Improved methods of producing RPE cells and compositions of RPE cells

Abstract

To provide improved methods of producing RPE cells and compositions of RPE cells.SOLUTION: The present invention provides improved methods for producing RPE cells from human embryonic stem cells or from other human pluripotent stem cells. The invention also relates to human retinal pigmented epithelial cells derived from human embryonic stem cells or other human multipotent or pluripotent stem cells. Here, hRPE cells derived from embryonic stem cells are molecularly distinct from adult and fetal-derived RPE cells, and are also distinct from embryonic stem cells. The hRPE cells described herein are useful for treating retinal degenerative diseases.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application Nos. 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. The disclosures of each of the above applications are incorporated herein by reference in their entirety. [Background technology]

[0002] The retinal pigment epithelium (RPE) is the layer of pigment cells just outside the neurosensory retina. This layer of cells provides nutrients to the retinal visual cells and covers them in association with the underlying choroid (a layer of blood vessels behind the retina). The RPE acts as a filter to determine what nutrients reach the retina from the choroid. In addition, the RPE provides a barrier between the retina and the choroid. Disruption of the RPE impedes retinal metabolism and causes retinal thinning. Retinal thinning can have serious consequences. For example, retinal thinning can cause "dry" macular degeneration, and can also result in inappropriate angiogenesis that can cause "wet" macular degeneration.

[0003] Due to the importance of the RPE in maintaining vision and retinal health, there has been a great deal of effort in studying the RPE and developing methods to generate RPE cells in vitro. RPE cells generated in vitro can be used to study the development of the RPE, to identify factors that cause the destruction of the RPE, or to identify drugs that can be used to stimulate the repair of endogenous RPE cells. In addition, RPE cells generated in vitro can be used themselves as a therapy to replace or repair all or part of damaged RPE cells in a patient. When used in this way, RPE cells may provide an approach to treat macular degeneration as well as other diseases and conditions caused in whole or in part by damage to the RPE.

[0004] The use of in vitro generated RPE cells for screening or therapeutic purposes depends on methods that can be used to generate large numbers of RPE cells in a systematic, directed manner. Such organized differentiation methods may offer significant advantages over previous mechanisms that were based on spontaneous differentiation of RPE cells, for example, from transformed cell lines or other sources. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a method for differentiating RPE cells from human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells. The method is used to generate large numbers of differentiated RPE cells for use in screening tests, to study the basic biology of RPE, and as a therapeutic method. As described herein, RPE cells differentiated from pluripotent stem cells, such as human embryonic stem cells, using this method are molecularly distinct from human embryonic stem cells as well as from adult and fetal RPE cells.

[0006] The present invention also provides preparations and pharmaceutical preparations of RPE cells derived from human pluripotent stem cells, which preparations of RPE cells are molecularly distinct from human embryonic stem cells, as well as from RPE cells of adult and fetal origin.

[0007] The present invention provides the first detailed molecular characterization of RPE cells differentiated from human embryonic stem cells.Detailed characterization includes comparison with RPE cells from other sources (such as adult RPE cells and fetal RPE cells) and human embryonic stem cells.This analysis not only provides a deeper understanding of RPE, but also reveals that RPE cells differentiated from human embryonic stem cells have distinct molecular characteristics that distinguish these cells from the RPE cells described above.

[0008] The present invention provides preparations of RPE cells, including preparations of substantially purified RPE cells. Exemplary RPE cells are differentiated from human pluripotent stem cells, such as human embryonic stem cells or iPS cells. RPE cells derived from human pluripotent stem cells can be used and formulated to treat retinal degenerative diseases. In addition, RPE cells derived from human pluripotent stem cells can be used in screening tests to identify agents that regulate RPE cell survival (in vitro and / or in vivo), to study maturation of RPE cells, or to identify agents that regulate maturation of RPE cells. Agents identified using such screening tests can be used in vitro or in vivo, and can provide additional therapeutics that can be used alone or in combination to treat retinal degenerative diseases.

[0009] The present invention provides an improved method for generating RPE cells from embryonic stem cells or other pluripotent stem cells. The method of the present invention can be used to generate differentiated RPE cells. Optionally, the level of maturation of differentiated RPE cells can be adjusted to generate differentiated RPE cells, mature RPE cells, or a mixture thereof, as assessed by pigmentation level. Also provided is an improved method for treating eye disorders. Specifically, these methods involve the use of RPE cells derived from human embryonic stem cells to treat or ameliorate the symptoms of eye disorders, particularly eye disorders that are caused or exacerbated in whole or in part by damage to or destruction of endogenous RPE layer.

[0010] In certain aspects, the present invention provides a method for generating a culture of retinal pigment epithelium (RPE) cells.In certain embodiments, the culture is a substantially purified culture that contains at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% differentiated RPE cells (at least 75% of the culture is differentiated RPE cells, regardless of the level of maturity).In certain embodiments, the substantially purified culture contains at least 30%, 35%, 40%, or 45% mature differentiated RPE cells.In certain embodiments, the substantially purified culture contains at least 50% mature differentiated RPE cells. In other embodiments, the substantially purified culture contains at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% mature differentiated RPE cells. In certain embodiments, the differentiated RPE cells are derived from human embryonic stem cells, human iPS cells, or other pluripotent stem cells.

[0011] In certain embodiments, the method comprises: a) providing human embryonic stem cells; b) culturing human embryonic stem cells as embryoid bodies in a nutrient-rich, low-protein medium, the medium optionally containing serum-free B-27 supplement; c) culturing the embryoid bodies as adherent cultures in a rich, low protein medium, the medium optionally containing serum-free B-27 supplement; d) culturing an adherent culture of the cells of (c) in a nutrient-rich, low-protein medium, the medium being serum-free and not containing B-27 supplement; e) culturing the cells of (d) in a medium capable of supporting the growth of a high density somatic cell culture, whereby RPE cells appear in the culture of cells; f) contacting the culture of (e) with an enzyme; g) selecting RPE cells from the culture and transferring the RPE cells into a separate culture containing a medium supplemented with growth factors to generate an enriched culture of RPE cells; and h) propagating the enriched culture of RPE cells to produce a substantially purified culture of RPE cells.

[0012] In certain other aspects, the present invention provides a method of generating mature retinal pigment epithelial cells (RPE), the method comprising: a) providing human embryonic stem cells; b) culturing human embryonic stem cells as embryoid bodies in a nutrient-rich, low-protein medium, the medium optionally containing serum-free B-27 supplement; c) culturing the human embryonic stem cells as adherent cultures in a nutrient-rich, low-protein medium, the medium optionally containing serum-free B-27 supplement; d) culturing an adherent culture of the cells of step (c) in a nutrient-rich, low-protein medium, the medium being serum-free and free of B-27 supplement; e) culturing the cells of (d) in a medium capable of supporting the growth of a high density somatic cell culture, whereby RPE cells appear in the culture of cells; f) contacting the culture of (e) with an enzyme; g) selecting the RPE cells and transferring the RPE cells into a separate culture containing a medium supplemented with growth factors to generate an enriched culture of RPE cells; h) propagating the enriched culture of RPE cells; i) culturing the enriched culture of RPE cells to produce mature RPE cells.

[0013] In any of the above-mentioned specific embodiments, the culture of substantially purified RPE cells can contain both differentiated RPE cells and mature differentiated RPE cells.The level of pigmentation may vary among mature RPE cells.However, mature RPE cells can be visually distinguished from RPE cells based on increased pigmentation level and more cylindrical shape.

[0014] In certain embodiments, the proportion of mature differentiated RPE cells in the culture can be reduced by decreasing the density of the culture. Thus, in certain embodiments, the method further comprises subculturing the population of mature RPE cells to generate a culture containing a smaller proportion of mature RPE cells.

[0015] In certain embodiments, the medium used when culturing cells as embryoid bodies can be selected from any medium suitable for culturing cells as embryoid bodies. For example, the skilled artisan can select from commercially available or proprietary media. Any medium that can support high density culture can be used, such as media for viral, bacterial, or eukaryotic cell culture. For example, the medium can be a high nutrient protein-free medium or a high nutrient low protein medium. For example, human embryonic stem cells can be cultured in MDBK-GM, OptiPro SFM, VP-SFM, EGM-2, or MDBK-MM. In certain embodiments, the medium can contain B-27 supplement.

[0016] In certain embodiments, the medium described herein can be supplemented with one or more growth factors.Growth factors that can be used include, for example, EGF, bFGF, VEGF, and recombinant insulin-like growth factor.The medium can also contain supplements such as heparin, hydrocortisone, ascorbic acid, serum (e.g., fetal bovine serum, etc.), or growth matrix (e.g., extracellular matrix from Matrigel (BD Biosciences) or gelatin, etc.).

[0017] In certain embodiments, mechanical or enzymatic methods are used to select RPE cells from the culture of non-RPE cells in the culture of embryoid bodies or to facilitate the subculture of adherent cells.Exemplary mechanical methods include, but are not limited to, titration with a pipette or cutting with a pulled needle.Exemplary enzymatic methods include, but are not limited to, any enzyme suitable for dissociating cells (e.g., trypsin, collagenase, dispase).In certain embodiments, non-enzymatic solutions are used to dissociate cells, such as solutions containing high EDTA, such as Hanks-based cell dissociation buffer.

[0018] In certain embodiments, for any of the relevant steps described above, the cells are cultured for about 3 to 45 days, such as 7 days, 7-10 days, 7-14 days, or 14-21 days. In certain embodiments, the cells are 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, or about 46 days. In certain embodiments, the cells are cultured for no more than about 45, 40, 35, 30, 25, 21, 20, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. Note that for each of the relevant method steps described above, the cells may be cultured for the same period of time at each step, or for different periods of time at one or more of the steps.

[0019] In certain embodiments, RPE cells are further cultured to generate a culture of mature RPE cells. Both RPE cells and mature RPE cells are differentiated RPE cells. However, mature RPE cells are characterized by an increased level of pigment compared to differentiated RPE cells. The maturity and level of pigmentation can be adjusted by increasing or decreasing the density of the culture of differentiated RPE cells. Thus, the culture of RPE cells can be further cultured to generate mature RPE cells. Alternatively, the density of the culture containing mature RPE cells can be decreased to decrease the proportion of mature differentiated RPE cells and increase the proportion of differentiated RPE cells.

[0020] The medium used for culturing RPE cells is any medium suitable for cell culture, and can be selected by those skilled in the art.For example, any medium that can support high-density culture, such as the medium for viral, bacterial, or animal cell culture, can be used.For example, the cells described herein can be cultured in VP-SFM, EGM-2, and MDBK-MM.

[0021] In any of the above specific embodiments, the culture of substantially purified RPE cells (with or without mature RPE cells) is frozen for storage. The cells may be stored by any suitable method known in the art, such as cryogenic freezing, and may be frozen at any temperature suitable for storing the cells. For example, the cells may be frozen at about -20°C, -80°C, -120°C, or any temperature suitable for storing the cells. The cryogenically frozen cells are stored in a suitable container and prepared for storage to reduce the risk of cell damage and maximize the chance of the cells surviving upon thawing. In other embodiments, the RPE cells are kept at room temperature or refrigerated, for example at about 4°C.

[0022] In any of the specific embodiments described above, the method is carried out in accordance with Good Manufacturing Practices (GMP). In any of the specific embodiments described above, the human embryonic stem cells from which the RPE cells are differentiated were derived in accordance with Good Manufacturing Practices (GMP). In any of the specific embodiments described above, the human embryonic stem cells from which the RPE cells are differentiated were derived from one or more blastomeres removed from an early embryo without destroying the remainder of the embryo.

[0023] In any of the specific embodiments described above, the method includes: 5 RPE cells, at least 5 x 10 5 RPE cells, at least 1 x 10 6 RPE cells, at least 5 x 10 6 RPE cells, at least 1 x 10 7 RPE cells, at least 2 x 10 7 RPE cells, at least 3 x 10 7 RPE cells, at least 4 x 10 7 RPE cells, at least 5 x 10 7 RPE cells, at least 6 x 10 7 RPE cells, at least 7 x 10 7 RPE cells, at least 8 x 10 7 RPE cells, at least 9 x 10 7 RPE cells, at least 1 x 10 8 RPE cells, at least 2 x 10 8 RPE cells, at least 5 x 10 8 RPE cells, at least 7 x 10 8 RPE cells or at least 1 × 10 9 The RPE cells are used to produce a preparation comprising RPE cells. In certain embodiments, the number of RPE cells in the preparation includes differentiated RPE cells, regardless of the level of maturity and regardless of the relative proportion of differentiated RPE cells and mature RPE cells. In other embodiments, the number of RPE cells in the preparation refers to either the number of differentiated RPE cells or mature RPE cells.

[0024] In certain embodiments, the method further comprises formulating the differentiated RPE cells and / or differentiated mature RPE cells to produce a preparation of RPE cells suitable for transplantation.

[0025] In another aspect, the present invention provides a method for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a preparation comprising RPE cells, and the RPE cells are derived from human embryonic stem cells, iPS cells, or other pluripotent stem cells.The condition characterized by retinal degeneration includes, for example, Stargardt's macular dystrophy, age-related macular degeneration (dry or wet), diabetic retinopathy, and retinitis pigmentosa.In certain embodiments, RPE cells are derived from human pluripotent stem cells by using one or more of the methods described herein.

[0026] In certain embodiments, the formulations were previously stored frozen and thawed prior to implantation.

[0027] In certain embodiments, the method of treatment further comprises administration of cyclosporine or one or more other immunosuppressive agents. When an immunosuppressive agent is used, the immunosuppressive agent may be administered systemically or locally and may be administered prior to, simultaneously with, or after administration of the RPE cells. In certain embodiments, immunosuppressive therapy continues for weeks, months, years, or indefinitely following administration of the RPE cells.

[0028] In certain embodiments, the method of treatment includes administration of a single dose of RPE cells. In other embodiments, the method of treatment includes 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, treatment may proceed in parallel, thereby requiring multiple administrations initially (e.g., a daily dose in the first week), followed by a reduction in frequency and dosage. Numerous treatment regimes are contemplated.

[0029] In certain embodiments, the administered RPE cells comprise a mixed population of differentiated RPE cells and mature RPE cells.In other embodiments, the administered RPE cells comprise a substantially purified population of either differentiated RPE cells or mature RPE cells.For example, the administered RPE cells can contain less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of other RPE cell types.

[0030] In certain embodiments, the RPE cells are formulated in a pharma- ceutically acceptable carrier or excipient.

[0031] In certain embodiments, the formulation comprising RPE cells is implanted in a suspension, matrix, or substratum. In certain embodiments, the formulation is administered by injection into the subretinal space of the eye. In certain embodiments, the formulation is administered by injection into the subretinal space of the eye. 4 ~about 10 6 The cells are administered to a subject. In certain embodiments, the method further comprises monitoring the effectiveness of the treatment or prevention by measuring electroretinogram response, optokinetic acuity threshold, or luminance threshold in the subject. The method may also comprise monitoring the effectiveness of the treatment or prevention by monitoring the immunogenicity of the cells or the migration of cells in the eye.

[0032] In a particular embodiment, the present invention provides a pharmaceutical preparation for treating or preventing a condition characterized by retinal degeneration, comprising an effective amount of RPE cells, wherein the RPE cells are derived from human embryonic stem cells or other pluripotent stem cells. The pharmaceutical preparation can be formulated in a pharma- ceutical acceptable carrier according to the route of administration. For example, the preparation can be formulated for administration to the subretinal space of the eye. The composition can be administered in an amount of at least 100 mg / ml. 4 , 10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 10 6 , 2×10 6 , 3×10 6 , 4×106 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , or 10 7 In certain embodiments, the composition may comprise at least 2×10 RPE cells. 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 In certain embodiments, the RPE cells may include mature RPE cells, and thus the cell number includes the sum of both differentiated RPE cells and mature differentiated RPE cells.

[0033] In another embodiment, the present invention provides a method for screening to identify agents that regulate the survival of RPE cells.For example, RPE cells differentiated from human embryonic stem cells can be used to screen agents that promote the survival of RPE.Identified agents can be used alone or in combination with RPE cells as part of a treatment plan.Alternatively, identified agents can be used as part of a culture method that enhances the survival of in vitro differentiated RPE cells.

[0034] In another embodiment, the present invention provides a method for screening to identify agents that regulate the maturation of RPE cells.For example, RPE cells differentiated from human ES cells can be used to screen agents that promote the maturation of RPE.

[0035] In any of the above specific embodiments, the method is carried out in accordance with Good Manufacturing Practice (GMP). In any of the above specific embodiments, the human embryonic stem cells or other pluripotent stem cells from which the RPE cells are differentiated were derived in accordance with Good Manufacturing Practice (GMP). In any of the above specific embodiments, the human embryonic stem cells from which the RPE cells are differentiated were derived from one or more blastomeres removed from an early embryo without destroying the remainder of the embryo.

