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

Abstract

To provide methods of enriching a retinal pigment epithelium cell population derived from stem cells.SOLUTION: A method for providing an enriched population of retinal pigment epithelial (RPE) cells comprises the steps for: a) obtaining a starting cell population comprising RPE cells; and b) enriching the starting cell population for RPE cells by removing therefrom cells that are positive for CD24, cells that are positive for CD56 and / or cells that are positive for CD90, thereby providing a RPE-enriched cell population that is enriched for RPE cells as compared to the starting cell population.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 215,272, filed on 8 September 2015, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 1. Technical field This invention generally relates to the field of stem cell biology. More specifically, it relates to a method for enriching a population of stem cell-derived retinal pigment epithelial cells.

[0003] 2. Explanation of related technologies The retina is a layered, photosensitive tissue that lines the inner surface of the eye. Photoreceptor cells in the retina (either rod or cone cells) directly sense light, converting chemical light signals into electrical events that trigger nerve impulses. The retinal pigment epithelium (RPE) is a layer of pigment cells that form the blood-retinal barrier. RPE cells play a crucial role in maintaining visual function and transporting ions, water, and metabolites from the subretinal space to the blood (Strauss et al., 2005). Furthermore, RPE cells establish the eye's immune privilege by secreting immunosuppressive factors. Damage to RPE cells can lead to retinal degeneration, loss of visual function, and blindness. Since several retinal disorders, including acute and age-related macular degeneration and Best's disease, involve RPE degeneration, cell replacement therapy is considered a treatment option to preserve vision (Buschholz et al., 2009).

[0004] Generally, stem cells are undifferentiated cells capable of giving rise to a range of mature, functional cells. For example, hematopoietic stem cells can give rise to any of the various terminally differentiated blood cells. Embryonic stem (ES) cells are pluripotent cells derived from embryos and have the ability to develop into any organ or tissue type, including RPE cells.

[0005] The creation of induced pluripotent stem cells (iPSCs) from somatic cells of adult mice, established in 2006, became a major breakthrough in stem cell research, drug discovery, disease models, and cell therapy (Takahashi et al., 2006). Human iPSCs can be differentiated into specific cell types and have the potential to produce patient-specific, immunotype-matched cells for regenerative medicine (Yu et al., 2007).

[0006] iPSCs are known to give rise to ophthalmic cells, including RPE cells (Hirami et al., 2009). However, to use iPSC-derived RPE cells for therapy, screening assays, retinal disease models, and RPE biology studies, it is necessary to remove contaminated cells from the differentiated RPE cell population and / or enrich the desired population. [Overview of the project]

[0007] This embodiment overcomes a major drawback of the art by providing a method for obtaining a population rich in retinal pigment epithelium (RPE) cells from a starting RPE cell population by removing CD24-positive cells, CD56-positive cells, and / or CD90-positive cells. In certain embodiments, the starting RPE cell population can be obtained from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0008] In one embodiment, a method is provided for providing a population rich in retinal pigment epithelial (RPE) cells, the method comprising (a) obtaining a starting cell population containing RPE cells, and (b) enriching the starting cell population by removing cells positive for CD24, cells positive for CD56, and / or cells positive for CD90 from the starting cell population to provide an RPE-rich cell population in which RPE cells are enriched compared to the starting cell population. In some aspects, the starting cell population is ungenetically engineered or unmodified. In some aspects, enriching the cell population does not involve genetically engineering the cells. Thus, in certain aspects, the RPE-rich cell population is ungenetically engineered or unmodified cells.

[0009] In some contexts, this method may further involve determining the level of enrichment of RPE cells in an RPE-rich population. In certain contexts, the level of enrichment is measured using retinal epithelium-specific markers. For example, retinal epithelium-specific markers may be BEST1, CRALBP, TYRP1, PMEL17, or MITF. In certain contexts, an RPE-rich cell population is one in which RPE cells are enriched compared to the starting cell population, as measured by BEST1 selection.

[0010] In certain contexts, an RPE-rich population is one in which at least 95%, 96%, 97%, 98%, or 99% of cells are RPE cells. In other contexts, an RPE-rich population is one in which cells are essentially pure RPE cells.

[0011] In certain aspects of the embodiment, the starting cell population is prepared from pluripotent stem cells. In further aspects, the pluripotent stem cells are induced pluripotent stem cells. For example, RPE cells may be human RPE cells.

[0012] In certain aspects, 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 sorting using magnetic beads or sorting using fluorescence. In certain aspects, CD24-positive cells, CD56-positive cells, and / or CD90-positive cells are removed using an antibody or aptamer that recognizes CD24, CD56, and / or CD90.

[0013] In certain aspects, the cell population is enriched for RPE cells by removing CD24-positive cells from this cell population. In other aspects, the cell population is enriched for RPE cells by removing CD56-positive cells from this cell population. In yet other aspects, the cell population is enriched for RPE cells by removing CD90-positive cells from this cell population. In further aspects, the cell population is enriched for RPE cells by removing CD90-positive cells, CD56-positive cells, and CD24-positive cells from this cell population.

[0014] The cell populations provided herein may essentially contain no contamination by non-RPE cells such as fibroblasts or undifferentiated pluripotent stem cells. In further aspects, the RPE cells are mouse or human RPE cells. In certain aspects, the RPE cells may be cryopreserved RPE cells.

[0015] The RPE cells produced by the methods herein can be used in any method and application currently known in the field of RPE cells. For example, a method for evaluating a compound can be provided that includes assaying the pharmacological or toxicological properties of the compound against RPE cells. Also provided can be a method for evaluating a method for evaluating a compound for its effect on RPE cells, the method comprising: a) contacting the RPE cells provided herein with the compound; and b) assaying the effect of the compound on the RPE cells.

[0016] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, the detailed description and specific examples, while illustrating preferred embodiments of the invention, are provided for illustrative purposes only, and various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0017] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0018] [Figure 1A] Image of an iPSC before it reached confluence. [Figure 1B] An example image of RPE on day 25 of differentiation. [Figure 1C] An example image of RPE at day 40 of differentiation. [Figure 1D] An example image taken on day 60 after transferring the cells to a culture medium containing RPE-MM on day 40. Brightfield imaging, 100x magnification.

[0019] [Figure 2A] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, before cell sorting of iPSC-derived RPE cell populations. [Figure 2B] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, before cell sorting of iPSC-derived RPE cell populations.

[0020] [Figure 3A-1]Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells. [Figure 3A-2] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells. [Figure 3B-1] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells. [Figure 3B-2] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells. [Figure 3C-1] Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells. [Figure 3C-2]Flow cytometry analysis of relevant markers, including MAP2, NES, PAX6, MITF, PMEL17, TYRP1, CRALBP, and BEST1, after cell sorting of iPSC-derived RPE cell populations to remove CD24-positive cells, CD24 and CD56-positive cells, CD24 and CD90-positive cells, and CD24, CD56, and CD90-positive cells.

