Pharmaceutical preparations of human RPE cells and uses thereof

Abstract

To provide the first description of hESC-derived cells transplanted into human patients, wherein results are reported for one patient with each of Stargardt's Macular Dystrophy (SMD) and Dry Age-Related Macular Degeneration (AMD).SOLUTION: Controlled hESC differentiation resulted in near-100% pure RPE populations. Immediately after surgery, hyperpigmentation was visible at a transplant site in both patients, with subsequent evidence that cells had attached and integrated into a native RPE layer. No signs of inflammation or hyperproliferation were observed. HESC-derived RPE cells showed no signs of rejection or tumorigenicity at a time of this report. Visual measurements suggest improvement in both patients.SELECTED DRAWING: Figure 1A-B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Application No. 61 / 559,521, filed November 14, 2011, U.S. Provisional Application No. 61 / 724,047, filed November 8, 2012, and U.S. Provisional Application No. 61 / 589,741, filed January 23, 2012, the entire contents of each of which are incorporated herein by reference.

[0002] Human embryonic stem cells (hESCs) are considered a promising source of replacement cells for regenerative medicine (1). Despite significant scientific advances, hESCs remain one of the most complex biological therapeutic entities proposed for clinical use to date (2). In addition to the dynamic complexity of their biology, numerous regulatory concerns, including the risk of teratoma formation and challenges associated with histoincompatibility, have hindered their clinical translation. Until further developments in cell reprogramming technologies, such as somatic cell nuclear transfer (3) or induced pluripotent stem cells (4, 5), diseases affecting the eye and other immune-privileged sites will likely represent the first pluripotent stem cell-based therapies in patients. It is well established that the subretinal space is protected by the blood-ocular barrier, 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 a variety of sight-threatening diseases, including dry age-related macular degeneration (AMD) and Stargardt macular dystrophy (SMD), the two leading causes of adult and early-onset blindness worldwide, respectively. While neither disease is currently treatable, there is evidence that transplantation of hESC-derived RPE can rescue photoreceptors and prevent vision loss in preclinical models of macular degeneration (7, 8). Among its functions, the RPE maintains photoreceptor health by recycling photopigments, delivering, metabolizing, and storing vitamin A, phagocytosing photoreceptor outer segments, transporting iron and small molecules between the retina and choroid, and absorbing stray light to enable better image resolution (9, 10). In the Royal College of Surgeons (RCS) rat, an animal model of visual deterioration due to RPE dysfunction, subretinal transplantation of hESC-derived RPE resulted in massive photoreceptor rescue and visual improvement (100% greater than untreated controls) without evidence of adverse pathology (7). The retinal pigment epithelium (RPE) is a pigmented cell layer located outside the neurosensory retina, between the underlying choroid (the vascular layer behind the retina) and the overlying retinal photoreceptors (e.g., photoreceptor rods and cones). The RPE is important for the function and health of photoreceptors and the retina. The RPE maintains photoreceptor function by recycling photopigments, delivering, metabolizing, and storing vitamin A, phagocytosing rod photoreceptor outer segments, transporting iron and small molecules between the retina and choroid, maintaining Bruch's membrane, and absorbing stray light to allow for better image resolution. Engelmann and Valtink (2004) "RPE Cell Cultivation." See also Graefe's Archive for Clinical and Experimental Ophthalmology 242(1):65-67; ​​Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, in STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009). Degeneration of the RPE can lead to retinal detachment, retinal dysplasia, or retinal atrophy, which are associated with several degenerative visual diseases resulting in photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava). See, e.g., WO 2009 / 051671. I want to be illuminated. [Background technology]

[0004] Certain subject matter, including RPE cells, methods of making RPE cell compositions, and uses thereof, are described in U.S. Pat. Nos. 7,736,896, now U.S. Pat. No. 11 / 186,720, filed July 20, 2005; 7,795,025, now U.S. Pat. No. 11 / 490,953, filed July 21, 2006; and 11 / 490,953, now U.S. Pat. No. 7,795,025, filed January 24, 2005; each of which is incorporated herein by reference in its entirety. No. 7,794,704, filed as U.S. Provisional Patent Application No. 1 / 041382; U.S. Provisional Patent Application No. 60 / 538,964, filed January 23, 2004; U.S. Provisional Patent Application No. 12 / 682,712, filed October 10, 2010; U.S. Provisional Patent Application No. 60 / 998,668, filed October 12, 2007; U.S. Provisional Patent Application No. 60 / 998,766, filed October 12, 2007; U.S. Provisional Patent Application No. 60 / 998,766, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 009908, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 009911, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 367038, filed July 23, 2010; U.S. Provisional Patent Application No. 61 / 414770, filed November 17, 2010; International Patent Application No. PCT / US11 / 45232, filed July 25, 2011; U.S. Provisional Patent Application No. PCT / US11 / 45232, filed December 14, 2010. No. 12 / 682712, filed Jan. 24, 2005; International Patent Application No. PCT / US05 / 02273, filed Jan. 24, 2005; International Patent Application No. PCT / US2010 / 57056, filed Nov. 17, 2010 (published as WO 2011 / 063005); and U.S. Provisional Patent Application No. 61 / 262,002, filed Nov. 17, 2009. [Prior art documents] [Non-patent literature]

[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 [Problem to be solved by the invention]

[0006] Transplantation of intact sheets and suspensions of primary RPE cells has previously been attempted in human subjects, with mixed results regarding 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 problem]

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

[0008] One year after treatment, no hyperproliferation, tumorigenicity, ectopic tissue formation, or overt rejection was observed in either patient at any time point. Detailed clinical and diagnostic laboratory evaluations were performed at multiple evaluations after transplantation. Due to their pluripotency, abnormal proliferation (or tumor formation) is considered a significant safety concern for stem cell-based therapies, particularly hESC-derived stem cell-based therapies; therefore, controlling hESC differentiation is critical. 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 in patients with SMD. This evidence included increased pigmentation at the RPE level within the transplanted area, beginning 1 week after transplantation and continuing throughout the follow-up period. The transplanted stem cell-derived RPE appeared to engraft in the appropriate location and adopt normal RPE morphology. Engraftment and increased pigmentation were not detected in patients with dry AMD. However, both patients showed some visual improvement at a 4-month follow-up period, which persisted for at least 1 year of follow-up.

[0010] As detailed below, the visual acuity of a patient with Stargardt disease improved from manual valve alone to 20 / 800 vision. Before treatment, the patient was unable to read any letters on the ETDRS visual acuity chart. However, two weeks after implantation, she was able to begin reading letters with the treated eye, which improved to five letters within one to three months and to 15 letters within one year (20 / 500 vision).

[0011] While several new medications are available to treat wet AMD, there are currently no proven cures for either dry AMD or Stargardt's disease. Despite the progressive nature of these conditions, both patients' vision appeared to improve after cell transplantation, even at the lowest doses. Applicants expect even more significant improvement if they treat patients earlier in the disease course, when more significant results might potentially be expected. Increasing the cell dose may also result in more significant improvement.

[0012] Human embryonic stem cells may provide an excellent source of replacement tissue by generating an unlimited number of healthy "juvenile" cells with potentially reduced immunogenicity. The eye is an immune-privileged site due to the protection of the subretinal space by the blood-eye barrier; as a result, only low, transient doses of immunosuppression were used. No signs of rejection or inflammation were observed in either patient, and doctors continue to monitor both patients.

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

[0014] hESC-derived RPE cells underwent extensive safety testing prior to transplantation. Cells were confirmed to be free of animal and human pathogens, and sensitive assays were performed to eliminate the presence of any undifferentiated hESCs, a risk factor for tumorigenesis, 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 demonstrate that the degree of RPE maturity and pigmentation can dramatically affect the subsequent attachment and growth of cells after transplantation.

[0015] Both trials are prospective, open-label studies that will evaluate the efficacy and safety of hESC-derived RPE cells at the primary study endpoint of 12 months following subretinal transplantation in patients with SMD and dry AMD. The trials are designed to determine safety and tolerability. Each trial will enroll 12 patients in cohorts of three patients each in an escalating dose format. Patients with both SMD and dry AMD received subretinal transplants 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 plurality of RPE cells have an average melanin content of less than 8 pg / cell. The RPE cells may be contained in a suspension, gel, colloid, substrate, matrix, scaffold, or graft.

[0017] The pharmaceutically acceptable carrier may comprise a sterile solution having an osmolality 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, which may comprise, consist of, or consist essentially of, per mL, 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), and hydrochloric acid and / or sodium hydroxide (to adjust the pH to about 7.4) in water.

[0018] The volume of the pharmaceutical composition may be about 100 μL to 1000 μL, or may be at least about 150 μL. The pharmaceutical composition contains about 1,000 to about 1×10 9The pharmaceutical composition may comprise about 333 live RPE cells / μL to about 2,000 live RPE cells / μL, about 444 live RPE cells / μL to about 1,766 live RPE cells / μL, about 333 live RPE cells / μL, about 444 live RPE cells / μL, about 666 live RPE cells / μL, about 888 live RPE cells / μL, about 999 live RPE cells / μL, or about 1,333 live RPE cells / μL.

[0019] The concentration of RPE cells in the pharmaceutical composition may be high enough so 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 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 product 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 may not be RPE cells.

[0021] The average melanin content of the RPE cells may be less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, less than 5 pg / cell, less than 4 pg / cell, less than 3 pg / cell, less than 2 pg / cell, and at least 0.1 pg / cell, and optionally at least 0.5 pg / cell or 1 pg / cell; 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+. 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 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 exemplary embodiments, no more than about 1 cell per 1 million cells, and optionally no more than 2 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 about 444 viable cells / μL to about 1,766 viable cells / μL when loaded into the needle or injection cannula. The concentration of viable RPE cells delivered from the needle or injection cannula may be about 333 viable cells / μL to about 1,333 viable cells / μL. The needle or injection cannula may have a diameter of about 0.3 mm to about 0.9 mm. The needle or injection cannula may have a diameter of about 0.5 mm to about 0.6 mm. The needle or injection cannula may include 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 (0.50mm (25g) x 28mm cannula with a 0.12mm (38g) x 5mm tip) or a Synergetics Angled 39g infusion cannula.

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

[0026] The RPE cells may be human cells.

[0027] The pharmaceutical composition may further comprise at least one angiogenesis inhibitor, which may be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with said RPE cells. Exemplary angiogenesis inhibitors include pepgatanib sodium; aflibercept; bevasiranib; rapamycin; AGN-745; vatalanib (vitalanib); pazopanib; NT-502; NT-503; PLG101; CPD791; anti-VEGF antibodies or functional fragments thereof; bevacizumab; ranibizumab; anti-VEGFR1 antibodies; anti-VEGFR2 antibodies; anti-VEGFR3 antibodies; 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); tyrosine kinase inhibitors (TKIs); TKIs that inhibit VEGFR-1 and / or VEGFR-2; sorafenib (Nexavar) );SU5416 (semaxinib);SU11248 / sunitinib (Sutent);vandetanib (ZD6474);Ly317615 (enzastaurin);anti-α5β1 integrin antibody or functional fragment thereof;volociximab;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 (asterisk indicates cyclization via a disulfide bond through the cysteine ​​residues) Taste); 2-methoxyestradiol; αVβ3 inhibitors; angiopoietin 2; antiangiogenic steroids and heparins; angiostatin; angiostatin-related molecules; anti-cathepsin S antibodies; antithrombin III fragments; 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); restin; soluble NRP-1; soluble VEGFR-1; SPARC; SU5416; suramin; tecogalan; tetrathiomolyb and / or α5β1 integrin; peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitors of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; IL-6 antagonists; anti-IL-6 antibodies; and any combination thereof, optionally in an amount sufficient to prevent or treat a proliferative (neovascular) eye disease.

[0028] 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 RPE cells producing one or more factors that inhibit angiogenesis. Exemplary factors that inhibit angiogenesis include at least one factor selected from the group consisting of: a fibronectin fragment or domain; anasterin; a specific anti-VEGF antibody or a functional fragment or domain thereof; a specific anti-VEGF receptor antibody or a functional fragment or domain thereof; a specific anti-α5β1 integrin antibody or a functional fragment or domain thereof; a VEGF fragment or domain; a VEGFR receptor fragment or domain; VEGF-Trap; and any combination thereof.

[0029] The production of the angiogenesis-inhibiting factor may be regulated by an RPE-specific promoter, which 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 include 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 cultured in multilayered populations or embryoid bodies for a period of time sufficient for pigmented epithelial cells to emerge during the culture. The period of time sufficient for pigmented epithelial cells to emerge 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, at least about 7 weeks, or at least about 8 weeks. The multilayered populations or embryoid bodies 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 an RPE cell population. The isolation may comprise enzymatically, chemically, or physically separating cells or cell clusters from the culture and selecting for pigmented epithelial cells or cell clusters that may comprise pigmented epithelial cells. The embryoid bodies may be cultured in suspension and / or as adherent cultures (e.g., in suspension followed by adherent culture). The embryoid bodies cultured as adherent cultures may be cultured in suspension and / or as adherent cultures (e.g., in suspension followed by adherent culture). The pluripotent stem cells may produce one or more outgrowths comprising primary 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 that may be selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The substrate may comprise Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

[0031] A pharmaceutical composition optionally comprising cells lacking substantial expression of one or more embryonic stem cell markers, which 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 expression of one or more RPE cell markers, which may comprise RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and / or tyrosinase.

[0033] The RPE cells may be produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to achieve the average melanin content.The RPE cells may be produced by a method comprising maintaining 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 feeder cells (MEFs) and human embryonic stem cells (hES).

[0035] The RPE may be produced by a method comprising culturing the RPE cells under conditions that increase expression of one or more α integrin subunits, such as, for example, 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 comprise 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 four passagings 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 immunotolerizing agent that may be administered to a subject in need thereof prior to, simultaneously with, subsequent to, and / or together with the RPE cells, including one or more of mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), daclizumab® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophemolate mofetil. That's fine.

[0038] In one aspect, the present disclosure provides a kit comprising a pharmaceutical composition as described above and a separate container containing 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 entire volume of the pharmaceutically acceptable diluent is combined with the entire plurality of RPE cells, the resultant plurality of RPE cells has the desired target concentration. The temperature of the pharmaceutically acceptable diluent may be about 0-10°C, optionally about 2-8°C. The temperature of the plurality of RPE cells or the pharmaceutically acceptable carrier containing the plurality of RPE cells may be about 0-10°C, optionally about 2-8°C.

[0039] The kit may further comprise at least one immunosuppressant or immunotolerizing agent, including one or more of those listed above, which may be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

[0040] The kit may further comprise one or more angiogenesis inhibitors, 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, subsequently 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, the cryopreserved RPE cells having an average maturity level at the time of freezing such that the RPE cells, which may be recovered following 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 representative 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 cryopreservation composition comprising a plurality of cryopreserved retinal pigment epithelial (RPE) cells, wherein the plurality of cryopreserved RPE cells may have an average melanin content of 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, e.g., about 5% to about 50% DMSO and about 30% to about 95% serum, which may optionally be fetal bovine serum (FBS). The cryopreservation medium may comprise about 90% FBS and about 10% DMSO.

[0044] Following thawing, the harvested RPE cells 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 cryopreservation composition may contain about 5,000 to about 1 x 10 live RPE cells at the time of freezing, such as about 200,000 to about 10,000,000, about 20,000 to about 50,000,000, about 250,000 to about 5,000,000, about 500,000 to about 4,000,000, or about 1,000,000 to about 4,000,000 live RPE cells at the time of freezing. 8 The tissue may comprise viable RPE cells.

[0046] The RPE cells recovered following 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 least about 3, 6, 9, or 12 months after freezing.

[0047] At least 85% of the cells that are viable upon thawing may remain viable upon storage 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 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.

[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 no more than about 1 cell per 1 million cells, and optionally no more than 2 cells per 9 million cells in said cryopreservation composition, may be positive for both OCT-4 and alkaline phosphatase (AP) expression.

[0053] The cryopreserved 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, subsequently 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 RPE cells producing one or more factors that inhibit angiogenesis. The one or more factors that inhibit angiogenesis include at least one factor selected from the group consisting of a fibronectin fragment or domain; anasterin; a specific anti-VEGF antibody or a functional fragment or domain thereof; a specific anti-VEGF receptor antibody or a functional fragment or domain thereof; a specific anti-α5β1 integrin antibody or a functional fragment or domain thereof; a VEGF fragment or domain; a VEGFR receptor fragment or domain; VEGF-Trap; and any combination thereof, regulated by an RPE-specific promoter, 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, which may be cultured in multilayered populations or embryoid bodies for a sufficient time for pigmented epithelial cells to emerge during the culture.

[0057] The time sufficient for pigmented epithelial cells to appear in 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 disclosure provides a method for producing retinal pigment epithelial (RPE) cells for use in a pharmaceutical product, 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 pharmaceutical formulation, wherein at the time of harvest, the harvested population of pigmented RPE cells has an average melanin content of less than 8 pg / cell.

[0059] At least 10 6 The RPE cells may be harvested for cryopreservation or pharmaceutical 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 most pigmented and least pigmented 5 percent of harvested RPE cells, and 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 disclosure provides a method for culturing RPE cells under adherent conditions to form a substantially monolayer culture of pigmented RPE cells having a cobblestone morphology; (b) passage the RPE cells at least once before the RPE cells reach an average melanin content of greater than 8 pg / cell; and (c) optionally, after one or more passages, cryopreserving the RPE cells. or harvesting the RPE cells for 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 membrane 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 a brown pigment dispersed in the cytoplasm; and (c) isolating and culturing the pigmented cells of (b) to generate a culture population containing RPE cells having an average pigment level of . Step (b) may comprise culturing the embryoid bodies to form an adherent culture. Step (a) may comprise overgrowing the culture of pluripotent cells, thereby forming a multilayered population. Step (a) may comprise culturing the pluripotent cells on a low-adherent substrate or culturing the pluripotent cells using a hanging drop method, thereby forming 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 RPE cells producing factors that inhibit angiogenesis, such as those identified above. 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 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, 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 in 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 steps (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 mechanically disrupting or isolating the culture, or contacting the culture with EDTA or EGTA, thereby preventing adhesion of the pigmented cells to the culture substrate. Pluripotent stem cells have a reduced HLA antigen complexity. 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] Following their formation, the embryoid bodies may be cultured as adherent cultures, for example to allow for outgrowth. The RPE cells may be positive for at least one RPE cell marker, including one or more of RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, or tyrosinase, or optionally PAX6 and bestrophin.

[0064] The method may involve culturing a cell under conditions that increase α integrin subunit expression, e.g., as described above. and culturing the RPE cells.

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

[0066] In one aspect, the present disclosure provides RPE cells derived from the provided human tissue that contain an average melanin content of less than 8 pg / cell, maintain their phenotype for at least about 1 month after transplantation, maintain their phenotype for at least about 1 month in culture, integrate into the host after transplantation, do not substantially proliferate after transplantation, may be phagocytositic, deliver and metabolize or store vitamin A, transport iron between the retina and choroid after transplantation, attach to Bruch's membrane after transplantation, absorb stray light after transplantation, have elevated alpha integrin subunit expression, have greater average telomere length compared to RPE cells derived from the provided human tissue, The present invention provides a pharmaceutical product comprising RPE cells suitable for treating retinal deterioration, which may have at least one of the following properties: a longer replicative lifespan in culture compared to RPE cells derived from provided human tissue; greater expression of one or more alpha integrin subunits compared to RPE cells derived from provided human tissue; a lower A2E content compared to RPE cells derived from provided human tissue; a lower lipofuscin content compared to RPE cells derived from provided human tissue; less UV damage accumulation compared to RPE cells derived from provided human tissue; or a greater number of phagosomes compared to RPE cells derived from provided human tissue. In one aspect, the present disclosure provides a pharmaceutical product comprising RPE cells suitable for treating retinal deterioration, which may contain an average melanin content of less than 8 pg / cell and may have at least one of the following properties: adhere to Bruch's membrane after transplantation; absorb stray light after transplantation; have a greater average telomere length than RPE cells derived from donated human tissue; have a longer replicative lifespan in culture than RPE cells derived from donated human tissue; have a lower A2E content than RPE cells derived from donated human tissue; have a lower lipofuscin content than RPE cells derived from donated human tissue; exhibit less UV damage accumulation than RPE cells derived from donated human tissue; or contain a greater number of phagosomes than RPE cells derived from donated human tissue.

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

[0068] The retinal degenerative condition may include choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt's disease, pigmented streaks, or myopic macular degeneration. The administering step may include intraocularly administering the RPE cells to an eye in need thereof. The intraocular administration may include injecting the RPE cells into the subretinal space. The intraocular administration may include injecting an aqueous solution, optionally an isotonic solution and / or saline solution, into the subretinal space, thereby forming a pre-bleb, and removing the aqueous solution before administering the RPE cells into the same subretinal space. The injection may be through a needle or injection cannula. The diameter of the needle or injection cannula may be about 0.3 mm to 0.9 mm, or about 0.5 to about 0.6 mm. The needle or injection cannula may have a tip with a diameter of about 0.09 mm to about 0.15 mm. The cannula may be a MEDONE POLYTIP® cannula 25 / 38g (0.12mm (38g) x 5mm tip 0.50mm (25g) x 28mm cannula) The efficacy of the treatment may be assessed by determining one or more of the following vision results: slit-lamp biomicroscopy, fundus photography, IVFA, and SD-OCT, and best-corrected visual acuity (BCVA). The method may improve BCVA and / or increase the number of readable letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart. The retinal degenerative condition may be dry AMD or Stargardt's disease.

[0069] The amount effective to treat 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, such as prednisolone or methylprednisolone, prior to or concurrently with the administration of the RPE cells. The subject may not be administered a corticosteroid within at least 3, 6, 12, 24, 48, 72, or 96 hours prior to or concurrently with the administration of the RPE cells. The subject may not be administered a corticosteroid prior to or concurrently with the administration of the RPE cells within at least 1 hour prior to or concurrently 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 following the administration of the RPE cells. The subject may not be administered a corticosteroid within at least 48 hours following 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 angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors that contribute to increased antioxidant activity, macular xanthophylls, long-chain omega-3 fatty acids, amyloid inhibitors, CNTF agonists, inhibitors of RPE65, factors that target A2E and / or lipofuscin accumulation, downregulators or inhibitors of photoreceptor function and / or metabolism, alpha-2 adrenergic receptor agonists, selective serotonin 1A agonists, factors that target C-5, membrane attack complex (C5b-9) and optionally other drusen components, immunosuppressants, and agents that prevent or treat 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 product may be used in the manufacture of a medicament for treating a retinal degenerative condition such as choroideremia, diabetic retinopathy, dry age-related macular degeneration, wet age-related macular degeneration, retinal detachment, retinitis pigmentosa, Stargardt's disease, pigmented streaks, 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 have a replicative lifespan that may be longer than the replicative lifespan of RPE cells obtained from other sources; an average telomere length that may be at least 30 percent of the telomere length of hESCs and / or human iPS cells (or the average of a population of hESCs and / or human iPS cells), or at least 40, 50, 60, 70, 80, or 90 percent of the telomere length of hESCs and / or human iPS cells; longer than 4 kb, or longer than 5, 6 an average final restriction fragment length (TRF) that may be longer than 7, 8, 9, 10, 11, 12, or 13 kb, or may be 10 kb or longer; an average lipofuscin content that is less than 50 percent of the average lipofuscin content of an equivalent number of RPE cells isolated from adult eyes, 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 adult eyes; 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 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 adult eyes; 5an average N-retinylidene-N-retinylethanolamine (A2E) content that may be less than 50 ng per 100,000 cells; a photoreceptor outer segment (POS) phagocytosis rate that may be at least 50 percent greater than the POS phagocytosis rate of an equivalent number of RPE cells isolated from adult eyes, or that may be at least 75, 100, 150, or 200 percent greater than the POS phagocytosis rate of an equivalent number of RPE cells isolated from adult eyes; a photoreceptor outer segment (POS) phagocytosis rate that is at least 20 percent of the total POS concentration after 24 hours, or that may be at least 25, 30, 25, 40, or 50 percent of the total POS concentration after 24 hours; reduced levels of oxidative stress and / or accumulated DNA damage compared to RPE cells isolated from an adult host; an average proteasome activity that may be at least 50 percent greater than the average proteosome activity of an equivalent number of RPE cells isolated from adult eyes, or that may be at least 60, 70, 80, 90, or 100 percent greater than the average proteosome activity of an equivalent number of RPE cells isolated from adult eyes; an average accumulation of ubiquitin conjugates that 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 that 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 adult eyes. [Brief explanation of the drawings]

