Methods for detecting rare subpopulations of cells and highly purified compositions of cells

Abstract

The objective of the present invention is to provide a method that can overcome the limitations of conventional methods for detecting stem cells, which have limited sensitivity due to background expression of stem cell genes in non-stem cells and due to cell loss during sample preparation, and that provides a highly sensitive method for detecting rare cell types in a cell population. The present invention provides a method for detecting the presence or absence of a target cell in a population of cells, comprising specific steps (a) to (e).

Description

[Technical Field]

[0001] Related Application Disclosure This application claims the benefit of U.S. Provisional Patent Application No. 61 / 367,038 (Attorney Docket No. 75820.160001), filed July 23, 2010, and U.S. Provisional Patent Application No. 61 / 414,770 (Attorney Docket No. 75820.160002), filed November 17, 2010, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Therapeutic uses of stem cells have profound potential to revolutionize medical treatment. Researchers have long desired to use embryonic stem cells and other pluripotent cell types as a source of cells for therapeutic transplantation. For example, embryonic stem cells can be differentiated into retinal pigment epithelial cells and transplanted into patients to prevent or treat retinopathies, as described in U.S. Patent Nos. 6,213,999; 6,213,999; and 6,213,999; U.S. Patent Application No. 12 / 682,712; U.S. Provisional Patent Applications Nos. 60 / 998,668, 60 / 998,766, 61 / 009,911, 61 / 009,908, and 61 / 262,002; U.S. Patent No. 6,213,999; and U.S. Patent No. 6,213,999; each of which is incorporated herein by reference in its entirety.

[0003] However, the self-renewal and plasticity of stem cells—the key attributes that confer such therapeutic promise—also raise safety concerns. A dramatic illustration of the potential dangers of using stem cells without adequate safety testing is the recent case of a “stem cell tourist” in Thailand who allegedly received intrarenal injections of stem cells—a treatment that was apparently untested and not approved by any regulatory agency. The patient experienced no apparent therapeutic benefit from the procedure; instead, he developed a mass at the injection site, necessitating kidney removal (Thirabanjasak et al., “Angiomyeloproliferative Lesions Following Autologous Stem Cell Therapy,” J Am Soc Nephrol. 2010 Jun 17). The study authors concluded that the mass arose from the transplanted stem cells. The case has been widely reported in major newspapers as a sobering cautionary tale that patients should not risk their safety and financial well-being by engaging in stem cell tourism, which is rampantly practiced by unscrupulous practitioners (Coghlan, “Deathrevives warnings about rogue stem cell clinics,” The New Scientist, June 17, 2010).

[0004] Even in therapeutic regimens using differentiated cell types derived from ES cells, the presence of a few residual ES cells can lead to the formation of tumors or teratomas. Some safety assurance can be obtained from administering cell preparations to animals (e.g., immunocompromised animals). However, animal studies alone are considered insufficient because human ES cells are more prone to teratomas in human hosts than in animal models.

[0005] One complementary approach addressing potential safety concerns is genetically engineering stem cells to express an inducible "suicide gene." The promise of these methods is that stem cells and their progeny can be selectively killed if their proliferation becomes unregulated (see, e.g., Schuldiner et al., "Selective ablation of human embryonic stem cells expressing a 'suicide' gene," Stem Cells, 2003;21(3):257-65). However, depending on the disease involved and the location and quantity of stem cell-derived tissue, inducing the death of transplanted cells may pose significant harm to the patient. Furthermore, in the case of patient-specific cell therapy, the use of this type of safety mechanism would be crucial for the potentially very labor-intensive genetic engineering of patient-specific cells, as well as for the validation of each patient-specific cell lineage with regard to safety, efficacy, and the absence of undesired genetic modifications. Although an inducible suicide gene may offer a therapeutic option after tumors or teratomas have begun to form, it is clearly preferable to completely avoid tumor or teratomas from forming. Furthermore, cells can be made resistant to the induction of a suicide gene, for example, by mutation or loss of the suicide gene, which disables the suicide gene and allows the cell to continue to be potentially harmful. Thus, a method for directly detecting stem cells in a cell population would also be desirable to provide assurance that undifferentiated stem cells are present at very low concentrations at best, or preferably completely absent.

[0006] Conventional methods for detecting stem cells have limited sensitivity that may not provide sufficient assurance of the absence of stem cells. For methods utilizing bulk cell extracts (e.g., RT-PCR and Western blot), sensitivity may inevitably be limited by background expression of stem cell genes in non-stem cells. For example, if stem cells are expressed at concentrations 1000-fold lower than non-stem cells, a bulk cell extract with a stem cell concentration of 1 in 1000 will have less than a two-fold increase in the gene product, which is below the noise threshold for many conventional detection methods. If a treatment regimen administers 500,000 cells, such methods will be unable to detect as many as 500 residual stem cells in the cell preparation. This number of stem cells is likely to be considered unsafe due to the potential for teratoma formation. Thus, these methods are most useful when the desired detection sensitivity does not exceed the fold difference in stem cell gene expression between stem cells and other cells in the population. Due to this insensitivity, such methods are considered insufficient to ensure patient safety.

[0007] Flow cytometry, another conventional stem cell detection method, can be somewhat more sensitive than RT-PCR for detecting stem cells because it considers individual cells rather than bulk extracts. However, cell preparation for flow cytometry involves steps that result in cell loss, such as cell permeabilization, washing, and sieving to ensure a single-cell suspension. This cell loss raises concerns about whether the population of cells reaching the flow cytometer is truly representative of the initial cell population. For example, stem cells tend to be relatively "sticky" compared to other cell types, which may result in preferential loss during the cell sieving step. Furthermore, due to their small size, small aggregates of stem cells may pass through the sieving membrane together and be miscounted as a single event in the flow cytometer, resulting in an undercount of stem cells in the preparation. Furthermore, flow cytometry assays typically require gating of cell populations to eliminate cell debris, cell aggregates, and nonspecific background "noise." Even when evaluating ungated cells, interpretation of results (up to where to "draw the line" regarding positive / negative cells) can depend on the subjective opinion of the operator, and while the majority of contaminating cells in a population can be easily detected by flow cytometry, minority cells will almost certainly fall into the "noise" category, even if they are numerous enough to pose a potential safety concern. That is, flow cytometry methods may also be insufficiently sensitive to ensure patient safety due to the possibility of missing or failing to completely count stem cells.

[0008] Thus, there is a need in the art for a sensitive method for detecting the presence of undifferentiated cells in a cell composition, and for a stem cell-inducing composition that is confirmed to be free of undifferentiated stem cells. Preferred methods are sensitive enough to detect stem cells even at low cell densities of less than 1 in 100,000, and further allow for the testing of every individual cell in a population without losing a significant proportion of cells during assay preparation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,795,025 [Patent Document 2] U.S. Patent No. 7,794,704 [Patent Document 3] U.S. Patent No. 7,736,896 [Patent Document 4] International Publication No. 2009 / 051671 [Patent Document 5] International Publication No. 2011 / 063005 Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides assay methods for sensitive detection of target-type cells within a larger population of other cell types. In preferred embodiments, the target cells will include rare cells, i.e., cells present in extremely low abundance in a cell population, preferably when the cell population is intended for cell therapy. In particularly preferred embodiments, these rare cells will include cells that may induce adverse reactions or disease if administered to a subject. Specific examples include virally infected (e.g., HIV, hepatitis, etc.) cells, other diseased or abnormal cells (e.g., cancerous, precancerous, and cancer stem cells), and certain immune cells, such as T lymphocytes, which, if administered to a recipient, may result in infection, disease, or other adverse reactions, such as adverse immune responses (e.g., GVHD), or may result in the proliferation of unwanted cells. For example, the methods may be used to confirm that a population containing donor cells, such as bone marrow or pancreatic cells derived from an autologous or allogeneic donor, intended for use in cell therapy or transplantation, does not harbor cells that may induce cancer, viral infection, or adverse immune responses. In preferred exemplified embodiments, examples are provided of the use of these methods to test for the presence of residual pluripotent cells in a population of cells intended for use in transplantation therapy. Experiments in which defined quantities of ES cells were added to a cell population demonstrated sufficient sensitivity to detect as few as five ES cells from a population of 600,000 cells, demonstrating the ability to detect target cells as rare as 0.0008% of the total cell population. Using exemplified embodiments of these methods, a skilled operator can examine numbers on the order of 1-10 million cells per hour. Because arbitrarily large numbers of cells can be examined, the methods have the potential for infinite sensitivity.

[0011] In one exemplary embodiment, the present disclosure provides a method for preparing a soluble polymeric material comprising the steps of:

[0012] (a) Preparing the cell population;

[0013] (b) applying a first stain and a second stain to the cell population, wherein the first stain detects a first marker indicative of target cells, and the second stain detects a second marker indicative of target cells, and wherein cells positive for the first stain are detectable under visible light and cells positive for the second stain are detectable under ultraviolet light;

[0014] (c) detecting cells that are positive for the first marker by observing the cell population under a microscope under visible light;

[0015] (e) observing the cells that are positive for the first marker under a microscope under ultraviolet light and determining whether the cells are positive for the first marker and the second marker; and

[0016] (f) identifying cells that are positive for the first marker and the second marker as target cells; The present invention provides a method for detecting the presence of a target cell in a cell population comprising:

[0017] The first stain may be observable under visible and ultraviolet light.

[0018] The first marker may be alkaline phosphatase.

[0019] The first stain may comprise a colored reagent or an antibody coupled directly or indirectly to an enzyme capable of producing a colored reagent.

[0020] The colored reagent may include gold particles, silver particles, or latex particles.

[0021] The first stain may comprise an antibody directly or indirectly coupled to gold particles, and the method may further comprise forming a silver precipitate on the gold particles.

[0022] The first marker may be selected from the group consisting of alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

[0023] The first stain may comprise a first enzyme selected from the group consisting of alkaline phosphatase, beta-galactosidase, and peroxidase.

[0024] The peroxidase may be horseradish peroxidase.

[0025] The first enzyme may be expressed by the target cell.

[0026] The first enzyme may be directly or indirectly coupled to the primary or secondary antibody.

[0027] The first stain may include alkaline phosphatase.

[0028] The first stain may further comprise an alkaline phosphatase substrate selected from the group consisting of naphthol AS-BI phosphate; 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT); BCIP reagent and INTX reagent; naphthol AS-BI and fast red violet LB; tetrazolium salts; diazo compounds; VECTOR® Red; VECTOR® Blue; VECTOR® Black; and p-nitrophenyl phosphate (pNPP).

[0029] The first stain may include peroxidase.

[0030] The first staining agent was further classified as 3,3',5,5'-tetramethylbenzidine (TMB); 3,3'-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol; 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); 2,3,5-triphenyltetrazolium chloride; 2-chloro-5,5-dimethyl-1,3-cyclohexanedione; ... Tetramethylbenzidine;3,3'-Diaminobenzidine tetrachloride;3-Nitrotetrazolium blue chloride;4-Aminophthalhydrazide;4-Chloro-1-naphthol;4-Chloro-7-nitrobenzofurazan;5-Aminosalicylic acid;Dicarboxidine dihydrochloride;Guaiacol;Hydrogen peroxide-urea adduct;Iodonitrotetrazolium chloride;Luminol;MTT formazan;N-(4-Aminobutyl)-N-ethylisoluminol ; N-(6-aminohexyl)-N-ethylisoluminol; nitrotetrazolium blue chloride; pyrogallol; tetranitro blue tetrazolium chloride; tetrazolium blue chloride indicator; tetrazolium violet; o-dianisidine; o-dianisidine dihydrochloride; o-phenylenediamine dihydrochloride; o-phenylenediamine free base; and trans-5-phenyl-4-pentenyl hydroperoxide.

[0031] The first stain may include beta-galactosidase.

[0032] The first stain may further comprise a beta-galactosidase substrate selected from the group consisting of 1-methyl-3-indolyl-β-D-galactopyranoside; 2-nitrophenyl β-D-galactopyranoside; 4-methylumbelliferyl β-D-galactopyranoside; 4-nitrophenyl β-D-galactopyranoside; 5-bromo-3-indolyl β-D-galactopyranoside; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside; 6-bromo-2-naphthyl β-D-galactopyranoside; 6-chloro-3-indolyl-β-D-galactopyranoside; fluorescein di(β-D-galactopyranoside); and resorufin β-D-galactopyranoside.

[0033] The target cell may be an embryonic stem cell, and the second marker may be selected from the group consisting of alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

[0034] The second stain may comprise a primary antibody, which may comprise a fluorescent label.

[0035] The second stain may further comprise a secondary antibody, which may comprise a fluorescent label.

[0036] Fluorescent labels include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790, fluorescein isothiocyanate (FITC), Texas Red, SYBR Green, DyLightFluors, green fluorescent protein (GFP), TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, and isophthalic acid. , cresyl fast violet, cresyl blue violet, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, TET (6-carboxy-2',4,7,7'-tetrachlorofluorescein), HEX (6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein), Joe (6-carboxy-4',5'- Dichloro-2',7'-dimethoxyfluorescein), 5-carboxy-2',4',5',7'- The compound may be selected from the group consisting of tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, Tamra (tetramethylrhodamine), 6-carboxyrhodamine, Rox (carboxy-X-rhodamine), R6G (rhodamine 6G), phthalocyanines, azomethines, cyanines (e.g., Cy3, Cy3.5, Cy5), xanthines, succinylfluoresceins, N,N-diethyl-4-(5'-azobenzotriazolyl)-phenylamine, aminoacridine, and quantum dots.

[0037] The cell population may comprise cells of a species selected from the group consisting of antelope, cow, camel, cat, mouse deer (mouse deer), chimpanzee, cow, deer, dog, giraffe, goat, guinea pig, hamster, hippopotamus, horse, human, mouse, non-human primate, ovine, boar, pig, pronghorn, rabbit, rat, macaque, rhinoceros, sheep, tapir, and ungulate.

[0038] The method may further comprise determining the approximate number of cells in the cell population.

[0039] At least 90% of the cells in the cell population may be examined under visible light to detect cells that are positive for the first marker, and each cell that is positive for the first marker may be examined under ultraviolet light to determine whether the cell is positive for the second marker.

[0040] The cell population may contain any number of cells depending on the intended use. For example, the cell population may contain at least 10 5 cells, at least 10 6 cells, at least 10 7 cells, at least 10 8 cells, at least 10 9 cells, at least 10 10 cells, or 10 5 ~10 10 The cell may contain 10 cells.

[0041] The first marker and the second marker may be embryonic stem cell markers.

[0042] The cell population may be produced by in vivo or in vitro differentiation of embryonic stem cells.

[0043] The target cells may be embryonic stem cells or induced pluripotent (iPS) cells.

[0044] The cell population may further comprise cells differentiated from embryonic stem cells or iPS cells.

[0045] The cells differentiated from embryonic stem cells or iPS cells may be RPE cells, for example, RPE cells may be produced by any of the methods disclosed in U.S. Patent Nos. 7,795,025, 7,794,704, and 7,736,896, U.S. Patent Application No. 12 / 682,712, WO / 2009 / 051671, and WO2011 / 063005, each of which is incorporated by reference in its entirety.

[0046] Target cells may be selected from specific types of cells, including, for example, adipocytes; bone marrow fibroblasts; cardiomyocytes; chondrocytes; differentiated RBC and WBC lineages; endothelial bone marrow fibroblasts; ectoderm; ectodermal precursors; embryoid bodies (EBs); embryonal carcinomas (ECs); embryonic stem cells (ESs); endoderm; endothelium; hematopoietic cells; hematopoietic stem cells (HSCs), satellite bodies, endothelial precursors; hepatocytes; keratinocytes; mesenchyme; mesenchymal stem cells (MSCs); mesoderm; MSC precursors; myoblasts; myocytes; neural precursors; neural stem cells; neurons; oligodendrocytes; osteoblasts; pancreatic epithelium; pancreatic islets; pancreatic precursors; skeletal muscle cells; smooth muscle; interstitial (mesenchymal) progenitor cells; and white blood cells (WBCs). The second marker and the second marker may be markers associated with the target cells, as shown in Table 1. Additionally, target cells may include cells involved in a particular disease, such as cancerous or pre-cancerous cells, or cancer stem cells that are desirable to eliminate from a cell population potentially for transplantation or cell therapy, such as bone marrow, or may include adult stem cells that provide cells of a particular lineage.

[0047] The method may further comprise, prior to step (a), culturing the cell population under conditions favoring maintenance cells of the target cell type, and the target cells may be embryonic stem cells or induced pluripotent (iPS) cells, and the culture conditions may comprise the presence of embryonic stem cell medium and / or mouse embryonic fibroblast feeder cells.

[0048] The method may further include plating a second cell population comprising the target cell type and analyzing the second cell population by the same method as the cell population of step (a) to define the detection limit of the method. Only cells of the target cell type may be added to the second population, or the second cell population may comprise a mixture of a first population of cells and a second population of cells, where the first population of cells is of the same type as the target cell type. The second population of cells may have essentially the same constituency as the cell population of step (a).The ratio of the number of cells of the first group of cells to the second group of cells is 1:10; 1:100; 1:1,000; 1:10,000; 1:100,000; 1:1,000,000; 1:10,000,000; 1:100,000,000; 1:1,000,000,000; between 1:10 and 1:100; between 1:10 and 1:1,000; between 1:10 and 1:10,000; between 1:10 and 1:100,000; between 1:10 and 1:1,000,000; between 1:10 and 1:10,000,000; between 1:10 and 1:100,000 between; between 1:10 and 1:1,000,000,000; between 1:100 and 1:1,000; between 1:100 and 1:10,000; between 1:100 and 1:100,000; between 1:100 and 1:1,000,000; between 1:100 and 1:10,000,000; between 1:100 and 1:100,000,000; between 1:100 and 1:1,000,000,000; between 1:1,000 and 1:10,000; between 1:1,000 and 1:100,000; between 1:1,000 and 1:10 ,000,000;between 1:1,000 and 1:100,000,000;between 1:1,000 and 1:1,000,000,000;between 1:10,000 and 1:100,000;between 1:10,000 and 1:1,000,000;between 1:10,000 and 1:100,000,000;between 1:10,000 and 1:1,000,000,000;between 1:100,000 and 1:1,000,000,000;between 1:100,000 and 1:1,000,000;between 1:100,0 between 1:1,000,000 and 1:100,000,000; between 1:1,000,000 and 1:10,000,000; between 1:1,000,000 and 1:100,000,000; between 1:1,000,000 and 1:1,000,000,000; between 1:1,000,000 and 1:1,000,000,000; between 1:10,000,000 and 1:100,000,000; between 1:10,000,000 and 1:1,000,000,000; and between 1:100,000,000 and 1:1,000,000,000.

[0049] The target cells may be selected from virus-infected cells, cancerous or pre-cancerous cells, cancer stem cells, and immune cells.

[0050] The composition putatively containing the target cells may include bone marrow, blood cells, or pancreatic cells.

[0051] The cell population suspected to contain the target cells may have been previously treated by cell sorting, irradiation, chemotherapy or other means to eliminate the target cells.

[0052] Another exemplary embodiment provides a composition comprising somatic cells derived from stem cells that may be essentially free of said stem cells.

[0053] Another exemplary embodiment provides a composition comprising cells and an indicator that indicates the number or proportion of target cells (e.g., stem cells) present, where the indicator is representative of the cells in the composition. The value of the indicator may be measured by applying the methods described herein to a cell population. The composition may include RPE cells, which may be differentiated from pluripotent stem cells such as embryonic stem cells, iPS cells, blastomeres, inner cell masses, or oocytes, which may be parthenogenetically activated. These pluripotent stem cells may be recombinant or genetically engineered (e.g., engineered to express a desired therapeutic protein or to eliminate expression of a gene involved in a genetic disorder such as macular degeneration). RPE cells may be formulated and used to treat retinal degenerative diseases. Furthermore, pluripotent stem cell-derived RPE cells may be used in screening assays to discover agents that modulate RPE cell survival (in vitro and / or in vivo), to study RPE cell maturation, or to discover agents that modulate RPE cell maturation. Materials discovered using such screening assays may be used in vitro or in vivo and may provide additional therapeutic agents that can be used alone or in combination with RPE cells to treat retinal degenerative diseases. In one embodiment, the composition may comprise a substantially purified preparation of human RPE cells differentiated from human pluripotent stem cells, wherein the RPE cells express RPE-65, bestrophin, PEDF, CRALBP, Otx2, and MITF at the mRNA and protein levels, and wherein the cells substantially lack expression of Oct-4, NANOG, and Rex1. In another embodiment, the RPE cells include differentiated RPE cells and mature differentiated RPE cells, wherein at least the mature differentiated RPE cells further express PAX2, pax-6, and tyrosinase at the mRNA and protein levels. In another embodiment, the RPE cells are differentiated from human ES cells or human iPS cells.

[0054] The composition comprises at least 10 5 cells, at least 10 6 cells, at least 10 7 cells, at least 10 8 cells, at least 10 9 cells, at least 10 10 cells, or 10 5 ~1010 The cell may comprise:

[0055] The composition may comprise cryopreserved cells. In one embodiment, the composition comprises at least about 10 4 The cryopreserved preparation may include a cryopreserved preparation comprising human RPE cells, wherein the preparation is a substantially purified preparation of human RPE cells derived from human pluripotent stem cells, and wherein the RPE cells express RPE-65, bestrophin, PEDF, CRALBP, Otx2, and MITF. In another embodiment, at least about 85% of the RPE cells remain viable after thawing. For example, RPE cells may be cryopreserved by a method comprising: (a) culturing RPE cells; (b) harvesting the RPE cells; (c) centrifuging the RPE cells; (d) resuspending the RPE cells in a 10% DMSO / 90% FBS solution; and (e) freezing the RPE cells.