[0021] [Figure 4A] Beta-catenin and F-actin staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. Beta-catenin staining is observed in the cytoplasm of untreated cells and in the membrane of treated cells. [Figure 4B] pERM (ezrin) and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. ERM staining is weak in the cytoplasm of untreated cells and strong in the cytoplasm of treated cells, while ZO1 staining is observed at tight junctions of the plasma membrane in both untreated and treated cells. [Figure 4C] RPE65 and ZO1 staining of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. RPE65 staining is weak in the cytoplasm of untreated cells and strong in the cytoplasm of treated cells. [Figure 4D] Transmission electron micrographs of untreated iPSC-RPE cells and iPSC-RPE cells treated with PGE2. Cells treated with PGE2 exhibit more extensive apical processes.

[0022] [Figure 5A] Beta-catenin staining of cells treated with IWP2+endo-IWR1, IWP2, or LiCl. In cells treated with IWP2 or IWP2+endo-IWR1, beta-catenin is observed on the cell membrane. In cells treated with LiCl, beta-catenin is observed in the nucleus, while in untreated cells, beta-catenin is observed in the cytoplasm. [Figure 5B]p27 staining of cells treated with IWP2+endo-IWR1, IWP2, or LiCl. Cells treated with IWP2 or IWP2+endo-IWR1 show higher p27 expression in the nucleus, suggesting that the cells have left the cell cycle. Untreated cells or cells treated with LiCl show weak p27 expression in the nucleus. [Figure 5C] RPE65 and ZO1 tight junctions in cells treated with IWP2+IWR1, IWP2, or LiCl. RPE65 staining is strong in the cytoplasm of IWP2+IWR1-treated and IWP2-treated cells, weak in untreated cells, and not observed in LiCl-treated cells. [Figure 5D] Electron micrographs of functional tight junctions in cells treated with IWP2+IWR1, IWP2, or LiCl.

[0023] [Figure 6A] RPE differentiation by multiple operators. The data represent the results of RPE differentiation performed by multiple operators using optimized protocols across three lineages, expressed as flow cytometry measurements using the RPE marker, retinaldehyde-binding protein (Cralbp) 1. [Figure 6B] Reproducibility of RPE differentiation protocols for various starting cell line populations, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD donor 3D, AMD donor 3C, and HLA cell line A. [Figure 6C-1] Reproducibility of RPE differentiation protocols across various starting cell line populations. The data shows the results of 109 differentiation cycles performed by 5 operators on 28 iPSC cell lines provided by 13 donors. While the purity of the cell population before purification varied, the percentage of Cralbep-positive cells increased to 90-100%. [Figure 6C-2]Reproducibility of RPE differentiation protocols across various starting cell line populations. The data shows the results of 109 differentiation cycles performed by 5 operators on 28 iPSC cell lines provided by 13 donors. While the purity of the cell population before purification varied, the percentage of Cralbep-positive cells increased to 90-100%. [Figure 6D] Reproducibility of RPE differentiation protocols across various starting cell line populations. The data shows the results of 109 differentiation cycles performed by 5 operators on 28 iPSC cell lines provided by 13 donors. While the purity of the cell population before purification varied, the percentage of Cralbep-positive cells increased to 90-100%.

[0024] [Figure 7A] The functionality of the barrier function of RPE cells generated using the RPE differentiation protocol is demonstrated by transepithelial potential (TEP) measurement of the ion gradient across the entire monolayer. [Figure 7B] Functionality of RPE cells treated with IWP2 or IWP2+endo-IWR2. [Figure 7C] Transepithelial electrical resistance (TER) and TEP (thin line) of untreated cells. [Figure 7D] Transepithelial electrical resistance (TER) and TEP (thin line) of PGE2-treated cells. [Figure 7E] Transepithelial electrical resistance (TER) and TEP (thin line) of IWP2+ endo-IWR1 treated cells. [Figure 7F] Functional response (TER) of cells matured in RPE-MM+PGE2 medium supplemented with 50 μM or 100 μM PGE2 during the period from day 54 to day 75 of the iPSC-derived differentiation protocol. Compared to iPSC-derived RPE cultured in RPE-MM+PGE2 medium supplemented with 50 μM PGE2 during the period from day 54 to day 75 of the differentiation protocol, TER measurements were gradually increased in cells differentiated in medium supplemented with 100 μM PGE2. This indicates that higher PGE2 concentrations promote maturation and functional efficiency of iPSC-derived RPE cultures. [Figure 7G]Purity of iPSC-derived RPE cells at day 75, matured in medium supplemented with 50 μM or 100 μM PGE2. This experiment was initiated from day 54 to day 75 of the iPSC-derived RPE differentiation protocol and is shown as a percentage of RPE marker expression. Significant expression of Pmel17, Tryp1, and Cralbp (RPE-specific markers) was observed in iPSC-derived RPE cultured with 50 μM PGE2. This indicates that PGE2 promotes iPSC-derived RPE differentiation over a certain concentration range. Expression of the Best1 marker (late-maturation RPE marker) was significantly higher in cells treated with 100 μM PGE2 compared to cells treated with 50 μM PGE2, indicating that increasing PGE2 concentration improves the purity and maturity of iPSC-derived RPE. [Modes for carrying out the invention]

[0025] This disclosure overcomes several major problems in current technology by providing a method for enriching a population of stem cell-derived retinal pigment epithelial (RPE) cells. While RPE cells can be derived from pluripotent stem cells such as ES cells and iPS cells, existing techniques have shown that even within stem cell-derived RPE populations, a certain amount of non-RPE cells (e.g., undifferentiated stem cells) are present. The present invention provides a method for separating and removing contaminated cells from a starting population of RPE cells. Cells contaminating an RPE cell population possess specific cell surface antigens, such as CD24, CD56, and / or CD90, which can be used to deplete contaminated non-RPE cells from the population. Therefore, by removing cells positive for one or more of these specific cell surface markers, an RPE-rich cell population with a higher proportion of RPE cells than the starting population can be obtained. Specific methodologies such as magnetically activated cell sorting (MACS®), fluorescence-activated cell sorting (FACS), or single-cell sorting are known in the art for separating various cell populations according to such surface antigens. In a preferred embodiment, this disclosure includes a sorting method, preferably using MACS, to deplete CD24, CD56, and / or CD90-positive cells from a starting RPE cell population in order to obtain an RPE-rich cell population. Thus, this method enables the production of a therapeutic RPE-rich cell population from stem cells, a regenerative source, more quickly and efficiently. Further embodiments and advantages of this disclosure are described below.

[0026] I. Definition The term "purified" does not imply absolute purity, but rather is intended as a relative term. Therefore, a purified population of cells is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure and essentially free of other cell types.

[0027] As used herein, “essentially” or “essentially not” with respect to a particular component means that the particular component is not intentionally formulated in the composition and / or is present only as a contaminant or in trace amounts. Accordingly, the total amount of a particular component resulting from unintended contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferably, the amount of a particular component present in the composition is at a level that cannot be detected by standard analytical methods.

[0028] As used herein, “a” or “an” may mean one or more. As used in conjunction with the term “comprising,” the words “a” or “an” may mean one or more.

[0029] In the claims, the term “or” means “and / or” unless it expressly means that only one of the options is being referred to, or that the options are mutually exclusive. Where used herein, “another” may mean at least a second or more.

[0030] Throughout this application, the term “approximately” is used to indicate that a certain value includes the inherent variation of the device’s error, the method used to determine the value, or the variation present among subjects.

