MACS-based purification of stem cell-derived retinal pigment epithelium

Abstract

Provided is a method for enrichment of a retinal pigment epithelium (RPE) cell colony derived from stem cells. Such method can include removing contaminating cells by depleting CD24-positive cells, CD56-positive cells and / or CD90-positive cells from the starting colony of RPE cells.

Description

[0001] This application is a divisional application, and its corresponding parent application has the application number 201680060869.2, the application date is September 7, 2019, and the invention title is "MACS-based purification of stem cell-derived retinal pigment epithelium".

[0002] This application claims priority to U.S. Provisional Application No. 62 / 215,272, filed September 8, 2015, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention generally relates to the field of stem cell biology. More specifically, it relates to a method for enriching a population of retinal pigment epithelial cells derived from stem cells. Background Technology

[0004] The retina is the light-sensitive layer of tissue located on the inner surface of the eye. Photoreceptor cells (rod or cone cells) in the retina are directly sensitive to light and convert chemical light signals into electrical activity that triggers nerve impulses. Retinal pigment epithelial cells (RPE) are a layer of pigment cells that form the blood-retinal barrier. RPE cells play a crucial role in maintaining visual function and in the transport of ions, water, and metabolic end products from the subretinal space to the bloodstream (Strauss et al., 2005). Furthermore, RPE cells establish the eye's immune privilege by secreting immunosuppressive factors. Disorders or damage to RPE cells can lead to retinal degeneration, loss of visual function, and blindness. Several retinal diseases, including acute and age-related macular degeneration and Best's disease, involve RPE degeneration; therefore, cell replacement therapy is a possible treatment option for preserving vision (Buchholz et al., 2009).

[0005] Typically, stem cells are undifferentiated cells that can produce a range of mature, functional cells. For example, hematopoietic stem cells can produce any different type of terminally differentiated blood cells. Embryonic stem (ES) cells originate from the embryo and are pluripotent, thus possessing the ability to develop into any organ or tissue type, including RPE cells.

[0006] The generation of induced pluripotent stem cells (iPS cells) from adult mouse somatic cells in 2006 provided a major breakthrough for stem cell research, drug development, disease models, and cell therapy (Takahashi et al., 2006). Human iPSCs can differentiate into specific cell types and have the potential to be patient-specific, immune-matched cells for regenerative medicine (Yu et al., 2007).

[0007] iPSCs have been shown to generate ocular cells, including RPE cells (Hirami et al., 2009). However, using iPSC-derived RPE cells for therapeutic, screening assays, retinal disease models, and RPE biology research requires removing contaminating cells and / or enriching the target population from the differentiated RPE cell population. Summary of the Invention

[0008] Embodiments of the present invention overcome major deficiencies in the art by providing a method for obtaining a rich population of retinal pigment epithelial (RPE) cells from a starting RPE cell population by removing CD24-positive, CD56-positive, and / or CD90-positive cells. In some embodiments, the starting RPE cell population may be obtained from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0009] In one embodiment, a method is provided for providing an enriched population of retinal pigment epithelial (RPE) cells, comprising (a) obtaining a starting cell population containing RPE cells and (b) enriching the RPE cells of said cell population by removing CD24-positive cells, CD56-positive cells, and / or CD90-positive cells therefrom, thereby providing an RPE-enriched cell population richer in RPE cells compared to the starting cell population. In some aspects, the starting cell population is ungenetically modified or genetically isolated. In some aspects, the enriched cell population does not include genetically modified cells. Therefore, in some aspects, the RPE-enriched cell population is ungenetically modified or genetically isolated.

[0010] In some aspects, the method may further include determining the enrichment level of RPE cells in the RPE-rich cell population. In some aspects, the enrichment level is determined by using retinal epithelium-specific markers. For example, retinal epithelium-specific markers may be BEST1, CRALBP, TYRP1, PMEL17, or MITF. In specific aspects, as determined by BEST1 sorting, the RPE-rich cell population is rich in RPE cells compared to the starting cell population.

[0011] In some respects, RPE-rich clusters are at least 95%, 96%, 97%, 98%, or 99% RPE cells. In other respects, RPE-rich clusters are essentially pure RPE cells.

[0012] In some aspects of the implementation scheme, the starting cell population is prepared from pluripotent stem cells. In other aspects, the pluripotent stem cells are induced pluripotent stem cells. For example, RPE cells can be human RPE cells.

[0013] In some respects, CD24-positive cells, CD56-positive cells, and / or CD90-positive cells are removed. For example, CD24-positive cells, CD56-positive cells, and / or CD90-positive cells can be removed by magnetic bead-based sorting or fluorescence-based sorting. In some respects, antibodies or aptamers that recognize CD24, CD56, and / or CD90 are used to remove CD24-positive cells, CD56-positive cells, and / or CD90-positive cells.

[0014] In some respects, RPE cells are enriched by removing CD24-positive cells. In other respects, RPE cells are enriched by removing CD56-positive cells. In yet another respect, RPE cells are enriched by removing CD90-positive cells. In yet another respect, RPE cells are enriched by removing CD90-positive, CD56-positive, and CD24-positive cells.

[0015] The cell populations presented in this article may be substantially free of contaminating non-RPE cells, such as fibroblasts or undifferentiated pluripotent stem cells. On the other hand, the RPE cells are mouse or human RPE cells. In a specific context, the RPE cells may be cryopreserved RPE cells.

[0016] RPE cells generated by the methods described herein can be used in any method and application of RPE cells known in the art. For example, methods for evaluating compounds can be provided, including determining the pharmacological or toxicological properties of the compounds on RPE cells. Methods for evaluating the effects of compounds on RPE cells can also be provided, comprising: a) contacting the RPE cells provided herein with the compound; and b) determining the effects of the compound on the RPE cells.

[0017] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while preferred embodiments of the invention have been indicated, the detailed description and specific examples are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description. Attached Figure Description

[0018] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments given herein.

[0019] Figure 1A-1DA) Image of iPSCs before confluence. B) Example image on day 25 of RPE differentiation. C) Example image on day 40 of RPE differentiation. D) Example 100x bright-field image on day 60 after rearranging the culture with RPE-MM on day 40.

[0020] Figure 2A-2B Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) prior to cell sorting of the iPSC-derived RPE cell population.

[0021] Figures 3A-3C Flow cytometry analysis of relevant markers (including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1) after sorting iPSC-derived RPE cell populations to remove CD24-positive, CD24-positive and CD56-positive, CD24-positive and CD90-positive, and CD24-positive, CD56-positive, and CD90-positive cells.

[0022] Figures 4A-4D A) β-catenin and F-actin staining in untreated iPSC-RPE cells and PGE2-treated iPSC-RPE cells. β-catenin staining is visible in the cytoplasm of untreated cells and on the membrane of treated cells. B) pERM (Ezrin) and ZO1 staining in untreated iPSC-RPE cells and PGE2-treated iPSC-RPE cells. ERM staining is lighter in the cytoplasm of untreated cells and darker in the cytoplasm of treated cells. ZO1 staining is visible at the tight junctions of the plasma membranes of both untreated and treated cells. C) RPE65 and ZO1 staining in untreated iPSC-RPE cells and PGE2-treated iPSC-RPE cells. RPE65 staining is lighter in the cytoplasm of untreated cells and darker in the cytoplasm of treated cells. D) Transmission electron micrographs of untreated iPSC-RPE cells and PGE2-treated iPSC-RPE cells. Cells treated with PGE2 have broader apical processes.

[0023] Figures 5A-5DA) β-catenin staining in cells treated with IWP2+endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2+endo-IWR1 showed β-catenin on the cell membrane. Cells treated with LiCl showed β-catenin in the nucleus, while untreated cells showed β-catenin in the cytoplasm. B) p27 staining in cells treated with IWP2+endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2+endo-IWR1 showed high p27 expression in the nucleus, indicating cell cycle exit. Cells treated with LiCl or untreated cells showed weak p27 expression in the nucleus. C) Tightly linked RPE65 and ZO1 in cells treated with IWP2+IWR1, IWP2, or LiCl. RPE65 was high in the cytoplasm of IWP2+IWR1 and IWP2 cells, low in untreated cells, and no staining was observed in LiCl-treated cells. D) Electron microscopic images of functional tight junctions in cells treated with IWP2+IWR1, IWP2, or LiCl.

[0024] Figures 6A-6D A) Multi-operator RPE differentiation. The data shown represent RPE differentiation settings using an optimized multi-operator protocol across three cell lines, as measured by flow cytometry via RPE-labeled retinaldehyde-binding protein 1 (Cralbp). B) Reproducibility of the RPE differentiation protocol is shown in different starting cell line populations including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, AMD Donor3C, and HLA lineage A. C) Reproducibility of the RPE differentiation protocol is shown in different starting cell line populations. The data represent 109 differentiations performed by 5 operators on 28 iPSC cell lines from 13 donors. The percentage of Cralbp-positive cells increased to 90–100% compared to different purities of the pre-purification cell populations.

[0025] Figures 7A-7GA) The functionality of barrier function in RPE cells generated using the RPE differentiation protocol was shown by transepithelial potential (TEP) measurements across the monolayer ion gradient. B) Functionality of RPE cells treated with IWP2 or IWP2+endo-IWR2. CE) Transepithelial resistance (TER) and TEP (fader lines) of untreated cells, PGE2-treated cells, and IWP2+endo-IWR1-treated cells. F) Functional response (TER) of cells matured using 50 μM PGE2 in RPE-MM+PGE2 medium relative to 100 μM PGE2 from day 54 to day 75 of the iPSC-derived differentiation protocol. Compared to iPSC-derived RPE cultured with 50 μM PGE2 in RPE-MM+PGE2 medium from day 54 to day 75 of the differentiation protocol, the TER gradually increased during differentiation at 100 μM. This indicates that increased PGE2 concentration promotes maturation and functional efficiency of iPSC-derived RPE cultures. G) The purity of iPSC-derived RPEs was expressed as the percentage expression of mature RPE markers on day 75 in cultures containing 50 μM PGE2 relative to 100 μM PGE2, from day 54 to day 75 of the iPSC-derived RPE differentiation protocol. The expression of Pmel17, Tryp1, and Cralbp (RPE-specific markers) was comparable to that of iPSC-derived RPEs cultured with 50 μM PGE2. This indicates that PGE2 promotes iPSC-derived RPE differentiation within a certain concentration range. Cells treated with 100 μM PGE2 showed significantly higher expression of the Best 1 marker (a late-mature RPE marker) compared to cells treated with 50 μM PGE2, suggesting that increasing the PGE2 concentration enhances the purity and maturity of iPSC-derived RPEs. Detailed Implementation

[0026] This disclosure overcomes several major problems of the prior art by providing a method for enriching stem cell-derived retinal pigment epithelial (RPE) cell populations. RPE cells can be derived from pluripotent stem cells, such as ES cells and iPS cells; however, even within stem cell-derived RPE populations using existing techniques, a certain amount of contaminating cells (e.g., undifferentiated stem cells) is found. This invention provides a method for isolating and removing contaminating cells from a starting RPE cell population. Cells contaminating an RPE cell population possess specific cell surface antigens that can be used to deplete contaminating non-RPE cells from the population, such as CD24, CD56, and / or CD90. Therefore, removing cells that are positive for one or more of these specific cell surface markers can produce an RPE-enriched cell population with a higher percentage of RPE cells than the starting population. Certain methodologies are known in the art, such as magnetically activated cell sorting (MACS). Fluorescence-activated cell sorting (FACS) or single-cell sorting, etc., are used to separate various cell populations based on their surface antigens. In a preferred embodiment, this disclosure includes methods using these sorting methods, preferably MACS, to obtain RPE-enriched cell populations from an initial RPE cell population that have depleted CD24, CD56, and / or CD90-positive cells. Therefore, the method of the present invention is more time- and cost-effective and enables the production of RPE-enriched cell populations from renewable sources such as stem cells for use in therapeutics. Other embodiments and advantages of this disclosure are described below.

[0027] I. Definition

[0028] The term “purification” does not require absolute purity; rather, it is a relative term. Thus, a purified cell population is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, or most preferably, substantially free of other cell types.

[0029] As used herein, "substantially" or "substantially free of" with respect to a particular component is used to indicate that the particular component was not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is in which the amount of the particular component is undetectable by standard analytical methods.

[0030] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used with the word "comprising," the word "a" or "an" may mean one or more.

[0031] The term “or” is used in the claims to mean “and / or” unless it is explicitly stated that it refers only to an alternative or that the alternatives are mutually exclusive, although this disclosure supports the definitions of referring only to an alternative and “and / or”. As used herein, “another” can mean at least a second or more.