[0076] [Figure 1A-B]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1C-D]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1E-F]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1G-H]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1I]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1J]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 1K]Figure 1. Characterization of RPE generated from hESC MA09. A: 6-well plate showing RPE pigmentation spots formed during embryoid body differentiation culture. B-H: Evaluation of molecular markers in thawed and prepared RPE. MITF and PAX6 (C-D) were evaluated in overnight cultures of freshly prepared cells and bestrophin / PAX6, and ZO-1 immunostaining was performed in 3-week-old cultures. B: HMC photomicrograph of 3-week-old prepared RPE showing a confluent cobblestone monolayer with moderate pigmentation established. C: Merged MITF / PAX6 (MITF is red in original, PAX6 is green in original). D: DAPI corresponding to MITF / PAX6; E: Merged bestrophin / PAX6, F: DAPI corresponding; G: ZO-1, H: DAPI corresponding. Note that nearly 100% of cells in C-H were positive for marker evaluation. Magnification, ×400 (B-H). I-q: PCR analysis showing upregulation of RPE markers and downregulation of hESC markers in thawed clinical RPE (right panels of each group, green in the original) compared to reference RPE lots (left panels of each group, blue in the original). Genes shown (from left to right) are bestrophin, Pax-6, MITF, RPE-65, NANOG, OCT-4, and SOX-2. J: FACS analysis showing phagocytosis of PhRodo bioparticles by hES-RPE at 37°C and 4°C (control). Untreated cells (black line in the original; leftmost curve), 4°C control cells (red line in the original; left portion of the curve slightly rising to the right of the untreated cell curve and right portion overlapping with the right portion of the untreated cell curve), and 37°C-treated cells (blue line in the original; rightmost curve) are shown. K: Normal female (46XX) karyotype of clinical RPE lots. [Figure 2A-C]Figure 2. Survival and integration of RPE generated from hESC-MA09 into NIH III mouse eyes after 9 months. Sections stained with anti-human mitochondria (A, red in original) and anti-human bestrophin (B, green in original). Note the precise colocalization of human mitochondrial and bestrophin staining in the same cell (C, merged A and B) and the absence of staining in mouse RPE (F, merged A, B, C, E). The box on the brightfield image (E) is magnified in "D" to show the morphology of the human RPE. Magnification 200x (A-C, E, F); D is an additional 4.5x magnification. [Figure 2D-F] Figure 2. Survival and integration of RPE generated from hESC-MA09 into NIH III mouse eyes after 9 months. Sections stained with anti-human mitochondria (A, red in original) and anti-human bestrophin (B, green in original). Note the precise colocalization of human mitochondrial and bestrophin staining in the same cell (C, merged A and B) and the absence of staining in mouse RPE (F, merged A, B, C, E). The box on the brightfield image (E) is magnified in "D" to show the morphology of the human RPE. Magnification 200x (A-C, E, F); D is an additional 4.5x magnification. [Figure 3A-B]Figure 3. Differences in attachment and growth of RPE cells with varying degrees of pigmentation. Micrographs show the attachment and behavior of lighter (A-C) and darker (D-F) RPE lots. G illustrates the growth rate of RPE cells from the darker (left panel of each group) and lighter (right panel of each group) RPE lots, showing the total number of cells per well for three consecutive days after seeding. A and D show the total cell counts 21 hours after seeding, B and E show the same cultures as A and D after removal of floating cells, and C and F show the same cultures 3 days after seeding. Note that 21 hours after thawing, the majority of cells in the lighter (A, B, G) RPE lots have attached, while in the darker (D, E, G) RPE lots, only a few cells have attached (arrows) and the majority of cells remain floating. By day 3 of culture, the lighter (C) RPE lot had established a higher number of cells and a confluent monolayer, while the darker (D, E, G) RPE lot remained subconfluent. Magnification: ×200. [Figure 3C-D] Figure 3. Differences in attachment and growth of RPE cells with varying degrees of pigmentation. Micrographs show the attachment and behavior of lighter (A-C) and darker (D-F) RPE lots. G illustrates the growth rate of RPE cells from the darker (left panel of each group) and lighter (right panel of each group) RPE lots, showing the total number of cells per well for three consecutive days after seeding. A and D show the total cell counts 21 hours after seeding, B and E show the same cultures as A and D after removal of floating cells, and C and F show the same cultures 3 days after seeding. Note that 21 hours after thawing, the majority of cells in the lighter (A, B, G) RPE lots have attached, while in the darker (D, E, G) RPE lots, only a few cells have attached (arrows) and the majority of cells remain floating. By day 3 of culture, the lighter (C) RPE lot had established a higher number of cells and a confluent monolayer, while the darker (D, E, G) RPE lot remained subconfluent. Magnification: ×200. [Figure 3E-F]Figure 3. Differences in attachment and growth of RPE cells with varying degrees of pigmentation. Micrographs show the attachment and behavior of lighter (A-C) and darker (D-F) RPE lots. G illustrates the growth rate of RPE cells from the darker (left panel of each group) and lighter (right panel of each group) RPE lots, showing the total number of cells per well for three consecutive days after seeding. A and D show the total cell counts 21 hours after seeding, B and E show the same cultures as A and D after removal of floating cells, and C and F show the same cultures 3 days after seeding. Note that 21 hours after thawing, the majority of cells in the lighter (A, B, G) RPE lots have attached, while in the darker (D, E, G) RPE lots, only a few cells have attached (arrows) and the majority of cells remain floating. By day 3 of culture, the lighter (C) RPE lot had established a higher number of cells and a confluent monolayer, while the darker (D, E, G) RPE lot remained subconfluent. Magnification: ×200. [Figure 3G] Figure 3. Differences in attachment and growth of RPE cells with varying degrees of pigmentation. Micrographs show the attachment and behavior of lighter (A-C) and darker (D-F) RPE lots. G illustrates the growth rate of RPE cells from the darker (left panel of each group) and lighter (right panel of each group) RPE lots, showing the total number of cells per well for three consecutive days after seeding. A and D show the total cell counts 21 hours after seeding, B and E show the same cultures as A and D after removal of floating cells, and C and F show the same cultures 3 days after seeding. Note that 21 hours after thawing, the majority of cells in the lighter (A, B, G) RPE lots have attached, while in the darker (D, E, G) RPE lots, only a few cells have attached (arrows) and the majority of cells remain floating. By day 3 of culture, the lighter (C) RPE lot had established a higher number of cells and a confluent monolayer, while the darker (D, E, G) RPE lot remained subconfluent. Magnification: ×200. [Figure 4]Images of the hESC-derived RPE transplant site. Color fundus photographs (A-C) of the left retina macula of a patient with SMD, pre- and postoperatively. The inner rectangular area bisects the border between the surgical transplant site and the corresponding macular atrophy not included in the surgical injection. A: Color image of the baseline macula, showing diffuse RPE and neurosensory macular atrophy. B: Color image of the macula 1 week after hESC-RPE transplant. Note the mild pigmentation, most evident in the area of ​​baseline RPE atrophy. This pigmentation increased at 6 weeks (C). Panels D and E show spectral-domain ocular coherence tomography (SD-OCT) and overlaid black-and-white photographs (Hiedelberg Engineering). The cross-section shown in panel E corresponds to the horizontal line (bright green in the original) indicated by the arrow in panel D. The dashed circle (red in the original) in panel E highlights what appear to be hESC-RPE cells settling or adhering onto the damaged native RPE layer. [Figure 5] Fluorescein angiography images of an AMD patient. No evidence of leakage is observed at different time intervals. A: Baseline early, B: Baseline late, C: 4 weeks early, D: 4 weeks late, E: 8 weeks early, F: 8 weeks late. (Selected images are decentered inferiorly to represent the implanted area.) [Figure 6] Fluorescein angiography images of a Stargardt patient. No evidence of leakage is observed at different time intervals. A: Baseline early, B: Baseline late, C: 4 weeks early, D: 4 weeks late, E: 8 weeks early, F: 8 weeks late. (Selected images are decentered inferiorly to represent the implanted area.) [Figure 7] OCT images of a Stargardt patient at different time intervals. No evidence of edema or subretinal fluid was observed in any of the images at different time intervals. (OCT corresponds to the selected implanted area.) Panel A: Baseline, B: 1 week, C: 4 weeks, D: 8 weeks. [Figure 8] Slit-lamp images of a patient with AMD. Panels A and B: One week postoperative. No evidence of anterior inflammation or corneal edema is observed. [Figure 9]Slit-lamp images of a Stargardt patient. Panels A and B: 1 week postoperative. No evidence of anterior inflammation or corneal edema is observed. [Figure 10] Goldmann visual field test performed in an AMD patient. Panel A: Baseline; Panel B: 6 weeks. A slightly smaller central scotoma is observed. [Figure 11] Goldmann visual field tests performed in a Stargardt patient. Panel A: Baseline; Panel B: 6 weeks. Minimal reduction in scotoma was observed. [Figure 12] Phagocytosis assay results for two lots of RPE cells produced using different media. RPE were produced using either MDBK media (Panel A) or EB-DM and RPE-GM / MM (Panel B). Results are presented as histograms from FACS analysis of cells cultured without fluorescent bioparticles ("Untreated"), negative control cells cultured with fluorescent bioparticles at 4°C ("4°C"), and cells cultured with fluorescent bioparticles at 37°C ("37°C"). [Figure 13A-C] Figure 13. Images of the hESC-RPE transplant site in a patient with Stargardt macular dystrophy. Pre- and postoperative color fundus photographs (A-C) of the patient's left retinal macula. The inner rectangular area bisects the border between the surgical transplant site and the corresponding macular atrophy not included in the surgical injection. (A) Color image of the baseline macula, showing diffuse RPE and neurosensory macular atrophy. (B) Color image of the macula 1 week after hESC-RPE transplant. Note the mild pigmentation, most evident in the area of ​​baseline RPE atrophy. This pigmentation increased at 6 weeks (C). (D-G) Color fundus photographs and SD-OCT images at baseline (D) and 3 months after transplant (F). The color images show increasing levels of RPE pigmentation from baseline to 3 months. Overlaid SD-OCT images (E, G) show increased pigmentation at the level of the RPE, normal monolayer RPE engraftment, and survival at 3 months (arrows) in the area adjacent to exposed Bruch's membrane, which lacks native RPE. hESC = human embryonic stem cells. RPE = retinal pigment epithelium. SD-OCT = spectral-domain ocular coherence tomography. [Fig. 13D-G] Figure 13. Images of the hESC-RPE transplant site in a patient with Stargardt macular dystrophy. Pre- and postoperative color fundus photographs (A-C) of the patient's left retinal macula. The inner rectangular area bisects the border between the surgical transplant site and the corresponding macular atrophy not included in the surgical injection. (A) Color image of the baseline macula, showing diffuse RPE and neurosensory macular atrophy. (B) Color image of the macula 1 week after hESC-RPE transplant. Note the mild pigmentation, most evident in the area of ​​baseline RPE atrophy. This pigmentation increased at 6 weeks (C). (D-G) Color fundus photographs and SD-OCT images at baseline (D) and 3 months after transplant (F). The color images show increasing levels of RPE pigmentation from baseline to 3 months. Overlaid SD-OCT images (E, G) show increased pigmentation at the level of the RPE, normal monolayer RPE engraftment, and survival at 3 months (arrows) in the area adjacent to exposed Bruch's membrane, which lacks native RPE. hESC = human embryonic stem cells. RPE = retinal pigment epithelium. SD-OCT = spectral-domain ocular coherence tomography. [Figure 14] Tabular summary of visual acuity changes after hESC-RPE transplantation in untreated eyes ("fellow eyes") of patients with Stargardt macular dystrophy and in RPE cell-injected eyes ("operated eyes") of patients with SMD. The operated eyes showed detectable improvement by ETDRS and BCVA, whereas no visual acuity changes were detected in the untreated eyes. hESC = human embryonic stem cells. RPE = retinal pigment epithelium. BCVA = best-corrected visual acuity. ETDRS = Early Treatment Diabetic Retinopathy Study visual acuity chart. [Figure 15] Two fundus photographs are shown, including the retina, optic disc, macula, and posterior pole of two additional Stargardt patients, each treated with 50,000 RPE cells derived from an hESC source. Each photograph shows the injection site and the area of ​​the bleb that formed upon injection of the RPE cell-containing solution. [Figure 16]Figures 16 and 17 (showing three fundus photographs each from two different additional SMD patients, taken at the indicated time points (i.e., baseline, 1 month, and 2 or 3 months) for each patient) demonstrate the establishment of an area within the injected bleb with an increasing patch of pigmented RPE cells, indicating engraftment and resurfacing of the retinal area with a new RPE layer. [Figure 17] Figures 16 and 17 (showing three fundus photographs each from two different additional SMD patients, taken at the indicated time points (i.e., baseline, 1 month, and 2 or 3 months) for each patient) demonstrate the establishment of an area within the injected bleb with an increasing patch of pigmented RPE cells, indicating engraftment and resurfacing of the retinal area with a new RPE layer. [Figure 18] The top panels of Figures 16 and 17 show the assessed visual acuity of the treated ("injected") and untreated ("uninjected") eyes of the patient shown in Figures 16 and 17. The vertical axis shows the Early Treatment Diabetic Retinopathy Study (ETDRS) score, and the horizontal axis shows the number of days postoperatively. [Figure 19] Visible light micrographs (migrographs) illustrate the expected pigmentation and morphology of RPE cultures generated from hESCs created without embryo destruction. The top three panels (A, B, C) show RPE generated from three different human iPS (hiPS) cell lines. The bottom three panels (D, E, F) show RPE generated from hESCs (cell lines designated D30469 and NED7) generated from biopsied blastomeres, while the remaining embryos were cryopreserved to maintain viability. [Figure 20] Long-term RPE engraftment in the same SMD patient is shown at an earlier time point in Figure 4. Fundus photographs taken at (A) baseline and (B) 1 year after RPE injection demonstrate the presence of pigmented cells, suggesting long-term engraftment of RPE cells that persists for at least 1 year after injection. [Figure 21] Peripheral ETDRS / BCVA scores of AMD patients up to one year after treatment. [Figure 22] Central ETDRS / BCVA scores of SMD patients up to 1 year after treatment. DETAILED DESCRIPTION OF THE INVENTION

[0077] This disclosure describes the initial results for two patients in a prospective clinical trial exploring the safety and tolerability of these hESC-derived RPE cells in patients with dry AMD and Stargardt disease. At the time of this report, hESC-derived RPE cells showed no signs of rejection or tumorigenicity. Visual measurements suggest improvement in both patients. These results suggest that hESCs may serve as a potentially safe and inexhaustible source of RPE for effectively treating a range of retinal degenerative diseases.

[0078] We also describe a method for generating hESC-derived RPE cell populations with advantageous properties. Controlling differentiation pathways, including the degree of gene and pigment expression, significantly improved cell survival, attachment, and growth after injection. Specifically, the data presented here demonstrate that the degree of RPE maturation and pigmentation dramatically impacts subsequent cell attachment and growth in vitro. These results illustrate the advantages that may be obtained by using cells differentiated from hESCs for therapeutic purposes compared with the use of primary cells. In addition to generating unlimited quantities of healthy "juvenile" cells with potentially reduced immunogenicity (20, 21), these results demonstrate that in vitro differentiation steps can be controlled at the cellular and molecular levels to ensure safety, identity, purity, and potency 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), the leading causes of blindness in developed countries. Preoperative and postoperative 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 site in both patients, with subsequent evidence that the cells had attached and integrated into the native RPE layer. No signs of inflammation or hyperproliferation were observed. Visual acuity measurements showed signs of improvement over the first 2 months. By the second week, the treatment eye of the AMD patient showed an improvement in best-corrected visual acuity (BCVA) from 20 / 500 before treatment to 20 / 200, with continued improvement on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart (20 / 200 to 20 / 320) and an increase in letter length. The SMD patient improved from a manual valve to an index valve during the same period; at 1 and 2 months, BCVA improved to 20 / 800. Before the RPE transplant, the patient was unable to read any letters on the ETDRS chart, but by the second week he began to read letters, which continued to improve over the course of the study (5 letters at 1 and 2 months).

[0081] The hESC-derived RPE cells showed no signs of rejection or tumorigenicity at the time of this report, and visual measurements demonstrate improvement in both patients.

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

[0083] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the present invention or in testing the present invention, and suitable methods and materials are described below. Materials, methods, and examples are intended to be illustrative only and are not intended to be limiting. The following terms and definitions are provided herein.

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

[0085] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" shall imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0086] As used herein, an "effective amount" broadly refers to the amount of a compound or cell that, when administered to a patient for treating a disease, is sufficient to effect treatment for such disease. An effective amount may be a prophylactically effective amount and / or a preventatively effective amount. An effective amount may be an amount effective to reduce, prevent the onset of signs / symptoms, reduce the severity of the onset of signs / symptoms, eliminate the onset of signs / symptoms, delay the progression of the onset of signs / symptoms, arrest the progression of the onset of signs / symptoms, and / or provide prophylaxis for the onset of signs / symptoms. An "effective amount" may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of this disclosure.

[0087] "Embryo" or "embryonic," as used herein, refers broadly to a developing cell mass that has not implanted within the uterine membrane of a maternal host. "Fetal 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] As used herein, "embryonic stem cells" (ES cells) broadly refer to cells derived from the inner cell mass of a blastocyst or morula that have been serially passaged as a cell line. ES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means of generating ES cells homozygous for HLA regions. ES cells may also refer to cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage resulting from the fusion of a sperm and an egg cell; nuclear transfer; parthenogenesis; or chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the plasma membrane to generate cells. Regardless of their origin or the particular method used to generate them, embryonic stem cells can be identified based on (i) their ability to differentiate into all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunodeficient animals. The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo (see, e.g., Chung et al., Cell Stem Cell. 2008 Feb 7;2(2):113-7; U.S. Patent No. 20060206953 (pre-grant publication); U.S. Patent No. 2008 / 0057041 (pre-grant publication), the entire contents of each of which are incorporated herein by reference). The term also includes cells produced by somatic cell nuclear transfer, even if non-embryonic cells are used in the process. ES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means of creating ES cells homozygous for HLA regions. ES cells are also cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, resulting from the fusion of sperm and an egg cell; nuclear transfer; parthenogenesis; or chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the plasma membrane to create cells. 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 generate RPE cells are derived and maintained according to GMP standards.

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

[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 membrane pigment epithelium. Macular degeneration diseases include, but are not limited to, age-related macular degeneration, North Carolina macular dystrophy, Thorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy, Best's disease, Malachialeventinis, Doyne's honeycomb choroidopathy, dominant drusen, and radial drusen.

[0091] "Pluripotent stem cells," as used herein, broadly refer to cells that are capable of long-term or substantially indefinite in vitro propagation while maintaining their undifferentiated state, exhibit a stable (preferably normal) karyotype, and have the capacity to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) under appropriate conditions.

[0092] "Pluripotent embryonic stem cells," as used herein, are cells that (a) can induce teratomas when transplanted into immunodeficient (SCID) mice; (b) can induce all three germ layers (e.g., ectoderm, mesoderm, and endoderm) (c) express at least one molecular embryonic stem cell marker (e.g., Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA The term "pluripotent stem cells" broadly refers to cells expressing the following antigens: 4 surface antigens, NANOG, TRA 1 60, TRA 1 81, SOX2, and REX1. As an additional example, 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 a blastocyst-stage embryo and embryonic stem cells derived from one or more blastomeres of a cleavage- or morula-stage embryo (optionally without destruction of the remainder of the embryo). Such embryonic stem cells can be generated from embryonic material 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), which are created 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 created 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 other embodiments, 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 other embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. iPS cells can typically be identified by the expression of the same markers as embryonic stem cells, although particular iPS cell lines may vary in their expression profile.

[0093] As used herein, the terms "RPE cells," "differentiated RPE cells," "ES-derived RPE cells," and "ES cell-derived RPE cells" are used interchangeably throughout to broadly refer to RPE cells differentiated from pluripotent stem cells, for example, using the methods disclosed herein. The terms are used inclusively to refer to differentiated RPE cells regardless of the level of cellular maturity and thus encompass RPE cells of various levels of maturity. RPE cells can be visually recognized by their cobblestone morphology and early 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, and based on the expression of RPE markers such as RPE 65, PEDF, CRALBP, and bestrophin. For example, cells may be considered positive for a given marker if the expected staining pattern is observed, such as nuclear localization of PAX6, plasma membrane localization of bestrophin in a polygonal pattern (showing bestrophin staining localized in a distinct line at the cell periphery), ZO-1 staining present in tight junctions that outline the cell in a polygonal pattern, and MITF staining detected as restricted to the nucleus. Unless otherwise specified, as used herein, RPE cells refer to RPE cells differentiated in vitro from pluripotent stem cells.

[0094] As used herein, the terms "mature RPE cells" and "mature differentiated RPE cells" may be used interchangeably throughout to broadly refer to the changes that occur following the initial differentiation of RPE cells. Specifically, RPE cells can be recognized in part by the initial appearance of pigment, but after differentiation, mature RPE cells can be recognized by advanced pigment formation.

[0095] "Planting efficiency," as used herein, refers to the fraction of recovered cells that remain viable and can adhere to a culture substrate upon thawing. For example, seeding efficiency can be determined by thawing, washing, and seeding (preferably onto gelatin); the total cell number is determined before seeding, and the viable cell number is determined after seeding. The seeding efficiency can be determined by measuring the viability of the cells in a 37°C water bath, and then calculating the seeding efficiency as the fraction of the total cells before seeding that are viable and adhere to the substrate after seeding. As a more specific example, seeding efficiency can be determined by thawing the cells in a 37°C water bath with constant agitation (e.g., for 1-2 minutes or a time sufficient for the cells to thaw), subsequently washing the cells three times with phosphate-buffered saline (or another suitable washing solution), measuring the total cell count, including viable and non-viable cells (e.g., using a hemocytometer), seeding the cells onto gelatin supplemented with growth medium (e.g., RPE-GM), culturing the cells (preferably at 37°C), allowing the cells to adhere to the gelatin for about 24 hours, and then measuring the viable cell count (e.g., using a hemocytometer, with trypan blue exclusion used as a viability measure); then, after seeding, dividing the viable cell count by the total cell number before seeding to determine the seeding efficiency.

[0096] As used herein, "pigment" broadly refers to any level of pigmentation, such as the pigmentation that occurs initially when RPE cells differentiate from ES cells. Pigmentation may vary depending on the cell density and maturity of the differentiated RPE cells. The pigmentation of RPE cells may be the same as that of average RPE cells after terminal differentiation of the RPE cells. The pigmentation of RPE cells may be greater than that of average RPE cells after terminal differentiation of the RPE cells. The pigmentation of RPE cells may be less than that of average RPE cells after terminal differentiation.

[0097] A "sign" of disease, as used herein, refers broadly to any abnormality indicative of disease detectable upon examination of a patient; an objective indicator of disease, as opposed to a symptom, which is a subjective indicator of disease.

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

[0099] "Therapy," "therapeutic," "treating," "treat," or "treatment," as used herein, broadly refers to treating a disease, arresting or reducing the progression of a disease or its clinical symptoms, and / or alleviating a disease, causing regression of the disease or its clinical symptoms. Therapy includes prophylaxis, prevention, treatment, cure, correction, reduction, alleviation, and / or providing relief from the disease, signs, and / or symptoms. Therapy includes alleviating signs and / or symptoms in patients with ongoing disease signs and / or symptoms (e.g., blindness, retinal deterioration). Therapy also encompasses "prophylaxis" and "prevention." Prophylaxis includes preventing the onset of a disease or reducing the incidence or severity of a disease in a patient following treatment of the disease in the patient. The term "alleviated," for purposes of therapy, broadly refers to a reduction in clinically significant signs and / or symptoms. Treatment includes treating recurrence or recurrent signs and / or symptoms (e.g., retinal degeneration, vision loss). Treatment always includes, but is not limited to, preventing the appearance of signs and / or symptoms, as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. Treatment includes treatment of chronic disease ("maintenance") and treatment of acute disease. For example, treatment includes treating or preventing recurrence or recurrence of signs and / or symptoms (e.g., blindness, retinal degeneration).

[0100] As used herein, the term "corticosteroids" refers to a class of steroid hormones that bind to the glucocorticoid receptor, including natural and artificial corticosteroids, analogs, etc. Exemplary corticosteroids include prednisolone, hydrocortisone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, fluticasone (including fluticasone propionate (FP)), budesonide, ciclesonide, mometasone and flunisolide.

[0101] RPE cell preparations and combination therapies The present disclosure provides RPE cell preparations, including RPE cells, substantially purified RPE cell populations, pharmaceutical products 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 natural 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, pharmaceutical products comprising human RPE cells, and cryopreserved preparations of human RPE cells. The preparations may be preparations comprising human embryonic stem cell-derived RPE 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 replicative lifespan that is longer than that of RPE cells obtained from other sources (e.g., cultures derived from donated human tissue, such as fetal, infant, child, adolescent, or adult tissue). Replicative lifespan may be assessed by measuring the number of population doublings in culture prior to replicative senescence. For example, the RPE cells of the preparation may have a replicative lifespan that is at least 10 percent longer than that of an RPE population derived from donated human tissue, and preferably at least 20, 30, 40, 50, 60, 70, 80, 90, 100 percent or more longer than that of an RPE population derived from donated 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 the hESCs and / or human iPS cells (or the average of the hESCs and / or human iPS cell population), preferably at least 40, 50, 60, 70, 80, or even 90 percent of the telomere length of the hESCs and / or human iPS cells (or the average of the hESCs and / or human iPS cell population). For example, the hESCs and / or human iPS cells (or the hESCs and / or human iPS cell population) may be the cells or cell population from which the RPE cells are differentiated.

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

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

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

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

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

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

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

[0111] The RPE cells of the preparation may have an average proteasome activity that is at least 50 percent greater than the average proteosome activity of an equivalent number of RPE cells isolated from the eyes of an adult human (i.e., a human adult patient aged 25-80 years, more preferably an adult aged 50-80 years), and more preferably at least 60, 70, 80, 90, or 100 percent of the average proteosome activity of an equivalent number of RPE cells isolated from the eyes of an adult human. Proteosome activity may be measured using, as illustrative and non-limiting examples, succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVY-AMC) for chymotrypsin-like activity, Nt-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 preparation may have an average accumulation of ubiquitin conjugates that is less than 50 percent of the average accumulation of ubiquitin conjugates in an equivalent number of RPE cells isolated from the eyes of an adult human (e.g., a human adult patient aged 25-80 years, more preferably an adult aged 50-80 years), and more preferably, less than 40, 30, 20, or 10 percent of the average accumulation of ubiquitin conjugates in an equivalent number of RPE cells isolated from an adult human eye. The accumulation of ubiquitin conjugates may be, by way of illustrative and non-limiting example, less than 50 percent of the average accumulation of ubiquitin conjugates in an equivalent number of RPE cells isolated from an adult human eye. 2008 vol. 49 no. 8 3622-3630.