[0031] In this specification, the term “cell” is used to mean a structural and functional unit of an organism that is capable of independently replicating, is enclosed by a membrane, and contains biomolecules and genetic material. As used herein, cells may be naturally occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0032] In this specification, the term “cell population” is used to mean a group of cells, typically a group of cells sharing a common type. A cell population may originate from a common progenitor cell or may contain more than one cell type. An “enriched” cell population is a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population) that contains a particular cell type in a greater proportion than that cell type in the starting population. A cell population may be enriched in one or more cell types, or may be depleted in one or more cell types.

[0033] In this specification, the term “stem cell” refers to a cell that, under appropriate conditions, can differentiate into a diverse range of specialized cell types, and under other appropriate conditions, self-replicates and remains essentially in an undifferentiated pluripotent state. The term “stem cell” also encompasses pluripotent cells, multipotent cells, progenitor cells, and precursor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them into a pluripotent state by expressing specific transcription factors associated with pluripotency. Such cells are called “induced pluripotent stem cells” or “iPSCs.”

[0034] The term "pluripotency" refers to the property of cells to differentiate into all other cell types of an organism, excluding extraembryonic or placental cells. Pluripotent stem cells can differentiate into all cell types of the three germ layers (e.g., ectoderm, mesoderm, and endoderm cell types) even after long-term culture. Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of a blastocyst. In other embodiments, pluripotent stem cells are induced pluripotent stem cells, which are produced by reprogramming somatic cells.

[0035] The term "differentiation" refers to the process by which unspecialized cells become more specialized cell types through changes in structural and / or functional properties. Mature cells typically possess altered cellular structures and tissue-specific proteins. In the context of this method, more specifically, it refers to the process by which human stem cells become retinal pigment epithelial (RPE) cells and exhibit characteristics that indicate the RPE cells are mature, terminally differentiated cells.

[0036] As used herein, “undifferentiated” means a cell that exhibits characteristic markers and morphological features of an undifferentiated cell, which is clearly distinguishable from terminally differentiated cells of embryonic or adult origin.

[0037] Embryoid bodies (EBs) are aggregates of pluripotent stem cells that can differentiate into endoderm, mesoderm, and ectoderm cells. When pluripotent stem cells aggregate and EBs can be cultured in a non-adherent suspension state, a spherical structure is formed.

[0038] "Isolated" cells are substantially separated or purified from other cells in a living organism or culture. Isolated cells may have a purity of, for example, at least 99%, at least 98%, at least 95%, or at least 90%.

[0039] An "embryo" is a cell mass obtained from one or more compartments of a fertilized egg or an activated oocyte with an artificially reprogrammed nucleus.

[0040] Embryonic stem (ES) cells are undifferentiated pluripotent cells obtained from an embryo at an early stage, such as the inner cell mass of the blastocyst, or undifferentiated pluripotent cells produced by artificial means (e.g., nuclear transfer), and are capable of giving rise to any more differentiated cell type of the embryo or adult, including germ cells (e.g., sperm and eggs).

[0041] Induced pluripotent stem cells (iPSCs) are cells produced by reprogramming cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be produced using somatic cells from a fetus, postnatal, neonatal, juvenile, or adult. Factors that may be used to reprogram somatic cells into pluripotent stem cells in certain embodiments include, for example, Oct4 (sometimes called Oct3 / 4), Sox2, c-Myc and Klf4, Nanog and Lin28. In some embodiments, to reprogram somatic cells into pluripotent stem cells, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors.

[0042] An "allele" refers to one of two or more forms of a gene. Diploid organisms like humans have two copies of each chromosome, so each chromosome can be said to have one allele.

[0043] The term "homozygous" is defined as having two identical alleles at a particular gene locus. The term "heterozygous" refers to having two different alleles at a particular gene locus.

[0044] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. Haplotypes may be based on combinations of single nucleotide polymorphisms (SNPs) and / or alleles of the major histocompatibility complex on a single chromosome.

[0045] As used herein, the term “haplotype-matched” defines cells (e.g., iPSC cells) that share one or more major histocompatibility locus haplotypes with the target being treated. The haplotype of the target can be readily determined using assays well known in the art. Haplotype-matched iPSC cells may be autologous cells or allogeneic cells. Autologous cells that have grown in tissue culture and essentially differentiated into RPE cells are haplotype-matched with the target.

[0046] "Substantially identical HLA types" means that the HLA types of the donor and the patient match to the extent that the transplanted cells, obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can be transplanted.

[0047] In this specification, “superdonor” refers to an individual homozygous for specific MHC class I and class II genes. These homozygous individuals can function as superdonors, and their cells, including tissues and other materials containing them, can be transplanted into individuals that are homozygous or heterozygous for that haplotype. Superdonors may be homozygous for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus / locus alleles, respectively.

[0048] In this specification, “feeder-free” or “feeder-independent” is used to mean a culture supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) instead of a feeder cell layer. Thus, differentiated pluripotent cells can be cultured and maintained in an undifferentiated and proliferative state using “feeder-free” or feeder-independent culture systems and media. In some cases, feeder-free cultures are grown using animal substrates (e.g., Matrigel®) or on substrates such as fibronectin, collagen, or vitronectin. These approaches make it possible to keep human stem cells essentially undifferentiated without using a mouse fibroblast “feeder layer.”

[0049] In this specification, “feeder layer” is defined as a cell coating layer applied to the bottom surface of a culture dish or the like. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can adhere.

[0050] When used in relation to culture media, extracellular matrices, or culture conditions, the terms “defined” or “fully defined” refer to culture media, extracellular matrices, or culture conditions in which the chemical composition and nearly all component amounts are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium includes a basal medium (e.g., Dulbecco’s Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Laboratory Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) to which recombinant albumin, chemically defined lipids, and recombinant insulin are added. An example of a fully defined medium is Essential 8® medium.

[0051] When used in relation to culture media, extracellular matrices, or culture conditions, the term "xeno-free (XF)" refers to culture media, extracellular matrices, or culture conditions that are essentially free of components derived from other animals. When culturing human cells, any protein from a non-human animal, such as a mouse, can be considered a xeno-component. In certain contexts, a xeno-free matrix may essentially be free of components derived from non-human animals and therefore may not contain mouse feeder cells or Matrigel®. Matrigel® is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, i.e., a tumor rich in extracellular matrix proteins including laminin (the main component), collagen IV, heparan sulfate proteoglycans, and entactin / nidogen.

[0052] In this specification, “KNOCKOUT® serum substitute” means a serum-free preparation for optimizing the proliferation and maintenance of undifferentiated cells (e.g., stem cells).

[0053] "Pre-confluent" refers to a cell culture where approximately 60-80% of the culture surface is covered by cells. Typically, pre-confluent means that about 70% of the culture surface is covered by cells.

[0054] The "retina" refers to the layered, light-sensitive tissue that covers the inner surface of the eye.

[0055] The "retinal pigment epithelium" refers to the single layer of pigment cells between the choroid, the blood vessel-filled layer, and the retina.

[0056] In this specification, "retinal lineage cells" refers to cells that can produce or differentiate into RPE cells.

[0057] In this specification, "retinal induction medium (RIM)" refers to a growth medium containing a WNT pathway inhibitor and a BMP pathway inhibitor that can differentiate PSCs into retinal lineage cells. RIM also contains a TGFβ pathway inhibitor.

