Method for preparing retinal pigment epithelial cells

Abstract

To provide a method for producing retinal pigment epithelial cells.SOLUTION: A method of producing retinal pigment epithelium (RPE) cells, comprising the steps of: (a) culturing a cell population of undifferentiated human pluripotent stem cells on an adherent surface selected from the group consisting of laminin, fibronectin, vitronectin, collagen I and collagen IV in a medium comprising nicotinamide as a differentiation-inducing agent and lacking activin A under feeder cell-free conditions to obtain differentiated cells; (b) culturing the differentiated cells on the adherent surface in a medium comprising nicotinamide and one or more members of the TGF β superfamily selected from the group consisting of TGF β 1, TGF β 3, and activin A to obtain RPE cells, wherein at least 50% of the cells in the population of cells are Oct4 + TRA-1-60 +.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION In some aspects, the present invention relates to methods for preparing retinal pigment epithelial cells from pluripotent stem cells. [Background technology]

[0002] Background of the Invention The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and the choriocapillaris. RPE cells play a crucial role in the maintenance and function of the retina and its photoreceptors. These roles include forming the blood-retinal barrier, absorbing stray light, providing nutrients to the neural retina, regenerating visual pigment, and uptake and recycling of desquamated photoreceptor outer segments.

[0003] Retinal tissue can degenerate for several reasons. These reasons are usually non-genetic, including arterial or venous blockage, diabetic retinopathy, and retinopathy of prematurity. Genetic diseases that also involve degeneration of retinal tissue include retinitis pigmentosa, retinoschisis, lattice degeneration, Best disease, and Stargardt disease. A common retinal degenerative condition is age-related macular degeneration (AMD). These conditions are characterized by progressive retinal degeneration.

[0004] RPE cells can potentially be used for cell replacement therapy of RPE, which degenerates in the aforementioned retinal diseases. They can also be used as a vehicle for gene transfer for the treatment of retinal degenerative diseases. These cells can also serve as in vitro models of retinal degenerative diseases, as tools for high-throughput screening of the therapeutic effects of small molecules, and for the discovery and testing of new drugs for retinal degenerative diseases. RPE cells can also be used for basic research into RPE development, maturation, characteristics, properties, metabolism, immunogenicity, function, and interactions with other cell types.

[0005] Human fetal and adult RPE have been used as alternative donor sources for allogeneic transplantation. However, practical challenges in obtaining sufficient tissue supplies and ethical concerns regarding the use of tissue from aborted fetuses have limited the widespread use of these donor sources. Given these limitations in the supply of adult and fetal RPE grafts, the potential for alternative donor sources has been explored. Human pluripotent stem cells offer significant advantages as a source of RPE cells for transplantation. Their pluripotent developmental potential may enable differentiation into bona fide functional RPE cells, and if endowed with the potential for unlimited self-renewal, they could serve as an unlimited donor source of RPE cells. Indeed, it has been demonstrated that human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) can differentiate into RPE cells in vitro after subretinal transplantation into the Royal College of Surgeons (RCS) rat model of retinal degeneration caused by RPE dysfunction, alleviating retinal degeneration and preserving visual function. Therefore, pluripotent stem cells may be an unlimited source for RPE cell production.

[0006] Current protocols for deriving RPE cells from pluripotent stem cells are laborious and time-consuming and produce only limited numbers of pigment cells. New methods are needed to produce RPE cells in sufficient quantities for use in clinical settings.

[0007] Background art includes WO2013 / 114360 (Patent Document 1), WO2008 / 129554 (Patent Document 2), and WO2013 / 184809 (Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2013 / 114360 [Patent Document 2] WO2008 / 129554 [Patent Document 3] WO2013 / 184809 Summary of the Invention

[0009] According to an aspect of the invention, there is provided a method of producing retinal pigment epithelial (RPE) cells, comprising: (a) culturing a population of undifferentiated human pluripotent stem cells on an adhesive surface in a medium containing a differentiation inducer to obtain differentiated cells, wherein at least 50% of the cells in the cell population are Oct4 + TRA-1-60 + and (b) culturing the differentiated cells on an adherent surface in a medium containing one or more members of the TGFβ superfamily to obtain RPE cells. A method is provided, comprising:

[0010] According to an aspect of the present invention, there is provided a method of treating a retinal disease in a subject in need thereof, comprising: (a) producing RPE cells according to the methods described herein; and (b) after harvesting, transplanting a therapeutically effective amount of RPE cells into the eye of the subject, thereby treating the disease. A method is provided, comprising:

[0011] According to an aspect of the present invention, there is provided a population of RPE cells produced according to the methods described herein.

[0012] According to aspects of the present invention, there is provided a method of treating a retinal disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of RPE cells described herein, thereby treating the retinal disease or disorder.

[0013] According to some embodiments of the invention, the method further comprises, prior to step (a), growing the population of undifferentiated human pluripotent stem cells on an adherent surface in the absence of a differentiation-inducing agent.

[0014] According to some embodiments of the present invention, the expanding step is carried out in the absence of a differentiation-inducing agent.

[0015] According to some embodiments of the invention, the method further comprises isolating the RPE cells from the adherent surface after step (b).

[0016] According to some embodiments of the present invention, the method further comprises culturing the isolated RPE cells on an additional adherent surface to produce an expanded RPE cell population.

[0017] According to some embodiments of the invention, the method further comprises the step of collecting the RPE cells.

[0018] According to some embodiments of the present invention, the isolating step is performed enzymatically.

[0019] According to some embodiments of the invention, the method further comprises growing the human pluripotent stem cells on human feeder cells prior to step (a).

[0020] According to some embodiments of the invention, the method further comprises growing the human pluripotent stem cells on human feeder cells prior to the step of growing on the adherent surface.

[0021] According to some embodiments of the present invention, more than 90% of the cells in the expanded RPE cell population are CRALBP. + PMEL17 + is.

[0022] According to some embodiments of the present invention, the adhesive surface is selected from the group consisting of laminin, fibronectin, collagen I, vitronectin, and collagen IV.

[0023] According to some embodiments of the present invention, the adhesive surface is laminin or vitronectin.

[0024] According to some embodiments of the present invention, the laminin is human laminin.

[0025] According to some embodiments of the present invention, the laminin is laminin-521.

[0026] According to some embodiments of the present invention, the additional adhesive surface is selected from the group consisting of gelatin, poly-d-lysine, laminin, collagen I and collagen IV.

[0027] According to some embodiments of the present invention, the additional adhesive surface is gelatin or poly-d-lysine.

[0028] According to some embodiments of the invention, the method is carried out for at least 3 weeks.

[0029] According to some embodiments of the present invention, the method further comprises the step of cryopreserving the RPE cells after collection.

[0030] According to some embodiments of the present invention, the cryopreserving step is carried out in a medium selected from the group consisting of 90% HS / 10% DMSO, CryoStor 2%, CryoStor 5% and CryoStor 10% and Stem Cell Banker.

[0031] According to some embodiments of the present invention, the human feeder cells comprise human cord fibroblasts.

[0032] According to some embodiments of the present invention, the human pluripotent stem cells include human embryonic stem cells.

[0033] According to some embodiments of the present invention, the differentiation inducer comprises nicotinamide.

[0034] According to some embodiments of the invention, the medium in step (a) lacks activin A.