[0113] Preferably, one or more angiogenesis inhibitors may be administered in combination with the RPE cell preparation in a therapeutically effective amount to prevent or treat an ocular disease, such as an angiogenesis-associated ocular disease. 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, bevasiranib, rapamycin, AGN-745, vatalanib (vitalanib), pazopanib, NT-502, NT-503, or PLG101, CPD791 (a di-Fab' polyethylene glycol (PEG) conjugate that inhibits VEGFR-2), anti-VEGF antibodies or functional fragments thereof (e.g., bevacizumab (Avastin®) or ranibizumab (Lucentis®)), or VEGF antagonists such as VEGF and / or VEGF receptor (VEGFR, e.g., VEGFR1 (FLT1, FLT), VEGFR2 (KDR, FLK1, VEGFR, CD309), VEGFR3 (FLT4, PCL)) inhibitors, such as peptides, peptidomimetics, small molecules, chemicals, or nucleic acids, like anti-VEGF receptor antibodies (e.g., IMC-1121(B) (a monoclonal antibody against VEGFR-2), or IMC-18F1 (an antibody against the extracellular binding domain of VEGFR-1)). Drugs include: Additional exemplary inhibitors of VEGF activity include fragments or domains of VEGFR receptors, such as VEGF-Trap (aflibercept), 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 VEGF receptors 1 and 2. Additional exemplary VEGF antagonists include sorafenib (Nexavar), These include tyrosine kinase inhibitors (TKIs), including TKIs that have been reported to inhibit VEGFR-1 and / or VEGFR-2, such as SU5416 (Semaxinib), SU11248 / Sunitinib (Sutent), and vandetanib (ZD6474). Additional exemplary VEGF antagonists include Ly317615 (Enzastaurin), which is believed to target downstream kinases involved in VEGFR signaling (protein kinase C).Additional exemplary angiogenesis inhibitors include an anti-α5β1 integrin antibody or functional fragment thereof (such as volociximab), a peptide, a peptidomimetic, a small molecule, 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 *(Arg-Cys-Asp-thioproline-Cys; the asterisk denotes cyclization by a disulfide bond through the cysteine ​​residue) or nucleic acids. Additional exemplary angiogenesis inhibitors include 2-methoxyestradiol, αVβ3 inhibitors, angiopoietin 2, antiangiogenic steroids and heparin, angiostatin, angiostatin-related molecules, anti-α5β1 integrin antibodies, anti-cathepsin S antibodies, antithrombin 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, and 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, suramin, tecogalan, tetrathiomolybdate, thalidomide, lenalidomide, thrombospondin, TIMP, TNP-470, TSP-1, TSP-2, vasostatin, VEGFR antagonist, VEGI, volociximab (also known as M200), anasterin such as fibronectin fragments (see Yi and Ruoslahti, Proc Natl Acad Sci US A. 2001 Jan 16;98(2):620-4), or any combination thereof. The agent is preferably in an amount sufficient to prevent or treat proliferative (neovascular) ocular diseases, such as choroidal neovascular membrane (CNV), which is associated with wet AMD and other retinal diseases. Additional exemplary angiogenesis inhibitors include IL-6 antagonists, such as lenvatinib (E7080), motesanib (AMG 706), pazopanib (Votrient), and anti-IL-6 antibodies. Additional exemplary angiogenesis inhibitors include fragments, mimetics, chimeras, fusions, analogs, and / or domains of any of the foregoing. Additional exemplary angiogenesis inhibitors include combinations of any of the foregoing. In an exemplary embodiment, the RPE cell preparation comprises an anti-VEGF antibody, such as bevacizumab, for example, about 0.1 mg to about 6.0 mg, for example, about 1.25 mg and about 2.5 mg per intraocular injection. In a further exemplary embodiment, the RPE cell preparation comprises one or more inhibitors of VEGF activity and one or more inhibitors of α5β1 integrin activity.

[0114] One or more anti-inflammatory agents may be administered in combination with the RPE cell preparation.Exemplary anti-inflammatory agents include glucocorticoids, nonsteroidal anti-inflammatory drugs, aspirin, ibuprofen, naproxen, cyclooxygenase (COX) enzyme inhibitors, aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, fluocinolone acetonide (e.g., ILUVIEN®), 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 nonsteroidal anti-inflammatory agent.

[0115] Furthermore, the patient may not be administered a corticosteroid prior to, concurrently with, and / or following administration of the RPE cell preparation. While not wishing to be limited by theory, the applicants hypothesize that corticosteroid administration interferes with RPE cell precipitation and / or engraftment. In certain preferred embodiments, the patient is not treated with prednisolone or methylprednisolone prior to, concurrently with, and / or following administration of the RPE cell preparation. In more preferred embodiments, the patient is not treated with prednisolone prior to, concurrently with, and / or following administration of the RPE cell preparation. For example, the patient may not be administered prednisolone or methylprednisolone or another corticosteroid within at least 3, 6, 12, 24, 48, 72, 96, or 120 hours or more prior to administration of the RPE cell preparation. Additionally, the patient may not receive prednisolone or methylprednisolone or another cortocosteroid for at least 3, 6, 12, 24, 48, 72, 96, or 120 hours or more following administration of the RPE cell preparation.

[0116] The patient may be administered a non-corticosteroid immunosuppressant prior to and / or following 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 that contribute 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] One or more macular xanthophylls (such as lutein and / or zeaxanthin) may be administered in combination with the RPE cell preparation.

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

[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) (a humanized monoclonal antibody against ABeta40 and ABeta42), or GSK933776 (GlaxoSmithKline) (an 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, which may be delivered by an intraocular device such as NT-501 (Neurotech)) may be administered.

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

[0123] In combination with the RPE cell preparation, one or more factors that target 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 metabolism downregulators or inhibitors, such as fenretinide and ACU-4429, may be administered.

[0125] One or more alpha 2 adrenergic receptor agonists, such as brimonidine tartrate, may be administered in combination with the RPE cell preparation.

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

[0127] In combination with the RPE cell preparation, one or more factors that target C-5, the membrane attack complex (C5b-9), and / or optional other drusen components may be administered, including, for example, complement factor D, C-3, C-3a, C5, and C5a inhibitors, and / or factor H agonists, such as 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).

[0128] One or more immunosuppressants, such as sirolimus (rapamycin), may be administered in combination with the RPE cell preparation.

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

[0130] When one or more agents (such as angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors that contribute to increased antioxidant activity, macular xanthophylls, long-chain omega-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, alpha-2 adrenergic receptor agonists, selective serotonin 1A agonists, factors targeting C-5, membrane attack complex (C5b-9) and / or optional other drusen components, immunosuppressants, agents that prevent or treat lipofuscin accumulation, etc.) are administered in combination with an RPE cell preparation, the agents may be administered simultaneously with, prior to, and / or subsequent to the RPE cell preparation. For example, the agents may be administered to the patient's eye during a procedure in which the RPE cell preparation is introduced into the patient's eye. Administration of the agent may begin prior to and / or continue after administration of the RPE cells to the patient's eye. For example, the agent may be provided in a solution, suspension, in sustained release form, and / or in a sustained release system (e.g., Allergan Novadur™ delivery system, NT-501, or another intraocular device or sustained release system).

[0131] RPE cell populations may contain differentiated RPE cells of various levels of maturity, or may be substantially pure with respect to differentiated RPE cells of a particular level of maturity. RPE cells may be a substantially purified preparation comprising RPE cells of various levels of maturity / pigmentation. For example, a substantially purified RPE cell culture may contain both differentiated RPE cells and mature differentiated RPE cells. Mature RPE cells may have variable pigmentation levels. However, mature RPE cells may be visually distinguishable from RPE cells based on their increased pigmentation levels and more columnar shape. A substantially purified RPE cell preparation may comprise RPE cells of different levels of maturity (e.g., differentiated RPE cells and mature differentiated RPE cells). In such cases, there may be variation across the preparation in the expression of markers indicative of pigmentation. The pigmentation of RPE cells in cell culture may be homogeneous. Furthermore, the pigmentation of RPE cells in cell culture may be heterogeneous, and an RPE cell culture may comprise both differentiated RPE cells and mature RPE cells. Preparations comprising RPE cells include preparations 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 preparations that are substantially pure with respect to non-RPE cell types, as well as with respect to RPE cells of other levels of maturity.

[0132] The percentage of mature differentiated RPE cells in the culture may be reduced by decreasing the culture density. Therefore, the method described herein may further comprise subculturing the population of mature RPE cells to generate a culture containing a lower percentage of mature RPE cells. The number of RPE cells in the preparation includes differentiated RPE cells regardless of the maturity level and regardless of the relative percentage of differentiated RPE cells and mature differentiated RPE cells. The number of RPE cells in the preparation refers to the number of either differentiated RPE cells or mature RPE cells. The preparation may comprise at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% differentiated RPE cells. The preparation 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 preparation may comprise a mixed population of differentiated and mature RPE cells.

[0133] The present disclosure provides cell cultures comprising human RPE cells that are pigmented and express at least one gene that is not expressed in non-human RPE cells. For example, such RPE cells may have substantially the same expression of RPE65, PEDF, CRALBP, and bestrophin as native human RPE cells, but the RPE cells may exhibit a maturity level with respect to expression of one or more of PAX2, Pax6, MITF, and / or tyrosinase. It may vary depending on the model. Note that the change in pigmentation after differentiation is also correlated 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.

[0134] The preparation may be substantially purified with respect to non-RPE cells, comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells. RPE cell preparations may be essentially free of non-RPE cells or 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% of non-RPE cell types. For example, RPE cell preparations have been shown to be approximately 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%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.08%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09 ... It may comprise less than 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 and with respect to RPE cells of other maturity levels. The preparation may be substantially purified with respect to non-RPE cells and enriched for mature RPE cells. For example, in an RPE cell preparation enriched for 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 enriched for differentiated RPE cells but not 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 comprises at least approximately 1 x 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 10The RPE cell preparation may comprise at least about 5,000 to 10,000, 50,000 to 100,000, 100,000 to 200,000, 200,000 to 500,000, 300,000 to 500,000, or 400,000 to 500,000 RPE cells. The RPE cell preparation may comprise at least about 20,000 to 50,000 RPE cells. The RPE cell preparation may also comprise at least about 5,000, 10,000, It may comprise 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 comprises at least approximately 1 x 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 The RPE cell preparation may comprise at least about 5,000 to 10,000, 50,000 to 100,000, 100,000 to 200,000, 200,000 to 500,000, 300,000 to 500,000, or 400,000 to 500,000 RPE cells / mL. The RPE cell preparation may comprise at least about 20,000 to 50,000 RPE cells / mL. The RPE cell preparation may also comprise 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 RPE cells / mL.

[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, forming a monolayer between the neurosensory retina and choroid of the patient receiving the cell transplant. The RPE cells may also provide nutrients to adjacent photoreceptors and phagocytose shed rod photoreceptor outer segments. Furthermore, the RPE cells described herein may have greater proliferation potential than cells derived from an ocular 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 lifespan than cells derived from an ocular donor.

[0140] Preparations comprising RPE cells may be prepared in accordance with Good Manufacturing Practice (GMP) (e.g., the preparation is GMP compliant) and / or current Good Tissue Practice (GTP) (e.g., the preparation 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 cultures may be at least about 1 x 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 The RPE cells may comprise:

[0142] RPE cells may be further cultured to generate mature RPE cell cultures. RPE cells may be matured, and the RPE cells may be further cultured, for example, in RPE-GM / MM or MDBK MM medium, until a desired level of maturation is achieved. This may be determined by monitoring the increase in pigmentation levels during maturation. As an alternative to RPE-GM / MM or MDBK MM medium, functionally equivalent or similar media may be used. Regardless of the specific medium used to mature RPE cells, the medium may optionally be supplemented with growth factors or drugs. Both RPE cells and mature RPE cells are differentiated RPE cells. However, mature RPE cells are characterized by increased pigment levels compared to differentiated RPE cells. The degree of maturation and the level of pigmentation may be adjusted by increasing or decreasing the culture density of the differentiated RPE cells. Thus, RPE cell cultures may be further cultured to generate mature RPE cells. Alternatively, the density of a culture containing mature RPE cells may be reduced to decrease the percentage of mature, differentiated RPE cells and increase the percentage of differentiated RPE cells.

[0143] RPE cells may be identified by comparing messenger RNA transcripts between such cells and cells derived in vivo. Cell aliquots are collected at various intervals during the differentiation of embryonic stem cells into RPE cells and assayed for expression of any of the markers described above. These characteristics distinguish 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, methods for cryopreserving RPE cells are also provided. RPE cells may be harvested, washed in buffer or medium, counted, concentrated (by centrifugation), formulated in a freezing medium (e.g., 90% FBS / 10% DMSO), or any combination of these procedures. For example, RPE cells may be seeded into several culture vessels and continuously expanded. RPE cells may be harvested and maintained in FBS at approximately 4°C while several flasks of RPE cells are combined into a single lot. RPE cells may also be washed at least 1, 2, 3, 4, or 5 times with a saline solution (e.g., DPBS). Furthermore, RPE cells may be cryopreserved after dystrophin has organized at the cell membrane and PAX6 expression has decreased. Furthermore, 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 number (e.g., 1 x 10 6 These may include: (cells / mL), expiration date (e.g., recommended date by which the vial should be used), manufacturing information (e.g., name and address), warnings, and storage instructions (e.g., store in liquid nitrogen).

[0148] The cryopreserved RPE cell preparations described herein may comprise at least about 50,000 to 100,000 RPE cells. The cryopreserved RPE cell preparations may also comprise at least about 20,000 to 500,000 RPE cells. The cryopreserved RPE cell preparations may also 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. Cryopreserved RPE cell preparations 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. Cryopreserved RPE cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1 x 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 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 The RPE cells of the cryopreserved RPE cell preparation may be mammalian RPE cells, including human RPE cells.

[0149] Further, the cryopreserved RPE cell preparations described herein may comprise at least about 50,000-100,000 RPE cells / mL. Cryopreserved RPE cell preparations may also comprise at least about 20,000-500,000 RPE cells / mL. Cryopreserved RPE cell preparations may also 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. Cryopreserved RPE cell preparations may contain 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 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 x 10 RPE cells / mL. 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 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 cryopreserved and then recovered from storage. The 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 maintain their viability and differentiation state after cryopreservation. Furthermore, 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. The cryopreserved formulation comprising RPE cells may be substantially free of DMSO.

[0151] Methods for generating RPE cells The cell populations analyzed by the subject methods 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 visual-related neural cells, such as interneurons (e.g., "relay" neurons of the inner nuclear layer (INL)) and amacrine cells. In addition, retinal cells, rods, cones, and corneal cells may be generated. Cells that provide the ocular vasculature may also be generated by the methods described herein.

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

[0153] The RPE cells described herein may be differentiated from pluripotent stem cells, such as human embryonic stem cells, and are molecularly distinct from embryonic stem cells, adult-derived RPE cells, and fetal-derived RPE cells. For example, the manufacturing process steps described herein may impart unique structural and functional properties to the final RPE cell product such that these cells closely resemble native RPE cells and differ 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 Nos. 7,736,896, 7,795,025, and 7,794,704, and International Publication Nos. WO / 2012 / 012803 and WO 2011 / 063005, the entire contents of which are incorporated herein by reference. RPE may be generated from pluripotent cells cultured as multilayered populations or embryoid bodies. For example, embryoid bodies may be formed by culturing pluripotent cells under non-adherent conditions, such as on a low-adherent substrate or in "hanging drops." In 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, the entire contents of which are incorporated herein by reference. Typically, embryoid bodies initially form as solid masses or clusters of pluripotent cells; over time, some embryoid bodies contain fluid-filled cavities; the former are referred to as "simple" EBs and the latter as "cystic" EBs. As previously reported by the applicants, cells in these EBs (both solid and cystic) can differentiate, increasing the number of RPE cells over time. Optionally, the EBs can then be cultured as adherent cultures to form outgrowths. Similarly, the applicants have previously reported that pluripotent cells that are overgrown to form multilayered cell masses can differentiate over time to form RPE cells. Once RPE cells are formed, they can be easily identified and isolated for further use based on their morphological characteristics, including pigmentation and a cobblestone appearance.

[0155] Pluripotent cells may be propagated 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 xeno-free culture and / or under feeder cell-free conditions. See Klimanskaya et al., Lancet. 2005 May 7-13;365(9471):1636-41; Richards et al., Stem Cells. 2003;21(5):546-56; US Pat. No. 7,410,798; Ilic et al., Stem Cells Dev. 2009 November;18(9):1343-5; Xu et al. Nat Biotechnol. 2001 October;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™ (a 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 a conditioned medium. For example, the conditioned medium may be conditioned by pluripotent cells, such as ES cells, iPS cells, feeder cells, or fetal cells, any of which may or may not be human.

[0156] During the generation of RPE, pluripotent cells may be cultured in the presence of a rho-associated protein kinase (ROCK) inhibitor. A ROCK inhibitor refers to any substance, such as a small molecule, siRNA, miRNA, or antisense RNA, that inhibits or reduces rho-associated kinase function or its signaling pathway in a cell. The term "ROCK signaling pathway" refers to any substance, as used herein, that inhibits or reduces rho-associated kinase function or its signaling pathway. As used herein, the term "ROCK inhibitor" may include any signal processor involved in a ROCK-related signaling pathway, such as the Rho-ROCK-myosin II signaling pathway, its upstream signaling pathway, or its downstream signaling pathway in a cell. An exemplary ROCK inhibitor that may be used is Stemgent's Stemolecule Y-27632, a rho-associated protein kinase (ROCK) inhibitor (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):681-6). Other ROCK inhibitors include, for example, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, and SB-772077-B. Doe et al., J. Pharmacol. Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. Pharmacol., 57:976-983, 2000; Nakajima et al., Cancer Chemother. Pharmacol., 52:319-324, 2003; and Sasaki et al., Pharmacol. Ther., 93:225-232, 2002, each of which is incorporated by reference as if set forth in its entirety. ROCK inhibitors may be utilized at concentrations and / or culture conditions known in the art, for example, as described in U.S. Patent Publication No. 2012 / 0276063 (pre-grant publication), the entire contents of which are incorporated by reference. For example, a ROCK inhibitor may have a concentration of about 0.05 to about 50 μM, e.g., 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 deducible therein, 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 in Matrigel™ or another substrate. Embryoid bodies may then be generated from the dissociated pluripotent cells without the use of trypsin, using EDTA, collagenase, or the like, or mechanically. Embryoid bodies may be formed in a medium comprising Y-27632 or another ROCK inhibitor. For example, a ROCK inhibitor may enhance cell viability in embryoid bodies generated from pluripotent cells cultured in Matrigel™ or another substrate. This may improve RPE cell yield.

[0158] An exemplary method for generating RPE cells includes the steps of: (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells as embryoid bodies in a nutrient-rich, low-protein medium, optionally comprising serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium, optionally comprising serum-free B27 supplement; (d) culturing the adherent culture cells of (c) in a nutrient-rich, low-protein medium without serum-free B27 supplement; and (e) supporting the growth of high-density somatic cell cultures. (d) culturing the cells of (d) in a medium capable of sustaining the growth of RPE cells, thereby resulting in RPE cells in the cell culture; (f) separating cells or cell clumps from the culture of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme, or another separation medium); (g) selecting 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; and (g) expanding the enriched culture of RPE cells to produce RPE cells. These method steps may be performed at least once to produce a substantially purified 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 RPE cells.

[0159] Additionally, the present disclosure also provides a method for culturing pluripotent stem cells, comprising the steps of: (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells in a nutrient-rich, low-protein medium, optionally comprising serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium, optionally comprising serum-free B27 supplement; (d) culturing the adherent culture cells of step (c) in a nutrient-rich, low-protein medium without serum-free B27 supplement; (e) culturing the cells of (d) in a medium capable of supporting the growth of high-density somatic cell cultures, whereby RPE cells emerge in the cell culture; (f) separating cells or cell clumps from the culture of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme, or another separation medium); (g) selecting RPE cells from the culture and transferring the RPE cells to a culture containing another medium supplemented with growth factors to generate an enriched culture of RPE cells; (h) expanding the enriched culture of RPE cells; and (I) culturing the enriched culture of RPE cells to generate mature RPE cells. These method steps may be performed at least once to produce a substantially purified culture of mature RPE cells, and 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 coupled procedures, the cells may be cultured for at least about 1-10 weeks. For example, the cells may be cultured for at least about 3-6 weeks. In any of the coupled procedures, the cells may be cultured for about 1-50 days, for example, at least about 1-3, 3-4, 7, 4-9, 7-10, 7-12, 8-11, 9-12, 7-14, 14-21, and 3-45 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 2-4 and 3-6 hours. In each of the above linked method steps, the cells may be cultured for the same period of time at each step, or for different periods of time at one or more of the steps. Additionally, any of the above linked method steps may be repeated to generate more RPE cells (e.g., scaled up to generate large numbers of RPE cells).

[0161] In the methods described herein, RPE cells may begin to differentiate among cells in the adherent culture of EBs. RPE cells may be visually recognized based on their cobblestone morphology and the early appearance of pigmentation. As 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 in embryoid body culture or to facilitate subculture of adherent cells. Exemplary mechanical methods include, but are not limited to, pipette titration or cutting with a pulled needle. Exemplary enzymatic methods include, but are not limited to, any enzyme suitable for cell separation (e.g., trypsin (e.g., trypsin / EDTA), collagenase (e.g., collagenase B, collagenase IV), dispase, papain, collagenase and dispase mixtures, collagenase and trypsin mixtures). Non-enzymatic solutions, such as solutions with a high EDTA content, such as Hank's-based cell separation buffer, may also be used to separate cells.

[0163] RPE cells may be differentiated from embryoid bodies. Isolating RPE cells from EBs allows them to be expanded in vitro in enriched culture. In human cells, RPE cells are differentiated from embryoid bodies. The RPE cells may be obtained from EBs grown for less than 0 days. Furthermore, RPE cells may arise 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 the pluripotent stem cells, embryoid bodies, and RPE cells may be removed and / or replaced with the same or different medium at any interval. For example, the medium may be removed and / or replaced after at least about 0-7 days, 7-10 days, 10-14 days, 14-28 days, or 28-90 days. Furthermore, the medium may be replaced at least daily, every other day, or at least every three days.

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

[0165] RPE cells may be selected from the isolated cells and cultured separately to produce 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. Additionally, several RPE markers are known, including 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 vitelloid macular dystrophy gene (VMD2); and pigment epithelium-derived factor (PEDF), a 48 kD secreted protein with anti-angiogenic properties. Messenger RNA transcripts of these markers may be assayed using PCR (e.g., RT-PCR) or Northern blot. Protein levels of these markers may also be assayed using immunoblotting techniques or Western blot.

[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. Evaluation may also be performed by examining in vivo function after transplantation of RPE cells into a suitable host animal (such as a human or non-human animal suffering from a spontaneous or induced retinal degenerative pathology) using, for example, behavioral tests, fluorescence angiography, histology, tight junction conductivity, or electron microscopy.

[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 (e.g., bFGF, heparin, hydrocortisone, vascular endothelial growth factor, recombinant insulin-like growth factor, ascorbic acid, or human epidermal growth factor). RPE cells may be phenotypically stable for extended periods (e.g., >6 weeks) in culture.

[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 also 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 derived. Pluripotent stem cells may be generated, 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, and embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destruction of the remainder of the embryo). Such Such embryonic stem cells can be produced from embryonic material produced by fertilization or by asexual means of reproduction, including somatic cell nuclear transfer (SCNT), parthenogenesis, cell reprogramming, and androgenesis. Furthermore, 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 derived.

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

[0171] After culturing EBs in suspension culture, the EBs may be transferred to generate adherent cultures. For example, EBs may be plated onto gelatin-coated plates in medium. When cultured as adherent cultures, EBs may be cultured in the same type of medium as when grown in suspension. When culturing cells as adherent cultures, B27 supplements may be added to the medium. Alternatively, the medium may be initially supplemented with B27 (e.g., for about 7 days or less), and then subsequently cultured in the absence of B27 for the remainder of the period as adherent cultures. EBs may be cultured as adherent cultures for at least about 14-28 days. However, in certain embodiments, EBs may be cultured as adherent cultures for less than about 14 days (less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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 the progeny of the inner cell mass (ICM) of a blastocyst, or cells derived from another source, and may remain pluripotent substantially indefinitely. hES cells may be derived from one or more blastomeres of an early cleavage stage embryo, optionally without disruption or damage to the embryo. hES cells may also be generated using nuclear transfer. hES cells may also be induced pluripotent cells (iPS cells), which are described in further detail below. Cryopreserved hES cells may also be used. hES cells may be cultured by any method known in the art, such as with or without 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. Stem Cell Information (Culture See the National Institute of Health website, 2010, for more information on human embryonic stem cells (hESCs). hES cells may be used and maintained according to GMP standards.

[0173] When cultured on a feeder layer under defined conditions, hES cells maintain a specific morphology and form flat colonies consisting of small, tightly packed cells with a high nucleus-to-cytoplasm ratio, clear cell boundaries, and sharp, refractile colony borders. hES cells express a set of molecular markers, including octamer-associated 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, which differentiate into defined lineages, hES cells in culture can be induced to differentiate. For example, hES cells can be differentiated into human RPE under defined conditions as 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. No. 10 / 100,0 ...

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

[0176] Induced pluripotent stem cells (iPS cells) Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells), which are created by reprogramming somatic cells through the expression or induction of expression of a combination of factors ("reprogramming factors"). iPS cells may be created using fetal, postnatal, neonatal, juvenile, or adult somatic cells. iPS cells may be obtained from cell banks. Alternatively, iPS cells may be created de novo (by methods known in the art) prior to initiating differentiation into RPE cells or another cell type. The creation of iPS cells may be the first step in generating differentiated cells. iPS cells may be specifically created using material from a particular patient or matched donor for the purpose of generating histocompatible RPE cells. iPS cells may be generated from cells that are substantially non-immunogenic in the intended recipient, such as from autologous cells or from cells that are 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 may be fibroblasts, such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblast cells. The somatic cells are reprogrammed by expressing at least 1, 2, 3, 4, or 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] Somatic cells may also be reprogrammed using combinatorial approaches, expressing reprogramming factors (e.g., using viral vectors, plasmids, etc.) and inducing reprogramming factor expression (e.g., using small organic molecules). Reprogramming factors may also be expressed in somatic cells by infection with viral vectors, such as retroviral or lentiviral vectors. Episomal plasmids may also be used. Reprogramming factors can be expressed in somatic cells using non-integrating vectors such as smids. For example, see Yu et al., Science. 2009 May 8; 324 (5928): 797-801, the entire contents of which are incorporated by reference. When non-integrating vectors are used to express reprogramming factors, electroporation, transfection, or vector-mediated somatic cell transformation can be used to express the factors in cells. For example, in mouse cells, the 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, the 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 are expressed in the cells, the cells may be cultured. Over time, cells with ES characteristics will appear in the culture dish. The cells may be selected and passaged, for example, based on ES morphology or on the expression of selectable or detectable markers. The cells may be cultured to generate cell cultures that resemble ES cells; these are putative iPS cells.

[0180] To confirm the pluripotency of 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 their ability to form teratomas when transplanted into SCID mice; or the cells may be evaluated for their ability to differentiate and give rise to cell types of all 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 be differentiated 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. RPE cells produced according to exemplary embodiments of the methods disclosed herein and in the related applications identified above may differ from those achievable by previous methods and from RPE cells from other sources. For example, the manufacturing process steps described herein may impart unique structural and functional properties to the final RPE cell product, as do cells from these isolated RPE cells obtained from other sources, such as fetal-derived RPE cells or RPE cell lines (e.g., ARPE19).

[0182] Furthermore, exemplary embodiments of the methods for generating RPE cells described herein do not tolerate ES cells, which cannot persist and pose an unacceptable contamination risk to RPE cell cultures and preparations.

[0183] Cells provided by the present disclosure include, but are not limited to, RPE, RPE progenitor cells, iris pigmented epithelial (IPE) cells, and other visually related neural cells such as interneurons (e.g., "relay" neurons of the inner nuclear layer (INL)) and amacrine cells. 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 resulting from retinal detachment, retinal dysplasia, pigmented streaks, myopic macular degeneration or retinal atrophy, or associated with several visual degenerative diseases resulting in photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava).