[0032] Throughout this application, the term “about” is used to indicate a value that includes the inherent variation of the error of the apparatus, the method used to determine that value, or the variation that exists between subjects under study.

[0033] The term "cell" is used herein to refer to the structural and functional unit of an organism that can replicate independently, is surrounded by a membrane, and contains biomolecules and genetic material. The cells used herein may be naturally occurring cells or artificially modified cells (e.g., fused cells, genetically modified cells, etc.).

[0034] The term "cell population" is used herein to refer to a group of cells that are typically of common types. A cell population may originate from a common progenitor cell or may contain more than one cell type. An "enriched" cell population refers to a cell population derived from a starting cell population (e.g., an ungraded, heterogeneous cell population) that contains a greater percentage of that particular cell type than the percentage of that particular cell type in the starting population. A cell population may be enriched or depleted for one or more cell types.

[0035] The term "stem cell" as used herein refers to a pluripotent cell that, under suitable conditions, can differentiate into a wide range of specialized cell types, and under other suitable conditions, can self-renew and remain essentially undifferentiated. The term "stem cell" also includes pluripotent cells, multipotent cells, progenitor cells, and precursor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells derived from bone marrow tissue, embryonic stem cells derived from embryonic tissue, or embryonic germ cells derived from fetal germ tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming them into a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs."

[0036] The term "pluripotent" refers to the property of a cell to differentiate into all other cell types (except extraembryonic or placental cells) within an organism. Even after extended culture, pluripotent stem cells are capable of differentiating into cell types from all three germ layers (e.g., ectoderm, mesoderm, and endoderm cell types). Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of the blastocyst. In other embodiments, pluripotent stem cells are induced pluripotent stem cells derived from somatic cells through reprogramming.

[0037] The term "differentiation" refers to the process by which undifferentiated cells transform into a more specialized type with changes in structural and / or functional properties. Mature cells typically possess altered cell structures and tissue-specific proteins. More specifically, in the context of the method of this invention, differentiation is the process by which human stem cells acquire cell types that exhibit characteristics indicative of retinal pigment epithelial (RPE) cells as mature, terminally differentiated cells.

[0038] As used in this article, “undifferentiated” refers to cells that exhibit characteristic markers and morphological features of undifferentiated cells (which clearly distinguish them from terminally differentiated cells of embryonic or adult origin).

[0039] "Embryonic bodies (EBs)" are aggregates of pluripotent stem cells that can differentiate into cells of the endoderm, mesoderm, and ectoderm. When pluripotent stem cells aggregate and suspend a non-adherent culture of EBs, they form spherical structures.

[0040] "Isolated" cells have been substantially separated or purified from other cells in an organism or culture. The isolated cells may be, for example, at least 99%, at least 98%, at least 95%, or at least 90% pure.

[0041] An "embryo" refers to a cluster of cells obtained through one or more divisions of a fertilized egg or an activated oocyte with an artificially reprogrammed nucleus.

[0042] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells that are obtained from the embryo at an early stage (e.g., the inner cell mass at the blastocyst stage) or produced artificially (e.g., by nuclear transfer) and can produce any differentiated cell type in the embryo or adult, including germ cells (e.g., sperm and eggs).

[0043] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells through the expression of a combination of expression factors (referred to herein as reprogramming factors) or a combination of inducing factors. iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed into pluripotent stem cells by expressing at least two, at least three, or four reprogramming factors.

[0044] An "allele" is one of two or more forms of a gene. Diploid organisms like humans have two copies of each chromosome, so each chromosome carries one allele.

[0045] The term "homozygous" is defined as having two identical alleles at a given locus. The term "heterozygous" refers to having two distinct alleles at a given locus.

[0046] A haplotype is a combination of alleles at multiple loci along a single chromosome. Haplotypes can be based on a set of single nucleotide polymorphisms (SNPs) and / or alleles in the major histocompatibility complex on a single chromosome.

[0047] As used herein, the term "haplotype-matched" is defined as a cell (e.g., iPSC cells) and the treated subject sharing one or more major histocompatibility loci haplotypes. The subject's haplotype can be readily determined using assays known in the art. Haplotype-matched iPSC cells can be autologous or allogeneic. Autologous cells that grow in tissue culture and differentiate into RPE cells are inherently haplotype-matched with the subject.

[0048] "Largely identical HLA types" means that the donor's HLA type matches the patient's type to such an extent that transplanted cells obtained by inducing differentiation of iPSCs from donor somatic cells can be transplanted when implanted into the patient.

[0049] “Super donors” are referred to in this paper as individuals homozygous for certain MHC class I and II genes. These homozygous individuals can serve as super donors, and their cells (including tissues and other material containing their cells) can be transplanted into individuals that are homozygous or heterozygous for that haplotype. Super donors can be homozygous for the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ loci, respectively.

[0050] This article uses the terms "feeder-free" or "feeder-independent" to refer to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as a substitute for the feeder cell layer. Therefore, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain undifferentiated and proliferating pluripotent cells. In some cases, feeder-free cultures utilize animal-based substrates (e.g., MATRIGEL). TM They can also grow on a matrix such as fibronectin, collagen, or fibronectin. These methods allow human stem cells to remain largely undifferentiated without requiring a "feeder layer" of mouse fibroblasts.

[0051] In this paper, a "feeder layer" is defined as a coating layer of cells, such as the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface for other cells, such as pluripotent stem cells, to attach to.

[0052] When used in relation to culture media, extracellular matrix, or culture conditions, the terms "determined" or "fully determined" refer to a culture medium, extracellular matrix, or culture condition in which the chemical composition and amounts of virtually all components are known. For example, a determined culture medium does not contain undetermined factors, such as in fetal bovine serum, bovine serum albumin, or human serum albumin. Typically, determined culture media include basal media supplemented with recombinant albumin, chemically determined lipids, and recombinant insulin (e.g., Dulbecco modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640 containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy). An exemplary fully determined culture medium is Essential 8. TM Culture medium.

[0053] When used in relation to culture media, extracellular matrix, or culture conditions, the term "heterogeneous-free (XF)" refers to a culture medium, extracellular matrix, or culture condition that is substantially free of components of xenogeneic animal origin. For culturing human cells, any proteins from non-human animals, such as mice, would be xenogeneic components. In some respects, a xenogeneic-free matrix can be substantially free of any components of non-human animal origin, thus excluding mouse feeder cells or MATRIGEL cells. TM MATRIGEL TM It is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (which is a tumor rich in extracellular matrix proteins containing laminin (the main component), collagen IV, heparan sulfate proteoglycan and endogenous / nestin).

[0054] KNOCKOUT TM "Serve substitutes" are referred to in this paper as serum-free formulations optimized for the growth and maintenance of undifferentiated cells such as stem cells in cultures.

[0055] "Pre-merging" refers to cell cultures in which approximately 60-80% of the culture surface is covered by cells. Typically, pre-merging refers to cultures in which approximately 70% of the culture surface is covered by cells.

[0056] The "retina" refers to the light-sensitive layer of tissue located on the inner surface of the eye.

[0057] "Retinal pigment epithelial cells" refers to the single layer of pigment cells between the choroid (a layer full of blood vessels) and the retina.

[0058] In this article, "retinal lineage cells" refers to cells that can produce or differentiate into RPE cells.

[0059] "Retinal Induction Medium (RIM)" in this article refers to a growth medium containing inhibitors of the WNT and BMP pathways that can induce PSC differentiation into retinal lineage cells. RIM also contains inhibitors of the TGFβ pathway.

[0060] “Retinal differentiation medium (RDM)” is defined in this paper as a culture medium containing inhibitors of the WNT pathway, BMP pathway, and MEK pathway for differentiating retinal cells. RDM also contains inhibitors of the TGFβ pathway.

[0061] “Retinal medium (RM)” is defined as a growth medium containing activin A and nicotinamide for culturing retinal cells.

[0062] In this article, "RPE maturation medium (RPE-MM)" refers to a culture medium containing taurine and hydrocortisone used to mature RPE cells. RPE-MM also contains triiodothyronine. RPE-MM may also contain PD0325901 or PGE2.

[0063] “Mature” RPE cells are referred to as RPE cells in this paper, which have downregulated expression of immature RPE markers such as Pax6 and upregulated expression of mature RPE markers such as RPE65.

[0064] In this article, RPE cell “maturation” refers to the process of regulating RPE developmental pathways to produce mature RPE cells. For example, regulating ciliary function can lead to RPE maturation.

[0065] The term "therapeutic effective amount" as used in this article refers to the amount of a compound that is sufficient to achieve such treatment when administered to a subject to treat a disease or condition.

[0066] In this paper, an "inducer" is defined as a molecule that regulates gene expression within a cell, such as activating a gene. Inducers can bind to either inhibitors or activators. Inducers work by inactivating inhibitors.

[0067] II. Pluripotent stem cells

[0068] A. Embryonic stem cells

[0069] ES cells originate from the inner cell mass of the blastocyst and possess high in vitro differentiation capacity. ES cells can be isolated by removing the outer trophoblast of the developing embryo and then culturing the inner cell mass on a feeder layer of non-growing cells. The replatened cells can continue to proliferate and generate new ES cell colonies, which can be removed, dissociated, replatened, and allowed to grow. This process of "passaging" undifferentiated ES cells can be repeated many times to generate cell lines containing undifferentiated ES cells (US Patent Nos. 5,843,780; 6,200,806; 7,029,913). ES cells possess proliferative potential while maintaining their pluripotency. For example, ES cells can be used to study cells and genes controlling cell differentiation. The pluripotency of ES cells, combined with genetic manipulation and selection, can be used for in vivo gene analysis studies by generating transgenic, chimeric, and knockout mice.

[0070] Methods for producing mouse ES cells are well known. In one method, preimplantation blastocysts from the 129 mouse strain are treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured in a medium containing fetal bovine serum on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts. Colonies of developing undifferentiated ES cells are passaged in the presence of fetal bovine serum on the mouse embryonic fibroblast feeder layer to generate an ES cell population. In some methods, mouse ES cells can be cultured without a feeder layer by adding the cytokine leukemia suppressor factor (LIF) to a serum-containing medium (Smith, 2000). In other methods, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0071] Human ES cells can be generated or derived from mammalian embryos at the fertilized egg or blastocyst stage, which are generated by fusing sperm and egg cells, nuclear transfer, parthenogenesis, or chromatin reprogramming, and subsequently incorporating the reprogrammed chromatin into the plasma membrane to produce embryonic cells via previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum, trophoblastic ectoderm cells are lysed and removed from the inner cell mass cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, cell masses from the inner cell mass are chemically or mechanically dissociated and replatened, and colonies with undifferentiated morphologies are selected by micropipette, dissociated, and replatened (US Patent No. 6,833,269). In some methods, human ES cells can be grown in the absence of serum by culturing ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, protein matrices such as MATRIGEL are used in the presence of a "conditioned" medium containing basic fibroblast growth factor. TM Cells can be cultured on laminin to grow human ES cells without a feeder cell layer (Xu et al., 2001).

[0072] Using the methods previously described (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000), ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As another example, established mouse ES cell lines include the CGR8 cell line derived from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0073] ES stem cells can be detected by protein markers, including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1.

[0074] B. Induced pluripotent stem cells

[0075] Pluripotency induction was initially achieved in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells with pluripotency-associated transcription factors. Pluripotent stem cells can remain undifferentiated and are capable of differentiating into almost any cell type. Using iPSCs avoids most of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous grafts may not require lifelong immunosuppressive therapy to prevent transplant rejection.