[0058] In this specification, "retinal differentiation medium (RDM)" is defined as a medium for differentiating retinal cells, comprising a WNT pathway inhibitor, a BMP pathway inhibitor, and a MEK inhibitor. RDM also includes a TGFβ pathway inhibitor.

[0059] "Retinal medium (RM)" is defined as a growth medium for retinal cell culture containing activin A and nicotinamide.

[0060] In this specification, “RPE-Maturation Medium (RPE-MM)” refers to a medium for maturing RPE cells, containing taurine and hydrocortisone. RPE-MM also contains triiodothyronine. RPE-MM may further contain PD0325901 or PGE2.

[0061] In this specification, "mature" RPE cells refer to cells in which the expression of immature RPE markers such as Pax6 is reduced, and the expression of mature RPE markers such as RPE65 is increased.

[0062] In this specification, "maturation" of RPE cells refers to a process in which the RPE developmental pathway is regulated to produce mature RPE cells. For example, regulation of ciliary function can lead to RPE maturation.

[0063] As used herein, “therapeutic effective dose” means the amount of a compound sufficient to provide treatment to a subject for the treatment of a disease or condition.

[0064] In this specification, “inducer” is defined as a molecule that regulates gene expression, such as by activating genes within a cell. Inducers may bind to repressors or activators. Inducers function by deactivating repressors.

[0065] II. Pluripotent stem cells A. Embryonic stem cells ES cells originate from the inner cell mass of a blastocyst and possess high differentiation potential in vitro. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner mass cells on a non-proliferating cell feeder layer. The reseeded cells continue to proliferate, generating new colonies of ES cells. These ES cells can be harvested, isolated, and reseeded for further proliferation. This process of “passaging” undifferentiated ES cells can be repeated many times to produce cell lines containing undifferentiated ES cells (U.S. Patents 5,843,780, 6,200,806, and 7,029,913). ES cells have the ability to proliferate while maintaining their pluripotency. For example, ES cells are useful in research on genes that control cells and cell differentiation. The pluripotency of ES cells, when combined with genetic manipulation and selection, can be used in in vivo genetic analysis studies through the generation of transgenic mice, chimeric mice, and knockout mice.

[0066] Methods for generating mouse ES cells are well known. In one method, preimplantation blastocysts from 129 mouse strains are treated with mouse antiserum to remove the trophectoderm, and then 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 then subcultured on the mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to produce a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding cytokine leukemia inhibitor (LIF) to serum-containing medium (Smith, 2000). In other methods, mouse ES cells can be grown in serum-free medium in the presence of osteomorphonectomy proteins and LIF (Inn et al., 2003).

[0067] Human ES cells can be produced or induced from zygote or blastocyst-stage mammalian embryos produced by sperm-egg cell fusion, nuclear transfer, pathogenicity, or chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane, as previously reported (Thomson and Marschel, 1998; Reubinoff et al., 2000). One method involves exposing human blastocysts to anti-human serum, lysing trophectoderm cells to remove them from the inner cell mass, and culturing these on a mouse embryo fibroblast feeder layer. Furthermore, cell masses derived from the inner cell mass are chemically or mechanically dissociated and reseeded, and colonies with undifferentiated morphology are selected using a micropipette, dissociated, and reseeded (U.S. Patent No. 6,833,269). Several methods allow ES cells to be grown without serum by culturing them on a fibroblast feeder layer in the presence of basic fibroblast growth factor (Amit et al., 2000). Alternatively, human ES cells can be grown without a feeder cell layer by culturing them on a protein matrix such as Matrigel® or laminin in the presence of a “conditioned” medium containing fibroblast growth factor (Shu et al., 2001).

[0068] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets (e.g., Thomson and Marschel, 1998; Thomson et al., 1995; Thomson and Odoriko, 2000), according to methods already reported, 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 a further example, an established mouse ES cell line is the CGR8 cell line, established from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown without a feeder layer if LIF is present.

[0069] ES stem cells can be detected by protein markers such as the 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.

[0070] B. Induced pluripotent stem cells The induction of pluripotency 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 through the introduction of transcription factors associated with pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into almost any cell type. The use of iPSCs avoids many of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive therapy to prevent graft rejection.

[0071] Any cell type except germ cells can be used as a starting material for iPSCs. For example, such cell types may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (U.S. 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. Undifferentiated progenitor cells (including somatic cell stem cells) and finally differentiated mature cells can also be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cells are themselves RPE cells, such as human RPE cells. The RPE cells may be adult or fetal RPE cells. Human ES cells can be differentiated into specialized cell types, and iPSCs can be grown under conditions in which human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, are known to be expressed.

[0072] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells (see, for example, Published U.S. Patent Applications 20090246875, 2010 / 0210014, 20120276636; U.S. Patent Nos. 8,058,065; 8,129,187; 8,278,620; PCT International Publication No. 2007 / 069666A1; and U.S. Patent No. 8,268,620, which are incorporated herein by reference). Nuclear reprogramming factors are generally used to produce 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 used.

[0073] Cells are generally treated with nuclear reprogramming materials, which are one or more factors or nucleic acids (including in vector-integrated forms) encoding these substances that can induce iPSCs from somatic cells. Nuclear reprogramming materials generally 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, Lin28 or Lin28b, or nucleic acids encoding Lin28 or Lin28b can also be used as further nuclear reprogramming materials. Nanog can also be used for nuclear reprogramming. Examples of reprogramming factors used in iPSC production, as disclosed in U.S. Patent Application No. 20120196360, include (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18).(1) Klf4 can be replaced with 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, HPV16E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV1 6E6, HPV16E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3 / 4, Klf4, Sox2, L-M yc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT;(11)Oct3 / 4 ,Esrrb,Sox2,L-Myc(Esrrb is replaced with Esrrg);(12)Oct3 / 4,Klf4,Sox2;(13)Oct3 / 4,Klf4,Sox2,TERT,SV40LT;(14)Oct3 / 4,Klf4,Sox2,TERT,HPVI6E6;(15)Oct3 / 4,Klf4,Sox2,TERT,HPV16E7;(16)Oct3 / 4,Klf4,Sox2,TERT,HPV16E6,HPV16E7;(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 may be replaced with Esrrg). Non-limiting examples include Oct3 / 4, Klf4, Sox2 and c-Myc. 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.Other examples include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are used. In other non-limiting examples, 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, embedded vectors such as systems using EBV elements can be used (U.S. Patent No. 8,546,140). In a further context, reprogramming proteins can be directly introduced into somatic cells by protein transduction. Reprogramming may further involve contacting cells with glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signaling inhibitors, leukemia inhibitors (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that virtually any iPS cells or cell lines can be used.

[0074] The mouse and human cDNA sequences of these nuclear reprogramming materials are available by reference to the NCBI accession number listed in International Publication No. 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming materials, or nucleic acids encoding these reprogramming materials, 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.

[0075] Induced iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, as described in U.S. Patent No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603. For mouse cells, they are cultured in a standard medium supplemented with leukemia suppressor (LIF), a differentiation inhibitor. For human cells, it is preferable to supplement with basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs can also be used, as is known to those skilled in the art.