[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 also be functional RPE cells, characterized by their ability to integrate into the retina upon corneal, subretinal, or other administration to a human or non-human animal.

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

[0187] In contrast, RPE cells may express ES cell markers. For example, the expression levels of ES cell genes Oct-4, NANOG, and / or Rex-1 may be approximately 100- to 1000-fold lower in RPE cells than in ES cells. For example, RPE cells may substantially lack expression of ES cell markers, including, but not limited to, octamer-associated 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, Rex-1, Sox2, TDGF-1, DPPA2, DPPA3 (STELLA), DPPA4, and / or DPPA5. Thus, RPE cells preferably substantially lack expression of Oct-4, NANOG, and / or Rex-1 compared to ES cells.

[0188] The RPE cells described herein may also exhibit elevated expression levels of α integrin subunits 1-6 or 9 compared to uncultured RPE cells or other RPE cell preparations. The RPE cells described herein may also exhibit elevated expression levels of α integrin subunits 1, 2, 3, 4, 5, or 9. The RPE cells described herein may be cultured under conditions that promote expression of α integrin subunits 1-6. For example, RPE cells may be cultured with an integrin activator, including, but not limited to, manganese and the activating monoclonal antibody (mAb) TS2 / 16. See Afshari, et al. Brain (2010) 133(2):448-464. RPE cells may be plated on laminin (1 μg / mL) and cultured with 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 the α integrin subunit.

[0189] The RPE cells may exhibit a normal karyotype, expressing RPE markers and not expressing hES markers.

[0190] The RPE cells described herein may also be identified and characterized based on the degree of cellular pigmentation. Changes in pigmentation can be controlled by the density at which RPE cells are cultured and maintained, and the time the RPE is maintained in culture. Rapidly dividing, differentiated RPE cells produce less pigment. In contrast, more slowly dividing or non-dividing RPE cells increase their pigmentation levels by adopting their characteristic polygonal or hexagonal shape and accumulating melanin and lipofuscin. For example, quiescent RPE cultures (e.g., due to confluence) typically increase their pigmentation levels over time. Thus, pigmentation accumulation serves as an indicator of RPE differentiation and increased pigmentation, with cell density serving as an indicator of RPE maturity. For example, mature RPE cells may be subcultured at lower densities to reduce pigmentation. In this context, mature RPE cells may be cultured to generate more immature RPE cells. Such The RPE cells remain differentiated RPE cells that express RPE differentiation markers.

[0191] The RPE cells described herein can maintain their phenotype for a long period outside of the body.For example, RPE cells can maintain their phenotype for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 generations.RPE cells can 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.RPE cells can maintain their phenotype for 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 after transplantation. After transplantation, RPE cells may maintain their phenotype throughout the recipient's lifetime. For example, after transplantation, 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. Furthermore, after transplantation, RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. Furthermore, after transplantation, 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 or more years.

[0193] Melanin content of RPE cell populations Exemplary embodiments of the present disclosure provide RPE cell populations with low or moderate average pigmentation levels, and pharmaceutical products comprising RPE cells with low or moderate average pigmentation levels.As detailed in the Examples below, applicants have shown that RPE cells with relatively lower pigmentation levels perform better in assays that evaluate the ability of cells to attach and survive.Without wishing to be limited by theory, it is believed that as RPE cells mature, they may have a reduced ability to form cell attachments, survive, and grow after cryopreservation, and this may be due to the increased pigmentation (melanin) levels contained in more mature RPE cells, and / or the increased other phenotypes of mature RPE that generally tend to be correlated with increased pigmentation (including, for example, changes in cytoskeleton, membrane composition, cell surface receptor expression, adhesion strength, nuclear structure, gene expression, or other phenotypes or phenotype combinations). Also, without wishing to be limited 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 increased levels of pigmentation (melanin) contained in more mature RPE cells and / or other phenotypes of mature RPE that generally tend to correlate with increased pigmentation.

[0194] The level of pigmentation may be measured as the average melanin per cell of a population, e.g., expressed as picograms per cell (pg / cell), and it will be understood that in general there may be some variation in the level of melanin in cells within a 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, e.g., 4 to 5 pg / cell, e.g., 0.1 to 7 pg / cell, 0.1 to 6 pg / cell, 0.1 to 5 pg / cell, 0.1 to 4 pg / cell, 0.1 to 3 pg / cell, 0.1 to 2 pg / cell, 0.1 to 1 pg / cell, 1 to 8 pg / cell, 1 to 7 pg / cell, 1 to 6 pg / cell, 1 to 5 pg / cell, 1 to 4 pg / cell, 1 to 3 pg / cell, 1 to 2 pg / cell, 2 to 6 pg / cell, 3 to 5 pg / cell, or 4.2 to 4.8 pg / cell. In further embodiments, the average melanin content may be less than 5 pg / cell, for example, 0.1 to 5 pg / cell, 0.2 to 5 pg / cell, 0.5 to 5 pg / cell, 1 to 5 pg / cell, 2 to 5 pg / cell, 3 to 5 pg / cell, 4 to 5 pg / cell, or 4.5 to 5 pg / cell.

[0195] Melanin content may be measured using a variety of methods, including those utilizing cell extracts, FACS-based methods, etc. See, 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, 1999; See, e.g., et al., Anal Biochem. 1973 Nov;56(1):91-9. For example, to determine the average melanin content, the number of cells in a representative sample may be measured, or the cells in the representative sample may be lysed, and the total melanin content of the cell lysate (e.g., from an NaOH-extracted cell pellet) may be measured (e.g., by spectrophotometry) and divided by the number of cells in the representative sample to obtain the average melanin content per cell. Optionally, the number of cells in the representative sample may be measured (e.g., counting cells that are positive for one or more markers of RPE and / or that exhibit morphological characteristics of RPE cells), ignoring non-RPE cells in the culture, 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 that excludes the most pigmented and least pigmented 5 percent of harvested RPE cells.

[0197] RPE populations with a desired average melanin content can be easily obtained. For example, the melanin content of non-dividing, metabolically active RPE (e.g., in confluent culture) tends to increase over time due to the accumulation of synthesized melanin, while the accumulated melanin is diluted by cell division, so that the melanin content in dividing cells is relatively low. See, for example, Dunn et al., Exp Eye Res. 1996 Feb; 62(2): 155-69. Therefore, RPE populations with a desired average melanin content can be obtained by selecting an appropriate growth history, such as maintaining the RPE population for a sustained period that results in the desired average melanin content, such as quiescent culture 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, including maintaining the RPE population for a period of time followed by dividing the cells again for a specified time or number of divisions (thereby reducing the average melanin content achieved in the quiescent population), can also be used to adjust the average melanin content. Melanin content may also be regulated by the use of various media and / or media supplements; for example, melanin accumulation in cultured RPE has been reported to be reduced 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-β 1 (1 to 100 U / ml) (Kishi et al., Curr Eye Res. 1998 May;17(5):483-6). Melanin content may also be increased by treating cells with zinc alpha-2-glycoprotein (ZAG) (see U.S. Pat. No. 7,803,750) and / or with 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. Pat. No. 5,998,423).Each of the foregoing documents is incorporated herein by reference in its entirety.

[0198] Alternatively or in addition to the above-mentioned method, RPE cells with desired average melanin content can be obtained through cell sorting, for example, using a flow cytometer. For example, melanin-containing cells can be detected by their light scattering characteristics, including increased side scattering and decreased forward scattering; these characteristics can be used to sort populations according to pigmentation level, thereby purifying populations with desired average melanin content. Boissy et al., Cytometry. 1989 November; 10(6): 779-87; Swope et al., J Invest Dermatol. 1997 September; 109(3): 289-95, the entire contents of which are incorporated herein by reference.

[0199] Modifying MHC genes in human embryonic stem cells to obtain RPE cells with reduced complexity Human embryonic stem (hES) cells (from which RPE may be derived, e.g., as described herein) may be derived from a human embryonic stem cell library. A 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 in a 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. A human embryonic stem cell library may comprise stem cells that are hemizygous, homozygous, or null-deficient for all MHC alleles present in a 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-deficient locus means that the gene is defective at that location (i.e., both alleles of the gene are deleted or inactivated).

[0200] hES cells may comprise an alteration in one of the sister chromosome alleles in the MHC complex of the cell. Genes in the MHC complex may be altered using a variety of methods for creating genetic alterations, such as gene targeting. The altered allele of the MHC complex in the cell may then be subsequently altered to become homozygous, such that the same allele is present on the sister chromosome. Methods such as loss of heterozygosity (LOH) may be used to genetically engineer cells to have homozygous alleles in the MHC complex. For example, one or more genes in a set of MHC genes from the parental allele may be targeted to create hemizygous cells. Other sets of MHC genes may be removed by gene targeting or LOH to create a null line. This null line may then be used as an embryonic cell line into which an HLA gene array or individual genes may be inserted to create a hemizygous or homozygous bank with an otherwise uniform genetic background. Stem cells null for all MHC genes may be generated by standard methods known in the art, such as gene targeting and / or loss of heterozygosity (LOH). See, for example, U.S. Patent Application Nos. 2004 / 0091936, 2003 / 0217374, and 2003 / 0232430, and U.S. Provisional Patent Application No. 60 / 729,173.

[0201] Therefore, the present disclosure relates to methods for obtaining RPE cells, including libraries of RPE cells with reduced MHC complexity. The RPE cells with reduced MHC complexity may be used to increase the cell supply available for therapeutic applications, potentially eliminating the difficulties associated with patient matching. Such cells may be derived from stem cells that have been modified to be hemizygous or homozygous for genes in the MHC complex.

[0202] The present disclosure provides a method for producing RPE cells ( The present disclosure also provides 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 therapy. 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 in a human population, and 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 in a human population.

[0203] Culture medium The methods described herein may use any medium capable of supporting cell culture, such as media for viral, bacterial, or eukaryotic cell culture. 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. Furthermore, the medium may also contain nutritional 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, the nutrient-rich low-protein medium may be any medium with a low protein content that supports cell growth in culture. For example, 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 mix, F-10 nutrient mix EGM-2, DMEM / F-12 medium, Medium 1999, or MDBK-MM. See also Table 1. Furthermore, 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 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 references to the protein percentage present in low-protein medium refer only to the medium and do not take into account, for example, the protein present in B-27 supplements. Therefore, when cells are cultured in low-protein medium and B-27 supplements, it is understood that the protein percentage present in the medium may be higher.

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

[0206] The growth factors, drugs, and other supplements described herein may be used alone or in combination with other factors, drugs, or supplements for inclusion in the medium. The factors, drugs, and supplements may be added to the medium immediately or at any time 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™ (basement membrane substrate), or gelatin); fibronectin; proteolytic fragments of fibronectin, laminin, thrombospondin, aggrecan, and syndezan.

[0208] The medium may be supplemented with one or more factors or agents.

[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, beta cellulin, beta interferon, brain-derived neurotrophic factor, cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemoattractant-1, endothelial cell growth supplement, eotaxin, epidermal growth factor, epidermal neutrophil-activating peptide-78, erythropoietin, estrogen, and the like. Estrogen receptor α, estrogen receptor β, fibroblast growth factor (recombinant acidic / basic heparin-stabilized), 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-α / MGSA, GRO-B, GRO-γ, HCC-1, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, heregulin-α (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 1β, macrophage inflammatory protein 2, macrophage inflammatory protein 3α, macrophage inflammatory protein 3β, monocyte chemotactic protein 1, monocyte chemotactic protein 2, monocyte chemotactic protein 3, neurotrophin 3, neurotrophin 4, NGF-β (human or Also included are ribosomal receptor agonists (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, thrombopoietin, transforming growth factor alpha, transforming growth factor beta 1, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, tumor necrosis factors (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 alpha, interferon alpha A / D, interferon beta, interferon gamma, interferon gamma-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 1 alpha.

[0211] The medium contains 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, α-melanocyte-stimulating hormone, chorionic gonadotropin, corticosteroid-binding globulin, corticosterone, dexamethasone, estriol, follicle-stimulating hormone, gastrin-1, glucagon, gonadotropins, hydrocortisone, insulin, insulin-like growth factor-binding protein, L-3,3',5'-triiodothyronine, L-3,3',5'-triiodothyronine, leptin, leutinizing hormone, L-thymidine, L-threonine ... Hormones and hormone antagonists may be supplemented, including, but not limited to, thyroxin, melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone-binding globulin, thyrotropin, thyrotropin-releasing factor, thyroxine-binding globulin, and vasopressin. The culture medium contained antibodies against low-density lipoprotein receptor, anti-progesterone receptor, intracellular antibody, anti-α-interferon receptor chain 2, anti-cc-chemokine receptor 1, anti-CD118, anti-CD119, anti-colony stimulating factor-1, anti-CSF-1 receptor / c-fins, anti-epidermal growth factor (AB-3), anti-epidermal growth factor receptor, anti-epidermal growth factor receptor, phospho-specific antibody, anti-epidermal growth factor (AB-1), anti-erythropoietin receptor, anti-estrogen receptor, anti-estrogen receptor C-terminal, anti-estrogen receptor B, anti-fibroblast growth factor receptor, anti-fibroblast growth factor, basic antibody, anti-γ-interferon receptor chain, anti-γ-interferon human recombinant, anti-GFR α-1 C-terminal, and anti-GFR α-2. Antibodies against various factors may be supplemented, including, but not limited to, C-terminal antibodies, anti-granulocyte colony-stimulating factor (AB-1) antibodies, anti-granulocyte colony-stimulating factor receptor antibodies, anti-insulin receptor antibodies, anti-insulin-like growth factor-1 receptor antibodies, anti-interleukin-6 human recombinant antibodies, anti-interleukin-1 human recombinant antibodies, anti-interleukin-2 human recombinant antibodies, anti-leptin mouse recombinant antibodies, anti-nerve growth factor receptor antibodies, anti-p60, chicken antibodies, anti-parathyroid hormone-like protein antibodies, anti-platelet-derived growth factor receptor antibodies, anti-platelet-derived growth factor receptor B antibodies, anti-platelet-derived growth factor α antibodies, anti-progesterone receptor antibodies, anti-retinoic acid receptor α antibodies, anti-thyroid hormone nuclear receptor antibodies, anti-thyroid hormone nuclear receptor α 1 / Bi antibodies, anti-transferrin receptor / CD71 antibodies, anti-transforming growth factor α antibodies, anti-transforming growth factor B3 antibodies, anti-tumor necrosis factor α antibodies, and anti-vascular endothelial growth factor antibodies.

[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 transplants described in the prior literature is low graft survival; for example, many cell transplant studies tend to result in rapid cell loss following transplantation (e.g., within the first week). This cell loss does not appear to be due to rejection of the transplanted cells, but rather to the failure of a certain percentage of cells to be retained at the transplant site. This lack of cell retention is most likely due to several factors, including failure of cells to attach to the underlying structure, lack of sufficient nutrients, or physical stress at the transplant site. Following this initial drop in cell numbers, cell survival at various time points after transplantation can vary significantly between studies. Thus, while some studies show a steady decline in numbers, others show that transplanted cells can reach a stable number. However, an important factor in considering transplant success is the percentage of recipients whose grafts survive following cell transplantation.

[0216] In contrast to previous preparations, the RPE cells in the preparations described herein may survive for a long period of time 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. Furthermore, 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. Furthermore, the RPE cells may survive throughout the lifespan of the transplant recipient. Furthermore, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of recipients of the RPE cells described herein may exhibit survival of transplanted RPE cells. Furthermore, the RPE cells described herein may successfully integrate into the RPE layer of a transplant recipient, forming a semi-continuous cell line and maintaining expression of key RPE molecular markers (e.g., RPE65 and bestrophin). The RPE cells described herein may also attach to Bruch's membrane to form a stable RPE layer in the transplant recipient. The RPE cells described herein are also substantially free of ES cells, and the transplant recipient does not exhibit abnormal proliferation or tumor formation at the transplant site.

[0217] The method for treating patients suffering from a condition associated with retinal degeneration may comprise the step of locally administering the composition of the present disclosure (for example, by intraocular injection or insertion of a substrate comprising the pharmaceutical agent of the present disclosure).Intraocular administration of the pharmaceutical agent of the present disclosure includes, for example, intravitreal, transcorneal, subconjunctival, subretinal, submacular (for example, by transfoveal submacular injection), juxtascleral, posterior scleral, and sub-Tenon's portion of the eye.See, for example, U.S. Patent No. 7,794,704; U.S. Patent No. 7,795,025; U.S. Patent No. 6,943,145; and U.S. Patent No. 6,943,153.

[0218] The present disclosure also provides a method for administering human RPE cells derived from embryonic stem cells with reduced complexity to a patient. This method may include the steps of: (a) identifying a patient in need of treatment involving the 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 RPE cell generating method of the present disclosure; (d) selecting RPE cells from the library that match the MHC proteins on the patient's cells; and (e) administering any of the cells from step (d) to the patient. This method may be performed at a regional center, such as a hospital, clinic, doctor's office, or other medical facility. Furthermore, if the RPE cells selected as a match for the patient are low in number, they may be expanded prior to treating the patient.

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

[0220] The specific treatment plan, route of administration, and concomitant therapy may be tailored based on the specific condition, the severity of the condition, and the overall health of the patient. Administration of a preparation comprising RPE cells may be effective in reducing the severity of symptoms and / or preventing further degeneration of the patient's condition. For example, administration of a pharmaceutical product comprising RPE cells may improve the patient's vision. In further specific embodiments, administration of RPE cells may be effective in completely reversing any blindness or other symptoms. Furthermore, administration of RPE cells may treat symptoms of endogenous RPE layer damage.

[0221] RPE cell medicine The RPE cells may be formulated with a pharmaceutically acceptable carrier. For example, the RPE cells may be administered alone or as a component of a pharmaceutical formulation. The test compound may be formulated for administration in any convenient manner for use in medicine. A pharmaceutical preparation suitable for administration may comprise RPE cells combined with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions, or emulsions, or sterile powders that may be reconstituted immediately before use into a sterile injectable solution or dispersion, which may contain antioxidants, buffers, bacteriostats, solutes, or suspending or thickening agents. An exemplary pharmaceutical product comprises RPE cells combined with ALCON® BSS PLUS (a balanced salt solution containing, per mL: 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 sodium phosphate dibasic, 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 treating human patients, such as pyrogen-free or essentially pyrogen-free and sterile. When administered, pharmaceuticals used in the present disclosure may be in a physiologically acceptable form that is pyrogen-free and sterile. Formulations comprising RPE cells used in the methods described herein may be in suspension, gel, or other suitable form. The formulation may be implanted in a solution, colloid, slurry, or mixture. Furthermore, 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. At the time of injection, cryopreserved RPE cells may also be resuspended in a commercially available balanced salt solution to achieve the desired osmolality and concentration for administration via subretinal injection. The formulation may also be administered to areas around the central macula that have not been completely lost due to disease, which may promote the attachment and / or survival of the administered cells.

[0223] Compositions of the present disclosure may include a rho-associated protein kinase (ROCK) inhibitor, such as Stemgent's Stemolecule Y-27632. For example, an exemplary composition includes RPE and a ROCK inhibitor, which may be 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 in a pharmaceutically acceptable eye drop solution by intraocular injection. For example, when administering the formulation by intravitreal injection, the solution may be concentrated so that a minimized volume can be delivered. The concentration for injection may be any effective and non-toxic amount depending on the factors described herein. The RPE cell pharmaceutical product for treating patients may contain at least about 10 4 RPE cell preparations for treating patients may be formulated at a dose of at least about 10 cells / mL. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10For example, the RPE cells may be formulated in a pharmaceutically acceptable carrier or excipient, formulated at a dose of individual RPE cells / mL.

[0225] The RPE cell medicines 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 medicines may comprise at least about 1 x 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 The RPE cell medicine may comprise at least about 1 x 10 RPE cells. 2 ~1×10 3 , 1×10 2 ~1×10 4 , 1×10 4 ~1×10 5 , or 1 × 10 3 ~1×10 6 The RPE cell medicine 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 medicine may comprise at least about 20,000 to 200,000 RPE cells in a volume of at least about 50 to 200 μL. Further, the RPE cell medicine 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, RPE cell number or RPE cell concentration may be determined by counting viable cells and excluding non-viable cells. For example, non-viable RPE may be detected by their inability to exclude vital dyes (e.g., trypan blue) or by using functional assays (e.g., ability to adhere to a culture substrate, phagocytosis, etc.). Furthermore, RPE cell number or RPE cell concentration may be determined. The degree may be determined by counting cells that express one or more RPE cell markers and / or excluding cells that express one or more markers indicative of cell types other than RPE.

[0227] The 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 to allow the cells to enter the affected area of ​​the eye, such as, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoridal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, supraclavicular space, pars plana, surgically induced avascular area, or macula.

[0228] RPE cells may be contained in a cell sheet. For example, a cell sheet comprising RPE cells may be prepared by culturing the RPE cells on a substrate onto which the cells can attach and grow at culture temperatures, and then release the intact cell sheet therefrom, such as a thermoresponsive polymer, such as a thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) transplantation surface, which changes surface properties upon temperature transition (e.g., by cooling below the lower critical solution temperature (LCST)), causing the release of the cultured cell sheet. (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, the entire contents of each of which are incorporated herein by reference. 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 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 a host organism, and is prepared, for example, by culturing cells on a substrate suitable for transplantation or by releasing cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. An exemplary substrate potentially suitable for transplantation may comprise gelatin (Hsiue et al. al., supra). Alternative substrates that may be suitable for transplantation include fibrin-based matrices. The cell sheets may be used in the manufacture of medicaments to prevent or treat retinal degenerative diseases. RPE cell sheets may be formulated for delivery to the eye of a subject in need thereof. For example, the cell sheets may be delivered to an eye in need thereof via subfoveal membrane resection to transplant the RPE cell sheet, or may be used to manufacture medicaments 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 infusion, the volume of the RPE cell pharmaceutical of the present disclosure may be 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 infusion, 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 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, 1 For example, the volume of a formulation of the present disclosure may be from at least about 10-50, 20-50, 25-50, or 1-200 μL. The volume of the formulations 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 x 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 4The formulation may comprise 2000 RPE cells per μL, 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 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® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, and tacrolimus. The immunosuppressant may be administered at a dose of at least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg / kg. If immunosuppressants are used, they may be administered systemically or locally, and they may be administered before, simultaneously with, or following administration of the RPE cells. Immunosuppressive therapy may be continued for weeks, months, years, or indefinitely following administration of the RPE cells. For example, following administration of the RPE cells, a patient may be administered 5 mg / kg of cyclosporine for 6 weeks.

[0232] The treatment for retinal degeneration may comprise the administration of a single dose of RPE cells. The treatment methods described herein may also comprise a course of treatment in which RPE cells are administered multiple times over a period of time. Exemplary treatment courses may comprise weekly, biweekly, monthly, quarterly, twice-yearly, or annual treatment. Alternatively, treatment may be stepwise, whereby multiple doses are administered initially (e.g., daily administration for the first week), followed by the need for smaller, less frequent doses.

[0233] When administered via intraocular injection, RPE cells may be delivered once or multiple times periodically throughout the patient's life. 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, more frequent administration may be desirable for certain conditions or disorders. When administered via an implant or device, RPE cells may be administered once, or once or multiple times periodically throughout the patient's life, depending on the needs of the particular patient and disease or condition being treated. Drug administration schedules that vary over time are similarly contemplated. For example, Initially, more frequent treatments may be required (e.g., daily or weekly treatments). Over time, as the patient's condition improves, less frequent treatments may be required, or even no further treatments may be necessary.

[0234] The methods described herein may further comprise monitoring the efficacy of the treatment or prevention in the subject by measuring electroretinogram response, optokinetic acuity threshold, or luminance threshold. The methods may also comprise monitoring the efficacy of the treatment or prevention by monitoring cellular immunogenicity or cell migration within the eye.

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

[0236] RPE cell provided herein can be human RPE cell.But please note that human cell can be used in animal model or animal patient as well as human patient.For example, human cell can be tested in mouse, rat, cat, dog or non-human primate model of retinal degeneration.Furthermore, human cell can be used therapeutically to treat animals that need it, such as in veterinary medicine.

[0237] Mode of administration The pharmaceutical agent may be formulated in a pharmaceutically acceptable carrier depending on the route of administration. For example, the formulation may be formulated to be administered to the subretinal space of the eye. The 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, the 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). As described above, the RPE cells may have a variety of possible configurations, such as individual cells, clumps, clusters, sheets, or any combination thereof, which may be contained in an aqueous carrier, gel, matrix, 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 implanted into the subretinal space using vitrectomy. Furthermore, at the time of injection, the RPE cells may be resuspended in a commercially available balanced salt solution (e.g., Alcon with BSS®) to achieve the desired osmolality and concentration for administration by subretinal injection.

[0239] Optionally, RPE cells may be administered by a method comprising a pars plana vitrectomy, such as a three-port pars plana vitrectomy. The method includes a small retinal incision. Prior to cell administration, a subretinal bleb may be formed ("pre-bleb"), e.g., by injection of saline or another suitable fluid, which may then be removed prior to cell administration. However, cells may also be administered without pre-blebbing. Cells may be administered in a bleb at the temporal fossa location. For example, the bleb may optionally be expanded within the arcade vessels. The bleb may be positioned so as not to detach the macular fovea.

[0240] Depending on the method of administration, RPE cells may be added to a buffered electrolyte-balanced aqueous solution; a buffered electrolyte-balanced aqueous solution supplemented with lubricating polymers, mineral oil or petrolatum-based ointments, 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 an implant or device. In certain embodiments, the device is a bioerodible implant for treating ocular diseases, comprising an active agent dispersed in a bioerodible polymer matrix, wherein at least about 75% of the active agent particles have a diameter of less than about 10 μm. The bioerodible implant may be sized for implantation into an ocular region. The ocular region may be any one or more of the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, epicorneal space, sclera, pars plana, surgically induced avascular region, macula, and retina. The bioerodible polymer may be, for example, poly(lactic-co-glycolic) acid (PLGA) copolymer, biodegradable poly(DL-lactic-co-glycolic acid) film, or PLLA / PLGA polymer matrix. The ratio of glycolic acid monomers in the polymer may be about 25 / 75, 40 / 60, 50 / 50, 60 / 40, 75 / 25, or more preferably about 50 / 50 weight percent. The PLGA copolymer may comprise about 20, 30, 40, 50, 60, 70, 80, or about 90 weight percent of the bioerodible implant. The PLGA copolymer may comprise about 30 to about 50 weight percent, preferably about 40 weight percent, of the bioerodible implant. RPE cells may also be implanted in conjunction with biocompatible polymers such as polylactic acid, poly(lactic-co-glycolic acid), 50:50 PDLGA, 85:15 PDLGA, and INION GTR® biodegradable membranes (biocompatible polymer blends). See U.S. Patent Nos. 6,331,313; 7,462,471; and 7,625,582.See also Hutala, et al. (2007) "In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: Direct contact studies." Journal of Biomedical Materials Research 83A(2):407-413; Lu, et al. (1998) J Biomater Sci Polym Ed 9:1187-205; and Tomita, et al. (2005) Stem Cells 23:1579-88.