[0056] Another exemplary embodiment comprises the steps of:

[0057] (a) Preparing the cell population;

[0058] (b) applying a first stain and a second stain to the cell population, wherein the first stain detects alkaline phosphatase and the second stain detects a marker indicative of embryonic stem cells, and wherein cells positive for the first stain are detectable under visible and ultraviolet light, and cells positive for the second stain are detectable under ultraviolet light;

[0059] (c) observing the cells of the cell population under a microscope under visible light to detect cells that are positive for the first marker;

[0060] (e) observing the cells that are positive for the first marker under a microscope under ultraviolet light, and determining whether the cells are positive for the first marker and the second marker. and

[0061] (f) identifying cells that are positive for the first marker and the second marker as embryonic stem cells; The present invention provides a method for detecting the presence of human embryonic stem cells in a cell population comprising:

[0062] The cell population may comprise RPE cells differentiated from pluripotent cells by the methods described in U.S. Patents 7,795,025, 7,794,704 and 7,736,896, U.S. Patent Application Serial No. 12 / 682,712, U.S. Provisional Patent Applications 60 / 998,668, 60 / 998,766, 61 / 009,911, 61 / 009,908 and 61 / 262,002, WO / 2009 / 051671 and WO2011 / 063005, each of which is incorporated by reference in its entirety.

[0063] In one embodiment, the target cells are embryonic stem cells, induced pluripotent stem (iPS) cells, adult stem cells, hematopoietic cells, fetal stem cells, mesenchymal stem cells, postpartum stem cells, multipotent stem cells, or embryonic germ cells. In another embodiment, the pluripotent stem cells may be mammalian pluripotent stem cells. In yet another embodiment, the pluripotent stem cells are Human pluripotent stem cells may be, but are not limited to, human embryonic stem (hES) cells, human induced pluripotent stem (iPS) cells, human adult stem cells, human hematopoietic stem cells, human fetal stem cells, human mesenchymal stem cells, human postpartum stem cells, human multipotent stem cells, or human embryonic germ cells. In another embodiment, the pluripotent stem cells may be an hES cell line listed in the European Human Embryonic Stem Cell Registry - hESCreg. In another embodiment, the hES cell line may be a blastomere-derived hES cell line, such as MA09, or other cell line derived by the methods described in U.S. Patents 7,893,315 or 7,838,727, each of which is incorporated herein by reference in its entirety.

[0064] In one embodiment, the invention provides the use of a pharmaceutical preparation of RPE cells in the manufacture of a medicament for the treatment of retinal degeneration.

[0065] In one embodiment, the invention provides a method for treating retinal degeneration comprising administering an effective amount of RPE cells described herein. In another embodiment, the retinal degeneration is due to congenital choroideremia, diabetic retinopathy, age-related macular degeneration, retinal detachment, retinitis pigmentosa, or Stargardt's disease.

[0066] In one embodiment, the preparation is implanted as a suspension, matrix, gel, colloid, scaffold, or substrate. In another embodiment, the preparation is administered by injection into the subretinal space of the eye.

[0067] In other embodiments, the effective amount is at least about 20,000-200,000 RPE cells, hi other embodiments, the effective amount is at least about 20,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, or 200,000 RPE cells.

[0068] In one embodiment, the method further comprises monitoring the efficacy of the method by measuring electroretinogram response, optometer acuity threshold, or luminosity threshold in the subject.

[0069] In one embodiment, the preparation is substantially free of ES cell, viral, bacterial, or fungal contamination. In another embodiment, the RPE cells can be incorporated into the retina by transplantation. In other embodiments, the RPE cells improve visual clarity after transplantation.

[0070] The present invention provides methods for the treatment of ocular disorders. In particular, these methods involve the use of RPE cells to treat or ameliorate the symptoms of ocular disorders, particularly ocular disorders caused or exacerbated in whole or in part by damage or destruction of the endogenous RPE layer.

[0071] In one embodiment, the RPE cells described herein are substantially free of genetic mutations that may result in retinal disorganization.

[0072] In one embodiment, RPE cells may be implanted with a biocompatible polymer such as polylactic acid, poly(lactic-co-glycolic acid), 50:50 PDLGA, 85:15 PDLGA, and INION GTR® biodegradable membrane (a blend of biocompatible polymers).

[0073] In another embodiment, the RPE cells adhere to Bruch's membrane after transplantation, become polarized, and integrate into the recipient's tissue.

[0074] In one embodiment, the RPE cells may improve visual clarity after transplantation, hi another embodiment, the RPE cells may substantially improve visual clarity after transplantation.

[0075] In one embodiment, the RPE cells express genes that include Oct-4, NANOG, Rex1, alkaline phosphatase, Sox2, TDGF 1, DPPA 2, and DPPA 4. The RPE cells lack substantial expression of embryonic stem cell markers, including but not limited to, RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, Pax 6, and tyrosinase. In another embodiment, the RPE cells express at least one gene, wherein expression of the at least one gene is increased in the RPE cells relative to expression in human ES cells. In one embodiment, the RPE cells exhibit increased alpha integrin subunit expression. In another embodiment, the alpha integrin subunit is alpha 1, 2, 3, 4, 5, 6, or 9. In yet another embodiment, expression is mRNA expression, protein expression, or both mRNA and protein expression.

[0076] The present invention provides a method for providing an RPE preparation to a clinical setting, comprising: (a) thawing a vial of cryopreserved RPE cells; (b) resuspending the RPE cells in medium; (c) centrifuging the RPE cells; (d) resuspending the RPE cells in medium; (e) dispensing the RPE cells into vials; and (f) transferring the RPE cells to a clinical setting. In one embodiment, the resuspension and centrifugation steps may be repeated at least 1, 2, 3, 4, or 5 times. In another embodiment, the RPE product is transported to a clinical setting within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after completion of step (e). In other embodiments, the vials may be labeled.

[0077] The present invention also provides a method for providing an RPE cell preparation for sale, comprising (a) producing RPE cells and (b) preparing the RPE cell preparation for transfer to a customer. In one embodiment, the method may comprise cryopreserving the RPE cells. In another embodiment, the method comprises providing the RPE cell preparation for sale. In other embodiments, the method comprises advertising the RPE cell preparation.

[0078] The present invention contemplates any combination of the above or below aspects and embodiments. For example, RPE cell preparations including any combination of differentiated RPE cells and mature RPE cells can be used in treating any of the conditions described herein. Similarly, the ocular methods described herein for producing RPE cells using human embryonic stem cells as starting material may similarly be performed using any human pluripotent stem cells as starting material. [Brief explanation of the drawings]

[0079] [Figure 1] Figure 1 shows representative micrographs showing the detection of hES cells (Oct-4 positive and alkaline phosphatase positive) from differentiated cells. GFP expression confirms the identity of hES cells.

[0080] [Figure 2A] RNA was extracted from mixed populations of RPE and hES cells (containing 0%, 0.01%, 0.1%, 1%, 10%, or 100% hES cells, as indicated). cDNA was then synthesized, and relative gene expression was assayed in triplicate; replicates were normalized to the beta-actin signal present in each sample. Gene expression profiling was performed by RT-qPCR using Applied Biosystems StepOne Plus software version 2.1 and TaqMan gene expression assays from Life Technologies, following the manufacturer's recommended cycling conditions for comparative relative quantification. Data are presented as the mean ± standard deviation of triplicates, with p values ​​determined by t-test. Results are shown for the expression of (A) OCT4, (B) Nanog, and (C) SOX2. [Figure 2B] Same as above. [Figure 2C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description Previous methods for detecting stem cells have had limited sensitivity due to background expression of stem cell genes in non-stem cells and cell loss during sample preparation. The present disclosure provides methods that can overcome these limitations and provide highly sensitive methods for detecting rare cell types in cell populations. Using these methods, an operator can examine numbers on the order of 1 to 10 million cells per hour and detect individual cells of a target cell type within a cell population. The illustrated embodiments may be implemented in an automated or semi-automated manner, which may further increase throughput. For example, when generating differentiated cells from ES cells, it may be desirable to determine whether any residual ES cells remain in the cell population, and a given lot of cells may be discarded if the proportion of ES cells exceeds some predetermined threshold. With only a few hours of labor, millions of representative cells from each lot of differentiated cells can be examined, providing greatly improved assurance of the absence of residual ES cells.

[0082] Because an arbitrarily large number of cells can be examined, method embodiments of the present invention have potentially infinite sensitivity.

[0083] In an exemplary embodiment, cells are plated at a high density, but most preferably no more than a monolayer (to simplify microscopic observation of the cells), which may be on a monolayer of feeder cells, and then stained for the presence of two or more characteristic markers of the target cell type, including a first marker detectable under visible light and a second marker detectable under ultraviolet light. The plated cells are then observed microscopically under visible light to detect cells expressing the first marker. Starting at one corner of the plated cells, the operator (or an automated system) scans across the entire plate and returns in overlapping tracks, ensuring that each cell is examined. Using visible light This allows unstained cells to be visualized, and the focal plane can be adjusted as needed to allow cells to remain in focus as the plated cells are moved across the field. The use of visible light also prevents photobleaching, which can occur under ultraviolet light. Scanning under visible light can also be performed at relatively low magnification, thereby increasing the number of cells observed in each microscope field and increasing throughput. Cells that are positive for the first marker are then examined under ultraviolet light to determine whether they also express a second marker. Marker selection can be based on their known expression in pluripotent cells: for example, alkaline phosphatase (which may be used with ES cells as a marker used to "scan" the entire plate with visible light) is known to persist longer in ES cells than other markers as they begin to differentiate. That is, if a cell is positive for alkaline phosphatase, it may be positive or negative for a second marker, and therefore, cells that are positive for both the first and second markers are considered hES cells. Preferably (as with AP and Oct-4 for hES cells), the first marker robustly stains target cells (even though it may stain other cells as well) to avoid missing the opportunity to observe cells that may be positive for the second marker but have low or undetectable expression of the first marker. Typically, positive cells are photographed so that comparison of photographs can avoid double counting of cells observed in overlapping fields. The total number of plated cells is determined by counting all cells in each of one or more representative microscopic fields and dividing the number of counted cells by the percentage of the total plated area counted. Finally, the number of detected target cells is expressed as a proportion of the total number of cells examined.

[0084] These markers are selected to distinguish the target cells from other cell types in the population, for example, if the target cells are hES cells and the other cell types in the population are human RPE cells, the first marker can be alkaline phosphatase and the second marker can be Oct-4. Other exemplary hES cell markers that may be used with these methods include Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand). The methods of the present invention utilize at least two markers and may utilize three, four, five, six, etc. markers, which may further confirm whether the cells are of the target type.

[0085] In additional exemplary embodiments, the target cells are cancer stem cells and are detected by staining for one or more of the markers disclosed in one of the following U.S. Patent Application Publications: 20100169990, 20090004205, 20080194022, 20080178305, 20080175870, 20080064049, 20070297983, 20070259969, 20070231325, 20070212737, 20070190647, 20060073125, 20060051325, 20040037815, and 20020119565, which are incorporated by reference in their entireties.

[0086] Preferably, the cells are plated under conditions that favor the maintenance of the target cell type. Plating under such conditions should improve the retention of the target cell type in culture and improve the sensitivity of the method. For example, if the target cells are hES cells, the cells may be plated under conditions known to maintain hES cells in a pluripotent state. Exemplary culture conditions include irradiation or pretreatment with mitomycin C. This involves plating the ES cells onto, or in medium conditioned by, feeder cells, which may be mitotically inactivated by other methods known in the art. Many suitable feeder cell types are known in the art, including, for example, primary fibroblast cultures such as murine embryonic fibroblasts (MEFs), human adult dermal fibroblasts, STO cells, and others. Culture conditions for feeder-free maintenance of ES cells are known in the art and may include Matrigel, laminin, adrenocorticotropic hormone, conditioned medium, or any combination thereof. Exemplary ES cell culture media, including feeder-free media, are described in Carpenter et al., Dev Dyn. 2004 Feb;229(2):243-58; Xu et al., Nat Biotechnol. 2001 Oct;19(10):971-4; Rosler et al., Dev Dyn. 2004 Feb;229(2):259-74; and Amit et al., Biol Reprod. 2004 Mar;70(3):837-45; WO 01 / 51616; U.S. Patent No. 6,800,480, each of which is incorporated by reference in its entirety. US Patent Application Publication No. 2009 / 0275128; US Patent Application Publication No. 2008 / 0064099; and US Patent Application Publication No. 2007 / 0160974.

[0087] Preferably, the method is validated to determine its sensitivity, for example, by "spiking experiments," in which a predetermined number of cells of the target cell type are added to a population, which are then stimulated with the desired method to detect cells of the target cell type. The use of a population containing a predetermined number of target cells allows for the determination of the minimum number of target cells that must be present before the assay detects them, or the detection limit of the assay, e.g., the minimum percentage of the population that must, on average, be of the target cell type. The detection limit may be expressed as a ratio or percentage indicating the minimum proportion of cells of the target cell type that can be detected in the assay (e.g., 5 in 600,000 cells or 0.0008%).

[0088] The method may include determining a correction factor that can be used to correct for the sensitivity or detection limit of the assay. For example, target cells may be lost during culture prior to performing the assay (e.g., the target cells may differentiate into other cell types). A predetermined number of cells of the target type may be plated under the same conditions as the spiking experiment, but in the absence of other types of cells in the population (although feeder cells may be present if they are part of the culture conditions). For example, if feeder cells of a species different from the target cells are present, the cells may be stained with a species-specific antibody that specifically recognizes cells of the target cell species to detect cells that have lost the target cell-specific marker. After this culture, the culture can be examined to determine the percentage of target cells that retain expression of the target cell marker used in the assay, thereby determining a "correction factor" that defines the relationship between the number of cells plated and the number that retain the phenotype used for detection. For example, if it is found that 50% of the target cells lose expression of the target cell marker assayed under these conditions, the effective number of target cells (e.g., used to calculate the detection limit in a spiking experiment) will be correspondingly reduced. That is, in a "spiking" experiment, if the assay can detect as few as 10 added target cells per million cells in the population, but it is shown that 50% of the target cells lose the detectable phenotype during culture, then the corrected limit of detection of the assay may be calculated to be 5 cells in 10 million to reflect the number of target cells that actually remain present in the population.

[0089] Furthermore, the method preferably employs sample preparation that maximizes the number of target cells that remain present in the population and retain expression of the target cell markers used for detection. Additional exemplary embodiments provide a method for identifying culture conditions that preserve expression of selected markers by target cells, which involves plating target cells under various culture conditions, identifying the percentage of target cells that retain expression of one or more markers characteristic of the target cells under each culture condition, and identifying the culture condition that retains a higher percentage as preserving the target cell expression of the selected markers. For example, the present disclosure provides a method for detecting hES cells by using hES cell markers (and thereby assaying the hES cell markers) to detect hES cells. It has been shown that the retention of sensitivity (sensitivity) is significantly improved by using hES cell culture conditions (culture on hES cell medium) rather than RPE cell culture conditions (absence of MEFs and medium in which hES cells differentiate).

[0090] The marker may be detected using at least one stain detectable under visible light and another marker detectable under ultraviolet light. The marker detectable under visible light may be detectable based on enzymatic activity. Examples of enzymes capable of producing a colored product detectable under visible light include alkaline phosphatase, peroxidase (e.g., horseradish peroxidase), and β-galactosidase. These enzymes may be expressed by the target cell or may be coupled to a molecule (e.g., a primary or secondary antibody) that binds to the target cell.

[0091] One marker detectable under visible light that may be used in embodiments of the present disclosure is alkaline phosphatase, which may be stained based on its enzymatic activity. The alkaline phosphatase may be endogenously expressed, coupled to an antibody, or both. Many substrates can be used for alkaline phosphatase staining to produce a product detectable under visible light, including naphthol AS-BI phosphate as the substrate and fast red dye as a colorimetric readout (e.g., using Stemgent® Alkaline Phosphatase (AP) Staining Kit II); bromo-chloro-isothiazolinone; and bromo-chloro-isothiazolinone. and thiazolyl phosphate (BCIP) and nitro blue tetrazolium (NBT / thiazolyl blue / nitroBT) (e.g., alkaline phosphatase blue microwell substrate (SIGMA-ALDRICH®)); BCIP reagent and INTX reagent (SIGMA Naphthol AS-BI and Fast Red Violet LB (e.g., Leukocyte Alkaline Phosphatase Kit (SIGMA-ALDRICH®)); precipitate-forming substrates are based either on the reduction of a tetrazolium salt or on the generation of a colored diazo compound (e.g., VECTOR® Red, VECTOR® Blue, Vector® Black, p-nitrophenyl phosphate, BCIP / NBTAP Substrate Kit, and 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium). Optionally, alkaline phosphatase inhibitors, such as levamisole ((S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole), or heat treatment may be used to inhibit unwanted background alkaline phosphatase activity.

[0092] Another marker detectable under visible light that may be used in embodiments of the present disclosure is a peroxidase, such as horseradish peroxidase (HRP), which may be stained based on its enzymatic activity. The peroxidase may be endogenously expressed, coupled to an antibody, or both. Peroxidase can catalyze the conversion of a chromogenic substrate into a colored molecule. Exemplary peroxidase substrates include 3,3',5,5'-tetramethylbenzidine (TMB); 3,3'-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol; 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); 2,3,5-triphenyltetrazolium chloride; 2-chloro-5,5-dimethyl-1,3-cyclohexanedione; 3,3',5,5'-tetramethylbenzidine; 3,3'-diaminobenzidine tetrachloride; and 3-nitrotetrazolium bromine. Chloride;4-Aminophthalhydrazide;4-Chloro-1-naphthol;4-Chloro-7-nitrobenzofurazan;5-Aminosalicylic acid;Dicarboxidine dihydrochloride;Guaiacol;Hydrogen peroxide-urea adduct;Iodonitrotetrazolium chloride;Luminol;MTT formazan;N-(4-Aminobutyl)-N-ethylisoluminol;N-(6-Aminohexyl)-N-ethylisoluminol;Nitrotetrazolium blue chloride;Pyrogallol;Tetranitrobluetetrazoliumchloride;Tetrazolium blue chloride indicator;Tetrazolium violet o-Dianisidine dihydrochloride; o-Phenylenediamine dihydrochloride; o-Phenylenediamine free base; and trans-5-phenyl-4-pentenyl hydroperoxide.

[0093] Another marker detectable under visible light that may be used in embodiments of the present disclosure is β-galactosidase, which may be stained based on its enzymatic activity. β-galactosidase may be endogenously expressed, coupled to an antibody, or both. β-galactosidase can catalyze the conversion of a chromogenic substrate into a colored molecule. Exemplary β-galactosidase substrates include 1-methyl-3-indolyl-β-D-galactopyranoside; 2-nitrophenyl β-D-galactopyranoside; 4-methylumbelliferyl β-D-galactopyranoside; 4-nitrophenyl β-D-galactopyranoside; 5-bromo-3-indolyl β-D-galactopyranoside; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside; 6-bromo-2-naphthyl β-D-galactopyranoside; 6-chloro-3-indolyl-β-D-galactopyranoside; fluorescein di(β-D-galactopyranoside); and resorufin β-D-galactopyranoside.

[0094] When an antibody is used as the staining component, the marker can be directly or indirectly coupled to the antibody. Examples of indirect coupling include avidin / biotin coupling, coupling via a secondary antibody, and combinations thereof. For example, cells may be stained with a primary antibody that binds to a target-specific antigen, and A secondary antibody that binds to the primary antibody or to a molecule coupled to the primary antibody can be coupled to a detectable marker. The use of indirect coupling can improve the signal-to-noise ratio, for example, by reducing background binding and / or by providing signal amplification.

[0095] Other stains detectable under visible light include particles containing gold, silver, or latex. For example, particles may be directly or indirectly coupled to primary or secondary antibodies or otherwise bound to target cells. Optionally, staining may be enhanced using immunogold-silver staining, in which cells are stained with antibodies coupled to colloidal gold, and gold particles are revealed using a silver precipitation method (Holgate et al., J Histochem Cytochem. 1983 Jul;31(7):938-44).

[0096] Illustrative embodiments of the methods of the present invention include fluorescent molecules such as ethidium bromide, SYBR Green, fluorescein isothiocyanate (FITC), DyLightFluors, green fluorescent protein (GFP), TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorene, and the like. Fluorescein, TET (6-carboxy-2',4,7,7'-tetrachlorofluorescein), HEX (6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein) Fluorescein), Joe (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-calcium Couplings to carboxyfluorescein, 5-carboxyrhodamine, Tamra (tetramethylrhodamine), 6-carboxyrhodamine, Rox (carboxy-X-rhodamine), R6G (rhodamine 6G), phthalocyanines, azomethines, cyanines (e.g., Cy3, Cy3.5, Cy5), xanthines, succinylfluoresceins, N,N-diethyl-4-(5'-azobenzotriazolyl)-phenylamine, and aminoacridine Other exemplary fluorescent molecules are disclosed in the patent literature [e.g., each of which is incorporated herein by reference in its entirety]. Quantum dots with various surface chemistries and fluorescent properties are disclosed in U.S. Patents 6,207,299, 6,322,901, 6,576,291, 6,649,138 (surface modification methods in which mixed hydrophobic / hydrophilic polymeric transfer agents are attached to the surface of quantum dots), 6,682,596, 6,815,064 (relating to alloy or mixed shells), and in the technical literature [e.g., "Alternative Routes toward High Quality CdSe Nanocrystals," (Qu et al., NanoLett., 1(6):333-337 (2001)], all of which are incorporated herein by reference. Quantum dots with various surface chemistries and fluorescent properties are disclosed in U.S. Patents 6,207,299, 6,322,901, 6,576,291, 6,649,138 (surface modification methods in which mixed hydrophobic / hydrophilic polymeric transfer agents are attached to the surface of quantum dots), 6,682,596, 6,815,064 (relating to alloy or mixed shells), and in the technical literature [e.g., "Alternative Routes toward High Quality CdSe Nanocrystals," (Qu et al., NanoLett., 1(6):333-337 (2001)], among others. Technologies (Troy, NY) and Quantum Dot Corporation (Hayward, Calif.). Quantum dots also include alloyed quantum dots, such as ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN. Alloyed quantum dots and methods for making them are disclosed, for example, in U.S. Patent Application Publication No. 2005 / 0012182 and PCT Publication No. WO 2005 / 001889.