[0035] According to some embodiments of the invention, the member of the TGFβ superfamily is selected from the group consisting of TGFβ1, TGFβ3 and activin A.

[0036] According to some embodiments of the present invention, the medium in step (b) comprises nicotinamide and activin A.

[0037] According to some embodiments of the invention, the method further comprises, after step (b), culturing the RPE cells in a medium comprising nicotinamide and lacking activin A.

[0038] According to some embodiments of the present invention, step (a) is carried out for at least 5 days.

[0039] According to some embodiments of the present invention, step (b) is carried out for at least one week.

[0040] According to some embodiments of the present invention, transplantation of differentiated RPE cells is performed into the subretinal space of the eye.

[0041] According to some embodiments of the present invention, RPE cells are transplanted in suspension or as a monolayer of cells immobilized on a matrix or substrate.

[0042] According to some embodiments of the present invention, the retinal disease or disorder is selected from at least one of retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophy, Stargardt's disease, RPE damage and retinal damage resulting from damage caused by any one of photoinjury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury or traumatic injury.

[0043] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [The present invention 1001] 1. A method for producing retinal pigment epithelial (RPE) cells, comprising: (a) culturing a population of undifferentiated human pluripotent stem cells on an adhesive surface in a medium containing a differentiation inducer to obtain differentiated cells, wherein at least 50% of the cells in the cell population are Oct4 + TRA-1-60 + and (b) culturing the differentiated cells on the adherent surface in a medium containing one or more members of the TGFβ superfamily to obtain RPE cells. A method comprising: [The present invention 1002] 1002. The method of claim 1001, further comprising, prior to step (a), growing the population of undifferentiated human pluripotent stem cells on an adherent surface in the absence of a differentiation-inducing agent. [The present invention 1003] 1002. The method of claim 10, wherein the proliferating step is carried out in the absence of a differentiation inducer. [The present invention 1004] 1001. The method of claim 1001, further comprising the step of isolating RPE cells from the adherent surface after step (b). [The present invention 1005] The method of claim 1004, further comprising culturing the isolated RPE cells on a further adherent surface to produce an expanded RPE cell population. [The present invention 1006] The method of claim 1005, further comprising the step of collecting RPE cells. [The present invention 1007] The method of claim 1004, wherein the isolating step is performed enzymatically. [The present invention 1008] 1001. The method of claim 1001, further comprising the step of growing the human pluripotent stem cells on human feeder cells prior to step (a). [The present invention 1009] 1003. The method of claim 1002, further comprising the step of growing the human pluripotent stem cells on human feeder cells prior to the step of growing on the adherent surface. [The present invention 1010] 1005. The method of claim 1005, wherein more than 90% of the cells in said expanded RPE cell population are CRALBP+PMEL17+. [The present invention 1011] The method of any of claims 1001 to 1010, wherein the adhesive surface is selected from the group consisting of laminin, fibronectin, vitronectin, collagen I and collagen IV. [The present invention 1012] The method of claim 1011, wherein the adhesive surface is laminin or vitronectin. [The present invention 1013] The method of claim 1012, wherein the laminin is laminin 521. [The present invention 1014] 1005. The method of claim 10, wherein the additional adhesive surface is selected from the group consisting of gelatin, poly-d-lysine, laminin, collagen I, and collagen IV. [The present invention 1015] The method of claim 1014, wherein the further adhesive surface is gelatin or poly-d-lysine. [The present invention 1016] 1001. The method of claim 1001, wherein the method is carried out for at least 3 weeks. [The present invention 1017] 1006. The method of claim 1006, further comprising the step of cryopreserving the RPE cells after collection. [The present invention 1018] 1017. The method of claim 1017, wherein the cryopreserving step is carried out in a medium selected from the group consisting of 90% HS / 10% DMSO, CryoStor 2%, CryoStor 5% and CryoStor 10%, and Stem Cell Banker. [The present invention 1019] 1009. The method of claim 10, wherein the human feeder cells comprise human cord fibroblasts. [The present invention 1020] 1020. The method of any one of claims 1001 to 1019, wherein the human pluripotent stem cells comprise human embryonic stem cells. [The present invention 1021] The method of any one of claims 1001 to 1019, wherein the differentiation inducer comprises nicotinamide. [The present invention 1022] 1021. The method of claim 1021, wherein the medium of step (a) lacks activin A. [The present invention 1023] The method of any of claims 1001 to 1019, wherein the member of the TGFβ superfamily is selected from the group consisting of TGFβ1, TGFβ3 and activin A. [The present invention 1024] The method of any of claims 1001 to 1019, wherein the medium in step (b) comprises nicotinamide and activin A. [The present invention 1025] 1024. The method of claim 1024, further comprising, after step (b), culturing the RPE cells in a medium containing nicotinamide and lacking activin A. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein step (a) is carried out for at least 5 days. [The present invention 1027] The method of any one of claims 1001 to 1026, wherein step (b) is carried out for at least one week. [The present invention 1028] 1. A method of treating a retinal disease in a subject in need thereof, comprising: (a) producing RPE cells according to any one of the methods of the present invention 1001 to 1027; and (b) after harvesting, transplanting a therapeutically effective amount of the RPE cells into the eye of the subject, thereby treating the disease. A method comprising: [The present invention 1029] The method of claim 1028, wherein transplantation of differentiated RPE cells is performed in the subretinal space of the eye. [The present invention 1030] The method of claim 1028, wherein the RPE cells are transplanted in suspension or as a monolayer of cells immobilized on a matrix or substrate. [The present invention 1031] 1028. The method of claim 1028, wherein the retinal disease or disorder is selected from at least one of retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophy, Stargardt's disease, RPE damage and retinal damage resulting from damage caused by any one of light injury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury or traumatic injury. [The present invention 1032] A population of RPE cells produced according to any of the methods of the present inventions 1001 to 1027. [The present invention 1033] A method for treating a retinal disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of RPE cells of the present invention 1032 to the subject, thereby treating the retinal disease or disorder. [Brief explanation of the drawings]

[0044] Several aspects of the present invention are described herein, by way of example only, with reference to the accompanying drawings and images. When referring in detail to the drawings, it is emphasized that the particulars shown are by way of example and for illustrative purposes of explaining aspects of the invention. In this regard, the detailed description taken in conjunction with the drawings will make apparent to those skilled in the art how aspects of the present invention may be practiced.

