growth. The membrane is substantially non-biodegradable, porous, and has a maximum thickness of approximately 11 μm. The membrane is preferably substantially planar, and its minimum dimension is preferably less than approximately 11 μm. The thickness within its dimensions may vary, but is preferably a thickness of 9 μm to 11 μm.

[0249] The membrane may have a maximum weight of approximately 1.5 mg / cm^2. More preferably, the weight of the membrane is 1.0 mg / cm^2 to 1.4 mg / cm^2. The minimum tensile strength of the membrane is preferably at least 100 bar, providing sufficient strength to enable proper handling during surgery. The maximum tensile strength is preferably 300 bar, allowing the membrane to be easily handled during surgery. The burst strength of the membrane is preferably at least 10 psi.

[0250] Preferably, the membrane is hydrophilic. This may confer excellent wetting ability to the membrane, allowing for easy attachment of cells and other desirable coatings.

[0251] The membrane preferably has a physiologically acceptable pH, such as a pH of 4 to 8 for example.

[0252] The membrane preferably comprises a coating on at least one side. The coating is preferably a protein or glycoprotein such as laminin, Matrigel (TM), collagen, fibronectin, and / or PLGA poly(lactic-co-glycolic acid). The coating may also comprise a pharmacological agent or biological agent bound to the coating component. For example, the coating may comprise a neurotrophic agent, an anti-inflammatory agent, or an angiogenesis inhibitor.

[0253] In particular, the coating preferably contains laminin, such as laminin-1 or fragments thereof, such as IgVAV. In particular, the coating may contain more laminin-1 than other proteins or glycoproteins. Preferably, the coating may consist of at least 30% or at least 40% of laminin, such as laminin-1. The coating may be applied so as to produce a concentration of laminin-1 of approximately 40 - 45 μg / cm^2 on the membrane.

[0254] Thus, the RPE may be provided on or cultured on a membrane that supports cell growth, the membrane comprising a substantially non-biodegradable and porous support layer coated on at least one side with a coating comprising laminin-1.

[0255] The membrane may be made of a hydrophilic polymer. Hydrophobic polymers made hydrophilic by irradiating the polymer with UV light may also be used. Exemplary polymers include polyesters such as polyethylene terephthalate and polybutylene terephthalate; polyurethanes and polyurea urethanes, especially those containing polycarbonate and polysiloxane, and those based on polyester or polyether; polyamides such as nylon; polyether esters such as Sympatex; polycarbonates such as Makrolon; polyacrylates such as Perspex; poly(tetrafluoroethene) (PTFE); polysiloxane; polyolefins such as polyethylene and polypropylene; and polyoxymethylene (POM) commonly known under the DuPont trade name Delrin. It is particularly preferred that the membrane is made of polyethylene terephthalate or polybutylene terephthalate. In another preferred embodiment, the membrane is made of polyester.

[0256] The membrane may be used to culture the RPE cell layer of the present disclosure. The membrane preferably may comprise a cell layer on the membrane. The cells may be any cells selected according to the membrane and the intended use of the cells.

[0257] The membrane and the cell layer are preferably at least 3 mm by 5 mm in length and width. Preferably, the membrane and the cell layer are at least 4 mm by 6 mm.

[0258] The membrane and the cell layer may be transplanted into the eye of a patient in need thereof, for example, in the treatment of age-related macular degeneration, retinal holes, macular distrophy, choroidemia, Leber Congenital Amarosis, Stargardt disease, and other retinal diseases or conditions.

[0259] Screening assay The present disclosure provides a method for screening and identifying agents that modulate RPE cell maturity. For example, RPE cells differentiated from human ES cells may be used to screen for agents that promote RPE maturation. The identified agents may be used alone or in combination with RPE cells as part of a treatment regimen. Alternatively, the identified agents may be used as part of a culture method that improves the survival of in vitro differentiated RPE cells.

[0260] RPE cells may be used as research tools in situations such as pharmaceutical, chemical, or biotech companies, hospitals, or academic or research institutions. Such uses include, for example, the use of RPE cells differentiated from embryonic stem cells in screening assays to identify agents that may be used to promote RPE survival in vitro or in vivo, or to promote RPE maturation, survival, and / or engraftment. The identified agents may be studied in vitro or in animal models to evaluate their possible use, for example, alone or in combination with RPE cells.

[0261] ​The present disclosure provides a method for identifying an agent that promotes RPE maturation, comprising the steps of providing RPE cells, contacting the RPE cells with an agent, evaluating the RPE cells for signs of maturity, and then, if the RPE cells exhibit signs of maturity in response to the agent, identifying an agent that promotes RPE maturation. Signs of maturity may be, as discussed herein, pigmentation levels, gene expression levels, and morphology.

Industrial Applicability

[0262] Certain aspects of the present disclosure relate to the production of RPE cells in commercial quantities. RPE cells may be produced on a large scale, stored if desired, and supplied to hospitals, clinicians, or other medical institutions.

[0263] Accordingly, certain aspects of the present disclosure relate to methods of generating, storing, and distributing RPE cells produced by the methods disclosed herein. Following RPE production, the RPE cells may be harvested, purified, and optionally stored prior to treatment of a patient. The RPE cells may optionally be patient-specific or may be specifically selected based on HLA or other immune profiles. For example, once a patient presents with signs such as, for example, diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, retinal atrophy, retinal detachment, retinal dysplasia, and Stargardt's disease (macular dystrophy), pigmentary streaks, or myopic macular degeneration, RPE cells can be ordered and provided in a timely manner. Accordingly, the present disclosure relates to a method for generating RPE cells for obtaining cells on a commercial scale, a cell preparation comprising RPE cells derived from said method, and a method for providing (i.e., generating, optionally storing, and selling) RPE cells to hospitals and clinicians. The production of differentiated RPE cells or mature differentiated RPE cells may be scaled up for commercial use.

[0264] The present disclosure also provides a method of conducting a pharmaceutical business, which may include the step of establishing a distribution system for distributing formulations for sale, or may include the step of establishing a sales group for marketing pharmaceuticals.

[0265] The present disclosure provides a method of providing RPE cells to hospitals, medical centers, and clinicians. The RPE cells generated by the methods disclosed herein are stored and ordered according to the requirements of hospitals, medical centers, or clinicians, and administered to patients in need of RPE cell therapy. Hospitals, medical centers, or clinicians order RPE cells based on patient-specific data, and the RPE cells are generated according to the patient's specifications and subsequently supplied to the ordering hospital or clinician. For example, after a particular RPE cell formulation is selected as suitable for a patient, it is then expanded to an amount suitable for the patient's treatment.

[0266] A further aspect of the present disclosure relates to an RPE cell library that can provide compatible cells to potential patient recipients. Accordingly, the present disclosure provides a method of conducting a pharmaceutical business that includes the step of providing an RPE cell preparation that is homozygous for at least one histocompatibility antigen, the cells being selected from a cell bank comprising an RPE cell library that may be expanded by the methods disclosed herein, each RPE cell preparation being hemizygous or homozygous for at least one MHC allele present within the human population, said RPE cell bank comprising cells that are hemizygous or homozygous for different sets of MHC alleles, respectively, with respect to another member of the cell bank. As described above, gene targeting or loss of heterozygosity may be used to create hemizygous or homozygous MHC allele stem cells that are used to derive RPE cells. As described above, gene targeting or loss of heterozygosity may be used to create hemizygous or homozygous MHC allele stem cells that are used to derive RPE cells.

[0267] The present disclosure also includes a method of obtaining or generating human ES cells (e.g., induced pluripotent (iPS) cells, or ES cells generated by somatic cell nuclear transfer, or ES cells generated by other reprogramming methods) from a patient or a tissue compatibility provider, and then creating and growing RPE cells derived from the ES cells. These RPE cells may be stored. Further, these RPE cells may be used to treat the patient from whom the ES was obtained, or a blood relative of that patient, or an individual with tissue compatibility.

[0268] The present disclosure demonstrates that human RPE cells may reliably differentiate and grow from human ES cells under well-defined and reproducible conditions, which represents an inexhaustible source of cells for patients with retinal degenerative disorders. The concentration of these cells is not limited by availability, but rather can be dosed according to the exact clinical requirements of an individual. Repeated infusions or transplants of the same cell population over a patient's lifetime are also possible by a physician if deemed necessary. Further, the ability to create a bank of HLA hES lines that match or have reduced complexity from which RPE cells can then be generated has the potential to reduce or eliminate the need for immunosuppressive agents and / or immunomodulatory protocols altogether.

Example

[0269] The present invention will now be generally described, which is included for illustrative purposes only of specific aspects and embodiments of the present invention and is not intended to limit the present invention. It will be more readily understood with reference to the following examples.

[0270] Further information regarding the results presented in the examples and / or additional experimental results are included in the accompanying manuscript included immediately prior to the claims of this application.

[0271] Example 1 Method Creation of hESC master cell bank The hESC lines used in these studies were previously described as MA09(22), derived from unused in vitro fertilization (IVF) embryos obtained with full informed consent and used in accordance with the Ethics Advisory Board and Institutional Review Board of Advanced Cell Technology. Using current Good Manufacturing Practice, MA09 seed cultures were thawed and grown through four consecutive passages on mitotically inactivated mouse embryonic fibroblasts (MEFs). A human embryonic stem cell master cell bank (hESC-MCB) was cryopreserved and confirmed to have a normal female (46,XX) karyotype and to be free of bacterial and mycoplasma contaminants, as well as human, bovine, porcine, and murine viruses. PCR analysis showed no changes or mutations in genes associated with macular degeneration, including CTRP5, EVLV4, RPE-65, VMD2, and ABCA4 (Table 3 below).