[0076] In certain embodiments, undefined conditions may be used. For example, pluripotent cells can be cultured on fibroblast feeder cells or on a medium exposed to fibroblast feeder cells in order to maintain stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division, as feeder cells. Alternatively, pluripotent cells may be cultured and maintained in an essentially undifferentiated state by using defined feeder-independent culture systems such as TESR® medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8® medium (Chen et al., 2011).

[0077] In some embodiments, iPSCs can be modified to express exogenous nucleic acids, such as by including a tyrosinase enhancer functionally linked to a nucleic acid sequence encoding a promoter and a first marker. The tyrosinase gene is available, for example, from accession number 22173, registered with Genbank® on January 1, 2013. This sequence is located at 5286971-5291691 (reverse orientation) on chromosome 7 of the C57BL / 6 mouse strain. A 4721 base pair sequence is sufficient for expression in RPE cells (see Murisier et al., Dev. Biol. 303:838-847, 2007, incorporated herein by reference). This construct is expressed in retinal pigment epithelial cells. Other enhancers can also 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, such as 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, it is advantageous to introduce the construct into cells. Suitable vectors for stable transduction include, but are not limited to, retroviral vectors, lentiviral vectors, and Sendai viruses.

[0078] Plasmids are designed with many objectives in mind, including achieving high copy numbers under controlled conditions, avoiding potential causes of plasmid instability within bacteria, and providing a means for selecting plasmids suitable for use in mammalian cells, including human cells. Particular attention is paid to two major requirements for plasmids for use in human cells. The first requirement is that they be suitable for maintenance and fermentation in E. coli and that large amounts of DNA can be produced and purified. The second requirement is that they are safe and suitable for use in human patients and animals. The first requirement necessitates high copy number plasmids that can be selected and stably maintained relatively easily during bacterial fermentation. The second requirement requires attention to elements such as selectable markers and other coding sequences. Plasmids encoding markers in some embodiments comprise (1) a high copy number origin, (2) a selection marker such as (but not limited to) a neo gene for antibiotic selection by kanamycin, (3) a transcription termination sequence such as a tyrosinase enhancer, and (4) a multicloning site for various nucleic acid cassette incorporation; and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. Numerous plasmid vectors for inducing protein-encoding nucleic acids exist and are known in the art. These include, but are not limited to, vectors disclosed in U.S. Patents 6,103,470; 7,598,364; 7,989,425; and 6,416,998, which are incorporated herein by reference.

[0079] The viral gene delivery system may be an RNA or DNA viral vector. The episomal gene delivery system may be a plasmid, an EBV (Epstein-Barr virus) based episomal vector, a yeast-based vector, an adenovirus-based vector, a Simian virus 40 (SV40) based episomal vector, a bovine papillomavirus (BPV) based vector, or a lentiviral vector.

[0080] 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 / sea mushroom luciferase or nanoluc), or other proteins. Markers may also be proteins (including secreted, cell surface, or cellular proteins; proteins synthesized or taken up by cells), nucleic acids (e.g., mRNA, or enzymatically active nucleic acid molecules), or polysaccharides. They may include any determinants of such cellular components that are specific to the marker of the cell type of interest and detectable by antibodies, lectins, probes, or nucleic acid amplification reactions. Markers can also be identified by biochemical assays or enzymatic assays or biological responses that depend on the function of the gene product. Nucleic acid sequences encoding these markers can be operably ligated to tyrosinase enhancers. Furthermore, other genes may also be included, such as genes that may affect RPE differentiation from stem cells or RPE function, or physiology or pathology. Therefore, in some embodiments, the nucleic acids include those encoding one or more of the following: 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.

[0081] 1. MHC haplotype matching The primary cause of immune rejection in allogeneic organ transplantation is the major histocompatibility complex (MHC). The MHC class I haplotypes (A, B, and C) and the MHC class II haplotypes (DR, DP, and DQ) are the three major MHC class II haplotypes. HLA loci are distributed across 4 Mb on chromosome 6 and exhibit a high degree of polymorphism. The ability to haplotype HLA genes within this region is clinically significant because it is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donor and recipient affects the clinical outcome of transplantation. HLA corresponding to MHC class I presents peptides from inside the cell, while HLA corresponding to MHC class II presents antigens from outside the cell to T lymphocytes. MHC haplotype incompatibility between the graft and host triggers an immune response to the graft, leading to rejection. Therefore, patients may be treated with immunosuppressants to prevent rejection. HLA-compatible stem cell lines may overcome the risk of immune rejection.

[0082] Because HLA is important in transplantation, HLA loci are typically classified serologically and by PCR to identify preferred donor-recipient pairs. Serological detection of HLA class I and class II antigens can be performed using complement-mediated lymphotoxicity tests with purified T or B lymphocytes. This procedure is primarily used to match HLA-A and HLA-B loci. Molecular-based histological typing is often more accurate than serological testing. HLA antigens can be identified using low-resolution molecular methods such as SSOP (sequence-specific oligonucleotide probe) methods, which test PCR products against a series of oligonucleotide probes, and these methods are now the primary methods for classifying HLA class II. High-resolution techniques such as SSP (sequence-specific primer) methods, which utilize allele-specific primers for PCR amplification, can identify specific MHC alleles.

[0083] When donor cells are HLA homozygous, that is, they contain identical alleles for each antigen-presenting protein, MHC compatibility between donor and recipient is significantly increased. Most individuals are heterozygous for MHC class I and class II genes, but certain individuals are homozygous for these genes. These homozygous individuals act as superdonors, and grafts generated from their cells can be transplanted into any individual that is homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently found in the population, these cells can be used for transplantation therapy in a large number of individuals.

[0084] Therefore, iPSCs can be produced from somatic cells of the subject being treated, or from somatic cells of another subject having the same or substantially the same HLA type as the patient. In one example, 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 another example, a somatic cell donor may be a superdonor, and thus RPE cells can be produced using iPSCs derived from an MHC homozygous superdonor. Thus, iPSCs derived from a superdonor can be transplanted into a subject that is either homozygous or heterozygous for its haplotype. For example, an iPSC may be homozygous for two HLA alleles, such as HLA-A and HLA-B. In this way, iPSCs generated from a superdonor may be used in the methods disclosed herein to produce RPE cells that are potentially "suitable" for a large number of potential recipients.

[0085] 2. Episome vectors In certain contexts, reprogramming factors are expressed from expression cassettes contained within one or more exogenous episomal gene elements (see U.S. Patent Publication 2010 / 0003757, incorporated herein by reference). Thus, iPSCs may not inherently contain exogenous gene elements such as retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extrachromosomal replication vectors (i.e., episomal vectors), which are vectors that can replicate episomatically to produce iPSCs that are essentially free of exogenous vectors or viral elements (see U.S. Patent No. 8,546,140, ​​incorporated herein by reference; U et al., 2009). Many DNA viruses, such as adenoviruses, Simian vacuolated virus 40 (SV40), or bovine papillomavirus (BPV), or budding yeast ARS (autonomous replication sequence)-containing plasmids, replicate extrachromosomally or episomatically in mammalian cells. These episomal plasmids inherently do not contain the drawbacks associated with vector integration (Bode et al., 2001). For example, as already defined, lymphocyte atrophic lateral herpesvirus, or EBV (Epstein-Barr virus), can replicate outside the chromosome and help deliver reprogramming genes to somatic cells. Useful EBV elements include OriP and EBNA-1, or their variants or functional equivalents. A further advantage of episomal vectors is that the exogenous elements are lost over time after introduction into cells, leading to self-persistent iPSCs that essentially do not contain these elements.