[0242] In another aspect, the present disclosure provides compositions comprising RPE located on a membrane and methods of using the compositions in the prevention or treatment of retinal diseases, disorders, or conditions. For example, the membrane may be the membrane described in U.S. Patent No. 20110236464 (pre-grant publication), the entire contents of which are incorporated herein by reference. The membrane may be substantially non-biodegradable and porous, with pores approximately 0.2 μm to 0.5 μm in diameter. For example, the pore size may be 0.3 μm to 0.45 μm. The use of a non-biodegradable membrane may ensure that, once implanted in the eye, 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.

[0243] The pore density may be approximately 1 x 10^7 to 3 x 10^8 pores per square cm, such as 5 x 10^7 and 1 x 10^8 pores per square cm. This density allows for a 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 be subject to angiogenesis from the rich choroidal bed. This has been shown in the rich vascular beds of the outer eye (Cassell et al., 2002; Patrick et al., 1999; Saxena et al., 1999; Peter et al., 1998), but only occurs when porosity is sufficient (Menger et al., 1990).

[0244] For example, the membrane's water permeability may exceed 50 x 10 msec Pa. Specifically, the membrane's water permeability may be approximately 33 mL / min / cm². This is equivalent to 801.21 x 10 msec Pa, eight times the water permeability of cadaveric Bruch's membrane in the macula of young individuals. This excess permeability is potentially useful because the artificial membrane may rely entirely on passive processes. Not only can it meet the needs of the overlying cells in terms of nutrient diffusion, but it preferably does not impede fluid transport from the basal side of the RPE layer, which would otherwise cause the RPE to detach from the polymer surface. Consistent with this prediction, it has been hypothesized that a decrease in the water permeability of Bruch's membrane in the elderly may cause 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 degradation.

[0246] Preferably, the membrane may be sealed by ultrasonic sealing, radio frequency sealing, or insert molding. This allows other layers to be attached to the membrane, such as a formulation or coating layer. For example, one may want to attach a more rigid biodegradable layer, such as PLGA, to provide rigidity to the membrane and aid in delivery. Alternatively, a layer containing a pharmacological or biological agent, or other cell-supporting layer, may be included.

[0247] The membrane preferably has a maximum thickness of approximately 11 μm. More preferably, the membrane thickness is between 9 μm and 11 μm. The membrane thickness may be selected to allow for nutrient diffusion and vascularization, and to allow for easy insertion of the membrane into the eye.

[0248] Thus, RPE may be provided or cultured on a cell growth-supporting membrane, which is substantially non-biodegradable, porous, and has a maximum thickness of approximately 11 μm. The membrane is preferably substantially planar, and its smallest dimension is preferably less than approximately 11 μm. The thickness of that dimension may vary, but is preferably between 9 μm and 11 μm thick.

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

[0250] Preferably, the membrane is hydrophilic, which may give it good wetting capabilities and allow for easy attachment of cells and other desired coatings.

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

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

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

[0254] Thus, RPE may be provided or cultured on a membrane that supports cell growth, the membrane comprising a substantially non-biodegradable, 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 polycarbonates and polysiloxanes, and polyester- or polyether-based; polyamides such as nylon; polyetheresters such as Sympatex; polycarbonates such as Makrolon; polyacrylates such as Perspex; poly(tetrafluoroethene) (PTFE); polysiloxanes; polyolefins such as polyethylene and polypropylene; and polyoxymethylene (POM), commonly known under the DuPont trademark Delrin. It is particularly preferred that the membrane be made of polyethylene terephthalate or polybutylene terephthalate. In another preferred embodiment, the membrane is made of polyester.

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

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

[0258] The membranes and cell layers may be transplanted into the eye of a patient in need thereof, for example, in the treatment of age-related macular degeneration, retinal tears, macular dystrophy, choroidemia, Leber Congenital Amarosis, Stargardt's disease, and other retinal diseases or conditions.

[0259] Screening assays The present disclosure provides a method for screening and identifying agents that regulate RPE cell maturation. 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 plan. Alternatively, the identified agents may be used as part of a culture method to improve the survival of differentiated RPE cells in vitro.

[0260] RPE cells may also be used as research tools in settings such as pharmaceutical, chemical, or biotechnology companies, hospitals, or academic or research institutions. Examples include 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 that may be used to promote RPE maturation, survival, and / or engraftment. Identified agents may be studied in vitro or in animal models to evaluate their potential 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 providing RPE cells, contacting the RPE cells with an agent, evaluating the RPE cells for signs of maturation, and then identifying an agent that promotes RPE maturation if the agent causes the RPE cells to exhibit signs of maturation. Signs of maturation may be pigmentation levels, gene expression levels, and morphology, as discussed herein. [Industrial Applicability]

[0262] Certain aspects of the present disclosure relate to the production of RPE cells in commercial quantities, which 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 for generating, storing, and distributing RPE cells produced by the methods disclosed herein. Following RPE generation, RPE cells may be harvested, purified, and optionally stored prior to patient treatment. RPE cells may optionally be patient-specific or specifically selected based on HLA or other immune profiles. For example, once a patient exhibits symptoms of, for example, diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, retinal atrophy, retinal detachment, retinal dysplasia, and Stargardt disease (fundus flava), pigmented streaks, or myopic macular degeneration, RPE cells can be ordered and provided in a timely manner. Thus, the present disclosure relates to methods for generating RPE cells for obtaining cells on a commercial scale, cell preparations comprising RPE cells derived from the methods, and methods 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 that comprises establishing a distribution system to distribute the formulation for sale or may include establishing a sales group to market the pharmaceutical product.

[0265] The present disclosure provides methods for providing RPE cells to hospitals, medical centers, and clinicians, and RPE cells produced by the methods disclosed herein can be stored and ordered at the request of a hospital, medical center, or clinician and administered to patients in need of RPE cell therapy. The hospital, medical center, or clinician orders RPE cells based on patient-specific data, and the RPE cells are produced according to the patient's specifications and subsequently supplied to the hospital or clinician that placed the order. For example, after a particular RPE cell preparation is selected as suitable for a patient, it is then expanded to a quantity suitable for treating the patient.

[0266] A further aspect of the present disclosure relates to RPE cell libraries that can provide compatible cells for potential patient recipients. Accordingly, the present disclosure provides a method of conducting a pharmaceutical business comprising the step of providing RPE cell preparations that are homozygous for at least one histocompatibility antigen, wherein the cells are 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 in the human population, and wherein the RPE cell bank is heterozygous for at least one MHC allele relative to another member of the cell bank. These comprise cells that are hemizygous or homozygous for different sets of genes, respectively. As described above, gene targeting or loss of heterozygosity may be used to generate hemizygous or homozygous MHC allele stem cells that are used to derive RPE cells.

[0267] The present disclosure also includes methods for 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 histocompatible donor, and then creating and expanding RPE cells derived from the ES cells. These RPE cells may be stored. These RPE cells may also be used to treat the patient from whom the ES cells were obtained, or a relative of the patient, or a histocompatible individual.

[0268] This disclosure demonstrates that human RPE cells can be reliably differentiated and expanded from human ES cells under well-defined, reproducible conditions, representing an inexhaustible cell source for patients with retinal degenerative disorders. The concentration of these cells is not limited by availability, but rather can be titrated to an individual's precise clinical requirements. Repeated infusions or transplants of the same cell population over the patient's lifetime are also possible, if deemed necessary by the physician. Furthermore, the ability to create banks of matched or reduced-complexity HLA hES lines from which RPE cells can be generated could potentially reduce or eliminate the need for immunosuppressants and / or immunomodulatory protocols altogether. [Example]

[0269] The invention will now be generally described, which will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

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

[0271] Example 1 method Creation of an hESC master cell bank The hESC line used in these studies, previously described as MA09 (22), was derived from unused in vitro fertilized (IVF) embryos obtained with full informed consent and used in accordance with Advanced Cell Technology's Ethics Advisory Board and Institutional Review Board. Using current good manufacturing practices, MA09 seed culture stocks were thawed and propagated through four serial passages on mitotically inactivated mouse embryonic fibroblasts (MEFs). The clinical hESC master cell bank (hESC-MCB) was cryopreserved and confirmed to have a normal female (46,XX) karyotype and to be free of bacterial and mycoplasmal contaminants, as well as human, bovine, porcine, and murine viruses. PCR analysis demonstrated no alterations or mutations in genes associated with macular degeneration, including CTRP5, EVLV4, RPE-65, VMD2, and ABCA4 (Table 3, below).

[0272] 1. Preparation of Retinal Pigment Epithelium Vials of hESC-MCB were thawed and expanded for three passages on mitomycin C-treated MEFs. Because the hESCs were co-cultured with animal cells, the differentiated derivatives were classified as xenotransplantation products and were subject to FDA guidelines for donor animal and product handling, testing, and archiving, as well as patient monitoring and registration (detailed in Example 2 below). After hESC expansion, the cells were sequentially induced to form embryoid bodies, and the cells This was followed by outgrowth and localized 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 trypsinization, the isolated cells were seeded, grown to confluence, and induced to redifferentiate for a total of three serial passages. Passage 2 RPE were cryopreserved and served as the starting material for preparing cells for clinical use.

[0273] Preclinical trials Human ESC-derived RPE cells were injected subretinal-ly into NIH III immunized nude mice (tumorigenicity and biodistribution studies) and into dystrophic RCS rats and ELOV4 mice (efficacy studies) as previously described (8). Detection of human cells in the injected eyes and other organs was performed by DNA Q-PCR designed to amplify human Alu Y DNA sequences and by paraffin section immunostaining 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 in-process testing and testing performed after thawing, final product formulation, and maturation culture to simulate the fate of transplanted cells in vitro. Potential bacterial, mycoplasma, mouse virus, and residual mouse DNA safety assessments were performed by WuXi Apptec, Inc., St. Paul, MN, according to standard protocols. Cytogenetic analysis for karyotype analysis, DNA fingerprinting for cell line authentication, and fluorescence in situ hybridization (FISH) were performed by Cell Lines Genetics, Madison, WI. Endotoxin testing was performed by Cape Cod Associates, Inc., East Falmouth, MA, on cryopreserved RPE formulated as the final product for clinical infusion. Quantitative immunohistochemical staining was performed using standard methods, and the percentage of positively stained cells was normalized for the number of DAPI-stained nuclei examined. Assessment of RPE purity and 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 assessed 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 with known cell numbers (as detailed in Example 2).

[0275] Cell preparation and injection A vial of cryopreserved MA09-RPE was thawed, washed three times by centrifugation, and diluted to 2 × 10 cells per 1 μL of BSS PLUS® (Alcon). 3The RPE cells were resuspended to a density of 0.000 viable cells / ml. A vial containing the appropriate volume of formulated RPE and a paired vial containing the appropriate volume of BSS PLUS® were delivered to the OR at 2-8°C. Immediately prior to injection, the two vials were reconstituted in a 1 mL syringe to obtain a loading cell density that would result in the delivery of the desired number of RPE cells (50,000 viable RPE cells into the subretinal space of each patient's eye). To ensure accurate delivery of the intended dose, the loading cell density was increased to compensate for the expected loss of viable RPE cells encountered during mixing, loading, and delivery through the cannula. This viable cell loss was measured as described in Example 3 below and was shown to be dependent on the cannula used. In these examples, a MEDONE POLYTIP® cannula 25 / 38 (0.50 mm (25 g) x 28 mm cannula with a 0.12 mm (38 g) x 5 mm tip) was used, with a loaded cell density of 444 viable cells / μL. , resulting in a predicted delivery of 336 + / - 40 viable cells / μL (N=6), resulting in an expected delivery of 50,400 viable RPE cells in a volume of 150 μL into the subretinal space of each patient's eye.

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

[0277] Transplantation and Rationale Pars plana vitrectomy was performed, which involved surgical guidance to separate the posterior vitreous from the optic nerve anterior to the posterior edge of the vitreous fundus. 5 × 10 vitreous cells in a volume of 150 μl were injected into a preselected area around the central macula that had not been completely obliterated by disease. 4Submacular injections of hESC-RPE cells were delivered. The transplantation site was carefully selected based on the presence of damaged but native RPE and overlying photoreceptors to optimize the probability of graft integration and the potential for photoreceptor cell rescue. Graft attachment within the pathoanatomic complex of a completely atrophied central macula is unlikely and does not mimic the condition of the central macula in earlier stages of degeneration, which may be the ultimate therapeutic target for stem cell-based regenerative transplantation strategies.

[0278] The immunosuppressive regimen includes low-dose tacrolimus (targeting blood levels of 3–7 ng / mL) and mycophemolate mofetil (MMF ranging from 0.25 g to 2 g orally daily) 1 week before surgery, continued for 6 weeks. At week 6, the regimen calls for discontinuation of tacrolimus and continuation of MMF for an additional 6 weeks.

[0279] result Characterization of the RPE Controlled hESC differentiation resulted in nearly 100% pure RPE (Figure 1). A single (9.6 cm) 6-well plate of pigmented tissue (Figure 1A) yielded approximately 1.5 × 10 8 RPE cells (e.g., up to 3 x 10 per patient) 6We generated 1000 cells (sufficient to treat 50 patients at a cell dose of 1000 cells). The cells exhibited typical RPE behavior, losing their pigmented cobblestone morphology during proliferation (after trypsinization); once confluence was reestablished, they redifferentiated into a monolayer of polygonal-cuboidal pigmented epithelium. Q-PCR showed that markers of pluripotency (Oct-4, NANOG, and SOX2) were significantly downregulated, while RPE markers RPE65, bestrophin, Pax6, and MITF were expressed at high levels (Figure 1B–F and Table 5). Immunostaining of mature cultures showed that bestrophin, a late marker of differentiated RPE, was organized membranously in the majority of cells before harvest; all (>99%) cells were positive for bestrophin and / or PAX6 (PAX6 became weaker or disappeared in more mature cells) and for the tight junction component ZO-1 (not shown). After cryopreservation, a vial of cells was thawed and prepared for transplantation. Staining for the retinal marker Pax6 and / or MITF (a marker for pigmented cells) confirmed 100% RPE purity (Figure 1C). To further validate the prepared cells, they were cultured for 2–3 weeks to allow growth and maturation until RPE morphology was established. Pax6 / bestrophin (Figure 1E) and ZO-1 (Figure 1G) immunostaining was similar to pre-harvest cultures, and a potency assay showed that >85% of cells phagocytosed bioparticle fragments (Figure 1J).

[0280] Safety Testing Because hESCs were exposed to animal cells and products, the MCB and RPE were extensively tested for animal and human pathogens. Cells were confirmed 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. Although the RPE manufacturing process was performed under conditions that do not support pluripotent cells, highly sensitive assays were performed to rule out the presence of any contaminating hESCs in the final RPE product. Examination of 2 / 9 million cell RPE samples (at P1 / P2) stained for Oct-4 and alkaline phosphatase did not indicate the presence of any pluripotent cells. Tumorigenicity, biodistribution, and toxicity studies performed in NIH-III mice did not indicate any adverse or safety issues in any animals. Furthermore, no tumors were observed in animals injected with 50,000–100,000 RPE cells supplemented with either 0.01%, 0.1%, or 1% undifferentiated hESC cells. Human RPE cell survival was confirmed in 100% of animal eyes up to 3 months and 92% of animals up to 9 months after injection (Table 6, below). Human RPE cells survived throughout the lifespan of the animals and integrated into the mouse RPE layer; although morphologically nearly indistinguishable from host RPE cells (Figure 2), they could be confirmed by immunostaining and expressed bestrophin in a typical basolateral manner (Figure 2B). Ki-67 staining showed low levels of proliferation 1–3 months after transplantation, but no Ki-67-positive cells were observed at 9 months, suggesting that the hESC-derived RPE had formed a mature, quiescent monolayer.

[0281] 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 believed to be critical for the success of this therapeutic strategy. A distinctive feature of hESC technology is the ability to control the degree of differentiation in vivo. The degree of RPE differentiation is manifested in a range of regulated genotypic and phenotypic manifestations, including the level of pigmentation. Cells maintained under similar conditions but harvested and cryopreserved at different time points exhibit varying levels of pigmentation. Figure 3 shows two representative lots of cryopreserved RPE harvested at apparently different pigment levels (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 prepared using clinical transplantation protocols. After extrusion through an injection cannula, cells were seeded onto gelatin-coated tissue culture plates to monitor attachment and subsequent growth. RPE cells from the lighter pigmented lot showed the least number of floating cells in overnight culture; the majority of cells attached and spread, exhibiting RPE behavior and morphology typical of this growth stage (Figure 3A). After 3 days in culture, RPE cell numbers were 4.0 × 10 seeded. 4 pieces to 10.6 x 10 4 In stark contrast, the more deeply pigmented RPE showed numerous floating cells; after 3 days in culture, only a small proportion of cells were attached and viable, with a marked decline in cell numbers (less than one-tenth of the more lightly pigmented lot [9.0 × 10]). 3 ]) (Figures 3F and 3G). These results suggest a strong correlation between RPE differentiation stage and in vitro attachment and growth capacity. The RPE lot used in this clinical study had a melanin content of 4.1 pg / cell and exhibited similar attachment and growth as the lighter pigmented lot. The stresses associated with freeze-thaw cycles, post-thaw washing, centrifugation, and formulation, as well as extrusion through the injection cannula, may, to some extent, be responsible for the observed differences between the lighter and more heavily pigmented lots.

[0282] clinical results The patient with SMD was a 26-year-old Caucasian woman with baseline best-corrected visual acuity (BCVA), a manual valve (HM), and inability to read any letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. No evidence of intraocular inflammation or hyperproliferation was reported at any time point following implantation. No signs of inflammation were detected. The absence of clinically detectable inflammation was supported by slit-lamp biomicroscopy, fundus photography, IVFA, and SD-OCT (detailed in Example 2 and Figures 8 and 9). Clinical increased pigmentation at the level of the RPE was observed beginning 1 week postoperatively and appeared to extend beyond the surgical implant site (Figure 4). Goldmann visual fields improved from baseline through 2 months after implantation (preoperative and postoperative visual fields are shown in Figures 10 and 11). At 2 weeks, BCVA was CF (ETDRS 1 letter), which continued to improve over the study period (ETDRS 5 letters [BCVA20 / 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 subjectively improved color vision and improved contrast and dark adaptation in the operated eye, with no changes in the fellow eye.

[0283] [Table 2]

[0284] The AMD patient was a 77-year-old Caucasian woman with a baseline BCVA of 21 letters (20 / 500) on ETDRS. Despite moderate noncompliance with the immunosuppressive regimen, no signs of intraocular inflammation or hyperproliferation were detected at any time point following implantation. The absence of clinically detectable inflammation was supported by slit-lamp biomicroscopy, fundus photography, IVFA, and SD-OCT (detailed in Example 2 and Figures 8 and 9). 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 (20 / 320) on ETDRS and remained stable through week 8. Goldmann visual fields, as measured by central scotoma, decreased slightly in size at week 8 compared to baseline.

[0285] Consideration The therapeutic use of human embryonic stem cells poses difficult technology transfer challenges. This report provides the first clinical evidence suggesting that hESC-derived cells can be safely transplanted into human patients. In this study, a low dose (5 × 10) of hESC-derived cells was used to generate the cells. 4 RPE cells (100 cells) 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 early-onset blindness, respectively, in developed countries.

[0286] To improve the probability of cell attachment to Bruch's membrane, we selected a submacular injection site where the macular complex (photoreceptors, Bruch's membrane, and RPE) was still present and potentially viable, thus increasing the predictability that transplanted cells would incorporate into the native RPE, potentially salvaging damaged perimacular tissue. Both patients tolerated the transplantation well, and at the time of this report, there was no evidence of postoperative inflammation, rejection, or tumorigenicity. Clinical and Laboratory Findings suggests that transplanted RPE cells may attach, integrate, and begin to influence the damaged native RPE.

[0287] Ongoing 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 visual acuity gains persist. It is anticipated that immune responses, if any, may be managed through methods known in the art, including immunosuppressive and / or tolerizing regimens. It is also anticipated that greater visual acuity gains may be achievable through the administration of greater numbers of RPE cells. Furthermore, administration of RPE cells is expected to delay or prevent vision loss associated with retinal degenerative conditions, including AMD, SMD, and the like.

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

[0289] The RPE starting material used in this study was a well-characterized hESC master cell bank, generated using a procedure optimized to reliably generate large numbers of pluripotent stem cells under controlled conditions. Although the RPE differentiation procedure is not permissive for supporting hESC survival, extensive preclinical safety testing confirmed that transplanted hESC-RPE did not induce ectopic tissue formation or tumors during the animal's lifetime. An immunofluorescence-based assay capable of detecting fewer than one undifferentiated hESC cell per million or more cells confirmed that the clinical RPE lot used in this study had no detectable pluripotent cells, representing a detection level five orders of magnitude lower than the hESC dose shown to induce tumors in in vivo studies. The creation of the hESC-MCB and the manufacturing of each RPE cell lot involve growth on a primary mouse embryonic fibroblast feeder layer. Therefore, hESC-RPE are classified as a xenotransplantation product and were subject to all testing and monitoring prescribed by FDA xenotransplantation guidelines to ensure the cells were free of mouse pathogens. RPE also underwent an extensive battery of safety tests to confirm the absence of microbial contaminants and viruses and were characterized by a variety of RPE-specific attributes, including phagocytic capacity, gene expression, morphological assessment, and immunohistochemical staining for RPE-specific markers. Prior to the initiation of these clinical trials, transplantation of hESC-RPE into dystrophic animals demonstrated the cells' ability to rescue photoreceptors and visual function in a dose-dependent manner.

[0290] This study was designed to test the safety and tolerability of hESC-RPE in patients with advanced SMD and dry AMD. To date, the cells transplanted into both patients appear to be free of abnormal proliferation, teratoma formation, graft rejection, or other adverse pathological reactions. Continued follow-up and further research are warranted. Ultimately, however, The goal of current treatment is to treat patients earlier in the disease course, potentially increasing the chances of photoreceptor and central vision salvage.

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

[0292] The characteristics of the clinical hESC Master Cell Bank (hESC-MCB) (from which the RPE cells used in Example 1 were generated) are shown in Table 3.

[0293] [Table 3-1]

[0294] [Table 3-2]

[0295] Mouse Embryonic Fibroblast (MEF) Master Cell Bank Because MA09-hRPE cells were in contact with non-human (mouse) cells in vitro, they were not in compliance with the April 2003 Guidance for Industry, “Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation.” According to the “Points to Consider on Xenogeneic Cell Therapy Medicinal Products in Humans” and “Points to Consider on Xenogeneic Cell Therapy Medicinal Products” (EMEA / CHMP / CPWP / 83508 / 2009), these human cells are defined as xenotransplant products. Charles River A breeding colony at Charles River 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 sealed room under extensive health monitoring. Donor animals were timed mated and isolated during pregnancy. 12 days after mating, a veterinarian performed a physical examination on all mice before sacrifice; animals were euthanized, leukocyte and plasma preparations were preserved, and blood was collected from each donor mouse for serological testing for mouse pathogens by Charles River Laboratories, Wilmington, MA. A board-certified veterinary pathologist performed necropsies on the carcasses and uterus of each donor animal and one embryo from each litter. Organs from each animal, along with plasma and cryopreserved leukocytes, are stored for at least 30 years (as required by EMEA / CHMP / CPWP / 83508 / 2009). MEFs are isolated and cultured as previously described (Klimanskaya and McMahon, 2005), frozen at P1, and stored in a mitomycin C-free environment. They were used at P2 after activation. To minimize the risk of introducing mouse viruses and other pathogens, MEFs were tested and characterized by WuXi AppTec, Inc. The testing specifications and results of lot MEF-08, used in hESC-MCB preparations and hRPE medical lots, 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 Deparaffinized sections were incubated in 0.1 M citrate buffer (pH 6.0) for 40 minutes in a steamer for antigen retrieval (bestrophin and human mitochondria) or for 30 minutes in a pressure cooker for Ki67. Antibody staining was performed as described above, except that in some cases biotin-conjugated secondary antibodies were used after blocking endogenous biotin using a kit from Vector (Burlingame, CA). The antibodies used were anti-bestrophin (rabbit, Abcam), anti-human mitochondria (mouse, Spring Bioscience), and anti-ki67 (rabbit, Abcam). Secondary antibodies were anti-mouse biotin, anti-mouse Cy3 (Jackson Immunoresearch), and anti-rabbit Alexa488 (Invitrogen), and Streptavidn-Cy3 was obtained from Jackson Immunoresearch. Sections of mouse teratomas formed by hESCs were used as positive controls for anti-human mitochondrial and Ki67, and sections of fixed, paraffin-embedded hRPE pellets were used as bestrophin positive controls. Negative controls were mouse rabbit and mouse IgG (Novus Biologicals).

[0302] q-RT-PCR RNA was extracted from the cell mixture using an RNeasy RNA isolation kit from Qiagen, resulting in a final volume of 30 μL RNA per sample. cDNA was then synthesized from 10 μL RNA using a Quantitect cDNA synthesis kit from Qiagen, resulting in a final volume of 20 μL cDNA. 1 μL of cDNA was then tested for relative gene expression in triplicate, normalized for 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 manufacturer's recommended cycle conditions for comparative Ct relative quantification. 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 replicates normalized to the β-actin signal present in each sample. Data are presented as the mean + / - SD of three replicates.

[0303] Phagocytosis assay Phagocytosis is characterized by the phospholipids pHro, which fluoresce when internalized in the low pH environment of intracellular phagosomes. The activity was assessed by a FACS-based assay using doe™ coli fluorescent bioparticles (Invitrogen). Bioparticles were prepared according to the manufacturer's instructions. Confluent RPE cells were cultured in CO2-independent medium (Invitrogen) with 50-200 μL of bioparticles per well of 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, harvested with trypsin, and analyzed by FACS, counting 10,000 events on a C6 flow cytometer.