[0272] Manufacture of retinal pigment epithelium Vials of hESC-MCB were thawed and grown for three passages on mitomycin C-treated MEFs. Since hESCs were co-cultured with animal cells, differentiated derivatives were classified as xenotransplantation products and were subject to FDA guidelines for donor animals and product processing, testing, and archiving, as well as for patients, monitoring, and registration (detailed in Example 2 below). After hESC growth, the cells were sequentially induced to form embryoid bodies, followed by cell proliferation and localization differentiation into pigmented RPE patches. The generation of RPE used in this example is detailed in Example 4 below. Pigmented patches were isolated with collagenase, and after purification and trypsin treatment, the dissociated cells were seeded and grown to confluence, inducing redifferentiation over a total of three consecutive passages. The second passage of RPE was cryopreserved and served as a starting material for preparing cells for clinical use. Preclinical trials

[0273] Preclinical trials As previously described, human ESC-derived RPE cells were subretinally injected into NIH III immunodeficient mice (tumorigenicity and biodistribution studies), dystrophic RCS rats, and Elov4 mice (efficacy studies) (8). Detection of human cells in the injected eyes and other organs was carried out by DNA Q-PCR designed to amplify the human Alu Y DNA sequence and by immunohistochemistry of paraffin sections for human mitochondria and human bestrophin (detailed in Example 2).

[0274] Cell characterization and safety testing RPE cells were evaluated for safety and characterized for several RPE-specific attributes at various time points, including during manufacturing process testing, and after thawing, final product formulation, and maturation culture to simulate the fate of transplanted cells in vitro. Safety evaluations for possible bacteria, mycoplasma, murine viruses, and residual murine DNA were performed by WuXi Apptec, Inc., St. Paul, MN, according to standard protocols. Cytogenetic analysis for karyotyping, DNA fingerprinting for cell line authentication, and fluorescence in situ hybridization (FISH) were performed by Cell Lines Genetics, Madison, WI. Endotoxin testing was performed on cryopreserved RPE formulated as the final product for clinical injection by Cape Cod Associates, Inc., East Falmouth, MA. Quantitative immunohistochemical staining was performed using standard methods and the percentage of positively stained cells was normalized to the number of DAPI-stained nuclei examined. Assessment of RPE purity and degree of differentiation was based on the percentage of cells stained for bestrophin, Pax6, ZO-1 and / or MITF. Screening to confirm the absence of pluripotency markers was performed by staining for OCT-4 and alkaline phosphatase. Phagocytosis (potency assay) was evaluated by quantitative fluorescence-activated cell sorting (FACS) analysis of RPE cultures exposed to PhRodo™ (Invitrogen) fluorescent bioparticles. Quantitative reverse transcription (q-RT) PCR assays were performed to confirm upregulation of RPE-specific genes (RPE-65, PAX-6, MITF, bestrophin) and downregulation of hESC-specific genes (OCT-4, NANOG, SOX-2). Melanin content per cell was measured spectrophotometrically in NaOH-extracted pellets of known cell number (detailed in Example 2).

[0275] Cell Formulation and Injection Vials of cryopreserved MA09-RPE were thawed, washed three times by centrifugation, and resuspended at 2×10 per 1 μL of BSS PLUS® (Alcon) 3Resuspended in the density of individual live cells / . A vial containing an appropriate volume of formulated RPE and a paired vial containing an appropriate volume of BSS PLUS® were delivered to the OR at 2 - 8°C. Immediately prior to injection, the two vials were back - titrated in a 1 mL syringe to obtain a loaded cell density that would result in delivery of the desired number of RPE cells (50,000 live RPE cells to the subretinal space of each patient's eye). To ensure accurate delivery of the intended dose, the loaded cell density was increased to compensate for the predicted loss of live RPE cells encountered during mixing, loading, and delivery through the cannula. This loss of live cells was measured as described in Example 3 below and was shown to depend on the cannula used. In these examples, a MEDONE POLYTIP® cannula 25 / 38 (0.12 mm (38 g) × 5 mm tip with 0.50 mm (25 g) × 28 mm cannula) was used and the loaded cell density was 444 live cells / μL , resulting in a predicted delivery of 336 + / - 40 live cells / μL (N = 6), and delivery of the expected 50,400 live RPE in a 150 μL volume into the subretinal space of each patient's eye.

[0276] Patient Selection Patients were selected based on several inclusion and exclusion criteria, including end - stage disease, absence of central visual field loss, other significant ophthalmic lesions, no history of cancer, current cancer screening, absence of contraindications to systemic immunosuppression, ability to undergo vitreoretinal surgery under monitored anesthesia care, and psychological suitability to participate in the first human clinical trial involving hESC - derived transplant tissue (Tables 7 and 8 below).

[0277] Transplantation and Rationale Pars plana vitrectomy, including surgical induction, was performed to separate the posterior vitreous from the optic nerve anterior to the posterior border of the vitreous base. 5×10 in a 150 μl volume was injected into a pre - selected area around the fovea of the retina that was not completely obliterated by the disease 4Subretinal injection of individual hESC-RPE cells was delivered. The transplant site was carefully selected based on the presence of the damaged but native RPE and the photoreceptors overlaying it, optimizing the probability of graft integration and the potential for photoreceptor cell rescue. Graft adhesion is unlikely in the completely atrophic macular center's pathoanatomic complex and does not mimic the macular center's state in earlier stages of degeneration, which may be the ultimate therapeutic target of a stem cell-based regenerative transplantation strategy.

[0278] The immunosuppression regimen included low-dose tacrolimus (targeting blood levels of 3 - 7 ng / mL) and mycophenolate mofetil (MMF in the range of 0.25 g - 2 g oral / day) starting 1 week before surgery and continued for 6 weeks. At week 6, the regimen called for discontinuation of tacrolimus and continuation of MMF for an additional 6 weeks.

[0279] Results Characterization of RPE Controlled hESC differentiation yielded nearly 100% pure RPE (Figure 1). A single (9.6 cm2) pigmented patch in a 6-well plate (Figure 1A) contained approximately 1.5×10 8 RPE cells (e.g., up to 3×10 6Generated (sufficient to treat 50 patients at the individual cell dose). The cells exhibited typical RPE behavior, losing their pigmented cobblestone morphology during proliferation (after trypsin treatment); once confluence was re-established, they redifferentiated into a monolayer of polygonal cuboidal pigmented epithelium. Q-PCR showed that the pluripotency markers (Oct-4, NANOG, and SOX2) were significantly downregulated, while the RPE markers RPE65, bestrophin, Pax6, and MITF were highly expressed (Figures 1B–F and Table 5). Immunostaining of the mature cultures showed that bestrophin, a late marker of differentiated RPE, was organized membranously in most of the cells before harvest; all (>99%) cells were positive for bestrophin and / or PAX6 (PAX6 becomes weaker or disappears in more mature cells) and for ZO-1, an adherens junction component (not shown). After cryopreservation, the cell vials were thawed and prepared for transplantation. Staining for the retinal marker Pax6 and / or MITF (a marker of pigmented cells) confirmed 100% RPE purity (Figure 1C). To further validate the prepared cells, they were cultured for 2–3 weeks to grow and mature until the RPE morphology was established. Pax6 / bestrophin (Figure 1E) and ZO-1 (Figure 1G) immunostaining was similar to that of the pre-harvest cultures, and the potency assay showed that >85% of the cells phagocytosed the bioparticle fragments (Figure 1J).

[0280] Safety test Since hESCs were exposed to animal cells and products, the MCBs and RPEs were extensively tested for animal and human pathogens. The cells were confirmed to be free of microbial contaminants, including animal and human pathogenic viruses, at all stages (Table 3 below). The final RPE product had a normal female (46, XX) karyotype (Figure 1K) and a DNA fingerprint profile consistent with the hESC line MA09. The RPE manufacturing process was carried out under conditions that did not support pluripotent cells, but sensitive assays were performed to exclude the presence of any contaminating hESCs in the final RPE product. Examination of 2 out of 9 million cell RPE samples (at P1 / P2) stained for Oct-4 and alkaline phosphatase showed no presence of any pluripotent cells. Tumorigenicity, biodistribution, and addition tests performed in NIH-III mice showed no harmful or safety issues in any of the animals. Furthermore, no tumors were observed in animals injected with 50,000 - 100,000 RPE cells supplemented with any of 0.01%, 0.1%, or 1% undifferentiated hES cells. Survival of human RPE cells was confirmed in the eyes of 100% of animals up to 3 months and 92% up to 9 months post-injection (Table 6 below). Human RPE survived over the lifetime of the animals and was incorporated into the mouse RPE layer; they were morphologically almost indistinguishable from host RPE cells (Figure 2), but they could be confirmed by immunostaining and expressed bestrophin in a typical basolateral pattern (Figure 2B). Ki-67 staining showed low levels of proliferation 1 - 3 months after transplantation, but no Ki-67 positive cells were seen at 9 months, suggesting that hESC-derived RPE formed a mature, quiescent monolayer.