[0036] In another embodiment, the present invention contemplates that the starting material or preparation thereof for generating RPE cells can be other types of human pluripotent stem cells instead of human embryonic stem cells.As an example, the present invention contemplates using the included induced pluripotent stem (iPS) cells as starting material for differentiating RPE cells using the methods described herein.Such iPS cells can be obtained from cell banks or can be previously derived.Alternatively, iPS cells can be newly generated before starting to differentiate into RPE cells.

[0037] In one embodiment, RPE cells or preparations differentiated from pluripotent stem cells, including iPS cells, are used in therapeutic methods.

[0038] The present invention also provides functional human retinal pigment epithelial (hRPE) cells terminally differentiated from human embryonic stem cells (hESCs) or other human pluripotent stem cells. In transplantation experiments in non-human primates, these hRPEs can be identified from other cells by their unique physical characteristics, such as unique mRNA and protein expression. Furthermore, hRPEs can treat retinal degeneration in affected animals when implanted into validated animal models of retinal degeneration. Thus, the hRPEs of the present invention are useful for treating patients suffering from various retinal degenerative disorders. The present invention thus provides a renewable source of hRPEs that can be produced and manufactured under GLP-like and GMP-compliant conditions for the treatment of visual degenerative diseases and disorders.

[0039] In certain embodiments, the present invention provides human retinal pigment epithelial cells derived from human embryonic stem cells, which are pigmented and express at least one gene that is not expressed in cells that are not human retinal pigment epithelial cells. In certain embodiments, the human retinal pigment epithelial cells are isolated from at least one protein, molecule, or other impurity found in their natural environment.

[0040] In another embodiment, the present invention provides a cell culture comprising human RPE cells derived from human embryonic stem cells or other pluripotent stem cells, which express at least one gene that is pigmented and is not expressed in non-human RPE cells.As used in this way, pigmentation refers to any level of pigmentation, for example, the level of pigmentation that first occurs when RPE cells are differentiated from ES cells.Pigmentation may vary depending on the cell density and maturity of differentiated RPE cells.However, when cells are referred to as pigmented, this term should be understood to refer to any and all levels of pigmentation.

[0041] In some embodiments, the cell culture comprises a substantially purified population of human RPE cells. A substantially purified population of hRPE cells is one in which the hRPE cells comprise at least about 75% of the cells in the population. In other embodiments, a substantially purified population of hRPE cells is one in which the hRPE cells comprise at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, or even greater than 99% of the cells in the population. In some embodiments, the pigmentation of the hRPE cells in the cell culture is uniform. In other embodiments, the pigmentation of the hRPE cells in the cell culture is heterogeneous, and the culture of RPE cells comprises both differentiated and mature RPE cells. The cell culture of the present invention comprises at least about 10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , or at least about 10 9 The cell may comprise hRPE cells.

[0042] The present invention provides human retinal pigment epithelial cells with various degrees of pigmentation.In certain embodiments, the pigmentation of human retinal pigment epithelial cells is the same as the average human pigment epithelial cells after terminal differentiation of hRPE cells.In certain embodiments, the pigmentation of human retinal pigment epithelial cells is more pigmented than the average human retinal pigment epithelial cells after terminal differentiation of hRPE cells.In certain other embodiments, the pigmentation of human retinal pigment epithelial cells is less pigmented than the average human retinal pigment epithelial cells after terminal differentiation.

[0043] In certain embodiments, the invention provides human RPE cells differentiated from ES cells or other pluripotent stem cells and expressing (at the mRNA and / or protein level) one or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F (1, 2, 3, 4, 5, or 6). In certain embodiments, gene expression is measured by mRNA expression. In other embodiments, gene expression is measured by protein expression. In certain embodiments, the RPE cells are The RPE cells do not substantially express ES-specific genes, such as Oct-4, alkaline phosphatase, nanog, and / or Rex-1. In other embodiments, the RPE cells express one or more (1, 2, or 3) of pax-2, pax-6, and / or tyrosinase. In certain embodiments, expression of pax-2, pax-6, and / or tyrosinase distinguishes mature differentiated RPE cells from differentiated RPE cells. In other embodiments, the RPE cells express one or more of the markers shown in Table 2, and the expression of one or more markers is upregulated in the RPE cells relative to their expression (if any) in human ES cells. In other embodiments, the RPE cells express one or more of the markers shown in Table 3, and the expression of one or more markers is downregulated in the RPE cells relative to their expression (if any) in human ES cells.

[0044] In certain embodiments, the present invention provides pharmaceutical preparations comprising human RPE cells derived from human embryonic stem cells or other pluripotent stem cells. 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or about 10 9 The cell may comprise hRPE cells.

[0045] In other embodiments, the present invention provides a cryopreserved formulation of RPE cells described herein. The cryopreserved formulation can be frozen for storage for days or years. The cells can be stored by any suitable method known in the art, such as cryogenic freezing, and can be frozen at any temperature suitable for storing cells. For example, the cells can be frozen at about -20°C, -80°C, -120°C, or any temperature suitable for storing cells. The cryopreserved cells are stored in a suitable container and prepared for storage to reduce the risk of cell damage and maximize the likelihood that the cells will survive thawing. In other embodiments, the RPE cells can be kept at room temperature or refrigerated, for example at about 4°C. The cryopreserved formulation of the compositions described herein can be frozen for at least about 10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or about 10 9 hRPE cells. In certain embodiments, the hRPE cells of the present invention are recovered from storage after cryopreservation. In certain embodiments, more than 65%, 70%, 75%, or more than 80% of the RPE cells remain viable after cryopreservation. In other embodiments, more than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the RPE cells remain viable after deep cryopreservation.

[0046] In another aspect, the present invention provides preparations of substantially purified human RPE cells having any combination of the structural, molecular, and functional characteristics described herein. Such preparations may be formulated as pharmaceutical preparations for administration and / or may be formulated for cryopreservation.

[0047] In another aspect, the present invention provides the use of human RPE cells as described herein in the manufacture of a medicament for treating a condition in a patient in need thereof. In another embodiment, the present invention provides the use of a cell culture comprising human RPE cells as described herein in the manufacture of a medicament for treating a condition in a patient in need thereof. In another embodiment, the present invention provides the use of a pharmaceutical preparation comprising human RPE cells as described herein in the manufacture of a medicament for treating a condition in a patient in need thereof. Conditions that may be treated include, but are not limited to, degenerative diseases of the retina, such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration, glaucoma, and diabetic retinopathy. In certain embodiments, the present invention provides a method of treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a preparation comprising RPE cells, wherein the RPE cells are derived from mammalian embryonic stem cells. Conditions characterized by retinal degeneration include, for example, Stargardt's macular dystrophy, age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa.

[0048] In another embodiment, the present invention provides a solution of human RPE cells derived from human embryonic stem cells or other pluripotent stem cells, the RPE cells having any combination of the characteristics described herein. Such a solution can be prepared by subjecting the RPE cells to at least about 10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or about 10 9 The solution may contain 100 hRPE cells. Such solutions are suitable for injection into a subject. Such solutions are suitable for cryopreservation as described herein. The invention also provides the use of these solutions for the manufacture of a medicament for treating a disease that can be treated by administration of RPE cells, such as, for example, a retinal degenerative disease of the eye.

[0049] In another embodiment, the RPE cells of the present invention are derived from human embryonic stem cells or other potent stem cells previously derived under GMP regulations. In one embodiment, the human ES cells are derived from one or more blastomeres of early cleavage stage embryos, optionally without destroying the embryo. In another embodiment, the human ES cells are derived from a library of human embryonic stem cells. In a particular embodiment, the library of human embryonic stem cells comprises stem cells that are hemizygous, homozygous, or nullzygous for at least one MHC allele present in the human population, and each member of the library of human embryonic stem cells is hemizygous, homozygous, or nullzygous for a different set of MHC alleles relative to the remaining members of the library. In a further embodiment, the library of human embryonic stem cells comprises stem cells that are hemizygous, homozygous, or nullzygous for all MHC alleles present in the human population. In certain other embodiments, the invention provides libraries of RPE cells that are each hemizygous, homozygous, or nullzygous for at least one MHC allele present in a human population, where each member of the library of RPE cells is hemizygous, homozygous, or nullzygous for a different set of MHC alleles relative to the remaining members of the library. In further embodiments, the invention provides libraries of human PRE cells that are hemizygous, homozygous, or nullzygous for all MHC alleles present in a human population.

[0050] In any of the above specific embodiments, the culture of substantially purified RPE cells (with or without mature RPE cells) is frozen for storage. The cells may be stored by any suitable method known in the art, such as cryogenic freezing, and may be frozen at any temperature suitable for storing the cells. For example, the cells may be frozen at about -20°C, -80°C, -120°C, or any temperature suitable for storing the cells. The cryogenically frozen cells are stored in a suitable container and prepared for storage to reduce the risk of cell damage and maximize the chance of the cells remaining viable upon thawing. In other embodiments, the RPE cells can be kept at room temperature or refrigerated, for example at about 4°C.

[0051] In any of the above specific embodiments, the human RPE cells are produced in accordance with Good Manufacturing Practices (GMP). In any of the above specific embodiments, the human embryonic stem cells from which the RPE cells are differentiated are derived in accordance with Good Manufacturing Practices (GMP). In any of the above specific embodiments, the human embryonic stem cells from which the RPE cells are differentiated are derived from one or more blastomeres that are removed from an early embryo without destroying the remainder of the embryo. Thus, the present invention provides GMP-compliant preparations of RPE cells, including substantially purified preparations of RPE cells. Such preparations are substantially free of viral, bacterial, and / or fungal contamination or infection.

[0052] In any of the above specific embodiments, the composition or preparation of RPE cells comprises at least 1×10 5 RPE cells, at least 5 x 10 5 RPE cells, at least 1 x 10 6 RPE cells, at least 5 x 10 6 RPE cells, at least 1 x 10 7 RPE cells, at least 2 x 10 7 RPE cells, at least 3 x 10 7 RPE cells, at least 4 x 10 7RPE cells, at least 5 x 10 7 RPE cells, at least 6 x 10 7 RPE cells, at least 7 x 10 7 RPE cells, at least 8 x 10 7 RPE cells, at least 9 x 10 7 RPE cells, at least 1 x 10 8 RPE cells, at least 2 x 10 8 RPE cells, at least 5 x 10 8 RPE cells, at least 7 x 10 8 RPE cells or at least 1 × 10 9 In certain embodiments, the number of RPE cells in the preparation comprises differentiated RPE cells, regardless of the level of maturity and regardless of the relative proportion of differentiated RPE cells and mature differentiated RPE cells. In other embodiments, the number of RPE cells in the preparation refers to the number of either differentiated RPE cells or mature RPE cells.

[0053] In certain embodiments, the method further comprises formulating the differentiated RPE cells and / or differentiated mature RPE cells to produce a preparation of RPE cells suitable for transplantation.

[0054] In another aspect, the present invention provides a method for treating or preventing the condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a preparation comprising RPE cells, wherein the RPE cells are derived from human pluripotent stem cells.In certain embodiments, the RPE cells are derived using any of the methods described herein.The condition characterized by retinal degeneration includes, for example, Stargardt's macular dystrophy, age-related macular degeneration (dry or wet), diabetic retinopathy, and retinitis pigmentosa.

[0055] In certain embodiments, the formulations were previously stored frozen and thawed prior to implantation.

[0056] In certain embodiments, the method of treatment further comprises administration of cyclosporine or one or more other immunosuppressants. In use, the immunosuppressants may be administered systemically or locally, and may be administered prior to, simultaneously with, or after administration of the RPE cells. In certain embodiments, the immunosuppressive therapy continues for weeks, months, years, or indefinitely after administration of the RPE cells.

[0057] In certain embodiments, the method of treatment comprises administration of a single dose of RPE cells. In other embodiments, the method of treatment comprises 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, treatment may proceed in parallel, thereby requiring multiple administrations initially (e.g., a daily dose in the first week), followed by a reduction in frequency and dosage. Numerous treatment regimes are contemplated.

[0058] In certain embodiments, the administered RPE cells comprise a mixed population of differentiated RPE cells and mature RPE cells.In other embodiments, the administered RPE cells comprise a substantially purified population of either differentiated RPE cells or mature RPE cells.For example, the administered RPE cells can contain less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of other RPE cell types.

[0059] In certain embodiments, the RPE cells are formulated in a pharma- ceutically acceptable carrier or excipient.

[0060] In certain embodiments, the formulation comprising RPE cells is implanted in a suspension, matrix, or substratum. In certain embodiments, the formulation is administered by injection into the subretinal space of the eye. In certain embodiments, the formulation is administered transcorneal. In certain embodiments, the formulation is administered about 10 4 ~about 10 6 In certain embodiments, the method comprises administering to the subject The method further includes monitoring the effectiveness of the treatment or prevention by measuring electroretinogram response, optokinetic acuity threshold, or luminance threshold in the eye. The method may also include monitoring the effectiveness of the treatment or prevention by monitoring the immunogenicity of cells or the migration of cells in the eye.

[0061] In a particular embodiment, the present invention provides a pharmaceutical preparation for treating or preventing a condition characterized by retinal degeneration, comprising an effective amount of RPE cells, wherein the RPE cells are derived from human embryonic stem cells or other pluripotent stem cells. The pharmaceutical preparation can be formulated in a pharma- ceutical acceptable carrier according to the route of administration. For example, the preparation can be formulated for administration to the subretinal space or cornea of ​​the eye. The composition can be administered in an amount of at least 100 mg / ml. 4 , 10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , or 10 7 In certain embodiments, the composition may comprise at least 2×10 RPE cells. 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 In certain embodiments, the RPE cells may include mature RPE cells, and thus the cell number includes the sum of both differentiated RPE cells and mature differentiated RPE cells.

[0062] In another embodiment, the present invention provides a method for screening to identify agents that regulate the survival of RPE cells.For example, RPE cells differentiated from human embryonic stem cells can be used to screen agents that promote the survival of RPE.Identified agents can be used alone or in combination with RPE cells as part of a treatment plan.Alternatively, identified agents can be used as part of a culture method that enhances the survival of in vitro differentiated RPE cells.

[0063] In another embodiment, the present invention provides a method for screening to identify agents that regulate the maturation of RPE cells.For example, RPE cells differentiated from human ES cells can be used to screen agents that promote the maturation of RPE.

[0064] In any of the above specific embodiments, the method is carried out in accordance with Good Manufacturing Practices (GMP). In any of the above specific embodiments, the human embryonic stem cells from which the RPE cells are differentiated are derived in accordance with Good Manufacturing Practices (GMP). In any of the above specific embodiments, the human embryonic stem cells from which the RPE cells are differentiated are derived from one or more blastomeres that are removed from an early embryo without destroying the remainder of the embryo.

[0065] In another embodiment, the present invention contemplates that the starting material or preparation thereof for generating RPE cells can be other types of human pluripotent stem cells instead of human embryonic stem cells.As an example, the present invention contemplates that induced pluripotent stem (iPS) cells, which contain hES characteristics, can also be used as starting material for differentiating RPE cells using the methods described herein.Such iPS cells can be obtained from cell banks or can be previously derived.Alternatively, iPS cells can be generated de novo before starting to differentiate into RPE cells.

[0066] In one embodiment, RPE cells or preparations differentiated from pluripotent stem cells, including iPS cells, are used in therapeutic methods.