[0045] [Figure 1]FIG. 1 is a pictorial representation of a process for inducing retinal pigment epithelial (RPE) cells in accordance with an embodiment of the present invention. [Figure 2] 2A-B are photographs showing RPE cells at the end of the differentiation stage (FIG. 2A) and at the end of the P0 stage (FIG. 2B). [Figure 3] 3A-C are graphs showing the purity of hESC-derived pigmented / polygonal cells produced without spheroid bodies, as measured by FACS analysis. [Figure 4] Figures 4A-D are graphs showing the purity of hESC-derived pigment cells produced without SB at the end of P2 after cryopreservation (Figures 4C-D) compared with undifferentiated embryonic stem cells (negative control; Figure 4A) and RPE cells produced by the action of SB and activin A and nicotinamide (positive control; Figure 4B). [Figure 5A] Figures 5A-C are representative phase-contrast images of RPE cells induced on vitronectin. Figure 5A—RPE cells at passage 1 (x10). [Figure 5B] Figures 5A-C are representative phase-contrast images of RPE cells induced on vitronectin. Figure 5B—RPE cells at passage 2 (x10). [Figure 5C] Figures 5A-C are representative phase-contrast images of RPE cells induced on vitronectin. Figure 5C—RPE cells at passage 2 (x20). [Figure 6A] Figures 6A-E show immunofluorescence images of RPE cells induced on vitronectin decorated with antibodies identifying RPE markers. The majority of cells in the images express RPE cell markers (×40). Figure 6A—Anti-bestrophin (counterstained with DAPI). [Figure 6B] Figures 6A-E show immunofluorescence images of RPE cells induced on vitronectin decorated with antibodies identifying RPE markers. The majority of cells in the images express RPE cell markers (×40). Figure 6B shows anti-CRALBP (counterstained with DAPI). [Figure 6C]Figures 6A-E show immunofluorescence images of RPE cells induced on vitronectin decorated with antibodies that recognize RPE markers. The majority of cells in the images express RPE cell markers (×40). Figure 6C—Anti-MITF. [Figure 6D] Figures 6A-E show immunofluorescence images of RPE cells induced on vitronectin decorated with antibodies identifying RPE markers. The majority of cells in the images express RPE cell markers (×40). Figure 6D shows anti-PEDF (counterstained with DAPI). [Figure 6E] Figures 6A-E show immunofluorescence images of RPE cells induced on vitronectin decorated with antibodies that recognize RPE markers. The majority of cells in the image express RPE cell markers (×40). Figure 6E—Anti-ZO-1. DETAILED DESCRIPTION OF THE INVENTION

[0046] Description of Certain Aspects of the Invention In some aspects, the present invention relates to methods for preparing retinal pigment epithelial cells from pluripotent stem cells.

[0047] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0048] Human embryonic stem cells have been proposed as a cell source for RPE cell production. Two general approaches have been used to obtain retinal pigment epithelial (RPE) cells from hESCs: spontaneous differentiation and induced differentiation.

[0049] In spontaneous differentiation, hESCs in flat colonies or embryoid bodies (EBs) are allowed to differentiate spontaneously into a population of cells containing pigmented RPE cells.

[0050] The differentiation of human embryonic stem cells (hESCs) into RPE cells is traditionally performed after the production of embryoid / spheroid bodies (SBs). See, e.g., U.S. Patent No. 8,956,866, the contents of which are incorporated herein by reference. However, this process is inherently variable, which prevents the scale-up required for an industrial process.

[0051] We now propose that RPE cells can be generated without initial differentiation as embryoid or spheroid bodies, as demonstrated by the recognition of the typical polygonal morphology of RPE following a differentiation protocol performed entirely under adherent conditions (Figure 2A-B).

[0052] FACS analysis performed after expansion of enzymatically isolated cells revealed that >99% of the cells expressed CRALBP. + PMEL17 + We demonstrated that this method produces highly purified RPE cell populations (Figure 3A-C).

[0053] Thus, according to a first aspect of the present invention there is provided a method of producing retinal pigment epithelial (RPE) cells, comprising the steps of: (a) culturing a population of undifferentiated human pluripotent stem cells on an adhesive surface in a medium containing a differentiation inducer to obtain differentiated cells, wherein at least 50% of the cells in the cell population are Oct4 + TRA-1-60 + and (b) culturing the differentiated cells on an adherent surface in a medium containing one or more members of the TGFβ superfamily to obtain RPE cells. A method is provided, comprising:

[0054] "Retinal pigment epithelial cells," "RPE cells," and "RPE," which may be used interchangeably when the context allows, refer to cells of a cell type that are functionally similar to the cell type of native RPE cells that form the pigment epithelial cell layer of the retina (e.g., when transplanted into the eye, they exhibit functional activity similar to that of native RPE cells).

[0055] According to one embodiment, RPE cell expresses at least one, two, three, four or five markers of mature RPE cell.Such markers include but are not limited to CRALBP, RPE65, PEDF, PMEL17, bestrophin and tyrosinase.Optionally, RPE cell can also express the marker of RPE progenitor cell, such as MITF.In another embodiment, RPE cell expresses PAX-6.

[0056] As used herein, the phrase "marker of mature RPE cells" refers to an antigen (e.g., a protein) that is increased (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in mature RPE cells compared to non-RPE or immature RPE cells.

[0057] As used herein, the phrase "marker of RPE progenitor cells" refers to an antigen (e.g., a protein) that is increased (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in RPE progenitor cells compared to non-RPE cells.

[0058] According to another embodiment, the RPE cells have a morphology similar to that of natural RPE cells that form the pigmented epithelial cell layer of the retina, ie, pigmented and with a characteristic polygonal shape.

[0059] According to yet another embodiment, RPE cells can treat diseases such as macular degeneration.

[0060] According to yet another embodiment, the RPE cells meet at least one, two, three, four or all of the requirements listed herein above.

[0061] As used herein, the term "stem cells" refers to cells (e.g., pluripotent or multipotent stem cells) that can remain in an undifferentiated state in culture for extended periods of time until induced to differentiate into other cell types (e.g., fully differentiated cells) that have a particular specialized function. Preferably, the term "stem cells" encompasses embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells.

[0062] According to certain embodiments, RPE cells are produced from pluripotent stem cells. Such cells can differentiate into any of the three germ layers: endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). According to certain embodiments, pluripotent stem cells can differentiate into cells of the embryo proper, such as embryonic stem cells and iPSCs (e.g., ESCs or iPSCs).

[0063] Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by genetic manipulation of somatic cells, for example, by retroviral introduction of transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 into somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells [Yamanaka S, Cell Stem Cell. 2007, 1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14. (Electronic version ahead of print); IH Park, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-872]. Other embryonic-like stem cells can be produced by nuclear transfer into oocytes, fusion with embryonic stem cells, or nuclear transfer into zygotes, provided the recipient cells are arrested in mitosis.

[0064] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm) or remain undifferentiated. The term "embryonic stem cells" can include cells obtained from embryonic tissues that form after conception (e.g., blastocysts), cells obtained from embryonic tissues that form before implantation of an embryo (i.e., preimplantation blastocysts), expanded blastocysts (EBCs) obtained from blastocysts at the postimplantation / pregastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before 10 weeks of gestation. Embryonic stem cells of some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are generally obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. For the isolation of human ES cells, the zona pellucida is removed from blastocysts, and the trophectoderm cells are then lysed and gently pipetted away from the intact inner cell mass (ICM). The ICM is then plated into tissue culture flasks containing an appropriate medium that allows for its proliferation. After 9–15 days, the ICM-derived outgrowths are dissociated into clumps by either mechanical dissociation or enzymatic digestion, and the cells are then replated onto fresh tissue culture medium. Colonies exhibiting undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then routinely split every 4–7 days.For further details regarding methods for preparing human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May: 18(5): 559; Thomson et al., [U.S. Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

[0065] It will be appreciated that commercially available stem cells may also be used in accordance with some embodiments of the present invention. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry [www.grants(dot)nih(dot)gov / stem_cells / registry / current(dot)htm]. Non-limiting examples of commercially available embryonic stem cell lines include HAD-C102, ESI, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNhem19, BJNhem20, SA001, SA001.