[0097] While the present disclosure provides exemplary embodiments illustrating the sensitive detection of human embryonic stem cells in differentiated RPE cell populations, these methods can be readily adapted to detect other cell types or to use species other than humans. For example, these methods may be used with other cell types, such as other stem cell types, cancer cells, feeder cells, heterologous cells, or any other cell type expressing a characteristic marker. Similarly, these methods may be used with human, non-human primate, antelope, cow, camel, cat, mouse deer (mouse deer), chimpanzee, cow, deer, dog, giraffe, goat, guinea pig, hamster, hippopotamus, horse, human, mouse, non-human primate, ovine, boar, pig, pronghorn, rabbit, rat, macaque, rhinoceros, sheep, tapir, and ungulate, or any other mammalian or non-mammalian cell type.

[0098] Target cells (e.g., stem cells) may be identified by the expression of one or more stem cell markers. It may be desirable to further ensure that cells expressing target cell markers are indeed target cells by testing for the expression of one or more target cell markers (e.g., two, three, four, etc.). For example, a "cocktail" of antibodies against different markers may each be coupled (directly or indirectly) to the same or different labels. As an example, a cocktail of antibodies against different markers may each contain a binding motif that binds to the same label (e.g., each may contain the same species of Fc recognized by the same secondary antibody, or each may be biotinylated and specifically bound by the same avidin-coupled label). Optionally, two or more different antibodies or cocktails of antibodies may be utilized. Preferably, cells are stained with at least two labels that are distinguishable from one another, allowing for the identification of cells expressing at least two different markers of the target cell type. Cells may also be stained with at least three, four, five, or more different labels that are distinguishable from one another, allowing for the detection of cells that express markers of more target cell types. In some cases, a cell may be identified as a cell of the target type if it expresses a preselected number of markers, or a particular preselected combination of markers.

[0099] Furthermore, markers of the target cell type need not be unique to the target cell, as long as they allow for the differentiation of the target cell from other cells in the population. For example, alkaline phosphatase may be used as a suitable marker for detecting hES cells, even though other cell types also express alkaline phosphatase within a population of RPE, as alkaline phosphatase is not normally expressed by RPE.

[0100] Exemplary embryonic stem cell markers include alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster specified 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

[0101] For some exemplary cell types, cells are considered positive for a given marker only if that marker exhibits a characteristic localization or pattern within the cell. For example, a cytoskeletal marker may be considered "positive" if present in the cytoskeleton and "negative" if there is any diffuse cytoplasmic staining. In such cases, cells may be cultured under appropriate conditions (e.g., adherent culture) to establish the characteristic localization or pattern within the cell. Suitable culture conditions and time for cytoskeletal assembly (or other processes for establishing subcellular organization), which may be necessary for robust detection of a given marker, are readily determined by those skilled in the art. Furthermore, markers can be readily selected that reduce or eliminate the need for adherent culture as a precondition for robust staining. For example, nonadherent cell populations may be stained after a cytospin-like procedure (in which cells lose their normal morphology while being crushed onto a slide by centrifugal force); suitable markers for use in such situations are well known or readily discoverable.

[0102] In addition to the stem cell markers described above, the methods of the present disclosure may be used with other cell types. Markers for other exemplary cell populations or target cell types that can be used in accordance with embodiments of the methods of the present disclosure are listed in Table 1 below.

[0103] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0104] The RPE cells may express RPE cell markers. For example, the expression levels of RPE cell genes RPE65, PAX2, PAX6, and tyrosinase, bestrophin, PEDF, CRALBP, Otx2, and MITF may be equivalent to those in naturally occurring RPE cells. The maturity level of RPE cells may be assessed by the expression of at least one of PAX2, PAX6, and tyrosinase, or the expression level of each of them.

[0105] On the other hand, RPE cells may not express ES cell markers. For example, the expression levels of ES cell genes Oct-4, NANOG, and / or Rex1 may be approximately 100-1000 times lower in RPE cells than in ES cells. For example, RPE cells may express octamer-binding transcription factor 4 (Oct-4, also known as POU5F1), stage-specific embryonic antigen (SSEA) 3, and other markers. and may substantially lack expression of ES cell markers, including, but not limited to, SSEA4, tumor rejection antigen (TRA)-1-60, TRA-1-80, alkaline phosphatase, NANOG, and Rex1. That is, compared to ES cells, RPE cells substantially lack expression of Oct-4, NANONG, and / or Rex1.

[0106] Preparation of RPE cells

[0107] The invention provides preparations of indicators indicative of the number or proportion of RPE cells and other sex cells present, where the value of the indicator may be determined by applying the methods described herein to a cell population representative of the cells in the preparation. The invention described herein provides RPE cells, substantially purified populations of RPE cells, pharmaceutical preparations comprising RPE cells, and cryopreserved preparations of RPE cells. The RPE cells described herein may be substantially free (e.g., "isolated") from at least one protein, molecule, or other contaminant present in its natural environment. RPE cells may be derived from mammalian, e.g., human, RPE cells. The present invention also provides human RPE cells, substantially purified populations of human RPE cells, pharmaceutical preparations comprising human RPE cells, and cryopreserved preparations of human RPE cells. The preparations may be human embryonic stem cell-derived RPE cells, preparations comprising human iPS cell-derived RPE cells, and substantially purified (with respect to non-RPE cells) preparations comprising differentiated ES cell-derived RPE cells.

[0108] A population of RPE cells may include differentiated RPE cells at various levels of maturity, or may be substantially pure with respect to differentiated RPE cells at a particular level of maturity. RPE cells may be a substantially purified preparation containing RPE cells at various levels of maturity / pigmentation. For example, a substantially purified culture of RPE cells may contain both differentiated RPE cells and mature differentiated RPE cells. The level of pigmentation among mature RPE cells may vary widely. However, mature RPE cells may be visually distinguishable from RPE cells based on their elevated levels of pigmentation and more columnar shape. A substantially purified preparation of RPE cells may include RPE cells at various levels of maturity (e.g., differentiated RPE cells and mature differentiated RPE cells). In such cases, there may be variability in the expression of markers indicative of pigmentation between preparations. The pigmentation of RPE cells in cell culture may be homogeneous. Furthermore, the pigmentation of RPE cells in cell culture may be heterogeneous, and a culture of RPE cells may include both differentiated RPE cells and mature RPE cells. Preparations containing RPE cells are substantially pure with respect to non-RPE cell types, but include preparations containing a mixture of differentiated RPE cells and mature differentiated RPE cells. Preparations containing RPE cells also include preparations that are substantially pure with respect to both non-RPE cell types and with respect to RPE cells at other levels of maturity.

[0109] The percentage of mature, differentiated RPE cells in the culture may be reduced by decreasing the density of the culture. That is, the methods described herein may further include subculturing a population of mature RPE cells to produce a culture containing a smaller percentage of mature RPE cells. The number of RPE cells in a preparation includes differentiated RPE cells, regardless of their level of maturity and regardless of the relative percentages of differentiated RPE cells and mature, differentiated RPE cells. The number of RPE cells in a preparation refers to the number of either differentiated RPE cells or mature RPE cells. The preparation may contain 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.

[0110] The present invention provides cell cultures comprising human RPE cells that are pigmented and express at least one gene 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. RPE cells may vary in their level of maturity with respect to the expression of one or more of PAX2, Pax6, MITF, and / or tyrosinase. Such variations in pigmentation after differentiation also correlate with changes in PAX2 expression. Mature RPE cells may be distinguished from RPE cells by their level of pigmentation and the expression levels of PAX2, Pax6, and / or tyrosinase. For example, mature RPE cells may have higher levels of pigmentation or higher levels of PAX2, Pax6, and / or tyrosinase expression compared to RPE cells.

[0111] The preparation may be substantially purified with respect to non-RPE cells and contain at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% RPE cells. For example, a substantially purified preparation of RPE cells may contain less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of non-RPE cell types. For example, an RPE cell preparation may contain less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, or 0.02% of non-RPE cell types. %, 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.

[0112] RPE cell preparations may be substantially pure with respect to both non-RPE cells and RPE cells of other levels of maturity. The preparation may be substantially purified with respect to non-RPE cells and enriched with respect to 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 with respect to 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.

[0113] RPE cell preparations should contain at least approximately 1 x 10 3 , 2x10 3 , 3x10 3 , 4x10 3 , 5x10 3 , 6x10 3 , 7x10 3 , 8x10 3 , 9x10 3 , 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x106 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 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, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 100,000, or 500,000 RPE cells.

[0114] RPE cell preparations should contain at least approximately 1 x 10 3 , 2x10 3 , 3x10 3 , 4x10 3 , 5x10 3 , 6x10 3 , 7x10 3 , 8x10 3 , 9x10 3 , 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x1 0 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x106 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 10 The RPE cell preparation may contain 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 cells / ml. The RPE cell preparation may contain at least about 20,000 to 50,000 RPE cells / ml. Alternatively, the RPE cell preparation may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 100,000 or 500,000 RPE cells per ml.

[0115] The preparations described herein may be substantially free of bacterial, viral or fungal contamination or infection, such as, but not limited to, the presence of HIV1, HIV2, HBV, HCV, CMV, HTLV1, HTLV2, parvovirus B19, Epstein-Barr virus, or herpesvirus 6. The preparations described herein may be substantially free of mycoplasma contamination or infection.

[0116] The RPE cells described herein may also act as functional RPE cells after transplantation, in which case the RPE cells form a monolayer between the neurosensory retina and the choroid in the patient receiving the transplanted cells. The RPE cells may also provide nutrients to adjacent photoreceptors and discard used photoreceptor outer segments through phagocytosis. Furthermore, the RPE cells described herein may age more slowly than cells derived from an ocular donor (e.g., the RPE cells are "younger" than those of an ocular donor). This allows the RPE cells described herein to have a longer lifespan than cells derived from an ocular donor.

[0117] Preparations containing RPE cells may be manufactured in accordance with Good Manufacturing Practices (GMP) (e.g., the preparation is GMP compliant) and / or current Good Tissue Practices (GTP) (e.g., the preparation may be GTP compliant). It may be prepared as follows.

[0118] RPE cell culture

[0119] The present invention also provides substantially purified cultures of RPE cells, including human RPE cells, and indicators of the number or percentage of pluripotent RPE cells present, wherein the value of the indicator may be determined by applying the methods described herein to a representative population of cells in the culture. 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 1x10 4、2x10 4 、3x10 4 、4x10 4 、5x10 4 、6x10 4 、7x10 4 、8x10 4 、9x10 4 、1x10 5 、2x10 5 、3x10 5 、4x10 5 、5x10 5 、6x10 5 、7x10 5 、8x10 5 、9x10 5 、1x10 6 、2x10 6 、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x 10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10, 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 10 The RPE cells may comprise:

[0120] RPE cells may be further cultured to produce a culture of mature RPE cells. RPE cells may be matured and brought to a desired level of maturity by further culturing the RPE cells, for example, in MDBKMM medium. This may be achieved by monitoring the increase in pigmentation levels during maturation. Functionally equivalent or similar media may be used as an alternative to MDBKMM medium. Regardless of the specific medium used to mature RPE cells, the medium may optionally be supplemented with growth factors or agents. Both RPE cells and mature RPE cells are differentiated RPE cells. However, mature RPE cells are characterized by increased levels of pigment compared to differentiated RPE cells. The level of maturation and pigmentation may be adjusted by increasing or decreasing the density of the culture of differentiated RPE cells. That is, mature RPE cells may be produced by further culturing the RPE cell culture. Alternatively, the density of the culture containing mature RPE cells may be decreased to decrease the percentage of mature, differentiated RPE cells and increase the percentage of differentiated RPE cells.

[0121] RPE cells may be identified by comparing the messenger RNA transcripts of such cells with those of cells derived in vivo. Aliquots of cells are harvested at various time intervals during the differentiation of embryonic stem cells into RPE cells and assayed for expression of any of the markers described above. These characteristics allow differentiated RPE cells to be distinguished.

[0122] 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.

[0123] The RPE cell cultures are prepared according to Good Manufacturing Practices (GMP) (e.g., the cultures are GMP compliant) and / or current Good Tissue Practices (GTP) (e.g., the cultures can be GTP compliant). You may.

[0124] Cryopreserved preparation of RPE cells

[0125] RPE cells may be frozen for storage. For example, a portion of the RPE cell population may be analyzed by the methods described herein to detect the presence of any residual hES cells in the population, and the remainder of the population may be cryopreserved to determine an approximate concentration of hES cells in the cell population. The frozen RPE cells may be associated with an indicator that indicates the number or concentration of hES cells detected in the population or that otherwise indicates the result of the assay, e.g., an indicator that indicates that the cells "passed" the test for hES cell contamination because the detected number or concentration of hES cells was below an established marketing threshold and therefore the cells are considered suitable for use.

[0126] RPE cells may be stored by any suitable method known in the art (e.g., deep freezing) and may be frozen at any temperature suitable for storing the 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, or any other temperature suitable for storing the cells. Cryogenically 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 cryopreserved immediately after differentiation, after in vitro maturation, or after a period of culture. RPE cells may also be maintained at room temperature or refrigerated, for example, at about 4°C.

[0127] Methods for cryopreserving RPE cells are also provided. RPE cells may be harvested, washed with buffer or medium, counted, concentrated (via centrifugation), formulated into freezing medium (e.g., 90% FBS / 10% DMSO), or subjected to any combination of these steps. For example, RPE cells may be seeded into several culture vessels and serially expanded. RPE cells may be harvested and maintained in FBS at approximately 4°C, and several flasks of RPE cells may be combined into a single lot. RPE cells may also be washed at least 1, 2, 3, 4, or 5 times with saline (e.g., DPBS). Additionally, RPE cells may be cryopreserved after dystrophin has been organized at the cell membrane and PAX6 expression has reached a low level. Additionally, 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., 1x10 cells), and cell number (e.g., 1x10 cells). 6 The label may include information such as the number of vials per mL, the number of vials per mL, the expiration date (e.g., the last date the vial should be consumed), manufacturing information (e.g., name and address), warnings, and storage instructions (e.g., store in liquid nitrogen).

[0128] The cryopreserved RPE cell preparations described herein may comprise at least about 50,000-100,000 RPE cells. The cryopreserved RPE cell preparations may also comprise at least about 20,000-500,000 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 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 RPE cells. Cryopreserved RPE cell preparations may contain at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x107 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 or 9x10 9 The RPE cells of the cryopreserved RPE cell population may be mammalian RPE cells, for example human RPE cells.

[0129] Additionally, the cryopreserved RPE cell preparations described herein may contain at least about 50,000-100,000 RPE cells / ml. The cryopreserved RPE cell preparations may also contain at least about 20,000-500,000 RPE cells / ml. Additionally, the cryopreserved RPE cell preparations may contain 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 / ml. The cryopreserved RPE cell preparation 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, 1x10 RPE cells / ml. 4 , 2x104 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 or 9x10 9The RPE cells of the cryopreserved RPE cell population may be mammalian RPE cells, for example human RPE cells.

[0130] The RPE cells of the present invention 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 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the RPE cells may retain viability and differentiation after cryopreservation. Furthermore, the RPE cells of the present invention 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 invention may 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 invention 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 invention may be cryopreserved for at least about 4 years and exhibit at least about 80% viability. The cryopreserved preparations comprising RPE cells are substantially free of DMSO.

[0131] Methods for producing RPE cells

[0132] The cell populations analyzed by the subject methods may be produced from pluripotent stem cells. Cell types that may be produced include, but are not limited to, RPE cells, RPE precursor cells, iris pigmented epithelial (IPE) cells, and other vision-related neural cells, such as interneurons (e.g., "relay" neurons of the inner nuclear layer (INL)) and amacrine cells. Additionally, retinal cells, rods, cones, and corneal cells may also be produced. Cells that provide the ocular vasculature may also be produced by the methods described herein.

[0133] Without being bound by any particular theory, the inventors have discovered that the methods described herein may induce the expression of paired box 6 (PAX6) transcription factor by signaling through FGF, EGF, WNT4, TGF-beta, and / or oxidative stress, MAP kinase, and potentially cJun terminal kinase pathways. PAX6 acts synergistically with PAX2 to induce terminal differentiation of mature RPE through coordination with MITF and Otx2, thereby transcribing RPE-specific genes such as tyrosinase (Try) and downstream targets such as RPE 65, bestrophin, CRALBP, and PEDF. See Figure 1 of WO2009 / 051671.

[0134] The RPE cells described herein may be differentiated from pluripotent stem cells, such as human embryonic stem cells, and may be molecularly distinguishable from embryonic stem cells, adult-derived RPE cells, and fetal-derived RPE cells. For example, the manufacturing process steps described herein may be used to ensure that these cells closely resemble native RPE cells and are not fetal-derived, relative to the final RPE cell product. They may be endowed with distinct structural and functional properties that make them distinguishable from conventional RPE cells or RPE cell lines (eg, APRE19).

[0135] Exemplary methods for producing RPE cells include (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells as embryoid bodies in a nutrient-rich, low-protein medium, wherein the medium optionally contains a serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium, wherein the medium optionally contains a serum-free B27 supplement; (d) culturing adherent cultures of the cells of (c) in a nutrient-rich, low-protein medium, wherein the medium does not contain a serum-free B27 supplement; and (e) supporting the growth of high-density somatic cell cultures. (d) generating RPE cells in cell culture by culturing the cells of (d) in a medium capable of dissociating the cells or cell aggregates from the medium of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme or another dissociation medium); (f) dissociating the cells or cell aggregates from the medium of (e), preferably mechanically or chemically (e.g., using a protease or other enzyme or another dissociation medium); (g) creating an enriched culture of RPE cells by selecting the RPE cells from the medium and transferring the RPE cells into another culture medium containing a medium supplemented with growth factors; and (g) producing RPE cells by expanding the enriched culture of RPE cells. These method steps may be performed at least once to produce a substantially purified culture of RPE cells. 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.

[0136] Furthermore, the present invention also provides for the production of RPE cells by (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells as embryoid bodies in a nutrient-rich, low-protein medium, wherein the medium optionally contains a serum-free B27 supplement; (c) culturing the embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium, wherein the medium optionally contains a serum-free B27 supplement; (d) culturing adherent cultures of the cells of (c) in a nutrient-rich, low-protein medium, wherein the medium does not contain a serum-free B27 supplement; and (e) culturing the cells of (d) in a medium capable of supporting the growth of high-density somatic cell cultures. (f) preferably mechanically or chemically dissociating the cells or cell aggregates from the medium of (e) (e.g., using a protease or other enzyme or another dissociation medium); (g) creating an enriched culture of RPE cells by selecting RPE cells from the medium and transferring the RPE cells into another culture medium containing a medium supplemented with growth factors; (h) expanding the enriched culture of RPE cells; and (i) producing mature RPE cells by culturing the enriched culture of RPE cells. These method steps may be performed at least once to produce a substantially purified culture of RPE cells. 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.

[0137] For any of the specified steps, the cells may be cultured for at least about 1 to 10 weeks. For example, the cells may be cultured for at least about 3 to 6 weeks. For any of the specified steps, the cells may be cultured for about 1 to 50 days, for example, at least about 1 to 3, 3 to 4, 7, 4 to 9, 7 to 10, 7 to 12, 8 to 11, 9 to 12, 7 to 14, 14 to 21, and 3 to 45 days. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days. The cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. For example, the cells may be cultured for 2-4 and 3-6 hours. For any of the above-specified method steps, the cells may be cultured for the same amount of time at each step, or for different amounts of time at one or more of the steps. Additionally, any of the steps of the methods specified above may be repeated to produce more RPE cells (eg, scaled up to produce large numbers of RPE cells).

[0138] In the methods described herein, RPE cells may be differentiated from cell populations in adherent cultures of EBs. RPE cells may be visually recognized based on their cobblestone morphology and initial appearance of pigmentation. As RPE cells continue to differentiate, clusters of RPE cells may be observed.

[0139] Mechanical or enzymatic methods may be used to select RPE cells from clusters of non-RPE cells in embryoid body cultures or to facilitate subculture of adherent cells. Exemplary mechanical methods include, but are not limited to, titration with a pipette or cutting with a needle. Exemplary enzymatic methods include, but are not limited to, any enzyme suitable for dissociating cells (e.g., trypsin (e.g., trypsin / EDTA), collagenase (e.g., collagenase B, collagenase IV), dispase, papain, a mixture of collagenase and dispase, or a mixture of collagenase and trypsin). Non-enzymatic solutions, such as solutions containing high EDTA, such as Hanks-based cell dissociation buffer, may also be used to dissociate cells.