[0281] The differentiation stage affects cell attachment and survival The attachment of transplanted cells to Bruch's membrane and their subsequent survival and integration into the host RPE layer are considered important for the success of this treatment strategy. A prominent feature of hESC technology is the ability to control the degree of differentiation in vivo. The degree of RPE differentiation manifests in a series of regulated genotype and phenotype expressions, including the level of pigment formation. Cells maintained under similar conditions but harvested and cryopreserved at different time points exhibit various levels of pigment formation. Figure 3 shows two representative lots of cryopreserved RPE harvested at seemingly different pigment levels (the melanin content was 4.8 ± 0.3 SD pg / cell and 10.4 ± 0.9 SD pg / cell for the lighter and darker pigmented lots, respectively). Cells from both RPE lots were processed and formulated using a clinical transplantation protocol. After extrusion through an injection cannula, the cells were seeded onto gelatin-coated tissue culture plates to monitor attachment and subsequent growth. RPE cells from the lighter pigmented lot showed a minimal number of floating cells in the overnight culture; most of the cells attached and spread, showing the typical behavior and morphology of RPE at this growth stage (Figure 3A). Three days after culture, the number of RPE cells increased from 4.0×10 4 cells seeded to 10.6×10 4 cells (Figure 3C and Figure 1G). In stark contrast, the darker pigmented RPE showed a large number of floating cells; three days after culture, only a small proportion of the cells attached and survived, and the cell number decreased significantly (less than one-tenth of the lighter pigmented lot [9.0×10 3 )(Figure 3F and Figure 3G). These results suggest a strong correlation between the differentiation stage of RPE and its in vitro attachment and growth ability. The RPE lot used in this clinical study had a melanin content of 4.1 pg / cell and showed attachment and growth similar to the lighter pigmented lot. The stress associated with freeze-thaw cycles, post-thaw washing, centrifugation, and formulation, as well as extrusion through an injection cannula, may, to some extent, account for the differences observed between the lighter and darker pigmented lots.

[0282] Clinical Results The SMD patient was a 26-year-old white female with a baseline best corrected visual acuity (BCVA) of hand motion (HM) and unable to read any letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart. At all time points following transplantation, no signs of intraocular inflammation or excessive proliferation were detected. The absence of clinically detectable inflammation was confirmed by slit lamp biomicroscopy, fundus photography, IVFA, and SD-OCT (detailed in Example 2 and Figures 8 and 9). Clinically detectable increased pigmentation at the level of the RPE began to be observed at 1 week postoperatively and appeared to spread outside the surgical transplant site (Figure 4). The Goldman visual field improved from baseline until 2 months after transplantation (preoperative and postoperative visual fields are shown in Figures 10 and 11). At 2 weeks, the BCVA was counting fingers (CF) (1 ETDRS letter), which continued to improve during the study period (5 ETDRS letters [BCVA 20 / 800] at 1 and 2 months) (Table 2). The patient was highly reliable and had worked as a graphic artist for many years. She reported subjective improvement in color vision, contrast, and dark adaptation in the operated eye and no changes in the other eye. None of the above symptoms of intraocular inflammation or excessive proliferation were detected at any time point following transplantation. The absence of clinically detectable inflammation was confirmed by slit lamp biomicroscopy, fundus photography, IVFA, and SD-OCT (detailed in Example 2 and Figures 8 and 9). Clinically detectable increased pigmentation at the level of the RPE began to be observed at 1 week postoperatively and appeared to spread outside the surgical transplant site (Figure 4). The Goldman visual field improved from baseline until 2 months after transplantation (preoperative and postoperative visual fields are shown in Figures 10 and 11). At 2 weeks, the BCVA was counting fingers (CF) (1 ETDRS letter), which continued to improve during the study period (5 ETDRS letters [BCVA 20 / 800] at 1 and 2 months) (Table 2). The patient was highly reliable and had worked as a graphic artist for many years. She reported subjective improvement in color vision, contrast, and dark adaptation in the operated eye and no changes in the other eye.

[0283] [Table 2]

[0284] The AMD patient is a 77-year-old white female, and the baseline BCVA is 21 letters on the ETDRS (20 / 500). Despite a moderate non-compliance with the immunosuppressive regimen, no signs of intraocular inflammation or excessive proliferation were detected at any time point following transplantation. The absence of clinically detectable inflammation was confirmed by slit-lamp biomicroscopy, fundus photography, IVFA, and SD-OCT (detailed in Example 2 and Figures 8 and 9). The OCT images are shown in Figures 4 and 7. At week 2, the ETDRS BCVA was 33 letters (20 / 200). At week 6, the BCVA was 28 letters on the ETDRS (20 / 320) and remained stable until week 8. The Goldman visual field measured by the central scotoma decreased slightly in size at week 8 compared to the baseline.

[0285] Discussion The therapeutic use of human embryonic stem cells poses challenges of difficult technology transfer. This report provides the first clinical evidence suggesting that hESC-derived cells can be safely transplanted into human patients. In this study, low-dose (5×10 4 cells) of RPE cells generated from hESCs were transplanted into the eyes of two patients with different forms of macular degeneration, dry AMD and SMD, which are the leading causes of adult and juvenile blindness, respectively, in developed countries.

[0286] To improve the probability of cell attachment to Bruch's membrane, a subfoveal injection site where the macular complex (photoreceptors, Bruch's membrane, and RPE) is still present and potentially viable was selected, thus increasing the predictability that transplanted cells will be incorporated into the native RPE and potentially rescue damaged perimacular tissue. Both patients tolerated the transplantation well, and at the time of this report, there were no signs of postoperative inflammation, rejection, or tumorigenicity. Clinical and laboratory findings suggest that the transplanted RPE cells may begin to attach, incorporate, and affect the damaged native RPE.

[0287] Continuous monitoring and evaluation of the patient may determine whether the transplanted hESC-RPs have reduced immunogenicity, whether they undergo rejection in the absence of long-term immunosuppression, and whether the observed vision improvement persists. The immune response, if any, is predicted to be manageable through methods known in the art, including immunosuppression and / or tolerance regimens. It is also anticipated that a greater increase in vision may be achievable through administration of a greater number of RPE cells. Additionally, administration of RPE cells is expected to delay or prevent vision loss associated with retinal pathologies such as AMD, SMD, etc.

[0288] Transplantation of intact sheets and suspensions of primary RPE cells has been attempted previously (11 - 19), but RPE derived from adult organ donors has limited proliferative capacity (23) and is also limited in its ability to differentiate in vitro, including its inability to express genes required for melanin biosynthesis using standard culture conditions (24). Clinically, adult RPE sheets transplanted into the subretinal space of AMD patients have failed to improve visual function (25). RPE derived from pre- and postnatal tissues has been successfully isolated and induced to grow and mature with attributes suggesting fully differentiated RPE in vitro (26 - 28), but such sources are extremely limited and vary in quality and proliferative capacity. In contrast to adult and fetal tissues, hESCs are characterized by their ability to proliferate indefinitely without senescence and provide a virtually unlimited source of "young" cells as starting material for differentiation. Another expected advantage of using progeny obtained from hESCs is the ability to control the in vitro differentiation stage to maximize post-transplant survival and functionality. Indeed, the data presented here show that the degree of RPE maturity and pigmentation dramatically affects subsequent cell attachment and proliferation in vitro.

[0289] The starting material for the RPE used in this study was a well-characterized hESC master cell bank created using procedures optimized to reliably generate large quantities of pluripotent stem cells under controlled conditions. The RPE differentiation procedure is non-permissive for the survival of hESCs, but extensive preclinical safety studies confirmed that transplanted hESC-RPE did not cause ectopic tissue formation or tumors during the lifetime of the animals. An immunofluorescence-based assay capable of detecting less than one undifferentiated hES cell in over a million cells confirmed that the clinical RPE lots used in this study had no detectable pluripotent cells, which corresponds to a detection level five orders of magnitude lower than the hESC doses shown to cause tumors in in vivo addition tests. The creation of the hESC-MCB and the manufacture of each RPE cell lot involve growth on a feeder cell layer of primary mouse embryonic fibroblasts. Thus, hESC-RPE is classified as a xenotransplantation product and is subject to all tests and monitoring as defined by the FDA xenotransplantation guidelines, ensuring that the cells are free of mouse pathogens. The RPE also underwent an extensive series of safety tests to confirm the absence of microbial contaminants and viruses and was characterized by a variety of RPE-specific attributes, including phagocytic ability, gene expression, morphological evaluation, and immunohistochemical staining for RPE-specific markers. Prior to the initiation of these clinical trials, transplantation of hESC-RPE into dystrophic animals demonstrated that the cells have the ability to rescue photoreceptors and visual function in a dose-dependent manner.

[0290] This study was designed to verify the safety and tolerability of hESC-RPE in patients with end-stage SMD and dry AMD. To date, there do not appear to be any abnormal growth, teratoma formation, graft rejection, or other adverse pathologic responses in the cells transplanted into either patient group. Continued follow-up and further studies are needed. However, the ultimate goal of treatment is to treat patients earlier in the disease course to potentially increase the likelihood of photoreceptor and central vision rescue.

[0291] Example 2 This example provides supplementary information and methods related to Example 1.

[0292] (From which the RPE cells used in Example 1 were generated) The characteristics of the clinical hESC master cell bank (hESC-MCB) are shown in Table 3.