[0304] Melanin measurement The RPE cell suspension was centrifuged at 160 x G for 5 minutes at room temperature, and a sample was removed for hemocytometer cell count. The pellet was resuspended in 1N NaOH, heated at 80°C for 10 minutes, vortexed, and absorbance was measured at 475 nm against a synthetic melanin (Sigma catalog no. 8631) standard curve ranging from 5 to 180 μg / mL. Samples were evaluated in triplicate, and data were normalized for total cell number 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 Adjusting cell density to ensure accurate dose delivery This study describes the determination of the impact of the loading and injection steps on the delivery of viable RPE. Specifically, this example shows that the loading and injection steps result in some loss of viable cells, that this loss can be easily assessed (and may vary with delivery protocol, e.g., depending on the particular injection cannula used), and that increasing cell concentration can compensate for this loss and allow the expected number of cells to be delivered. Furthermore, we show 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 end-product RPE cells at 2000 viable cells / μL to obtain the desired cell density to be injected; (2) the addition of a 1 ml injection syringe (BD (3) Gentle mixing of the RPE cells and BSS-Plus using an 18g blunt fill needle (BD) fitted with a LUER-LOK™. (4) Extrusion of 150 μL of the formulated RPE cells from the filling syringe through the injection cannula.

[0315] Maintenance of RPE cells in Alcon BSS BSS-Plus® on ice has been demonstrated to be stable for more than 4 hours, provided the cells are formulated at a concentration of 1000 cells / µL or greater. Under these conditions, there is no detectable loss in viable cell count. To ensure cell integrity, a precise volume of final RPE cell product is delivered to the operating room at 2000 viable cells / µL. Each tube of RPE cells is accompanied by a second tube containing a precise volume of cold BSS-Plus, which is added to the cells and mixed immediately prior to injection. Pre-aliquoted RPE cells and BSS-Plus are delivered to the OR in sterile microcentrifuge tubes at 2-8°C.

[0316] Study 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] Cells extruded through the MedOne cannula and control cells formulated but not extruded through a cannula were centrifuged, resuspended in RPE growth medium, and seeded at 10,000 cells per well in a gelatin-coated, full-area 96-well plate. For comparison, portions of the same cell preparation were loaded and passed through a Synergetics cannula (39 ga rigid microinjection cannula, angled), processed, and seeded as described above. Four days after seeding, cells were trypsinized and counted. Table 10 below shows the mean cell counts + / - SD for the three cell counts.

[0321] [Table 10]

[0322] Subsequent seeding and growth of cells extruded through either cannula was comparable to control cells not extruded through a cannula.

[0323] Test 2 - Synergetics, Inc. Infusion Cannula, Angled, 39g RPE cells from a lot with properties similar to the intended clinical RPE lot were thawed, processed, and formulated in cold BSS-Plus as described in Example 1. After thawing and formulation, a total of 2.6 million viable cells were recovered. Starting viability was 97%, and the number of cells recovered after thawing and compounding was typical for this lot. Cells were diluted in cold BSS-Plus to a starting concentration of 375 viable cells / μL 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 Synergetics, Inc. 39ga rigid microinjection cannula, angled, was attached to the syringe containing the cells. Gently tapping the syringe plunger administered 150µL of cells through the injection cannula. Total injection time was 2-3 minutes. Cells were collected into a sterile tube and viable cell count assessed. Across a series of eight injections, the average number of viable cells delivered was 238 + / - 25 viable cells / μL, or approximately 100 viable cells less than intended for delivery as the minimum cell dose in this study (50,000 cells per eye).

[0324] Thus, Study 2 demonstrated that loaded RPE cells extruded through a Synergetics cannula resulted in a predictable loss in cell density at the lowest intended cell dose range (loading densities of 375 viable cells / μL tested). The average loss in viable cell density was 38.4 + / - 6.8% (N=8). Loading cell density can be increased accordingly to compensate for the expected loss and thus ensure accurate delivery of the intended number of viable RPE cells (such as 50,000 cells / eye in this study).

[0325] Study 3 - MedOne POLYTIP® Cannula 23 / 38 and Synergetics 39ga Rigid Microinfusion Cannula, Angled Study 3 was performed on the RPE lot used for patient administration in Example 1 above. In this study, RPE cells were loaded at 25% higher dose-to-delivery cell density to compensate for expected losses in loading the syringe and infusing through the MedOne cannula. The same 25% corrected loading density was used to validate the Synergetics cannula.

[0326] When loaded with cells formulated 25% higher than the lowest target dose (444 viable cells / μL vs. 333 cells / μL delivered), the MedOne cannula delivered 336 + / - 40 viable cells / μL. Similarly, when loaded with cells formulated 25% higher than the lowest target dose (1776 viable cells / μL vs. 1333 cells / μL delivered), the MedOne cannula delivered 1433 + / - 187 viable cells / μL. Results of the lowest cell dose injection using the Synergetics cannula confirmed that an additional increase in loaded cell density of 100 viable cells / μL achieved the target dose at lower densities.

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

[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 viable RPE / eye), 311 μL of cold BSS-Plus was dispensed into the cell-containing tube, bringing the final volume to 400 μL at 444 cells / μL. This density was 25% higher than the intended delivery density of 333 cells / μL, and required no mixing with the fill needle, syringe loading, or extrusion through the MedOne cannula. Compensation for anticipated losses that occur during delivery through the

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

[0330] The microcentrifuge tube containing the diluted cells was capped and gently tapped with one finger to promote mixing. A blunt fill needle (90 μL void volume) was attached to a 1 mL BD syringe, and the cells were gently triturated in the blunt fill needle 1-2 times, 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 injection cannula was attached (MedOne 38g or Synergenic 39g). Approximately 150 μL of cells were dispensed into the microcentrifuge tube. Each dispensed aliquot was assessed 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 compensated 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 minimal dose delivery through a MedOne or Synergetics cannula, cells The cells were diluted with RPE growth medium, centrifuged, and seeded at 40,000 cells per well in gelatin-coated, full-area 96-well plates. Non-cannulated control cells, taken from the same tube as the cannulated cells, were treated and seeded similarly. 24 hours after seeding, all cells had attached under all conditions tested, and no floating cells, indicative of cell death or impaired seeding efficiency, were observed.

[0335] Cells extruded through the MedOne cannula, cells extruded through the Synergetic cannula, and control cells formulated but not extruded through a cannula were centrifuged, resuspended in RPE growth medium, and seeded at 40,000 cells per well into gelatin-coated full-area 96-well plates. Three days after seeding, cells were trypsinized and counted. Table 13 below shows the mean cell counts + / - 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 showed typical RPE morphology with actively dividing cells. No differences were observed between the control and cannula-injected cells.

[0336] [Table 13]

[0337] In summary, RPE cells in cold BSS-Plus are more stable at concentrations above 1000 cells / µL, and the final product can be resuspended in cold BSS-Plus at 2000 cells / µL in a capped microcentrifuge tube to load the cannula 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 containing the RPE cells at a precise volume of 2000 viable cells / µL, and one vial containing the precise volume of cold BSS-Plus to be added to bring the cell density to the injection density (i.e., a density that accounts for viable cell loss during loading and extrusion through the cannula, such as 25% higher than the final target dose, taking into account viable cell loss through the MedOne cannula). If concentrations higher than 1,000 cells / µL or higher than 2,000 cells / µL are to be loaded into the cannula, the dilution step may be omitted and cells may instead be delivered to the operating room in cold BSS-Plus at the desired concentration.

[0338] Customized formulation loading densities and corresponding doses for the MedOne cannula are shown in Table 14. Similar customizations are easily specified 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 were used in the studies described in Example 1.

[0341] Embryoid body differentiation medium (EB-DM), composed of Knockout™ DMEM supplemented with Glutamax, non-essential amino acids, 2-mercaptoethanol, and Knockout™ serum replacement, was used from the initiation of embryoid body formation through the time of pigmented spot harvest and isolation, i.e., throughout embryoid body formation, outgrowth, and subsequent pigmented spot formation. Each batch of EB-DM consisted 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) consisted of one part EB-DM (described in the preceding paragraph) and one part DMEM (high glucose), FBS, and Glutamax. This medium was used following RPE cell derivation from pigmented spots in the subsequent growth and maintenance of RPE from passage 0 through passage 2 to the time of harvest of the final bulk product. Each batch of RPE-GM / MM consisted of 100 mL of EB-DM, 90 mL of DMEM high glucose, 10 mL of fetal bovine serum (FBS) (Hyclone), and 1 mL of Glutamax-I.

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

[0344] RPE lots generated using this medium passed all in-process quality tests, including morphological assessment, 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 generated using MDBK-GM and MDBK-MM medium (Sigma-Aldrich), OptiPRO-SFM, or VP-SFM.

[0345] RPE lots were produced using EB-DM (instead of MDBK-GM or OptiPRO-SFM) from the time of embryo body formation until the time of pigmented spot harvest. After pigmented spot harvest and trypsinization, passage 0 RPE cells were subsequently seeded in RPE-GM (EGM-2 medium) (as defined above) and then switched to RPE Growth / Maintenance Medium instead of MDBK-MM or VP-SFM. Alternatively, RPE may be directly seeded into RPE-GM / MM and allowed to grow and differentiate for the duration of the passage. After an appropriate level of differentiation was observed, RPE cells at passage 0 were harvested and split two more times in these media until final harvest and cryopreservation of the bulk product at passage 2.

[0346] The data below summarizes the in-process testing of five sublots of RPE maintained in EB-DM from the time of embryoid body formation until the time of pigmented spot harvest. At this point, pigmented 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 confluence and subsequently switched to RPE Growth / Maintenance Medium to promote differentiation for passages 0, 1, and 2. Lots B3B and B3A were treated similarly, except at passages 1 and 2, respectively, when they were maintained exclusively in RPE-GM / MM for the entire duration of the passage. Thus, all lots were maintained in RPE-GM / MM once confluence was reached until appropriate levels of differentiation were observed. After passage 2, RPE cells were cryopreserved as bulk product. Lots maintained in EGM-2 for the initial growth phase and subsequently switched to RPE-GM / MM, or maintained in RPE growth / maintenance medium for the entire duration of several passages, were similar except for the slightly more rapid growth rate observed in EGM-2 medium. All lots passed morphological assessment at passages 0, 1, and 2; passing criteria included typical epithelial, cobblestone morphology, and moderate pigmentation. RPE marker expression was detected by indirect immunofluorescence using the following primary antibodies (dilutions of approximately 1:100–1:1000, empirically determined 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 (ab3201).

[0347] Secondary antibodies were used at a 1:500 dilution (or other indicated dilution) in 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 for RPE markers was performed to assess purity using PAX6 and MITF; bestrophin and PAX6 combined; and ZO-1 alone. RPE maturation was assessed by measuring the percentage of RPE staining positive for bestrophin. Immunostaining was performed at four time points during RPE cell preparation: (1) RPE were stained for bestrophin, PAX6, and ZO-1 before harvest at passage 1 and seeding passage 2; (2) RPE were stained for bestrophin, PAX6, and ZO-1 before harvest and cryopreservation at passage 2; (3) RPE bulk product was thawed and formulated as described in Example 1 and resuspended in BSS-PLUS at 1,000 viable cells / μL. Cells were then diluted in RPE-GM, centrifuged at 1000 RPM, resuspended, and seeded at 100,000–300,000 cells per well in gelatin-coated 4-well plates and cultured for 1–2 days before staining with MITF and PAX6; (4) RPE bulk product was thawed and formulated as described in Example 1 and resuspended in BSS-PLUS at 1,000 viable cells / μL. Cells were then diluted in RPE-GM, centrifuged at 1000 RPM, resuspended, and seeded in gelatin-coated 4-well plates at 100,000–300,000 cells per well and cultured for 1–2 days before staining with MITF and PAX6; (5) RPE bulk product was thawed and formulated as described in Example 1 and resuspended in BSS-PLUS at 1,000 viable cells / μL. Well plates were seeded at 50,000–200,000 cells per well and maintained until confluent prior to staining, at which point cultures were switched to RPE-MM and maintained until moderate pigmentation and cobblestone morphology were observed, at which point cultures were stained for PAX6, bestrophin, and ZO-1. Briefly, cells were washed 2-3 times with Ca2+- and Mg2+-free PBS (Gibco #14190), fixed with 2% paraformaldehyde for 10 min, washed 2x with PBS, incubated with 0.1% NP-40 substitute solution (Sigma #74388) in PBS for 15 min, washed 2x with PBS, and incubated with blocking solution (10% normal goat serum (Jackson Immunoresearch #005-000-121), 16% paraformaldehyde (freshly prepared or frozen aliquots) (Electron Microscopy Sciences #15710) prepared at a working concentration of 2% in PBS) for 30 min to overnight. Cells were then incubated with primary antibodies (up to two antibodies per well, using primary antibodies from different species) in blocking solution for 1–2 hours at room temperature or overnight at 4°C, washed with PBS, and washed three times with agitation (10–15 minutes each wash) in PBS-Tween solution (Ca2+-, Mg2+-free PBS (Gibco #14190) supplemented with 0.5% Tween 20 (Sigma #P7949)). Samples were then incubated with secondary antibodies and washed in the same manner as for the primary antibodies. After removal of the final wash, one to two drops of Vectashield with DAPI were added, and cells were examined and counted on an inverted fluorescence microscope. Three to six random fields containing a minimum of 1,000 nuclei were photographed at 20x magnification in all channels. Photographs were merged and images 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 the expected staining pattern was observed, such as nuclear localization of PAX6, a polygonal pattern of plasma membrane localization of bestrophin (showing bestrophin staining localized in a distinct line at the cell periphery), ZO-1 staining present in tight junctions outlining the cell in a polygonal pattern, and MITF staining detected in the nucleus. The percentage of cells positive for each marker or marker combination was determined by counting positive cells in the merged image and determining the total number of cells by counting 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 from each lot are shown in Table 16 and demonstrate that RPE genes were upregulated and ES cell genes were downregulated, as expected.

[0351] [Table 16]

[0352] Cryopreserved RPE cells produced using the above-mentioned media formulations (RPE-GM / MM and EB-DM), as well as previously produced using other media (MDBK-GM and MDBK-MM), were tested for their phagocytic ability. For this test, cryopreserved RPE cells were thawed and seeded in RPE growth / maintenance medium. RPE cells from this lot, generated using EB-DM during embryoid body formation and pigmented spot formation and RPE-GM / MM during RPE maturation, were trypsinized and seeded in RPE-GM / MM. Both cultures were grown to confluence and maintained until differentiation in RPE-GM / MM. They were then tested for their ability to phagocytose fluorescent bioparticles (Invitrogen catalog no. P35361), which fluoresce when internalized in the acidic environment of RPE cell phagosomes. Cells were cultured with the fluorescent bioparticles at 37°C to allow phagocytosis or at 4°C as a negative control. Fluorescence intensity shifts indicative of bioparticle phagocytosis were detected by FACS for cells cultured at 37° C. (FIG. 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 demonstrate phagocytosis in a high percentage of cells in both lots of RPE cells maintained on RPE-GM / MM, further demonstrating the suitability of using RPE-GM / MM for RPE cell growth and maturation.

[0355] Example 5 Additional Exemplary Methods of RPE Induction Using the methods of this example, additional hESC lines were generated without destroying embryos. These additional hESC lines were specifically iPS cells (especially iPS cells generated using non-integrating episomal vectors) and NED ("No Embryo Destruction") hES cells generated from biopsied blastomeres from which the embryos from which they were derived were subsequently cryopreserved to maintain viability. NED cells were generated as described by Chung et al. (Cell Stem Cell. 2008 Feb 7;2(2):113-7), the entire contents of which are incorporated by reference.

[0356] hESCs were grown on Matrigel™ diluted according to the manufacturer's instructions for TESR-1 medium (Stem Cell Technologies, Inc.). RPE were generated from embryoid bodies (EBs) or multilayered hESC cultures as previously described (Klimanskaya et al., Cell Stem Cells 6:217-245 (2004), the entire contents of which are incorporated by reference); EBs were cultured in suspension and then seeded for outgrowth prior to RPE harvest. However, EB formation from hESCs cultured on Matrigel™ was observed to be less efficient, with cells exhibiting lower rates of successful aggregation and reduced viability. The following protocol modifications improved EB formation efficiency.

[0357] hESCs were overgrown beyond the time they would normally be passaged, allowing colonies to become "thicker," i.e., slightly raised and / or multi-layered. For EB formation, hESCs were dissociated into single-cell suspensions using mechanical scraping, collagenase I, accutase, collagenase with accutase, or collagenase followed by accutase and an EDTA-based dissociation buffer. These methods allowed hESC colonies to be detached without dissociating into single cells. Trypsin, which tends to readily produce single-cell suspensions under typical conditions, was not utilized.

[0358] The isolated hESCs were then cultured on ultra-low attachment plates to form EBs. Optionally, other methods, such as hanging drop culture, may be used for EB formation. Typically, hESCs from one to three wells of a six-well culture dish were cultured in 2 to 7 ml of medium in one to two wells of a low-attachment plate. 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 2-3 days of culture in EB medium, during EB formation, the EB medium was supplemented with 10 micromolar Stemolecule Y-27632 from Stemgent, a rho-associated protein kinase (ROCK) inhibitor (see Watanabe et al., Nat Biotechnol. 2007 Jun;25(6):681-6, the entire contents of which are incorporated by reference). The use of the ROCK inhibitor improved cell viability, particularly for hESCs obtained using EDTA or enzymatic dissociation. The use of the ROCK inhibitor was optional for mechanically scraped hESCs, which survived well without it.

[0359] Seven to 12 days after EB formation, EBs were plated onto gelatin-coated plates for outgrowth. RPE were easily identified by their epithelial morphology (cobblestone appearance) and pigmentation.

[0360] RPE were also generated from multilayered cultures of hESCs grown on Matrigel™ essentially as previously described (Klimanskaya et al., 2004, supra), except that the cells were cultured on Matrigel™ instead of feeder cells. Briefly, hESCs were overgrown in mTESR-1 medium on Matrigel™ until hESC colonies formed multilayer structures (approximately 10-14 days of culture), at which point the medium was replaced with EB medium (as described above). ROCK inhibitor was optionally added. Although included in the medium, it is not essential for efficient RPE formation and recovery. RPE cells were easily identified by their epithelial morphology (cobblestone 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 multilayered cultures exhibited a "freckled" appearance, containing dark areas visible to the naked eye. Microscopic examination confirmed that these dark areas were composed of RPE cells, identifiable by their characteristic pigmentation and cobblestone epithelial morphology. The resulting RPE cell cultures are shown in Figure 19. Following differentiation from hESCs, RPE cells were isolated by either mechanical or enzymatic dissociation.

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

[0363] Patients were not given any corticosteroids immediately prior to surgery. Surgery was performed under general or local anesthesia with or without awake sedation at the surgeon's discretion.

[0364] 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, vials were removed from the liquid nitrogen freezer and then placed in a 37°C water bath with constant agitation for 1-2 minutes until thawed. The vials were 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. The cell suspension was centrifuged again, and if more than one cryovial was thawed, the pellets were pooled. The volume was brought to a final volume of 10 mL in BSS-PLUS and centrifuged a third time. The supernatant was completely aspirated, and the cells were brought to a final volume of approximately 150 μL of BSS-PLUS per mL of thawed cells (lower volumes can be used if a more concentrated suspension is desired). A sample was removed and a viable cell count was performed. The total viable cell count was determined, and the appropriate volume of BSS-PLUS was added to achieve a target viable cell concentration, such as 2,000 viable cells per μL. The appropriate volume of the formulated product was transferred to a 0.5 mL sterile microcentrifuge tube, and a sample was removed for archiving, viability determination, Gram staining, and sterility testing. A paired 0.5 mL sterile microcentrifuge tube containing the correct volume of BSS-Plus was also prepared and labeled. The paired vial was stored at 2–8°C for no more than 4 hours in preparation for final mixing and implantation in the operating room.

[0365] A standard three-port pars plana vitrectomy was performed on the patient. A small retinotomy was performed, and then a fluid injection system through the vitrectomy device was used to infuse BSS Plus into the subretinal space until a small neurosensory retinal detachment occurred. The surgeon ensured that a bleb was created at the temporal fovea. The bleb optionally extended into the arcade vessels, but did not detach the central macula / fovea. If the bleb was observed to extend toward the central macula, the surgeon had the option to discontinue and perform another retinotomy at that location, following the same rules. The subretinal BSS Plus was then removed.

[0366] The preloaded cannula was then inserted and cells were infused into the created cavity in a volume of 150 μL over approximately 1 minute. Direct visual monitoring was performed to ensure accurate cannula positioning. The exact location of the bleb was recorded by photograph or (preferably) video through an operating microscope to accurately correlate postoperative findings with the location of the bleb. did.

[0367] A suspension containing the desired number of hESC-derived RPE cells (e.g., 50,000, 100,000, 150,000, or 200,000 cells) in 150 μL of BSS Plus was implanted. Cells were infused for approximately 1 minute. The cannula was held in place for an additional minute to avoid reflux. At the surgeon's discretion, air-fluid exchange was optionally performed, for example, if the retinotomy enlarged. The incision was then closed using standard procedures. Patients then recovered from anesthesia while maintained in a supine position for 6 hours.

[0368] Corticosteroids were not permitted for 48 hours following the procedure. If necessary, topical or systemic nonsteroidal anti-inflammatory drugs were permitted to manage postoperative discomfort.

[0369] Example 7 Stability of cryopreserved RPE cell preparations This example demonstrates that cryopreserved RPE cells pass release criteria and remain suitable for use when tested at 6 and 12 months after freezing. Accordingly, cryopreserved RPE cells have been shown to maintain their function and product attributes for 12 months after freezing. 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, or more years).

[0370] Cryopreserved RPE samples were generated as described in Example 4 above, frozen in cryopreservation medium (90% FBS, 10% DMSO) 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, cryopreserved cells were thawed and washed as described in Example 6 (briefly, thawed in a 37°C water bath, the exterior of the container was washed with 70% ethanol, and the cells were washed to remove the cryopreservation medium). After thawing, the cells were tested to confirm product stability. Each release criteria was met, as shown in Table 18 below.

[0371] [Table 18]

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

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

[0374] [Table 19]

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

[0376] In additional experiments, RPE cell final products were formulated at 2,000 viable cells / μL and refrigerated for various periods of time prior to extrusion through a MedOne REF 3233 POLYTIP® cannula 23 / 38. In this study (data shown in Table 20), RPE cell lots (that had passed bulk product release testing for clinical use) were thawed and processed as described above. Cells were resuspended and stored in BSS-Plus at a density of 2,000 viable cells / μL and then kept on ice. A cell density of greater than 1,000 viable cells / μL was selected to promote cell survival during refrigeration in BSS-Plus. At this point, twenty-one 89 μL cell aliquots containing 177,600 total viable cells were dispensed into final product capped microcentrifuge tubes. 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, resulting in a final volume of 400 μL at 444 cells / μL. This density is 25% higher than the intended delivery density of 333 cells / μL and compensates for expected losses during mixing in 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) and 22 μL of cold BSS-Plus was dispensed into the cell-containing tube, resulting in a final volume of 400 μL at 1,776 cells / μL. This density is 25% higher than the intended delivery density of 1,333 cells / μL and compensates for anticipated losses during mixing in 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 (90 μL void volume) was attached to a 1 mL BD syringe, and the cells were gently triturated in the blunt fill needle 1-2 times, 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 38 g injection cannula was attached. Approximately 150 μL of cells were dispensed into the microcentrifuge tube. Each dispensed aliquot was assessed for cell density and viability by trypan blue exclusion. Time after compounding was the time (in minutes) elapsed since resuspension of the cells at 2,000 viable cells / μL in cold BSS-Plus. These data are shown in Table 20 for cells delivered at the indicated concentrations.

[0379] [Table 20]

[0380] The data show that the viable cell count of the final product RPE cells extruded through the infusion cannula does not decrease when refrigerated for the time period tested. The viable cell density observed more than 240 minutes (4 hours) after formulation supports an expiration time of at least 4 hours. In this study, RPE cells in BSS-Plus were stored within the final product seal on ice. A calibration probe was subsequently used to assess the temperature of the BSS-Plus in the microcentrifuge tube stored on ice and measured 3°C.

[0381] Further experiments examined the viability of the prepared RPE cells for up to 6 hours. Viability was assessed at time of preparation (time 0) and after 4 and 6 hours of refrigeration (2-8°C). The RPE lot used in this study was manufactured and cryopreserved using procedures, processes, and materials described for GMP manufacturing. Cryovials of RPE cells were thawed and formulated according to the procedures described in Example 6 above. Cells were assessed for viable cell count at time of preparation (time 0) and after 4 and 6 hours of refrigeration (2-8°C). Cells were seeded and cultured at time 0, 4, and 6 for further subsequent purity and potency assessment. At each seeding time point (time 0, 4, and 6 hours), purity was assessed by MITF and PAX6 immunostaining, and phagocytosis of fluorescent particles was assessed by FACS analysis.

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

[0383] [Table 21]

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

[0385] The initial cell densities at time 0 of 2,590 cells / μL and 1,700 cells / μL are lumped together to form a medical formulation with a target of 2,000 viable cells / μL. No loss in viable cell count was observed over the range of starting cell densities tested at 6 hours of refrigeration.

[0386] Example 9 This example provides initial treatment results for two additional patients with Stargardt's disease. Each patient was treated with 50,000 RPE cells derived from hESC sources (as described in Example 1) using the RPE transplantation method described in Example 6 above. Fundus photographs, including the retina, optic 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 RPE cells ( FIG. 15 ).

[0387] Further fundus photographs show the establishment of areas within the injected bleb with increasing pigmented RPE cell patches in two SMD patients (Figures 16 and 17). These results suggest engraftment and resurfacing of retinal areas with a new RPE layer.

[0388] Visual acuity was also assessed for the treated eyes of the patients shown in Figure 16. The vertical axis represents the Early Treatment Diabetic Retinopathy Study (ETDRS) score, and the horizontal axis represents postoperative days.

[0389] These results demonstrate stable engraftment of RPE cells that persists for at least 3 months after treatment. Visual acuity in the treated eyes returned to baseline levels 14 days after treatment and remained above baseline until day 84, the final time point shown.

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

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

[0392] For the AMD patient, the peripheral ERTDS-BVCA scores are graphically depicted in Figure 21. The patient's peripheral ERTDS-BVCA score decreased from an initial baseline value of 21 to 0 1 and 3 days after surgery, but returned to at least baseline levels 7 days after surgery and remained above baseline thereafter. One year after treatment, the patient's peripheral ERTDS-BVCA score was 34.