[0140] RPE cells may be differentiated from embryoid bodies. RPE cells can be propagated in vitro in enriched media by isolating them from EBs. In the case of human cells, RPE cells may be obtained from EBs grown for less than 90 days. Furthermore, RPE cells may be derived from human EBs grown for at least about 7-14 days, 14-28 days, 28-45 days, or 45-90 days. The medium used to culture pluripotent stem cells, embryoid bodies, and RPE cells may be removed and / or replaced with the same or different medium at any time interval. For example, the medium may be removed and / or replaced at least about 0-7 days, 7-10 days, 10-14 days, 14-28 days, or 28-90 days. Furthermore, the medium may be changed at least daily, every other day, or at least every three days.

[0141] To enrich for RPE and establish substantially purified cultures of RPE cells, RPE cells may be dissociated from each other and from non-RPE cells using mechanical and / or chemical (including enzymatic) methods, and the RPE cell suspension may then be transferred to new medium and new culture vessels to form an enriched population of RPE cells.

[0142] RPE cells may be selected from dissociated cells and cultured separately to produce substantially purified cultures of RPE cells. 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 exist, including cellular retinaldehyde-binding protein (CRALBP), a cytoplasmic protein present in apical microvilli; RPE65, a cytoplasmic protein involved in retinoid metabolism; bestrophin, a product of the Best vitelliform macular degeneration gene (VMD2); and pigment epithelium-derived factor (PEDF), a 48 kD secreted protein with antiangiogenic 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 immunoblot techniques or Western blot.

[0143] RPE cells may also be selected based on cell function, e.g., phagocytosis of wasted rod and cone outer segments (or phagocytosis of another substrate, e.g., polystyrene beads), absorption of stray light, vitamin A metabolism, retinal regeneration, and tissue repair. Assessment may also be performed to assess in vivo function following RPE cell transplantation into a suitable host animal (e.g., a human or non-human animal suffering from a naturally occurring or induced state of retinal degeneration) using, e.g., behavioral tests, fluorescence angiography, histology, and the like. This may be done by testing using texture analysis, the conductivity of the adhesive bond, or by evaluation using an electron microscope.

[0144] The enriched clusters of RPE cells may be cultured in a suitable medium, such as EGM2 medium. This, and functionally equivalent or similar media, 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 in culture for extended periods (e.g., greater than 6 weeks).

[0145] pluripotent stem cells

[0146] The methods described herein may use differentiated cells (e.g., RPE cells) produced 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- or morula-stage embryo (optionally without destruction of the remainder of the embryo). Such embryonic stem cells may be formed from embryonic material produced by fertilization or asexual means, such as 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.

[0147] Pluripotent stem cells (e.g., hES cells) may be cultured in suspension to produce embryoid bodies (EBs). The embryoid bodies may be cultured in suspension for approximately 7-14 days. However, in certain embodiments, the EBs may be cultured in suspension for less than 7 days (7, 6, 5, 4, 3, 2, or less than 1 day) or for more than 14 days. The EBs may be cultured in medium supplemented with B27 supplement.

[0148] After culturing EBs in suspension culture, the EBs may be transferred to form adherent cultures. For example, the EBs may be plated on gelatin-coated plates in medium. When cultured as adherent cultures, the EBs may be cultured in the same type of medium as when grown in suspension. When culturing cells as adherent cultures, the medium may not be supplemented with B27 supplements. Alternatively, the medium may be supplemented with B27 initially (e.g., within about 7 days) but then cultured in the absence of B27 for the remainder of the adherent culture period. 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.

[0149] human embryonic stem cells

[0150] Human embryonic stem (hES) cells may be used as pluripotent stem cells in the methods described herein. Human embryonic stem cells (hES) include the progeny of the inner cell mass (ICM) of a blastocyst, or cells from another source, and may maintain pluripotency virtually indefinitely. hES cells may be derived from one or more blastomeres of an early cleavage stage embryo, optionally without destroying or damaging the embryo. hES cells may be generated using nuclear transfer. hES cells may also be induced pluripotent cells (iPS cells), which are described in more detail below. Cryopreserved hES cells may also be used. hES cells may be cultured in any manner known in the art, for example, with or without feeder cells. For example, hES cells may be cultured in MDBKGM, hESC medium, INVITROGEN®, or other media. hESCs may be cultured in standard stem cell medium, OptiProSFM, VPSFM, EGM2, or MDBKMM. See, for example, Stem Cell Information (Culture of human Embryonic Stem Cells (hESC)) [NIH website, 2010]. hESCs are cultured in accordance with GMP standards. may be used and maintained.

[0151] When grown in culture on feeder layers under defined conditions, hES cells maintain a specific morphology, forming flat colonies of small, tightly packed cells with a high nuclear to cytoplasmic ratio, distinct cell-cell boundaries, and sharp, refractile colony boundaries. hES cells express a series of molecular markers, such as octamer-binding protein (Oct-4, also known as Pou5f1), stage-specific embryonic antigen (SSEA) 3 and SSEA 4, tumor rejection, and endothelial cell death. They express the transcription factors (TRA)-1-60, TRA-1-80, alkaline phosphatase, NANOG, and Rex1. Similar to cells of the ICM that 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 described herein.

[0152] Human embryonic stem cells that may be used include, but are not limited to, MA01, MA04, MA09, ACT4, MA03, H1, H7, H9, and H14. Additional exemplary cell lines include NED1, NED2, NED3, NED4, and NED5. Reference may also be made to the Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that may be used is MA09 cells. The isolation and preparation of MA09 cells has previously been described in Klimanskaya et al. (2006) "Human Embryonic Stem Cell Lines Derived from Single Blastomeres." Nature 444:481-485.

[0153] hES cells may initially be co-cultured with murine pulmonary feeder cells (MEF) cells. The co-culture may be seeded with hES cells after the MEF cells have been mitotically inactivated by exposure to mitomycin C, thereby preventing the MEFs from expanding in culture. Furthermore, the hES cell culture may be examined microscopically, and colonies containing non-hES cell morphology may be picked and discarded, for example, using a stem cell cutting tool, laser ablation, or other means. Typically, after the point of harvesting hES cells for seeding for embryoid body formation, no additional MEF cells are used in the process. The time between MEF removal and RPE cell harvest as described herein may be a minimum of at least 1, 2, 3, 4, or 5 passages in MEF-free cell culture, and at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days. The time between MEF removal and RPE cell harvest may also be at least about 3 passages and at least about 80-90 days in MEF-free cell culture. For the methods of production described herein, the culture and preparation of PE cells described herein may be substantially free of mouse embryonic fibroblasts (MEFs) and human embryonic stem cells (hES).

[0154] Induced pluripotent stem cells (iPS cells)

[0155] Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) formed by reprogramming somatic cells by expressing or inducing expression of a combination of factors ("reprogramming factors"). iPS cells may be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. iPS cells may be obtained from cell banks. Alternatively, iPS cells may be generated de novo by methods known in the art before initiating differentiation into RPE cells or other cell types. iPS cell generation may be an initial step in the production of differentiated cells. iPS cells may be specifically generated using material obtained from a particular patient or matched donor, with the goal of generating tissue-matched RPE cells. iPS cells are produced from cells that are substantially non-immunogenic in the intended recipient, e.g., from autologous cells or from cells that are histocompatible with the intended recipient. can be produced.

[0156] Induced pluripotent stem cells may be produced 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 somatic cells. The somatic cells are reprogrammed by expression at least one, two, three, four, or five times. The reprogramming factors may be selected from Oct3 / 4, Sox2, NANOG, Lin28, c-Myc, 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 factor.

[0157] Somatic cells can also be reprogrammed using combinatorial methods, in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules). For example, reprogramming factors can be expressed in somatic cells by infection with a viral vector, such as a retroviral vector or a lentiviral vector. Reprogramming factors can also be expressed in somatic cells using a non-integrating vector, such as an episomal plasmid. When reprogramming factors are expressed using a non-integrating vector, the factors can be expressed using electroporation, transfection, or transformation of somatic cells with the vector. For example, in mouse cells, expression of four factors (Oct3 / 4, Sox2, c-myc, and Klf4) using an integrating viral vector is sufficient to reprogram somatic cells. In human cells, expression of four factors (Oct3 / 4, Sox2, NANOG, and Lin28) using an integrating viral vector is sufficient to reprogram somatic cells.

[0158] After expressing the reprogramming factors 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 a selectable or detectable marker. Culturing the cells may produce a culture of cells that resemble ES cells, which become putative iPS cells.

[0159] To confirm the pluripotency of iPS cells, cells can be tested in one or more pluripotency assays.For example, cells can be tested for the expression of ES cell markers; cells can be evaluated for the ability to form teratomas when transplanted into SCID mice; cells can be evaluated for the ability to differentiate and generate the cell types of all three germ layers.After pluripotent iPS cells are obtained, they can be used to produce RPE cells.

[0160] Manipulation of MHC genes in human embryonic stem cells to obtain low-complexity differentiated cells

[0161] Human embryonic stem (hES) cells may be derived from a library of human embryonic stem cells. The library of human embryonic stem cells may comprise stem cells, each of which is hemizygous, homozygous, or nullzygous for at least one MHC allele present in a human population, where each member of the library of stem cells is hemizygous, homozygous, or nullzygous for a different set of MHC alleles relative to the remainder of the library. The library of human embryonic stem cells may comprise stem cells that are hemizygous, homozygous, or nullzygous for all MHC alleles present in a human population. In the context of the present invention, stem cells that are homozygous for one or more histocompatibility antigen genes encompass cells that are nullzygous for one or more (and in some embodiments, all) such genes. Nullzygous for a locus means that the gene is null at that locus (i.e., both alleles of the gene are deleted or inactivated).

[0162] hES cells may contain modifications to one of the sister chromosome alleles in the cell's MHC complex. Genes in the MHC complex may be modified using various methods for producing gene modifications, such as gene targeting. Furthermore, the modified MHC complex allele in the cell may then be engineered to be homozygous, so that the same allele is present on the sister chromosome. Cells may be engineered to have homozygous alleles in the MHC complex using methods such as loss of heterozygosity (LOH). For example, hemizygous cells can be generated by targeting one or more genes in a set of MHC genes derived from parental alleles. Other sets of MHC genes can be removed by gene targeting or LOH to create null lines. These null lines can then be used as embryonic cell lines with drop arrays of HLA genes or individual genes to create hemizygous or homozygous banks with otherwise uniform genetic backgrounds. Stem cells that are null for all MHC genes may be produced by standard methods known in the art, such as gene targeting and / or loss of heterozygosity (LOH). See, for example, U.S. Patent Application Publications 2004 / 0091936, 2003 / 0217374, and 2003 / 0232430, and U.S. Provisional Patent Application 60 / 729,173.

[0163] Thus, the present invention relates to methods for obtaining differentiated cells (e.g., RPE cells), including libraries of differentiated cells with reduced MHC complexity. Differentiated cells with reduced MHC complexity may be used to increase the supply of cells available for therapeutic use, as this may eliminate difficulties associated with patient matching. Such cells may be derived from stem cells engineered to be hemizygous or homozygous for genes in the MHC complex.

[0164] The present invention also provides libraries of differentiated cells (e.g., RPE cells and / or RPE lineage cells), in which several lineages of ES cells are selected and differentiated into differentiated cells. These differentiated cells may be used for patients in need of cell therapy. The present invention also provides libraries of differentiated cells, each of which is hemizygous, homozygous, or nullzygous for at least one MHC allele present in a human population, wherein each member of the library of differentiated cells is hemizygous, homozygous, or nullzygous for a different set of MHC alleles relative to the remainder of the library. The present invention provides libraries of human differentiated cells that are hemizygous, homozygous, or nullzygous for all MHC alleles present in a human population.

[0165] Culture medium

[0166] Any medium capable of supporting high-density culture, such as a medium for culturing viruses, bacteria, or eukaryotic cells, may be used in the methods described herein. For example, the medium may be a high-nutrient protein-free medium or a high-nutrient low-protein medium. Additionally, the medium may also include nutritional components such as albumin, B27 supplement, ethanolamine, fetuin, glutamine, insulin, peptone, purified lipoprotein material, sodium selenite, transferrin, vitamin A, vitamin C, or vitamin E. For example, a nutrient-rich low-protein medium may be any medium that supports cell growth in culture and has a low protein content. For example, a nutrient-rich low-protein medium includes, but is not limited to, MDBK GM, OptiPro SFM, VPSFM, DMEM, RPMI Medium 1640, IDMEM, MEM, F-12 Nutrient Mix, F-10 Nutrient Mix EGM2, DMEM / F-12 Medium, Medium 1999, or MDBKMM. See also Table 2. Additionally, the nutrient-rich, low-protein medium may be a medium that does not support the growth or maintenance of embryonic stem cells.

[0167] When using low-protein media, the media should be at least approximately 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% The low-protein medium may contain 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). However, it should be noted that when referring to the percentage of protein present in a low-protein medium, it refers to the medium alone and does not account for protein present in, for example, a B27 supplement. That is, it is understood that when cells are cultured in a low-protein medium and a B27 supplement, the percentage of protein present in the medium may be higher.

[0168] Low-protein or protein-free medium is supplemented with a serum-free B27 supplement. 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-threonine (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 medium supplemented with B27, protein-free refers to the medium before the addition of B27.

[0169] The growth factors, agents, and other supplements described herein may be used alone or in combination with other factors, agents, or supplements for inclusion in the medium. The factors, agents, and supplements may be added to the medium immediately, at any time during cell culture, or thereafter.

[0170] The medium may also contain supplements such as heparin, hydrocortisone, ascorbic acid, serum (e.g., fetal bovine serum), or growth matrix (e.g., bovine corneal epithelium-derived extracellular matrix, MATRIGEL® (basement membrane matrix) or gelatin), fibronectin, proteolytic fragments of fibronectin, laminin, thrombospondin, aggrecan, and syndesan.

[0171] The medium may be supplemented with one or more factors or agents.

[0172] 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 invention also include 6Ckine (recombinant), activin A, α-interferon, alpha-interferon, amphiregulin, angiogenin, beta-endothelial cell growth factor, betacellulin, beta-interferon, brain-derived auxotrophic factor, cardiotrophin 1, ciliary auxotrophic factor, cytokine-derived neutrophil chemoattractant 1, endothelial cell growth supplement, eotaxin, epidermal growth factor, epidermal neutrophil-activating peptide 78, erythropoietin, estrogen receptor-α, estrogen receptor-β, fibroblast growth factor (acidic / basic, heparin-stabilized, recombinant), FLT3 / FLK2 ligand (FLT3 ligand), gamma-interferon, glial cell lineage-derived auxotrophic factor, GlyHisLys, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, GRO-alpha / MGSA, GROB, GRO-gamma, HCC1, heparin IGF-binding epidermal growth factor-like growth factor, hepatocyte growth factor, heregulin alpha (EGF domain), insulin growth factor binding protein 1, insulin-like growth factor binding protein 1 / IGF1 complex, insulin-like growth factor, insulin-like growth factor II, 2.5S nerve growth factor (NGF), 7SNGF, macrophage inflammatory protein 1β, macrophage inflammatory protein 2, macrophage inflammatory protein 3α, macrophage inflammatory protein 3β, monocyte chemotactic protein 1, monocyte chemotactic protein Sex protein 2, monocyte chemotactic protein 3, neurotrophin 3, neurotrophin 4, NGF beta (human or rat recombinant), oncostatin M (human or mouse recombinant), pituitary extract, placental growth factor, oncostatin endothelial growth factor, oncostatin 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 These include 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.

[0173] Agents that may be used in accordance with the present invention include cytokines such as interferon-α, interferon-αA / D, interferon-β, interferon-γ, interferon-γ-inducible protein 10, interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, interleukin 15, interleukin 17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony-stimulating factor, and macrophage inflammatory protein 1-α.

[0174] The medium may be supplemented with hormones and hormone antagonists, including, but not limited to, 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-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, luteinizing hormone, L-thyroxine, melatonin, MZ4, oxytocin, parathyroid hormone, PEC60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone-binding globulin, thyroid-stimulating hormone, thyrotropin-releasing factor, thyroxine-binding globulin, and vasopressin.The culture medium contained antibodies against low-density lipoprotein receptor, anti-progesterone receptor, intracellular antibody, anti-alpha interferon receptor chain 2, anti-cc chemokine receptor 1, anti-CD118, anti-CD119, anti-colony stimulating factor 1, anti-CSF1 receptor / c-fins, anti-epidermal growth factor (AB3), anti-epidermal growth factor receptor, anti-epidermal growth factor receptor phospho-specific antibody, anti-epidermal growth factor (AB1), 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, anti-gamma interferon receptor chain, anti-gamma interferon human recombinant, anti-GFR alpha 1 C-terminal, anti-GFR alpha 2 C-terminal, anti-granulocyte colony-stimulating factor (AB1), and anti-granulocyte colony-stimulating factor receptor. Antibodies against various factors may be supplemented, including, but not limited to, anti-insulin receptor antibody, anti-insulin-like growth factor 1 receptor antibody, anti-interleukin-6 human recombinant antibody, anti-interleukin-1 human recombinant antibody, anti-interleukin-2 human recombinant antibody, anti-leptin mouse recombinant antibody, anti-nerve growth factor receptor antibody, anti-p60, chicken antibody, anti-parathyroid hormone-like protein antibody, anti-oncostatin growth factor receptor antibody, anti-oncostatin growth factor receptor B antibody, anti-oncostatin growth factor-alpha antibody, anti-progesterone receptor antibody, anti-retinoic acid receptor-alpha antibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroid hormone nuclear receptor-alpha 1 / Bi antibody, anti-transferrin receptor / CD71 antibody, anti-transformation growth factor-alpha antibody, anti-transformation growth factor B3 antibody, anti-tumor necrosis factor-alpha antibody, and anti-vascular endothelial growth factor antibody.

[0175] Exemplary culture media suitable for use in the methods described herein are listed in Table 2.

[0176] [Table 2-1] [Table 2-2]

[0177] Retinal pigment epithelium (RPE)

[0178] The retinal pigment epithelium (RPE) is the outer pigmented cell layer of the neurosensory retina, between the underlying choroid (the layer of blood vessels behind the retina) and the overlying retinal visual cells (e.g., photoreceptors—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; phagocytosis of rod photoreceptor outer segments; transporting iron and small molecules between the retina and choroid; maintaining Bruch's membrane; and absorbing stray light to provide good visual resolution. See Engelmann and Valtink (2004) “RPE Cell Cultivation.” Graefe's Archive for Clinical and Experimental Ophthalmology 242(1): 65-67; ​​also Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, in Stem Cell ANTHOLOGY 335-346 (Bruce Carlson ed., 2009).

[0179] Mature RPE cells are characterized by their cobblestone morphology of black pigmented cells and by RPE cell markers such as cellular retinaldehyde-binding protein (CRALBP), a 36 kD cytoplasmic retinaldehyde-binding protein present in apical microvilli (Eisenfeld et al., (1985) Experimental Research 41(3):299-304); RPE65, a 65 kD cytoplasmic protein involved in retinoid metabolism (Ma et al., (2001) Invest Opthalmol Vis Sci. 42(7):1429-35; Redmond (2009) Exp Eye Res. 88(5):846-847); bestrophin, a membrane protein encoding the Best vitelliform macular degeneration gene (VMD2). A localized 68 kD product (Marmorstein et al., (2000) PNAS 97(23): 12758-12763) and derived from pigment epithelium Physiological endocrine dehydrogenase (PEDF), a 48 kD secreted protein with antiangiogenic properties (Karakousis et al., (2001) Molecular Vision 7:154-163; Jablonski et al., (2000) The Journal of Neuroscience 20(19): 7149‐7157).

[0180] Retinal diseases

[0181] Degeneration of the RPE can lead to retinal detachment, retinal dysplasia, or retinal transplantation, which is associated with many vision-altering diseases that result in photoreceptor damage and blindness, such as congenital choroideremia, diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava). WO2009 / 051671.

[0182] Congenital choroideremia. Congenital choroideremia is an X-linked recessive retinal degenerative disorder caused by mutations in the CHM gene, which encodes Rab escort protein 1 (REP-2), resulting in degeneration of the choriocapillaris, retinal pigment epithelium, and photoreceptors of the eye. Genetics Home Reference (US National Library of Medicine) [October 17, 2010].

[0183] Diabetic Retinopathy. Diabetic retinopathy is the most common diabetic eye disease and the leading cause of blindness in the United States. Diabetic retinopathy is caused by changes in the blood vessels of the retina. While surgery can generally control or slow diabetic retinopathy, it cannot be cured, and patients are usually left with visual impairment. Therefore, improved treatments for diabetic retinopathy are needed. "Diabetic Retinopathy" (MayoClinic.org) [February 11, 2010].

[0184] Macular Degeneration. Age-related macular degeneration (AMD) is the most common cause of legal blindness in the United States and Europe. Atrophy of the RPE beneath the macula and the development of choroidal neovascularization (CNV) secondary to central vision loss. An early sign of AMD is deposits (drusen) between the retinal pigment epithelium and Bruch's membrane. Central geographic atrophy ("dry AMD") results from atrophy of the retinal pigment epithelium layer beneath the retina, which causes vision loss through loss of photoreceptors (rods and cones) in the central portion of the eye. Neovascular or exudative AMD ("wet AMD") ultimately causes vision loss due to abnormal blood vessel growth in the choriocapillaris (choroidal neovascularization) through Bruch's membrane, which leads to leakage of blood and proteins beneath the macula. Bleeding, leakage, and scarring from these vessels, if left untreated, ultimately cause irreversible damage to photoreceptors and rapid vision loss. Current treatments for macular degeneration include antiangiogenic therapy with ranibizumab (LUCENTIS®) or bevacizumab (AVASTIN®), photocoagulation (laser surgery), photodynamic therapy with verteporfin (VISUDYNE®), and surgical removal of submacular hemorrhages. "Macular Degeneration" (MayoClinic.org) [October 2010]. However, the goal of these treatments is to prevent further vision loss and, unfortunately, cannot reverse existing damage. Therefore, a cure for macular degeneration is highly desirable.