[0293] [Table 3-1]

[0294] [Table 3-2]

[0295] Mouse embryonic fibroblast (MEF) master cell bank Since the MA09-hRPE cells were in contact with non-human (mouse) cells in vitro, in accordance with the Guidance for Industry “Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans” and “Points to Consider on Xenogeneic Cell Therapy Medicinal Products” (EMEA / CHMP / CPWP / 83508 / 2009), these human cells are defined as xenotransplantation products. Charles River The breeding colony at Laboratories (Kingston Facility, Stoneridge, NY, USA) was used as the source of MEF cells. This AAALAC (Association for Assessment and Accreditation of Laboratory Care International)-accredited facility houses a closed colony of CD-1 specific pathogen-free (SPF) mice in a barrier-maintained room under extensive health monitoring. Donor animals were time-mated and isolated during pregnancy. Twelve days after mating and prior to sacrifice, a physical health examination was performed on all mice by a veterinarian; the animals were euthanized, and leukocyte and plasma preparations were stored. Blood was collected from each donor mouse for serological testing for murine pathogens by Charles River Laboratories, Wilmington, MA. A qualified veterinary pathologist performed necropsies on the carcass and uterus of each donor animal and one embryo from each littermate. Organs from each animal were stored, along with plasma and cryopreserved leukocytes, for at least 30 years (as defined by EMEA / CHMP / CPWP / 83508 / 2009). As previously described, MEFs were isolated and cultured (Klimanskaya and McMahon, 2005), frozen at passage 1, and used at passage 2 after mitomycin C inactivation. To minimize the risk of introducing murine viruses and other pathogens, the MEFs were tested and characterized by WuXi AppTec, Inc. The testing specifications and results for lot MEF-08 used in the hESC-MCB formulation and the hRPE medicinal lot are presented in Table 4. After activation, it was used at passage 2. To minimize the risk of introducing murine viruses and other pathogens, the MEFs were tested and characterized by WuXi AppTec, Inc. The testing specifications and results for lot MEF-08 used in the hESC-MCB formulation and the hRPE medicinal lot are presented in Table 4.

[0296]

Table 4-1

[0297]

Table 4-2

[0298] DNA Q-PCR of human DNA Detection of human DNA content in mouse tissues was performed by AltheaDX, Inc., San Diego, CA, using a Taqman assay for Alu Y sequences with a sensitivity of 1 human cell per 150,000 mouse cells.

[0299] [Table 5]

[0300] Immunostaining of cells Cells in 4-well or 6-well plates were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min and then incubated for 1 h. Cells were permeabilized in 0.1% NP-40 substitute (Sigma) in PBS for 10 minutes, blocked with 10% goat serum in PBS for at least 1 hour, and incubated with primary antibodies overnight at 4°C. Cells were then washed three times for 15 minutes in 0.1% Tween / PBS, incubated with secondary antibodies for 1 hour at room temperature, washed as above, and mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Stained cells were examined under an inverted fluorescence microscope (Nikon). The antibodies used were bestrophin (Novus Biologicals), PAX6 (Covance), MITF (Abcam), ZO-1-FITC (Invitrogen), OCT-4 (Santa Cruz Biotechnologies), anti-mouse-Alexa 594 (Invitrogen), anti-rabbit-FITC (Jackson Immunoresearch), anti-mouse-Alexa 488 (Invitrogen), and anti-rabbit-Alexa 594 (Invitrogen). Alkaline phosphatase activity was detected using the Vector Blue kit (Vector Laboratories).

[0301] Immunostaining of mouse tissue sections For antigen retrieval (Bestrophin and human mitochondria), deparaffinized sections were incubated in 0.1 M citrate buffer (pH 6.0) in a steamer for 40 minutes or for Ki67 in a pressure cooker for 30 minutes. Antibody staining was performed as described above, and in some cases, an endogenous biotin blocking kit from Vector (Burlingame, CA) was used followed by a biotin-conjugated secondary antibody. The antibodies used were anti-Bestrophin (Abcam, rabbit), anti-human mitochondria (mouse, Spring Bioscience), and anti-Ki67 (rabbit, Abcam). The secondary antibodies were anti-mouse-biotin, anti-mouse-Cy3 (Jackson Immunoresearch), anti-rabbit-Alexa488 (Invitrogen), and Streptavidn-Cy3 was obtained from Jackson Immunoresearch. Mouse teratoma sections formed by hESCs were used as positive controls for anti-human mitochondria and Ki67, and fixed and paraffin-embedded hRPE pellet sections were used as a Bestrophin positive control. Negative controls were mouse rabbit and mouse IgG (Novus Biologicals).

[0302] q-RT-PCR RNA was extracted from the cell mixture using the RNeasy RNA isolation kit from Qiagen, resulting in a final volume of 30 μL of RNA per sample. Next, cDNA was synthesized from 10 μL of RNA using the Quantitect cDNA synthesis kit from Qiagen, resulting in a final volume of 20 μL of cDNA. Next, 1 μL of cDNA was tested for relative gene expression in triplicate replicate assays normalized to the β-actin signal present in each sample. Applied Biosystems Gene expression profiling was performed using StepOne Plus software version 2.1 and TaqMan gene expression assays from Life Technologies, following the cycling conditions for comparative Ct relative quantification recommended by the manufacturer. qRT-PCR assays for hES markers (NANOG, OCT4, and SOX2) and hRPE markers (RPE-65, PAX-6, MITF, and bestrophin) were normalized to the level of expression observed in 100% hES cell samples, which served as the zero set point (RQ = relative quantification). Relative gene expression was assayed in triplicate replicate tests normalized to the β-actin signal present in each sample. Data are presented as the mean + / - SD of three replicate tests.

[0303] Phagocytosis assay Phagocytosis was evaluated by a FACS-based assay using pHrodo (trademark) E. coli fluorescent bioparticles (Invitrogen). The bioparticles were prepared according to the manufacturer's instructions. Confluent RPE was cultured in CO2-independent medium (Invitrogen) with 50 - 200 μL of bioparticles per well in a 4-well plate at 37 °C for 16 - 20 hours. Negative control plates were cultured at 4 °C. Cells were examined under a microscope, collected by trypsin, counted for 10,000 events on a C6 flow cytometer, and analyzed by FACS.

[0304] Melanin measurement The RPE cell suspension was centrifuged at 160×G for 5 minutes at room temperature to remove samples for hemocytometer cell counting. The pellet was resuspended in 1N NaOH, heated at 80 °C for 10 minutes, vortexed, and the absorbance was measured at 475 nm by comparison with a synthetic melanin (Sigma catalog number 8631) standard curve in the range of 5 - 180 μg / mL. Samples were evaluated in triplicate and data were normalized to the total number of cells extracted.

[0305] ​

Table 6

[0306]

Table 7-1

[0307]

Table 7-2

[0308]

Table 7-3

[0309]

Table 7-4

[0310]

Table 8-1

[0311]

Table 8-2

[0312]

Table 8-3

[0313] Example 3 Adjustment of cell density to ensure delivery of the correct dosage This study describes the determination of the impact of loading and injection procedure steps on the delivery of live RPE. Specifically, in this example, it shows that the loading and injection procedures result in some loss of live cells, that this loss can be readily evaluated (and may vary depending on the delivery protocol, such as depending on the particular injection cannula used), and that this loss can be compensated for by increasing the cell concentration to enable delivery of the expected number of cells. Further, it was shown that cell seeding and growth are not significantly adversely affected following loading and extrusion through two cannulas.

[0314] These studies incorporate the entire loading and injection procedure, including: (1) the final addition of cold BSS-Plus to the concentrated final product RPE cells at 2000 live cells / μL to obtain the desired cell density to be injected. (2) The gentle mixing of RPE cells and BSS-Plus using an 18g blunt fill needle (BD) attached to a 1 ml injection syringe (BD LUER-LOK™). (3) The extrusion of 150 μL of the formulated RPE cells from the filled syringe through the injection cannula.

[0315] The maintenance of RPE cells in Alcon BSS BSS-Plus® on ice was demonstrated to be unchanged for over 4 hours provided that the cells are formulated at a concentration of 1000 cells / μL or greater. Under these conditions, there is no detectable loss in the number of live cells. To ensure cell integrity, the correct volume of RPE final product cells is delivered to the operating room at 2000 live cells / μL. Each tube of RPE cells is accompanied by a second tube containing the correct volume of cold BSS-Plus that is added to and mixed with the cells immediately prior to injection. The pre-dispensed RPE cells and BSS-Plus are delivered to the OR in sterile microcentrifuge tubes at 2-8°C.

[0316] Test 1 - MEDONE POLYTIP® Cannula 23 / 38 RPE cells with characteristics similar to those of the intended clinical RPE lot were thawed, processed, and formulated in cold BSS-Plus as described in Example 1. A total of 4.1 million viable cells were recovered after thawing and formulation. Starting viability was 91%, and the number of cells recovered after thawing and formulation was typical for this lot. Cells were diluted to the indicated starting concentration in cold BSS-Plus and stored on ice. Cells were then gently triturated using an 18g blunt fill needle (BD) attached to a 1mL syringe (BD LUER-LOK™). Approximately 200µL of cells were transferred through the fill needle and into the syringe. The fill needle was removed, and a MEDONE POLYTIP® 23 / 38 cannula was attached to the syringe containing the cells. 150µL of cells were administered through the infusion cannula by gently tapping the syringe plunger. The total infusion time was 2-3 minutes. Cells were harvested into sterile tubes and viable cell counts were assessed.

[0317] Test 1 showed that RPE cells loaded and extruded through the MedOne cannula were The results demonstrated a predictable loss in delivered cell density across the range of cell densities tested (295-1144 viable cells / µL). The mean loss in viable cell density was 22.8 + / - 7.0% (N=6). The results are shown in Table 9 below.

[0318] [Table 9]

[0319] A decrease in the number of cells delivered through the infusion cannula was observed at all cell densities tested, ranging from 295 to 1144 viable cells / µL. The percentage decrease in cell density appears to be generally consistent across the range tested. The percentage decrease observed at the two lowest densities tested (199 and 296) was more variable, likely reflecting the accuracy of cell counting at these lower cell densities.