[0086] Other extrachromosomal vectors include vectors based on other lymphoproliferative herpesviruses. Lymphoproliferative herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids as part of their natural life cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpesvirus. Exemplary lymphoproliferative herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV), herpesvirus thymili (HS), and Marek's disease virus (MDV). Other episome-based vector sources are also possible, such as yeast ARS, adenovirus, SV40, or BPV.

[0087] C. Somatic cell nuclear transfer Pluripotent stem cells can be prepared by somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of donor nuclei into spindle-free oocytes. In one method, donor fibroblast nuclei derived from rhesus monkey dermal fibroblasts are introduced into the cytoplasm of mature metaphase II rhesus monkey ophthalmocellular cells (Birun et al., 2007) by electrofusion. The fused oocytes are activated by exposure to ionomycin and then incubated until the blastocyst stage. Subsequently, the inner cell mass of selected blastocysts is cultured to produce embryonic stem cell lines. Embryonic stem cell lines exhibit the morphology of normal ES cells, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0088] III.Retinal pigment epithelial cells RPE cells are produced in the manner disclosed herein. The cells of the retina that directly sense light are photoreceptor cells. Photoreceptors are photosensitive neurons in the outer part of the retina and can be either rod cells or cone cells. Photoreceptor cells convert the incident light energy, focused by a lens during the process of light transmission, into electrical signals, which are sent to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells, including cone cells and rod cells. Cone cells are adapted to detect fine detail, central and color vision, and function well in bright light conditions. Rod cells are responsible for peripheral vision and scotopic vision. Nerve signals from cone and rod cells are processed by other neurons in the retina.

[0089] The retinal pigment epithelium acts as a barrier between the bloodstream and the retina and interacts closely with photoreceptors in maintaining visual function. It consists of a single layer of hexagonal cells densely packed with melanin granules that absorb light energy reaching the retina. The main functions of these specialized RPE cells include the transport of nutrients such as glucose, retinol, and fatty acids from the blood to photoreceptors; the transport of water, metabolites, and ions from the subretinal space to the blood; protection against light absorption and photo-oxidation; re-isomerization of all-trans-retinol to 11-cis-retinal; phagocytosis of photoreceptor cell membranes; and the secretion of factors essential for the structural integrity of the retina.

[0090] The retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, the best vitiligo macular dystrophy gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with many visual alteration conditions, including retinal pigment epithelial detachment, malformations, atrophy, retinopathy, pigmentary retinitis, macular dystrophy, or degeneration.

[0091] Retinal pigment epithelial (RPE) cells can be characterized based on pigment deposition, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually recognized by their cobblestone-like morphology and the early appearance of their pigment. Furthermore, differentiated RPE cells have transepithelial resistance / TER and transepithelial potential / TEP throughout the monolayer (TER > 100 ohms / cm²). 2 (TEP > 2mV), it transports liquid and CO2 from the apical to the basal side, regulating the polar secretion of cytokines.

[0092] RPE cells express several proteins that can function as markers detected using methodologies such as immunocytochemistry, Western blotting, flow cytometry, and enzyme-linked immunoassay (ELISA). For example, RPE-specific markers may include cellular retinal aldehyde-binding protein (CRALBP), microophthalmopathy-associated transcription factor (MITF), tyrosinase-associated protein 1 (TYRP-1), retinal pigment epithelium-specific 65kDa protein (RPE65), premelanosome protein (PMEL17), bethroffin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). RPE cells do not express the embryonic stem cell markers Oct-4, nanog, or Rex-2 (at detectable levels). Specifically, the expression of these genes is approximately 1 / 100 to 1 / 1000 lower in RPE cells than in ES cells or iPSC cells, as assessed by quantitative RT-PCR.

[0093] RPE cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers used in standard amplification methods with publicly available sequence data (Genbank®). A tissue-specific marker is considered positive if its expression level, detected at the protein or mRNA level, is at least 2, 3, 4, 5, 6, 7, 8, or 9 times higher. More specifically, a tissue-specific marker is considered positive if its expression level is 10, 20, 30, 40, or 50 times higher than that of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0094] Dysfunction, damage, and loss of RPE cells are the cause of many ophthalmic diseases and disorders, including age-related macular degeneration (AMD), hereditary macular degeneration including Best's disease, and retinitis pigmentosa. One possible treatment for such diseases is transplanting RPE cells into the retinas of people who require such treatment. It is hypothesized that supplementing RPE cells through such transplantation can slow or halt the progression of the disease, reverse retinal deterioration, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining RPE cells directly from human donors and embryos is difficult.

[0095] A. Induction of RPE cells from PSC embryoid bodies iPSCs reprogrammed with well-known reprogramming factors have the potential to generate ophthalmic cells of the nervous system, including RPE cells (Hirami et al., 2009). PCT Publication 2014 / 121077, which is incorporated herein by reference in its entirety, discloses a method for inducing the expression of retinal progenitor cell markers by treating embryoid bodies (EBs) produced from iPSCs with Wnt and Nodal antagonists in a suspension culture system. This publication discloses a method for inducing RPE cells from iPSCs through a process of differentiating iPSC EBs into a culture enriched with RPE cells. For example, embryoid bodies (EBs) are produced from iPSCs by adding a rho-related coiled-coil kinase (ROCK) inhibitor and culturing in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. Furthermore, in a tissue culture system coated with Matrigel (trademark), differentiated RPE cells are formed in a second medium that does not contain basic fibroblast growth factor (bFGF) but contains a Nodal pathway inhibitor, approximately 20 ng to 90 ng of Noggin, and approximately 1 to 5% of a knockout serum substitute. The differentiated RPE cells are cultured in a third medium containing ACTIVIN and WNT3a. Subsequently, human RPE cells are produced by culturing the RPE cells in RPE medium containing approximately 5% fetal serum, a reference WNT inhibitor, a non-reference WNT inhibitor, and inhibitors of the Sonic Hedgehog and FGF pathways.

[0096] Using EBs to produce differentiated cells has several drawbacks. For example, EB production is an inconsistent and non-reproducible process due to variations in efficiency. The size and shape of EBs produced from iPSCs or ES cells are not homogeneous, and EBS production involves rate-limiting centrifugation. This disclosure provides a method that enables the large-scale production of iPSC or ES-derived cells required for clinical, research, or therapeutic applications without using EBs.

[0097] B. Induction of RPE cells from substantially single PSC cells In some embodiments, methods are provided for producing RPE cells from essentially single-cell suspensions of pluripotent stem cells (PSCs), such as human iPSCs. In some embodiments, the PSCs are cultured to a degree that does not reach confluence in order to prevent any cell aggregation. In certain aspects, the PSCs are dissociated by incubation with a cell dissociation enzyme such as Trypsin® or Tryple®. PSCs can also be dissociated into essentially single-cell suspensions by pipetting. Furthermore, after dissociation into single cells, brevistatin (e.g., about 2.5 μM) can be added to the culture medium to increase the viability of the PSCs without causing the cells to adhere to the culture vessel. ROCK inhibitors can be used instead of brevistatin to improve the viability of PSCs after dissociation into single cells.