[0185] Retinitis Pigmentosa (RP). Retinitis pigmentosa (RP) is a group of genetic disorders that damages the photoreceptors (e.g., rods and cones) in the retina, affecting approximately 1.5 million people worldwide. For example, autosomal recessive RP is caused by mutations in the cis-retinaldehyde binding protein or RPE65. The progression of RP is slow and varies from patient to patient. All RP patients suffer from some degree of vision loss, typically accompanied by early night blindness, followed by tunnel vision, and some experience complete vision loss. "Retinitis Pigmentosa," American Optometric Association (October 2010). Treatment with Vitamin A and Lutein Although some studies have shown that treatments can slow the progression of RP, there is no effective treatment.

[0186] Retinal detachment. Rhegmatogenous retinal detachment, exudative, serous or secondary retinal detachment, and tractional retinal detachment. Retinal detachment, including membrane separation, is an eye disorder in which the retina separates from the underlying layer of its supporting tissue, which can result in vision loss and blindness. See Ghazi and Green (2002) Eye 16:411-421; Facts About Retinal Detachment [NEI Health Information] (October 2010). Treatments for retinal detachment are desirable.

[0187] Stargardt Disease (Fundus Flavorum). Stargardt disease (Fundus Flavorum) is a type of macular degeneration, including both autosomal recessive and dominant forms, that causes progressive loss of central vision in both eyes. See Gass and Hummer (1999) Retina 19(4): 297-301 and Aaberg (1986) Tr. Am. Ophth. Soc. LXXXIV: 453-487. Currently, treatment for Stargardt disease is There is no law.

[0188] RPE cells in medicine

[0189] Given the importance of the RPE in maintaining vision and retinal health, the RPE and methods for producing RPE cells in vitro would be of great benefit. See Lund et al. (2001) Progress in Retinal and Eye Research 20(4):415-449. See, e.g., Gouras et al. (2002) Investigative Ophthalmology & Visual Science 43(10):3307-311. The study involved transgenic RPE65 - / - Gouras described the transplantation of RPE cells from normal mice into mice (a mouse model of retinal degeneration). - / - Treumer et al. (2007) Br J Opthalmol 91: 349-353 disclose that acetaminophen slowed retinal degeneration in mice, but after 3.7 weeks its beneficial effects began to wane. described successful transplantation of autologous RPE-choroidal sheets after removal of subfoveal choroidal neovascularization (CNV) in patients with age-related macular degeneration (AMD), although this procedure only resulted in a modest increase in mean visual acuity.

[0190] Furthermore, RPE cells have been suggested as a possible therapy for treating Parkinson's disease, a chronic degenerative disorder of the brain. The disease is caused by the degeneration of specialized neuronal cells in the basal ganglia region. The death of dopaminergic neurons leads to decreased synthesis of dopamine, an important neurotransmitter, in Parkinson's disease patients. Ming and Le (2007) Chinese Medical Journal 120(5): 416-420 is beneficial for treating Parkinson's disease They suggest transplanting RPE cells from ocular donors into the striatum of Parkinson's disease patients to provide neurotrophic and anti-inflammatory cytokines.

[0191] However, human donor-derived RPE cells pose several challenges. First, there is a shortage of eye donors, and current demand exceeds the capacity of donated eye tissue. Second, human donor-derived RPE cells may be contaminated with pathogens and may have genetic defects. Third, donated RPE cells are derived from cadavers, which may not be of sufficient quality for transplantation. For example, cadaver-sourced RPE may have age-related vascular changes, including senescence. Furthermore, RPE cells derived from fetal tissue exhibit extremely low proliferative capacity. Furthermore, cadaver-sourced RPE cells vary greatly from batch to batch and must be characterized for safety before transplantation. See, for example, Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, in STEM SELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009). The safety of RPE cells is also important. Human donor sources can also be subject to donor consent issues and regulatory constraints, complicating the collection and use of RPE cells for therapy. In AMD, patients and elderly patients also suffer from degeneration of Bruch's membrane, complicating RPE cell transplantation. See Gullapalli et al. (2005) Exp Eye Res. 80(2): 235-48.

[0192] Autologous RPE cells (obtained from the patient's "good" eye) have also been tried in limited trials. Although these methods have been used, they have proven unsatisfactory. Autologous cells have fundamental limitations because they carry the same genetic predisposition that may have led to the development of AMD. See, for example, Binder et al. (2007) Progress in Retinal and Eye Research 26(5): 516-554. Furthermore, autologous RPE cells have limited proliferative capacity (especially since AMD most frequently occurs in older patients), which limits their utility in therapeutic applications (e.g., RPE cells may not engraft well and are unlikely to persist long enough for more complete restoration of vision).

[0193] Embryonic stem cell-derived RPE cells (hESC-RPE cells)

[0194] Human embryonic stem cells (hES) are considered a promising source of alternative RPE cells for clinical use. See Idelson et al. (2009) Cell Stem Cell 5:396-408. However, their use as a therapeutic agent remains hampered by a number of issues, including the risk of teratoma formation and the need for potent immunosuppressants to overcome problems due to immune rejection. For example, Wang et al. (2010) Transplantation differentiated mouse embryonic stem cells into RPE cells. and then in a mouse model of retinitis pigmentosa (Rpe65 rd12 / Rpe rd12 The authors described a study in which RPE cell transplants were performed in mice (C57BL6 mice) and transplanted into Rpe65 mice. rd12 / Rpe rd12 The mice showed no significant visual recovery over a period of 7 months, which was complicated by retinal detachment and tumors.

[0195] Furthermore, the transition from basic research to clinical application is guided by current Good Manufacturing Practices (GMP) and current Good Tissue Practices (GTP), both of which are collectively referred to as current Good Manufacturing Practices (GMP) and current Good Tissue Practices (GTP), as set forth by the U.S. Food and Drug Administration. Manufacturing processes are constrained by the need to comply with established guidelines. In the context of clinical manufacturing of cell therapy products such as hES cell-derived RPE, GTP governs donor consent, traceability, and infectious disease screening, while GMP pertains to facilities, processes, testing, and practices to produce a product that is consistently safe and effective for human use. Lu et al., Stem Cells 27: 2126-2135 (2009). Thus, a systematic and straightforward way to produce large numbers of RPE cells suitable for use in transplantation therapy is desirable.

[0196] Patents and applications owned by the assignee of the present application include U.S. Patents 7,795,025, 7,794,704, and 7,736,896, U.S. Patent Application Serial No. 12 / 682,712, and November 2010 application entitled "Methods of Producing human RPE Cells and Pharmaceutical Preparations of human RPE Cells," each of which is incorporated by reference in its entirety. PCT application PCT / US10 / 57056 (now published as WO2011 / 063005) filed on January 17, 2011 (Attorney Docket No. 75820.001020) and U.S. Provisional Patent Application No. 61 / 262,002 (Attorney Docket No. 75820.001000) filed on November 17, 2009 disclose methods for producing RPE cells via differentiation of embryonic stem cells.

[0197] Treatment method

[0198] RPE cells produced by the methods described herein and pharmaceutical preparations comprising RPE cells may be used for cell therapy. The present invention provides a method for treating conditions involving retinal degeneration, comprising administering an effective amount of a pharmaceutical preparation comprising RPE cells, wherein the RPE cells are derived from pluripotent stem cells in vitro. Conditions involving retinal degeneration include, for example, congenital choroideremia, diabetic retinopathy, retinal atrophy, retinal detachment, retinal dysplasia, and retinitis pigmentosa. The RPE cells described herein can also be used to treat conditions such as age-related macular degeneration (dry or wet), neurofibromatosis, and glaucoma. The RPE cells described herein may also be used in methods for treating macular degenerations, including, but not limited to, Caroline's macular degeneration, Sorsby's basal dystrophy, Stargardt's disease, pattern dystrophy, Best's disease, Malattia-Leventetinese, Doyne's honeycomb choroidopathy, dominant drusen, and radial drusen. It may be used in methods for treating Parkinson's disease (PD).

[0199] A common feature of cell transplantation is low graft viability; for example, many cell transplantation studies tend to experience cell loss shortly after transplantation (e.g., within the first week). This loss is thought to be due to a failure to retain a certain percentage of cells at the transplant site, rather than rejection of the transplanted cells. This failure to retain cells is likely due to a number of factors, including the inability of cells to attach to underlying structures, lack of adequate nutrients, or physical stress at the transplant site. After this initial drop in cell numbers, cell viability at various time points after transplantation can vary greatly from study to study. That is, some studies show a steady decline in numbers, while others show that transplanted cells are able to reach a stable number. However, an important factor when considering transplant success is the percentage of recipients with viable grafts after cell transplantation.

[0200] In contrast to previous preparations, the RPE cells in the pharmaceutical preparations described herein may survive for extended periods after 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 for the lifetime of the recipient of the transplant. Furthermore, at least about 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 the transplanted RPE cells. Furthermore, the RPE cells described herein successfully integrate into the RPE layer in the transplant recipient, forming a semi-continuous line of cells and may retain expression of important RPE molecular markers (e.g., RPE65 and bestrophin). The RPE cells described herein also bind to Bruch's membrane and form a stable RPE layer in the transplant recipient. Furthermore, the RPE cells described herein are substantially free of coexisting ES cells, and the transplant recipient does not exhibit aberrant growth or tumor formation at the transplant site.

[0201] Methods for treating patients suffering from conditions associated with retinal degeneration can include topically administering the compositions of the present invention (e.g., by intraocular injection or insertion of a matrix containing the pharmaceutical preparation of the present invention). Intraocular administration of the pharmaceutical preparation of the present invention includes, for example, intravitreal, transcorneal, subconjunctival, juxtascleral, retroscleral, and subtendinous delivery. See, e.g., U.S. Patents 7,794,704; 7,795,025; 6,943,145; and 6,943,153.

[0202] The present invention also provides a method of administering to a patient human RPE cells that have been derived from low-complexity embryonic stem cells, the method comprising: (a) administering to the patient a treatment comprising administering to the patient human RPE cells; (b) identifying the MHC proteins expressed on the surface of the patient's cells; (c) providing a library of low MHC complexity human RPE cells produced by the method for producing RPE cells of the present invention; (d) selecting RPE cells from the library that match the MHC proteins on the patient's own cells; and (e) administering any of the cells from step (d) to the patient. This method may be performed in local facilities, such as clinics, clinics, doctors' offices, and other medical facilities. Additionally, RPE cells selected as a match for a patient, if stored as small numbers of cells, may be expanded prior to administration to the patient.

[0203] Prior to transplantation, the RPE cells may be cultured under conditions that increase expression of alpha integrin subunits 1-6 or 9 compared to uncultured RPE cells or other RPE cell preparations. The RPE cells described herein express alpha integrin subunits 1, 2, 3, The RPE cells may be cultured to increase the expression levels of alpha integrin subunits 1-6. The RPE cells described herein may be cultured under conditions that promote expression of alpha integrin subunits 1-6. For example, the RPE cells may be cultured with integrin activators, including, but not limited to, manganese and the activating monoclonal antibody (mAb) TS2 / 16. See Afshari et al., Brain (2010) 133(2): 448-464.

[0204] The particular dosing regimen, route of administration, and adjuvant therapy may be tailored based on the particular condition, the severity of the condition, and the patient's overall health. Administration of a pharmaceutical preparation comprising RPE cells may be effective to reduce the severity of symptoms and / or prevent further degeneration in the patient's condition. For example, administration of a pharmaceutical preparation comprising RPE cells may improve the patient's vision. Furthermore, in certain embodiments, administration of RPE cells may be effective to completely reverse vision loss or any of the other symptoms. Furthermore, administration of RPE cells may treat symptoms of damage to the endogenous RPE layer.

[0205] Pharmaceutical preparation of RPE cells

[0206] The RPE cells may be formulated using a pharmaceutically acceptable carrier. For example, the RPE cells may be administered alone or as a component of a pharmaceutical formulation. The subject compounds may be formulated for administration in any convenient manner for use in medicine. Pharmaceutical preparations suitable for administration may include the RPE cells in combination 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 into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes, suspending agents, or thickening agents.

[0207] For administration, pharmaceutical preparations for use in the present invention may be in a pyrogen-free, physiologically acceptable form. Preparations containing RPE cells used in the methods described herein may be implanted as a suspension, gel, colloid, slurry, or mixture. Furthermore, the formulations are desirably encapsulated or injected in a viscous form into the vitreous humor for delivery to the site of retinal or choroidal damage. Additionally, at the time of injection, cryopreserved RPE cells may be reconstituted with a commercially available balanced salt solution to achieve the desired osmolality and concentration for administration by subretinal injection.

[0208] The RPE cells of the present invention may be delivered in a pharmaceutically acceptable ophthalmic formulation by intraocular injection. When administering the formulation by intravitreal injection, for example, the solution may be concentrated so that a minimal volume is delivered. The concentration for injection may be any amount that is effective and non-toxic, depending on the factors described herein. Pharmaceutical preparations of RPE cells for treatment of patients include those containing at least about 10 4 RPE cell preparations for treatment of patients may be formulated at a dose of at least about 10 RPE cells / ml of cells. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8, 10 9 or 10 10 For example, the RPE cells may be formulated in a pharmaceutically acceptable carrier or extract.

[0209] Pharmaceutical preparations of RPE cells 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. Pharmaceutical preparations of RPE cells may comprise at least about 1 x 10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x108 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 10 The pharmaceutical preparation of RPE cells may comprise at least about 1 x 10 RPE cells. 2 ~1x10 3 , 1x10 2 ~1x10 4 , 1x10 4 ~1x10 5 or 1x10 3 ~1x10 6 Pharmaceutical preparations of RPE cells may contain 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, pharmaceutical preparations of RPE cells may contain at least about 20,000 to 200,000 RPE cells in a volume of at least about 50 to 200 μL. Furthermore, pharmaceutical preparations of RPE cells may contain at least about 180,000 RPE cells in a volume of at least about 150 μL.

[0210] The RPE cells may be formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle that allows the preparation to remain in contact with the ocular surface for a sufficient period of time to allow the cells to penetrate the affected area of ​​the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, supracortical space, pars plana, area of ​​surgically induced blood vessels, or macula.

[0211] The volume of the preparation administered according to the methods described herein may depend on factors such as the mode of administration, the number of RPE cells, the age and weight of the patient, and the type and severity of the disease being treated. When administered by injection, the volume of a pharmaceutical preparation of RPE cells of the invention 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 injection, the volume of a pharmaceutical preparation of RPE cells of the present invention is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 , 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 μL (microliters). For example, the volume of a preparation of the invention may be at least about 10 to 50, 20 to 50, 25 to 50, or 1 to 200 μL.The volume of the formulations of the invention may be at least about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μL.

[0212] For example, the preparation may be at least about 1 x 10 3 , 2x10 3 , 3x10 3 , 4x10 3 , 5x10 3 , 6x10 3 , 7x10 3 , 8x10 3 , 9x10 3 , 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 The preparation may contain 2000 RPE cells per μL, for example 100,000 RPE cells per 50 μL, or 180,000 RPE cells per 90 μL.

[0213] The method for treating retinal degeneration may further include the administration of 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. When immunosuppressants are used, they may be administered systemically or locally and may be administered before, simultaneously with, or after administration of RPE cells. Immunosuppressant therapy may continue for weeks, months, years, or indefinitely after administration of RPE cells. For example, the patient may receive 5 mg / kg of cyclosporine for six weeks after administration of the RPE cells.

[0214] Methods for treating retinal degeneration may involve administration of a single dose of RPE cells. Treatment methods described herein may also involve a course of treatment in which RPE cells are administered multiple times over a period of time. Exemplary courses of treatment may include weekly, biweekly, monthly, quarterly, twice-yearly, or annual treatment. Alternatively, a stepwise treatment may be used, requiring multiple doses initially (e.g., daily dosing for the first week) followed by fewer and less frequent doses thereafter.

[0215] When administered via intraocular injection, RPE cells may be delivered one or more times periodically throughout the patient's lifetime. For example, RPE cells may be delivered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desired for a particular condition or disorder. When administered via an implant or device, RPE cells may be administered once or one or more times periodically throughout the patient's lifetime, as needed for the particular patient and disorder or condition being treated. Similarly, it is contemplated that treatment regimens may change over time. 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 treatment may no longer be necessary.

[0216] The methods described herein may further include monitoring the efficacy or prevention of treatment by measuring the electroretinogram response, optometer acuity threshold, or luminosity threshold in the subject. The methods may also include monitoring the efficacy or prevention of treatment by monitoring the immunogenicity of the cells or the migration of the cells in the eye.

[0217] RPE cells may be used in the manufacture of a medicament for treating retinal degeneration. The present invention also encompasses the use of preparations containing RPE cells in the treatment of blindness. For example, preparations containing human RPE cells may be used to treat retinal degeneration associated with many vision-changing diseases that result in photoreceptor damage and blindness, 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). The preparation may contain at least about 5,000 to 500,000 RPE cells (e.g., 100,000 RPE cells), which may be administered to treat many vision-changing diseases that result in photoreceptor damage and blindness, such as retinal degeneration associated with diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava).

[0218] The RPE cells provided herein may be human RPE cells. However, it should be noted that human cells may be used in human patients as well as animal models or animal patients. For example, human cells may be tested in mouse, rat, cat, dog, or non-human primate models of retinal degeneration. Furthermore, human cells may be used to treat animals in need of treatment, such as in veterinary medicine.

[0219] Mode of administration

[0220] The pharmaceutical preparation may be formulated in a pharmaceutically acceptable carrier according to the route of administration. For example, the preparation may be formulated for subretinal administration. The preparation containing RPE cells may be administered to one eye or both eyes of the same patient. Administration to both eyes may be sequential or simultaneous. For example, the preparation containing RPE cells may be formulated as a suspension, solution, slurry, gel, or colloid.

[0221] The RPE cells of the present invention may be administered locally by injection (e.g., intravitreal injection) or as part of a device or implant (e.g., implant). For example, the preparation may be administered by injection into the subretinal space of the eye. Additionally, the preparation may be administered transcorneally. For example, the cells of the present invention may be implanted into the subretinal space via vitrectomy. Additionally, at the time of injection, the RPE cells may be resuspended in a commercially available balanced salt solution to achieve the desired osmolarity and concentration for administration by subretinal injection.

[0222] Depending on the method of administration, the RPE cells may be added to a buffered electrolyte-balanced aqueous solution containing a lubricious polymer, a mineral oil or petrolatum-based ointment, other oils, liposomes, cyclodextrin, sustained-release polymers, or a gel.

[0223] Matrices for use with RPE cells

[0224] The methods described herein include administering the RPE cells of the present invention as an implant or device. In certain embodiments, the device is a bioerodible implant for treating an ocular medical condition, comprising an active agent dispersed within a biodegradable polymer matrix, wherein at least about 75% of the particles of the active agent have a diameter of less than about 10 μm. The bioerodible implant is sized for implantation in an ocular region. The ocular region may be any one or more of the anterior chamber, posterior chamber, vitreous, choroid, suprachoroidal space, conjunctiva, subconjunctival space, episcleral space, intracorneal space, epicorneal space, sclera, pars plana, surgically induced vascular region, macula, and retina. The biodegradable polymer may be, for example, poly(lactic-co-glycolic) acid (PLGA) copolymer, biodegradable poly(DL-lactic-co-glycolic acid) film, or PLLA / PLGA polymer substrate. The ratio of lactic acid to glycolic acid monomers in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, or 75 / 25 weight percent, more preferably about 50 / 50. The PLGA polymer 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 of the bioerodible implant, preferably about 40 weight percent. RPE cells may be implanted in combination with biocompatible polymers such as polylactic acid, poly(lactic-co-glycolic acid), 50:50 PDLGA, 85:15 PDLGA, and INIONGTR® biodegradable membranes (a blend of biocompatible polymers). See U.S. Patents 6,331,313; 7,462,471; and 7,625,582. Also, Hutala et al. (2007) “In vitro biocompatibility of degradable biopolymersin cell line cultures from various ocular tissues: Direct contact studies.” Journal of Biomedical Materials Research 83A(2): 407-413; Lu et al. (1998) J BiomaterSci Polym Ed 9: 1187-205; and Tomita et al. (2005) Stem Cells 23: 1579-88.

[0225] Screening assays

[0226] The present invention provides a method for screening to identify agents that modulate the maturation of RPE cells. For example, RPE cells differentiated from human ES cells may be used in screening for agents that promote RPE maturation. The identified agents may be used alone or in combination with RPE cells as part of a treatment regimen. Alternatively, the identified agents may be used as part of a culture method to improve the viability of in vitro differentiated RPE cells.

[0227] RPE cells may be used as research tools in settings such as pharmaceutical, chemical, or biochemical industries, pathology, or academic or research institutions. Such uses include, for example, the use of RPE cells differentiated from embryonic stem cells in screening assays to identify agents that may be used to promote RPE survival or promote RPE maturation in vitro or in vivo. Identified agents may be tested in vitro or in animal models to evaluate their potential use, for example, alone or in combination with RPE cells.

[0228] The present invention provides a method for identifying an agent that promotes RPE maturation, comprising providing RPE cells, contacting the RPE cells with an agent, assessing the RPE cells for signs of maturation, and then identifying the agent that promotes RPE maturation if the agent induces the RPE cells to exhibit signs of maturation, which may be levels of pigmentation, levels of gene expression, and morphology, as discussed herein.