[0076] Besides germ cells, any cell type can be used as the starting point for iPSCs. For example, cell types can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of reprogrammed somatic cells (US Patent No. 8,741,648). There are no restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected; even undifferentiated progenitor cells (including adult stem cells) and ultimately differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cells themselves are RPE cells, such as human RPE cells. RPE cells can be adult or fetal RPE cells. iPSCs can be grown under conditions known to differentiate human ES cells into specific cell types and express human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0077] Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Induced pluripotent stem cells can be readily generated by those skilled in the art, see, for example, published U.S. Patent Application No. 20090246875, published U.S. Patent Application No. 2010 / 0210014; published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,278,620; PCT Publication No. WO 2007 / 069666 A1 and U.S. Patent No. 8,268,620, which are incorporated herein by reference. Typically, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0078] Cells are treated with nuclear reprogramming substances, which are typically one or more factors capable of inducing iPSCs from somatic cells or nucleic acids (including those integrated into vectors) encoding these substances. Nuclear reprogramming substances typically include at least Oct3 / 4, Klf4, and Sox2 or nucleic acids encoding these molecules. Functional inhibitors of p53, L-myc or nucleic acids encoding L-myc, and Lin28 or Lin28b or nucleic acids encoding Lin28 or Lin28b can be used as additional nuclear reprogramming substances. Nanog can also be used for nuclear reprogramming.As disclosed in U.S. Patent Application No. 20120196360, exemplary reprogramming factors for generating iPSCs include (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced by Sox1, Sox3, Sox15, Sox17 or Sox18; Klf4 can be replaced by Klf1, Klf2 or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3 / 4,Klf4,Sox2,L-Myc,Lin28,SV40LT; (9)Oct3 / 4,Klf4,Sox2,L-Myc,Lin28,TERT,SV40LT; (10 (11) Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (12) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg); (13) Oct3 / 4, Klf4, Sox2; (14) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (15) Oct3 / 4, Klf4, Sox2, TERT, HP VI 6E6; (15) Oct3 / 4,Klf4,Sox2,TERT,HPV16 E7; (16) Oct3 / 4,Klf4,Sox2,TERT,HPV16 E6,HPV16 E7; (17) Oct3 / 4,Klf4,Sox2,TERT,Bmil; (18) Oct3 / 4,Klf4,Sox2,Lin28; (19) Oct3 / 4,Klf4,Sox2,Lin28,SV40LT; (20) Oct3 / 4,Klf4,Sox2,Lin28,TERT,SV40LT; (21) Oct3 / 4,Klf4,Sox2,SV40LT; or (22) Oct3 / 4,Esrrb,Sox2 (Esrrb can be replaced by Esrrg). In a non-restricted instance, Oct3 / 4, Klf4, Sox2, and c-Myc are used.In other embodiments, Oct4, Nanog, and Sox2 are used, see, for example, U.S. Patent No. 7,682,828, which is incorporated herein by reference. These factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other instances, these factors include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting instances, Oct3 / 4, Klf4, c-Myc, and Sox2 are used. In other non-limiting instances, Oct3 / 4, Klf4, Sox2, and Sal4 are used. Factors such as Nanog, Lin28, Klf4, or c-Myc can improve reprogramming efficiency and can be expressed from several different expression vectors. For example, an integrative vector such as an EBV element-based system can be used (U.S. Patent No. 8,546,140). Alternatively, reprogrammed proteins can be directly introduced into somatic cells via protein transduction. Reprogramming may also involve contacting cells with one or more signal transduction receptors, including glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor β (TGF-β) receptor inhibitors or signal transduction inhibitors, leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These modulators may include small molecules, repressive nucleotides, expression cassettes, or protein factors. It is anticipated that virtually any iPS cell or cell line can be used.

[0079] The mouse and human cDNA sequences of these nuclear reprogramming substances are available with reference to the NCBI accession number mentioned in WO 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming substances or nucleic acids encoding such reprogramming substances are known in the art and are disclosed, for example, in published U.S. Patent Application No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0080] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. As described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603, iPSCs can be used with various culture media and techniques for culturing pluripotent stem cells, more specifically embryonic stem cells. In the case of mouse cells, culture is carried out by adding leukemia suppressor factor (LIF), a differentiation inhibitor, to the ordinary culture medium. In the case of human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods known to those skilled in the art for culturing and maintaining iPSCs can be used.

[0081] In some embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or on a culture medium already exposed to fibroblast feeder cells to maintain stem cells in an undifferentiated state. In some embodiments, cells are cultured in coexistence with mouse embryonic fibroblasts (as feeder cells) treated with radiation or antibiotics to terminate cell division. Alternatively, defined feeder cell-independent culture systems (e.g., TESR) may be used. TM Culture medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8 TM Pluripotent cells were cultured in a culture medium (Chen et al., 2011) and maintained in a largely undifferentiated state.

[0082] In some implementations, the iPSC can be modified to express a foreign nucleic acid, for example, to include a tyrosinase enhancer operatively linked to a promoter and a nucleic acid sequence encoding a first marker. Tyrosinase genes, for example, are available in GENBANK as of January 1, 2013. The sequence is disclosed in accession number 22173. It is aligned with chromosome 7 at positions 5286971-5291691 (reverse) in mouse strain C57BL / 6. The 4721-base-pair sequence is sufficient for expression in RPE cells; see Murisier et al., Dev. Biol. 303:838-847, 2007, which is incorporated herein by reference. This construct is expressed in retinal pigment epithelial cells. Other enhancers may be used. Other RPE-specific enhancers include D-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP, and RAB27A. Suitable promoters include, but are not limited to, any promoter expressed in retinal pigment epithelial cells, including tyrosinase promoters. The construct may also include other elements such as a ribosome binding site (internal ribosome binding sequence) for translation initiation and a transcription / translation terminator. Generally, transfection of cells with the construct is advantageous. Suitable vectors for stable transfection include, but are not limited to, retroviral vectors, lentiviral vectors, and Sendai virus.

[0083] Plasmids have been designed for multiple objectives, such as achieving regulated high copy numbers and avoiding potential causes of plasmid instability in bacteria, and providing tools for plasmid selection that is compatible with mammalian cells, including human cells. Particular attention has been paid to the dual requirements of plasmids for human cells. First, they are suitable for maintenance and fermentation in *E. coli*, allowing for the production and purification of large quantities of DNA. Second, they are safe and suitable for use in human patients and animals. The first requirement necessitates high copy number plasmids that can be selected relatively easily and remain stable during bacterial fermentation. The second requirement necessitates attention to elements such as selectivity markers and other coding sequences. In some embodiments, plasmids encoding markers consist of: (1) a high copy number origin of replication, (2) a selectivity marker, such as, but not limited to, a neo gene selected for antibiotic use with kanamycin, (3) a transcription termination sequence, including a tyrosinase enhancer, and (4) a multiple cloning site for incorporation into various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operablely linked to a tyrosinase promoter. Many plasmid vectors are known in the art for inducing nucleic acids encoding proteins. These include, but are not limited to, the carriers disclosed in U.S. Patent Nos. 6,103,470, 7,598,364, 7,989,425, and 6,416,998, which are incorporated herein by reference.

[0084] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Free gene delivery systems can be plasmids, free vectors based on Epstein-Barr virus (EBV), yeast-based vectors, adenovirus-based vectors, free vectors based on simian virus 40 (SV40), bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0085] The markers include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase or alkaline phosphatase or firefly / renen luciferase or nanoluciferase), or other proteins. The markers can be proteins (including secreted, cell surface, or internal proteins; synthetic or taken up by the cell); nucleic acids (e.g., mRNA or enzyme-active nucleic acid molecules); or polysaccharides. This includes determinants of any such cellular components that are specific to the cell type of interest and can be detected by antibodies, lectins, probes, or nucleic acid amplification reactions. The markers can also be identified by biochemical or enzymatic assays or biological reactions depending on the function of the gene product. The nucleic acid sequences encoding these markers can be operatively linked to tyrosinase enhancers. Additionally, other genes may be included, such as those that may affect stem cell differentiation into RPE or RPE function, physiology, or pathology. Therefore, in some implementations, the nucleic acid encoding one or more of the following is included: MITF, PAX6, TFEC, OTX2, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOLV4, ADAM10, CD55, CD59, and ARMS2.

[0086] 1. MHC haplotype matching

[0087] The major histocompatibility complex (MHC) is a primary cause of immune rejection in allogeneic organ transplants. There are three major class I MHC haplotypes (A, B, and C) and three major class II MHC haplotypes (DR, DP, and DQ). The HLA locus is highly polymorphic and located on 4 Mb of chromosome 6. The ability to haplotype HLA genes within this region is clinically important because this region is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donor and recipient can influence clinical transplant outcomes. HLA corresponding to class I MHC presents peptides intracellularly, while HLA corresponding to class II MHC presents antigens extracellularly to T lymphocytes. MHC haplotype incompatibility between graft and host triggers an immune response against the transplant and leads to its rejection. Therefore, immunosuppressants can be used to treat patients to prevent rejection. HLA-matched stem cell lines can overcome the risk of immune rejection.

[0088] Due to the importance of HLA in transplantation, HLA loci are typically identified through serological and PCR typing to determine favorable donor-recipient pairs. Serological testing for HLA class A and class II antigens can be performed using complement-mediated lymphocytotoxicity assays utilizing purified T or B lymphocytes. This procedure is primarily used to match HLA-A and -B loci. Molecular-based tissue typing is generally more accurate than serological testing. Low-resolution molecular methods such as SSOP (Sequence-Specific Oligonucleotide Probe) methods (where PCR products are tested against a series of oligonucleotide probes) can be used to identify HLA antigens, and these methods are currently the most commonly used for class II HLA typing. High-resolution techniques such as SSP (Sequence-Specific Primer) methods (which use allele-specific primers for PCR amplification) can identify specific MHC alleles.

[0089] If the donor cells are HLA homozygous, meaning they contain the same alleles for every antigen-presenting protein, MHC compatibility between the donor and recipient is significantly increased. Most individuals are heterozygous for MHC classes I and II genes, but some are homozygous for these genes. These homozygous individuals can serve as super-donors, and grafts produced from their cells can be transplanted into all individuals homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a high frequency of that haplotype in the population, these cells can be used for transplant therapy on a large number of individuals.

[0090] Therefore, iPSCs can be generated from somatic cells of the subject to be treated or from another subject with the same or substantially the same HLA type as the patient. In one case, the donor's major HLA (e.g., the three major loci of HLA-A, HLA-B, and HLA-DR) is identical to the recipient's major HLA. In some cases, the somatic cell donor may be a super-donor; thus, iPSCs derived from a homozygous MHC super-donor can be used to generate RPE cells. iPSCs from a super-donor can then be transplanted into a subject who is homozygous or heterozygous for that haplotype. For example, iPSCs may be homozygous at two HLA alleles, such as HLA-A and HLA-B. Thus, iPSCs generated from a super-donor can be used in the methods disclosed herein to generate RPE cells that can potentially "match" a large number of potential recipients.

[0091] 2. Free carrier

[0092] In some respects, reprogramming factors are expressed from expression cassettes contained in one or more exogenous free genetic elements (see U.S. Patent Publication 2010 / 0003757, incorporated herein by reference). Thus, iPSCs can be substantially free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared using extrachromosomal replication vectors (i.e., free vectors) that are capable of free replication so that the iPSCs are substantially free of exogenous vectors or viral elements (see U.S. Patent No. 8,546,140, ​​incorporated herein by reference; Yu et al., 2009). Many DNA viruses, such as adenoviruses, simian vacuolating virus 40 (SV40), bovine papillomavirus (BPV), or budding yeast ARS (autonomous replication sequence), contain plasmids that replicate extrachromosomally or free in mammalian cells. These free plasmids inherently do not have all of these disadvantages associated with integration vectors (Bode et al., 2001). For example, lymphoherpesvirus-based vectors, including Epstein-Barr virus (EBV) as defined above, can replicate extrachromosomally and help deliver reprogrammed genes into somatic cells. Useful EBV elements are OriP and EBNA-1, or variants or functional equivalents thereof. Another advantage of free vectors is that exogenous elements are lost over time after being introduced into cells, resulting in self-sustaining iPSCs being essentially devoid of these elements.

[0093] Other extrachromosomal vectors include other lymphoherpesvirus-based vectors. Lymphoherpesviruses are herpesviruses that replicate in lymphoblasts (such as human B lymphoblasts) and become plasmids in part of their natural life cycle. Herpes simplex virus (HSV) is not a “lymphotrophic” herpesvirus. Exemplary lymphoherpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); squirrel monkey herpesvirus (HSV); and Marek's disease virus (MDV). Other sources of isolate-based vectors are also envisioned, such as yeast ARS, adenovirus, SV40, or BPV.

[0094] C. Somatic cell nuclear transfer

[0095] Pluripotent stem cells can be prepared via somatic cell nuclear transfer. Somatic cell nuclear transfer involves transferring a donor cell nucleus into a non-spindle oocyte. In one approach, a donor fibroblast nucleus derived from rhesus monkey skin fibroblasts is introduced into the cytoplasm of a mid-mature II rhesus macaque oocyte from the non-spindle stage via electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to inosine and then incubated until the blastocyst stage. The inner cell mass of selected blastocysts is then cultured to generate embryonic stem cell lines. These embryonic stem cell lines exhibit normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0096] III. Retinal pigment epithelial cells

[0097] The method disclosed herein generates RPE cells. The cells in the retina that are directly sensitive to light are photoreceptor cells. Photoreceptors are photoreceptor neurons located outside the retina; they can be rod-shaped or cone-shaped. During phototransduction, photoreceptor cells convert the energy of incident light focused by a lens into electrical signals, which are then transmitted to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells: cone and rod cells. Cone cells are suited for detecting details, central and color vision, and function normally in bright light. Rod cells are responsible for peripheral and dim light vision. Neural signals from rod and cone cells are processed by other neurons in the retina.