[0066] According to a particular embodiment, the embryonic stem cell line is HAD-C102 or ESI.

[0067] In addition, ES cells have been used to generate embryonic stem cells from mice (Mills and Bradley, 2001), golden hamsters (Doetschman et al., 1988, Dev Biol. 127: 224-7), rats (Iannaccone et al., 1994, Dev Biol. 163: 288-92), rabbits (Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33), and several livestock species (Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci U S A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0068] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days post-fertilization, prior to gastrulation. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., with acidic Tyrode's solution (Sigma Aldrich, St. Louis, MO, USA)) to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos at least 9 days post-fertilization and no later than 14 days (i.e., prior to the gastrulation event) using standard embryonic stem cell culture methods.

[0069] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed during this process.

[0070] EG cells are prepared from primordial germ cells (PGCs) obtained from fetuses at approximately 8-11 weeks of gestation (in the case of human fetuses) using laboratory techniques known to those skilled in the art. Genital ridges are isolated and dissected into small clumps, which are then separated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks containing appropriate medium. The cells are cultured with daily changes of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages. For further details regarding methods for preparing human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0071] Yet another method for preparing ES cells is by parthenogenesis, a process in which the embryo is also not destroyed.

[0072] Current ES culture methods are primarily based on the use of feeder cell layers, which secrete factors necessary for stem cell proliferation while simultaneously inhibiting their differentiation. Culture is typically performed on solid surfaces, such as surfaces coated with gelatin or vimentin. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEFs), mouse embryonic fibroblasts (MEFs), mouse fetal fibroblasts (MFFs), human embryonic fibroblasts (HEFs), human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFMs), human fetal skin cells (HFSs), human adult skin cells, human foreskin fibroblasts (HFFs), human umbilical cord fibroblasts, human cells obtained from the umbilical cord or placenta, and human bone marrow stromal cells (hMSCs). Growth factors may be added to the culture medium to maintain ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGFβ. In another embodiment, agents may be added to the culture medium to maintain hESCs in a naive, undifferentiated state. See, for example, Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

[0073] Feeder-cell-free systems have also been used to culture ES cells. These systems utilize a matrix supplemented with serum replacement, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimeras) instead of a feeder cell layer. Stem cells can be grown on a solid surface, such as an extracellular matrix (e.g., Matrigel®, laminin, or vitronectin), in the presence of media such as Lonza L7, mTeSR, StemPro, XFKSR, E8, or Nutristem. Unlike feeder-based cultures, which require co-proliferation of feeder cells and stem cells and can result in mixed cell populations, stem cells grown in feeder-free systems easily detach from the surface. The media used to grow stem cells contain factors that effectively inhibit differentiation and promote their growth, such as MEF-conditioned medium and bFGF.

[0074] After optional expansion, pluripotent ESCs are subjected to induced differentiation on an adherent surface (without intermediate spheroid or embryoid body production).

[0075] Thus, in accordance with this aspect of the invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells subjected to differentiation induction on an adherent surface are undifferentiated ESCs and express markers of pluripotency. For example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells are Oct4 + TRA-1-60 + The undifferentiated ESCs preferably express other markers of pluripotency, such as NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-β, SSEA-3, SSEA-4 and / or TRA-1-81.

[0076] In one exemplary differentiation protocol, undifferentiated embryonic stem cells are differentiated into RPE cell lineages on an adherent surface using a first differentiation inducer, and then further differentiated into RPE cells using a member of the transforming growth factor-β (TGFβ) superfamily (e.g., TGFβ1, TGFβ2, and TGFβ3 subtypes, and homologous ligands including activins (e.g., activin A, activin B, and activin AB), nodular anti-Müllerian hormone (AMH), several bone morphogenetic proteins (BMPs), e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth differentiation factors (GDFs). According to a particular embodiment, the member of the transforming growth factor-β (TGFβ) superfamily is activin A, e.g., 20-200 ng / ml, e.g., 100-180 ng / ml activin A.

[0077] According to a particular embodiment, the first differentiation inducer is nicotinamide (NA), for example 1-100 mM, 5-50 mM, 5-20 mM, for example 10 mM nicotinamide (NA).

[0078] According to another embodiment, the first differentiation inducer is 3-aminobenzamide.

[0079] NA, also known as "niacinamide," is an amide derivative form of vitamin B3 (niacin) that is thought to preserve and improve beta-cell function. NA has the chemical formula CHNO. NA is essential for growth and the conversion of food to energy and is used in the treatment of arthritis and the treatment and prevention of diabetes. TIFF2026009985000002.tif42128

[0080] According to certain embodiments, the nicotinamide is a nicotinamide derivative or a nicotinamide mimetic. As used herein, the term "nicotinamide (NA) derivative" refers to a compound that is a chemically modified derivative of natural NA. In one embodiment, chemical modification can be the substitution of the pyridine ring of the basic NA structure (via the carbon or nitrogen member of the ring) through the nitrogen or oxygen atom of the amide moiety. When substituted, one or more hydrogen atoms can be replaced by a substituent, and / or the substituent can be bonded to the N atom to form a tetravalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In another embodiment, chemical modification can be the deletion or substitution of a group, for example, to form a thiobenzamide analog of NA, all of which are understood by those skilled in the art of organic chemistry. Derivatives related to the present invention also include nucleoside derivatives of NA (e.g., nicotinamide adenine). A variety of NA derivatives have been described, some of which are related to the inhibitory activity of PDE4 enzyme (WO03 / 068233; WO02 / 060875; GB2327675A) or as VEGF receptor tyrosine kinase inhibitors (WO01 / 55114).For example, the method for preparing 4-aryl-nicotinamide derivatives (WO05 / 014549).Other exemplary nicotinamide derivatives are disclosed in WO01 / 55114 and EP2128244.

[0081] Nicotinamide mimetics include modified forms of nicotinamide and chemical analogs of nicotinamide that reproduce the effect of nicotinamide in the differentiation and maturation of RPE cells from pluripotent cells.Exemplary nicotinamide mimetics include benzoic acid, 3-aminobenzoic acid and 6-aminonicotinamide.Another class of compounds that can act as nicotinamide mimetics is the inhibitor of poly(ADP-ribose) polymerase (PARP).Exemplary PARP inhibitors include 3-aminobenzamide, iniparib (BSI 201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827 and BMN-673.

[0082] According to a particular embodiment, differentiation is performed as follows: (a) culturing ESCs in a medium containing a first differentiation inducer (e.g., nicotinamide); and (b) Culturing the cells obtained from step (a) in a medium containing a member of the TGFβ superfamily (e.g., activin A) and a first differentiation inducer (e.g., nicotinamide).

[0083] Preferably, step (a) is carried out in the absence of a member of the TGFβ superfamily.

[0084] The above protocol may be continued by culturing the cells obtained in step (b) in a medium containing a first differentiation inducer (e.g., nicotinamide) but lacking a member of the TGFβ superfamily (e.g., activin A). This step is herein referred to as step (b). * ) is called

[0085] The above protocol is described in more detail below according to further aspects.