[0229] Commercial Applications and Methods

[0230] A particular aspect of the present invention relates to the production of RPE cells to reach commercial quantities: RPE cells may be produced on a large scale, stored as needed, and supplied to clinics, physicians, or other medical institutions.

[0231] In certain aspects, the present invention relates to methods for producing, storing, and distributing RPE cells produced by the methods disclosed herein. Following production of RPE cells, the 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 immunological profiles. For example, when a patient presents with indications such as diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, retinal atrophy, retinal detachment, retinal dysplasia, and Stargardt's disease (fundus flava), RPE cells may be ordered and provided without delay. Accordingly, the present invention relates to RPE cell production methods for obtaining cells on a commercial scale, cell preparations containing RPE cells derived from the methods, and methods for providing (i.e., producing, optionally storing, and selling) RPE cells to hospitals and physicians. Production of differentiated RPE cells or mature differentiated RPE cells may be scaled up for commercial use.

[0232] The present invention also provides a method of conducting a pharmaceutical business that includes establishing a distribution system for distributing the preparations for sale, or may include establishing a sales group for selling the pharmaceutical preparations.

[0233] The present invention provides a method for supplying RPE cells to hospitals, medical centers, and physicians, whereby RPE cells produced by the methods disclosed herein are stored, ordered upon request by the hospital, medical center, or physician, and administered to patients in need of RPE cell therapy. Hospitals, medical centers, and physicians can order RPE cells based on patient specific data, and administer RPE cells to patients in need of RPE cell therapy. The cells are produced according to the patient's specifications and then supplied to the hospital or physician that placed the order. For example, after a particular RPE cell preparation is selected as suitable for a patient, it is expanded to reach a quantity suitable for patient treatment.

[0234] Another aspect of the present invention relates to a library of RPE cells that can provide matched cells to potential patient recipients. Accordingly, the present invention provides a method of practicing pharmaceutical practice comprising the steps of providing a preparation of RPE cells that are homozygous for at least one histocompatibility antigen, wherein the cells are selected from a bank of such cells that comprises a library of RPE cells that may be expanded by the methods disclosed herein; wherein each RPE cell preparation is hemizygous or homozygous for at least one MHC allele present in a human population; and wherein the RPE cell bank contains cells that are hemizygous or homozygous for a different set of MHC alleles relative to other members of the bank of cells. As described above, gene targeting or loss of heterozygosity may be used to generate hemizygous or homozygous MHC allele stem cells used to derive RPE cells.

[0235] The present invention also encompasses methods for obtaining human ES cells from a patient and then generating and expanding ES cell-derived RPE cells. These RPE cells may be stored and used to treat the patient from whom the ES cells were obtained, or a relative of that patient.

[0236] This disclosure demonstrates that human RPE cells can be reliably differentiated and expanded from human ES cells under well-defined and reproducible conditions, providing an inexhaustible source of cells for patients with retinal degenerative disorders. The concentration of these cells would not be limited by availability, but rather would be titrated to the precise clinical needs of the individual. Repeated infusions or transplants of the same cell population over the patient's lifetime would also be possible, if deemed necessary by the physician. Furthermore, the ability to generate banks of matched or low-complexity HLA-hES lineages from which RPE cells could be produced could potentially reduce or eliminate the need for immunosuppressants and / or immunomodulatory protocols.

[0237] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of, or testing of, the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0238] To further clarify the present invention, the following terms and definitions are provided herein.

[0239] As used in this description and throughout the claims that follow, no distinction will be made between the singular and plural unless the context clearly dictates otherwise. Furthermore, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0240] Throughout the specification, the use of "comprises" or variations thereof, such as "comprising," should be understood to refer to the inclusion of a stated integer or group of integers, but not to the exclusion of any other integer or group of integers.

[0241] "Effective amount," as used herein, broadly refers to the amount of a compound or cells that, when administered to a patient for treating a disease, is sufficient to effect successful treatment for that 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, slow the progression of the onset of signs / symptoms, prevent the progression of the onset of signs / symptoms, and / or prevent the onset of signs / symptoms. The "effective amount" may vary depending on the disease and its severity, and the age, weight, medical history, susceptibility, and pre-existing conditions of the patient to be treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of the present invention.

[0242] "Embryo" or "embryonic," as used herein, broadly refers to a developing mass of cells that has not been implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell that has been isolated from or contained within an embryo. It also includes blastomeres obtained as early as the two-cell stage, and aggregated blastomeres.

[0243] "Embryonic stem cells" (ES cells), as used herein, broadly refer to cells derived from the inner cell mass of a blastocyst or morula that have been serially passaged as a cell lineage. ES cells may be derived by fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means of generating ES cells that are homozygous for the HLA region. ES cells also refer to cells derived from a mammalian embryo at the zygote, blastomere, or blastocyst stage produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or by the production of cells by chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane. Embryonic stem cells are defined as cells derived from the inner cell mass of a blastocyst or morula that have been serially passaged as a cell lineage. ES cells may be derived from a mammalian embryo at the zygote, blastomere, or blastocyst stage that have been produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or by the production of cells by chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane. Embryonic stem cells are defined as cells derived from the inner cell mass of a blastocyst or morula that have been serially passaged as a cell lineage. They can be distinguished based on (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunocompromised animals. The term also encompasses cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo. The term also encompasses cells produced by somatic cell nuclear transfer, even if non-embryonic cells are used in the process. ES cells may be derived by fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by any means that generates ES cells homozygous for the HLA region. ES cells are also cells derived from zygote, blastomere, or blastocyst-stage mammalian embryos produced by sperm-egg fusion, nuclear transfer, parthenogenesis, or by producing cells by chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane. Human embryonic stem cells of the present invention include, but are not limited to, MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. In certain embodiments, human ES cells used to produce RPE cells are derived and maintained according to GMP standards.

[0244] "Embryo-derived cells (EDC)" means cells derived from embryos or tissues of the As used herein, the term "EDC" broadly refers to morula-derived cells, blastocyst-derived cells, such as those from the inner cell mass, embryonic shield, or primitive ectoderm, and other pluripotent stem cells of early embryos, such as primitive endoderm, ectoderm, and mesoderm, and their subsequent organisms. "EDC" also encompasses cell masses derived from blastomeres and aggregated single blastomeres, or embryos derived from various stages of development, but excludes human embryonic stem cells that have been passaged as cell lineages.

[0245] "Macular degeneration," as used herein, refers to a disease characterized by progressive loss of central vision associated with abnormalities of Bruch's membrane, the neural retina, and the retinal pigment epithelium. Macular degeneration diseases include, but are not limited to, age-related macular degeneration, North Carolina macular degeneration, Sorsby's basal dystrophy, Stargardt's disease, pattern dystrophy, Best's disease, Malattia-Leventetinase, Doyne's honeycomb choroidopathy, dominant drusen, and radial drusen.

[0246] "Pluripotent stem cells," as used herein, broadly refer to cells that are capable of long-term or virtually indefinite proliferation in vitro, that maintain their undifferentiated state, that exhibit a stable (preferably normal) karyotype, and that have the ability to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) under appropriate conditions.

[0247] "Pluripotent embryonic stem cells," as used herein, are those that (a) are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; (b) have the potential to differentiate into cell types of all three germ layers (e.g., ectoderm, mesoderm, and endoderm cell types); and (c) express at least one molecular embryonic stem cell marker (e.g., expressing Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, NANOG, TRA160, TRA181, SOX2, REX1). 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 by fertilization or from embryonic material produced by asexual means, such as somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. Other exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells), which are generated by reprogramming somatic cells by expressing or inducing expression of a combination of factors (referred to herein as reprogramming factors). iPS cells may be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, 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 discovered 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 distinguished by the expression of the same markers as embryonic stem cells, although specific iPS cell lines may vary in their expression profile.

[0248] As used herein, the terms "RPE cells," "differentiated RPE cells," and "ES-derived RPE cells" may be used interchangeably throughout to broadly refer to RPE cells differentiated from pluripotent stem cells using the methods of the present invention. The terms are used to refer to differentiated RPE cells regardless of the level of genetic maturity of the cells and, therefore, may encompass RPE cells of various levels of maturity. RPE cells can be visually recognized by their cobblestone-like morphology and early pigmentation. RPE cells can also be molecularly distinguished based on the substantial lack of expression of embryonic stem cell markers such as Oct-4 and NANOG, and the expression of RPE markers such as RPE65, PEDF, CRALBP, and bestrophin. Thus, unless otherwise specified, RPE cells, as used herein, refer to RPE cells differentiated in vitro from pluripotent stem cells.

[0249] 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 after the initial differentiation of RPE cells. In particular, RPE cells may be recognized in part based on their initial pigment appearance, but after differentiation, mature RPE cells may be recognized based on their enhanced pigmentation.

[0250] "Pigmented," as used herein, broadly refers to any level of color. This refers to pigmentation, for example, pigmentation that occurs early 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 similar to that of average RPE cells after terminal differentiation of RPE cells. The pigmentation of RPE cells may be more pigmented than that of average RPE cells after terminal differentiation of RPE cells. The pigmentation of RPE cells may be less pigmented than that of average RPE cells after terminal differentiation.

[0251] A "sign" of disease, as used herein, refers broadly to any abnormality indicative of disease that is detectable upon examination of a patient; i.e., an objective indicator of disease, as opposed to a symptom, which is a subjective indicator of disease.

[0252] A "symptom" of disease, as used herein, refers to any morbid phenomenon or deviation from normal in structure, function, or sensation experienced by a patient and that is indicative of disease.

[0253] "Treatment," "therapeutic," "treating," or "treatment," as used herein, broadly refers to treating a disease, halting or reducing the occurrence of a disease or its clinical symptoms, and / or alleviating a disease or inducing regression of a disease or its clinical symptoms. Treatment includes prevention, prevention, treatment, cure, correction, reduction, relief, and / or amelioration of a disease, signs, and / or symptoms. Treatment includes the reduction of signs and / or symptoms in a patient with ongoing signs and / or symptoms of a disease (e.g., blindness, retinal deterioration). Treatment also includes "prophylaxis" and "prevention." Prevention includes preventing a disease from occurring after treatment of a disease in a patient, or reducing the occurrence or severity of a disease in a patient. The term "reduced," for purposes of treatment, broadly refers to a clinically significant reduction in signs and / or symptoms. Treatment includes treating recurrent or recurrent signs and / or symptoms (e.g., retinal degeneration, vision loss). Therapy includes, but is not limited to, preventing the appearance of signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms (e.g., blindness, retinal degeneration).

[0254] The present invention will now be described in more detail with reference to the following examples, which are illustrative only and should not be construed as limiting the invention described above. [Example]

[0255] Having generally described the invention, this will be more readily understood by reference to the following examples, which are intended only to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0256] Example 1

[0257] In preclinical animal studies, we have searched for the presence of teratomas / tumors, likely arising from hES cells, in human retinal pigment epithelial (hRPE) cell products. When animals were injected with 100% hES cells, teratomas were detected (microscopically) within 4 weeks. However, when hRPE cells were spiked with 1% or less hES cells, no tumors were detected. The maximum acceptable percentage of hES cells in the product was at least an order of magnitude lower than the concentration of hES cells that results in teratomas in mice, i.e., less than 0.1%. Ideally, it would be possible to accurately measure the concentration of hES cells even at much lower levels, or preferably to demonstrate the absence of hES cells in hRPE cell products. As noted above, conventional methods (e.g., RT-PCR, Western blot, and flow cytometry) can detect approximately 500,000 differentiated cells. It is not sensitive enough to determine whether there are a few residual hES cells in the population of cells.

[0258] Example 2

[0259] This example describes a sensitive assay for the detection of pluripotent hES cells in a cell population. Using this assay, cells differentiated from hES cells were examined to determine whether there were residual hES cells in the population. This assay can provide valuable safety information, as residual hES cells may form tumors upon transplantation into a recipient.

[0260] This example demonstrates that two distinct aspects of the assay (each used independently of the other) contribute to the sensitivity of the assay: first, plating under conditions that support the maintenance of hES cells to minimize cell differentiation, which can result in loss of hES cell marker expression; and second, development of a methodology that allows for the evaluation of each cell in the population by a skilled operator capable of observing approximately 1-10 million cells per hour.

[0261] At present, the scientific literature accepts that pluripotent hES cells (capable of forming teratomas in vivo) express a set of known molecular markers, and that expression of Oct-4 is important for maintaining pluripotency, and that loss of this marker is associated with cell differentiation. Any cells expressing this marker at detectable levels may be considered pluripotent and therefore undesirable in the final product. Cells that have lost Oct-4 expression can be considered no longer pluripotent. In this assay, cells were first examined for positive alkaline phosphatase staining under visible light and then examined for Oct-4 expression by immunofluorescence analysis.

[0262] Human retinal pigment epithelium (hRPE) was obtained by differentiating human pulmonary stem (hES) cells as described in U.S. Patent 7,736,896, U.S. Patent Application Serial No. 12 / 682,712, and U.S. Patent Application Serial No. 61 / 262,002. hRPE cells were differentiated from the MA09 hES cell line, which originated from a single human blastomere as described in U.S. Patent 7,893,315. These cells were designated MA09-hRPE.

[0263] The sensitivity of the assay to detect hES cells in RPE cell cultures was measured. In these experiments, mixtures of MA09-hRPE and hES cells containing 0.001%, 0.01%, 0.1%, 1%, or 10% hES (approximately 200,000 cells total) were prepared and seeded in triplicate. Cells were seeded in either of two different culture conditions: RPE culture conditions (specifically, plating on gelatin-coated 4-well plates in EGM®-2 (Lonza Biologics, Basel, Switzerland) medium according to standard procedures for culturing hRPE cells) or hES cell culture conditions (in this experiment, plating on MEFs, conditions that support the growth and maintenance of hES cells using conditioned hES-GM; see Table 2). The hES cells used were GFP-positive hES cell lines (H1 or WA01). Using this GFP-positive hES cell line, it was possible to detect the presence of hES cell-derived cells, whether the cells remained pluripotent or differentiated. Following these initial experiments with H1, similar results were obtained with another hES cell line, MA09, which did not express GFP but could be detected using an anti-human nuclei antibody that stained human cells (including hES cells) but not MEFs (of murine origin).

[0264] After plating, cells were incubated to allow attachment to the substrate, then fixed with 2% paraformaldehyde, permeabilized with 0.1% NP-40 substitute (which may be performed immediately after fixation or within several weeks, e.g., approximately 24 hours), and stained for alkaline phosphatase (AP) and Oct-4. AP was detected using VECTOR® Blue (Vector Laboratories), which provides staining detectable under visible light, according to the manufacturer's instructions. Oct-4 was detected using immunofluorescence (essentially the same method as described in Example 7 below). Using a low-magnification objective on a fluorescence microscope, an initial scan of the entire well was performed for blue staining of cells indicative of alkaline phosphatase activity, observing each cell on the plate in a repeating pattern of up, right, edge, down, left to edge, down, right, etc., under transmitted visible light. Because cells were observed under visible light, unstained cells were also visible, and the microscope focus was adjusted as necessary to correct for any vertical deviations during scanning, ensuring that cells remained in the focal plane. Once blue-stained cells were observed, they were reexamined under UV light at higher magnification (x20 objective) for Oct-4 staining and then for GFP. Because the duration of UV observation was kept low according to this method, Oct-4 and GFP staining did not photobleach. Representative photomicrographs are shown in Figure 1. The upper panel shows GFP-positive (left) and Oct-4-positive (right) cells mixed with RPE cells at a 1:10 ratio (10%). DAPI staining (lower left panel) shows all cells (both RPE and hES) present in the field, and the lower right panel shows alkaline phosphatase staining. 200x magnification.

[0265] Because AP staining is detectable under visible light, every cell in the population (200,000 cells in this example) could be rapidly examined as a primary screen. Cells that were AP-positive were then examined for Oct-4 and GFP expression under ultraviolet light. Cells positive for AP, Oct-5, and GFP are pluripotent, whereas cells positive for GFP but negative for Oct-4 are differentiated progeny of pluripotent cells. Some AP-positive cells were GFP- and Oct-4-negative, demonstrating that AP can be used to rapidly identify potential hES cells, which can then be examined for a second hES cell marker (Oct-4 in this example) to identify hES cells. Because the added hES cells were constitutively GFP-positive, we interpreted the detected cells that were AP-positive but negative for Oct-4 and GFP as not originating from the added hES cells but rather as resulting from scattered AP-positive cells within the MEF population.

[0266] When cells were plated under RPE culture conditions, we observed that over 50% of the GFP-positive cells were Oct-4 negative, confirming that RPE culture conditions barely support the pluripotent state of hES cells even after short culture periods. However, all Oct-4-positive cells also expressed alkaline phosphatase and GFP, which allowed us to conclude that Oct-4 expression is sufficient to distinguish hES cells.

[0267] However, when cells were plated under hES cell culture conditions, Oct-4 expression was maintained in a much higher percentage of GFP-positive cells, and it was possible to detect Oct-4-positive hES cells in cultures spiked with as little as 0.001% hES cells, consistent with our observation that culture conditions for RPE cell growth are not conducive to the maintenance of hES cell cultures but instead induce hES cell differentiation (see Example 3 below).

[0268] Alkaline phosphatase is more sensitive than Oct-4, detecting only 5 cells out of approximately 178,000. Therefore, alkaline phosphatase assay was selected as the primary screening method. However, because AP is not a unique marker for hES cells, Therefore, its expression identifies potential hES cells, which can be confirmed by more definitive markers such as Oct-4. False AP positivity has been observed (GFP or Oct-4 negative), and Oct-4 is used as a confirmatory assay. Using 0.001% hES cells, a total of 5 cells in 3 wells stained positive for alkaline phosphatase and were GFP positive. Thus, using these methods, the detection limit for immunofluorescence detection of hES cells in RPE cell cultures was concluded to be 5 / 600,000 cells (0.0008%).

[0269] Example 3

[0270] This example describes the results of in vitro spiking studies, the purpose of which was to show that even a high percentage of all added hESCs differentiated under RPE culture conditions over the course of 6 weeks or 2 passages, which is less than the time it takes to isolate RPE at the time of harvest for cryopreservation from hESCs or undifferentiated EBs.

[0271] In vitro spiking experiments were performed using MA09 and H1-GFP cells. Cultures contained a mixture of hESCs and RPE cells, with 1%, 10%, or 20% hESC. Cell populations were assessed by staining and Q-PCR at 3 weeks (p1) and 6 weeks (p2). Microscopic observation of the cultures revealed that hESCs almost immediately began to differentiate, forming 3D structures that were easily visible because they disrupted the integrity of the RPE monolayer. By the end of the culture period, Oct-4-positive cells were no longer observed, although some alkaline phosphatase staining persisted. AP staining was attributed to differentiated hESCs, because differentiation can lead to the formation of various cell types, some of which may be alkaline phosphatase-positive.

[0272] [Table 3]

[0273] Freshly prepared suspensions of hRPE and hES cells mixed at the above ratios were plated into gelatin-coated 6-well and 4-well plates in EGM-2 medium according to standard procedures for culturing hRPE cells. EGM-2 medium supports the growth of hRPE cells. The 4-well control plate was evaluated for the initial appearance of these cultures by fixation with 2% paraformaldehyde or harvesting (for qPCR analysis) on day 3. The remaining 6-well plates were cultured to confluence, at which point the medium was changed to MDBK-MM, which is used for maintaining hRPE cells. After approximately 3 weeks, the cultures were either fixed or harvested (for qPCR) or passaged into 6-well or 4-well plates. The plates were cultured for an additional 3 weeks. The cultures were then fixed or harvested for qPCR. This simulated the manufacturing process from isolation of differentiated RPE colonies to the final product.

[0274] In Experiment 1, the hES cells added to the cultures were MA09 cells. Fixed plates were stained using indirect immunofluorescence for the hES cell marker Oct-4, followed by an Alexa-conjugated secondary antibody (identified as red emitting) and DAPI for nuclear visualization, and examined under an inverted fluorescence microscope. Alkaline phosphatase activity was measured on the same plates using a VECTOR® Blue kit (Vector Laboratories, Burlingame, CA). Viable cultures were further examined for monolayer irregularities under a phase-contrast microscope or using Hoffman Modulation Optics (HMC).

[0275] In Experiment 2, hES cells added to the cultures were H1-GFPp cells, which constitutively express GFP. These hES cells and their progeny are easily detectable by their GFP expression, allowing for the detection of ES cells throughout their entire culture period, regardless of whether they remain pluripotent or have differentiated. qPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) to assay for the expression of Oct-4, Nanog, Rex-1, and Sox2. Beta-actin gene expression was used for normalization.

[0276] Experiment 3 was the same as experiment 2, except that MA09-RPE cells were passaged one more time in culture before being mixed with hESH1-GFPp cells.