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

[0394] These results demonstrate improved visual acuity 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 treatment of exudative macular degeneration:a 3-year follow-up.Arch Ophthalmol 2006;124,183-188. 18.Thumann G,Aisenbrey S,Schraermeyer U,et al.Transplantation of autologous iris pigment epithelium after removal of choroidal neovascular membranes.Arch Ophthalmol 2000;118,1350-1355. 19.Berger AS,Tezel TH,Del Priore LV,Kaplan HJ.Photoreceptor transplantation in retinitis pigmentosa:short-term follow-up.Ophthalmology 2003;110,383-391. 20.Drukker M,Katchman H,Katz G,et al.Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells Stem Cells 2006;24,221-229. 21.Okamura RM,Lebkowski J,Au M,Priest CA,Denham J,Majumdar AS.Immunological properties of human embryonic stem cell-derived oligodendrocyte progenitor cells.J Neuroimmunol 2007;192,134-144. 22.Klimanskaya I,Chung Y,Becker S,et al.Human embryonic stem cell lines derived from single blastomeres.Nature 2006;444,481-485. 23.Tezel TH,Del Priore LV.Serum-free media for culturing and serially-passaging of adult human retinal pigment epithelium.Exp Eye Res 1998;66,807-815. 24.Lu F,Zhou X,Hu DN,et al.Expression of melanin-related genes in cultured adult retinal pigment epithelium and uveal melanoma cells.Mol Vis 2007;13,2066-2072. 25.Tezel TH,Del Priore LV,Berger AS,et al.Adult retinal pigment epithelial transplantation in exudative age-related macu lar degeneration.Am J.Ophthalmol 2007;142,584-595. 26.Song MK,Lui GM.Propagation of fetal human RPE cells:preservation of original culture morphology after serial passage.J Cell Physiol 1990;143,196-203. 27.Gamm DM,Wright LS,Capowski EE,et al.Regulation of prenatal human retinal neurosphere growth and cell fate potential by retinal pigment epithelium and Mash1.Stem Cells 2008;26,3182-3193. 28.Maminishkis A,Chen S,Jalickee S,et al.Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.Invest Ophthalmol Vis Sci.2006 ;47,3612-3624.

[0396] All publications, patents, and patent applications are herein incorporated by reference in their entireties as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. U.S. Provisional Patent Application No. 60 / 998,766, filed October 12, 2007; U.S. Provisional Patent Application No. 60 / 998,668, filed October 12, 2007; U.S. Provisional Patent Application No. 61 / 009,908, filed January 2, 2008; and U.S. Provisional Patent Application No. 61 / 009,911, filed January 2, 2008, each of which is incorporated by reference in its entirety. Additionally, the disclosure of International Publication No. WO 2009 / 051671 is incorporated by reference in its entirety.

[0397] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. a plurality of retinal pigment epithelial (RPE) cells; a pharmaceutically acceptable carrier and 10. A pharmaceutical composition comprising: a) a ...

2. The pharmaceutical composition of claim 1 , wherein the RPE cells are contained in a suspension, gel, colloid, substrate, matrix, scaffold, or graft.

3. 3. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier comprises a sterile solution having an osmolality of about 290 mOsm / kg to about 320 mOsm / kg, or about 300 mOsm / kg to 310 mOsm / kg, or about 305 mOsm / kg.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutically acceptable carrier comprises a balanced salt solution.

5. 5. The pharmaceutical composition of claim 4, wherein the balanced salt solution comprises, consists of, or consists essentially of 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), and hydrochloric acid and / or sodium hydroxide (to adjust the pH to about 7.4) in 1 mL of water.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the volume of the pharmaceutical composition is about 100 μL to 1000 μL, or at least about 150 μL.

7. The pharmaceutical composition comprises from about 1,000 to about 1 x 10 ９ The pharmaceutical composition of any one of claims 1 to 6, comprising live RPE cells.

8. 8. The pharmaceutical composition of any one of claims 1-7, wherein the pharmaceutical composition comprises about 333 viable RPE cells / μL to about 2,000 viable RPE cells / μL, 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.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the concentration of RPE cells in the pharmaceutical composition is sufficiently high such that no more than about 30% of the RPE cells lose viability within 60 minutes, and optionally no more than about 10% of the RPE cells lose viability within 4 hours.

10. 10. The pharmaceutical composition of claim 9, wherein the RPE cell concentration is 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.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the medicament comprises 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.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the mean 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.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein at least 50%, at least 60%, at least 70%, or at least 80% of the cells in the pharmaceutical composition are bestrophin+.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition are PAX6+ and / or MITF+.

15. 15. The pharmaceutical composition of any one of claims 1-14, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition are PAX6+ and / or bestrophin+.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition are ZO-1+.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein at least 50%, at least 60%, or at least 70% of the cells in the pharmaceutical composition are PAX6+ and bestrophin+.

18. 18. The pharmaceutical composition of any one of claims 1-17, wherein at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition are PAX6+.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein no more than about 1 cell per million cells, and optionally no more than about 2 cells per 9 million cells in the pharmaceutical composition are positive for both OCT-4 and alkaline phosphatase (AP) expression.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein a needle or injection cannula contains at least some of the RPE cells.

21. 21. The pharmaceutical composition of claim 20, wherein the concentration of the RPE cells is from about 400 viable cells / μL to about 2,000 viable cells / μL when loaded into the needle or infusion cannula.

22. 22. The pharmaceutical composition of claim 20 or 21, wherein the concentration of viable RPE cells delivered from the needle or injection cannula is from about 333 viable cells / μL to about 1,333 viable cells / μL or from about 444 viable cells / μL to about 1,766 viable cells / μL.

23. 23. The pharmaceutical composition of any one of claims 20 to 22, wherein the needle or injection cannula diameter is from about 0.3 mm to about 0.9 mm.

24. The needle or injection cannula diameter is about 0.5 mm to about 0.6 mm.

23. The pharmaceutical composition according to any one of claims 22.

25. 25. The pharmaceutical composition of any one of claims 20 to 24, wherein the needle or injection cannula comprises a tip having a diameter of about 0.09 mm to about 0.15 mm.

26. 26. The pharmaceutical composition of any one of claims 20 to 25, wherein the cannula is a MEDONE POLYTIP® cannula 25 / 38g (0.50mm (25g) x 28mm cannula with a 0.12mm (38g) x 5mm tip) or a Synergetics Angled 39g infusion cannula.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the RPE cells comprise cryopreserved and thawed RPE cells.

28. The pharmaceutical composition of any one of claims 1 to 27, wherein the RPE cells are human cells.

29. 29. The pharmaceutical composition of any one of claims 1 to 28, further comprising at least one angiogenesis inhibitor, administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with said RPE cells.

30. and wherein the one or more angiogenesis inhibitors are selected from the group consisting of pepgatanib sodium; aflibercept; bevasiranib; rapamycin; AGN-745; vitalanib; pazopanib; NT-502; NT-503; PLG101; CPD791; an anti-VEGF antibody or 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 VEGFR; 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 (Nexa BARL); SU5416 (semaxinib); SU11248 / sunitinib (Sutent); vandetanib (ZD6474); Ly317615 (enzastaurin); anti-α5β1 integrin antibody or functional fragment thereof; volociximab; 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 ＊ (Arg-Cys-Asp-thioproline-Cys (asterisk indicates cyclization by a disulfide bond through the cysteine ​​residue); 2-methoxyestradiol; αVβ3 inhibitors; angiopoietin 2; antiangiogenic steroids and heparin; angiostatin; angiostatin-related molecules; anti-cathepsin S antibodies; antithrombin III fragments; 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; matocrit; Metalloproteinase inhibitors; 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 antagonists; VEGI; Volociximab (M200); Fibronectin fragment or domain; Anastellin; Lenalidomide 30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition is selected from the group consisting of: vatinib (E7080); motesanib (AMG706); pazopanib (Votrient); inhibitors of VEGF; inhibitors of VEGFR1; inhibitors of VEGFR2; inhibitors of VEGFR2; inhibitors of α5β1 integrin; peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitors of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; IL-6 antagonists; anti-IL-6 antibodies; and any combination thereof, optionally in an amount sufficient to prevent or treat a proliferative (neovascular) eye disease.

31. The pharmaceutical composition of any one of claims 1 to 30, wherein the RPE cells are genetically modified.

32. The pharmaceutical composition of any one of claims 1 to 31, wherein the RPE cells are generated from pluripotent cells.

33. The pharmaceutical composition of any one of claims 1 to 32, wherein the RPE cells are generated from pluripotent cells that have been genetically modified.

34. 34. The pharmaceutical composition of claim 31 or 33, wherein the genetic manipulation results in the production by the RPE cells of one or more factors that inhibit angiogenesis.

35. 35. The pharmaceutical composition of claim 34, wherein the one or more factors that inhibit angiogenesis comprise at least one factor selected from the group consisting of a fibronectin fragment or domain; anasterin; a specific anti-VEGF antibody or a functional fragment or domain thereof; a specific anti-VEGF receptor antibody or a functional fragment or domain thereof; a specific anti-α5β1 integrin antibody or a functional fragment or domain thereof; a VEGF fragment or domain; a VEGFR receptor fragment or domain; VEGF-Trap; and any combination thereof.

36. The pharmaceutical composition according to any one of claims 34 to 35, wherein the production of the factor that inhibits angiogenesis is regulated by an RPE-specific promoter.

37. 37. The pharmaceutical composition of claim 36, wherein the RPE-specific promoter is selected from the group consisting of the RPE65 promoter, the cathepsin D proximal promoter, and the VMD2 promoter.

38. 38. The pharmaceutical composition of any one of claims 32 to 37, wherein the pluripotent stem cells are positive for one or more markers comprising OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

39. 39. The pharmaceutical composition of any one of claims 32 to 38, wherein the pluripotent cells are human pluripotent cells cultured in multilayered populations or embryoid bodies for a sufficient time for pigmented epithelial cells to emerge in culture.

40. 40. The pharmaceutical composition of claim 39, wherein the time sufficient for pigmented epithelial cells to appear in the culture comprises 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.

41. 39. The multilayered population or embryoid bodies are cultured in a medium comprising DMEM. or 40. A pharmaceutical composition according to any one of claims 1 to 40.

42. 42. The pharmaceutical composition of claim 41, wherein the medium comprises, consists essentially of, or consists of EB-DM.

43. 43. The pharmaceutical composition of any one of claims 39 to 42, wherein the pigmented epithelial cells are isolated and cultured, thereby generating a population of RPE cells.

44. 44. The pharmaceutical composition of claim 43, wherein the isolating step comprises enzymatically, chemically, or physically separating cells or cell clusters from the culture to select pigmented epithelial cells or cell clusters comprising pigmented epithelial cells.

45. The pharmaceutical composition of any one of claims 39 to 44, wherein the embryoid bodies are cultured in suspension.

46. The pharmaceutical composition of any one of claims 39 to 45, wherein the embryoid bodies are cultured as adherent cultures.

47. 47. The pharmaceutical composition of claim 46, wherein the embryoid bodies cultured as adherent cultures give rise to one or more outgrowths comprising pigmented epithelial cells.

48. The pharmaceutical composition of any one of claims 32 to 47, wherein the pluripotent stem cells have a reduced HLA antigen complexity.

49. The pharmaceutical composition of any one of claims 32 to 48, wherein the pluripotent cells are cultured on a substrate prior to RPE generation.

50. 50. The pharmaceutical composition of claim 49, wherein the substrate is selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof.

51. 50. The pharmaceutical composition of claim 49, wherein the substrate comprises Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

52. 52. The pharmaceutical composition of any one of claims 1 to 51, comprising cells that substantially lack one or more embryonic stem cell markers.

53. 53. The pharmaceutical composition of claim 52, wherein the one or more embryonic stem cell markers comprise OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

54. 54. The pharmaceutical composition of any one of claims 1 to 53, wherein the RPE cells are positive for one or more RPE cell markers.

55. the one or more RPE cell markers are RPE65, CRALBP, PEDF, 55. The pharmaceutical composition of claim 54, comprising bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and / or tyrosinase.

56. 56. The pharmaceutical composition of any one of claims 1 to 55, wherein the RPE cells are produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to achieve said average melanin content.

57. 57. The pharmaceutical composition of any one of claims 1 to 56, wherein the RPE cells are produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to establish bestrophin expression in at least 50% of the RPE cells.

58. 58. The pharmaceutical composition of any one of claims 1 to 57, wherein the pharmaceutical composition is substantially free of mouse embryonic feeder cells (MEFs) and human embryonic stem cells (hES).

59. 59. The pharmaceutical composition of any one of claims 1 to 58, wherein the RPE cells are produced by a method comprising culturing the RPE cells under conditions that increase expression of one or more alpha integrin subunits.

60. 60. The pharmaceutical composition of claim 59, wherein the one or more alpha integrin subunits comprise one or more of alpha integrin subunit 1, alpha integrin subunit 2, alpha integrin subunit 3, alpha integrin subunit 4, alpha integrin subunit 5, alpha integrin subunit 6, or alpha integrin subunit 9.

61. 61. The pharmaceutical composition of any one of claims 59 or 60, wherein the conditions comprise exposure to manganese, exposure to an anti-CD29 antibody, exposure to monoclonal antibody HUTS-21, exposure to monoclonal antibody mAb TS2 / 16, and / or at least about four passages of the RPE cells.

62. 62. The pharmaceutical composition of any one of claims 1 to 61, wherein the RPE cells meet at least one of the criteria listed in Table 5.

63. 63. The pharmaceutical composition of any one of claims 1 to 62, wherein the RPE cells are manufactured in accordance with Good Manufacturing Practice (GMP).

64. 64. The pharmaceutical composition of any one of claims 1 to 63, further comprising at least one immunosuppressive or immunotolerizing agent, administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

65. 65. The pharmaceutical composition of claim 64, wherein the immunosuppressive or tolerizing agent comprises one or more of mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporin A), daclizumab® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil.

66. A kit comprising the pharmaceutical composition of any one of claims 1 to 65 and a separate container containing a volume of a pharmaceutically acceptable diluent sufficient to dilute the plurality of RPE cells to a desired target concentration.

67. 67. The kit of claim 66, wherein the volume of the pharmaceutically acceptable diluent is such that when the entire volume of the pharmaceutically acceptable diluent is combined with the entire plurality of RPE cells, it results in the plurality of RPE cells having the desired target concentration.

68. 68. The kit of any one of claims 66 to 67, wherein the temperature of the pharmaceutically acceptable diluent is about 0 to 10°C, optionally about 2 to 8°C.

69. 69. The kit of any one of claims 66 to 68, wherein the temperature of the plurality of RPE cells or the pharmaceutically acceptable carrier containing the plurality of RPE cells is about 0 to 10°C, optionally about 2 to 8°C.

70. 70. The kit of any one of claims 66 to 69, further comprising at least one immunosuppressive or immunotolerizing agent to be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

71. 71. The kit of claim 70, wherein the immunosuppressive or tolerizing agent comprises one or more of mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporin A), daclizumab® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil.

72. 72. The kit of any one of claims 66 to 71, further comprising one or more angiogenesis inhibitors to be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

73. and wherein the one or more angiogenesis inhibitors are selected from the group consisting of pepgatanib sodium; aflibercept; bevasiranib; rapamycin; AGN-745; vitalanib; pazopanib; NT-502; NT-503; PLG101; CPD791; an anti-VEGF antibody or 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 VEGFR; 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 (Nexa BARL); SU5416 (semaxinib); SU11248 / sunitinib (Sutent); vandetanib (ZD6474); Ly317615 (enzastaurin); anti-α5β1 integrin antibody or functional fragment thereof; volociximab; 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 ＊ (Arg-Cys-Asp-thioproline-Cys (asterisk indicates cyclization by a disulfide bond through the cysteine ​​residue); 2-methoxyestradiol; αVβ3 inhibitors; angiopoietin 2; antiangiogenic steroids and heparin; angiostatin; angiostatin-related molecules; anti-cathepsin S antibodies; antithrombin III fragments; 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); restin; soluble NRP-1; soluble VEGFR-1; SPARC; SU5416; suramin; tecogalan; tetrathiomolybdate; thalidomide; lenalidomide; thrombospondin; TIMP; TNP-470; TSP-1; TSP-2; vasostatin; VEGFR antagonists; VEGI; volociximab (M200); phi 73. The kit of claim 72, wherein the compound is selected from the group consisting of: bronectin fragments or domains; anasterin; lenvatinib (E7080); motesanib (AMG706); pazopanib (Votrient); inhibitors of VEGF; inhibitors of VEGFR1; inhibitors of VEGFR2; inhibitors of VEGFR2; inhibitors of α5β1 integrin; peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitors of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; IL-6 antagonists; anti-IL-6 antibodies; and any combination thereof, optionally in an amount sufficient to prevent or treat a proliferative (neovascular) eye disease.

74. A cryopreserved 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 recovered following thawing have a seeding efficiency of at least about 60%.

75. 75. The cryopreserved composition of claim 74, wherein the seeding efficiency is at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

76. 76. The cryopreserved composition of claim 74 or 75, wherein said average maturation level is determined by measuring the average melanin content of a cell population representative of said plurality of cryopreserved RPE cells.

77. 77. The cryopreserved composition of any one of claims 74 to 76, wherein the average melanin content of the plurality of cryopreserved RPE cells is less than 8 pg / cell.

78. A cryopreserved composition comprising a plurality of cryopreserved retinal pigment epithelial (RPE) cells; wherein the plurality of cryopreserved RPE cells have an average melanin content of less than 8 pg / cell.

79. 79. The cryopreservation composition of any one of claims 74 to 78, wherein the cells are contained in a cryopreservation medium.

80. 80. The cryopreservation composition of claim 79, wherein the cryopreservation medium comprises one or more of DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), propylene glycol, and sucrose.

81. 81. The cryopreservation composition of any one of claims 79-80, wherein the cryopreservation medium comprises about 5% to about 50% DMSO and about 30% to about 95% serum, optionally wherein the serum is fetal bovine serum (FBS).

82. 80. The cryopreservation composition of claim 79, wherein the cryopreservation medium comprises about 90% FBS and about 10% DMSO.

83. 83. The cryopreservation composition of any one of claims 78 to 82, wherein the RPE cells recovered following thawing have a seeding efficiency of at least about 60%.

84. 84. The cryopreserved composition of claim 83, wherein the seeding efficiency is at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

85. The cryopreserved composition has a density of about 5,000 to about 1 x 10 at the time of freezing. ８ 85. The cryopreservation composition of any one of claims 74 to 84, comprising live RPE cells.

86. 86. The cryopreservation composition of any one of claims 74 to 85, wherein the cryopreservation composition comprises 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 viable RPE cells at the time of freezing.

87. 87. The cryopreservation composition of any one of claims 74 to 86, wherein the RPE cells recovered following thawing 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 least about 3, 6, 9, or 12 months after freezing.

88. 88. The cryopreservation composition of any one of claims 74-87, wherein at least 85% of the cells that are viable upon thawing remain viable upon storage 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.

89. 89. The cryopreservation composition of any one of claims 74-88, wherein the cryopreservation composition comprises 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.

90. 90. The cryopreservation composition of any one of claims 74-89, wherein the RPE cells have an average melanin content of 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.

91. 91. The cryopreservation composition of any one of claims 74 to 90, wherein at least 50%, at least 60%, at least 70%, or at least 80% of the cells in the cryopreservation composition are bestrophin+.

92. 92. The cryopreservation composition of any one of claims 74-91, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition are PAX6+ and / or MITF+.

93. 93. The cryopreservation composition of any one of claims 74-92, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition are PAX6+ and / or bestrophin+.

94. 93. Claims 74-93, wherein at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition are ZO-1+. The cryopreservation composition according to any one of the preceding claims.

95. 95. The cryopreserved cryopreservation composition of any one of claims 74 to 94, wherein at least 50%, at least 60%, or at least 70% of the cells in the cryopreservation composition are PAX6+ and bestrophin+.

96. 96. The cryopreservation composition of any one of claims 74-95, wherein at least 90%, at least 95%, or at least 99% of the cells in the cryopreservation composition are PAX6+.

97. 97. The cryopreservation composition of any one of claims 74-96, wherein no more than about 1 cell per million cells, and optionally no more than about 2 cells per 9 million cells in the cryopreservation composition are positive for both OCT-4 and alkaline phosphatase (AP) expression.

98. 98. The cryopreservation composition of any one of claims 74 to 97, further comprising at least one angiogenesis inhibitor, administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

99. and wherein the one or more angiogenesis inhibitors are selected from the group consisting of pepgatanib sodium; aflibercept; bevasiranib; rapamycin; AGN-745; vitalanib; pazopanib; NT-502; NT-503; PLG101; CPD791; an anti-VEGF antibody or 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 VEGFR; 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 (Nexa BARL); SU5416 (semaxinib); SU11248 / sunitinib (Sutent); vandetanib (ZD6474); Ly317615 (enzastaurin); anti-α5β1 integrin antibody or functional fragment thereof; volociximab; 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 ＊ (Arg-Cys-Asp-thioproline-Cys (asterisk indicates cyclization by a disulfide bond through a cysteine ​​residue); 2-methoxyestradiol; αVβ3 inhibitors; angiopoietin 2; antiangiogenic steroids and heparin; angiostatin; angiostatin-related molecules; anti-cathepsin S antibodies; antithrombin III fragments; 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; Me Th-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; Volociximab (M200); Fibronectin fragment or domain; Anastellin; Lenvatinib (E7080); Motesanib (AMG706); Pazopanib (Votrient) 99. The cryopreserved composition of claim 98, wherein the cryopreserved composition is selected from the group consisting of: an inhibitor of VEGF; an inhibitor of VEGFR1; an inhibitor of VEGFR2; an inhibitor of VEGFR2; an inhibitor of α5β1 integrin; a peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitor of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; an IL-6 antagonist; an anti-IL-6 antibody; and any combination thereof, optionally in an amount sufficient to prevent or treat a proliferative (neovascular) eye disease.

100. 100. The cryopreservation composition of any one of claims 74 to 99, wherein the RPE cells are genetically modified.

101. The cryopreservation composition of any one of claims 74 to 100, wherein the RPE cells are generated from pluripotent cells.

102. 102. The cryopreservation composition of any one of claims 74 to 101, wherein the RPE cells are generated from pluripotent cells that have been genetically modified.

103. 103. The cryopreservation composition of claim 101 or 102, wherein the genetic manipulation results in the production by the RPE cells of one or more factors that inhibit angiogenesis.

104. The cryopreserved composition of claim 103, wherein the one or more factors that inhibit angiogenesis comprise at least one factor selected from the group consisting of a fibronectin fragment or domain; anasterin; a specific anti-VEGF antibody or a functional fragment or domain thereof; a specific anti-VEGF receptor antibody or a functional fragment or domain thereof; a specific anti-α5β1 integrin antibody or a functional fragment or domain thereof; a VEGF fragment or domain; a VEGFR receptor fragment or domain; VEGF-Trap; and any combination thereof.

105. A cryopreserved composition according to any one of claims 103 to 104, wherein the production of said factor that inhibits angiogenesis is regulated by an RPE-specific promoter.

106. The cryopreservation composition of claim 105, wherein the RPE-specific promoter is selected from the group consisting of the RPE65 promoter, the cathepsin D proximal promoter, and the VMD2 promoter.

107. 107. The cryopreservation composition of any one of claims 101-106, wherein the pluripotent stem cells are positive for one or more markers comprising OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

108. 108. The cryopreservation composition of any one of claims 101-107, wherein the pluripotent cells are human pluripotent cells cultured in multilayered populations or embryoid bodies for a sufficient time for pigmented epithelial cells to emerge in culture.

109. The cryopreservation composition of claim 108, wherein the time sufficient for pigmented epithelial cells to appear in the culture comprises 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.

110. 110. The cryopreservation composition of any one of claims 108 or 109, wherein the multilayered population or embryoid bodies are cultured in a medium comprising DMEM.

111. 111. The cryopreservation composition of claim 110, wherein the medium comprises, consists essentially of, or consists of EB-DM.

112. 112. The cryopreserved composition of any one of claims 108 to 111, wherein the pigmented epithelial cells are isolated and cultured, thereby resulting in a population of RPE cells.

113. The cryopreservation composition of claim 112, wherein the isolating step comprises enzymatically, chemically, or physically separating cells or cell clusters from the culture to select pigmented epithelial cells or cell clusters comprising pigmented epithelial cells.

114. 114. The cryopreservation composition of any one of claims 108 to 113, wherein the embryoid bodies are cultured in suspension.

115. 115. The cryopreservation composition of any one of claims 108 to 114, wherein the embryoid bodies are cultured as adherent cultures.

116. 116. The cryopreservation composition of claim 115, wherein the embryoid bodies cultured as adherent cultures give rise to one or more outgrowths comprising pigmented epithelial cells.

117. 117. The cryopreservation composition of any one of claims 108 to 116, wherein the pluripotent stem cells have reduced HLA antigen complexity.

118. 118. The cryopreservation composition of any one of claims 108 to 117, wherein the pluripotent cells are cultured on a substrate prior to RPE production.

119. 119. The cryopreservation composition of claim 118, wherein the substrate is selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof.

120. 119. The cryopreservation composition of claim 118, wherein the substrate comprises Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

121. 121. The cryopreservation composition of any one of claims 74 to 120, comprising cells that lack substantial expression of one or more embryonic stem cell markers.

122. 122. The composition of claim 121, wherein the one or more embryonic stem cell markers comprise OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-80.

123. The composition of any one of claims 74 to 122, wherein the RPE cells are positive for one or more RPE cell markers.

124. the one or more RPE cell markers are RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and / or 124. The composition of claim 123, comprising tyrosinase.

125. 125. The composition of any one of claims 74 to 124, wherein the RPE cells are produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to achieve said average melanin content.

126. 126. The composition of any one of claims 74 to 125, wherein the RPE cells are produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to establish bestrophin expression in at least 50% of the RPE cells.

127. 127. The composition of any one of claims 74-126, wherein the composition is substantially free of mouse embryonic feeder cells (MEFs) and human embryonic stem cells (hES).

128. 128. The composition of any one of claims 74 to 127, wherein the RPE cells are produced by a method comprising culturing the RPE cells under conditions that increase expression of one or more alpha integrin subunits.

129. The composition of claim 128, wherein the one or more alpha integrin subunits comprise one or more of alpha integrin subunit 1, alpha integrin subunit 2, alpha integrin subunit 3, alpha integrin subunit 4, alpha integrin subunit 5, alpha integrin subunit 6, or alpha integrin subunit 9.