[0086] Step (a): Once a sufficient number of ESCs are obtained (e.g., after growth on feeders), the process begins. ESCs are typically removed from the feeder culture (e.g., by using collagenase A, dispase, TrypLE select, or EDTA) and plated onto a feeder-free adhesive substrate (e.g., laminin, vitronectin, fibronectin, collagen I, and collagen IV). According to a specific embodiment, the feeder-free adhesive substrate is laminin, e.g., laminin 521, or vitronectin (e.g., Millipore CC080 Lot LV1689930). The undifferentiated embryonic stem cells may be further expanded on the adhesive substrate for 1 to 10 days, or may be passaged and expanded for a longer period (more than 10 days), before initiating differentiation induction in a medium that prevents differentiation (e.g., medium containing bFGF and TGFβ). Differentiation induction is then initiated by changing the medium with a medium containing a first differentiation inducer (e.g., nicotinamide). The medium does not contain the levels of TGFβ superfamily members (activin A, bFGF, and TGFβ) used in the second differentiation step. In one embodiment, the medium is completely devoid of TGFβ superfamily members. In another embodiment, the level of TGFβ superfamily members in the medium is less than 20 ng / ml, less than 10 ng / ml, less than 1 ng / ml, or even less than 0.1 ng / ml.

[0087] Exemplary concentrations of nicotinamide are 1-100 mM, 5-50 mM, 5-20 mM, for example, 10 mM.

[0088] This step can be carried out for at least 1 day, more preferably 2 days, 3 days, 1 week or even 14 days. Preferably, the cells are not cultured in the presence of nicotinamide (and in the absence of activin) at this step for more than 3 weeks.

[0089] In one embodiment, when cells are cultured on an adhesive substrate, such as laminin or vitronectin, the atmospheric oxygen conditions are 20%. The atmospheric oxygen conditions may be manipulated to have an oxygen content of less than about 20%, less than 15%, or less than 10%, more preferably less than about 9%, less than about 8%, less than about 7%, or less than about 6%, and even more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%).

[0090] According to a particular embodiment, cells are cultured on an adherent substrate, first under normal atmospheric oxygen conditions and then under subnormal atmospheric oxygen conditions.

[0091] Preferably, once the undifferentiated ESCs are plated onto the adherent substrate, the undifferentiated ESCs are not removed until pigment cells are observed.

[0092] Step (b): After the first differentiation induction step (step a; i.e., culturing in the presence of nicotinamide (e.g., 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM)), halved cells are obtained. Such halved cells (also referred to herein as differentiated cells) have differentiated along the retinal pigment epithelial pathway. Such cells may express markers of neural progenitor cells, i.e., early markers of RPE cells.

[0093] The differentiated cells are then subjected to a further differentiation step on the adhesive substrate, i.e., cultured in the presence of nicotinamide (e.g., 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM) and activin A (e.g., 20-200 ng / ml or 100-200 ng / ml, e.g., 140 ng / ml, 150 ng / ml, 160 ng / ml, or 180 ng / ml). This step can be carried out for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, 1 week to 8 weeks, 1 week to 4 weeks, e.g., at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks.

[0094] According to a particular embodiment, this step is carried out for about 2 weeks. This differentiation step can be carried out in low or normal atmospheric oxygen conditions, as detailed herein above.

[0095] Step (b*): After the second differentiation induction step (i.e., culturing on an adherent substrate in the presence of nicotinamide and activin A; step (b)), the further differentiated cells are optionally subjected to a subsequent differentiation step on an adherent substrate, i.e., culturing in the presence of nicotinamide (e.g., 1 to 100 mM, 5 to 50 mM, 5 to 20 mM, e.g., 10 mM) and in the absence of a member of the TGFβ superfamily, e.g., activin A.

[0096] In one embodiment, the medium is completely devoid of members of the TFGβ superfamily, hi another embodiment, the level of TFGβ superfamily members in the medium is less than 20 ng / ml, less than 10 ng / ml, less than 1 ng / ml, or even less than 0.1 ng / ml.

[0097] This step can be carried out for at least 1 day, 2 days, 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, or even 4 weeks. Preferably, this step is carried out for about 1 week. This differentiation step can also be carried out under low or normal atmospheric oxygen conditions, as detailed herein above.

[0098] The differentiation process is generally carried out until at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 6%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells on the plate are pigment cells.

[0099] The basal medium in which ESCs differentiate is any cell culture medium known in the art for supporting cell growth in vitro, generally a medium containing a defined base solution containing salts, sugars, amino acids, and any other nutrients necessary to maintain cells in a viable state in culture. According to certain embodiments, the basal medium is not a conditioned medium. Non-limiting examples of commercially available basal media that can be utilized in accordance with the present invention include Nutristem (without bFGF and TGFβ for ESC differentiation, and with bFGF and TGFβ for ESC expansion), Neurobasal™, KnockOut SR Xeno-Free Medium, KO-DMEM, DMEM, DMEM / F12, Cellgro™ Stem Cell Expansion Medium, or X-Vivo™. The basal medium may be supplemented with various agents known in the art of cell culture. The following is a non-limiting reference to various supplements that can be included in the culture system used in accordance with the present disclosure: - medium containing serum or serum replacement, such as but not limited to knock out serum replacement (KOSR), Nutridoma-CS, TCH™, N2, N2 derivatives or B27, or a combination; - extracellular matrix (ECM) components, such as, but not limited to, fibronectin, laminin, collagen, and gelatin, in which case the ECM may be used to carry one or more members of the TGFβ superfamily of growth factors; -Antibacterial agents, such as, but not limited to, penicillin and streptomycin; and - Non-essential amino acids (NEAAs), neurotrophins known to play a role in promoting survival of SCs in culture, such as but not limited to BDNF, NT3, NT4.

[0100] According to a preferred embodiment, the medium used to differentiate ESCs is Nutristem medium (Biological Industries, 05-102-1A or 05-100-1A).

[0101] According to a particular embodiment, differentiation of ESCs is carried out under xeno-free conditions.

[0102] According to one embodiment, the proliferation / growth medium is devoid of xenogeneic impurities, i.e. does not contain animal-derived components such as serum, animal-derived growth factors and albumin. Thus, according to this embodiment, the culture is carried out in the absence of xenogeneic impurities.

[0103] Other methods for culturing ESCs under xeno-free conditions are provided in U.S. Patent Application No. 20130196369, the contents of which are incorporated herein in their entirety.

[0104] The preparation comprising RPE cells may be prepared in accordance with Good Manufacturing Practice (GMP) (e.g., the preparation is a GMP-compliant preparation) and / or current Good Tissue Practice (GTP) (e.g., the preparation may be a GTP-compliant preparation).

[0105] During the differentiation process, the differentiation state of the embryonic stem cells can be monitored. Cell differentiation can be determined based on the examination of cell- or tissue-specific markers known to indicate differentiation.

[0106] Tissue / cell-specific markers can be detected using immunological techniques well known in the art [Thomson JA et al., (1998). Science 282: 1145-7]. Examples include, but are not limited to, flow cytometry for membrane-bound or intracellular markers, immunohistochemical techniques for extracellular and intracellular markers, and enzyme immunoassays for secreted molecular markers.

[0107] After the differentiation steps described hereinabove, a mixed cell population containing both pigmented and non-pigmented cells is obtained.

[0108] According to one embodiment of this aspect of the invention, all cells of the mixed cell population are removed from the plate.

[0109] This can be done enzymatically (e.g., using trypsin (TrypLE Select)). According to this aspect of the invention, at least 10%, 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70% of the cells removed from the culture (and subsequently expanded) are non-pigmented cells. In one embodiment, all the cells are removed from the plate (e.g., enzymatically) and then purified (e.g., using a strainer, e.g., a 40 μm strainer) and only then expanded.