[0277] Test 1 results

[0278] Because there is no way to positively identify MA09 embryonic stem cells in culture, the primary purpose of this experiment, in which MA09 hES cells were added to cultures of hRPE cells, was to determine whether cells staining for Oct-4 or alkaline phosphatase activity (which are specific for ES cells) could be detected in these mixed cultures. Results showed that cells staining positive for both Oct-4 and alkaline phosphatase (double staining) were detected at 3 days, but none clearly stained positive for embryonic stem cell markers at 3 weeks. These double-positive staining cells at 3 days were mostly observed in typical ES-like colonies and had ES-like morphology. Microscopic examination of all cultures revealed that in 100% (positive control), the hES cells behaved in a typical embryonic stem cell manner; i.e., they differentiated to form three-dimensional structures, with some forming free-floating, embryoid-like aggregates. In contrast, pure RPE cultures (negative controls) exhibited typical RPE proliferation behavior: they were elongated and hypopigmented during the proliferation phase, and after monolayer establishment and medium change to MDBK-MM, they regained the polygonal, pigmented appearance of epithelium. However, in all mixed cultures (1%, 10%, and 20% hESCs), the presence of two distinct cell types was readily apparent, ranging from some irregularities in the monolayer resembling differentiated derivatives of hESCs to large, budded embryoid bodies and other 3D structures typical of differentiated hESCs. Although several clusters of alkaline phosphatase-positive cells were observed in the 10% and 20% cultures, the morphology of these stained cells was quite different from that of ES cells, with fibroblast-like cells or small groups of cells present within the 3D differentiated aggregates of ESCs. In such three-dimensional aggregates, the level of nonspecific antibody binding, or "noise," was higher than in cell monolayers, but we did not associate any of the brightly stained patches with DAPI-stained nuclei that would indicate positive staining for Oct-4.

[0279] Results of Tests 2 and 3

[0280] Findings from both of these studies are considered together, as they were identical with both cultures of hRPE cells. At day 2, GFP-positive cells were detected with hES cell colony morphology and detectable Oct-4 expression by immunohistochemistry, confirming the presence of ES cells in the RPE cultures. hES cells grew and differentiated, forming highly prominent, three-dimensional aggregates in the RPE cultures that were readily detectable (both by outgrowth morphology and by proliferating GFP-positive cell clusters). Morphological observations confirming the presence of differentiated hES cells were also observed at a 1% hES:RPE ratio. However, by the end of 3 weeks, 100% of the hES cell cultures lacked detectable Oct-4 in the nuclei, except for occasional single cells (consistent with our previous observation of rare Oct-4-positive cells in 3-week-old differentiated hES cell cultures). Although the specific properties of these differentiated cells were not investigated, they appeared to be well differentiated by this time point.

[0281] Tables 4-8 summarize the morphological observations and staining results for these cultures. In these tables, - means not detectable, ? means signal is detectable but not reliably or specifically, + means signal is detectable, ++ means signal is very strong, and +++ means signal is strong and specific.

[0282] [Table 4]

[0283] [Table 5]

[0284] [Table 6]

[0285] [Table 7]

[0286] [Table 8]

[0287] conclusion

[0288] hESCs and their derivatives are detectable by microscopy in both early and late cultures of RPE cells, even when added at a 1% concentration. Colonies of hES cells (well differentiated) are visible as early as day 3, when the RPE cells are still flat and subconfluent. After 3 weeks in culture, microscopy reveals large, easily detectable clusters of hESC-derived cells that are quite distinct from RPE morphology.

[0289] At day 3, some of the hESCs present in the cultures still remained pluripotent, but by day 22, the results depended on the proportion of hES cells in the initial culture. In cultures that initially contained 20% or less hES cells, all cells had differentiated by day 22. However, in cultures that initially contained 100% hES cells, a few Oct-4-positive cells remained detectable at day 22.

[0290] Oct-4 staining is detectable on day 3. In 1% hESC cultures, Oct-4 positive cells were more difficult to detect, but at 10% and 20%, these cells were easily detectable.

[0291] When H1-GFPp cells were used, the GFP marker was present at all stages of the experiment and was easily detectable in 10% and 20% hESCs on day 3. On day 22, as the hESC derivatives proliferated, detection was quite easy at all different concentrations.

[0292] Example 4

[0293] This example describes further observations made when the mixed population of hES cells and RPE cells described in Example 3 was continued in culture.

[0294] After 3 weeks in culture at passage 2, cells were either harvested in RLT buffer for Q-PCR or fixed with 2% PFA and stained for Oct-4. Alkaline phosphatase activity was detected using VECTOR® Blue stain according to the manufacturer's instructions. Samples were mounted in VECTASHIELD® with DAPI and examined / photographed.

[0295] A few areas of blue staining (indicating the presence of alkaline phosphatase-reactive products) were observed in some samples. However, none of these cells were Oct-4 positive, and no other Oct-4-positive cells were detected in any of the fields examined. These results indicate that ES cells persisted in the cultures. The observed alkaline phosphatase staining is likely the result of differentiation of ES cells that gave rise to other cell types with alkaline phosphatase activity.

[0296] GFP-positive cells were present in all samples examined, and their morphology indicated various ES cell derivatives, but did not resemble ES cells.

[0297] The positive control (MA09 hES cells cultured for 3 days) showed very high levels of Oct-4 staining and alkaline phosphatase activity.

[0298] conclusion

[0299] hES cells mixed with RPE cells and cultured in RPE medium differentiated but continued to proliferate in a differentiated state. Several additional tests were performed to detect possible ES cell contamination. It is necessary to implement this.

[0300] For early cultures (2-3 days) of RPE cells stained for Oct-4 / alkaline phosphatase, the presence of single cells or small clusters should be easily detectable because at this stage the cells are subconfluent and growing as a monolayer and have not deposited sufficient amounts of extracellular matrix to form background. For immunofluorescence studies, a sample size of at least 200,000 cells, preferably in triplicate, is considered to provide greater sensitivity.

[0301] As RPE cultures grow and mature, the presence of hES cells can produce irregularities in the monolayer ranging from single cells with non-epithelial morphology to large 3D aggregates that may harbor undifferentiated ES cells. Single cells with non-RPE morphology may represent cross-differentiated or aged RPE or retinal precursors, whereas large aggregated 3D aggregates typical of differentiated ES cell morphology suggest the presence of hES cells in the culture.

[0302] Example 5

[0303] It is widely accepted that hES cells differentiate very rapidly when grown under conditions that do not support their pluripotent state (e.g., on feeder cells, a defined matrix, or a defined medium). This differentiation is associated with the loss of molecular markers of pluripotency, such as Oct-4 and alkaline phosphatase. Because RPE culture conditions do not support the undifferentiated growth of hES cells, it was hypothesized (and these experiments confirmed) that by the time the mixture of initially pluripotent hES cells with RPE cells was fixed, many hES cells had already lost or downregulated Oct-4 expression, rendering it undetectable.

[0304] To quantify the effect of culture conditions on the sensitivity of hES cell detection, a low concentration of H1-GFPp cells was mixed with MA09-hRPE cells and analyzed to determine whether they favored maintaining their pluripotent state. Cells were plated under either optimal or inappropriate conditions. Specifically, cells were cultured in either hES cell medium on mouse embryonic fibroblast feeder cells (MEFs) or in endothelial culture medium (EGM-2) supplemented with 2% FBS on gelatin. EGM-2 is a medium that supports RPE proliferation but does not maintain the pluripotent state of hES cells. To reduce the time required for differentiation to occur in the medium, cells were fixed after only 16 hours of culture. Cells were then stained with alkaline phosphatase and Oct-4 and examined microscopically as described in Example 2. For cell populations plated in ES cell medium on feeder cells, approximately 80% of GFP-positive cells were positive for AP and Oct-4. In contrast, for cell populations plated in EGM-2 medium, only 20% of GFP-positive cells were positive for AP and Oct-4. From these results, the inventors concluded that plating under conditions that favor the maintenance of the pluripotent state can greatly improve the sensitivity of detecting hES cells in a cell population.

[0305] Example 6

[0306] This example demonstrates the relatively low sensitivity of real-time PCR in detecting rare cell subpopulations.

[0307] method

[0308] qRT-PCR LOD was evaluated using a mixture of MA09-RPE and MA09 hES cells. Cryopreserved MA09-RPE cells were thawed and counted, and hES cells were analyzed. MA09 was also thawed and counted (using alkaline phosphatase staining to determine the number of pluripotent hES cells present). Mixtures of 400,000 cells were then formed such that 100%, 10%, 1%, 0.1%, 0.01%, or 0% of the cells were hES cells, with the remainder being MA09-RPE. RNA was extracted from the cell mixtures using the RNeasy RNA isolation kit from Qiagen, resulting in a final volume of 30 μL of RNA per sample. cDNA was then synthesized from 10 μL of RNA using the Quantitect cDNA synthesis kit from Qiagen, resulting in a final volume of 20 μL of cDNA. 1 μL of cDNA was then tested for relative gene expression in triplicates, normalized to the beta-actin signal present in each sample. Gene expression analysis was performed using Applied Chromatography with software version 2.1. Biosystems StepOne Plus and TaqMan gene expression assays from Life Technologies were used, following the manufacturer's recommended cycling conditions for comparative Ct relative quantification.

[0309] qRT-PCR assays for Nanog, Oct-4, and SOX2 Expression levels were normalized to those observed in 100% hES cell samples, which are used as the zero set point in the graphs shown below (RQ = relative quantification). The mean downregulation of Nanog (2.51 log), Oct-4 (2.73 log), and SOX2 (1.63 log) for unspiked (0% hES) RPE samples compared to 100% hES controls is consistent with previous data for this lot. Downregulation of all hES markers was dramatically reduced in RPE cells spiked with 1% and 10% hES cells.

[0310] result

[0311] Figures 2A–C graphically show the relative gene expression (detected by qRT-PCR) for (A) Oct-4; (B) Nanog; and (C) SOX2. Statistically significant differences between the RPE control (0% hES) and the 0.1% spiked samples are evident for Nanog and Oct-4. However, at the 0.1% spike dose, the degree of SOX2 downregulation is comparable to that observed in the 100% RPE sample. Expression of all three hES cell markers in the 0.001% spiked sample is indistinguishable from that observed in the 100% RPE control. Therefore, the qRT-PCR assay appears unable to detect potential hES cell contamination at the 0.01% level.

[0312] In this study, the LOD of qRT-PCR for two of the three hES markers (Nanog and Oct-4) was at the 0.1% hES cell level. However, depending on the degree of downregulation for a particular lot of RPE, it is questionable whether qRT-PCR even at this level provides a consistent and reliable indicator of hES cell contamination. In conclusion, qRT-PCR for hES expression is 3–4 orders of magnitude less sensitive than the immunohistochemistry assay described above.

[0313] Example 7

[0314] This example further demonstrates the high sensitivity of detecting rare hESCs within RPE cell populations using this staining method. These experiments measure the sensitivity of the assay for detecting undifferentiated (pluripotent) hESCs in RPE populations. RPE were spiked with distinct, relatively low numbers of hESCs (both H1 and MA09 in different experiments) on MEFs in hESC medium for a minimal amount of time to allow the cells to attach and expand, thereby allowing them to remain adherent during the immunostaining procedure. These conditions (MEFs, conditioned hESC-GM medium, minimal exposure to RPE) preserved the pluripotency of the hESCs. After 14-16 hours, plates were fixed and stained and then examined. Per well examined, 1000 cells were cultured. Nuclei (DAPI staining) per field were counted in several randomly selected fields relative to the total cell number. Because AP staining is expressed in other cell types in addition to pluripotent cells, AP+ / Oct4- cells were considered to have originated from MEFs; that is, each AP-positive cell was tested for Oct-4, and double-positive cells were counted as hESCs. Although there was a slight loss of pluripotent cells to differentiated types, as determined by comparison of Oct-4 and GFP, this loss was minimal under conditions optimized for these hESCs (though the loss was much greater in the absence of MEFs and in RPE medium).

[0315] method

[0316] MA09 hES and RPE cells were prepared at a ratio of 8 hES cells to 10 million RPE cells. A negative control consisted of RPE cells without added hES cells. Two million RPE cells, containing an average of 1.6 hES cells, were suspended in hES-conditioned cell culture medium and seeded into 6-well plates containing 500,000 feeder cells (mitomycin C-treated MEFs) to promote the maintenance of hES pluripotency. Cells were cultured for approximately 16 hours before fixation and staining. After 14–18 hours, the medium was removed, the cultures were washed with PBS, permeabilized with 0.1% NP-40 (10 minutes), and washed again. Nonspecific binding was blocked with 10% normal goat serum (30–60 minutes). Primary antibody against Oct-4 was added, followed by washing and incubation in a secondary fluorescently labeled antibody. To verify the staining conditions and to exclude any nonspecific staining due to MEFs present in the culture, a control assay was performed using double staining with anti-human nuclei (anti-HuNu) antibody and Oct-4. Cultures were stained in the presence of alkaline phosphatase (blue) using AP staining (Labs). After the AP color change was complete, the stain was removed, the cells were washed, and stained with DAPI (a fluorescent DNA stain).

[0317] When using MA09 cells lacking GFP or other internal markers, we included a control of hES cells alone (no RPE) on MEFs stained with the same markers plus anti-human nuclei. Because hES cells tend to differentiate in the absence of the mutual support of surrounding hES cells, this control experiment allowed us to determine an upper limit for the number of undifferentiated hES cells that could remain after plating. In these control experiments, the same number of hES cells was plated in the same plate area on MEFs in hES cell medium (conditioned hES-GM; see Table 2), but in the absence of RPE cells. Cultures were then stained with anti-human nuclei (anti-HuNu) antibody, allowing for detection of human cells on top of the MEFs, and the remaining undifferentiated hES cells were counted. These counts measured how many undifferentiated hES cells would remain when plated at low density but under optimal culture conditions (plated in hES medium on MEFs). The number of undifferentiated hES cells detected in these control plates was used as the denominator when calculating the percentage of undifferentiated cells detected. When a theoretically 5 hES cells per 600,000 were introduced, at least two double positives were detected. When such small numbers of contaminating cells are introduced, the "noise" effect can be significant because cell loss during manipulations such as pipetting is constant and does not correlate with the decreasing percentage of contaminating cells.

[0318] result

[0319] As discussed above, a significant proportion of hES cells may lose Oct-4 staining during the course of culture (24 hours) prior to immunostaining assays. The loss of Oct-4 staining was attributed to maintaining the culture in RPE culture medium prior to staining. Additional testing confirmed a similar loss of Oct-4 expression in MA09 hES cells during the course of culture (approximately 18 hours). Thus, depending on the assay conditions, a certain percentage of initially pluripotent hES cells may lose Oct-4 staining. It is believed that by the time they are fixed for immunostaining, the cells are downregulated with respect to Oct-4 expression, thereby attenuating the sensitivity of detecting hES cells in the original cell population.

[0320] To minimize loss of Oct-4 expression, assay conditions were modified to incorporate the use of MEF feeder cells and hES-conditioned culture medium, thereby enhancing the persistence of hES pluripotency markers during the course of the assay. AP was retained as a primary screening tool because a positive signal (blue cell staining) was observed in bright field, allowing the operator to maintain focus. Scanning cells for fluorescence, on the other hand, requires viewing the cells under fluorescent light, which may result in cells moving out of the focal plane and potentially resulting in missed positive signals. Furthermore, AP staining allows the operator to examine every cell among millions, pinpointing AP-positive cells for subsequent testing to confirm Oct-4 expression.

[0321] These studies confirm that this method exhibits excellent sensitivity for detecting the rare subpopulation of pluripotent MA09 hES cells within the RPE cell population.

[0322] For each of the two studies reported in this example, all cells in 4-5 wells of a 6-well plate were first examined for AP (blue) staining. Any AP-positive cells were then reexamined for red fluorescence, indicative of the presence of Oct-4. Cell staining was examined using the same scanning method as described in the previous example, i.e., blue-stained cells were searched for in bright field and a "right-bottom-left-bottom repeat" movement from the top left of the plate was continued until the right-most bottom field was examined. In this manner, every blue-stained AP-positive cell was examined for Oct-4 (red) staining. To determine the total number of cells examined per well, three randomly selected fields (0.26 mm) were scanned. 2 The number of DAPI-stained nuclei in the micrographs (600 mm / field) was counted, and the average cells per field was multiplied by the total number of fields per well (960 mm per well).2 / 0.26mm per field of view 2 ) = 6 multiplied by 3692 fields per well. Note that each well also contained approximately 500,000 MEFs, so a reference well containing only MEFs was set up, and MEF nuclei were also stained with DAPI and counted to obtain an accurate number of RPE / hES cells excluding MEFs. Alternatively, the number of human cells counted per well (as opposed to mouse-derived MEFs) can be determined by counting cells stained with a human-specific marker such as HuNu.

[0323] In these studies, a positive response was defined as a minimum of double-stained cells in spiked RPE preparations at a calculated ratio of 1.6MA09 hES cells per 2 million RPE cells. The LOD for detecting contaminating hES is therefore at a level of 0.00008%. Note that this is an order of magnitude more sensitive than previously reported GFP-hES assays. At least two factors contribute to the increased sensitivity: 1) the use of MEF and hES-conditioned medium to maintain hES pluripotency, and 2) the increased number of cells tested, which increases the sensitivity of the assay.

[0324] The results of these studies are summarized in Table 9 below. [Table 9]

[0325] In an additional study, two blinded operators examined wells containing 100% RPE cells and RPE cells spiked with 0.0001% hES. All operators identified all double-positive (Oct-4 / AP staining) cells in the spiked wells, whereas no hES staining was observed by either operator in the 100% RPE control.

[0326] Example 8

[0327] This example describes another exemplary method for detecting target cells in a population. Target cells are detected using a first antibody coupled directly or indirectly to alkaline phosphatase (AP) and a second antibody coupled to a fluorescent label. This method may be used to detect rare cell types that do not express AP (or express insufficient AP for robust detection). This method may also be used to detect rare cells that endogenously express AP (e.g., embryonic stem cells); in such cases, the AP-coupled antibody is expected to increase AP staining intensity. The first and second antibodies specifically bind to different markers of the rare cell type. For example, if the target cell type is an ES cell, the antibody specifically binds to two different ES cell markers, such as alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster specified 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

[0328] The coupling of the antibody to the AP and fluorescent label, respectively, may be done directly or indirectly, for example, via secondary antibodies, actin / biotin affinity, and other methods of indirect coupling. Cells that bind to the first antibody can then be visualized under visible light by staining the cells for alkaline phosphatase activity. The cell population is then pre-coated. As described in the Examples, AP-positive cells are identified by examining them under visible light; then, cells that are also labeled by the second antibody are detected by examining them under ultraviolet light. Cells that bind to both the first and second antibodies are identified as cells of the target type. Optionally, the cell population is plated under conditions that favor the growth and / or survival of the target cell type. Optionally, an estimate of the total number of cells to be examined may be made, for example, by counting the number of cells in a random field and scaling up to the total area counted, and a predetermined minimum number of cells may be examined to ensure a desired level of sensitivity. If the culture conditions include the presence of feeder cells, the cell population may be counted in the presence of a label that distinguishes cell populations derived from the feeder cells (e.g., a species-specific or lineage-specific antibody, where the feeder cells are of a different species or type from the cell population), which may be performed in duplicate wells.

[0329] Example 9

[0330] This example describes another illustrative method for detecting target cells within a population. A cell population is stained to detect two different target cell markers using first and second stains that are visually distinguishable from one another. For example, cells may be labeled with first and second antibodies, each coupled directly or indirectly to two different enzymes (e.g., selected from alkaline phosphatase, beta-galactosidase, and a peroxidase, such as horseradish peroxidase) that catalyze reactions that give visible products. The cells are then contacted with substrates for the two different enzymes that catalyze reactions that give visible products, where the substrates are selected so that the products are different colors from one another. Alternatively, the products may be the same color as one another but selected to have distinguishable staining patterns (e.g., one coupled to an antibody that stains the nucleus and the other coupled to an antibody that stains the plasma membrane). As an example, one of the enzymes may be alkaline phosphatase and its substrate may be Vector Red or Vector Blue (which produce red and blue products, respectively), and the other enzyme may be peroxidase and its substrate may be 3,3'-diaminobenzidine (DAB) (which produces a dark brown product). Another exemplary enzyme that may be used is beta-galactosidase and its substrate X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) (which produces a blue product), which may be readily used with peroxidase / DAB (brown product) or AP / Vector Red (red product). Other suitable enzyme / substrate combinations that produce visually distinguishable products are readily discovered through routine experimentation. An advantage of this method is that rare cells may be detected using only visible light, without the need for examination under fluorescent light. Additionally, if desired, a further fluorescent label (e.g., coupled to an antibody against a third marker) can be utilized to further identify detected cells (i.e., labeled with the first and / or second label) by examination under ultraviolet light.

[0331] While the present invention has been described by way of examples and preferred embodiments, the terms used herein are terms of description and not of limitation. Changes may be made in light of the appended claims without departing from the scope and spirit of the invention in its broader aspects. While the invention has been described herein with reference to specific means, materials, and embodiments, it is to be understood that the invention is not limited to those particulars. The invention also extends to all equivalent structures, means, and uses that fall within the scope of the appended claims.

[0332] 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, and such equivalents are intended to be encompassed by the claims set forth below.

[0333] Each document cited herein (including all patents, patent applications and other publications) are incorporated by reference herein in their entireties to the same extent as if each individual was specifically and individually indicated to be incorporated by reference herein. The disclosures of each of the following U.S. provisional patent applications are incorporated herein by reference: 60 / 998,766, filed October 12, 2007; 60 / 998,668, filed October 12, 2007; 61 / 009,908, filed January 2, 2008; and 61 / 009,911, filed January 2, 2008. Additionally, the disclosure of WO2009 / 051671 is incorporated herein in its entirety by reference.