[0320] The cells extruded through the MedOne cannula and the control cells that were formulated but not extruded through the cannula were centrifuged, resuspended in RPE growth medium, and seeded at 10,000 cells per well in a gelatin-coated Fluarea 96-well plate. For comparison, a portion of the same cell preparation was loaded into a Synergetics cannula (39ga rigid microinjection cannula, Angled) and passed through, and then processed and seeded as described above. Four days after seeding, the cells were trypsinized and counted. Table 10 below shows the mean cell count + / - SD for three cell counts.

[0321]

Table 10

[0322] Subsequent seeding and growth of the cells extruded through either cannula were equivalent to those of the control cells not extruded through the cannula.

[0323] Test 2 - Synergetics, Inc. injection cannula, Angled, 39g RPE cells from a lot with characteristics similar to the intended clinical RPE lot were thawed and processed, and formulated in cold BSS-Plus as described in Example 1. After thawing and formulation, a total 2.6 million live cells were recovered. The starting viability was 97%, and the number of cells recovered after thawing and formulation was typical for this lot. The cells were diluted in cold BSS-Plus to a starting concentration of 375 live cells / μL and stored on ice. Next, using an 18g blunt fill needle (BD) attached to a 1mL syringe (BD LUER-LOK™), the cells were gently triturated. Approximately 200L of cells were transferred into the syringe through the fill needle. The fill needle was removed, and a Synergetics, Inc. 39ga rigid microinjection cannula, Angled was attached to the syringe containing the cells. The plunger of the syringe was gently tapped to administer 150L of cells through the injection cannula. The total time of injection was 2 - 3 minutes. The cells were collected in a sterile tube and the number of live cells was evaluated. Over a series of 8 injections, the average number of live cells delivered was 238 + / - 25 live cells / μL, or approximately 100 fewer live cells than the delivery intended as the minimum cell dose for this study (50,000 cells per eye).

[0324] Therefore, in Test 2, it was demonstrated that RPE cells extruded through the loaded Synergetics cannula resulted in a predictable loss in cell density within the intended minimum cell dose range (a loaded density of 375 live cells / μL was tested). The average loss in live cell density was 38.4 + / - 6.8% (N = 8). The loaded cell density was increased accordingly to compensate for the expected loss and thus ensure accurate delivery of the intended number of live RPE (such as 50,000 cells / eye as in this study).

[0325] Test 3 - MedOne POLYTIP® cannula 23 / 38 and Synergetics 39ga rigid microinjection cannula, Angled Test 3 was performed on the RPE lot used for patient administration in Example 1 above. In this test, RPE cells were loaded at 25% higher than the dose-to-dose delivery cell density to compensate for the expected losses during syringe loading and injection through the MedOne cannula. The Synergetics cannula was verified using the same 25% compensatory loading density.

[0326] When loaded with cells formulated 25% higher than the minimum target dose (333 cells / μL delivered out of 444 live cells / μL), the MedOne cannula delivered 336 + / - 40 live cells / μL. Similarly, when loaded with cells formulated 25% higher than the minimum target dose (1,333 cells / μL delivered out of 1776 live cells / μL), the MedOne cannula delivered 1433 + / - 187 live cells / μL. The results of the lowest cell dose injection using the Synergetics cannula confirmed that an additional increase in the loaded cell density of 100 live cells / μL achieved the target dose at low density.

[0327] Eight vials (16 million cells total) were thawed and processed as described above (three centrifugations), and all procedures were performed at room temperature. The yield was 3.78 million cells with 95% viability (23.6% recovery, similar to previous thaw). The cells were resuspended in cold BSS-Plus to a storage and transport density of 2 million live cells / ml (2,000 live cells / μL) and then stored on ice. A cell density above 1 million cells / mL was selected to promote cell survival during refrigeration in BSS-Plus. Twenty-one 89 μL aliquots containing 177,600 total live cells were dispensed into microcentrifuge tubes with final product caps.

[0328] Cell aliquots were stored on ice until final dilution, at which point syringe loading and extrusion through the cannula were performed. For low-dose delivery (50,000 live RPE / eye), 311 μL of cold BSS-Plus was dispensed into the tube containing the cells, and the final volume was brought to 400 μL at a density of 444 cells / μL. This density is 25% higher than the intended delivery density of 333 cells / μL and compensates for the expected losses that occur during mixing with the fill needle, syringe loading, and delivery through the MedOne cannula. During delivery.

[0329] For high-dose delivery (200,000 live RPE / eye), two 89 μL aliquots of cells were pooled into one tube (356,000 cells), 22 μL of cold BSS-Plus was dispensed into the tube containing the cells, and the final volume was brought to 200 μL at a density of 1,776 cells / μL. This density is 25% higher than the intended delivery density of 1,333 cells / μL and compensates for the expected losses that occur during mixing with the fill needle, syringe loading, and delivery through the MedOne cannula.

[0330] The microcentrifuge tubes containing the diluted cells were capped and gently tapped with one finger to facilitate mixing. A blunt fill needle (dead volume 90 μL) was attached to a 1 mL BD syringe, and the cells were gently triturated 1 - 2 times within the blunt fill needle while taking care to minimize contact with the syringe. The syringe was filled with approximately 200 μL of cells. The blunt needle was removed and an infusion cannula was attached (MedOne 38g or Synergenic 39g). Approximately 150 μL of cells was dispensed into the microcentrifuge tube. Each dispensed aliquot was evaluated for cell density and viability by trypan blue exclusion. These results are summarized in Tables 11 and 12 below.

[0331]

Table 11

[0332]

Table 12

[0333] Increasing the initial loading density by 25% above the target dose effectively corrected for the loss of cell density encountered during loading and extrusion through the MedOne cannula. At the lowest dose administered, for a target delivery of 333 viable cells / μL, the MedOne cannula delivered an average cell density of 336 + / - 40 viable cells / μL (N = 6). At the highest cell density delivered (1333 viable cells / μL), the MedOne cannula delivered 1433 + / - 187 viable cells / μL (N = 3).

[0334] After delivery of the lowest dose through the MedOne or Synergetics cannula, the cells were diluted in RPE growth medium, centrifuged, and seeded at 40,000 cells per well in a gelatin-coated full area 96-well plate. Control cells not injected with the cannula, taken from the same tube as the cells extruded through the cannula, were treated and seeded similarly. Twenty-four hours after seeding, under all conditions tested, all cells had attached and no floating cells indicating cell death or seeding efficiency impairment were observed.

[0335] Cells extruded through the MedOne cannula, cells extruded through the Synergetic cannula, and control cells formulated but not extruded through the cannula were centrifuged, resuspended in RPE growth medium, and seeded at 40,000 cells per well in a gelatin-coated full area 96-well plate. Three days after seeding, the cells were trypsinized and counted. Table 13 below shows the mean cell number + / - SD. These results indicate that subsequent seeding and growth were not adversely affected by extrusion through either cannula. Control and MedOne cannula-injected cells were examined microscopically two days after seeding into culture, and typical RPE morphology with active cell division was shown. No difference was observed between control and cannula-injected cells.

[0336]

Table 13

[0337] In summary, RPE cells in cold BSS-Plus are more stable at concentrations exceeding 1000 cells / μL, and the final product can be resuspended in cold BSS-Plus at 2000 cells / μL in a microcentrifuge tube with a final product lid, such that a cannula can be loaded with a dose of 300,000 cells in a volume of up to 150 μL. After processing in a GMP clean room, two microcentrifuge tubes at 2 - 8 °C can be delivered to the operating room: one vial contains an exact volume of RPE cells at 2000 live cells / μL, and one vial contains an exact volume of cold BSS-Plus at a density such that when added to the cells, the density at which the cells are infused (i.e., a density 25% higher than the final target dose considering loss of live cells during loading and extrusion through the cannula, such as the loss of live cells by, for example, a MedOne cannula) is achieved. If a concentration higher than 1000 cells / μL or higher than 2000 cells / μL is loaded into the cannula, the dilution step can be omitted, and instead, the cells in cold BSS-Plus at the desired concentration can be delivered to the operating room.

[0338] The customized filling density and corresponding dose for the MedOne cannula are shown in Table 14. Similar customizations can be readily identified for the Synergetics cannula or another cannula or delivery system.

[0339]

Table 14

[0340] Example 4 RPE Differentiation from ES Cells This example describes the differentiation of RPE from hESCs. The resulting RPE was used in the tests described in Example 1.

[0341] Embryoid body differentiation medium (EB-DM) was composed of KnockOut™ DMEM supplemented with Glutamax, non-essential amino acids, 2-mercaptoethanol, and KnockOut™ Serum Replacement, and was used from the start of embryoid body formation until the time of harvesting and separating the pigmented patches, i.e., throughout embryoid body formation, proliferation, and subsequent pigmented patch formation. Each batch of EB-DM was composed of 250 mL of KnockOut™ DMEM, 3 mL of Glutamax-I, 3 mL of non-essential amino acids, 0.3 mL of 2-mercaptoethanol, and 38 mL of KnockOut™ Serum Replacement.

[0342] RPE growth / maintenance medium (RPE-GM / MM) was composed of 1 part of EB-DM (described in the preceding paragraph), 1 part of DMEM (high glucose), FBS, and Glutamax. This medium was used for the subsequent growth and maintenance of RPE from passage 0 to passage 2 after induction of RPE cells from the pigmented patches until the time of harvesting of the final bulk product. Each batch of RPT-GM / MM was composed of 100 mL of EB-DM, 90 mL of DMEM high glucose, 10 mL fetal bovine serum (FBS) (Hyclone), and 1 mL of Glutamax-I.