[0110] Furthermore, at least 10%, 20% or 30% of the cells removed from the culture system (and subsequently expanded) are pigment cells.

[0111] Preferably, at least 50%, 60%, 70%, 80%, 90%, 95%, 100% of the total cells in the culture system are removed (and subsequently expanded).

[0112] According to another embodiment, the non-pigmented cells are first mechanically isolated and removed from the culture plate, and then the pigmented cells are removed.

[0113] The cells may be subjected to a filtration process before growth and after harvesting. Thus, for example, the cells may be filtered through a 10-100 μm strainer (e.g., a 40 μm strainer).

[0114] RPE cells can be grown on extracellular matrices such as gelatin, collagen I, collagen IV, laminin, and poly-D-lysine. For growth, cells can be cultured in serum-free KOM, serum-containing medium (e.g., DMEM + 20%), or Nutristem medium (06-5102-01-1A, Biological Industries). Under these culture conditions, after passage under appropriate conditions, the ratio of pigmented cells to non-pigmented cells increases, resulting in a purified RPE cell population. Such cells exhibit the polygonal morphology and pigmentation characteristic of RPE cells.

[0115] RPE cells can be grown in suspension (with or without microcarriers) or in monolayers. Growth of mixed cell populations in monolayer or suspension cultures may be transformed into large-scale growth in bioreactors by methods well known to those skilled in the art.

[0116] According to one embodiment, the expansion step is carried out for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks or even 10 weeks. Preferably, the expansion step is carried out for 1 to 10 weeks, more preferably 2 to 10 weeks, more preferably 3 to 10 weeks, more preferably 4 to 10 weeks or 4 to 8 weeks.

[0117] According to yet another embodiment, the RPE cells are passaged at least once during the expansion stage, at least twice during the expansion stage, at least three times during the expansion stage, at least four times during the expansion stage, or at least five times during the expansion stage.

[0118] The RPE cell populations produced according to the methods described herein can be characterized according to several different parameters.

[0119] Thus, for example, the resulting RPE cells may be polygonal and pigmented.

[0120] Harvesting of the expanded RPE cell population may be performed using methods known in the art (eg, using an enzyme such as trypsin).

[0121] After collection, the expanded RPE cell population may optionally be cryopreserved using methods known in the art. Examples of media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, CryoStor 10%, 5% and 2%, Stem Cell Banker, and Prime XV® FreezIS.

[0122] It will be appreciated that the cell populations disclosed herein are devoid of undifferentiated human embryonic stem cells. According to one embodiment, fewer than 1 in 250,000 cells are Oct4 cells, as measured, for example, by FACS. + TRA-1-60 + The cells also have downregulated (more than 5,000-fold) expression of GDF3 or TDGF as measured by PCR.

[0123] The RPE cells of this aspect of the invention do not express other embryonic stem cell markers, which may include OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-β, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-81.

[0124] The RPE preparation may be substantially purified relative to non-RPE cells and contain at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells. The RPE cell preparation may be essentially free of non-RPE cells or may consist of RPE cells. For example, a substantially purified RPE cell preparation may contain less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of non-RPE cell types. For example, RPE cell preparations have been shown to be approximately 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.03%. It may contain less than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% non-RPE cells.

[0125] RPE cell preparation can be substantially pure for both non-RPE cells and RPE cells of other maturity levels.Preparation can be substantially purified for non-RPE cells and enriched for mature RPE cells.For example, in RPE cell preparation enriched for mature RPE cells, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% of RPE cells are mature RPE cells.Preparation can be substantially purified for non-RPE cells and enriched for differentiated RPE cells but not mature RPE cells. For example, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the RPE cells may be differentiated RPE cells rather than mature RPE cells.

[0126] The preparations described herein can be substantially free of bacterial, viral, or fungal contamination or infection, including, but not limited to, the presence of HIV I, HIV 2, HBV, HCV, HAV, CMV, HTLV 1, HTLV 2, parvovirus B19, Epstein-Barr virus or herpesvirus 1 and 2, SV40, HHV 5, 6, 7, 8, CMV, polyomavirus, HPV, enterovirus. The preparations described herein can be substantially free of mycoplasma contamination or infection.

[0127] Another method for characterizing the cell populations disclosed herein is by marker expression. Thus, for example, at least 80%, 85%, 90%, 95%, or 100% of the cells express bestrophin 1, as measured by immunostaining. According to one embodiment, 85-100% of the cells express bestrophin.

[0128] According to another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express microphthalmia-associated transcription factor (MITF), e.g., 85-100% of the cells express MITF.

[0129] According to another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97% or 100% of the cells express paired box gene 6 (PAX-6) as measured by immunostaining or FACS.

[0130] According to another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express cellular retinaldehyde-binding protein (CRALBP) as measured by immunostaining, e.g., 85-100% of the cells express CRALBP.

[0131] According to another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express retinal pigment epithelium-specific protein 65 kDa (RPE65) as measured by immunostaining, e.g., 85-100% of the cells express RPE65.

[0132] RPE cells express, and commonly co-express, markers indicative of terminal differentiation, such as bestrophin 1, pre-melanosome protein (PMEL17), CRALBP and / or RPE65.

[0133] After the proliferation stage, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 or even 100% of the cells are CRALBP. + PMEL17 + A cell population containing RPE cells is obtained.

[0134] Those skilled in the art will appreciate that RPE cell induction is highly beneficial. RPE cells can be used as an in vitro model for the development of new drugs to promote their survival, regeneration, and function. RPE cells can be useful for high-throughput screening of compounds with toxic or regenerative effects on RPE cells. RPE cells can be used to identify mechanisms, new genes, soluble factors, or membrane-bound factors that are important for the development, differentiation, maintenance, survival, and function of photoreceptor cells.

[0135] RPE cells can also serve as an unlimited source of RPE cells for transplantation, replacement, and support of dysfunctional or degenerated RPE cells in retinal degeneration. Furthermore, genetically modified RPE cells can also serve as vectors for carrying and expressing genes in the eye and retina after transplantation.

[0136] Ocular conditions for which RPE cells may serve as therapeutic agents include, but are not limited to, retinal diseases or disorders generally associated with retinal dysfunction, retinal injury, and / or loss of the retinal pigment epithelium. A non-limiting list of conditions that may be treated according to the present invention includes retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), dry AMD, Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophies and other dystrophies of the RPE, Stargardt's disease, RPE and retinal damage resulting from damage caused by any one of light injury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury, or traumatic injury.

[0137] Subjects that may be treated include primates (including humans), dogs, cats, ungulates (e.g., horses, cattle, swine (e.g., pigs)), birds, and other subjects. Commercially important human and non-human animals (e.g., livestock) are of particular interest. Exemplary mammals that may be treated include dogs; cats; horses; cattle; sheep; rodents, etc., and primates, particularly humans. Non-human animal models, particularly mammals, such as primates, rats, rabbits, etc., may also be used for experimental investigations.