[0098] The retinal pigment epithelium (RPE) acts as a barrier between blood flow and the retina and interacts closely with the photoreceptors in maintaining visual function. RPE cells consist of a single layer of hexagonal cells (comprised of tightly packed melanin granules that absorb light energy reaching the retina). The main functions of these specialized RPE cells include: transporting nutrients such as glucose, retinol, and fatty acids from the blood to the photoreceptors; transporting water, metabolic end products, and ions from the subretinal space to the blood; absorbing light and preventing photo-oxidation; the re-isomerization of all-trans retinol to 11-cis-retinaldehyde; phagocytosis of the shed photoreceptor membrane; and secreting various essential factors for the structural integrity of the retina.

[0099] Retinal pigment epithelial cells express markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, Best's vitrectomyces macular dystrophy gene (VMD2), and pigment epithelial cell-derived factor (PEDF). Retinal pigment epithelial dysfunction is associated with many visual impairment conditions, such as retinal pigment epithelial detachment, developmental abnormalities, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy, or macular degeneration.

[0100] Retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually identified by their cobblestone morphology and initial appearance of pigmentation. Additionally, differentiated RPE cells possess transepithelial resistance (TER) and transepithelial potential (TEP) across the monolayer (TER > 100 Ω·cm²; TEP > 2 mV), transport fluids and CO₂ from the apical to the basal side, and regulate the polarization secretion of cytokines.

[0101] Retinal pigment epithelium (RPE) cells express several proteins that can be used as markers for detection using methods such as immunocytochemistry, Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers may include: cellular retinaldehyde-binding protein (CRALBP), microocular-associated transcription factor (MITF), tyrosinase-associated protein 1 (TYRP-1), retinal pigment epithelium-specific 65 kDa protein (RPE65), promelanosome protein (PMEL17), bestrophin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-2. Specifically, when assessed by quantitative RT-PCR, the expression of these genes in ES cells or iPSC cells is approximately 100–1000 times higher than that in RPE cells.

[0102] Sequence-specific primers can be used in standard amplification methods, such as reverse transcriptase polymerase chain reaction (RT-PCR), Northern blotting, or dot blot hybridization, to utilize publicly available sequence data (GENBANK). RPE cell markers were detected at the mRNA level. Expression of tissue-specific markers detected at the protein or mRNA level was considered positive if the protein or mRNA level was at least 2, 3, 4, 5, 6, 7, 8, or 9 times higher than that of control cells such as undifferentiated pluripotent stem cells or other unrelated cell types, and more particularly 10, 20, 30, 40, 50 times or higher.

[0103] Dysfunction, damage, and loss of retinocytes (RPE cells) are contributing factors to many eye diseases and conditions, including age-related macular degeneration (AMD), hereditary macular degeneration (including Best's disease), and retinitis pigmentosa. A potential treatment for these diseases is to transplant RPE cells into the retina of individuals who require this treatment. It is hypothesized that supplementing RPE cells through transplantation could delay, halt, or reverse degeneration, improve retinal function, and prevent blindness caused by this condition. However, obtaining RPE cells directly from human donors and embryos presents a challenge.

[0104] A. RPE cells derived from the embryoids of PSCs

[0105] iPSCs reprogrammed with well-known reprogramming factors can generate ocular cells of neuronal lineages, including RPE cells (Hirami et al., 2009). Such a method is disclosed in PCT Publication No. 2014 / 121077, which is incorporated herein by full reference, in which embryoids (EBs) derived from iPSCs are treated in suspension culture with Wnt and Nodal antagonists to induce the expression of retinal progenitor cell markers. This publication discloses a method for deriving RPE cells from iPSCs by differentiating iPSC EBs into cultures highly enriched with RPE cells. For example, embryoids are generated from iPSCs by adding a rho-associated coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. Furthermore, EBs are plated in a second medium in a MATRIGEL medium. TM On the coated tissue cultures, the second medium did not contain basic fibroblast growth factor (bFGF), contained a Nodal pathway inhibitor, contained approximately 20 ng to approximately 90 ng of head protein, and contained approximately 1% to approximately 5% knockout serum substitute to form differentiated RPE cells. The differentiated RPE cells were cultured in a third medium containing ACTIVIN and WNT3a. The RPE cells were then cultured in RPE medium containing approximately 5% fetal serum, a typical WNT inhibitor, an atypical WNT inhibitor, and Sonic Hedgehog and an FGF pathway inhibitor to generate human RPE cells.

[0106] Using EBs to generate differentiated cell types has several drawbacks. For example, EB generation is an inconsistent and non-reproducible process, varying in efficiency. EBs generated from iPSCs or ES cells are not uniform in size and shape, and EB generation also involves rate-limited centrifugation. This disclosure provides a method for the large-scale production of iPSC- or ES-derived cells independent of EBs for clinical, research, or therapeutic applications.

[0107] B. RPE cells derived from essentially single-cell PSCs

[0108] In some embodiments, methods are provided for generating RPE cells from a suspension of essentially single cells of pluripotent stem cells (PSCs) such as human iPSCs. In some embodiments, the PSCs are cultured before confluence to prevent any cell aggregates. In some aspects, this is achieved by using, for example, trypsin... TM Or Tryple TMThe exemplified cell dissociation enzyme incubation is used to dissociate PSCs. PSCs can also be dissociated into a suspension of essentially single cells by pipetting. Alternatively, Blebbistatin (e.g., about 2.5 μM) can be added to the culture medium to increase PSC survival after dissociation into single cells, while preventing cell adhesion to the culture vessel. ROCK inhibitors, as alternatives to Blebbistatin, can be used to increase PSC survival after dissociation into single cells.

[0109] To effectively distinguish between RPE cells and single-cell PSCs, accurate counting of input density can increase RPE differentiation efficiency. Therefore, single-cell suspensions of PSCs are typically counted before inoculation. This is done, for example, using a hematology counter or an automated cell counter (such as VICELL). Single-cell suspensions of PSCs (or TC20) can be used. Cells can be diluted to a density of approximately 10,000 to 500,000 cells / mL, approximately 50,000 to 200,000 cells / mL, or approximately 75,000 to 150,000 cells / mL. In a non-limiting example, in a fully defined culture medium such as ESSENTIAL 8... TM (E8 TM The single-cell suspension of PSCs was diluted in the culture medium to a density of approximately 100,000 cells / mL.

[0110] Once a single-cell suspension of PSCs is obtained at a known cell density, the cells are typically seeded into suitable culture containers, such as tissue culture plates like flasks, 6-well, 24-well, or 96-well plates. Culture containers used for culturing cells can include, but are not limited to: flasks, flasks for tissue culture, dishes, culture dishes, dishes for tissue culture, multi-dish, microplates, microplates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, and cell stacks. Chambers, culture bags, and roller flasks, as long as they are capable of culturing stem cells within them. Depending on the culture requirements, cells can be cultured in volumes of at least 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derived thereof. In one embodiment, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a bioactive environment to allow cells to proliferate. The volume of the bioreactor can be at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters or any range derived therefrom.

[0111] In some respects, PSCs such as iPSCs are plated at cell densities suitable for efficient differentiation. Typically, cells are plated at a density of approximately 1,000 to approximately 75,000 cells / cm². 2 For example, approximately 5,000 to approximately 40,000 cells / cm² 2 Cell density seeding. In a 6-well plate, cells can be seeded at a cell density of approximately 50,000 to approximately 400,000 cells per well. In exemplary methods, cells are seeded at cell densities of approximately 100,000, approximately 150,000, approximately 200,000, approximately 250,000, approximately 300,000, or approximately 350,000 cells per well, for example, approximately 200,000 cells per well.

[0112] PSCs, such as iPSCs, are typically cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. Preferred cell adhesion proteins include, for example, extracellular matrix proteins such as fibronectin, laminin, collagen, and / or fibronectin, which can be used to coat culture surfaces as a means of providing solid support for pluripotent cell growth. The term "extracellular matrix" is well known in the art. Its components include one or more of the following proteins: fibronectin, laminin, fibronectin, tendin, nestin, platelet-reactive protein, elastin, gelatin, collagen, fibrin, layering protein, ankyrin, chondrone-adhesion protein, connexin, osteosialin, osteocalcin, osteopontin, epineclin, hyalin, crude fibroregulatory protein, epidermal integrin, and hindbrain protein. In exemplary methods, PSCs are grown on culture plates coated with fibronectin or fibronectin. In some embodiments, the cell adhesion proteins are human proteins.

[0113] Extracellular matrix (ECM) proteins can be of natural origin and purified from human or animal tissues, or they can be genetically engineered recombinant proteins or naturally synthesized proteins. ECM proteins can be complete proteins or in the form of natural or engineered peptide fragments. Examples of ECM proteins that can be used in cell culture substrates include laminin, collagen I, collagen IV, fibronectin, and fibronectin. In some embodiments, the matrix composition comprises a peptide fragment of synthetically produced fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is heterologous. For example, in a heterologous-free matrix for culturing human cells, human-derived matrix components can be used, where any non-human animal components can be excluded.

[0114] In some aspects, the total protein concentration in the matrix composition can be from about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is from about 1 μg / mL to about 300 μg / mL. In a more preferred embodiment, the total protein concentration in the matrix composition is from about 5 μg / mL to about 200 μg / mL.

[0115] Cells such as RPE cells or PSCs can be cultured with the nutrients necessary to support the growth of each specific cell population. Typically, cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain the pH. The medium may also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, and inorganic salts. Exemplary growth media contain minimally invasive media supplemented with various nutrients, such as Dulbecco Modified Eagle Medium (DMEM) or ESSENTIAL 8. TM (E8 TM Culture media are used to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Eagle (MEM) Alpha medium, Dulbecco modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. Additionally, minimal essential media may be supplemented with additives such as equine, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, growth media may contain “knockout serum substitutes,” which herein refers to serum-free formulations optimized for the growth and maintenance of undifferentiated cells, such as stem cells, in cultures. For example, KNOCKOUT is disclosed in U.S. Patent Application No. 2002 / 0076747. TM Serum substitutes, this application is incorporated herein by reference. Preferably, PSCs are cultured in a fully defined and feeder-free medium.

[0116] Therefore, single-cell PSCs are typically cultured in a fully defined medium after plating. In some cases, approximately 18-24 hours after inoculation, the medium is aspirated and replaced with fresh medium such as E8. TM Culture medium is added to the culture. In some respects, single-cell PSCs are cultured in a fully defined culture medium for about 1, 2, or 3 days. Preferably, single-cell PSCs are cultured in a fully defined culture medium for about 2 days before differentiation.

[0117] In some embodiments, the culture medium may or may not contain any alternative to serum. Serum alternatives may include suitable albumin-containing materials (e.g., lipid-rich albumin, albumin alternatives such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. For example, serum alternatives can be prepared by the method disclosed in International Publication No. WO 98 / 30679. Alternatively, any commercially available material can be used more conveniently. Commercially available materials include KNOCKOUT. TM Serum substitutes (KSR), chemically determined concentrated lipids (Gibco), and GLUTAMAX TM (Gibco).

[0118] Other culture conditions may be appropriately defined. For example, the culture temperature may be about 30 to 40°C, such as at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39°C, but is not specifically limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration may be about 1 to 10%, such as about 2 to 5%, or any range therefrom. The oxygen tension may be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range therefrom.

[0119] a. Differentiation culture medium

[0120] retinal induction culture medium

[0121] After single-cell PSCs adhere to the culture plate, the cells are preferably cultured in retinal induction medium to initiate the differentiation into retinal lineage cells. Retinal induction medium (RIM) contains WNT pathway inhibitors and induces PSC differentiation into retinal lineage cells. RIM also contains TGFβ pathway inhibitors and BMP pathway inhibitors. An exemplary RIM medium is shown in Table 3.