130. 130. The composition of any one of claims 128 or 129, wherein the conditions comprise exposure to manganese, exposure to an anti-CD29 antibody, exposure to monoclonal antibody HUTS-21, exposure to monoclonal antibody mAb TS2 / 16, and / or at least about four passages of the RPE cells.

131. 131. The composition of any one of claims 74 to 130, wherein the RPE cells meet at least one of the criteria listed in Table 5.

132. The composition of any one of claims 74 to 131, wherein the RPE cells are manufactured in accordance with Good Manufacturing Practice (GMP).

133. 133. The composition of any one of claims 74 to 132, further comprising at least one immunosuppressive or immunotolerizing agent, administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

134. 134. The composition of claim 133, wherein the immunosuppressive or tolerizing agent comprises one or more of mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporin A), daclizumab® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil.

135. A kit comprising the composition of any one of claims 74 to 134 and a separate container comprising a volume of a pharmaceutically acceptable diluent sufficient to dilute the plurality of RPE cells to a desired target concentration.

136. The kit of claim 135, wherein the volume of the pharmaceutically acceptable diluent is such that when the entire volume of the pharmaceutically acceptable diluent is combined with the entire plurality of RPE cells, it results in the plurality of RPE cells having the desired target concentration.

137. 137. The kit of any one of claims 135 to 136, further comprising at least one immunosuppressive or immunotolerizing agent to be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

138. 138. The kit of claim 137, wherein the immunosuppressive or tolerizing agent comprises one or more of mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, basiliximab® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporin A), daclizumab® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, rituximab® (anti-CD20 antibody), sirolimus, tacrolimus, and mycophenolate mofetil.

139. 139. The kit of any one of claims 135 to 138, further comprising one or more angiogenesis inhibitors to be administered to a subject in need thereof prior to, simultaneously with, subsequently to, and / or together with the RPE cells.

140. and wherein the one or more angiogenesis inhibitors are selected from the group consisting of pepgatanib sodium; aflibercept; bevasiranib; rapamycin; AGN-745; vitalanib; pazopanib; NT-502; NT-503; PLG101; CPD791; an anti-VEGF antibody or 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 VEGFR; 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 (Nexa BARL); SU5416 (semaxinib); SU11248 / sunitinib (Sutent); vandetanib (ZD6474); Ly317615 (enzastaurin); anti-α5β1 integrin antibody or functional fragment thereof; volociximab; 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 ＊ (Arg-Cys-Asp-thioproline-Cys (asterisk indicates cyclization by a disulfide bond through the cysteine ​​residue); 2-methoxyestradiol; αVβ3 inhibitors; angiopoietin 2; antiangiogenic steroids and heparin; angiostatin; angiostatin-related molecules; anti-cathepsin S antibodies; antithrombin III fragments; 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; matocrit; Metalloproteinase inhibitors; 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 antagonists; VEGI; Volociximab (M200); Fibronectin fragment or domain; Anastellin; Lenalidomide 140. The kit of claim 139, wherein the inhibitor is selected from the group consisting of: vatinib (E7080); motesanib (AMG706); pazopanib (Votrient); an inhibitor of VEGF; an inhibitor of VEGFR1; an inhibitor of VEGFR2; an inhibitor of VEGFR2; an inhibitor of α5β1 integrin; a peptide, peptidomimetic, small molecule, chemical, and / or nucleic acid inhibitor of VEGF, VEGFR1, VEGFR2, VEGFR3, and / or α5β1 integrin; an IL-6 antagonist; an anti-IL-6 antibody; and any combination thereof, optionally in an amount sufficient to prevent or treat a proliferative (neovascular) eye disease.

141. (a) culturing RPE cells under adherent conditions to form a substantially monolayer culture of pigmented RPE cells having a cobblestone morphology; (b) selecting and isolating RPE cells from the culture for cryopreservation or pharmaceutical formulation; 1. A method of producing retinal pigment epithelial (RPE) cells for use in a pharmaceutical product, comprising:

142. For cryopreservation or formulation, at least 10 ６ 142. The method of claim 141, wherein RPE cells are isolated.

143. 143. The method of claim 141 or 142, wherein the RPE cells are generated from pluripotent stem cells, which are optionally human embryonic stem cells or human iPS cells.

144. 144. The method of any one of claims 141 to 143, wherein the average melanin content is determined for a population of cells excluding the most pigmented and least pigmented 5 percent of isolated RPE cells.

145. 156. The method of any one of claims 141 to 155, wherein the average melanin content 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.

146. (a) culturing RPE cells under adherent conditions to form a substantially monolayer culture of pigmented RPE cells having a cobblestone morphology; (b) passage the RPE cells at least once before the RPE cells reach an average melanin content of greater than 8 pg / cell; (c) optionally, after one or more passages, harvesting the RPE cells for cryopreservation or pharmaceutical formulation; 1. A method for producing retinal pigment epithelial (RPE) cells for use in a pharmaceutical product, comprising:

147. (a) culturing pluripotent stem cells, optionally human embryonic stem cells or human iPS cells, to form embryoid bodies (EBs), or culturing pluripotent stem cells to form a multi-layered population; (b) culturing the multilayered cell population or EBs for a time sufficient for the emergence of pigmented cells comprising a brown pigment dispersed in the cytoplasm; (c) isolating and culturing the pigmented cells of (b) to produce a culture population containing RPE cells having an average pigment level of less than 8 pg / cell; 1. A method for producing retinal pigment epithelial (RPE) cells, comprising:

148. 148. The method of claim 147, wherein step (b) comprises culturing the embryoid bodies to form adherent cultures.

149. 148. The method of claim 147, wherein step (a) comprises overgrowing the pluripotent cell culture, thereby forming a multilayered population.

150. 150. The method of any one of claims 147 to 149, wherein step (a) comprises culturing the pluripotent cells on a low-adhesion substrate or culturing the pluripotent cells using a hanging drop method, thereby forming embryoid bodies from the pluripotent cells.

151. 151. The method of any one of claims 147 to 150, wherein the pluripotent stem cells are 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.

152. The method of any one of claims 147 to 150, wherein the pluripotent stem cells are human ES cells or human iPS cells.

153. 153. The method of any one of claims 147 to 152, wherein the pluripotent stem cells are genetically modified.

154. 154. The method of claim 153, wherein the genetic manipulation results in the production by the RPE cells of a factor that inhibits angiogenesis.

155. The method of claim 154, wherein the one or more factors that inhibit angiogenesis comprise at least one factor selected from the group consisting of a fibronectin fragment or domain; anasterin; a specific anti-VEGF antibody or a functional fragment or domain thereof; a specific anti-VEGF receptor antibody or a functional fragment or domain thereof; a specific anti-α5β1 integrin antibody or a functional fragment or domain thereof; a VEGF fragment or domain; a VEGFR receptor fragment or domain; VEGF-Trap; and any combination thereof.

156. 156. The method of any one of claims 147 to 155, wherein the medium in which the embryoid bodies are formed in step (a) and / or in which the pigmented cells are cultured in step (c) comprises DMEM.

157. 156. The method of claims 147-155, wherein the medium in which the embryoid bodies are formed in step (a) and / or in which the pigmented cells are cultured in step (c) comprises, consists essentially of, or consists of EB-DM.

158. 157. The method of any one of claims 147-156, wherein the medium in which the pigmented cells are cultured in step (c) comprises EB-DM.

159. 156. The method of claims 147-155, wherein the medium in which the pigmented epithelial cells are cultured in step (c) comprises, consists essentially of, or consists of RPE-GM / MM.

160. 147 - Claim 147, wherein the culturing period in step (b) is 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.

159. The method of any one of claims 159 to 159.

161. 161. The method of any one of claims 147-160, wherein the medium used in step (a), (b), or (c) is EB-DM, RPE-GM / MM, MDBK-GM, OptiPro SFM, VP-SFM, EGM-2, or MDBK-MM.

162. 162. The method of any one of claims 147-161, wherein step (c) comprises 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 comprises mechanically disrupting or isolating the culture, or comprises contacting the culture with EDTA or EGTA, thereby disrupting attachment of the pigmented cells to the culture substrate.

163. 163. The method of any one of claims 147 to 162, wherein the pluripotent stem cells have reduced HLA antigen complexity.

164. 164. The method of any one of claims 147 to 163, wherein the RPE cells lack substantial expression of one or more embryonic stem cell markers.

165. 165. The method of claim 164, wherein said one or more embryonic stem cell markers are Oct-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, DPPA-2, and / or DPPA-4.

166. 166. The method of any one of claims 147 to 165, wherein the RPE cells are positive for at least one RPE cell marker.

167. 167. The method of claim 166, wherein the at least one RPE cell marker comprises one or more of RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, or tyrosinase, or optionally PAX6 and bestrophin.

168. 168. The method of any one of claims 147 to 167, further comprising culturing the RPE cells under conditions that increase alpha integrin subunit expression.

169. The method of claim 168, wherein the alpha integrin subunit is 1-6 or 9.

170. 170. The method of claim 168 or 169, wherein the conditions comprise exposure to manganese, exposure to an antibody to CD29, or at least about four passages of the RPE cells.

171. 171. The method of claim 170, wherein the antibody against CD29 is a monoclonal antibody, optionally HUTS-21 or TS2 / 16.

172. 172. The method of any one of claims 147 to 171, wherein the medium used to grow the enriched culture of RPE cells does not support the growth or maintenance of undifferentiated pluripotent stem cells.

173. 173. The method of any one of claims 147 to 172, wherein the RPE cells meet at least one of the criteria listed in Table 5.

174. 174. The method of any one of claims 147 to 173, wherein the method is carried out in accordance with Good Manufacturing Practice (GMP).

175. 175. The method of any one of claims 147 to 174, wherein the EBs are formed in the presence of a rho-associated protein kinase (ROCK) inhibitor.

176. The method of claim 175, wherein the ROCK inhibitor is Y-27632.

177. 177. The method of claim 175 or 176, wherein the pluripotent cells are cultured on Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) prior to RPE formation.

178. (a) providing a multilayered population of human embryonic stem (hES) cells; (b) culturing said multilayered population of hES cells under conditions that do not maintain the undifferentiated state of said hES cells for a time sufficient to allow the appearance of putative human RPE cells comprising a brown pigment dispersed within the cytoplasm; (c) selecting one or more of said putative human RPE cells from the culture of step (b); (d) culturing the human RPE cells obtained in step (c) to form a culture containing cells that are bestrophin+, exhibit a characteristic cobblestone polygonal epithelial appearance, and comprise dispersed brown pigment within the cytoplasm, thereby producing an enriched population of human RPE cells; 1. A method for producing an enriched population of human retinal pigment epithelial (RPE) cells, comprising:

179. (a) providing a culture of human ES (hES) cells; (b) culturing the hES cells to generate one or more embryoid bodies; (c) culturing said one or more embryoid bodies for a time sufficient for the appearance of putative human RPE cells within at least one of said one or more embryoid bodies, said putative human RPE cells comprising a brown pigment dispersed within the cytoplasm, thereby forming one or more embryoid bodies containing putative human RPE cells; (d) selecting and isolating one or more of said embryoid bodies containing putative human RPE cells from the culture of step (c) to obtain human RPE cells; (e) culturing the human RPE cells obtained in step (d) to form a culture containing cells that are bestrophin+, exhibit a characteristic cobblestone polygonal epithelial appearance, and comprise a brown pigment dispersed within the cytoplasm, thereby producing an enriched population of human RPE cells; 1. A method for producing an enriched population of human retinal pigment epithelial (RPE) cells, comprising:

180. 180. The method of claim 178 or 179, wherein the culturing in step (b) comprises culturing in medium lacking exogenously added FGF.

181. 181. The method of claim 180, wherein the culturing step of step (b) comprises culturing in a medium lacking exogenously added LIF.

182. The culturing step of step (b) is carried out in a medium lacking exogenously added PLASMANATE® (an aqueous solution containing 5 g of plasma protein per 100 mL, buffered with sodium carbonate, stabilized with 0.005 M sodium caprylate and 0.004 M acetyltryptophan, the plasma protein comprising approximately 88% normal human albumin, 12% alpha and beta globulins, and less than 1% gamma globulins, with sodium 145 mEq / L, potassium 0.25 mEq / L, and chloride 100 mEq / L).

182. The method of claim 181, comprising culturing the cells in a medium containing 100% ethanol at 25° C. / L.

183. 184. The method of any one of claims 178-183, wherein the culture period of step (b) is about 6 weeks.

184. 184. The method of any one of claims 178-183, wherein the culture period in step (b) is from about 4 weeks to about 5 months, from about 7 weeks to about 4 months, from about 3 months to about 5 months, or from about 6 weeks to about 8 weeks.

185. 184. The method of any one of claims 178 to 183, wherein the culture period in step (b) is from about 3 months to about 5 months.

186. 186. The method of any one of claims 178 to 185, wherein the resulting RPE cell culture contains RPE cells that are bestrophin+, CRALBP+, PEDF+, and RPE65+.

187. 187. The method of any one of claims 186, wherein the resulting RPE cell culture has an absence of at least one ES cell marker selected from the group consisting of Oct4 and Sox2.

188. 188. The method of any one of claims 178 to 187, wherein prior to step (b), the hES cells are cultured in the presence of exogenously added FGF.

189. 189. The method of any one of claims 178 to 188, wherein prior to step (b), the hES cells are cultured in the presence of exogenously added FGF and LIF.

190. 189. The method of any one of claims 178-188, wherein prior to step (b), the hES cells are cultured in the presence of exogenously added FGF, PLASMANATE® (an aqueous solution containing 5 g of plasma protein per 100 mL, buffered with sodium carbonate, stabilized with 0.005 M sodium caprylate and 0.004 M acetyltryptophan, the plasma proteins comprising approximately 88% normal human albumin, 12% alpha and beta globulins, and less than 1% gamma globulins, containing 145 mEq / L sodium, 0.25 mEq / L potassium, and 100 mEq / L chloride), and a fibroblast feeder layer.

191. 191. The method of any one of claims 178 to 190, which produces a population of RPE cells containing an average melanin content of less than 8 pg / cell.

192. 192. The method of claim 191, wherein the average melanin content 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.

193. 193. The method of any one of claims 191 or 192, further comprising the step of maintaining the RPE cells as quiescent cells for a time sufficient to obtain the melanin content.

194. 194. The method of any one of claims 179 to 193, wherein the pluripotent cells are cultured on a substrate prior to RPE generation.

195. 195. The method of claim 194, wherein the substrate is selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof.

196. 195. The method of claim 194, wherein the substrate comprises Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

197. 197. The method of any one of claims 179 to 196, wherein the EBs are formed in the presence of a rho-associated protein kinase (ROCK) inhibitor.

198. The method of claim 197, wherein the ROCK inhibitor is Y-27632.

199. 200. The method of claim 197 or 198, wherein the pluripotent cells are cultured on Matrigel™ (a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) prior to the RPE formation.

200. 200. The method of any one of claims 178 to 199, wherein the resulting RPE cell culture contains RPE cells that are Pax6+.

201. 201. The method of any one of claims 178 to 200, wherein the resulting RPE cell culture contains RPE cells that are Pax6-.

202. A pharmaceutical product comprising RPE cells produced by the method of any one of claims 178 to 201.

203. containing an average melanin content of less than 8 pg / cell, maintain their phenotype for at least about one month after transplantation; maintain their phenotype for at least about one month in culture; Incorporated into the host after implantation, virtually no growth after transplantation, are phagocytositic, deliver, metabolize, or store vitamin A; Transporting iron between the retina and choroid after transplantation After transplantation, it adheres to Bruch's membrane. Absorbs stray light after transplantation, have elevated α integrin subunit expression, have a greater average telomere length than RPE cells derived from donated human tissue have a longer replicative lifespan in culture compared to RPE cells derived from donated human tissue; have greater expression of one or more alpha integrin subunits compared to RPE cells derived from the provided human tissue; have a lower A2E content compared to RPE cells derived from donated human tissue; have lower lipofuscin content compared to RPE cells derived from donated human tissue. do, exhibit less accumulated UV damage compared to RPE cells derived from donated human tissue; or Contains a greater number of phagosomes compared to RPE cells derived from donated human tissue 2. A pharmaceutical product comprising RPE cells suitable for the treatment of retinal deterioration, said RPE cells having at least one of the following properties:

204. containing an average melanin content of less than 8 pg / cell, After transplantation, it adheres to Bruch's membrane. Absorbs stray light after transplantation, have a greater average telomere length than RPE cells derived from donated human tissue have a longer replicative lifespan in culture compared to RPE cells derived from donated human tissue; have a lower A2E content compared to RPE cells derived from donated human tissue; have a lower lipofuscin content compared to RPE cells derived from donated human tissue; exhibit less accumulated UV damage compared to RPE cells derived from donated human tissue; or Contains a greater number of phagosomes compared to RPE cells derived from donated human tissue 2. A pharmaceutical product comprising RPE cells suitable for the treatment of retinal deterioration, said RPE cells having at least one of the following properties:

205. 205. The pharmaceutical product of any one of claims 202 to 204, wherein the average melanin content 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.

206. 140. A pharmaceutical product comprising thawed cryopreserved RPE cells according to any one of claims 74 to 134, or cryopreserved RPE cells contained in a kit according to any one of claims 135 to 140, wherein the RPE cells are in a pharmaceutically acceptable carrier.

207. A pharmaceutical product comprising the RPE cells of the composition or kit of any one of claims 1 to 73.

208. A pharmaceutical product according to any one of claims 202 to 207 for use in treating retinal degeneration.

209. 209. The pharmaceutical product of any one of claims 202 to 208, comprising RPE cells in an amount effective to prevent or treat retinal degeneration resulting from Stargardt's disease, dry or wet age-related macular degeneration (AMD), choroideremia, retinitis pigmentosa, retinal detachment, retinal dysplasia, retinal atrophy, pigmented streaks, or myopic macular degeneration in a patient in need thereof.

210. 210. The pharmaceutical product of any one of claims 202 to 209, wherein the formulation is formulated for implantation in an injectable form such as a suspension, gel, or colloid.

211. 211. The pharmaceutical product of any one of claims 202-210, wherein the formulation is formulated for implantation with a substrate, matrix, scaffold, or graft.

212. 212. The pharmaceutical product of any one of claims 202 to 211, wherein the formulation is formulated for administration to the subretinal space of the eye.

213. The formulation comprises at least about 10 ３ ~10 ９ RPE cells, about 10,000 to about 10 ６ 213. The pharmaceutical product of any one of claims 202 to 212, comprising about 25,000 to about 400,000 RPE cells, or about 50,000 to about 200,000 RPE cells.

214. The pharmaceutical product of any one of claims 202 to 213, wherein the RPE cells lack substantial expression of one or more embryonic stem cell markers.

215. The pharmaceutical product of claim 214, wherein the one or more embryonic stem cell markers comprise Oct-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, DPPA-2, and / or DPPA-4.

216. The pharmaceutical product of any one of claims 202 to 215, wherein the RPE cells are positive for at least one RPE cell marker.

217. The pharmaceutical product of claim 216, wherein the at least one RPE cell marker comprises at least one of RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, or tyrosinase.

218. The pharmaceutical product of any one of claims 202 to 217, wherein the RPE cells exhibit increased alpha integrin subunit expression.

219. The pharmaceutical agent of claim 208, wherein the alpha integrin subunit is alpha 1, 2, 3, 4, 5, 6, or 9.

220. 220. The pharmaceutical product of any one of claims 202 to 219, wherein the RPE cells meet at least one of the criteria listed in Table 5.

221. 221. The pharmaceutical product of any one of claims 202 to 220, wherein the preparation comprises at least about 75% RPE cells.

222. 222. The pharmaceutical product of any one of claims 202 to 221, wherein the formulation is substantially free of viral, bacterial, and / or fungal contamination.

223. 223. The pharmaceutical product of any one of claims 202 to 222, wherein the formulation is formulated in a pharmaceutically acceptable carrier.

224. 224. The pharmaceutical product of any one of claims 202 to 223, wherein the formulation is formulated for administration to the eye.

225. The pharmaceutical product of claim 224, wherein the formulation is formulated for administration to the subretinal space.

226. The pharmaceutical product of any one of claims 202 to 225, wherein the RPE cells are functional RPE cells that are capable of incorporating into the retina upon transplantation.

227. 227. The pharmaceutical product of any one of 202-226, wherein the pharmaceutical product is substantially free of mouse embryonic fibroblasts (MEF) and human embryonic stem cells (hES).

228. 202-227, wherein the formulation is in accordance with Good Manufacturing Practice (GMP).

229. A method of treating a retinal degenerative condition or other condition in which RPE cell transplantation is therapeutically desirable, comprising administering to the eye of a subject in need thereof a pharmaceutical product comprising the RPE cells of the composition or kit of any one of claims 1 to 140, a pharmaceutical product of any one of claims 202 to 228, or RPE cells produced according to the method of any one of claims 147 to 201, in an amount effective to treat the retinal degenerative condition or other condition in which RPE cell transplantation is therapeutically desirable.

230. 230. The method of claim 229, wherein the retinal degenerative condition comprises choroideremia, diabetic retinopathy, age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt's disease, pigmented streaks, or myopic macular degeneration.

231. 231. The method of claim 229 or 230, wherein the administering step comprises administering the RPE cells intraocularly to an eye in need thereof.

232. 232. The method of claim 231, wherein the intraocular administration comprises injecting the RPE cells into the subretinal space.

233. The method of claim 232, wherein the intraocular administration comprises the steps of injecting an aqueous solution, optionally an isotonic solution and / or a saline solution, into the subretinal space, thereby forming a pre-bleb, and removing the aqueous solution before administering the RPE cells into the same subretinal space as the aqueous solution.

234. 233. The method of claim 232 or 232, wherein the injection is via a needle or injection cannula.

235. 235. The method of claim 234, wherein the needle or injection cannula diameter is about 0.3 mm to 0.9 mm or about 0.5 to about 0.6 mm.

236. 236. The method of any one of claims 234-235, wherein the needle or injection cannula comprises a tip having a diameter of about 0.09 mm to about 0.15 mm.

237. 237. The method of any one of claims 234-236, wherein the cannula is a MEDONE POLYTIP® cannula 25 / 38g (0.50mm (25g) x 28mm cannula with a 0.12mm (38g) x 5mm tip).

238. 238. The method of any one of claims 229 to 237, wherein the effectiveness of treatment is assessed by determining visual outcomes by one or more of slit lamp biomicroscopy, fundus photography, IVFA, and SD-OCT, and best corrected visual acuity (BCVA).

239. 239. The method of any one of claims 229 to 238, which produces an improvement in best corrected visual acuity (BCVA) and / or increases the number of readable letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart.

240. 240. The method of claim 239, wherein the retinal degenerative condition is dry AMD or Stargardt's disease.

241. 241. The method of any one of claims 229-240, wherein the amount effective to treat the retinal degenerative condition is 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.

242. 242. The method of claim 241, wherein the amount effective to treat the retinal degenerative condition is 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.

243. 243. The method of any one of claims 229 to 242, wherein a corticosteroid is not administered to the subject prior to or concurrently with the administration of the RPE cells.

244. 243. The method of any one of claims 229-242, wherein a corticosteroid is not administered to the subject at least 3, 6, 12, 24, 48, 72, or 96 hours prior to or concurrently with the administration of the RPE cells.

245. 245. The method of any one of claims 229 to 244, wherein a corticosteroid is not administered to the subject at least one hour prior to, immediately prior to, or simultaneously with, the administration of the RPE cells.

246. 246. The method of any one of claims 229-245, wherein the administration of the RPE cells is not followed by administration of a corticosteroid to the subject within at least 12, 24, 48, 72, or 96 hours.

247. 246. The method of any one of claims 229 to 245, wherein the administration of the RPE cells is followed by administration of a corticosteroid to the subject within at least 48 hours.

248. 248. The method of any one of claims 229-247, wherein the RPE cells are administered to the patient in combination with one or more agents selected from the group consisting of angiogenesis inhibitors, antioxidants, antioxidant cofactors, other factors that contribute to increased antioxidant activity, macular xanthophylls, long chain omega-3 fatty acids, amyloid inhibitors, CNTF agonists, inhibitors of RPE65, factors that target A2E and / or lipofuscin accumulation, down-regulators or inhibitors of photoreceptor function and / or metabolism, alpha2-adrenergic receptor agonists, selective serotonin 1A agonists, factors that target C-5, membrane attack complex (C5b-9) and optionally other drusen components, immunosuppressants, and agents that prevent or treat lipofuscin accumulation.

249. 249. The method of claim 248, wherein the one or more agents are administered to the patient simultaneously with, prior to, and / or subsequent to the RPE cell preparation.

250. Use of a composition, kit, or pharmaceutical product described in any one of claims 1 to 140 or 202 to 228 in the manufacture of a medicament for treating a retinal degenerative condition or other condition in which transplantation of RPE cells is therapeutically desirable.

251. The use of claim 250, wherein the retinal degeneration condition comprises choroideremia, diabetic retinopathy, dry age-related macular degeneration, wet age-related macular degeneration, retinal detachment, retinitis pigmentosa, Stargardt's disease, pigmented streaks, or myopic macular degeneration.

252. 202. The method of any one of claims 147-201, wherein 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.

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

229. The composition, kit, or pharmaceutical product of any one of claims 1 to 140 or 202 to 228, exhibiting one or more of the following properties:

254. The RPE cells a longer replicative lifespan compared to that of RPE cells obtained from other sources; 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 human eye, or less than 40, 30, 20, or 10 percent of the average lipofuscin content of an equivalent number of RPE cells isolated from an adult human eye; Equivalent numbers of RPE isolated from adult eyes a mean N-retinylidene-N-retinylethanolamine (A2E) content that is less than 50 percent of the mean A2E content of the cells, or less than 40, 30, 20, or 10 percent of the mean A2E content of an equivalent number of RPE cells isolated from an adult human eye; 10 ５ an average N-retinylidene-N-retinylethanolamine (A2E) content of less than 50 ng per (100,000) cells; a phagocytosis rate of photoreceptor outer segments (POS) that is at least 50 percent greater than the phagocytosis rate of POS in an equivalent number of RPE cells isolated from adult eyes (isolated adult eyes), or at least 75, 100, 150, or 200 percent greater than the phagocytosis rate of POS in an equivalent number of RPE cells isolated from adult eyes (isolated adult eyes); or Reduced levels of oxidative stress and / or accumulated DNA damage compared to RPE cells isolated from adult hosts 229. The composition, kit, or pharmaceutical product of any one of claims 1 to 140 or 202 to 228, exhibiting one or more of the following properties:

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