[0138] The RPE cells produced as described herein can be transplanted into various target sites of the eye of the target.According to one embodiment, the transplantation of RPE cells is transplanted into the subretinal space of the eye, which is the usual anatomical location of RPE (between photoreceptor outer segments and choroid).In addition, depending on the migration ability and / or positive paracrine effect of cells, transplantation into other eye compartments can be considered, including the vitreous cavity, inner or outer retinal layer, periphery of retina and choroid.

[0139] The number of viable cells that can be administered to a subject is 50,000 to 5 x 10 per injection. 6 or 50,000 to 500,000.

[0140] The cells are generally formulated in a carrier (e.g., an isotonic solution and / or saline solution), such as BSS plus™. The carrier may optionally contain additional factors to aid in RPE engraftment, integration, survival, efficacy, etc.

[0141] Transplantation can be carried out by various techniques known in the art.The method of carrying out RPE transplantation is described in, for example, U.S. Patent No. 5,962,027, U.S. Patent No. 6,045,791 and U.S. Patent No. 5,941,250 and Eye Graefes Arch Clin Exp Opthalmol March 1997; 235(3): 149-58; Biochem Biophys Res Commun February 24, 2000; 268(3): 842-6; Opthalmic Surg February 1991; 22(2): 102-8. Methods for performing corneal transplants are described, for example, in U.S. Patent No. 5,755,785 and Eye 1995; 9 (Pt 6 Su): 6-12; Curr Opin Opthalmol August 1992; 3 (4): 473-81; Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; Ophthalmology April 2000; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999; 43 (6): 502-8. When primarily utilizing paracrine effects, cells can also be delivered to and maintained in a semipermeable container in the eye, which also reduces exposure of the cells to the host immune system (Neurotech USA CNTF delivery system; PNAS March 7, 2006 vol. 103 (10) 3896-3901).

[0142] The administering step can include intraocular administration of the RPE cells to an eye in need thereof. Intraocular administration can include injection of the RPE cells into the subretinal space.

[0143] According to one embodiment, transplantation is performed by delivering the cells into the subretinal space after pars plana vitrectomy through a small retinal opening or by direct injection.

[0144] RPE cells can be transplanted in various forms. For example, RPE cells can be introduced into the target site in the form of a cell suspension, with a matrix, or attached to a matrix or membrane, extracellular matrix or substrate, such as a cytodegradable polymer, or a combination thereof. RPE cells can also be transplanted (co-transplanted) with other retinal cells, such as photoreceptors.

[0145] The effectiveness of treatment may be assessed by various measures of visual and ocular function and structure, including best-corrected visual acuity (BCVA), retinal sensitivity measured by perimetry or microperimetry in dark-adapted and light-adapted states, full-field, multifocal, focal, or pattern electroretinogram (ERG), contrast sensitivity, reading speed, color vision, clinical biomicroscopy, fundus photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), infrared and multicolor imaging, fluorescein or ICG angiography, and additional measures used to assess visual function and ocular structure.

[0146] The subject may be administered a corticosteroid, such as prednisolone or methylprednisolone, Predforte, prior to or concurrently with administration of the RPE cells.

[0147] According to another embodiment, the subject is not administered a corticosteroid, such as prednisolone or methylprednisolone, Pred Forte, prior to or concurrently with administration of the RPE cells.

[0148] Immunosuppressant drugs may be administered to the subject prior to, concurrently with, and / or after treatment.

[0149] Immunosuppressants may belong to the following classes: Glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), agents acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Further drugs include interferons, opioids, TNF-binding proteins, mycophenolates, and small biological agents.

[0150] Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS1 L1X1MAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX1MAB® (anti-CD20 antibody), sirolimus, tacrolimus, and / or mycophenolate mofetil.

[0151] Antibiotics may be administered to the subject prior to, concurrently with, and / or after treatment. Examples of antibiotics include ofloxacin, gentamicin, chloramphenicol, tobrex, vigamox, or any other topical antibiotic preparation approved for ophthalmic use.

[0152] As used herein, the term "about" refers to ±10%.

[0153] The terms "comprise," "comprising," "include," "including," "having," and their conjugations mean "including, but not limited to."

[0154] The term "consisting of" means "including and limited to."

[0155] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts, provided that the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0156] As used herein, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0157] When a range of values ​​is given herein, it is meant to include any stated value (fractional or integer) within the stated range. A "range between" a first stated value and a second stated value and a "range from" a first stated value to a second stated value are used interchangeably herein and are meant to include the first and second stated values ​​and all fractional and integer values ​​therebetween.

[0158] As used herein, the term "method" refers to ways, means, techniques and methods for accomplishing a given task, including, but not limited to, ways, means, techniques and methods known to or readily developed by those skilled in the art of chemistry, pharmacology, biology, biochemistry and medicine.

[0159] As used herein, the term "treating" includes negating, substantially inhibiting, slowing, or reversing the progression of a condition, substantially ameliorating the clinical or outward symptoms of a condition, or substantially preventing the appearance of clinical or outward symptoms of a condition.

[0160] It will be understood that certain features of the invention that are described in the context of separate embodiments for ease of understanding may also be provided in combination in a single embodiment. Conversely, various aspects of the invention that are described in the context of a single embodiment for brevity may also be provided separately, or in any suitable subcombination, or with any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0161] Various embodiments and aspects of the present invention, as delineated hereinabove and as defined in the claims below, find experimental support in the following examples. [Example]

[0162] Reference is now made to the following examples which, together with the above detailed description, illustrate, in a non-limiting manner, certain aspects of the present invention.

[0163] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III, Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series," Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998); methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology," Volumes I-III, Coligan, JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994);Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are widely described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985);"Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984);"Animal Cell Culture" Freshney, RI, ed. (1986);"Immobilized Cells and Enzymes" IRL Press, (1986);"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al.See, "Strategies for Protein Purification and Characterization—A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference as if fully set forth. Other general references are provided throughout this document; the techniques therein are believed to be well known in the art and are provided for the convenience of the reader; all information contained therein is incorporated herein by reference.

[0164] Example 1 Induction of RPE cells (laminin) without spheroid formation material and method HAD-C 102 hESCs were manually expanded on human umbilical cord feeders (hUCF; code 008) in center-well plates, then passaged using collagenase onto feeder-free laminin 521 (5 μg / ml for 2 hours at 37°C; Biolamina, Lam-521)-coated flasks and grown in Nutristem™ (Biological Industries, 05-110-1A) containing HSA medium containing bFGF and TGFβ to produce RPE cells. On day 6 of hESC growth on laminin-521, differentiation was initiated and the medium was changed to Nutristem Minus (without bFGF and TGFβ; Biological Industries, 06-5102-01-1A) containing 10 mM nicotinamide (Sigma, N-5535) for 1 week (medium was changed on day 9), followed by Nutristem Minus containing 10 mM nicotinamide and 140 ng / ml activin A (Peprotech, G-120-14E) for 2 weeks, and then to Nutristem Minus containing 10 mM nicotinamide for approximately 1 week, at which time patches of pigmented cells were observed. Cells were then harvested using TrypLE select (Invitrogen, 12563-011), filtered through a 40 μm strainer, and centrifuged. Viable cells were counted and seeded into wells (of a 6-well plate) coated with three recombinant human gelatins (rhGelatin, Fibrogen, RhG100-001) in the presence of DMEM (HyClone, SH30081) containing 20% ​​human serum (Akron, AK9905) for 3 days and in the presence of Nutristem Minus for 11 days. Viable cells were then collected at the end of passage 0 (P0, day 10) and seeded into T75 flasks without rhGelatin in 20% human serum medium for up to 3 days and in the presence of Nutristem Minus for up to 11 days. This process was repeated once more, and cells at the end of passage 2 were collected and cryopreserved. A summary of the process can be seen in Figure 1.