[0067] The present invention contemplates any combination of the above or below described aspects and embodiments. For example, preparations of RPE cells, including any combination of differentiated RPE cells and mature RPE cells, may be used to treat any of the diseases and conditions described herein. Similarly, the methods described herein for generating RPE cells using human embryonic stem cells as a starting material can similarly be carried out using any human pluripotent stem cells as a starting material. The present invention provides, for example, the following items. (Item 1) 1. A method for producing a substantially purified culture of retinal pigment epithelial (RPE) cells, comprising: a) providing human embryonic stem cells; b) culturing the human stem cells as embryoid bodies in a nutrient-rich, low-protein medium; c) culturing said embryoid bodies as adherent cultures in a nutrient-rich, low-protein medium; d) culturing an adherent culture of the cells of (c) in a nutrient-rich, low-protein medium, the medium being serum-free and lacking B-27 supplement; e) culturing the cells of (d) in a medium capable of supporting the growth of a high density somatic cell culture, whereby RPE cells appear in the culture of said cells; f) contacting the culture of (e) with an enzyme; g) selecting the RPE cells from the culture and transferring the RPE cells into a separate culture containing a medium supplemented with growth factors to generate an enriched culture of RPE cells; h) propagating the enriched culture of RPE cells to produce a culture of substantially purified RPE cells; A method comprising: (Item 2) The method of claim 1, wherein the RPE cells are further cultured to generate a culture of mature RPE cells. (Item 3) 3. The method according to item 1 or 2, wherein the medium of (b) and / or (c) contains serum-free B-27 supplement. (Item 4) 3. The method of claim 2, wherein the cells are cultured in a medium selected from the group consisting of VP-SFM, EGM-2, and MDBK-MM. (Item 5) The method according to item 2, wherein the growth factor (g) is selected from the group consisting of EGF, bFGF, VEGF, and recombinant insulin-like growth factor. (Item 6) 3. The method of claim 2, wherein the medium contains one or more of heparin, hydrocortisone, or ascorbic acid. (Item 7) 7. The method according to any one of items 1 to 6, wherein the cells of (b) are cultured for 7 to 14 days. (Item 8) 8. The method according to any one of items 1 to 7, wherein the cells of (c) are cultured for 7 days. (Item 9) 9. The method according to any one of items 1 to 8, wherein the cells of (d) are cultured for 7 to 10 days. (Item 10) 10. The method according to any one of items 1 to 9, wherein the cells of (e) are cultured for 14 to 21 days. (Item 11) The medium (b) was selected from MDBK-GM, OptiPro SFM, and VP SFM. The method according to any one of items 1 to 10, wherein the method is selected from the group consisting of: (Item 12) The method according to any one of items 1 to 11, wherein the medium (c) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 13) The method according to any one of items 1 to 12, wherein the medium (d) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 14) The method according to any one of items 1 to 13, wherein the medium (e) is selected from the group consisting of MDBK-MM, OptiPro SFM, and VP SFM. (Item 15) The method according to any one of items 1 to 14, wherein the medium (g) is selected from the group consisting of EGM-2 and MDBK-MM. (Item 16) 16. The method according to any one of items 1 to 15, wherein the growth factor (g) is selected from the group consisting of EGF, bFGF, VEGF, and recombinant insulin-like growth factor. (Item 17) 17. The method according to any one of items 1 to 16, wherein the medium in (g) contains one or more of heparin, hydrocortisone, or ascorbic acid. (Item 18) 18. The method according to any one of items 1 to 17, wherein the enzyme (f) is selected from the group consisting of trypsin, collagenase, and dispase. (Item 19) 19. The method of any one of items 1 to 18, wherein the hES cells have reduced HLA antigen complexity. (Item 20) 20. The method according to any one of items 1 to 19, wherein the method is carried out in accordance with Good Manufacturing Practice (GMP). (Item 21) 1. A method for generating mature retinal pigment epithelial (RPE) cells, comprising: a) providing human embryonic stem cells; b) culturing said human embryonic stem cells as embryoid bodies in a nutrient-rich, low-protein medium; c) culturing said human embryonic stem cells as adherent cultures in a nutrient-rich, low-protein medium; d) culturing an adherent culture of the cells of step (c) in a nutrient-rich, low-protein medium, the medium being serum-free and free of B-27 supplement; e) culturing the cells of (d) in a medium capable of supporting the growth of a high density somatic cell culture, whereby RPE cells appear in the culture of said cells; f) contacting the culture of (e) with an enzyme; g) selecting the RPE cells and transferring the RPE cells into a separate culture containing a medium supplemented with growth factors to generate an enriched culture of RPE cells; h) propagating the enriched culture of RPE cells; i) culturing the enriched culture of RPE cells to produce mature retinal pigment epithelial cells to produce mature RPE cells; A method comprising: (Item 22) 22. The method according to item 21, wherein the medium (b) and / or (c) contains serum-free B-27 supplement. (Item 23) 23. The method according to item 21 or 22, wherein the cells of (b) are cultured for 7 to 14 days. (Item 24) 24. The method according to any one of items 21 to 23, wherein the cells of (c) are cultured for 7 days. (Item 25) 25. The method according to any one of items 21 to 24, wherein the cells of (d) are cultured for 7 to 10 days. (Item 26) 26. The method according to any one of items 21 to 25, wherein the cells of (e) are cultured for 14 to 21 days. (Item 27) 27. The method according to any one of items 21 to 26, wherein the medium (b) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 28) 28. The method according to any one of items 21 to 27, wherein the medium (c) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 29) 29. The method according to any one of items 21 to 28, wherein the medium (d) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 30) 30. The method according to any one of items 21 to 29, wherein the medium (e) is selected from the group consisting of MDBK-MM, OptiPro SFM, and VP SFM. (Item 31) The method according to any one of items 21 to 30, wherein the medium (g) is selected from the group consisting of EGM-2 and MDBK-MM. (Item 32) 32. The method according to any one of items 21 to 31, wherein the growth factor (g) is selected from the group consisting of EGF, bFGF, VEGF, and recombinant insulin-like growth factor. (Item 33) 33. The method according to any one of items 21 to 32, wherein the cells of (i) are cultured in a medium selected from the group consisting of VP-SFM, EGM-2, and MDBK-MM. (Item 34) The method according to any one of Items 21 to 33, wherein the medium (g) or (i) contains one or more of heparin, hydrocortisone, and ascorbic acid. (Item 35) 35. The method according to any one of items 21 to 34, wherein the enzyme (f) is selected from the group consisting of trypsin, collagenase, and dispase. (Item 36) 36. The method of any one of items 21 to 35, wherein the hES cells have reduced HLA antigen complexity. (Item 37) 37. The method according to any one of items 21 to 36, wherein the method is carried out in accordance with Good Manufacturing Practice (GMP). (Item 38) 13. A method for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a formulation comprising RPE cells, wherein the RPE cells are derived from human pluripotent stem cells by the method according to any one of items 1 to 37 or 53 to 76. (Item 39) 39. The method of claim 38, wherein the condition is selected from the group consisting of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. (Item 40) 40. The method of claim 38 or 39, wherein the formulation is implanted in a suspension, matrix, or substrate. (Item 41) 41. The method of any one of items 38 to 40, wherein the formulation is administered into the subretinal space of the eye by injection. (Item 42) about 10 4 ~about 10 6 42. The method of any one of items 38 to 41, wherein RPE cells are administered to the subject. (Item 43) 43. The method according to any one of items 38 to 42, further comprising monitoring the effectiveness of the treatment or prevention by measuring electroretinogram response, optokinetic acuity threshold, or luminance threshold in the subject. (Item 44) 13. A pharmaceutical preparation for treating or preventing a condition characterized by retinal degeneration, comprising an effective amount of RPE cells, wherein the RPE cells are derived from human pluripotent stem cells by the method according to any one of items 1 to 37 or 53 to 76. (Item 45) 45. The formulation of item 44, wherein the condition is selected from the group consisting of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. (Item 46) 46. ​​The formulation of item 44 or 45, wherein the composition is formulated for administration to the subretinal space of the eye. (Item 47) 47. The formulation according to any one of items 44 to 46, wherein the composition is in the form of a suspension, matrix or substrate. (Item 48) The composition comprises at least 10 4 48. The preparation according to any one of items 44 to 47, comprising RPE cells. (Item 49) The composition comprises at least 10 5 49. The preparation according to any one of items 44 to 48, comprising RPE cells. (Item 50) The composition comprises at least 10 6 50. The preparation according to any one of items 44 to 49, comprising RPE cells. (Item 51) 51. The preparation of any one of items 44 to 50, wherein the RPE cells comprise mature RPE cells. (Item 52) 52. The preparation of any one of items 44 to 51, wherein the RPE cells consist essentially of mature RPE cells. (Item 53) 1. A method for producing a substantially purified culture of retinal pigment epithelial cells (RPE), comprising: a) providing human pluripotent stem cells; b) culturing the human pluripotent stem cells in a nutrient-rich, low-protein medium; c) culturing the human pluripotent stem cells as adherent cultures in a nutrient-rich, low-protein medium; d) culturing an adherent culture of the cells of (c) in a nutrient-rich, low-protein medium, the medium being serum-free and lacking B-27 supplement; e) culturing the cells of (d) in a medium capable of supporting the growth of a high density somatic cell culture, whereby RPE cells appear in the culture of said cells; f) contacting the culture of (e) with an enzyme; g) selecting the RPE cells from the culture and transferring the RPE cells into a separate culture containing a medium supplemented with growth factors to generate an enriched culture of RPE cells; h) propagating the enriched culture of RPE cells to produce a culture of substantially purified RPE cells; A method comprising: (Item 54) 54. The method of claim 53, wherein the RPE cells are further cultured to generate a culture of mature RPE cells. (Item 55) 55. The method of claim 53 or 54, wherein the human pluripotent stem cells are induced pluripotent stem (iPS) cells. (Item 56) 56. The method of claim 55, wherein in step (b), the human pluripotent stem cells are cultured as aggregates. (Item 57) 57. The method according to any one of items 53 to 56, wherein the human pluripotent stem cells are human embryonic stem cells. (Item 58) 58. The method of claim 57, wherein in step (b), the human embryonic stem cells are cultured as embryoid bodies. (Item 59) 59. The method according to any one of items 53 to 58, wherein the medium (b) and / or (c) contains serum-free B-27 supplement. (Item 60) 55. The method of claim 54, wherein the cells are cultured in a medium selected from the group consisting of VP-SFM, EGM-2, and MDBK-MM. (Item 61) 55. The method according to item 54, wherein the growth factor (g) is selected from the group consisting of EGF, bFGF, VEGF, and recombinant insulin-like growth factor. (Item 62) 55. The method of claim 54, wherein the medium contains one or more of heparin, hydrocortisone, or ascorbic acid. (Item 63) 63. The method according to any one of items 53 to 62, wherein the cells of (b) are cultured for 7 to 14 days. (Item 64) 64. The method according to any one of items 53 to 63, wherein the cells of (c) are cultured for 7 days. (Item 65) 65. The method according to any one of items 53 to 64, wherein the cells of (d) are cultured for 7 to 10 days. (Item 66) 66. The method according to any one of items 53 to 65, wherein the cells of (e) are cultured for 14 to 21 days. (Item 67) 67. The method according to any one of items 53 to 66, wherein the medium (b) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 68) 68. The method according to any one of items 53 to 67, wherein the medium (c) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 69) The method according to any one of items 53 to 68, wherein the medium (d) is selected from the group consisting of MDBK-GM, OptiPro SFM, and VP SFM. (Item 70) 70. The method according to any one of items 53 to 69, wherein the medium (e) is selected from the group consisting of MDBK-MM, OptiPro SFM, and VP SFM. (Item 71) The method according to any one of items 53 to 70, wherein the medium (g) is selected from the group consisting of EGM-2 and MDBK-MM. (Item 72) 72. The method according to any one of items 53 to 71, wherein the growth factor (g) is selected from the group consisting of EGF, bFGF, VEGF, and recombinant insulin-like growth factor. (Item 73) 73. The method according to any one of Items 53 to 72, wherein the medium in (g) contains one or more of heparin, hydrocortisone, or ascorbic acid. (Item 74) 74. The method according to any one of items 53 to 73, wherein the enzyme (f) is selected from the group consisting of trypsin, collagenase, and dispase. (Item 75) 75. The method of any one of items 53 to 74, wherein the pluripotent cells have reduced HLA antigen complexity. (Item 76) 76. The method according to any one of items 53 to 75, wherein the method is carried out in accordance with Good Manufacturing Practice (GMP). (Item 77) A method for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a formulation comprising RPE cells, wherein the RPE cells are derived from human induced pluripotent stem (iPS) cells. (Item 78) 78. The method of claim 77, wherein the condition is selected from the group consisting of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. (Item 79) 79. The method of claim 77 or 78, wherein the formulation is implanted in a suspension, matrix, or substrate. (Item 80) 80. The method of any one of items 77-79, wherein the formulation is administered into the subretinal space of the eye by injection. (Item 81) about 10 4 ~about 10 6 81. The method of any one of items 77 to 80, wherein RPE cells are administered to the subject. (Item 82) 82. The method according to any one of items 77 to 81, further comprising monitoring the effectiveness of the treatment or prevention by measuring the electroretinogram response, optokinetic acuity threshold, or luminance threshold in the subject. (Item 83) A pharmaceutical formulation for treating or preventing a condition characterized by retinal degeneration, comprising an effective amount of RPE cells, wherein the RPE cells are derived from human induced pluripotent stem (iPS) cells. (Item 84) 84. The formulation of item 83, wherein the condition is selected from the group consisting of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. (Item 85) 85. The formulation of item 83 or 84, wherein the composition is formulated for administration to the subretinal space of the eye. (Item 86) 86. The formulation according to any one of items 83 to 85, wherein the composition is in the form of a suspension, matrix or substrate. (Item 87) The composition comprises at least 10 4 87. The preparation of any one of items 83 to 86, comprising RPE cells. (Item 88) The composition comprises at least 10 5 88. The preparation of any one of items 83 to 87, comprising RPE cells. (Item 89) The composition comprises at least 10 6 89. The preparation of any one of items 83 to 88, comprising RPE cells. (Item 90) 90. The preparation of any one of items 83 to 89, wherein the RPE cells comprise mature RPE cells. (Item 91) 91. The preparation of any one of items 83 to 90, wherein the RPE cells consist essentially of mature RPE cells. (Item 92) A preparation of substantially purified human RPE cells, wherein the RPE cells express one or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 93) 93. The preparation of item 92, wherein the preparation comprises at least 75% RPE cells. (Item 94) Item 95. The preparation of item 92, wherein the RPE cells comprise at least 30% mature RPE cells. 95. The preparation of any one of items 92 to 94, wherein the RPE cells express two or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 96) 96. The preparation of item 95, wherein the RPE cells express three or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 97) 97. The preparation of item 96, wherein the RPE cells express four or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 98) 98. The preparation of item 97, wherein the RPE cells express five or more of RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 99) 99. The preparation of item 98, wherein the RPE cells express RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 100) 99. The preparation of any one of items 92-99, wherein the RPE cells lack substantial expression of ES cell genes Oct-4, nanog, and / or Rex-1. (Item 101) 101. The formulation of any one of items 92 to 100, wherein the expression is mRNA expression. (Item 102) 101. The formulation of any one of items 92 to 100, wherein the expression is protein expression. (Item 103) 101. The formulation of any one of items 92 to 100, wherein the expression includes both mRNA and protein expression. (Item 104) 101. The preparation of any one of items 92 to 100, wherein the RPE cells express one or more of pax-2, pax-6, and tyrosinase. (Item 105) The preparation according to any one of items 92 to 100, wherein the preparation comprises mature RPE cells, and the mature RPE cells express pax-2, pax-6, and tyrosinase. 106. The preparation of any one of items 92 to 105, wherein the RPE cells express one or more of the genes listed in Table 2, and wherein expression of the one or more genes is increased in the RPE cells compared to expression in human ES cells. (Item 107) 107. The preparation of any one of items 92 to 106, wherein the RPE cells express one or more of the genes listed in Table 3, and expression of the one or more genes is decreased in the RPE cells compared to expression in human ES cells. (Item 108) The preparation contains at least 1×10 5 108. The preparation of any one of items 92 to 107, comprising RPE cells. (Item 109) The preparation contains at least 1×10 6 109. The formulation of item 108, comprising RPE cells. (Item 110) 101. The preparation according to any one of items 92 to 109, wherein the preparation is differentiated from human ES cells. (Item 111) 101. The preparation according to any one of items 92 to 109, wherein the preparation is differentiated from human induced pluripotent stem cells. (Item 112) The preparation according to any one of items 92 to 109, wherein the preparation is differentiated from human pluripotent stem cells using the method according to any one of items 1 to 37 or 53 to 76. (Item 113) 113. The formulation according to any one of items 92 to 112, wherein the formulation is substantially free of viral, bacterial, and / or fungal contamination or infection. (Item 114) The formulation according to any one of items 92 to 113, wherein the formulation complies with GMP. (Item 115) Formulated in a pharma- ceutical acceptable carrier, according to any one of items 92 to 114. The formulation described. (Item 116) 116. The formulation of item 115, formulated for administration to the eye. (Item 117) 17. The formulation of item 116, formulated for administration to the subretinal space or cornea. (Item 118) 18. The preparation of any one of items 92 to 117, wherein the RPE cells are functional RPE cells that can be integrated into the retina upon transplantation. (Item 119) At least 1×10 5 1. A cryopreserved preparation comprising human RPE cells, said preparation being a preparation of substantially purified human RPE cells derived from human pluripotent stem cells, said RPE cells expressing RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F. (Item 120) 120. The preparation of item 119, wherein the human RPE cells are derived from human embryonic stem cells. (Item 121) 120. The preparation of claim 119, wherein the human RPE cells are derived from induced pluripotent stem cells. (Item 122) 122. The preparation of any one of items 119 to 121, wherein at least 65% of the RPE cells remain viable after thawing. (Item 123) 19. Use of a formulation according to any one of items 92 to 118 in the manufacture of a medicament for treating a condition in a patient in need thereof. (Item 124) 13. A method for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of the formulation according to any one of items 92 to 118, wherein the RPE cells are derived from human pluripotent stem cells. (Item 125) 125. The method of claim 124, wherein the condition is selected from the group consisting of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. (Item 126) 126. The method of claim 124 or 125, wherein the formulation is implanted in a suspension, matrix, or substrate. (Item 127) 127. The method of any one of items 124 to 126, wherein the formulation is administered into the subretinal space of the eye by injection. (Item 128) about 10 4 ~about 10 6 128. The method of any one of items 124 to 127, wherein RPE cells are administered to the subject. (Item 129) The method according to any one of items 124 to 128, further comprising monitoring the effectiveness of the treatment or prevention by measuring electroretinogram response, optokinetic acuity threshold, or luminance threshold in the subject. (Item 130) A preparation of substantially purified human RPE cells differentiated from human pluripotent stem cells, wherein the RPE cells express RPE-65, bestrophin, PEDF, CRALBP, and Otx2 at the mRNA and protein levels, and the cells lack substantial expression of Oct-4, nanog, and Rex-1. (Item 131) The RPE cells include differentiated RPE cells and mature differentiated RPE cells, and include at least 131. The preparation of item 130, wherein the differentiated RPE cells further express pax-2, pax-6, and tyrosinase at the mRNA and protein levels. (Item 132) 132. The preparation of claim 130 or 131, wherein the RPE cells are differentiated from human embryonic stem cells. (Item 133) 132. The preparation of claim 130 or 131, wherein the RPE cells are differentiated from human induced pluripotent stem cells. (Item 134) The preparation according to any one of items 130 to 133, wherein the RPE cells include differentiated RPE cells and mature differentiated RPE cells, and at least the differentiated RPE cells further express pax-2, pax-6, and tyrosinase at the mRNA and protein levels. [Brief description of the drawings]