[0122] The RIM may include DMEM and F12 in approximately a 1:1 ratio. In an exemplary method, the RIM contains a WNT pathway inhibitor, such as CKI-7, a BMP pathway inhibitor, such as LDN193189, and a TGFβ pathway inhibitor, such as SB431542. For example, the RIM contains approximately 5 nM to approximately 50 nM, such as approximately 10 nM, of LDN193189, approximately 0.1 μM to approximately 5 μM, such as approximately 0.5 μM, of CKI-7, and approximately 0.5 μM to approximately 10 μM, such as approximately 1 μM, of SB431542. Additionally, the RIM may contain knockout serum substitutes, such as approximately 1% to approximately 5% of MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin-like growth factor 1 (IGF1). Preferably, the IGF1 is animal-free IGF1 (AF-IGF1) and is contained in the RIM at a concentration of about 0.1 ng / mL to about 10 ng / mL, such as about 1 ng / mL. The culture medium is aspirated daily and replaced with fresh RIM. Cells are typically cultured in the RIM for about 1 to about 5 days, such as about 1, 2, 3, 4 or 5 days, such as about 2 days, to generate retinal lineage cells.

[0123] retinal differentiation culture medium

[0124] Retinal lineage cells can then be cultured in retinal differentiation medium (RDM) for further differentiation. RDM contains inhibitors of the WNT pathway, BMP pathway, TGFβ pathway, and MEK. In one embodiment, RDM contains an inhibitor of the WNT pathway, such as CKI-7, an inhibitor of the BMP pathway, such as LDN193189, an inhibitor of the TGFβ pathway, such as SB431542, and an inhibitor of MEK, such as PD0325901. Alternatively, RDM may contain inhibitors of the WNT pathway, BMP pathway, TGFβ pathway, and bFGF. Typically, the concentrations of WNT pathway inhibitors, BMP pathway inhibitors, and TGFβ pathway inhibitors in RDM are higher than those in RIM, for example, about 9 to about 11 times, or for example, about 10 times, the concentrations in RIM. In an exemplary method, the RDM contains about 50 nM to about 200 nM of LDN193189, such as about 100 nM; about 1 μM to about 10 μM of CKI-7, such as about 5 μM; about 1 μM to about 50 μM of SB431542, such as about 10 μM; and about 0.1 μM to about 10 μM of PD0325901, such as about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM. An exemplary RDM culture medium is shown in Table 3.

[0125] Typically, RDM contains approximately a 1:1 ratio of DMEM and F12, knockout serum substitute (e.g., approximately 1% to approximately 5%, such as approximately 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., approximately 1 ng / mL to approximately 50 ng / mL, such as approximately 10 ng / mL). In certain methods, cells are given fresh RDM daily after aspirating the culture medium from the previous day. Cells are typically cultured in RDM for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, such as approximately 7 days, to derive differentiated retinal cells.

[0126] retinal culture medium

[0127] Next, the differentiated retinal cells can be further differentiated by culturing them in retinal culture medium (RM). The retinal culture medium contains activator A and may additionally contain nicotinamide. The RM may contain about 50 to about 200 ng / mL, for example about 100 ng / mL, of activator A and about 1 mM to about 50 mM, for example about 10 mM, of nicotinamide. Alternatively, the RM may contain other TGF-β pathway activators, such as GDF1 and / or WNT pathway activators, such as WAY-316606, IQ1, QS11, SB-216763, BIO (6-bromoindorubin-3'-oxime) or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Alternatively, the RM may additionally contain WNT3a. An exemplary RM culture medium is shown in Table 3.

[0128] RM may contain approximately a 1:1 ratio of DMEM and F12, approximately 1% to approximately 5% (e.g., approximately 1.5%) of knockout serum substitutes, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The culture medium can be changed daily at room temperature in the RM. Cells are typically cultured in the RM for approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days, such as approximately 10 days, to derivatize differentiated RPE cells.

[0129] RPE Maturation Medium

[0130] To further differentiate RPE cells, it is preferable to culture the cells in RPE maturation medium (RPE-MM). Exemplary RPE-MM media are shown in Table 3. RPE maturation medium may contain approximately 100 μg / mL to approximately 300 μg / mL, for example, approximately 250 μg / mL taurine; approximately 10 μg / L to approximately 30 μg / L, for example, approximately 20 μg / L hydrocortisone; and approximately 0.001 μg / L to approximately 0.1 μg / L, for example, approximately 0.013 μg / L triiodothyronine. Additionally, RPE-MM may contain MEMα, N-2 supplement, MEM non-essential amino acids (NEAA), and sodium pyruvate, as well as fetal bovine serum (e.g., approximately 0.5% to approximately 10%, for example, approximately 1% to approximately 5%). The medium may be changed every other day using room temperature RPE-MM. Cells are typically cultured in RPE-MM for approximately 5 to approximately 10 days, e.g., approximately 5 days. The cells can then be dissociated, for example, using a cell dissociation enzyme, and then seeded and cultured for an additional period of time, such as approximately 5 to 30 days, or for example, approximately 15 to 20 days, to further differentiate into RPE cells. In a further embodiment, RPE-MM does not include a WNT pathway inhibitor. RPE cells can be cryopreserved at this stage.

[0131] b. Maturation of RPE cells

[0132] RPE cells can then be cultured in RPE-MM for a period of time to mature. In some embodiments, RPE cells are grown in the wells of plates such as 6-well, 12-well, 24-well, or 10 cm plates. RPE cells can be maintained in RPE medium for about 4 to about 10 weeks, for example, about 6 to 8 weeks, such as 6, 7, or 8 weeks. In an exemplary method for the continuous maturation of RPE cells, the cells can be cultured with cell-dissociating enzymes such as TRYPLE. TM Dissociation in the middle, and on biodegradable scaffold components, such as in specialized SNAPWELL. TM The design involves reseeding RPE-MM cells with a MEK inhibitor such as PD0325901 for approximately 1 to 2 weeks. Alternatively, RPE-MM cells may contain a bFGF inhibitor instead of a MEK inhibitor. A method for culturing RPE cells on a degradable scaffold is taught and described in PCT Publication No. WO2014 / 121077, which is incorporated herein by reference in its entirety. Briefly, the main component of this method is CORNING. COSTAR SNAPWELL TM Plates, bio-inert O-rings, and biodegradable scaffolds. SNAPWELL TM The plate provides the structure and platform for the biodegradable scaffold. Creating microporous membranes on the top and base sides is ideal for providing support to the scaffold and isolating different sides of the cell's polarization layer. SNAPWELLTM The ability of the insert to separate the membrane allows the insert's support rings to be used as anchors for the scaffold. The resulting differentiated, polarized, and confluent functional RPE cell monolayers can be cryopreserved at this stage (e.g., in heterologous CS10 medium).

[0133] In some implementations, mature RPE cells can be further developed into a functional RPE cell monolayer, exhibiting intact RPE tissue, by continuing culture in an RPE-MM containing other chemicals or small molecules that promote RPE maturation. For example, these small molecules are primary ciliate inducers such as prostaglandin E2 (PGE2) or afedipine. PGE2 can be added to the culture medium at a concentration of about 25 μM to about 250 μM, for example, about 50 μM to about 100 μM. Alternatively, the RPE-MM can contain typical WNT pathway inhibitors. Typical inhibitors of the WNT pathway are N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylene-2H-isoindol-2-yl)-N-8-quinolinylbenzamide (endo-IWR1). Cells can be cultured in this medium for a further period, for example, approximately 1 to 5 weeks, or approximately 2 to 4 weeks, to obtain a mature and functional RPE cell monolayer. Therefore, the method disclosed herein provides mature RPE cells from a single-cell suspension of pluripotent cells, which can be consistently regenerated on a large scale for clinical applications.

[0134] c. Cryopreservation of RPE cells

[0135] Retinal pigment epithelial cells generated by the methods disclosed herein can be cryopreserved, see, for example, PCT Publication No. 2012 / 149484A2, which is incorporated herein by reference. Cells can be cryopreserved with or without a substrate. In several embodiments, storage temperatures are about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges thereof. In some embodiments, lower temperatures are used for the storage (e.g., maintenance) of cryopreserved cells. In several embodiments, liquid nitrogen (or other similar liquid coolant) is used to store the cells. In a further embodiment, cell storage is for more than about 6 hours. In another embodiment, cell storage is for about 72 hours. In several embodiments, cell storage is for 48 hours to about one week. In other embodiments, cell storage is for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In a further embodiment, cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Cells can also be stored for longer periods. Cells can be cryopreserved separately or cryopreserved on a substrate, such as any of the substrates disclosed herein.

[0136] In some embodiments, additional cryoprotectants may be used. For example, cells may be cryopreserved in a cryopreservation solution containing one or more cryoprotectants such as DM80, or serum albumin such as human or bovine serum albumin. In some embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In a specific embodiment, the solution contains 2.5% DMSO. In another specific embodiment, the solution contains 10% DMSO.

[0137] For example, cells can be cooled at about 1°C per minute during cryopreservation. In some embodiments, the cryopreservation temperature is about -80°C to about -180°C, or about -125°C to about -140°C. In some embodiments, cells are cooled to 4°C and then cooled at about 1°C per minute. Cryopreserved cells can be transferred to the gas phase of liquid nitrogen before thawing and use. In some embodiments, for example, once the cells reach about -80°C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be performed using a cryostat with a controlled rate. Cryopreserved cells can be thawed, for example, at a temperature of about 25°C to about 40°C, typically at a temperature of about 37°C.

[0138] d. Inhibitors

[0139] WNT pathway inhibitors

[0140] WNTs are a highly conserved family of secreted signaling molecules that regulate cell-cell interactions and are associated with the Drosophila segmental polar gene wingless. In humans, the WNT gene family encodes 38–43 kDa cysteine-rich glycoproteins. WNT proteins possess hydrophobic signaling sequences, conserved asparagine-linked oligosaccharide co-sequences (see, for example, Shimizu et al., Cell Growth Differ 8: 1349–1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote cytoplasmic β-catenin stabilization, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of specific WNT proteins has been shown to be associated with certain cancers.

[0141] The term "WNT inhibitor" in this document refers to WNT inhibitors in general. Therefore, a WNT inhibitor is any inhibitor of WNT family protein members, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16. Some embodiments of the methods of this invention relate to WNT inhibitors in differentiation media. Examples of suitable WNT inhibitors known in the art include N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]acetamide (IWP4), and 2-phenoxybenzoic acid [(5-methyl-2-furanyl)methylene]hydrazide (PNU) 74654) 2,4-Diamino-quinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiaro[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH 535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridyl]benzamide (TAK 715), Dickkopf-associated protein 1 (DKK1), and secretory coil-associated protein 1 (SFRP1). Furthermore, WNT inhibitors can include antibodies against WNT, dominant-negative variants of WNT, and siRNAs and antisense nucleic acids that inhibit WNT expression. Inhibition of WNT can also be achieved using RNA-mediated interference (RNAi).

[0142] BMP pathway inhibitors

[0143] Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily. BMPs are considered to constitute a key set of morphogenetic signals that coordinate the entire body's architecture. The important physiological functions of BMP signaling are highlighted by the various roles of dysregulated BMP signaling in pathological processes.

[0144] BMP pathway inhibitors typically include general BMP signaling inhibitors or inhibitors specific to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Exemplary BMP inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidin dihydrochloride (Dorsomorphin), 4-[6-[4-[1-methylethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347).

[0145] TGFβ pathway inhibitors

[0146] Transforming growth factor β (TGFβ) is a secreted protein that controls proliferation, cell differentiation, and other functions in most cells. It is a cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-β exists in at least three isoforms, called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a protein superfamily known as the transforming growth factor β superfamily, which includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1.

[0147] Generally, TGFβ pathway inhibitors can include any inhibitor of TGFβ signaling. For example, TGFβ pathway inhibitors include 4-[4-(1,3-benzo[1,3-dioxacyclopenten-5-yl]-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridyl)-1H-imidazol-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride. Salt hydrate (SB-505124), 4-(5-benzo[1,3]dioxacyclopenten-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxacyclopenten-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Lefty factor, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide (A) 83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide (D 4476), 4-[4-[3-(2-pyridyl)-1H-pyrazol-4-yl]-2-pyridyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-pyridyl)-1H-pyrazol-4-yl]-quinoline (LY 364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazol-1-ethanol (R 268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-diazanaphthalene (RepSox).

[0148] MEK inhibitors

[0149] MEK inhibitors are chemicals or drugs that inhibit mitogen-activated protein kinases MEK1 or MEK2. They can be used to affect the MAPK / ERK pathway. Examples of MEK inhibitors include N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyridino[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), and 6-(4-bromo-2-fluoroaniline) 6-(2-hydroxyethoxy)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119) and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).

[0150] bFGF inhibitors

[0151] Basic fibroblast growth factor (also known as bFGF, FGF2, or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the basement membrane and the subendothelial extracellular matrix of blood vessels. Additionally, bFGF is a common component of human ESC culture medium, in which it is essential for maintaining cells in an undifferentiated state.