[0165] result At the end of the differentiation process, patches of pigmented cells were produced (Figure 2A, yellow arrows). After harvesting all cells with TrypLE Select, filtering through a 40 μm strainer, and one expansion step, cells with polygonal morphology covered the entire cell culture plate (Figure 2B, end of P0).

[0166] At the end of P1, cells were tested for identity / purity using a CRALBP / PMEL17 double-staining FACS assay. As shown in Figure 3A-C, over 99% of cells were double-positive for CRALBP and PMEL17.

[0167] At the end of P2, cells were tested for identity / purity using a CRALBP / PMEL17 double-staining FACS assay. As shown in Figure 4A-D, over 99% of cells were double-positive for CRALBP and PMEL17.

[0168] Example 2 Induction of RPE cells (vitronectin) without spheroid formation material and method HAD-C 102 hESCs were manually expanded on human umbilical cord feeders (hUCF; code 008) in center-well plates, then passaged with collagenase onto flasks coated with feeder-free vitronectin (5–10 μg / 200 ml PBS per well of a 6-well plate) and grown in Nutristem™ (Biological Industries, 05-110-1A) containing HSA medium containing bFGF and TGFβ to produce RPE cells. In some experiments, undifferentiated hESCs were passaged with collagenase and replated on vitronectin in the same medium for further expansion. On day 6 or 7 of hESC growth on vitronectin, differentiation was initiated and the medium was changed to Nutristem Minus (without bFGF and TGFβ; Biological Industries, 06-5102-01-1A) containing 10 mM nicotinamide (Sigma, N-5535) for 2 weeks (medium was changed on day 9), followed by Nutristem Minus containing 10 mM nicotinamide and 140 ng / ml activin A (Peprotech, G-120-14E) for 2 weeks, and then to Nutristem Minus containing 10 mM nicotinamide for approximately 1 week, at which time patches of pigmented cells were observed. Entire cultures were harvested using Triple Select. Viable cells were counted and seeded into wells (of a 6-well plate) coated with three recombinant human gelatins (rhGelatin, Fibrogen, RhG100-001) in the presence of DMEM (HyClone, SH30081) containing 20% ​​human serum (Akron, AK9905) for up to 3 days and in the presence of Nutristem Minus for up to 11 days. Viable cells were then collected at the end of passage 0 (P0, day 10) and seeded into T75 flasks without rhGelatin containing 20% ​​human serum medium for 3 days and in the presence of Nutristem Minus for 11 days. This process was repeated once more, and cells at the end of passage 2 were collected and cryopreserved. The experiment was repeated four times (twice under hypoxia and twice under normoxia).

[0169] result At the end of the differentiation process, patches of pigmented cells were produced. The entire culture was harvested by enzymatic digestion and replated for further culture and expansion under conditions that promote RPE cell proliferation. Cells with polygonal morphology covered the entire cell culture plate. The majority of the cells harbored intense pigments (Figures 5A-C). Immunostaining showed that the majority of the cells expressed RPE cell markers, such as bestrophin, CRALBP, MITF, PEDF, and ZO-1 (Figures 6A-E).

[0170] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0171] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. Section headings, to the extent that they are used, should not be construed as necessarily limiting.

Claims

1. 1. A method for producing retinal pigment epithelial (RPE) cells, comprising: (a) culturing a cell population of undifferentiated human pluripotent stem cells on an adhesive surface selected from the group consisting of laminin, fibronectin, vitronectin, collagen I, and collagen IV in a medium containing nicotinamide as a differentiation inducer and lacking activin A, and under feeder cell-free conditions, to obtain differentiated cells, wherein at least 50% of the cells in the cell population are Oct4 + TRA-1-60 + and (b) culturing the differentiated cells on the adherent surface in a medium containing nicotinamide and one or more members of the TGFβ superfamily selected from the group consisting of TGFβ1, TGFβ3, and activin A to obtain RPE cells. A method comprising:

2. The method of claim 1, further comprising, prior to step (a), growing the population of undifferentiated human pluripotent stem cells on an adherent surface in the absence of a differentiation-inducing agent.

3. The method of claim 2, wherein the expanding step is carried out in the absence of a differentiation-inducing agent.

4. The method of claim 1, further comprising isolating the RPE cells from the adherent surface after step (b).

5. The method of claim 4, further comprising culturing the isolated RPE cells on an additional adhesive surface to produce an expanded RPE cell population.

6. The method of claim 5, further comprising the step of collecting the RPE cells.

7. 5. The method of claim 4, wherein the isolating step is performed enzymatically.

8. 2. The method of claim 1, further comprising growing the human pluripotent stem cells on human feeder cells prior to step (a).

9. 3. The method of claim 2, further comprising growing the human pluripotent stem cells on human feeder cells prior to growing on the adherent surface.

10. More than 90% of the cells in the expanded RPE cell population expressed CRALBP. + PMEL17 + 6. The method of claim 5, wherein

11. 10. The method of claim 1, wherein the adhesive surface is laminin or vitronectin.

12. 12. The method of claim 11, wherein the laminin is laminin 521.

13. 6. The method of claim 5, wherein the additional adhesive surface is selected from the group consisting of gelatin, poly-d-lysine, laminin, collagen I, and collagen IV.

14. 14. The method of claim 13, wherein the additional adhesive surface is gelatin or poly-d-lysine.

15. The method of claim 2, wherein the growing step is carried out for at least 3 weeks.

16. The method of claim 6, further comprising the step of cryopreserving the RPE cells after collection.

17. 17. The method of claim 16, wherein the cryopreserving step is performed in a medium selected from the group consisting of 90% HS / 10% DMSO, CryoStor 2%, CryoStor 5% and CryoStor 10%, and Stem Cell Banker.

18. 10. The method of claim 9, wherein the human feeder cells comprise human umbilical cord fibroblasts.

19. The method of any one of claims 1 to 18, wherein the human pluripotent stem cells comprise human embryonic stem cells.

20. 2. The method of claim 1, further comprising, after step (b), culturing the RPE cells in a medium containing nicotinamide and lacking activin A.

21. 21. The method of any one of claims 1 to 20, wherein step (a) is carried out for at least 5 days.

22. 22. The method of any one of claims 1 to 21, wherein step (b) is carried out for at least one week.

23. 23. Use of RPE cells prepared according to the method of any one of claims 1 to 22 in the manufacture of a medicament for the treatment of a retinal disease.

24. 24. The use of claim 23, wherein the RPE cells are in suspension or as a monolayer of cells immobilized on a matrix or substrate.

25. 24. The use of claim 23, wherein the retinal disease or disorder is selected from at least one of retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophy, Stargardt's disease, RPE damage and retinal damage resulting from damage caused by any one of light injury, laser injury, inflammatory injury, infectious injury, radiation injury, neovascular injury or traumatic injury.

26. 23. A population of RPE cells produced according to the method of any one of claims 1 to 22.

27. 27. Use of the RPE cells of claim 26 in the manufacture of a medicament for the treatment of a retinal disease or disorder.

Images