Claims

1. The following steps: (a) providing a cell population; (b) applying a first stain and a second stain to the cell population, wherein the first stain detects a first marker whose expression is indicative of the presence of a target cell, and the second stain detects a second marker whose expression is also indicative of the presence of the same target cell, and wherein the first stain is detectable under visible light and the second stain is detectable under ultraviolet light; (c) detecting any cells that are positive for the first marker by observing the cell population microscopically under visible light; (d) microscopically observing the cells that are positive for the first marker under ultraviolet light and determining whether any of the detected cells are positive for the first marker and the second marker; and (e) identifying the first marker and any cells positive for the first marker as target cells; 1. A method for detecting the presence or confirming the absence of a target cell in a cell population, comprising:

2. The following steps: (a) providing a cell population; (b) applying a first stain and a second stain to the cell population, wherein the first stain detects a first marker whose expression is indicative of a target cell, and the second stain detects a second marker whose expression is indicative of the same target cell, wherein the first stain is detectable under visible light, and the second stain is detectable under visible light, and wherein the first stain and the second stain are visually distinguishable; (c) detecting any cells that are positive for the first marker by observing the cell population microscopically under visible light; (d) detecting any cells that are positive for the second marker by microscopic observation of the cells of the cell population under visible light; and (e) identifying as target cells any cells that are positive for the first marker and the second marker from the cells detected in (c) and (d); 1. A method for detecting the presence or confirming the absence of a target cell in a cell population, comprising:

3. 3. The method of claim 1 or 2, further comprising removing or isolating the detected target cells from the cell population.

4. 3. The method of claim 1 or 2, wherein the cell population is derived from one or more pluripotent cells.

5. The method according to claim 1 or 2, which is carried out on a plurality of cell populations derived from one or more pluripotent cells.

6. The method of claim 5, wherein said cell populations are derived from pluripotent cells of different HLA types.

7. 3. The method of claim 1 or 2, wherein said first stain is observable under visible and ultraviolet light.

8. The method of any one of claims 1 to 7, wherein said first marker comprises alkaline phosphatase expressed by said target cell.

9. The method of any of claims 1 to 7, wherein said first stain comprises an antibody that specifically binds to said first marker, and said antibody is directly or indirectly coupled to alkaline phosphatase.

10. 10. The method of claim 8 or 9, wherein the first stain comprises an alkaline phosphatase substrate selected from the group consisting of naphthol AS-BI phosphate; 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT); BCIP reagent and INTX reagent; naphthol AS-BI and fast red violet LB; tetrazolium salts; diazo compounds; VECTOR® Red; VECTOR® Blue; VECTOR® Black; and p-nitrophenyl phosphate (pNPP).

11. 11. The method of claim 10, wherein the first stain comprises Vector Red or Vector Blue.

12. 12. The method of any of claims 1 or 7 to 11, wherein said second stain comprises a primary antibody that specifically binds to said second marker, and said primary antibody is directly or indirectly coupled to a fluorescent label.

13. 13. The method of claim 12, wherein the second marker is selected from the group consisting of alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, Lin28, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

14. 14. The method of claim 13, wherein the target cell is an embryonic stem cell.

15. 15. The method of claim 14, wherein said second marker is selected from the group consisting of Oct-4 and Nanog.

16. The method according to any one of claims 12 to 15, wherein the primary antibody comprises a fluorescent label.

17. The method of any one of claims 12 to 15, wherein the second staining further comprises a secondary antibody.

18. 18. The method of claim 17, wherein the secondary antibody comprises a fluorescent label.

19. The fluorescent label is Alexa Fluor350, Alexa Fluor405, Alexa Fluor430, Alexa Fluor488, Alexa Fluor514, Alexa Fluor532, Alexa Fluor546, Alexa Fluor555, Alexa Fluor568, Alexa Fluor594, Alexa Fluor610, Alexa Fluor633, Alexa Fluor635, Alexa Fluor647, Alexa Fluor660, Alexa Fluor680, Alexa Fluor700, Alexa Fluor750 and Alexa Fluor 790, fluorescein isothiocyanate (FITC), Texas Red, SYBR Green, DyLightFluors, green fluorescent protein (GFP), TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid Acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, TET (6-carboxymethyl-2- ... Fluorescein), HEX (6-carboxy-2', 19. The method of claim 16 or 18, wherein the compound is selected from the group consisting of N,N-diethyl-4-(5'-azobenzotriazolyl)-phenylamine, ...

20. 20. The method of claim 19, wherein the fluorescent label comprises Alexa Fluor 488.

21. 12. The method of any of claims 2 to 11, wherein the second stain comprises a primary antibody that specifically binds to the second marker, and the primary antibody is directly or indirectly coupled to a reagent that is observable under visible light.

22. 22. The method of claim 21, wherein the reagent observable under visible light comprises gold particles, silver particles, or latex particles.

23. 12. The method of any of claims 2 to 11, wherein the second stain comprises a primary antibody that specifically binds to the second marker, and the primary antibody is directly or indirectly coupled to an enzyme that catalyzes a reaction that produces a product observable under visible light.

24. 24. The method of claim 23, wherein the enzyme is selected from the group consisting of alkaline phosphatase, beta-galactosidase, and peroxidase.

25. 25. The method of claim 24, wherein the peroxidase comprises horseradish peroxidase.

26. The peroxidase was reacted with 3,3',5,5'-tetramethylbenzidine (TMB); 3,3'-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol; 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); 2,3,5-triphenyltetrazolium chloride; 2-chloro-5,5-dimethyl-1,3-cyclohexanedione; 3,3',5,5'-tetramethylbenzidine; 3,3'-diaminobenzidine tetrachloride; 3-nitrotetrazolium blue chloride; 4-aminophthalhydrazide; 4-chloro-1-naphthol; 4-chloro-7-nitrobenzofurazan; 5-aminosalicylic acid; dicarboxydihydrochloride; 25. The method of claim 23 or 24, further comprising contacting the sample with a peroxidase substrate selected from the group consisting of: o-dianisidine; guaiacol; hydrogen peroxide-urea adduct; iodonitrotetrazolium chloride; luminol; MTT formazan; N-(4-aminobutyl)-N-ethylisoluminol; N-(6-aminohexyl)-N-ethylisoluminol; nitrotetrazolium blue chloride; pyrogallol; tetranitro blue tetrazolium chloride; tetrazolium blue chloride indicator; tetrazolium violet; o-dianisidine; o-dianisidine dihydrochloride; o-phenylenediamine dihydrochloride; o-phenylenediamine free base; and trans-5-phenyl-4-pentenyl hydroperoxide.

27. 25. The method of claim 24, wherein the enzyme comprises beta-galactosidase.

28. The beta-galactosidase was prepared by the method of:

28. The method of claim 27, further comprising contacting the compound with a beta-galactosidase substrate selected from the group consisting of: 2-nitrophenyl β-D-galactopyranoside; 4-methylumbelliferyl β-D-galactopyranoside; 4-nitrophenyl β-D-galactopyranoside; 5-bromo-3-indolyl β-D-galactopyranoside; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside; 6-bromo-2-naphthyl β-D-galactopyranoside; 6-chloro-3-indolyl-β-D-galactopyranoside; fluorescein di(β-D-galactopyranoside); and resorufin β-D-galactopyranoside.

29. 12. The method of any of claims 2 to 11, wherein the second stain comprises a primary antibody that specifically binds to the second marker, the primary antibody being directly or indirectly coupled to gold particles, and the method further comprises forming a silver precipitate on the gold particles.

30. 30. The method of any of claims 21 to 29, wherein the second marker is selected from the group consisting of alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

31. 31. The method of claim 30, wherein the target cell is an embryonic stem cell.

32. 32. The method of claim 31, wherein said second marker is selected from the group consisting of Oct-4 and Nanog.

33. 33. The method of any of claims 1-2 or 12-32, wherein the first stain comprises a first primary antibody that specifically binds to the second marker, and the first primary antibody is coupled directly or indirectly to a first reagent that is observable under visible light.

34. 34. The method of claim 33, wherein the first reagent observable under visible light comprises gold particles, silver particles, or latex particles.

35. 33. The method of any of claims 1-2 or 12-32, wherein the first stain comprises a first primary antibody that specifically binds to the first marker, and the first primary antibody is coupled directly or indirectly to a first enzyme that catalyzes a reaction that produces a product observable under visible light.

36. 36. The method of claim 35, wherein the first enzyme is selected from the group consisting of alkaline phosphatase, beta-galactosidase, and peroxidase.

37. 37. The method of claim 36, wherein the peroxidase to which the first enzyme is coupled comprises horseradish peroxidase.

38. The peroxidase to which the first enzyme is coupled can be catalyzed by 3,3',5,5'-tetramethylbenzidine (TMB); 3,3'-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol; 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); 2,3,5-triphenyltetrazolium chloride; 2-chloro-5,5-dimethyl-1,3-cyclohexyl Benzidine; 3,3',5,5'-tetramethylbenzidine; 3,3'-diaminobenzidine tetrachloride; 3-nitrotetrazolium blue chloride; 4-aminophthalhydrazide; 4-chloro-1-naphthol; 4-chloro-7-nitrobenzofurazan; 5-aminosalicylic acid; Dicarboxidine dihydrochloride; Guaiacol; Hydrogen peroxide-urea adduct; Iodonitrotetrazolium chloride; Luminol; MTT formazan; N-(4-aminobutyl)-N-ethylisoluminol; N-(6-aminohexyl) 38. The method of claim 36 or 37, further comprising contacting the sample with a peroxidase substrate selected from the group consisting of: (cyl)-N-ethylisoluminol; nitrotetrazolium blue chloride; pyrogallol; tetranitro blue tetrazolium chloride; tetrazolium blue chloride indicator; tetrazolium violet; o-dianisidine; o-dianisidine dihydrochloride; o-phenylenediamine dihydrochloride; o-phenylenediamine free base; and trans-5-phenyl-4-pentenyl hydroperoxide.

39. 36. The method of claim 35, wherein the first enzyme comprises beta-galactosidase.

40. The beta-galactosidase to which the first enzyme is coupled is treated with 1-methyl-3-indolyl-β-D-galactopyranoside; 2-nitrophenyl β-D-galactopyranoside; 4-methylumbelliferyl β-D-galactopyranoside; 4-nitrophenyl β-D-galactopyranoside; 5-bromo-3-indolyl β-D-galactopyranoside; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; 28. The method of claim 27, further comprising contacting the β-galactosidase with a beta-galactosidase substrate selected from the group consisting of: 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside; 6-bromo-2-naphthyl-β-D-galactopyranoside; 6-chloro-3-indolyl-β-D-galactopyranoside; fluorescein di(β-D-galactopyranoside); and resorufin β-D-galactopyranoside.

41. 33. The method of any of claims 1-2 or 12-32, wherein the first stain comprises a primary antibody that specifically binds to the first marker, the primary antibody being directly or indirectly coupled to gold particles, and the method further comprises forming a silver precipitate on the gold particles.

42. 42. The method of any of claims 33 to 41, wherein the first marker is selected from the group consisting of alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

43. 43. The method of claim 42, wherein the target cell is an embryonic stem cell.

44. 44. The method of claim 43, wherein the second marker is selected from the group consisting of Oct-4, Lin28, Sox2, Klf4, n- or c-Myc, and Nanog.

45. 45. The method of any of claims 42 to 44, wherein the first marker is different from the second marker.

46. The target cells are embryonic stem cells, and the second marker is alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen- 4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), germ cell specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, cluster designated 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

47. 10. The method of any of the preceding claims, wherein the cell population comprises cells of a species selected from the group consisting of antelope, cattle, camel, cat, mouse deer, chimpanzee, cow, deer, dog, giraffe, goat, guinea pig, hamster, hippopotamus, horse, human, mouse, non-human primate, ovine, wild boar, pig, pronghorn, rabbit, rat, macaque, rhinoceros, sheep, tapir, and ungulate.

48. 48. The method of claim 47, wherein the cell population comprises human cells.

49. 10. The method of any preceding claim, further comprising determining the approximate number of cells in the cell population.

50. 10. The method of any one of the preceding claims, wherein cells positive for said first marker are detected by examining at least 90% of the cells in said cell population under visible light.

51. 51. The method of claim 50, wherein each cell that is positive for said first marker is tested to determine whether the cell is positive for said second marker.

52. The cell population is at least 10 ５ cells, at least 10 ６ cells, at least 10 ７ cells, at least 10 ８ cells, at least 10 ９ cells, at least 10 １０ cells, or 10 ５ ~10 １０ 10. The method of any one of the preceding claims, comprising cells.

53. 10. The method of claim 1, wherein the first marker and the second marker are embryonic stem cell markers.

54. 10. A method according to any one of the preceding claims, wherein said population of cells is produced by in vivo or in vitro differentiation of embryonic stem cells.

55. 10. The method of any one of the preceding claims, wherein the target cells are pluripotent cells, embryonic stem cells, or induced pluripotent (iPS) cells.

56. 56. The method of claim 55, wherein the cell population further comprises cells differentiated from pluripotent cells, embryonic stem cells, or induced pluripotent (iPS) cells.

57. 10. A method according to any preceding claim, wherein the cell population is cultured under conditions favouring the growth or maintenance of the target cell type.

58. 58. The method of claim 57, wherein the target cell type is an embryonic stem cell.

59. 59. The method of claim 58, wherein said culture conditions comprise culturing said cell population on feeder cells.

60. 60. The method of claim 59, wherein the feeder cells are MEFs.

61. The method according to any one of claims 56 to 60, wherein the cells differentiated from embryonic stem cells or iPS cells are retinal pigment epithelial (RPE) cells.

62. 62. The cell of claim 61, wherein the RPE cell is human.

63. The RPE cells are subjected to the following steps: (a) providing pluripotent stem cells; (b) culturing the pluripotent stem cells to form a multilayered population of pluripotent stem cells or an embryoid body comprising pluripotent stem cells; (c) culturing the multilayered populations or embryoid bodies for a period of time sufficient for RPE cells to appear in the cell culture; and (d) isolating the RPE cells from the culture; 63. The method of claim 61 or 62, wherein the composition is produced by a method comprising:

64. 64. The method of claim 63, 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.

65. The method of claim 63, wherein the pluripotent stem cells are human ES cells or human iPS cells.

66. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 7 to 10 days.

67. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 2 weeks.

68. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 3 weeks.

69. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 4 weeks.

70. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 5 weeks.

71. 66. The method of any one of claims 63 to 65, wherein the duration of step (c) is at least about 6 weeks.

72. 72. The method of any one of claims 61 to 71, wherein said RPE cell culture is substantially free of mouse embryonic feeder cells (MEF) and human embryonic stem cells (hES).

73. 73. The method of any one of claims 61 to 72, wherein the medium used to grow the enriched culture of RPE cells does not support the growth or maintenance of undifferentiated pluripotent stem cells.

74. Target cells include adipocytes, bone marrow fibroblasts, cardiomyocytes, chondrocytes, differentiated RBC and WBC lineages, endothelial bone marrow fibroblasts, ectoderm, ectodermal precursors, embryoid bodies (EBs), embryonic carcinomas (ECs), embryonic stem cells (ESs), endoderm, endothelium, hematopoietic cells, hematopoietic stem cells (HSCs), satellite bodies, endothelial precursors, hepatocytes, keratinocytes, mesenchymal, mesenchymal stem cells (MSCs), mesoderm, MSC precursors, myoblasts, myocytes, neural precursors, neural stem cells, neurons, oligodendrocytes, osteoblasts, 75. The method of any one of claims 1 to 74, wherein the cells are selected from the group consisting of pancreatic epithelium; pancreatic islets; pancreatic progenitors; skeletal muscle cells; smooth muscle; interstitial (mesenchymal) progenitor cells; and white blood cells (WBCs).

75. 72. The method of claim 71, wherein the first marker and the second marker are markers associated with said target cell as set forth in Table 1.

76. 10. The method of any of the preceding claims, further comprising culturing the cell population under conditions favoring maintenance cells of the target cell type prior to step (a).

77. 77. The method of claim 76, wherein the target cell is an embryonic stem cell or an induced pluripotent (iPS) cell.

78. 78. The method of claim 77, wherein the culture conditions comprise embryonic stem cell medium and the presence of mouse embryonic fibroblast feeder cells.

79. 10. The method of any one of the above claims, further comprising plating a second cell population comprising the target cell type and analyzing the second cell population by the same method as the cell population of step (a) to define the detection limit of the method.

80. 80. The method of claim 79, wherein said second cell population consists of or consists essentially of said target cell type.

81. 80. The method of claim 79, wherein said second cell population comprises a mixture of a first group of cells and a second group of cells, said first group of cells being of the same type as the target cell type.

82. 82. The method of claim 81, wherein said second group of cells has essentially the same constituency as said cell population of step (a).

83. the ratio of the number of cells in said first group of cells and said second group of cells is 1:10; １：１００；１：１，０００； １：１０，０００； １：１００，０００；１：１，０００，０００； １：１０，０００，０００； １：１００，０００，０００； １：１，０００，０００，０００； Between 1:10 and 1:100; Between 1:10 and 1:1,000; Between 1:10 and 1:10,000; Between 1:10 and 1:100,000; Between 1:10 and 1:1,000,000; Between 1:10 and 1:10,000,000; Between 1:10 and 1:100,000,000; Between 1:10 and 1:1,000,000,000; Between 1:100 and 1:1,000; Between 1:100 and 1:10,000; Between 1:100 and 1:100,000; Between 1:100 and 1:1,000,000; Between 1:100 and 1:10,000,000; Between 1:100 and 1:100,000,000; Between 1:100 and 1:1,000,000,000; Between 1:1,000 and 1:10,000; Between 1:1,000 and 1:100,000; Between 1:1,000 and 1:1,000,000; Between 1:1,000 and 1:10,000,000; Between 1:1,000 and 1:100,000,000; Between 1:1,000 and 1:1,000,000,000; Between 1:10,000 and 1:100,000; Between 1:10,000 and 1:1,000,000; Between 1:10,000 and 1:10,000,000; Between 1:10,000 and 1:100,000,000; Between 1:10,000 and 1:1,000,000,000; Between 1:100,000 and 1:1,000,000; Between 1:100,000 and 1:10,000,000; Between 1:100,000 and 1:100,000,000; Between 1:100,000 and 1:1,000,000,000; Between 1:1,000,000 and 1:10,000,000; Between 1:1,000,000 and 1:100,000,000; Between 1:1,000,000 and 1:1,000,000,000; Between 1:10,000,000 and 1:100,000,000; １：１０，０００，０ 83. The method of claim 81 or 82, wherein the ratio is selected from the group consisting of: between 1:100 and 1:1,000,000,000; and between 1:100,000,000 and 1:1,000,000,000.

84. 84. The method of any one of claims 79 to 83, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of target cells in the second cell population are detected as positive for the first marker and the second marker of target cells.

85. 85. The method of any one of claims 79 to 84, further comprising plating a third cell population of the target cell type under the same culture conditions and for the same period of time as the cell population of step (a), and detecting and counting cells of the target cell type in the third cell population, thereby determining the proportion of cells of the target cell type that maintain expression of the first and second markers of the target cells under the culture conditions.

86. The method of claim 85, further comprising calculating the detection limit of the method using the second cell population while using the population of cells of the target cell type maintained under the culture conditions as a correction factor to determine the expected number of cells of the target cell type in the second cell population.

87. 87. The method of any one of claims 79 to 86, wherein said target cells in said second cell population and / or said third cell population express another marker that identifies the target cells or their progeny.

88. 88. The method of claim 87, wherein the other marker comprises GFP or another fluorescent protein expressed by the cell.

89. 10. The method according to any one of the preceding claims, wherein the target cells are selected from virus-infected cells, cancerous or pre-cancerous cells, cancer stem cells and immune cells.

90. 90. The method of claim 89, wherein the composition suspected of containing the target cells is a bone marrow, blood cell, or pancreatic cell.

91. 90. The method of claim 89, wherein the cell population suspected of containing the target cells has been previously treated by cell sorting, irradiation, chemotherapy, or other means to eliminate the target cells.

92. A composition comprising somatic cells derived from said stem cells that is essentially free of said stem cells.

93. A composition comprising cells and an indicator indicating the number or proportion of stem cells present, wherein the value of the indicator is measured using a method according to any of the above claims applied to a cell population representative of the cells in the composition.

94. At least 10 ５ cells, at least 10 ６ cells, at least 10 ７ cells, at least 10 ８ cells, at least 10 ９ cells, at least 10 １０ cells, or 10 ５ and 10 １０ 94. The composition of claim 93, comprising cells between .

95. 95. The composition of any of claims 92 to 94, comprising cryopreserved cells.

96. The following steps: (a) providing a cell population; (b) applying a first stain and a second stain to the cell population, wherein the first stain is wherein said first stain detects alkaline phosphatase and said second stain detects a marker indicative of embryonic stem cells, and cells positive for said first stain are detectable under visible and ultraviolet light, and cells positive for said second stain are detectable under ultraviolet light; (c) examining the cell population under visible light to detect cells that are positive for the first marker. Microscopic observation of the cells of the group; (e) microscopically observing the cells that are positive for the first marker under ultraviolet light; and determining whether the cells are positive for the first marker and the second marker; and (f) identifying cells that are positive for the first marker and the second marker as embryonic stem cells. and identifying; A method for detecting the presence of human embryonic stem cells in a cell population comprising:

97. A method for detecting the presence of human embryonic stem (hES) cells in a cell population, comprising plating the cell population in hES cell medium on feeder cells, applying a stain that detects a marker indicative of hES cells, and identifying cells that are positive for the marker.

98. The method of any preceding claim, further comprising applying a third stain to the cell population, wherein the third stain detects a third marker whose expression indicates the presence of a target cell; detecting any cells that are positive for the third marker by microscopically observing the cells of the cell population; and identifying any cells that are positive for the first marker, the second marker, and the third marker as target cells.

99. 99. The method of claim 98, wherein the third stain is visually distinguishable from the first stain and from the second stain.

100. 100. The method of claim 98 or 99, wherein the third stain is detectable under visible light or ultraviolet light.

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