[0068] [Figure 1] Schematic model showing the developmental ontogeny of human RPE cells derived from human embryonic stem cells. [Diagram 2] FIG. 1 is a graph showing a comparison of gene expression between hES cells and human embryonic stem cell-derived RPE cells by quantitative real-time reverse transcription PCR (qPCR). [Diagram 3] FIG. 1 is a graph showing a comparison of gene expression between ARPE-19 cells (a pre-derived RPE cell line) and human embryonic stem cell-derived RPE cells by quantitative real-time reverse transcription PCR (qPCR). [Figure 4] Graph showing gene expression comparison between fetal RPE cells and human embryonic stem cell-derived RPE cells by quantitative real-time reverse transcription PCR (qPCR). [Diagram 5] Graph showing a comparison of gene expression between mature RPE cells and hES cells by quantitative real-time reverse transcription PCR (qPCR). [Figure 6] Photomicrographs showing Western blot analysis of hES-specific and RPE-specific markers. Embryonic stem cell-derived RPE cells (lane 1) derived from hES cells (lane 2) did not express the hES-specific proteins Oct-4, Nanog, and Rex-1. However, embryonic stem cell-derived RPE cells expressing RPE-specific proteins included RPE65, CRALBP, PEDF, bestrophin, PAX6, and Otx2. Actin was used as a protein loading control. [Figure 7]Graph showing major components of the microarray gene expression profile. Component 1, representing 69% of the variability, represents cell type, while component 2 represents cell lineage (i.e., genetic variability). Near-linear variance in gene expression profiles characterizes the developmental ontogeny of hRPE derived from hES cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] In order that the invention described herein may be fully understood, a detailed description of the invention is set forth below. Various embodiments of the invention will now be described in detail and may be further illustrated by the examples provided.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.Methods and materials similar or equivalent to those described herein can be used in the present invention or testing of the present invention, but suitable methods and materials are described below.Materials, methods, and examples are illustrative only and are not intended to be limiting.

[0071] All publications, patents, patent publications and applications, and other documents mentioned herein are incorporated by reference in their entirety.

[0072] To further clarify the present invention, the following terms and definitions are provided herein.

[0073] As used throughout the description of this specification and the claims that follow, the meanings of "a," "an," and "the" include the plural meanings unless the context clearly indicates otherwise. Also, as used in the description of this specification, the meaning of "in" includes "in" and "on," unless the context clearly indicates otherwise.

[0074] Throughout this specification, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of a specified integer or group of integers and not the exclusion of any other integer or group of integers.

[0075] "Embryo" or "embryonic" refers to a growing mass of cells that has not been implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained in an embryo. It also includes blastomeres obtained as early as the two-cell stage, and aggregated blastomeres.

[0076] The term "embryonic stem cell" refers to a cell derived from an embryo. More specifically, it refers to a cell isolated from the inner cell mass of a blastocyst or morula, and to a cell that has been serially passaged as a cell line. The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo. The term also includes cells generated by somatic cell nuclear transfer, even when non-embryonic cells are used in the process.

[0077] The term "human embryonic stem cell" (hES cell) is used herein as it is used in the art. This term includes cells derived from the inner cell mass of a human blastocyst or from a morula that has been serially passaged as a cell line. hES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means of generating hES cells with homozygosity in the HLA region. Human ES cells are also derived from zygote, blastomere, or blastocyst stage mammalian embryos generated by fusion of sperm and egg cells, nuclear transfer, parthenogenesis, or by reprogramming chromatin to generate cells and subsequent integration 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, the human ES cells used to generate RPE cells are derived and maintained according to GMP standards. Regardless of their source or the particular method used to generate them, human embryonic stem cells can be identified 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 generate teratomas when transplanted into immunocompromised animals.

[0078] The term "human embryo-derived cells" (hEDC) refers to cells derived from the morula, cells derived from the blastocyst, including cells of the inner cell mass, embryonic shield, or epiblast, or other totipotent or pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm and their derivatives, as well as blastomeres and cell masses derived from aggregated single blastomeres, or embryos derived from various developmental stages, but excluding human embryonic stem cells that have been passaged as cell lines.

[0079] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells (a) are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice, and (b) are capable of differentiating into cell types of all three germ layers (e.g., ectoderm, mesoderm, and blastocyst). (c) express one or more markers of embryonic stem cells (e.g., express Oct-4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). 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, as well as embryonic stem cells derived from one or more blastomeres of a cleavage or morula stage embryo (optionally without destroying the remainder of the embryo). Such embryonic stem cells can be generated from embryonic material generated by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgeny. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) that are generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPS cells can be generated using fetal, prenatal, 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 Oct-4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct-4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0080] The terms "RPE cells", "differentiated RPE cells", "ES-derived RPE cells", and "human RPE cells" are used interchangeably throughout to refer to RPE cells differentiated from human embryonic stem cells using the methods of the present invention. The terms are generally used to refer to differentiated RPE cells regardless of the level of maturity of the cells, and thus can encompass RPE cells at various levels of maturity. Differentiated RPE cells can be visually recognized by their cobblestone morphology and early appearance of pigment. Differentiated RPE cells can also be molecularly identified based on the lack of substantial expression of embryonic stem cell markers such as Oct-4 and nanog, and based on the expression of RPE markers such as RPE-65, PEDF, CRALBP, and bestrophin. It should be noted that when referring to other RPE-like cells, they generally refer specifically to adult, fetal, or APRE-19 cells. Thus, unless otherwise specified, RPE cells, as used herein, refer to RPE cells differentiated from human embryonic stem cells.

[0081] The terms "mature RPE cells" and "mature differentiated RPE cells" are used interchangeably throughout to refer to changes that occur following initial differentiation of RPE cells. Specifically, RPE cells can be recognized in part based on the initial appearance of pigment, but following differentiation, mature RPE can be recognized based on enhanced pigmentation. Pigmentation following differentiation does not indicate a change in the RPE state of the cells (e.g., the cells are still differentiated RPE cells). Rather, the change in pigment following differentiation corresponds to the density at which the RPE cells are cultured and maintained. Thus, mature RPE cells have increased pigmentation, which accumulates following initial differentiation. Mature RPE cells are more pigmented than RPE cells, but RPE cells have some level of pigmentation. Mature RPE cells can be subcultured at a lower density where pigmentation is reduced. In this context, mature RPE cells can be cultured to generate RPE cells. Such RPE cells are differentiated RPE cells that still express markers of RPE differentiation. Thus, in contrast to the early appearance of pigmentation that occurs when RPE cells begin to differentiate, the changes in pigmentation after differentiation are phenomenological and do not necessarily represent a part of RP. It does not reflect the retrodifferentiation of cells that have deviated from the E fate. Note that the change in pigmentation after differentiation also correlates with the expression of pax-2. Note that when referring to other RPE-like cells, they generally refer specifically to adult, fetal, or APRE-19 cells. Thus, unless otherwise specified, RPE cells, as used herein, refer to RPE cells differentiated from human embryonic stem cells.

[0082] "APRE-19" refers to cells of a pre-derived human RPE cell line. APRE-19 cells arise spontaneously and are not derived from a human embryo or embryonic stem cells.

[0083] overview The present invention provides formulations and compositions comprising human retinal pigmented epithelial (RPE) cells derived from human embryonic stem cells or other human pluripotent stem cells. The RPE cells are pigmented, at least to some extent. The RPE cells do not express (at any appreciable level) the embryonic stem cell markers Oct-4, nanog, or Rex-1. Specifically, expression of these ES genes in RPE cells is about 100-1000 times lower than in ES cells, as assessed by quantitative RT-PCR. The RPE cells express one or more of RPE65, CRALBP, PEDF, bestrophin, MitF, and / or Otx2, both at the mRNA and protein levels. In certain other embodiments, the RPE cells express one or more of Pax-2, Pax-6, MitF, and tyrosinase, both at the mRNA and protein levels. In any of the above-mentioned specific embodiments, the RPE cells are mature RPE cells with increased pigmentation compared to differentiated RPE cells.

[0084] The present invention provides human RPE cells, cell cultures comprising a population of substantially purified human RPE cells, pharmaceutical preparations comprising human retinal pigment epithelial cells, and cryopreserved preparations of human RPE cells. In certain embodiments, the present invention provides the use of human RPE cells in the manufacture of a medicament for treating a condition in a patient in need thereof. Alternatively, the present invention provides the use of cell cultures in the manufacture of a medicament for treating a condition in a patient in need thereof. The present invention also provides the use of a pharmaceutical preparation in the manufacture of a medicament for treating a condition in a patient in need thereof. In any of the above, the preparations comprising RPE cells may comprise differentiated RPE cells of various levels of maturity, or may be substantially pure with respect to differentiated RPE cells of a particular level of maturity. In any of the above specific embodiments, the preparations comprising RPE cells are prepared in accordance with Good Manufacturing Practice (GMP) (e.g., the preparations are GMP compliant). In certain embodiments, the preparations comprising RPE cells are substantially free of bacterial, viral, or fungal contamination or infection.

[0085] Human RPE cells (derived from embryos or other pluripotent stem cells) can be identified and characterized based on their structural characteristics. Specifically, and in certain embodiments, these cells are unique in that they can be identified or characterized based on the expression or lack of expression of one or more markers (when assessed at gene level or protein level). For example, in certain embodiments, differentiated ES-derived RPE cells can be identified or characterized based on the expression of one or more of the markers RPE-65, bestrophin, PEDF, CRALBP, Otx2, and Mit-F (e.g., cells can be characterized based on the expression of at least one, at least two, at least three, at least four, at least five, or at least five of these markers). Additionally or alternatively, ES-derived RPE cells can be identified or characterized based on the expression of PAX2, tyrosinase, and / or PAX6. Additionally or alternatively, the hRPE cells also express one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) markers analyzed in any of Tables 1-3 (as assessed at the gene or protein level). The genes can be identified or characterized based on their expression or lack of expression.

[0086] Additionally or alternatively, ES-derived RPE cells can be identified and characterized based on the degree of cellular pigmentation. Rapidly dividing differentiated hRPE cells are lightly pigmented. However, when cell density reaches maximum capacity or when hRPE cells specifically mature, hRPE cells adopt their characteristic phenotype of hexagonal shape and increase in pigmentation levels by accumulating melanin and lipofuscin. Thus, early accumulation of pigmentation serves as an indicator of RPE differentiation, and increased pigmentation in relation to cell density serves as an indicator of RPE maturity.

[0087] Preparations that include RPE cells include preparations that are substantially pure for non-RPE cell types, but contain a mixture of differentiated RPE cells and mature differentiated RPE cells. Preparations that include RPE cells also include preparations that are substantially pure for both non-RPE cell types and other levels of RPE cells.

[0088] For any of the above-mentioned embodiments, the present invention contemplates that the RPE cells (characterized as described above) may be derived from human pluripotent stem cells, such as iPS cells and embryonic stem cells. In certain embodiments, the RPE cells are derived from human pluripotent stem cells using any of the methods described herein.

[0089] Differentiation of RPE cells Embryonic stem (ES) cells can be maintained in vitro indefinitely in an undifferentiated state and can be differentiated into virtually any cell type, providing an unlimited supply of restored histocompatible cells for transplantation therapy. The problem of immune rejection can be solved by nuclear transfer and parthenogenetic techniques. Thus, human embryonic stem (hES) cells are useful for research on human cell differentiation and can be considered as a potential source for transplantation therapy. To date, differentiation of human and mouse ES cells into numerous cell types has been reported (reviewed by Smith, 2001), including cardiomyocytes (Kehat et al., 2001; Mummery et al., 2003; Carpenter et al., 2002), neural cells and neural precursors (Reubinoff et al., 2000; Carpenter et al., 2001; Schuldiner et al., 2001), adipocytes (Bost et al., 2002; Aubert et al., 1999), hepatocyte-like cells (Rambhatla et al., 2003), hematopoietic cells (Chadwick et al., 2003), oocytes (Hubner et al., 2003), thymocyte-like cells (Lin RY et al., 2003), pancreatic islet cells (Kahan, 2003), and osteoblasts (Zur Nieden et al., 2003).

[0090] The present invention provides for the differentiation of human ES cells into specialized cells within the neural lineage, the retinal pigment epithelium (RPE). The RPE is a dense pigmented epithelial monolayer between the choroid and the neural retina. The RPE acts as part of the barrier between the bloodstream and the retina. Its functions include phagocytosis of shed rod and cone outer segments, absorption of stray light, metabolism of vitamin A, regeneration of retinoids, and tissue repair (Grierson et al., 1994; Fisher and Reh, 2001; Marmorstein et al., 1998). The RPE can be recognized by the cobblestone-like cellular morphology of black pigmented cells. In addition, there are several known markers of the RPE, including cellular retinaldehyde-binding protein (CRALBP), a cytoplasmic protein also found in apical microvilli (Bunt-Milam and Saari, 1983), RPE65, a cytoplasmic protein involved in retinal metabolism (Ma et al., 2001; Redmond et al., 1998), bestrophin (VMD2, Marmorstein et al., 2000), the product of the Best vitelloid macular dystrophy gene, and pigment epithelium-derived factor (PEDF), a 48 kD secreted protein with antiangiogenic properties (Karakousis et al., 2001; Jablonski et al., 2000).

[0091] The RPE plays a key role in maintaining photoreceptors, and various RPE dysfunctions in vivo are associated with several vision-altering diseases, such as RPE detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy, or degeneration, including age-related macular degeneration, which can lead to photoreceptor damage and blindness. Due to its wound-healing ability, the RPE has been widely studied for transplantation therapy applications. Transplantation of the RPE has been shown to have a good potential for vision restoration in several animal models and in humans (Gouras et al., 2002; Stanga et al., 2002; Binder et al., 2002; Schraermeyer et al., 2001; reviewed by Lund et al., 2001). In recent years, another promising application field for RPE transplantation has been proposed and has reached the stage of clinical trials. As these cells secrete dopamine, they can be used to treat Parkinson's disease (Subramanian, 2001). However, even in immune privileged eyes, there is a problem of graft rejection, which hinders the progress of this technique when using allogeneic transplantation. Another problem is that adult RPE exhibits very low proliferation capacity, so it relies on fetal tissue. The present invention reduces the possibility of graft rejection and removes the dependency on the use of fetal tissue.

[0092] As a source of immunocompatible tissue, hES cells hold promise for transplantation therapy, since the problem of immune rejection can be overcome by nuclear transfer techniques.The use of new differentiated derivatives of human ES cells, including retinal pigment epithelium-like cells and neural progenitor cells, and the use of differentiation systems to generate them, offers an attractive potential supply of RPE and neural progenitor cells for transplantation.

[0093] Therefore, one aspect of the present invention is to provide an improved method of generating RPE cells derived from human embryonic stem cells, which can be purified and / or isolated. Such cells are useful for the therapy of retinal degenerative diseases, such as retinitis pigmentosa, macular degeneration, and other eye disorders. The cell types that can be generated using the present invention include, but are not limited to, RPE-like cells and RPE precursors. The cells that can also be generated include iris pigment epithelial (IPE) cells and other vision-related nerve cells, such as interneuron cells (e.g., "relay" nerve cells of the inner nuclear layer (INL)) and maculin cells. In addition, retinal cells, rods, cones, and corneal cells can be generated. In another embodiment of the present invention, cells that provide the vasculature of the eye can also be generated.