[0152] The term "bFGF inhibitor" in this article refers to general bFGF inhibitors. For example, bFGF inhibitors include, but are not limited to, N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), and 1-tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido [2,3-d]pyrimidin-7-yl]urea (PD161570), 6-(2,6-dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one dihydrochloride (PD166285), N-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD166866) and MK-2206.

[0153] IV. Uses of retinal pigment epithelial cells

[0154] Some aspects provide methods for generating RPE or RPE-rich cell populations that can be used for many important research, development, and commercial purposes.

[0155] In some respects, the methods disclosed herein result in at least or about 10 6 10 7 10 8 5x10 8 10 9 10 10 A cell population of 1,000 cells (or any range thereof) contains at least or about 90% (e.g., at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or any range thereof) RPE cells.

[0156] In some aspects, the starting cells used in the method of the present invention may include at least or about 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 Individual cells or any range from which they can be derived. The starting cell population may have at least or about 10,100 cells. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Inoculation density of cells / ml or any range thereof.

[0157] RPE cells generated by the methods disclosed herein can be used in any method and application of RPE cells currently known in the art. For example, methods for evaluating compounds can be provided, including determining the pharmacological or toxicological properties of the compounds on RPE cells. Methods for evaluating the effects of compounds on RPE cells can also be provided, comprising: a) contacting the RPE cells provided herein with the compound; and b) determining the effects of the compound on the RPE cells.

[0158] A. Screening of test compounds

[0159] RPE cells can be commercially used to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of these cells and their various progeny. For example, the test compound can be a chemical compound, a small molecule, a peptide, a growth factor, a cytokine, or other biological factor.

[0160] In one implementation, the method includes contacting RPE cells with a test agent and determining whether the test agent modulates the activity or function of RPE cells within a population. In some applications, screening assays are used to identify agents that regulate RPE cell proliferation or alter RPE cell differentiation. Screening assays can be performed in vitro or in vivo. Methods for screening and identifying ophthalmic agents or RPE agents include those suitable for high-throughput screening. For example, RPE cells can be positioned or placed in culture dishes, flasks, roller flasks, or culture plates (e.g., single multiwell culture dishes or plates such as 8, 16, 32, 64, 96, 384, and 1536-well multiwell culture plates or dishes), optionally in a defined location, for the identification of potential therapeutic molecules. Screenable libraries include, for example, small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0161] Other screening applications involve testing the effects of drug compounds on the maintenance or repair of retinal tissue. This screening is conducted because compounds are designed to have pharmacological effects on cells, or because compounds designed to have other effects may have unintended side effects on this type of tissue.

[0162] B. Treatment and Transplantation

[0163] Other implementation schemes can also provide RPE cells to enhance the maintenance and repair of eye tissues for any condition requiring it, including retinal degeneration or significant damage.

[0164] To determine the suitability of the cell composition for therapeutic application, the cells can first be tested in a suitable animal model. On one hand, the ability of RPE cells to survive in vivo and maintain their phenotype is assessed. The cell composition is administered to immunodeficient animals (e.g., nude mice or animals with immunodeficiency induced chemically or by radiation). After a period of growth, tissue is harvested, and the presence of pluripotent stem cell-derived cells is evaluated.

[0165] Many animals can be used to test the suitability of RPE cell compositions. For example, the Royal College of Surgeons (RCS) rat is a well-known model of retinal dystrophy (Lund et al., 2006). Additionally, RPE cell fitness and survival can be determined by transplantation (e.g., subcutaneously or subretinal) into matrix gels in immunodeficient animals such as NOG mice (Kanemura et al., 2014).

[0166] The human RPE cells described herein, or pharmaceutical compositions containing these cells, can be used to manufacture remedies for patients with this need. RPE cells can be cryopreserved in advance. In some aspects, the disclosed RPE cells are derived from iPSCs and can therefore be used to provide “personalized medicine” for patients with eye diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations, differentiate into RPEs, and engineered to form RPE tissue. This RPE tissue can be used to replace endogenously degenerated RPEs from the same patient. Alternatively, iPSCs derived from healthy donors or HLA-homozygous “super donors” can be used. RPE cells can be treated in vitro with certain factors such as pigment epithelial-derived factor (PEDF), transforming growth factor (TGF)-β, and / or retinoic acid to create an anti-inflammatory and immunosuppressive environment in vivo.

[0167] Introducing RPE cells obtained using the methods disclosed herein can treat or prevent a variety of ocular conditions. Conditions include retinal diseases or conditions commonly associated with retinal dysfunction or degradation, retinal damage, and / or loss of retinal pigment epithelium. Treatable conditions include, but are not limited to, degenerative retinal diseases such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration (such as age-related macular degeneration), glaucoma, and diabetic retinopathy. Other conditions include Lebers' congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, rotator cholestasis, choroidal dystrophy, pattern dystrophy, other RPE malnutrition, and RPE and retinal damage caused by any of light, laser, inflammation, infection, radiation, neovascularization, or traumatic injury. In some embodiments, methods for treating or preventing conditions characterized by retinal degeneration are provided, comprising administering an effective amount of a composition containing RPE cells to a subject in need. These methods may include selecting a subject with one or more of these conditions and administering a therapeutically effective amount of RPE cells sufficient to treat the condition and / or improve its symptoms. RPE cells can be transplanted in various forms. For example, RPE cells can be introduced to the target site in the form of a cell suspension, or adhere to the matrix, extracellular matrix, or substrate such as a biodegradable polymer in monolayer or combination form. RPE cells can also be transplanted together with other retinal cells (co-transplantation), such as with a photoreceptor. In some embodiments, the RPE cells are generated from iPSCs of the subject to be treated, and are therefore autologous. In other embodiments, the RPE cells are generated from an MHC-matched donor.

[0168] In some embodiments, RPE cells can be used as autologous RPE grafts for subjects suitable for regenerative medicine. RPE cells can be transplanted in combination with other retinal cells, such as photoreceptor cells. Transplantation of RPE cells generated by the disclosed methods can be performed using various techniques known in the art. For example, methods for performing RPE transplantation are described in U.S. Patent Nos. 5,962,027 and 6,045,791, each of which is incorporated herein by reference in its entirety. According to one embodiment, transplantation is performed via vitrectomy followed by delivery of cells to the subretinal space through a small retinal opening or by direct injection. RPE cells can be introduced to the target site in the form of a cell suspension, adhering to a matrix such as an extracellular matrix, or provided on a substrate such as a biodegradable polymer. RPE cells can also be transplanted together with other cells (co-transplantation), such as retinal cells with photoreceptors. Therefore, compositions comprising RPE cells obtained by the methods disclosed herein are provided. In some embodiments, these RPE cells include a tyrosinase enhancer operably linked to a promoter and a labeled nucleic acid. In other embodiments, the RPE cell also includes a second constitutive promoter operatively linked to a nucleic acid encoding a second label.

[0169] Pharmaceutical compositions for generating RPE cells using the methods disclosed herein. These compositions may include at least about 1 × 10 3 1 × 10 RPE cells, approximately 1 × 10 4 1 × 10 RPE cells, approximately 1 × 10 5 1 × 10 RPE cells, approximately 1 × 10 6 1 × 10 RPE cells, approximately 1 × 10 7 1 × 10 RPE cells, approximately 1 × 10 8 One RPE cell or approximately 1 × 10 9 RPE cells. In some embodiments, the composition is a substantially pure (as opposed to non-RPE cells) formulation comprising differentiated RPE cells produced by the methods disclosed herein. Compositions comprising a scaffold (e.g., a polymeric carrier and / or extracellular matrix) and an effective amount of RPE cells produced by the methods disclosed herein are also provided. For example, the cells are provided as a monolayer. The matrix material is generally physiologically acceptable and suitable for in vivo application. For example, physiologically acceptable materials include, but are not limited to, absorbable and / or non-absorbable solid matrix materials such as the small intestinal submucosa (SIS), cross-linked or uncross-linked alginate, hydrolyzed colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, wool, and bioadhesives.

[0170] Suitable polymer supports also include porous networks or sponges formed from synthetic or natural polymers, as well as polymer solutions. For example, the matrix may be a polymer network or sponge, or a polymer hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polymers of polysaccharides such as alginate and hyaluronic acid. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohol.

[0171] Polymers capable of forming stretchable ionicly or covalently cross-linked hydrogels can be used. Hydrogels are substances formed when organic polymers (natural or synthetic) are cross-linked via covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure that traps water molecules to form a gel. Examples of materials that can be used to form hydrogels include ionicly cross-linked polysaccharides such as alginate, polyphosphazene, and polyacrylate, or block copolymers such as PLURON1CS. TM Or TETRON1CS TM This is a cross-linked polyethylene oxide-polypropylene glycol block copolymer, produced by temperature or H2 cross-linking. Other materials include proteins such as fibroin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0172] Optionally, the pharmaceutical composition may be packaged in a suitable container with written instructions for the desired purpose, such as reconstructing RPE cell function to improve diseases or abnormalities in retinal tissue. In some embodiments, RPE cells generated by the methods of this disclosure may be engineered to form RPEs that can be used to replace degenerated RPEs in a subject with this need.

[0173] C. Allocation of funds for commercial, therapeutic, and research purposes

[0174] In some embodiments, a reagent system is provided comprising a group of cells or a combination of cells containing an RPE-enriched cell population present at any point during the manufacturing, dispensing, or use process. The cell assembly comprises any combination of the cell population described herein with undifferentiated pluripotent stem cells or other differentiated cell types, generally sharing the same genome. Each cell type may be packaged together at the same or different times, in the same facility or at different locations, under the control of the same or different entities with a shared commercial relationship, or packaged in different containers.

[0175] Optionally, the pharmaceutical composition may be packaged in a suitable container and have written instructions for use for the desired purpose, such as reconstructing RPE cell function to improve diseases or injuries of ocular tissues.

[0176] V. Reagent Kit

[0177] In some embodiments, a kit is provided that may include one or more culture media and components, for example, for generating RPE cells. Where appropriate, the reagent system may be packaged as an aqueous culture medium or lyophilized form. The container device of the kit will typically include at least one vial, test tube, flask, bottle, syringe, or other container device in which the components can be placed, and preferably suitably in aliquots. When the kit contains multiple components, it will typically also include a second, third, or other additional container in which other components can be placed individually. However, various combinations of components may be contained in vials. The components of the kit may be provided as a dry powder. When reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in another container device. These kits typically also include a device for tightly sealing the kit components for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials are retained. The kit may also include instructions for use, such as in printed or electronic formats, such as digital formats.

[0178] VI. Examples

[0179] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention, and therefore can be considered as constituting preferred modes of practice. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the disclosed specific embodiments and similar or analogous results can still be obtained without departing from the spirit and scope of the invention.

[0180] Example 1 - Preparation of the initial pluripotent stem cell population

[0181] The starting population of RPE cells can be derived from pluripotent stem cells, such as ES cells and iPSCs. In exemplary methods, RPE cells are derived from human iPSCs reprogrammed from somatic cells using methods known in the art, such as U.S. Patent Nos. 8,546,140, ​​8,741,648, 8,691,574, U.S. Patent Application Publication No. 20090246875, U.S. Patent No. 8,278,104, 9,005,967, 8,058,065, 8,129,187, PCT Publication No. WO 2007 / 069666A1, U.S. Patent Nos. 8,183,038, and 8,268,620, which are incorporated herein by reference. For example, nuclear programming factors Oct4, Sox2, c-Myc, and Klf4 are used to generate pluripotent stem cells from somatic cells. In another exemplary approach, pluripotent stem cells are generated from somatic cells using nuclear programming factors Oct4, Sox2, Nanog, Lin28, L-Myc, and the SV40 large T antigen.

[0182] Incubate iPSCs in a fully defined culture medium such as ESSENTIAL 8. TM (E8 TM In culture medium without mouse or human feeder layers, iPSCs were grown on plates coated with glassnein. Glassnein was diluted 1:200 in DPBS without calcium or magnesium, the culture plates were coated with glassnein, and incubated at room temperature for approximately 1 hour. iPSCs were separated before confluence and overgrowth was not permitted to prevent unhealthy and / or differentiated cells. Figure 1A ).

[0183] To derive RPE cells, iPSCs were dissociated into a single-cell suspension to remove any aggregates or embryoids. To obtain the single-cell suspension, cells were washed with DPBS and incubated at 37°C with a cell dissociation enzyme such as TRYPLE. TM Incubate for approximately 10 minutes. Then separate the cells using a serum pipette and collect the cell suspension in a conical tube. If the cells are not separated by gentle aspiration, the culture can be incubated for a longer time, such as 2-3 minutes. To collect all cells, sterilize at room temperature (E8). TM Wash the culture vessel with culture medium, then add the medium to the tube containing the cell suspension. Additionally, add Blebbistatin (e.g., 2.5 μM) to the E8. TM The culture medium was used to increase PSC survival after dissociation into single cells, while ensuring the cells did not adhere to the culture vessel. To collect the cells, they were centrifuged at 400×g for approximately 5 minutes, the supernatant was aspirated, and the cells were resuspended in an appropriate volume of E8 medium. TM In the culture medium.