[0343] RPE cells induced and cultured in these media expressed the molecular markers RPE bestrophin, CRALBP, and RPE65. PEDF had phagocytic ability and rescued visual function in RCS rats.

[0344] RPE lots prepared using the above media passed all in-process quality tests, including morphological evaluation, immunohistochemical staining, and q-RT-PCR for upregulation of RPE genes and downregulation of hES cell gene expression. Yield and cell purity were comparable to RPE cells previously prepared using MDBK-GM and MDBK-MM media (Sigma Aldrich), OptiPRO-SFM, or VP-SFM.

[0345] From the time of embryoid body formation until the time of pigment spot harvesting, a lot of RPE was produced using EB-DM (instead of MDBK-GM or OptiPRO-SFM). After harvesting and trypsinizing the pigment spots, the passage 0 RPE cells were subsequently seeded into RPE-GM (EGM-2 medium) as defined above, and then switched to RPE growth / maintenance medium instead of MDBK-MM or VP-SFM. As an alternative, RPE may be seeded directly into RPE-GM / MM and grown and differentiated throughout the duration of the passage. After an appropriate level of differentiation was observed, the passage 0 RPE cells were harvested and further split two more times in these media until the final harvest and cryopreservation of the passage 2 bulk product.

[0346] The following data shows an overview of the manufacturing process test of five sublots of RPE maintained in EB-DM from the time of embryoid body formation until the time of pigment spot harvesting. At this point, the pigment spots were harvested from different wells on different days, trypsinized, and seeded as passage 0 RPE. Lots B1A, B2A, and B2B were seeded in EGM-2 medium until confluent, followed by a switch to RPE growth / maintenance medium, which promoted differentiation over passages 0, 1, and 2. Lots B3B and B3A were treated similarly except for passages 1 or 2 when maintained only in RPE-GM / MM throughout the duration of the passage. Thus, all lots were maintained in RPE-GM / MM until the appropriate level of differentiation was observed upon reaching confluence. After the end of passage 2, the RPE cells were cryopreserved as a bulk product. Lots maintained in EGM-2 for the initial growth phase followed by a switch to RPE-GM / MM, or lots maintained in RPE growth / maintenance medium throughout the duration of several passages, were similar except for a slightly more rapid growth rate observed in EGM-2 medium. All lots passed the morphological evaluation at passages 0, 1, and 2, and the acceptance criteria included typical epithelial, cobblestone morphology, and moderate pigmentation. RPE marker expression was detected by indirect immunofluorescence using the following primary antibodies (dilutions were approximately 1:100 to 1:1000 and were determined empirically for each antibody batch). Bestrophin - mouse monoclonal; Novus Biologicals (#NB 300-164); PAX6 - Covance, rabbit polyclonal (PRB-278P); ZO-1 - Invitrogen; mouse monoclonal (339100); ZO-1 - Invitrogen; rabbit polyclonal (61-7300); ZO-1-FITC - Invitrogen; mouse monoclonal (339111); MITF - mouse monoclonal, Abcam (ab320).

[0347] The secondary antibodies were used at 1:500 dilution (or other indicated dilution) in the blocking solution and were as follows: Alexa Fluor 488 anti-mouse, Invitrogen #A11001; Alexa Fluor 488 anti-rabbit, Invitrogen #A11008; Alexa Fluor 594 anti-mouse, Invitrogen #A11032; Alexa Fluor 594 anti-rabbit, Invitrogen #A11012; goat anti-mouse Cy3 conjugate (Jackson Immunoresearch catalog number 115-165-146), used at 1:200.

[0348] Immunostaining of the RPE marker was performed to evaluate purity by the combinations of PAX6 and MITF; bestrophin and PAX6, and ZO-1 alone. RPE maturation was evaluated by measuring the percentage of bestrophin-positive stained RPE. Immunostaining was performed at four time points during the production of RPE cells: (1) prior to harvest of passage 1 and seeded passage 2, RPE was stained for bestrophin, PAX6, and ZO-1; (2) prior to harvest and cryopreservation of passage 2, RPE was stained for bestrophin, PAX6, and ZO-1; (3) the RPE bulk product was thawed and formulated as described in Example 1, resuspended in BSS-PLUS at 1,000 live cells / μL. The cells were then diluted in RPE-GM, centrifuged at 1000 RPM, resuspended and seeded at 100,000 - 300,000 cells per well onto gelatin-coated 4-well plates and cultured for 1 - 2 days prior to staining with MITF and PAX6; (4) the RPE bulk product was thawed and formulated as described in Example 1, resuspended in BSS-PLUS at 1,000 live cells / μL. The cells were then diluted in RPE-GM, centrifuged at 1000 RPM, resuspended and Well plates were seeded with 50,000 - 200,000 cells per well and maintained until confluent prior to staining. At this point, the culture was switched to RPE-MM and maintained until moderate pigmentation and a cobblestone morphology were observed, at which point the cultures were stained for PAX6, bestrophin, and ZO-1. Briefly, cells were washed 2 - 3 times with PBS without Ca2+ and Mg2+ (Gibco #14190), fixed with 2% paraformaldehyde for 10 minutes, washed with 2×PBS, incubated with a 0.1% NP-40 substitute solution in PBS (Sigma #74388) for 15 minutes, washed with 2×PBS, and incubated with a blocking solution (10% normal goat serum (Jackson Immunoresearch #005-000-121), 16% paraformaldehyde prepared at a working concentration of 2% in PBS (freshly made or frozen aliquots) (Electron Microscopy Sciences #15710)) for 30 minutes to overnight. Next, the cells were incubated with primary antibodies in the blocking solution (up to 2 antibodies per well using primary antibodies from different species) for 1 - 2 hours at room temperature or overnight at 4°C, washed with PBS, and washed 3 times (each wash for 10 - 15 minutes) with agitation in a PBS-Tween solution (PBS without Ca2+ and Mg2+ (Gibco #14190) with 0.5% Tween 20 (Sigma #P7949) added). The samples were then incubated with secondary antibodies and washed in the same manner as the primary antibodies. After removal of the final wash solution, 1 - 2 drops of DAPI-containing Vectashield were added and the cells were examined and counted on an inverted fluorescence microscope. Three to six random fields containing at least 1000 nuclei were photographed at 20× magnification in all channels. The photographs were merged and the image was adjusted as needed to visualize which cells were negative for bestrophin and PAX6, or negative for PAX6 and MITF, and negative for ZO-1.Cells were considered positive for a given marker if predicted staining patterns were observed, such as nuclear localization of PAX6, plasma membrane localization of bestrophin in a polygonal pattern (showing localization of bestrophin staining as distinct lines around the cell periphery), ZO-1 staining present during tight junctions outlining the cell contour of the polygonal pattern, and MITF staining detected by nuclear confinement. The percentage of cells positive for each marker or marker combination was determined by counting the positive cells in the merged image and determining the total number of cells by counting the nuclei from the unmerged DAPI-stained image.

[0349]

Table 15

[0350] Furthermore, mRNA expression was detected by q-RT-PCR as described in Example 1. The results obtained from each lot are shown in Table 16, demonstrating that the RPE genes were upregulated and the ES cell genes were downregulated as predicted.

[0351]

Table 16

[0352] RPEs produced using the above-described media formulations (RPE-GM / MM and EB-DM), and cryopreserved RPE cells previously produced using other media (MDBK-GM and MDBK-MM), were tested for their phagocytic ability. In this test, cryopreserved RPE was thawed and seeded in RPE growth / maintenance medium. RPE cells from this lot, created using EB-DM during embryoid body formation and pigment spot formation and RPE-GM / MM during RPE maturation, were trypsinized and seeded similarly in RPE-GM / MM. Both cultures were grown to confluence and maintained in RPE-GM / MM until differentiation, after which their ability to phagocytose fluorescent biological particles (Invitrogen catalog number P35361), which fluoresce when internalized in the acidic environment of the phagosomes of RPE cells, was examined. Cells were cultured at 37 °C with the fluorescent biological particles to allow phagocytosis, or at 4 °C as a negative control. For cells cultured at 37 °C, the shift in fluorescence intensity indicative of bioparticle phagocytosis was detected by FACS (Figure 12). Statistical integration of the peaks yields the percentage of phagocytosis-positive cells for each lot and culture temperature.

[0353]

Table 17

[0354] These results show phagocytosis in a high percentage of cells in both lots of RPE cells maintained on RPE-GM / MM, further demonstrating the appropriateness of using RPE-GM / MM for the growth and maturation of RPE cells.

[0355] Example 5 Additional Exemplary Methods of RPE Induction Using the methods of this example, additional hESC lines generated without destroying the embryo Differentiated RPE was generated, and these additional hESC lines were, in particular, iPS cells (especially those generated using non-integrating episomal vectors), and NED ("no embryo destruction") hES cells generated from biopsied blastomeres from which embryos were obtained and subsequently cryopreserved while maintaining viability. NED cells were generated as described in Chung et al. (Cell Stem Cell. 2008 Feb 7;2(2):113-7), which is incorporated by reference in its entirety.

[0356] hESCs were grown on Matrigel™ diluted according to the manufacturer's instructions for use with TESR-1 medium (Stem Cell Technologies, Inc.). RPE was generated from embryoid bodies ("EBs") or multilayer hESC cultures as previously described (Klimanskaya et al., Cell Stem Cells 6:217-245 (2004), which is incorporated by reference in its entirety); EBs were seeded for proliferation prior to RPE harvest after suspension culture. However, EB formation from hESCs cultured on Matrigel™ was observed to be less efficient, with cells showing a lower success rate of aggregation and reduced viability. The following protocol modifications improved EB formation efficiency.