[0098] To efficiently differentiate RPE cells from single PSC cells, accurately measuring the input density can improve the differentiation efficiency of RPE cells. Therefore, it is common practice to count single-cell suspensions of PSCs before seeding. For example, single-cell suspensions of PSCs are counted by a hemocytometer or an automated cell counter such as VICELL® or TC20. Cells can be diluted to cell densities of approximately 10,000 to 500,000 cells / mL, 50,000 to 200,000 cells / mL, or 75,000 to 150,000 cells / mL. In a non-limiting example, single-cell suspensions of PSCs are diluted to a density of approximately 100,000 cells / mL using a well-defined medium such as Essential 8 (E8®) medium.

[0099] Once a single-cell suspension of PSCs at a known concentration is obtained, the cells are typically seeded into appropriate culture vessels such as flasks, 6-well, 24-well, or 96-well tissue culture plates. Culture vessels used for culturing cells include flasks, tissue culture flasks, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as they are capable of culturing stem cells. Cells can be cultured in volumes of at least approximately 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 from which can be derived, as required for culture. In certain embodiments, the culture vessel may be a bioreactor, meaning any ex vivo device or system that supports a biologically active environment from which cells can grow. A bioreactor may have a volume of at least or approximately 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 from which can be derived.

[0100] In certain situations, PSCs, such as iPSCs, are seeded at a cell density appropriate for efficient differentiation. Generally, this density is approximately 5,000 to 40,000 cells / cm³. 2 Approximately 1,000 to 75,000 cells / cm² 2 Cells are seeded at the following cell densities. In a 6-well plate, cells can be seeded at a cell density of approximately 50,000 to 400,000 cells per well. In an exemplary method, cells are seeded at cell densities of approximately 100,000, 150,000, 200,000, 250,000, 300,000, or 350,000 cells per well, such as approximately 20,000 cells per well.

[0101] PSCs (such as iPSCs) are generally cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins such as vitronectin, laminin, collagen, and / or fibronectin, which can be used to coat the culture surface as a means of providing a solid support layer for the proliferation of pluripotent cells. The term "extracellular matrix" is a recognized term in the art and includes one or more of the following: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, ancholine, chondronectin, binding proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and karin. In an exemplary method, PSCs are grown on culture plates coated with vitronectin or fibronectin. In some embodiments, cell adhesion proteins are human proteins.

[0102] Extracellular matrix (ECM) proteins may be of natural origin, purified from human or animal tissue, or may be genetically engineered recombinant proteins or synthesized in a natural system. ECM proteins may be whole proteins or in the form of natural or modified peptide fragments. Examples of ECM proteins that can be used as a matrix for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition includes a peptide fragment of synthesized fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is heterogeneous. For example, in a heterogeneous matrix for culturing human cells, human-derived matrix components may be used, and non-human animal components may be excluded.

[0103] In some aspects, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In a more preferred embodiment, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0104] Cells such as RPE cells or PSCs can be cultured with the nutrients necessary to support the growth of each specific cell population. Cells are generally cultured in growth media containing a carbon source, a nitrogen source, and buffers to maintain pH. The media 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 include minimal essential media such as Dulbecco's Modified Eagle Medium (DMEM) or Essential 8 (E8)® medium, supplemented with various nutrients such as non-essential amino acids and vitamins to enhance the proliferation rate of stem cells. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium (MEM) Alpha, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. Furthermore, minimal essential media may be supplemented with additives such as horse, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other examples, the growth medium may include a “knockout serum substitute,” which is a serum-free formulation optimized for growing and maintaining undifferentiated cells, such as stem cells, in culture. Knockout (registered trademark) serum substitutes are disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference. It is preferable to culture the PSCs in a fully defined feeder-free medium.

[0105] Therefore, generally, single-cell PSCs are seeded and cultured in a fully defined medium. In certain situations, the medium is aspirated approximately 18-24 hours after seeding, and fresh medium, such as E8® medium, is added to the culture. In certain situations, single-cell PSCs are cultured in a fully defined medium for approximately 1, 2, or 3 days after plating. It is preferable to culture single-cell PSCs in a fully defined medium for approximately 2 days before allowing the differentiation process to proceed.

[0106] In some embodiments, the culture medium may or may not contain any serum substitute. Examples of serum substitutes include albumin (such as lipid-rich albumin, recombinant albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by the method disclosed in International Publication No. 98 / 30679. Alternatively, any commercially available material can be used more simply. Commercially available materials include KNOCKOUT® serum substitution (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX® (Gibco).

[0107] Other culture conditions can be determined as appropriate. For example, the culture temperature can be approximately 30-40°C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not limited to these. In one embodiment, cells are cultured at 37°C. The CO2 concentration can be approximately 1-10%, for example, about 2-5%, or any range within that range that can be derived. The oxygen partial pressure can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range derived therefrom.

[0108] a. Differentiation medium Retinal induction medium After single-cell PSCs attach to a culture plate, it is preferable to culture the cells in retinal induction medium to initiate the differentiation process into retinal lineage cells. Retinal induction medium (RIM) contains WNT pathway inhibitors and can induce differentiation of PSCs into retinal lineage cells. RIM further contains TGFβ pathway inhibitors and BMP pathway inhibitors. A typical composition of one RIM medium is shown in Table 3.

[0109] RIM may contain DMEM and F12 in a ratio of approximately 1:1. In exemplary cases, RIM may contain WNT pathway inhibitors (e.g., CKI-7), BMP pathway inhibitors (e.g., LDN193189), and TGFβ pathway inhibitors (e.g., SB431542). For example, RIM may contain approximately 5 nM to approximately 50 nM (e.g., approximately 10 nM) of LDN193189, approximately 0.1 μM to approximately 5 μM (e.g., approximately 0.5 μM) of CKI-7, and approximately 0.5 μM to approximately 10 μM (e.g., approximately 1 μM) of SB431542. Furthermore, RIM may contain approximately 1% to approximately 5% of knockout serum substitutes, MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplements, B-27 supplements, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, IGF1 is animal-free IGF1 (AF-IGF1) and is included in the RIM at a concentration of about 0.1 ng / mL to about 10 ng / mL (e.g., about 1 ng / mL). The culture medium is aspirated daily and replaced with fresh RIM. The cells are cultured in the RIM for about 1 to about 5 days, for example, about 1, 2, 3, 4, or 5 days, for example, about 2 days, to produce retinal lineage cells.

[0110] Retinal differentiation medium Next, retinal lineage cells can be cultured in retinal differentiation medium (RDM) for further differentiation. RDM comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In one embodiment, RDM comprises a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD0325901). Alternatively, RDM may contain a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor. Generally, the concentrations of Wnt pathway inhibitors, BMP pathway inhibitors, and TGFβ pathway inhibitors are about 9 to 11 times (e.g., about 10 times) higher in RDM compared to RIM. In an exemplary method, RDM contains approximately 50 nM to approximately 200 nM (e.g., approximately 100 nM) of LDN193189, approximately 1 μM to approximately 10 μM (e.g., approximately 5 μM) of CKI-7, approximately 1 μM to approximately 50 μM (e.g., approximately 10 μM) of SB431542, and approximately 0.1 μM to approximately 10 μM (e.g., approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM) of PD0325901. One exemplary RDM medium is shown in Table 3.