[0184] To effectively distinguish RPE cells from single-cell iPSCs, automated cell counters such as VICELL are used. TM Accurately count the input density of single-cell iPSCs, and at room temperature E8 TM Dilute to approximately 1×10 in the culture medium 5 Cell suspension at 100 cells / mL. Once a single-cell suspension of iPSCs has been obtained at a known cell density, the cells are spread into suitable culture containers, such as 6-well plates coated with teicoplanin. Cells are seeded at a density of approximately 200,000 cells per well and placed in a humidified incubator at 37°C. After approximately 18–24 hours, the culture medium is aspirated and fresh E8 culture medium is introduced. TM Culture medium was added to the culture. After inoculation, the cells were cultured in E8 culture medium. TM Incubate in the culture medium for about 2 days to allow for proper adhesion to the plate.

[0185] Example 2 - Differentiating iPSCs into RPE cells

[0186] Once the single-cell iPSCs, seeded at an appropriate cell density as in Example 1, are cultured for approximately 2 days, they are cultured in various differentiation media to derive RPE cells. On day 3, E8 cells are aspirated. TM The culture medium was then added to room temperature retinal induction medium (RIM) (e.g., Table 3). In short, RIM contains approximately a 1:1 ratio of DMEM and F12, knockout serum substitutes, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. Additionally, RIM contains inhibitors of the WNT pathway, BMP pathway, TGFβ pathway, and insulin-like growth factor 1 (IGF1). The culture medium was aspirated daily, and fresh RIM was added to the cells. Cells were cultured in RIM for approximately 2 to 4 days.

[0187] Cells were then cultured in retinal differentiation medium (RDM) for approximately 7 to 14 days. In brief, RDM (Table 2) contained approximately a 1:1 ratio of DMEM and F12, knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. Additionally, RDM contained WNT pathway inhibitors (e.g., CKI-7), BMP pathway inhibitors (e.g., LDN193189), TGFβ pathway inhibitors (e.g., SB431542), and MEK inhibitors (e.g., PD325901). The concentrations of WNT, BMP, and TGFβ pathway inhibitors in RDM were 10 times higher than those in RIM. The medium was aspirated daily, and room-temperature RDM was added to the cells to generate differentiated retinal cells.

[0188] To derive RPE cells, the cells were then cultured in retinal medium (RM) for 7 to 10 days. RM contains approximately a 1:1 ratio of DMEM and F12, knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. Additionally, RM contains nicotinamide and activin A. The medium was changed daily with room temperature RM to generate RPE cells.

[0189] To mature RPE cells, they were cultured in RPE maturation medium (RPE-MM) for 5–10 days. RPE-MM (Table 2) contains MEMα, fetal bovine serum, N-2 supplement, MEM NEAA, and sodium pyruvate. Additionally, RPE-MM contains taurine, hydrocortisone, and 3,3',5-triiodo-L-thyroxine (TTL). Figure 1C The culture medium was changed every other day using room temperature RPE-MM. Cells were then dissociated with cell dissociation enzymes and reseeded onto polinecin-coated plates. At this stage, the derived PRE cells could be cryopreserved in heterologous CS10 medium. To continue RPE maturation, the plated cells were cultured for approximately 15 days.

[0190] Example 3 - Maturation of RPE cells

[0191] To allow the RPE cells generated in Example 2 to continue maturing, the cells are subjected to cell dissociation enzymes such as TRYPLE. TM Dissociation in the medium and in RPE-MM with MEK inhibitors such as PD325901 in a dedicated SNAPWELL TM Reseeding was performed on the designed biodegradable scaffold assembly for 1-2 weeks. This resulted in a differentiated, polarized, and confluent monolayer of functional RPE cells. Figure 1D It can be cryopreserved at this stage in heterologous CS10 medium.

[0192] Mature RPE cells further develop into a functional RPE cell monolayer by continued culture in RPE-MM containing additional small molecules such as primary ciliate inducers like PGE2 or afenidimycin, which function as intact RPE tissue. Unbound by theory, these primary ciliate inducers inhibit the classical WNT pathway, induce cell cycle exit in cells, and induce apical-basal polarization in the RPE monolayer. Alternatively, RPE maturation can be induced by typical WNT pathway inhibitors such as IWP2 and endo-IWR1, which also induce cell cycle exit in RPE cells to promote RPE maturation. Cells are then cultured in this medium for another 2–3 weeks to obtain a mature and functional RPE cell monolayer. Therefore, the method disclosed herein provides mature RPE cells derived from pluripotent cells that can be regenerated on a large scale and consistently for clinical applications.

[0193] Example 4 - Cryopreservation of RPE Cells

[0194] For the cryopreservation of differentiated RPE cells in Example 2, the culture medium was aspirated and the cells were washed twice with Dulbecco phosphate-buffered saline (DPBS). The cells were then incubated with cell dissociation enzymes, and the cell suspension was pipetted into conical tubes. The cells were centrifuged, the supernatant was aspirated, and the cells were resuspended in RPE-MM at room temperature. The cell suspension was then filtered through a STERIFLIP cell filter, and the cells were counted. Next, the cells were centrifuged and densified at an appropriate density (e.g., 1 × 10⁻⁶). 7 (cells / mL) resuspended in cold CryoStor In CS10, the cell suspension was aliquoted into pre-labeled cryovials, placed in a freezing container, and transferred to a -80°C freezer for 12–24 hours. The vials were then transferred to liquid nitrogen for storage.

[0195] Example 5 - MACS depletion of contaminated non-RPE cells and enrichment of the RPE cell starting population by depletion of CD24, CD56 and / or CD90

[0196] The RPE cell population obtained in Example 2 or 3 may contain residual contaminating non-RPE cells and immature RPE cells (collectively referred to as "contaminating cells"), both of which can be isolated and removed to produce a mature RPE-enriched cell population. This can be achieved through various methods such as magnetically activated cell sorting (MACS). Fluorescence-activated cell sorting (FACS) or single-cell sorting removes contaminating cells from the culture. MACS, known in the art for separating various cell populations based on their surface antigens, is used. The method separates contaminated cells from the desired, more mature RPE cells.

[0197] Contaminating cells in the RPE cell initiation population possess specific cell surface markers that can be used to isolate contaminating cells from the desired mature RPE cells. For example, CD24, CD56, and / or CD90 are cell surface antigens expressed on (but not limited to) pluripotent stem cells and other neural cell types. CD24 is a glycoprotein expressed on the surface of pluripotent stem cells, some B lymphocytes, and differentiated neuroblasts. CD56, or neural cell adhesion molecule (NCAM), is a glycoprotein expressed on the surface of neurons and natural killer cells. CD90, or Thy-1, is a marker expressed on the surface of various stem cells and neurons. The expression of CD24, CD56, and / or CD90 is lost during stem cell differentiation into many mature cell types, including RPE cells. Therefore, removing CD24, CD56, and / or CD90-positive cells leads to the depletion of residual contaminating cells.

[0198] To implement the separation technique, it is ideal to dissociate the initial population of RPE cells into a single-cell suspension for sorting (e.g., MACS). For previously cryopreserved cells, the cells must be thawed and replated. To obtain a single-cell suspension from cells in adherent culture, the cells are washed (e.g., with DPBS) and a cell dissociation enzyme (e.g., TRYPLE) is added. TM After incubating the cells at 37°C for approximately 5 minutes, the container was gently tapped to separate the neuronal clusters. The cells were washed twice in DPBS and then treated with a cell dissociation enzyme (e.g., Tryple). TM After incubating the cells at 37°C for approximately 30 minutes, the cell suspension was collected in RPE-MM plating medium and centrifuged at 400 x g for 5 minutes. The cell pellet was resuspended in RPE-MM plating medium and filtered through a cell filter (e.g., a 20 μM steriflip cell filter) to dissociate any remaining cell clusters. The cell suspension was counted (e.g., using a ViCell counter) to obtain cell concentration. The counted cell suspension provides a single-cell suspension that can be used for sorting or flow cytometry purity determination.

[0199] To remove contaminating cells from the RPE cell starting population, CD24-positive, CD56-positive, and / or CD90-positive cells were depleted using MACS. After dissociating cells from the RPE cell starting population into single-cell suspensions, the cells were resuspended in MACS buffer, for example at 1 × 10⁻⁶. 7 Cells / mL. Table 3 includes an exemplary MACS buffer. Next, stain the cells with anti-CD24 antibody, anti-CD56 antibody, and / or anti-CD90 antibody (each diluted 1:500) and incubate at 4°C for 20 minutes to allow the antibodies to bind to the antigens on the cells. The antibodies used should be labeled with a tag that can bind a secondary antibody (e.g., FITC). After incubation, add 20 mL of MACS buffer and centrifuge the cells at 400 x g for 5 minutes. Resuspend the cell pellet in 20 mL of MACS buffer, mix vigorously, and centrifuge at 400 x g for 5 minutes to remove any unbound antibodies. Resuspend the cell pellet in MACS buffer (e.g., 1.11 x 10⁻⁶ cells / mL). 8 Add microbeads coated with a secondary antibody (e.g., anti-FITC) diluted (1:10) to a concentration of cells / mL, and incubate the cells at 4°C for 20 minutes. After incubation, wash the cells with MACS buffer to remove unbound microbeads, and then add up to 1.25 × 10⁻⁶ cells / mL. 8Cells were resuspended in 500 μL of MACS buffer. The cell suspension was transferred to an LD column placed in a strong magnetic field, retaining cells expressing antigens CD24, CD56, and / or CD90 attached to the microbeads. The LD column was washed twice with MACS buffer. Unlabeled cells not expressing antigens CD24, CD56, and / or CD90 were passed through and collected. For further characterization and culture, the collected unlabeled cell suspension was centrifuged (400 × g, 5 min) and re-seeded in RPE-MM plating medium, and a portion of the cell suspension was used for flow cytometry purity determination. Thus, MACS cell sorting resulted in the depletion of CD24, CD56, and / or CD90-positive cells in the RPE-enriched cell population. It should be noted that the use of this method is not limited to the starting population generated by the method detailed in Example 2, and can be used to remove contaminating cells from RPE populations generated by other methods, such as, but not limited to, those described in U.S. Applications Nos. 12 / 523,444 and 14 / 405,730.

[0200] Table 1: Overview of MACS Cell Sorting

[0201]

[0202] The percentage of RPE-labeled positive cells before sorting is those cells present in the initial RPE cell population of Example 2. Depletion of the combination of CD24-positive and CD56-positive cells resulted in a higher enrichment of RPE cells than depletion of CD24-positive cells alone. Depletion of CD24-positive, CD56-positive, and CD90-positive cells resulted in over 99% RPE cells in the cell population.

[0203] Example 6 - Flow cytometry purity determination for characterizing RPE-enriched cell populations

[0204] RPE cells were characterized by relevant marker groups, including BEST1, CRALBP, TYRP1, PMEL17, MAP2, NES, and MITF, before and after MACS sorting (e.g., before and after sorting). Flow cytometry purity assays were performed to obtain measurements (Figures 2 and 3) of the percentage of each marker-positive cell before and after removal of CD24-positive, CD56-positive, and / or CD90-positive cells by MACS (Table 1).

[0205] Flow cytometry purity assays were performed to determine the percentage of RPE cells obtained using the sorting method described in this disclosure. Aliquots of cell suspensions (2 × 10⁻⁶) were collected from the MACS analysis. 6Cells were centrifuged at 400×g for 3 minutes in 5 mL FACS tubes (for each sample). The cell pellet was resuspended in 1 mL of staining agent (e.g., Live-Dead Red) and incubated in the dark at room temperature for 15 minutes. After incubation, 2 mL of wash buffer was added and the cells were centrifuged at 400×g for 3 minutes to remove any unbound staining agent. The cell pellet was resuspended in fixation buffer and incubated in the dark at room temperature for 15 minutes. After incubation, 2 mL of wash buffer was added, and the cells were centrifuged at 400×g for 3 minutes, and the supernatant was decanted. The cell pellet was resuspended in 2 mL of wash buffer to prepare 1 x 10n cells. 6 100 μL of cells / mL suspension was prepared, and 200 μL of cell suspension was transferred to FACS tubes. 2 mL of Perm buffer was added to each tube, and the cells were centrifuged at 400 × g for 3 min. The primary antibody for the RPE-specific marker was diluted in Perm buffer, and 100 μL of the diluted antibody solution was added to each tube. After incubation overnight in the dark at 4 °C, the cells were washed twice with 2 mL of Perm buffer. The secondary antibody solution was added to each tube, and the cells were incubated in the dark at room temperature for 1–2 h. After incubation, the cells were washed twice with Perm buffer, centrifuged (400 × g, 3 min), and resuspended in 100 μL of wash buffer for flow cytometry analysis. Flow cytometry analysis was performed using methods known to those skilled in the art, such as U.S. Patent No. 8,682,810 and Herzenberg et al., 2006, which are incorporated herein by reference, to obtain the percentage of cells positive for each test marker (Table 1). Flow cytometry purity assays showed that MACS sorting produced RPE cell-rich clusters (95–99%) from depleted CD24, CD56, and / or CD90-positive contaminated cells, as determined by BEST1 labeling, compared to the starting cell population (78.6%).