[0094] Human embryonic stem cells are the starting material for this method. Embryonic stem cells may be cultured in any manner known in the art, such as in the presence or absence of feeder cells. In addition, human ES cells generated using any method can be used as starting material for generating RPE cells. For example, human ES cells may be derived from a blastocyst stage embryo, which is the product of in vitro fertilization of an egg and a sperm. Alternatively, human ES cells may be derived from one or more blastomeres removed from an early cleavage stage embryo, optionally without destroying the rest of the embryo. In yet another embodiment, human ES cells may be generated using nuclear transfer. As starting material, previously cryopreserved human ES cells can be used.

[0095] In the first step of this method for generating RPE cells, human embryonic stem cells are cultured as embryoid bodies. Embryonic stem cells may be pelleted, resuspended in medium, or plated and cultured on culture dishes (e.g., low-attachment culture dishes). Cells may be cultured in any medium sufficient for high-density cell growth, such as commercially available media for viral, bacterial, insect, or animal cell culture. In certain embodiments, a rich, low-protein medium is used (e.g., MDBK-GM medium, containing about 150 mg / ml (0.015%) of animal-derived protein). When a low-protein medium is used, the medium may contain, for example, about 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 1. ... %, 0.2%, 0.1%, 0.05%, 0.02%, 0.016%, 0.015%, or even 0.010% or less of animal-derived protein. Note that the statement of the percentage of protein present in the low protein medium refers only to the medium and does not include proteins present in, for example, B-27 supplement. Therefore, it should be understood that when cells are cultured in low protein medium and B-27 supplement, the percentage of protein present in the medium may be higher.

[0096] In certain embodiments, a rich, protein-free medium (e.g., MDBK-MM medium) is used. Other examples of media include, for example, OptiPro SFM, VP-SFM, and EGM-2. Such media may contain nutritional components such as insulin, transferrin, sodium selenite, glutamine, albumin, ethanolamine, fetuin, peptone, purified lipoprotein material, vitamin A, vitamin C, and vitamin E.

[0097] In certain embodiments, cell cultures in either low-protein or protein-free media are supplemented with serum-free B-27 supplement (Brewer et al., Journal of Neuroscience Research 35:567-576(1993)). Nutritional components of B-27 supplement include biotin, L-carnitine, corticosterone, ethanolamine, D+-galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, triiodo-1-thyronine (T3), DL-α-tocopherol (vitamin E), DL-α-tocopherol acetate, bovine serum albumin, catalase, insulin, superoxide dismutase, and transferrin. When cells are cultured in protein-free media supplemented with B-27, protein-free refers to the media before B-27 is added.

[0098] The medium may also contain supplements such as heparin, hydrocortisone, ascorbic acid, serum (such as fetal bovine serum), or a growth matrix (such as Matrigel (BD Biosciences) or an extracellular matrix from gelatin).

[0099] In the present method of the invention, RPE cells are differentiated from embryoid bodies. Isolation of RPE cells from EBs (embryoid bodies) allows for the expansion of RPE cells in enriched cultures in vitro. In human cells, RPE cells can be obtained from EBs grown for less than 90 days. In certain embodiments of the invention, RPE cells arise in human EBs grown for 7-14 days. In other embodiments, RPE cells arise in human EBs grown for 14-28 days. In another embodiment, RPE cells can be identified and isolated from human EBs grown for 28-45 days. In another embodiment, RPE cells arise in human EBs grown for 45-90 days. The medium used to culture the embryonic stem cells, embryoid bodies, and RPE cells may be removed and / or replaced with the same or different medium at any interval. For example, the medium may be removed and / or replaced after 0-7 days, 7-10 days, 10-14 days, 14-28 days, or 28-90 days. In certain embodiments, the medium is replaced at least every day, every other day, or at least every 3 days.

[0100] In certain embodiments, RPE cells differentiated from EBs are washed (e.g., with trypsin / EDTA, collagenase B, collagenase IV, or dispase) and dissociated. In certain embodiments, a non-enzymatic solution, such as a solution containing high EDTA, such as Hanks-based cell dissociation buffer, is used to dissociate the cells.

[0101] RPE cells are selected from the dissociated cells and cultured separately to generate a culture of substantially purified RPE cells. The RPE cells are selected based on characteristics associated with the RPE cells. For example, RPE cells can be recognized by their cobblestone cell morphology and pigmentation. In addition, there are several known markers for RPE, including cellular retinaldehyde-binding protein (CRALBP), a cytoplasmic protein also found in apical microvilli (Bunt-Milam and Saari, 1983), RPE65, a cytoplasmic protein involved in retinal metabolism (Ma et al., 2001; Redmond et al., 1998), bestrophin (VMD2, Marmorstein et al., 2000), the product of the Best vitelloid macular dystrophy gene, and pigment epithelium-derived factor (PEDF), a 48 kD secreted protein with antiangiogenic properties (Karakousis et al., 2001; Jablonski et al., 2000). Alternatively, RPE cells can be selected based on cell functions, such as those by (Grierson et al., 1994; Fisher and Reh, 2001; Marmorstein et al., 1998), including phagocytosis of shed rod and cone outer segments, absorption of stray light, metabolism of vitamin A, regeneration of retinoids, and tissue repair. Evaluation may also be performed using behavioral tests, fluorescent angiography, histology, tight junction conductivity, or using evaluation using electron microscopy. Another embodiment of the present invention is a method of identifying RPE cells by comparing the messenger RNA transcripts of such cells with cells induced in vivo. In certain embodiments, aliquots of cells are harvested at various intervals during differentiation of embryonic stem cells to RPE cells and assayed for expression of any of the markers mentioned above. These characteristics distinguish differentiated RPE cells.

[0102] The medium for RPE cell culture may be supplemented with one or more growth factors or drugs. Growth factors that may be used include, for example, EGF, FGF, VEGF, and recombinant insulin-like growth factor. Other growth factors that may be used in the present invention include 6Ckine (recombinant), activin A, αA-interferon, α-interferon, amphiregulin, angiogenin, B-endothelial cell growth factor, β-cellulin, B-interferon, brain-derived neurotrophic factor, C10 (recombinant), cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemoattractant-1, endothelial cell growth supplement, eotaxin, epidermal growth factor, epithelial neutrophil activating peptide-78, erythropoietin, esophageal cancer cell line, erythropoietin ... strogen receptor-α, estrogen receptor-B, fibroblast growth factor (acidic / basic, heparin stabilized, recombinant), FLT-3 / FLK-2 ligand (FLT-3 ligand), gamma interferon, glial cell line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, GRO-α / MGSA, GRO-B, GRO-γ, HCC-1, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, heregulin-α (EGF domain), binding protein-1 Insulin-like growth factor complex, Insulin-like growth factor binding protein-1 / IGF-1 complex, Insulin-like growth factor, Insulin-like growth factor II, 2.5S nerve growth factor (NGF), 7S-NGF, Macrophage inflammatory protein-1B, Macrophage inflammatory protein-2, Macrophage inflammatory protein-3α, Macrophage inflammatory protein-3B, Monocyte chemotactic protein-1, Monocyte chemotactic protein-2, Monocyte chemotactic protein-3, Neurotrophin-3, Neurotrophin-4, N GF-B (human or rat recombinant), oncostatin M (human or mouse recombinant), pituitary extract, placental growth factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, pleiotrophin, RANTES, stem cell factor, stromal cell-derived factor 1B / pre-B cell growth stimulating factor, thrombopoietin, transforming growth factor alpha, transforming growth factor-B1, transforming growth factor-B2, transforming growth factor-B3, transforming growth factor-B5, tumor necrosis factor (alpha and B), and vascular endothelial growth factor.Agents that may be used in accordance with the present invention include interferon-αA, 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-1. 0, interleukin-11, interleukin-12, interleukin-13, interleukin-15, interleukin-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony-stimulating factor, and macrophage inflammatory protein-1α.

[0103] Medicaments according to the invention also include hormones and hormone antagonists, such as 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, α-melanocyte stimulating hormone, chorionic gonadotropin, corticosteroid binding globulin, corticosterone, dexamethasone, estriol, follicle stimulating hormone, gastrin 1, glucagon, gonadotropins, hydrocortisone, insulin, insulin-like growth factor binding protein, L-3,3',5'-triiodothyronine, L-3,3',5-triiodothyronine, leptin, luteinizing hormone, L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone binding globulin, thyrotropin, thyrotropin releasing factor, psychyloxine binding globulin, and vasopressin.

[0104] In addition, agents according to the invention include extracellular matrix components such as fibronectin, proteolytic fragments of fibronectin, laminin, thrombospondin, aggrecan, and syndezan.

[0105] The agents according to the invention are anti-low density lipoprotein receptor antibodies, anti-progesterone receptor, intrabody, anti-alpha interferon receptor chain 2 antibody, anti-cc chemokine receptor 1 antibody, anti-CD118 antibody, anti-CD119 antibody, anti-colony stimulating factor-1 antibody, anti-CSF-1 receptor / c-fins antibody, anti-epidermal growth factor (AB-3) antibody, anti-epidermal growth factor receptor antibody, anti-epidermal growth factor receptor, phosphospecific antibody, anti-epidermal growth factor (AB-1) antibody, anti-erythropoietin receptor antibody, anti-estrogen receptor antibody, anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor-B antibody, anti-fibroblast growth factor receptor antibody, anti-fibroblast growth factor, basic antibody, anti-gamma interferon receptor chain antibody, anti-gamma interferon human recombinant antibody, anti-GFRα-1 C-terminal antibody, anti-GFRα-2 Also included are antibodies against various factors, such as C-terminal antibodies, anti-granulocyte colony-stimulating factor (AB-1) antibodies, anti-granulocyte colony-stimulating factor receptor antibodies, anti-insulin receptor antibodies, anti-insulin-like growth factor-1 receptor antibodies, anti-interleukin-6 human recombinant antibodies, anti-interleukin-1 human recombinant antibodies, anti-interleukin-2 human recombinant antibodies, anti-leptin mouse recombinant antibodies, anti-nerve growth factor receptor antibodies, anti-p60, chicken antibodies, anti-parathyroid hormone-like protein antibodies, anti-platelet-derived growth factor receptor antibodies, anti-platelet-derived growth factor receptor-B antibodies, anti-platelet-derived growth factor-α antibodies, anti-progesterone receptor antibodies, anti-retinoic acid receptor-α antibodies, anti-thyroid hormone nuclear receptor antibodies, anti-thyroid hormone nuclear receptor-α1 / Bi antibodies, anti-transferrin receptor / CD71 antibodies, anti-transforming growth factor-α antibodies, anti-transforming growth factor-B3 antibodies, anti-tumor necrosis factor-α antibodies, and anti-vascular endothelial growth factor antibodies.

[0106] The growth factors, drugs, and other supplements described herein may be used alone or in combination with other factors, drugs, or supplements. The factors, drugs, and supplements may be added to the medium immediately after cell culture or at any time after cell culture.

[0107] In certain embodiments, RPE cells are further cultured to produce the culture of mature RPE cells.The medium used to culture RPE cells can be any medium suitable for growing high-density cell culture as described herein.For example, the cells described herein can be cultured in VP-SFM, EGM-2, and MDBK-MM.

[0108] Certain operative combinations of the above-mentioned inventive features are described in more detail below.

[0109] In certain embodiments, a culture of pre-derived human embryonic stem cells is provided. The hES cells can be pre-derived, for example, from blastocysts (produced by fertilization or nuclear transfer) or from early cleavage stage embryos (optionally without destroying the remainder of the embryo). The human ES cells are cultured as suspension cultures to generate embryoid bodies (EBs). The embryoid bodies are cultured in suspension for about 7-14 days. However, in certain embodiments, the EBs can be cultured in suspension for less than 7 days (less than 7, 6, 5, 4, 3, 2 days, or less than 1 day), or for more than 14 days. The EBs can be cultured in medium, optionally supplemented with B-27 supplement.

[0110] To generate an adherent culture, EBs can be cultured in medium on gelatin-coated plates and then transferred. For example, EBs can be plated and cultured in medium. When cultured as an adherent culture, EBs can be cultured in the same type of medium as when grown in suspension. In certain embodiments, when cells are cultured as an adherent culture, the medium is not supplemented with B-27 supplement. In other embodiments, the medium is supplemented with B-27 supplement initially (e.g., for about 7 days or less), but then cultured as an adherent culture in the absence of B-27 for the remainder of the period. EBs can be cultured as an adherent culture for about 14-28 days. However, in certain embodiments, EBs can be cultured for less than 14 days (less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or less than 1 day), or for more than 28 days.

[0111] RPE cells begin to differentiate from cells within the adherent culture of EBs. RPE cells can be visually recognized based on their cobblestone morphology and early appearance of pigmentation. As RPE cells continue to differentiate, clumps of RPE cells can be observed.

[0112] To enrich for RPE cells and to establish substantially purified RPE cells, RPE cells are differentiated from each other and from non-RPE cells using mechanical and / or chemical methods. The suspension of RPE cells can then be transferred to fresh medium and fresh culture vessels to provide an enriched population of RPE cells.

[0113] Enriched cultures of RPE cells can be cultured in a suitable medium, such as EGM-2 medium, which, or a functionally equivalent or similar medium, may be supplemented with one or more growth factors or agents (e.g., bFGF, heparin, hydrocortisone, vascular endothelial growth factor, recombinant insulin-like growth factor, ascorbic acid, human epidermal growth factor).

[0114] In the embodiment where RPE cells are matured, the RPE cells can be further cultured, for example, in MDBK-MM medium until a desired level of maturation is achieved. This can be determined by monitoring the increase in pigmentation levels during maturation. As an alternative to MDBK-MM medium, functionally equivalent or similar media can be used. Regardless of the particular medium used to mature RPE cells, the medium can be optionally supplemented with one or more growth factors or drugs.

[0115] Cultures of RPE cells, and thus preparations of RPE cells prepared from these cultures, may be substantially pure RPE cells, containing less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% non-RPE cells. In certain embodiments, the substantially purified (relative to non-RPE cells) culture contains RPE cells at various levels of maturity, hi other embodiments, the culture is substantially pure with respect to both non-RPE cells and RPE cells at different levels of maturity.

[0116] For any of the above-mentioned embodiments, the present invention contemplates that the RPE cells (characterized as described above) may be derived from human pluripotent stem cells, such as iPS cells and embryonic stem cells. In certain embodiments, the RPE cells are derived from human pluripotent stem cells using any of the methods described herein.

[0117] Preparation of differentiated pluripotent stem cell-derived RPE cells The present invention provides a preparation of RPE cells derived from human pluripotent stem cells. In certain embodiments, the preparation is a preparation of RPE cells derived from human embryonic stem cells. In certain embodiments, the preparation is a preparation of RPE cells derived from human iPS cells. In certain embodiments, the preparation is a substantially purified (relative to non-RPE cells) preparation comprising differentiated ES-derived RPE cells. By substantially purified relative to non-RPE cells, it is meant that the preparation comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even greater than 99% RPE cells. In other words, a substantially purified preparation of RPE cells contains less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of non-RPE cell types. In certain embodiments, the RPE cells in such substantially purified preparations contain RPE cells of various maturity / pigmentation levels. In other embodiments, the RPE cells are substantially pure with respect to both non-RPE cells and RPE cells of other maturity levels. In certain embodiments, the preparation is substantially purified with respect to non-RPE cells and enriched for mature RPE cells. Enriched with respect to mature RPE cells means that at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the RPE cells are mature RPE cells. In other embodiments, the preparation is substantially purified with respect to non-RPE cells and enriched for differentiated RPE cells, but not mature RPE cells. By enriched, it is meant that at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the RPE cells are differentiated RPE cells rather than mature RPE cells. In certain embodiments, the mature RPE cells are distinguished from RPE cells by one or more of the following: level of pigmentation, level of expression of Pax-2, Pax-6, and / or level of expression of tyrosinase. In certain embodiments, the preparation comprises at least 1×103 RPE cells, 5 x 10 3 RPE cells, 1 x 10 4 RPE cells, 5 x 10 4 RPE cells, 1 x 10 5 RPE cells, 2 x 10 5 RPE cells, 3 x 10 5 RPE cells, 4 x 10 5 RPE cells, 5 x 10 5 RPE cells, 6 x 10 5 RPE cells, 7 x 10 5 RPE cells, 8 x 10 5 RPE cells, 9 x 10 5 RPE cells, 1 x 10 6 RPE cells, 5 x 10 6 RPE cells, 6 x 10 6 RPE cells, 7 x 10 6 RPE cells, 8 x 10 6 RPE cells, 9 x 10 6 RPE cells, 1 x 10 7 RPE cells, 5 x 10 7 RPE cells, 1 x 10 8 RPE cells, 1 x 10 9 RPE cells or 1 × 10 9 Contains more than 10 RPE cells.