[0357] hESCs were overgrown beyond the normal passage time so that the colonies were "thicker", i.e., slightly raised / or had a multilayer structure. For EB formation, hESCs were detached without dissociating them into a single cell suspension using mechanical scraping, collagenase I, accutase, collagenase with added accutase, or collagenase followed by accutase, an EDTA-based dissociation buffer. These methods allowed the hESC colonies to be peeled off without dissociating them into single cells. Trypsin, which typically tends to readily generate a single cell suspension under normal use conditions, was not utilized.

[0358] Next, the isolated hESCs were cultured on ultra-low attachment plates to form EBs. Optionally, other methods such as hanging drops may be used for EB formation. Typically, hESCs from 1 to 3 wells of a 6-well culture dish were cultured in 2 to 7 ml of medium in 1 to 2 wells of a low attachment plate. The cells were cultured in EB medium (knockout high glucose DMEM, 1% non-essential amino acid solution, 2 mM GlutaMAX I, 0.1 mM β-mercaptoethanol, and 13% serum replacement (SR, Invitrogen)). During the first 2 to 3 days of culture in EB medium while the EBs were forming, the EB medium was supplemented with 10 micromolar concentration of Stemgent's Stemolecule Y-27632, a rho-associated protein kinase (ROCK) inhibitor (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):681-6, which is incorporated by reference in its entirety). The use of the ROCK inhibitor improved cell viability, particularly for hES cells obtained using EDTA or enzymatic dissociation. The use of the ROCK inhibitor was optional for mechanically scraped hESCs that survived well without it.

[0359] Seven to twelve days after EB formation, the EBs were seeded onto gelatin-coated plates for proliferation. RPEs were easily identified by their epithelial morphology (cobblestone appearance) and pigmentation.

[0360] RPEs were also generated from multilayer cultures of hESCs grown on Matrigel (TM), essentially as previously described (Klimanskaya et al., 2004, supra), except that the cells were cultured on Matrigel (TM) instead of on feeder cells. Briefly, hESCs were overgrown in mTESR-1 medium on Matrigel (TM) until the hESC colonies formed a multilayer structure (cultured for approximately 10 to 14 days), at which point the medium was replaced with EB medium (as described above). The ROCK inhibitor was optional. It is contained in the medium but is not essential for efficient RPE formation and recovery. The RPEs were easily identified by their epithelial morphology (paving stone-like appearance) and pigmentation. The medium was changed every 1 - 2 days until pigmented RPE cells were observed (typically within 4 - 5 weeks).

[0361] The resulting EBs or multi-layered cultures exhibited a "freckled" appearance containing dark regions visible to the naked eye. Microscopic examination confirmed that these dark regions were composed of RPE cells distinguishable by their characteristic pigmentation and paving stone-like epithelial morphology. The resulting RPE cell cultures are shown in Figure 19. After differentiation from hESCs, the RPE cells were isolated by either mechanical or enzymatic dissociation.

[0362] Example 6 RPE transplantation method For cell transplantation into patients with dry age-related macular degeneration (AMD) and Stargardt macular dystrophy (SMD), the following method was used.

[0363] Immediately before surgery, no corticosteroids were administered to the patients. The surgery was performed under general or local anesthesia, with or without conscious sedation at the discretion of the surgeon.

[0364] The cells for transplantation were provided as a frozen suspension stored in the vapor phase of a liquid nitrogen storage system (approximately -140 °C). To prepare the cells for administration, the vial was removed from the liquid nitrogen freezer and then placed in a 37 °C water bath and continuously agitated for 1 - 2 minutes until thawed. The vial was then sprayed with 70% isopropanol and allowed to dry. The contents of each vial (1 mL of cryopreservation medium containing 1 million cells at the time of freezing) were transferred to a 50 mL conical tube and washed with 40 mL of serum-free DMEM. The cells were centrifuged and each pellet was resuspended in 40 mL of BSS-PLUS. If the cell suspension was centrifuged again and two or more cryopreservation vials were thawed, the pellets were combined and pooled. The volume was adjusted to a final volume of 10 mL in BSS PLUS and centrifuged a third time. The supernatant was completely aspirated and the cells were resuspended to a final volume of approximately 150 μL of BSS PLUS per 1 mL of thawed cells (a lower volume could be used if a more concentrated suspension was desired). Samples were taken and the number of viable cells was counted. The total number of viable cells was measured and an appropriate volume of BSS-PLUS was added to obtain a target viable cell concentration such as 2,000 viable cells per μL. An appropriate volume of the prepared product was transferred to a 0.5 mL sterile microcentrifuge tube and samples were taken for archiving, viability determination, Gram staining, and sterility testing. A pair of 0.5 mL sterile microcentrifuge tubes containing the correct volume of BSS-Plus were also prepared and labeled. The paired vials were stored at 2 - 8 °C for no more than 4 hours in preparation for the final mixing and transplantation in the operating room.

[0365] Standard three-port trans pars plana vitrectomy was performed on the patient. A small retinotomy was made and then an infusion of BSS Plus into the subretinal space was performed using a fluid infusion system through the vitrectomy device until a small neurosensory retinal detachment occurred. The surgeon ensured that a bleb was created at the temporal position. The bleb could optionally extend into the arcade vessels, but the central retinal macula / fovea was not detached. If the bleb was observed to expand towards the central retinal macula, the surgeon had the option to follow the same protocol at that location, stop, and perform another retinotomy. The BSS Plus injected subretinally was then removed.

[0366] Next, a preloaded cannula was inserted, and cells were infused into the created space over approximately 1 minute in a volume of 150 μL. Visual monitoring was performed to ensure accurate cannula positioning. To accurately correlate postoperative findings with the position of the bleb, the exact position of the bleb was recorded by photography or (preferably) video through the operating microscope. did.

[0367] A suspension containing the desired number of hESC-derived RPE cells (e.g., 50,000, 100,000, 150,000, or 200,000) in 150 μL of BSS Plus was transplanted. The cells were infused over approximately 1 minute. The cannula was kept in position for an additional 1 minute to avoid reflux. Air-fluid exchange was optionally performed at the discretion of the surgeon, for example, if the retinotomy was enlarged. The incision was then closed using standard procedures. The patient then recovered from anesthesia but was maintained in the dorsal recumbent position for 6 hours.

[0368] For 48 hours following the procedure, corticosteroid administration was not permitted. If necessary, the use of local or systemic non-steroidal anti-inflammatory agents was permitted to manage postoperative discomfort.

[0369] Example 7 Stability of cryopreserved RPE cell preparations This example demonstrates that cryopreserved RPE cells meet the release criteria and remain suitable for use when tested at 6 and 12 months after freezing. Based on this, cryopreserved RPE has been shown to maintain their functions and product attributes for up to 12 months after cryopreservation. Cryopreserved RPE cells are expected to remain suitable for transplantation for many years after freezing (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more).

[0370] Cryopreserved RPE samples were generated as described in Example 4 above and frozen in a cryopreservation medium (10% DMSO in 90% FBS) in liquid nitrogen and stored in the vapor phase of a liquid nitrogen storage system (approximately -140 °C). After 6 or 12 months of storage, the cryopreserved cells were thawed and washed as described in Example 6 (briefly, thawed in a 37 °C water bath, the outside of the container was washed with 70% ethanol, and the cells were washed to remove the cryopreservation medium). After thawing, the cells were subjected to inspection to confirm the stability of the product. As shown in Table 18 below, each release criterion was met.

[0371]

Table 18

[0372] Example 8 Stability of Formulated RPE Cells This example demonstrates that when RPE cells are maintained at 2 - 6 °C, they remain suitable for use for at least 4 hours after preparation for thawing and administration.

[0373] Cryopreserved cells were thawed and formulated as described in Example 6 above and stored at 2 - 8 °C in the final product container (sterile microcentrifuge tube with 0.5 mL gasket). Table 19 shows the mean viability percentage ± SD by trypan blue exclusion for two lots tested at the time of formulation and after 4 hours of refrigeration (3 final formulations per lot).

[0374]

Table 19

[0375] These data showed that the formulated RPE cells maintained cell viability up to 4 hours after preparation.

[0376] In additional experiments, the RPE cell final product was formulated at 2,000 viable cells / μL and refrigerated for various times prior to extrusion through a MedOne REF 3233 POLYTIP® cannula 23 / 38. In this test (data shown in Table 20), an RPE cell lot (which passed the release test for the bulk product for clinical use) was thawed and processed as described above. The cells were resuspended and stored at a density of 2,000 viable cells / μL in BSS-Plus and then kept on ice. A cell density above 1,000 live cells / μL was selected to promote cell survival during refrigeration in BSS-Plus. At this point, 21 cell aliquots of 89 μL, containing a total of 177,600 viable cells, were dispensed into microcentrifuge tubes with final product caps. The cell aliquots were stored on ice until final dilution, at which point syringe loading and extrusion through the cannula were performed. For low-dose delivery, 311 μL of cold BSS-Plus was dispensed into the tube containing the cells, and the final volume was made 400 μL at a density of 444 cells / μL. This density is 25% higher than the intended delivery density of 333 cells / μL to compensate for the expected losses that occur during mixing with the fill needle, syringe loading, and delivery through the MedOne cannula.

[0377] For high-dose delivery, two 89 μL aliquots of cells were pooled into one tube (356,000), 22 μL of cold BSS-Plus was dispensed into the tube containing the cells, and the final volume was made 400 μL at a density of 1,776 cells / μL. This density is 25% higher than the intended delivery density of 1,333 cells / μL to compensate for the expected losses that occur during mixing with the fill needle, syringe loading, and delivery through the MedOne cannula.