[0206] Example 7 - Alternative methods for differentiating RPE cells

[0207] Regarding the methods described in Examples 2 and 3, including 1 μM PD0325901 in the culture medium during certain time windows from day 2 after iPSC plating until the end of the differentiation process (including after MACS culture) can improve the purity of the RPE population (meaning a reduction in contaminated cells) and the maturation of the resulting RPE population. It has been shown that including 1 μM PD 0325901 in both the RDM and RPE-MM (approximately day 42 to 50) of the RPE process described herein can improve the purity and maturity of the RPE population.

[0208] Example 8 - Alternative methods for differentiating RPE cells

[0209] Regarding the methods described in Examples 2 and 3, reducing the percentage of fetal bovine serum in RPE-MM and RPE-MM plated media from 5% to 0.5-1% can improve the purity of the RPE population (meaning a reduction in contaminated cells) and the maturation of the resulting RPE population.

[0210] Example 9 - Functional properties of mature RPE cells

[0211] To analyze mature RPE cells generated by PGE2 treatment, immunostaining of RPE monolayers was performed using ZO1 staining and transmission electron microscopy of iPSC-RPE cells. Figure 4D Confirm the tightly connected hexagonal structure. Figures 4A-4C Staining showed that PGE2-treated RPE cells had decreased β-catenin and increased RPE65. Additionally, treatment with IWP2+endo-IWR1 or IWP2 also resulted in a decrease in β-catenin. Figure 5A ) and RPE65 increase ( Figure 5C The combination of IWP2 and endo-IWR1 was found to be more effective than either IWP2 or endo-IWR1 alone. Therefore, treatment with PGE2, IWP2, or IWP2 + endo-IWR1 resulted in mature RPE cells.

[0212] To measure the barrier function of RPE cells generated by the method of this invention, transepithelial potential (TEP) is measured by an ion gradient across a monolayer, generated by an ion pump that regulates the energy of channels passing through the cell. Transepithelial resistance (TER) is measured primarily by the resistance of substances passing through the paracellular space through the fine structure of tight junctions. Figure 7A ).

[0213] The function of mature RPE cells treated with IWP2 or endo-IWR1 was also characterized. TEP and TER measurements of untreated RPE cells and RPE cells treated with PGE2 or IWP2+endo-IWR1 showed increased function in the treated mature RPE cells. Figures 7C-7E ).

[0214] Next, the study tested whether increasing the PGE2 concentration in RPE-MM+PGE2 medium from 50 μM to 100 μM improved the purity of the RPE population (i.e., a reduction in contaminated cells) and the maturity of the resulting RPE population. To determine the maturity and functionality of cultures treated with 50 μM and 100 μM PGE2, barrier function in terms of transepithelial electrical resistance (TER) was measured. Figure 7FTo compare resistance to substances passing through the cell periphery, as explained in Example 9. To determine the percentage of pure RPE cells obtained after treating iPSC-derived RPE cultures in RPE-MM+PGE2 medium with 50 μM or 100 μM, flow cytometry purity was determined for RPE-specific markers. Figure 7G As described in Example 6, higher concentrations of the primary inducer PGE2 promoted the purity and maturity of the RPE population during iPSC-derived RPE differentiation.

[0215] Example 10 - Reproducibility of the RPE differentiation method

[0216] To test the reproducibility of the RPE differentiation process, three iPSC cell lines were differentiated into RPE cells by multiple operators. Figure 6A The mean purity of the obtained RPE cells was characterized by measuring the RPE-labeled retinaldehyde-binding protein 1 (Crap1bp) by flow cytometry (Table 2). The RPE differentiation process was found to be highly reproducible across different starting cell populations and different operators. Furthermore, reproducibility was confirmed by RPE differentiation from different starting cell lines, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD Donor3D, and HLA lineage A. Figure 6B HLA line A (21525.102) is an iPSC cell line generated from donors homozygous for HLA-A*01 and HLA-B*08, which can provide 11.38% of the US population with a beneficial match. Furthermore, RPE has also been successfully generated using this method from an iPSC line called HLA line C (21526.101) homozygous for HLA-A*03 and HLA-B*07, which can potentially provide 7.63% of the US population with a beneficial match. HLA line A (21525.102) and HLA line C (21526.101), homozygous for HLA-A and HLA-B as described above, were provided by Cellular Dynamics International, Inc. Additionally, reproducibility was further confirmed in 109 RPE differentiations performed on 28 iPSC cell lines from 13 donors by measuring the percentage of Cralbp-positive cells before and after purification. Figure 6C -D). Although the percentage of Cralbp-positive cells varies after RPE differentiation, MACS purification consistently results in purity exceeding 95%, and in most cases, purity close to 100%. Therefore, the RPE differentiation method of the present invention has a significant advantage over any method of generating RPE cells from embryoids because it provides more consistent and reproducible results across donor genotypes and different operators.

[0217] Table 2: RPE Differentiation among Multi-Operator

[0218]

[0219] Example 11 - Materials and Methods

[0220] The materials used in Examples 1-10 are shown in Table 3.

[0221] Table 3: Exemplary Culture Medium Components

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] Flow cytometry wash buffer was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium-free). The buffer was sterilized by filtration and can be stored at 4°C for up to 4 weeks.

[0234] Flow cytometry perm buffer was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium-free). 1 g of saponin was added and mixed thoroughly. The buffer was sterilized by filtration and can be stored at 4°C for up to 4 weeks.

[0235] Live-Dead Red staining for flow cytometry was prepared by diluting Live-Dead staining agent 1:1000 in DPBS (i.e., without calcium and magnesium). For each 1×10⁻⁶ cells to be analyzed... 6 Prepare 1 mL of staining solution per cell. Prepare the staining solution fresh before use.

[0236] Flow cytometry fixation buffer was prepared by adding 1 mL of 36.5% formaldehyde to 8.1 mL of DPBS (i.e., calcium and magnesium-free). For each 1 × 10⁻⁶ cells to be measured... 6 Prepare 1 mL of staining agent for cells. Prepare the buffer fresh before use.

[0237] Based on this disclosure, all methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations in the steps or order of the steps of the methods can be made without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for those described herein to obtain the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0238] References

[0239] The following references, to the extent that they provide exemplary procedural supplements or other details that supplement those set forth herein, are expressly incorporated herein by reference.

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[0283] Yu et al., Science, 318:1917-1920, 2007.

[0284] In summary, the present invention provides the following implementation schemes:

[0285] 1. A method for providing an enriched population of retinal pigment epithelial (RPE) cells, comprising:

[0286] a) Obtain a starting cell population containing RPE cells; and

[0287] b) The RPE cells of the starting cell population are enriched by removing CD24-positive cells, CD56-positive cells and / or CD90-positive cells, thereby providing an RPE-enriched cell population that is richer in RPE cells than the starting cell population.

[0288] 2. The method of implementation scheme 1 further includes determining the enrichment level of RPE cells in the RPE enrichment cluster.

[0289] 3. The method of embodiment 2, wherein the enrichment level is determined by using retinal epithelium-specific markers selected from BEST1, CRALBP, TYRP1, PMEL17 and MITF.

[0290] 4. The method of implementation scheme 1, wherein, as determined by BEST1 sorting, the RPE-enriched cell population is rich in RPE cells compared to the starting cell population.

[0291] 5. The method of implementation scheme 1, wherein the RPE-rich cluster is at least 95% RPE cells.

[0292] 6. The method of implementation scheme 5, wherein the RPE-rich cluster is at least 99% RPE cells.

[0293] 7. The method of implementation scheme 6, wherein the RPE-rich clusters are substantially pure RPE cells.

[0294] 8. The method of implementation scheme 1, wherein the RPE cells are human RPE cells.

[0295] 9. The method of implementation scheme 1, wherein the starting cell population is prepared from pluripotent stem cells.

[0296] 10. The method of embodiment 9, wherein the pluripotent stem cell is an induced pluripotent stem cell.

[0297] 11. The method of implementation scheme 1, wherein CD24-positive cells, CD56-positive cells and / or CD90-positive cells are removed by magnetic bead-based sorting or fluorescence-based sorting.

[0298] 12. The method of implementation scheme 1, wherein antibodies or aptamers that recognize CD24, CD56 and / or CD90 are used to remove CD24-positive cells, CD56-positive cells and / or CD90-positive cells.

[0299] 13. The method of implementation scheme 1, wherein the RPE-enriched cell population is not genetically modified.

[0300] 14. The method of implementation scheme 1, wherein step b) is performed without genetic modification of the starting cell population.

[0301] 15. The method of implementation 1, wherein the initial cell population is enriched with RPE cells by removing CD24-positive cells therefrom.

[0302] 16. The method of implementation 1, wherein the initial cell population is enriched with RPE cells by removing CD56-positive cells therefrom.

[0303] 17. The method of implementation 1, wherein the initial cell population is enriched with RPE cells by removing CD90-positive cells therefrom.

[0304] 18. The method of implementation scheme 1, wherein the initial cell population is enriched with RPE cells by removing CD90-positive cells, CD56-positive cells and CD24-positive cells therefrom.

Claims

1. A cell population enriched in retinal pigment epithelium (RPE), prepared by a method comprising the following steps: a) Obtaining a starting cell population comprising RPE cells, wherein the starting cell population is prepared from induced pluripotent stem cells; and b) Enriching the RPE cells of the starting cell population by depleting the following cells from the starting cell population: (i) CD24-positive cells, or (ii) CD56-positive cells, or (iii) CD24-positive cells and CD56-positive cells, or (iv) CD24-positive cells and CD90-positive cells, or (v) CD56-positive cells and CD90-positive cells, or (vi) CD24-positive cells, CD56-positive cells, and CD90-positive cells. This provides an RPE-enriched cell population that is richer in RPE cells with downregulated expression of Pax6 and upregulated expression of RPE65 compared to the starting cell population, wherein the RPE-enriched cell population is at least 90% pure RPE cells when determined using TYRP1 and / or PMEL17 as markers.

2. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is at least 95% RPE cells.

3. The RPE-enriched cell population according to claim 2, wherein the RPE-enriched cell population is at least 99% RPE cells.

4. The RPE-enriched cell population according to claim 3, wherein the RPE-enriched cell population is pure RPE cells.

5. The RPE-enriched cell population according to claim 1, wherein the RPE cells are human RPE cells.

6. The RPE-enriched cell population of claim 1, wherein CD24-positive cells, CD56-positive cells, or CD90-positive cells are depleted by magnetic bead-based sorting or fluorescence-based sorting.

7. The RPE-enriched cell population according to claim 1, wherein CD24-positive cells, CD56-positive cells, or CD90-positive cells are depleted by using antibodies or aptamers that recognize CD24, CD56, or CD90, respectively.

8. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is not genetically modified.

9. The RPE-enriched cell population according to claim 1, wherein step b) of the method is performed without genetic modification of the starting cell population.

10. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching RPE cells of the starting cell population by depleting CD24-positive cells from the starting cell population.

11. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching RPE cells of the starting cell population by depleting CD56-positive cells from the starting cell population.

12. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching the RPE cells of the starting cell population by depleting CD24-positive and CD90-positive cells from the starting cell population.

13. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching the RPE cells of the starting cell population by depleting CD90-positive cells, CD56-positive cells and CD24-positive cells from the starting cell population.

14. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching the RPE cells of the starting cell population by depleting CD24-positive and CD56-positive cells from the starting cell population.

15. The RPE-enriched cell population according to claim 1, wherein the RPE-enriched cell population is prepared by enriching the RPE cells of the starting cell population by depleting CD56-positive and CD90-positive cells from the starting cell population.

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