[0118] In certain embodiments, ES-derived RPE cells do not express ES cell markers. For example, the expression of ES cell genes Oct-4, nanog, and / or Rex-1 in RPE cells is about 100-1000 times lower than that in ES cells when assessed by quantitative RT-PCR. Thus, compared to ES cells, RPE cells are substantially negative for Oct-4, nanog, and / or Rex-1 gene expression.

[0119] In certain embodiments, ES-derived RPE cells express one or more of RPE65, bestrophin, PEDF, CRALBP, Otx2, and MitF at mRNA and protein levels.In certain embodiments, RPE cells express two or more, three or more, four or more, five or more, or six of these markers.In certain embodiments, RPE cells additionally or alternatively express one or more (one, two, or three) of pax-2, pax-6, and tyrosinase at mRNA and protein levels.In other embodiments, the maturity level of RPE cells is evaluated by one or more (one, two, or three) of pax-2, pax-6, and tyrosinase.

[0120] In certain embodiments, ES-derived RPE cells express, at the mRNA and protein level, one or more (1, 2, 3, 4, 5, 6, 7, 8, or 9) of the RPE-specific genes (pax-6, pax-2, RPE65, PEDF, CRALBP, bestrophin, mitF, Otx-2, and tyrosinase) listed in Table 1, as well as one or more (1, 2, 3, or 4) of the neural retina genes (CHX10, NCAM, nestin, beta-tubulin) listed in Table 1. However, the RPE cells do not substantially express the ES cell-specific genes Oct-4, nanog, and / or Rex-1 (e.g., expression of ES cell-specific genes is 100-1000-fold less than in RPE cells as assessed by quantitative RT-PCR).

[0121] In certain embodiments, ES-derived RPE cells express one or more of the genes listed in Table 2 (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, , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more than 48), and expression of one or more genes is increased in RPE cells compared to the level of expression in human ES cells (if any). Alternatively, or in addition, ES-derived RPE cells express one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 25) of the genes listed in Table 3 at the mRNA and / or protein level, but the expression of the one or more genes in the RPE cells is reduced (including reduced to nearly undetectable levels) compared to the level of expression in human ES cells.

[0122] In certain embodiments, the preparation of substantially purified RPE cells comprises RPE cells with different levels of maturity (e.g., differentiated RPE cells and mature differentiated RPE cells).In such cases, there may be variability throughout the preparation in terms of the expression of markers that indicate pigmentation.For example, such RPE cells may have substantially the same expression of RPE65, PEDF, CRALBP and bestrophin.RPE cells may vary in terms of the expression of one or more of pax-2, pax-6, mitF and / or tyrosinase depending on the level of maturity.

[0123] In certain embodiments, the ES-derived RPE cells are stable, terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. In certain embodiments, the ES-derived RPE cells are functional RPE cells.

[0124] In certain embodiments, ES-derived RPE cells are administered to the patient at the time of corneal, subcorneal, or other transplantation. or by their ability to accumulate in the retina upon administration to an animal.

[0125] The formulations are produced according to GMP standards. Thus, in certain embodiments, the formulations are GMP-compliant formulations. In other embodiments, the formulations are substantially free of viral, bacterial, and / or fungal infection and contamination.

[0126] In certain embodiments, the preparation is cryopreserved for storage and later use. Thus, the present invention provides a cryopreserved preparation comprising substantially purified RPE cells. The cryopreserved preparation is formulated in a suitable excipient to maintain cell viability during and after cryopreservation. In certain embodiments, the cryopreserved preparation contains at least 1×10 3 RPE cells, 5 x 10 3 RPE cells, 1 x 10 4 RPE cells, 5 x 10 4 RPE cells, 1 x 10 5 RPE cells, 2 x 10 5 RPE cells, 3 x 10 5 RPE cells, 4 x 10 5 RPE cells, 5 x 10 5 RPE cells, 6 x 10 5 RPE cells, 7 x 10 5 RPE cells, 8 x 10 5 RPE cells, 9 x 10 5 RPE cells, 1 x 10 6 RPE cells, 5 x 10 6 RPE cells, 6 x 10 6 RPE cells, 7 x 10 6 RPE cells, 8 x 10 6 RPE cells, 9 x 10 6 RPE cells, 1 x 10 7 RPE cells, 5 x 10 7 RPE cells, 1 x 10 8 RPE cells, 1 x 10 9 RPE cells or 1 × 109 The cryopreserved preparation of RPE cells comprises over 100 RPE cells. The cryopreserved preparation may have the same purity level for non-RPE cells and / or various maturity levels for RPE cells as detailed above. In certain embodiments, at least 65% of the RPE cells in the cryopreserved preparation of RPE cells maintain viability after thawing. In other embodiments, at least 70%, 75%, 80%, 85%, 90%, 81%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the RPE cells in the cryopreserved preparation of RPE cells maintain viability after thawing.

[0127] The RPE cells provided herein are human cells.However, it should be noted that human cells can be used in animal models or animal patients in addition to human patients.For example, human cells can be tested in rat, dog, or non-human primate models of retinal degeneration.In addition, human cells can be used therapeutically to treat animals in need thereof, such as in veterinary medical settings.

[0128] The formulation may be formulated as a pharmaceutical preparation prepared in a pharma- ceutical acceptable carrier or excipient. Suitable formulations are specifically formulated for ocular (e.g., subretinal, corneal, ocular, etc.) administration.

[0129] In any of the specific embodiments described above, the RPE cells are derived from human pluripotent stem cells, such as human embryonic stem cells or human iPS cells. The present invention contemplates that any of the preparations described herein may be derived from suitable human pluripotent stem cells.

[0130] Formulations that include any one or more of the above-mentioned attributes are contemplated.

[0131] The present invention contemplates that any of the above-mentioned preparations of RPE cells, including substantially purified preparations and preparations having certain minimal numbers of RPE cells, may be used in the treatment of any of the indications described herein.Furthermore, RPE cells differentiated using any of the methods described herein may be used in the treatment of any of the indications described herein.

[0132] RPE cell-based therapies The pharmaceutical products comprising RPE cells and RPE cells produced by the methods described herein and / or having the characteristics of the RPE cells described herein include RPE cells, The RPE cells may be used for cell-based therapy that is required or improves treatment. The following section describes the method of using RPE cells provided by the present invention to treat various conditions that may benefit from RPE cell-based therapy. The specific treatment plan, route of administration, and any supplemental therapy are tailored based on the specific condition, the severity of the condition, and the overall health of the patient. In addition, in certain embodiments, administration of RPE cells may be effective for the complete recovery of any blindness or other symptoms. In other embodiments, administration of RPE cells may be effective for reducing the severity of the condition and / or preventing further degeneration of the patient's condition. The present invention contemplates that administration of a formulation comprising RPE cells may be used to treat any of the above or below conditions, including reducing the severity of the condition in whole or in part. In addition, administration of RPE cells may be used to help treat any symptoms of damage to the endogenous RPE layer.

[0133] The present invention contemplates that RPE cells derived using any of the methods described herein, including preparations comprising RPE cells, can be used in the treatment of any of the indications described herein.Furthermore, the present invention contemplates that any of the preparations comprising RPE cells described herein can be used in the treatment of any of the indications described herein.

[0134] Retinitis pigmentosa is an inherited disease in which visual receptors are gradually destroyed through aberrant genetic programming. Some forms cause total blindness at a relatively young age, while other forms show characteristic "osteophyte" retinal changes with mild vision destruction. Approximately 1.5 million people worldwide are affected by the disease. Two genetic defects that cause autosomal recessive retinitis pigmentosa have been found in genes expressed exclusively in the RPE. One is due to an RPE protein (cis-retinaldehyde binding protein) involved in vitamin A metabolism. The other involves another protein unique to the RPE, RPE65. The present invention provides methods and compositions for treating either of these forms of retinitis pigmentosa by administration of RPE cells.

[0135] In another embodiment, the present invention provides methods and compositions for treating disorders associated with retinal degeneration, including macular degeneration.

[0136] A further aspect of the present invention is the use of RPE cells for the treatment of eye diseases, including inherited and acquired eye diseases. Examples of acquired or inherited eye diseases are age-related macular degeneration, glaucoma, and diabetic retinopathy.

[0137] Age-related macular degeneration (AMD) is the most common cause of legal blindness in Western countries. Atrophy of the subfoveal retinal pigment epithelium and the development of choroidal neovascularization (CNV) secondarily result in loss of central vision. For the majority of patients with subfoveal CNV and geographic atrophy, there is currently no treatment available to prevent loss of central vision. An early sign of AMD is deposits (crystals) between the retinal pigment epithelium and Bruch's membrane. During the disease, sprouting of choroidal blood vessels into the subretinal space of the macula occurs. This leads to a decline in central vision and reading ability.

[0138] Glaucoma is the name given to a group of diseases in which pressure inside the eye increases abnormally. This leads to a restriction of the visual field and a general diminution of the ability to see. The most common form is primary glaucoma, which is divided into two forms: chronic open-angle and acute angle-closure glaucoma. Secondary glaucoma can be caused by infection, tumor, or injury. The third type, hereditary glaucoma, usually originates from a developmental disorder during pregnancy. The aqueous humor in the eye is under a certain pressure that is necessary for the optical properties of the eye. This intraocular pressure, usually 15-20 millimeters of mercury, is controlled by the equilibrium between the production of aqueous humor and the outflow of aqueous humor. In glaucoma, the aqueous humor in the angle of the anterior chamber is released into the eye. The outflow of the aqueous is blocked, thus increasing the pressure inside the eye. Glaucoma usually develops at middle or advanced age, but hereditary forms and diseases are often seen in children and adolescents. The pressure in the eye is only slightly elevated, and there are no further obvious symptoms, but gradual damage occurs, especially the limitation of the visual field. In contrast, angle closure causes pain, redness, dilated pupils, and severe vision impairment. The cornea becomes cloudy and the pressure in the eye increases significantly. As the disease progresses, the visual field becomes increasingly narrow, but can be easily detected using an ophthalmic instrument, a perimeter. Chronic glaucoma generally responds well to topically administered drugs that increase the outflow of aqueous humor. Systemic active substances may be given to reduce the production of aqueous humor. However, drug treatment is not always successful. If drug therapy fails, laser therapy or conventional surgery is used to create a new outflow of aqueous humor. Acute glaucoma is a medical emergency. If the intraocular pressure is not reduced within 24 hours, permanent damage will occur.

[0139] Diabetic retinopathy occurs in cases of diabetes mellitus. It can lead to thickening of the basement membrane of vascular endothelial cells as a result of protein glycosylation. It is the cause of early vascular sclerosis and the formation of microaneurysms. These vascular changes lead to diabetic retinopathy after a few years. The vascular changes cause hypoperfusion of the capillary area. This leads to lipid deposits (hard exudates) and vascular proliferation. The clinical course differs in different patients with diabetes mellitus. In age-related diabetes (type II diabetes), microaneurysms appear first. Then, due to impaired capillary perfusion, hard and soft exudates appear in the retinal parenchymal tissue, as well as punctate hemorrhages. In the later stages of diabetic retinopathy, fatty deposits are lined up coronally around the macula (retinitis circumferentialis). These changes are often accompanied by edema at the posterior pole of the eye. If the edema involves the macula, there is a sudden and severe loss of vision. The main problem in type I diabetes is the proliferation of blood vessels in the fundus area. The standard therapy is laser coagulation of the affected areas of the fundus. Laser coagulation is first performed locally on the affected areas of the retina. If the white spot persists, the scope of laser coagulation is expanded. The center of the retina with the sharpest vision, i.e. the spot and the papillomacular bundle, cannot be coagulated, since the procedure results in the destruction of parts of the retina that are most important for vision. If proliferation has already occurred, it is often necessary to press the lesion very densely on the basis of the proliferation. This is accompanied by the destruction of areas of the retina. The result is the loss of a corresponding field of vision. In type I diabetes, timely laser coagulation is often the only chance to save the patient from blindness.

[0140] In certain embodiments, the RPE cells of the present invention may be used to treat disorders of the central nervous system. RPE cells may be transplanted into the CNS. To date, many different cell types have been used in Parkinson's disease patients in animal experiments or clinical studies. An example is fetal cells obtained from the human fetal brain. Already, fetal cells from the ventral midbrain or dopaminergic neurons have been transplanted in clinical studies on over 300 Parkinson's disease patients (for an overview, see Alexi T, Borlongan CV, Faull RL, Williams CE, Clark RG, Gluckman PD, Hughes 2006). PE (2000) “Neuroprotective strategies for "Ganglia degeneration: Parkinson's and Huntington's basal diseases" Prog Neurobiol 60:409 470). A number of different cell types, including non-neuronal cells, such as cells from the adrenal cortex, Sertoli cells on the gonads or glomus cells from the carotid body, fibroblasts, or astrocytes, have been used in Parkinson's disease patients or animal models to replace dopamine either spontaneously or after gene transfer (Alexi et al. 2000, supra). The survival rate of transplanted fetal dopaminergic neurons was 58%, which was sufficient to produce a modest improvement in signs and symptoms (Alexi et al. 2000, supra).

[0141] In the last few years, neural stem cells from adult vertebrate brains have been isolated, expanded in vitro, and reimplanted into the CNS, after which they have been differentiated into pure neurons. However, their function in the CNS remains unclear. Neural progenitor cells have also been used for gene transfer (Raymon HK, Thode S, Zhou J, Friedman GC, Pardinas JR, Barrere C, Johnson RM, Sah DW (1999) "Immortalized human dorsal neural stem cells: a review"). Root ganglion cells differentiate into neurons with nociceptive properties” J Neurosci 19:5420 5428). Schwann cells overexpressing NGF and GDNF were neuroprotective in models of Parkinsonism (Wilby MJ, Sinclar SR, Muir EM, Zietlow R, Adcock KH, Horellou P, Rogers JH, Dunnett SB, Fawcett JW(1999) “A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival in mice” and fiver outgrowth from embryonic nigral nertons grafted to the striatum and to the lesioned substantia nigra" J Neurosci 19:2301 2312). Another aspect of the present invention is therefore the use of pigment epithelial cells for the treatment of neurological diseases, in particular diseases of the nervous system, preferably diseases of the CNS, especially Parkinson's disease.

[0142] One example of a common disease of the CNS is Parkinson's disease, a chronic degenerative disorder of the brain. The disease is caused by the degeneration of specialized nerve cells in the basal ganglia region. The death of dopaminergic nerve cells leads to reduced synthesis of the important neurotransmitter dopamine in Parkinson's patients. The standard treatment is medical therapy with L-dopa, which is metabolized to dopamine in the basal ganglia, where it takes over the function of the missing endogenous neurotransmitter. However, L-dopa therapy loses its activity after several years.

[0143] Animal models of retinitis pigmentosa that may be treated or used to test the efficacy of RPE cells generated using the methods described herein include rodents (rd mice, RPE-65 knockout mice, tabby mice, RCS rats), cats (Abyssinian cats), and dogs (cone degeneration "cd" dogs, progressive rod-cone degeneration "prcd" dogs, early retinal degeneration "erd" dogs, rod-cone dysplasia 1, 2, and 3 "rcd1, rcd2, and rcd3" dogs, photoreceptor dysplasia "pd" dogs, and Briard "RPE-65" dogs).

[0144] Another embodiment of the present invention is a method for inducing RPE lineage or precursors of RPE cells with enhanced ability to prevent neovascularization. Such cells can be generated by aging somatic cells from a patient to shorten telomerase until at least 10% of the normal replicative life span of the cells has elapsed, and then using the somatic cells as donor cells for nuclear transfer to generate cells that overexpress angiogenesis inhibitors such as pigment epithelium-derived factor (PEDF / EPC-1). Alternatively, such cells can be genetically modified with exogenous genes that inhibit neovascularization.

[0145] The present invention contemplates that preparations of RPE cells differentiated from human pluripotent stem cells (e.g., human embryonic stem cells, iPS cells, or other pluripotent stem cells) can be used to treat any of the above-mentioned diseases or conditions, as well as damage to the endogenous RPE layer. These diseases can be treated with preparations of RPE cells that include a mixture of differentiated RPE cells at various levels of maturity, as well as with preparations of differentiated RPE cells that are enriched for mature differentiated RPE cells or differentiated RPE cells. Treatment may also be with a preparation of RPE cells.

[0146] Mode of administration The RPE cells of the invention can be administered locally, systemically, locally, such as by injection (e.g., intravitreal injection) or as part of a device or implant (e.g., a sustained release implant). For example, the cells of the invention can be transplanted into the subretinal space by using a vitrectomy procedure.