[0378] The microcentrifuge tube containing the diluted cells was capped and gently tapped with one finger to promote mixing. A blunt fill needle (void volume 90 μL) was attached to a 1 mL BD syringe, and the cells were gently triturated 1 - 2 times (1 - 2 times) in the blunt fill needle while taking care to minimize contact with the syringe. The syringe was filled with approximately 200 μL of cells. The blunt needle was removed and a MedOne 38g infusion cannula was attached. Approximately 150 μL of cells were dispensed into the microcentrifuge tube. Each dispensed aliquot was evaluated for cell density and viability by trypan blue exclusion. The time after formulation was the time (in minutes) elapsed from the resuspension of the cells at 2,000 live cells / μL in cold BSS - Plus. These data are shown in Table 20 for the cells delivered at the indicated concentrations.

[0379]

Table 20

[0380] The data indicate that when refrigerated over the time tested, the viable cell count of the final product RPE cells extruded through the infusion cannula did not decrease. The viable cell density observed at times greater than 240 minutes (4 hours) after formulation supports a minimum 4 - hour shelf - life. In this study, RPE cells in BSS - Plus were stored in the final product seal on ice. Subsequently, the temperature of the BSS - Plus in the microcentrifuge tube stored on ice was evaluated using a calibrated probe and measured at 3°C.

[0381] Further experiments tested the viability of the formulated RPE cells up to 6 hours. The viability was adjusted Evaluations were performed at the time of formulation (0 hours) and after 4 and 6 hours of refrigeration (2 - 8°C). The RPE lots used in this study were manufactured and cryopreserved using the procedures, processes, and materials described for GMP manufacturing. Cryopreserved vials of RPE cells were thawed and formulated according to the procedure described in Example 6 above. Cells were evaluated for viable cell number at the time of formulation (0 hours) and after 4 and 6 hours of refrigeration (2 - 8°C). For subsequent purity and potency assessments, cells at 0, 4, and 6 hours were seeded and cultured. At each seeding time point (0, 4, and 6 hours), purity was evaluated by MITF and PAX6 immunostaining, and phagocytosis of fluorescent particles was evaluated by FACS analysis.

[0382] Viable cell density was determined by counting trypan blue - excluding cells with a hemocytometer. Data are the mean + / - SD of counts performed on 4 hemocytometer chambers. Results are shown in Table 21 below.

[0383]

Table 21

[0384] Temperature readings of the GMP storage refrigerator storing the formulated cells confirmed that the temperature remained at 6°C throughout the experiment.

[0385] Starting viable cell densities of 2,590 cells / μL and 1,700 cells / μL at 0 hours are considered together as 2,000 viable cells / μL for a medical preparation. No loss of viable cell number was observed over the range of starting cell densities tested during 6 - hour refrigeration.

[0386] Example 9 This example provides the initial treatment results for two additional patients with Stargardt’s disease. The two patients were each treated with 50,000 RPE cells derived from hESC (described in Example 1) using the RPE transplantation method described in Example 6 above. Fundus photographs of the retina, optic nerve disc, macula, and posterior pole of the two Stargardt patients show the injection site and the bleb area created upon injection of the solution containing the RPE cells (Figure 15).

[0387] Additional fundus photographs show the establishment of areas within the injection blebs with increased pigmented RPE cell patches for the two SMD patients (Figures 16 and 17). These results suggest engraftment and surface reorganization of the retinal area by the new RPE layer.

[0388] For the treated eye of the patient shown in Figure 16, visual acuity was also evaluated. The vertical axis shows the Early Treatment Diabetic Retinopathy Study (ETDRS) score, and the horizontal axis shows the number of days post - surgery.

[0389] These results show stable engraftment of the RPE cells lasting at least three months after treatment. The visual acuity of the treated eye returned to the baseline level at 14 days after treatment and continued to be above baseline until day 84, the final time point shown.

[0390] Example 10 One - year patient evaluation AMD patients and SMD patients were evaluated over a one - year period after RPE treatment as described in Example 1 above.

[0391] Fundus photography of the SMD patients demonstrated the presence of pigmented cells in the treated eyes one year after treatment (Figure 20B). In contrast, at the pre - treatment baseline, pigmented cells were undetectable (Figure 20A). These results show long - term engraftment of the RPE maintained for at least one year after treatment.

[0392] For AMD patients, the peripheral ETDRS / BVCA scores are graphed in Figure 21. The patients' peripheral ERTDS-BVCA scores decreased from an initial baseline value of 21 to 0 one and three days after surgery, but returned to at least the baseline level seven days after surgery and then continued to exceed the baseline. At one year after treatment, the patients' peripheral ERTDS-BVCA scores were 34.

[0393] In SMD patients, at one year after treatment, the central ETDRS / BVCA score was 15. The peripheral scores are graphed in Figure 22. The patients' peripheral ERTDS-BVCA scores increased from an initial baseline value of 0 to 1 two weeks after surgery and then continued to increase to a value of 15 at one year after treatment.

[0394] These results demonstrate vision improvement in both AMD and SMD patients due to RPE cell administration, which was maintained for at least one year after treatment.

[0395] References 1.Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 2008;282:1145-1147. 2.Fink DW, Bauer SR. Stem cell-based therapies: Food and Drug Administration product and pre-clinical regulatory considerations. In: Lanza R, Hogan B, Melton D, Pedersen R, Thomas, ED, Thomson J, Wilmut I, eds. Essentials of Stem Cell Biology. San Diego: Academic Press / Elsevier, 2009:619-630. 3.Lanza RP,Chung HY Yoo JJ,et al.Generation of histocompatible tissues using nuc lear transplantation.Nature Biotechnology 2002;20,689 - 696. 4.Takahashi K,Tanabe K,Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 2007;131,861-872. 5.Kim D,Kim,CH,Moon JI,et al.Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.Cell Stem Cells 2009;4,472-476. 6.Kaplan HJ,Tezel TH,Berger AS,Del Priore LV.Retinal transplantation.In:Streilein JW,ed.Immune Response and the Eye.Chem Immunol.Basel:Karger,1999:207-219. 7.Lund RD,Wang S,Klimanskaya I,et al.Human embryonic stem cell-derived cells rescue visual function in dystrophic rats.Cloning and Stem Cells 2006;8,189-199. 8.Lu B,Malcuit C,Wang S,et al.Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration.Stem Cells 2009;21,2125-2135. 9.Sparrow JR,Hicks D,Hamel CP.The retinal pigment epithelium in health and disease.Curr Mol Med 2010;10,802-823. 10.Strauss O.The retinal pigment epithelium in visual function.Physiol Rev 2005;85,845-881. 11.Binder S.,et al.Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration:a prospective trial.Invest Ophthalmol Vis Sci 2004;45,4151-4160. 12.Algvere PV,Berglin L,Gouras P,Sheng Y.Transplantation of fetal retinal pigment epithelium in age-related macular degeneration with subfoveal neovascularization.Graefes Arch Clin Exp Ophthalmol 1994;232,707-716. 13.Kaplan HJ,Tezel TH,Berger AS,Del Priore LV.Retinal transplantation.Chem Immunol 1999;73,207-219. 14.Binder S,Stolba U,Krebs I,et al.Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration:a pilot study.Am J Ophthalmol 2002;133,215-225. 15.MacLaren RE,Bird AC,Sathia PJ,Aylward GW.Long-term results of submacular surgery combined with macular translocation of the retinal pigment epithelium in neovascular age-related macular degeneration.Ophthalmology 2005;112,2081-2087. 16.Lappas A,Weinberger AW,Foerster AM,Kube T,Rezai KA,Kirchhof B.Iris pigment epithelial cell translocation in exudative age-related macular degeneration.A pilot study in patients.Graefes Arch Clin Exp Ophthalmol 2000;238,631-641. 17.Aisenbrey S,Lafaut BA,Szurman P,et al.Iris pigment epithelial translocation in the treatmen...

Claims

A method for generating retinal pigment epithelial (RPE) cells for use as a therapeutic agent for retinal degenerative conditions, comprising: (a) culturing a population of RPE cells; (b) measuring the average melanin content in the population; and (c) harvesting the population as a therapeutic agent for retinal degenerative conditions when the average melanin content measured in (b) is between 0.1 and 8 pg / cell.

2. The method according to claim 1, wherein the population is harvested in (c) when the average melanin content measured in (b) is between 1 and 5 pg / cell, between 3 and 5 pg / cell, between 4 and 5 pg / cell, or between 4.2 and 4.8 pg / cell.

3. The method according to claim 1 or 2, wherein step (b) comprises: (i) lysing a population of RPE cells; and (ii) measuring the absorbance at 475 nm in comparison with a melanin standard curve.

4. In (i), the RPE cells are lysed by exposing the cells to NaOH and high temperature, preferably, wherein the NaOH has a concentration of 1 N, the high temperature is about 80 °C, the RPE cells are exposed to NaOH and elevated temperature for about 10 minutes, or a combination thereof, The method according to claim 3.

5. The method according to any one of claims 1 to 4, wherein the RPE cells are human cells, are generated in vitro from pluripotent stem cells, or a combination thereof.

6. At least 50%, at least 60%, at least 70%, or at least 80% of the harvested RPE cells are bestrophin+; At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the harvested RPE cells are PAX6+; At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the harvested RPE cells are MITF+; At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the harvested RPE cells are ZO-1+; Less than 1% of the cells in the harvested population are not RPE cells; or a combination thereof, The method according to any one of claims 1 to 5.

Images