[0147] Depending on the method of administration, RPE cells can be added to buffered and electrolyte-balanced aqueous solutions, buffered and electrolyte-balanced aqueous solutions with lubricating polymers, mineral oil or petrolatum-based ointments, other oils, liposomes, cyclodextrins, sustained release polymers, or gels. These formulations can be administered topically to the eye 1-6 times per day for up to the life of the patient.

[0148] In certain embodiments, the method of treating a patient suffering from a condition related to retinal degeneration comprises administering the composition of the present invention locally (e.g., by intraocular injection or insertion of a sustained release device that releases the composition of the present invention) by local means or by systemic administration (e.g., by a route of administration that allows for in vivo systemic absorption or allows drug accumulation in the bloodstream and then distribution throughout the body, including but not limited to intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular routes). Intraocular administration of the composition of the present invention includes, for example, delivery to the vitreous, transcorneal, subconjunctival, juxtascleral, posterior scleral, and subtenon's portion of the eye. See, for example, U.S. Patent Nos. 6,943,145, 6,943,153, and 6,945,971, the contents of which are incorporated herein by reference.

[0149] The RPE cells of the present invention may be delivered in a pharma- ceutical acceptable ophthalmic formulation by intraocular injection. When administering the formulation by intravitreal injection, for example, the solution should be concentrated so that a minimal volume can be delivered. The concentration for injection may be any amount that is effective and non-toxic, based on the factors described herein. In some embodiments, RPE cells for treatment of patients are administered at about 10 4In another embodiment, the RPE cells for treatment of a patient are formulated at a dose of about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 Formulated at a dose of cells / mL.

[0150] The RPE cells may be formulated for delivery in a pharma- ceutically acceptable ophthalmic vehicle such that the composition is maintained in contact with the ocular surface for a period of time sufficient to permit the cells to penetrate the affected portion of the eye, such as, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / cilia, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, epicorneal space, pars plana, surgically induced avascular area, or macula. Products and systems, such as delivery vehicles, that include the agents of the invention, particularly those formulated as pharmaceutical compositions (as well as kits that include such delivery vehicles and / or systems), are also contemplated as part of the invention.

[0151] In certain embodiments, the therapeutic method of the present invention comprises administering the RPE cells of the present invention as an implant or device. In certain embodiments, the device is an in vivo degradable 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 in vivo degradable implant is sized for implantation within an ocular region. The ocular region can be any one or more of the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, superior corneal space, sclera, pars plana, surgically induced avascular region, macula, and retina. The biodegradable polymer can be, for example, poly(lactide-co-glycol). In certain embodiments, the ratio of lactic acid to glycolic acid monomers in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, 75 / 25 weight percent, more preferably about 50 / 50. In addition, the PLGA copolymer can be about 20, 30, 40, 50, 60, 70, 80 to about 90 weight percent of the in vivo degradable implant. In certain preferred embodiments, the PLGA copolymer can be about 30 to about 50 weight percent, preferably about 40 weight percent, of the in vivo degradable implant.

[0152] The volume of the composition administered according to the methods described herein also depends on factors such as the method of administration, the number of RPE cells, the age and weight of the patient, and the type and severity of the disease being treated. For example, when administered orally as a liquid, the volume of the liquid containing the composition of the present invention can be about 0.5 milliliters to about 2.0 milliliters, about 2.0 milliliters to about 5.0 milliliters, about 5.0 milliliters to about 10 milliliters, or about 10 milliliters to about 50 milliliters. When administered by injection, the volume of the liquid containing the composition of the present invention can be about 5.0 microliters to about 50 microliters, about 50 microliters to about 250 microliters, about 250 microliters to about 1 milliliter, about 1 milliliter to about 5 milliliters, about 5 milliliters to about 25 milliliters, about 25 milliliters to about 100 milliliters, or about 100 milliliters to about 1 liter.

[0153] When administered by intraocular injection, the RPE cells can be delivered one or more times periodically throughout the patient's life. For example, the RPE cells can be delivered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. When administered by implant or device, the RPE cells can be delivered one or more times periodically throughout the patient's life, depending on the needs of the particular patient and disorder or condition being treated. Similarly, treatment regimens that change over time are contemplated. For example, more frequent treatments (e.g., daily or weekly treatments) may be required initially. Over time, as the patient's condition improves, less frequent treatments may be required or no further treatments may be required.

[0154] In certain embodiments, the patient is also administered immunosuppressive therapy, either prior to, concurrently with, or after administration of the RPE cells. Immunosuppressive therapy may be required throughout the patient's lifespan or for a shorter period of time.

[0155] In certain embodiments, the RPE cells of the present invention are formulated with a pharma- ceutical acceptable carrier. For example, the RPE cells can be administered alone or as a component of a pharmaceutical composition. The subject compounds may be formulated for administration in any convenient manner for use in human medicine. In certain embodiments, pharmaceutical compositions suitable for parenteral administration may include RPE cells in combination with one or more pharma-ceutical acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into a sterile injectable solution or dispersion, and may contain antioxidants, buffers, bacteriostatic agents, water for formulation with the intended recipient's blood, suspending agents, or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyhydric alcohols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0156] The composition of the present invention may contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, sorbic acid, etc. Also, it may be preferable to include isotonic agents such as sugar, sodium chloride, etc. in the composition. In addition, sustained absorption of injectable pharmaceutical forms can be brought about by including one or more agents that delay absorption, such as aluminum monostearate and gelatin.

[0157] When administered, the therapeutic compositions for use in the present invention will, of course, be in a pyrogen-free, physiologically acceptable form. Additionally, the compositions may desirably be encapsulated or injected into the vitreous humor in a viscous form for delivery to the site of retinal or choroidal injury.

[0158] Engineering MHC genes in human embryonic stem cells to obtain reduced complexity RPE cells The human embryonic stem cells used as the starting point for the method of generating RPE cells of the present invention may be derived from a library of human embryonic stem cells, each of which is hemizygous or homozygous for at least one MHC allele present in a human population. In certain embodiments, each member of the library of stem cells is hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library. In certain embodiments, the library of stem cells is hemizygous or homozygous 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 include 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. Stem cells that are nullzygous for MHC genes can be generated by standard methods known in the art, such as, for example, gene targeting and / or loss of heterozygosity (LOH). See, for example, U.S. Patent Publication Nos. US20040091936, US20030217374, and US20030232430, and U.S. Provisional Application No. 60 / 729,173, the entire disclosures of which are incorporated herein by reference.

[0159] Thus, the present invention relates to a method for obtaining RPE cells, including a library of RPE cells with reduced MHC complexity.The reduced MHC complexity RPE cells will eliminate the problems associated with patient matching, thus increasing the supply of cells available for therapeutic use.Such cells can be derived from stem cells that are engineered to be hemizygous or homozygous for genes of the MHC complex.

[0160] Human embryonic stem cells may contain modifications to one of the alleles of the sister chromosome of the MHC complex of the cell. Various methods for generating gene modifications, such as gene targeting, may be used to modify the genes in the MHC complex. Furthermore, the altered alleles of the MHC complex in the cell may then be engineered to be homozygous, such that the same alleles are present on the sister chromosomes. Methods such as loss of heterozygosity (LOH) may be used to engineer cells to have homozygous alleles in the MHC complex. For example, one or more genes in a set of MHC genes from a parental allele may be targeted to generate hemizygous cells. The other set of MHC genes may be removed by gene targeting or LOH to create a null line. This null line may further be used as an embryonic cell line in which the sequence of HLA cells or individual cells is interrupted to create a hemizygous or homozygous bank with another uniform genetic background.

[0161] In one embodiment, the members of the library are homozygous for at least one HLA gene. A library of ES cell lines is used to derive RPE cells according to the method of the present invention. In another embodiment, the present invention provides a library of RPE cells (and / or RPE lineage cells), in which some lines of ES cells are selected and differentiated into RPE cells. These RPE cells and / or RPE lineage cells can be used for patients who need cell-based therapy.

[0162] Thus, certain embodiments of the present invention relate to a method of administering reduced complexity human RPE cells derived from embryonic stem cells to a patient in need thereof. In certain embodiments, the method includes: (a) identifying a patient in need of treatment involving administering human RPE cells to the patient; (b) identifying MHC proteins expressed on the surface of the patient's cells; (c) providing a library of reduced MHC human RPE cells produced by the method for generating RPE cells of the present invention; (d) selecting RPE cells from the library that match the MHC proteins for the patient's cells; and (e) administering any of the cells from step (d) to the patient. The method may be performed in a centralized location, such as a hospital, clinic, doctor's office, or other medical facility. Furthermore, the RPE cells selected as matching the patient may be expanded before the patient's treatment if only a small number of cells have been stored.

[0163] Other commercial applications and methods Certain aspects of the present invention relate to the production of RPE cells that reach commercial quantities. In certain embodiments, RPE cells are produced on a large scale, stored as needed, and supplied to hospitals, clinicians, or other medical facilities. RPE cells can be ordered and provided in a timely manner, for example, when a patient shows symptoms of Stargardt's macular dystrophy, age-related macular degeneration, or retinitis pigmentosa. Thus, the present invention relates to a method of producing cells to achieve commercial cell scale, a cell preparation comprising RPE cells derived from the method, as well as a method of providing (i.e., producing, optionally storing, and selling) RPE cells to hospitals and clinicians.

[0164] Thus, certain aspects of the present invention relate to methods of producing, storing and distributing RPE cells produced by the methods disclosed herein. After RPE production, the RPE cells may be harvested, purified and optionally stored prior to treatment of the patient. The RPE cells may be patient-specific or specifically selected based on HLA or other immunological profiles.

[0165] Thus, in certain embodiments, the present invention provides methods for supplying RPE cells to hospitals, medical facilities, and clinicians, whereby RPE cells generated by the methods described herein can be stored, ordered upon request of a hospital, medical facility, or clinician, and administered to patients in need of RPE cell therapy. In alternative embodiments, a hospital, medical facility, or clinician orders RPE cells based on patient-specific data, generates the RPE cells according to the patient's medical condition, and subsequently supplies the RPE cells to the hospital or clinician that placed the order.

[0166] In certain embodiments, the method of differentiating RPE cells from human embryonic stem cells is carried out according to Good Manufacturing Practice (GMP).In certain embodiments, the initial derivation or generation of human embryonic stem cells is also carried out according to Good Manufacturing Practice (GMP).Cells may be tested at one or more times throughout the differentiation protocol to ensure that there is no virus, bacteria or fungal infection or contamination.Similarly, human embryonic stem cells used as starting material may be tested to ensure that there is no virus, bacteria or fungal infection or contamination.

[0167] In certain embodiments, the generation of differentiated RPE cells or mature differentiated RPE cells comprises: For example, the method can be scaled up to produce at least 1×10 5 pieces, 5×10 5 pieces, 1×10 6 pieces, 5×10 6 pieces, 1×10 7 pieces, 5×10 7 pieces, 1×10 8 pieces, 5×10 8 pieces, 1×10 9 pieces, 5×10 9 pcs or 1×10 10 can be used to generate RPE cells.

[0168] A further aspect of the invention relates to a library of RPE cells that can provide matched cells to a potential patient recipient. Thus, in one embodiment, the invention provides a method of conducting a pharmaceutical business, comprising providing an RPE cell preparation that is homozygous for at least one histocompatibility antigen, where the cells are selected from a bank of such cells that comprises a library of RPE cells that can be expanded by the methods described herein, where each RPE cell preparation is hemizygous or homozygous for at least one MHC allele present in the human population, and where the bank of RPE cells comprises cells that are hemizygous or homozygous, respectively, for a different set of MHC alleles relative to other members in the bank of cells. As described above, gene targeting or loss of heterozygosity can be used to generate hemizygous or homozygous MHC allele stem cells that are used to derive RPE cells. In one embodiment, a particular RPE cell preparation that will be suitable for a patient is selected and then expanded until a quantity suitable for the treatment of the patient is reached. The method of conducting a pharmaceutical business can also include the steps of establishing a distribution system for distributing the preparation for sale, or establishing a sales group for marketing the pharmaceutical preparation.

[0169] Another aspect of the present invention relates to the use of the RPE cells of the present invention as a research tool in the environment of pharmaceutical, chemical, or biotechnology companies, hospitals, or academic or research institutions. Such uses include the use of RPE cells differentiated from embryonic stem cells in screening tests to identify agents that can be used to promote RPE survival or promote RPE maturation, for example, in vitro or in vivo. Identified agents can be studied in vitro or in animal models to evaluate their potential use, for example, alone or in combination with RPE cells.

[0170] The present invention also includes a method of obtaining human ES cells from a patient, and then generating and expanding RPE cells derived from the ES cells. These RPE cells can be stored. In addition, these RPE cells can be used to treat the patient from whom the ES cells were obtained, or a relative of the patient.

[0171] As the above-mentioned methods and uses relate to the treatment, pharmaceutical preparation, and storage of RPE cells, the present invention also relates to a solution of RPE cells suitable for such uses. Accordingly, the present invention relates to a solution of RPE cells suitable for injection into a patient. Such a solution may comprise cells formulated in a physiologically acceptable liquid (e.g., saline, buffered saline, or balanced salt solution). The number of cells in the solution is at least about 10 2 ~about 10 9 In other embodiments, the number of cells in the solution can be less than about 10 1 pieces, 10 2 pieces, 5×10 2 pieces, 10 3 pieces, 5×10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pcs or 10 8 pieces~approx. 5×10 2 pieces, 10 3 pieces, 5×10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pcs or 10 9 The range can be any number of doses, where the upper and lower limits are independently selected, provided that the lower limit is always less than the upper limit. Further, the cells can be administered in a single dose or multiple doses.

[0172] The cells provided by the methods described herein can be used immediately or can be frozen for days or years. Thus, in one embodiment, the invention provides a cryopreserved preparation of RPE cells, the cryopreserved preparation being capable of being frozen for at least about 10 1 pieces, 102 pieces, 5×10 2 pieces, 10 3 pieces, 5×10 3 pieces, 10 4 pieces, 5×10 4 pieces, 10 5 pieces, 5×10 5 Pieces or 10 6 The cryopreserved preparation contains at least about 5×10 cells. 6 pieces, 10 7 pieces, 5×10 7 pieces, 10 8 pieces, 5×10 8 pieces, 10 9 pieces, 5×10 9 Pieces or 10 10 The RPE cell in the preparation may further comprise a cell.Similarly, a method for cryopreserving RPE cells is provided.RPE cells can be cryopreserved immediately after differentiation, after in vitro maturation, or after a period of time in culture.The RPE cells in the preparation may comprise a mixture of differentiated RPE cells and mature RPE cells.

[0173] Other pluripotent cells The above discussion focuses on the preparation and method of using human embryonic stem cells as starting material for making specific RPE cells, as well as the RPE cells differentiated from human embryonic stem cells.However, the above detailed method and method can also be used to generate RPE cells using other types of human pluripotent stem cells as starting material.Therefore, the present invention intends that any of the above or below aspects and embodiments of the present invention can also be applied to the method and method of the RPE cells differentiated from other types of human pluripotent stem cells.In particular, it should be noted that if induced pluripotent stem (iPS) cells have the characteristics of embryonic stem cells, such cells can be used to generate RPE cells that are identical or substantially identical to the RPE cells differentiated from embryonic stem cells.

[0174] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are functionally defined as stem cells that (a) can induce teratomas when transplanted into immune-deficient (SCID) mice, (b) can differentiate into all three cell types of the germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types), and (c) express one or more markers of embryonic stem cells (e.g., express Oct-4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). 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, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo). Such embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, cellular reprogramming, and male reproduction. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPS cells can be generated using somatic cells from fetuses, pre-born infants, neonates, juveniles, or adults. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct-4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct-4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors.In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0175] Embryonic stem cells are one example of a pluripotent stem cell. Another example is an induced pluripotent stem (iPS) cell.

[0176] In certain embodiments, the pluripotent stem cells are embryonic stem cells or cells derived from an embryo. In other embodiments, the pluripotent stem cells are induced pluripotent stem cells. In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells generated by expressing or inducing expression of one or more reprogramming factors in somatic cells. In certain embodiments, the somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or pulmonary fibroblasts. In other embodiments, the somatic cells are not fibroblasts, but rather non-fibroblast somatic cells. In certain embodiments, the somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In other embodiments, the somatic cells are reprogrammed by expressing at least four, at least five, or at least six reprogramming factors. In certain embodiments, the reprogramming factors are selected from Oct3 / 4, Sox2, Nanog, Lin28, c-Myc, and Klf4. In other embodiments, the set of programming factors expressed includes at least one, at least two, at least three, or at least four of the reprogramming factors listed above, and optionally includes one or more other reprogramming factors. In certain embodiments, at least one, at least two, at least three, or at least four of the above or other reprogramming factors are induced by contacting the somatic cell with one or more agents, such as small organic molecule agents, that induce expression of one or more reprogramming factors. In certain embodiments, the somatic cell is reprogrammed using a combinatorial approach in which one or more reprogramming factors are expressed (e.g., using a viral vector, a plasmid, etc.) and expression of one or more reprogramming factors is induced (e.g., using a small organic molecule).

[0177] In certain embodiments, the reprogramming factors are expressed in somatic cells by infection with a viral vector, such as a retroviral or lentiviral vector. In other embodiments, the reprogramming factors are expressed in somatic cells using a non-integrative vector, such as an episomal plasmid. When the reprogramming factors are expressed using a non-integrative vector, the factors can be expressed in the cells using electroporation, transfection, or transformation of somatic cells with the vector.

[0178] In certain embodiments, the pluripotent stem cells are developed from somatic cells, and the somatic cells are selected from embryonic, fetal, neonatal, neonatal, juvenile, or adult cells.

[0179] Methods for generating iPS cells by expressing or inducing the expression of reprogramming factors are known in the art. Briefly, somatic cells are infected, transfected, or transduced with an expression vector that expresses the reprogramming factors. In mice, the expression of four factors (Oct3 / 4, Sox2, c-myc, and Klf4) using an integrative viral vector was sufficient to reprogram somatic cells. In humans, the expression of four factors (Oct3 / 4, Sox2, Nanog, and Lin28) using an integrative viral vector was sufficient to reprogram somatic cells. However, the expression (or induction of expression) of fewer factors or other reprogramming factors may be sufficient. In addition, the use of an integrative vector is only one mechanism for expressing reprogramming factors in cells. Other methods can be used, including, for example, the use of a non-integrative vector.

[0180] In certain embodiments, at least one, at least two, at least three, or at least four of the above or other reprogramming factors are induced by contacting the somatic cell with one or more agents, such as small organic molecule agents, that induce expression of the one or more reprogramming factors. In certain embodiments, the somatic cell is contacted with a somatic cell in which one or more reprogramming factors are expressed (e.g., using a viral vector, a plasmid, etc.) and ( The cells are reprogrammed using a combinatorial approach in which expression of one or more reprogramming factors is induced (eg, using small organic molecules).

[0181] Once reprogramming factors are expressed in the cells, the cells are cultured. Over time, cells with ES characteristics appear in the culture dish. The cells can be collected and cultured, for example, based on ES morphology or based on the expression of selectable or detectable markers. The cells are cultured to generate a culture of ES cell-like cells. These cells are putative iPS cells.

[0182] To confirm the pluripotency of iPS cells, the cells can be tested in one or more pluripotency assays. For example, the cells can be tested for the expression of ES cell markers. The cells can be evaluated for their ability to generate teratomas when transplanted into SCID mice. The cells can be evaluated for their ability to differentiate and generate cell types of all three germ layers.

[0183] Once pluripotent iPS cells are obtained (either freshly derived or from a bank or stock of previously induced cells), such cells can be used to generate RPE cells.

[0184] In certain embodiments, the generation of iPS cells is the first step in the generation of RPE cells. In other embodiments, pre-induced iPS cells are used. In certain embodiments, iPS cells are generated using material from a specific patient or matched donor, specifically for tissue-matched RPE cells. In certain embodiments, iPS cells are universal donor cells that are substantially non-immunogenic.

[0185] The present invention will now be more fully described with reference to the following examples, which are merely illustrative and should not be construed as limiting the invention described above. EXAMPLES

[0186] The present invention will now be generally described and more readily understood by reference to the following examples, which are merely illustrative of certain aspects and embodiments of the present invention and are not intended to limit the present invention.

[0187] Pluripotency of embryonic stem cells is maintained in part by a delicate reciprocal balance between two transcription factors, Oct4 (Pou5fl) and Nanog. During ES cell differentiation, expression of these genes is downregulated, and recent evidence suggests that this is due to hypermethylation of the genes encoding these proteins. Loss of one or both of these genes results in transcriptional activation of genes associated with cell differentiation.

[0188] The retinal pigmented epithelium (RPE) arises from the neuroectoderm and lies adjacent to the neural retina and choroid, providing a barrier between the vasculature and the retina. Data provided herein show that RPE cells are genetically and functionally distinct from the surrounding photoreceptors after terminal differentiation, although the cells may share common precursors.

[0189] This model shows that the unique elements of our claimed culture method act through FGF, EGF, WNT4, TGF-β, and / or oxidative stress to signal MAP kinase and latent C-Jun terminal kinase pathways to induce expression of paired box 6 (PAX6) transcription factor. PAX6, in cooperation with Mit-F and Otx2, promotes the transcription of RPE-specific genes such as tyrosinase (Tyr) and downstream targets such as RPE-65, bestrophin, CRALBP, and PEDF. It functions synergistically with AX2 to direct terminal differentiation of mature RPE.

[0190] To characterize developmental stages during the differentiation process of human embryonic stem cells (hESc) into retinal pigmented epithelium (RPE), multiple assays were used to identify the expression levels of key genes for each representative developmental stage. Multiple genes were found to be uniquely expressed as mRNA and protein in RPE cells. For example, PAX6 was found to function synergistically with PAX2, via cooperation with Mit-F and Otx2, to transcribe RPE-specific genes such as tyrosinase (Tyr), as well as downstream targets such as RPE-65, bestrophin, CRALBP, and PEDF, to terminally differentiate mature RPE. Importantly, the RPE-specific signature of mRNA and protein expression was distinct not only from hHS cells, but also from fetal RPE and ARPE-19. The RPE cells described herein expressed multiple genes that were not expressed in hES cells, fetal RPE cells, or ARPE-19 cells (Figures 3, 4, and 6). The unique expression of mRNA and proteins in the RPE cells of the present invention constitutes a set of markers that make these RPE cells distinct from cells in the prior art, such as hES cells, ARPE-19 cells, and fetal RPE cells.

[0191] Example 1: Differentiation and culture of RPE Cryopreserved hES cells were thawed and placed in suspension culture on Lo-bind Nunclon Petri dishes in MDBK-growth medium (Sigma-SAFC Biosciences) or OptimPro SFM (Invitrogen) supplemented with L-glutamine, penicillin / streptomycin, and B-27 supplement. hES cells were previously derived from single blastomeres biopsied from early cleavage stage human embryos. The remaining human embryos were not destroyed. Two hES cell lines derived from single blastomeres, MA01 and MA09, were used. Cells were cultured as embryoid bodies (EBs) for 7–14 days.

[0192] After 7–14 days, EBs were plated and cultured on tissue culture plates coated with gelatin from pig skin. EBs were grown as adherent cultures for an additional 14–28 days in MDBK-growth medium or OptimPro SFM supplemented with L-glutamine and penicillin / streptomycin but without B-27 supplement.

[0193] RPE cells become visible among the cells in adherent cultures of EBs and are recognized by their cobblestone cell morphology and pigmentation development.

[0194] Example 2: Isolation and propagation of RPE As differentiated RPE cells continue to appear in the adherent cultures, differentiated RPE clumps become visibly evident based on cell shape. Frozen collagenase IV (20 mg / ml) was thawed and diluted to 7 mg / ml. Collagenase IV was applied to the adherent cultures containing the RPE clumps (1.0 ml for each well in a 6-well plate). Over a period of approximately 1-3 hours, collagenase IV dissociated the cell clumps. An enriched suspension of RPE cells was obtained by dissociating the RPE clumps from other cells in the culture. The enriched RPE cell suspension was removed from the culture plate and transferred to a 100 mm tissue culture dish with 10 ml of MEF medium. The pigmented clumps are transferred by a stem cell cutting tool (Swemed-Vitrolife) into the wells of a 6-well plate containing 3 ml of MEF medium. After all clumps have been removed, the pigmented cell suspension is transferred to a 15 ml conical tube containing 7 ml MEF medium and centrifuged at 1000 rpm for 5 minutes. The supernatant is removed. 5 ml of a 1:1 mixture of 0.25% trypsin and cell dissociation buffer is added to the cells. The cells are incubated at 37°C for 10 minutes. The cells are dispersed by pipetting with a 5 ml pipette until few clumps remain. 5 ml MEF medium is added to the cells and centrifuged at 1000 rpm for 5 minutes. The supernatant is removed and the cells are incubated for 10 minutes at 37°C for 10 minutes in EGM-2 culture. The cells are plated onto gelatin-coated plates in medium (Cambrex) divided into 1 / 3 of the original culture.

[0195] Cultures of RPE cells were expanded by continuing to grow in EGM-2 medium. Cells were passaged as needed at ratios of 1:3 to 1:6 using a 1:1 mixture of 0.25% trypsin-EDTA and cell dissociation buffer.

[0196] To enrich for mature differentiated RPE cells, cells were grown to near confluence in EGM-2, and then the medium was changed to MDBK-MM (SAFC Biosciences) to support further maturation of RPE cells.

[0197] Example 3: Expression of RPE-specific mRNA as measured by quantitative real-time reverse transcription PCR (qPCR) To characterize developmental stages during the differentiation process of human embryonic stem cells (hES) into retinal pigmented epithelium (RPE), multiple assays were used to identify the expression levels of key genes for each representative developmental stage. qPCR was developed to provide quantitative and relative measurements of multiple cell type-specific mRNA transcripts of interest in the RPE differentiation process. qPCR was used to determine genes that are uniquely expressed in human embryonic stem cells, in neural retinal cells during eye development, and in RPE cells differentiated from human embryonic stem cells. Genes by cell type are shown in Table 1.

[0198] [Table 1] hES-specific genes were determined to include Oct-4(POU5F1), Nanog, Rex-1, TDGF-1, SOX-2, and DPPA-2. Neuroectoderm / neural retina-specific genes include CHX10, NCAM, nestin, and β-tubulin. In contrast, RPE cells differentiated from human embryonic stem cells were found to uniquely express PAX-6, PAX-2, RPE-65, PEDF, CRALBP, bestrophin, MitF, Otx-2, and Tyr by qPCR measurements.

[0199] As evident from the qPCR data, hES-specific genes are significantly downregulated (almost 1000-fold) in hES-derived RPE cells, whereas RPE- and neuroectoderm-specific genes are significantly upregulated (about 100-fold) in hES-derived RPE cells.

[0200] In addition, qPCR analysis of fully mature RPE showed high levels of expression of the RPE-specific markers RPE65, tyrosinase, PEDF, bestrophin, MitF and Pax6. This finding elaborates on the ontogeny described above and supports the Pax- 13 2-induced regulation, and downstream activation of genes associated with terminally differentiated RPE.

[0201] Example 4: Expression of RPE-specific proteins identified by Western blot analysis To validate the above qPCR results and to identify proteins that are uniquely expressed in RPE cells, a subset of hES- and RPE-specific markers were selected as candidates for assay by Western blotting, thereby translating the messages detected by PCR. Western analysis provides an absolute measure of the reliability of other assays by semiquantitative (by densitometry) and qualitative data. The results are shown in Figure 6. Actin was used as a protein loading control.

[0202] RPE cells derived from hES cells did not express the hES-specific proteins Oct-4, Nanog, and Rex-1, but they expressed RPE65, CRALBP, PEDF, Bestrophin, PAX6, and Otx2. Thus, these proteins are good markers of RPE cells differentiated from hES cells. In contrast, APRE-19 cells showed an indeterminate pattern of proteomic marker expression.

[0203] Example 5: Microarray gene expression profiling of RPE cells Manually purified, in vitro hES cell differentiated hRPE undergo significant morphological events in culture during the expansion phase. Single cell suspensions plated and cultured in thin media are depigmented, increasing the surface area of ​​the cells. hRPE cells retain this morphology during expansion as the cells divide rapidly. However, when cell density reaches maximum capacity, RPE adopt their characteristic phenotype of a hexagonal shape and increase pigmentation levels by accumulating melanin and lipofuscin.

[0204] The level of pigmentation played a major role in our pharmaceutical studies in the RCS rat model. Therefore, we performed global gene expression analysis via microarrays on hRPE cells derived from both single blastomere-derived hES cell lines MA01 and MA09. In addition, fetal RPE, ARPE-19, and retinoblastoma cell lines were analyzed as controls.

[0205] Our data show that this phenotypic change is driven by changes in the global gene expression patterns of these cells, specifically with regard to the expression of PAX6, PAX2, Otx2, MitF and Tyr.

[0206] Figure 7 shows a plot of the main components of the distribution of samples based on the fewest genes that explain the variability between each sample. Component 1, which represents 69% of the variability, represents cell type, while component 2 represents cell lineage (i.e., genetic variability). As can be clearly seen, the near linear distribution of gene expression profiles characterizes the developmental ontogeny of hRPE derived from hES cells.

[0207] Based on ANOVA analysis involving each hES cell line against its RPE counterpart, we selected the 100 highest and lowest expressed genes and performed computational analysis to select genes related to pluripotency and eye development. Upregulated genes are shown in Table 2. Downregulated genes are shown in Table 3.

[0208] [Table 2-1]

[0209] [Table 2-2]

[0210] [Table 2-3]

[0211] [Table 2-4]

[0212] [Table 2-5]

[0213]

Table 2-6

[0214]

Table 2-7

[0215]

Table 2-8

[0216]

Table 2-9

[0217]

Table 2-10

[0218]

Table 3-1

[0219]

Table 3-2

[0220]

Table 3-3

[0221] The present disclosure also demonstrates that RPE cells differentiated by the methods described herein express multiple genes that are not expressed in hES cells, fetal RPE cells, or ARPE-19 cells. The unique molecular fingerprint of mRNA and protein expression in the ES cell-derived RPE cells of the present invention constitutes a set of markers, such as RPE-65, bestrophin, PEDF, CRABLP, Otx2, Mit-F, PAX6, and PAX2, that make these RPE cells distinct from prior art cells such as hES cells, ARPE-19 cells, and fetal RPE cells.

[0222] Example 6: Rescue of visual function using RPE cells derived from embryonic stem cells Certain retinal diseases are characterized by degeneration of the retinal pigment epithelium (RPE), resulting in the loss of photoreceptors. Examples include Stargardt's macular dystrophy in humans and genetically determined dystrophy in Royal College of Surgeons (RCS) rats. Such processes may also play a role in macular degeneration, which affects over 10 million people in the United States alone.

[0223] We investigated the conditions under which highly characterized RPE cells derived from embryonic stem cell lines and manufactured under GMP-compliant conditions could optimally rescue visual function in RCS rats. MAO1 and MAO9 derived RPE cells were injected into the subretinal space of postnatal day 23 (P23) RCS rats and oral cyclosporine A immunosuppression was continued postoperatively. Functional efficacy was tested by recorded threshold optokinetic acuity and luminance thresholds from the superior colliculus. All treated eyes were compared to sham-injected and untreated eyes. These functional evaluations were followed by histological considerations.

[0224] The experimental results showed a clear dose-response in RCS rats. 4 Administration of a formulation containing 2 × 10 RPE cells demonstrated only slightly better visual motion thresholds than sham animals, whereas administration of a formulation containing 2 × 10 5 Formulations containing 5×10 RPE cells showed improved performance over controls. 5 Formulations containing RPE cells provided superior performance that was sustained over time: at P60, animals performing at 0.48 c / d were significantly (p<0.001) better (0.26 c / d) than sham animals with some treated eyes, showed normal thresholds (0.6 c / d), and at best exceeded 0.5 c / d at P90 (sham and untreated animals showed values ​​of 0.16 c / d, a level indicating substantial visual impairment).

[0225] Superior colliculus recordings at P94 also showed extremely low luminance threshold responses in RPE cell-injected eyes, with some individual recordings falling within the normal range. Histological studies showed that the donor cells were arranged as a semi-continuous layer of pigmented cells immediately inside the endogenous host RPE. Donor RPE cells were positive for RPE65 and bestrophin, indicating that the transplanted cells were RPE cells and that the cells maintained their post-transplant cell fate.

[0226] In addition, transplanted animals maintained photoreceptor thickness compared to control animals: photoreceptors in RPE-treated animals were 4-5 cells thick in the rescued area, compared to a single layer in sham and untreated controls.

[0227] The results show that well-characterized RPE cells derived from embryonic stem cells and produced under GMP-compliant conditions survive after transplantation into the subretinal space of RCS rats, do not migrate into the retina, and continue to express molecular hallmarks of the RPE. Most importantly, they achieve remarkable rescue of visual function in a dose-dependent manner in an animal model of photoreceptor degeneration. The data suggest that these cells may limit and / or reverse the deterioration of vision that accompanies RPE-driven photoreceptor degeneration in human disease.

[0228]

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[0230]

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[0231]

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[0232]

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[0237] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

[Claim 1] The invention described herein.

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