[0111] Generally, RDM contains DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute (approximately 1% to 5%, e.g., approximately 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., approximately 1 ng / mL to 50 ng / mL, e.g., approximately 10 ng / mL). In certain methods, cells are given fresh RDM daily after aspirating the culture medium, starting from the day before culture. Generally, cells are cultured in RDM for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days (e.g., approximately 7 days) to induce differentiated retinal cells.

[0112] Retinal culture medium Next, differentiated retinal cells can be further differentiated by culturing them in retinal medium (RM). Retinal medium contains activin A and may also contain nicotinamide. RM may contain approximately 50–200 ng / mL (e.g., approximately 100 ng / mL) of activin A and approximately 1 mM–50 mM (e.g., approximately 10 mM) of nicotinamide. Alternatively, RM may contain other TGF-β activators such as GDF1, and / or WNT pathway activators such as WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindilbine-3'-oxime), or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Alternatively, RM may further contain WNT3a. One example of RM medium is shown in Table 3.

[0113] RM may contain DMEM and F12 in a ratio of approximately 1:1, approximately 1% to approximately 5% (e.g., approximately 1.5%) of knockout serum substitute, MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The medium can be changed daily in RM at room temperature. Cells are generally cultured in RM for approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days, for example, approximately 10 days, to induce differentiated RPE cells.

[0114] RPE maturation medium For further differentiation of RPE cells, it is preferable to culture the cells in RPE maturation medium (RPE-MM). An example of RPE-MM medium is shown in Table 3. RPE-Maturation medium contains approximately 100 μg / mL to approximately 300 μg / mL (e.g., approximately 250 μg / mL) of taurine, approximately 10 μg / L to approximately 30 μg / L (e.g., approximately 20 μg / L) of hydrocortisone, and approximately 0.001 μg / L to approximately 0.1 μg / L (e.g., approximately 0.013 μg / L) of triiodothyronine. Furthermore, RPE-MM may contain MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), and sodium pyruvate, and fetal bovine serum (approximately 0.5% to approximately 10%, e.g., approximately 1% to approximately 5%). The medium may be changed every other day with RPE-MM at room temperature. Generally, cells are cultured in RPE-MM for about 5 to 10 days, for example, about 5 days. Afterward, the cells are dissociated using cell dissociation enzymes, re-seeded, and the culture period is further extended to about 5 to 30 days (for example, about 15 to 20 days) to further differentiate them into RPE cells. In a further embodiment, RPE-MM does not contain WNT pathway inhibitors. RPE cells can be cryopreserved at this stage.

[0115] b. Maturation of RPE cells Subsequently, RPE cells can be continuously cultured in RPE-MM to allow them to mature. In some embodiments, RPE cells are grown in wells 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, e.g., about 6 to 8 weeks, e.g., 6, 7, or 8 weeks. An exemplary method for the continuous maturation of RPE cells involves dissociating the cells with a cell-dissociating enzyme such as TRYPLE®, re-seeding them on a special, degradable scaffold assembly such as a SNAPWELL® design, and culturing them for about 1 to 2 weeks in RPE-MM containing a MEK inhibitor such as PD0325901. Alternatively, RPE-MM may contain a bFGF inhibitor instead of a MEK inhibitor. A method for culturing RPE cells on a degradable scaffold is taught in PCT Publication WO2014 / 121077, which is incorporated herein by reference in its entirety. In short, the main components of this method are Corning® Costar® Snapwell® plates, bioinert O-rings, and a biodegradable scaffold. The Snapwell® plate provides the structure and platform for the biodegradable scaffold. The microporous membranes forming the apical and basal sides not only provide support for the scaffold but are also ideal for isolating the polarized layers of cells from their respective sides. The ability of the Snapwell® insert to peel the membrane allows the support ring of the insert to be used as an anchor for the scaffold. The resulting differentiated, polar, and confluent monolayer functional RPE cells can be cryopreserved at this stage (e.g., using heterogeneous component-free CS10 medium).

[0116] In some embodiments, mature RPE cells can be further developed into a monolayer of functional RPE cells that function as intact (complete) RPE tissue by serially culturing them in RPE-MM with additional chemicals or small molecules that promote RPE maturation. For example, these small molecules include primary ciliary inducers such as prostaglandin E2 (PGE2) or affidicorin. PGE2 can be added to the culture medium at concentrations of about 25 μM to about 250 μM, for example, about 50 μM to about 100 μM. Alternatively, RPE-MM may contain canonical WNT pathway inhibitors. Exemplary canonical WNT pathway inhibitors are N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). Cells can be cultured in this medium for an additional period, e.g., about 1 to 5 weeks, e.g., about 2 to 4 weeks, to obtain a mature and functional RPE cell monolayer. Thus, the method disclosed herein provides mature RPE cells for clinical use that can be replicated on a large scale and consistently from a single-cell suspension of pluripotent cells.

[0117] c. Cryopreservation of RPE cells Retinal pigment epithelial cells produced by the methods disclosed herein can be cryopreserved (see, for example, PCT Publication 2012 / 149484A2, incorporated herein by reference). Cells can be cryopreserved with or without a substrate. In some embodiments, storage temperatures include 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 therebetween. In some embodiments, low temperatures are used for preserving (e.g., maintenance) cryopreserved cells. In some embodiments, cells are preserved using liquid nitrogen (or other similar liquid coolant). In further embodiments, cells are preserved for longer than about 6 hours. In further embodiments, cells are preserved for about 72 hours. In some embodiments, cells are preserved for 48 hours to about 1 week. In yet another embodiment, cells are preserved 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 on a substrate such as any substrate disclosed herein.

[0118] In some embodiments, cryoprotective agents may be used in addition. For example, cells can be cryopreserved in a cryopreservation solution containing one or more cryoprotective substances such as DM80, human or bovine serum albumin, or other serum albumins. In certain 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%, 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 certain embodiments, the solution contains 2.5% DMSO. In yet another particular embodiment, the solution contains 10% DMSO.

[0119] Cells can be cooled at a rate of approximately 1°C / min, for example, during cryopreservation. In some embodiments, the cryopreservation temperature is approximately -80°C to approximately -180°C, or approximately -125°C to approximately -140°C. In some embodiments, cells are cooled to 4°C at a rate of 1°C / min before freezing. Cryopreserved cells can be thawed and used by transferring them to the vapor phase of liquid nitrogen. In some embodiments, for example, when the cells reach approximately -80°C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be performed using a rate-controlled freezer. Cryopreserved cells can be thawed at a temperature of approximately 25°C to approximately 40°C, typically at approximately 37°C.

[0120] d. Inhibitors WNT pathway inhibitors WNTs are a family of highly conserved secretory signaling molecules that regulate intercellular interactions and are associated with wingless, a polarity gene in the Drosophila segment. Human WNT family genes encode 38-43 kDa cysteine-rich glycoproteins. WNT proteins possess a hydrophobic signaling sequence, a conserved asparagine-binding oligosaccharide consensus sequence (see, e.g., Shimizu et al., Cell Growth Differ 8:1349-1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote cytoplasmic beta-catenin stabilization, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain types of cancer.

[0121] In this specification, the term "WNT inhibitor" basically means a WNT inhibitor. Therefore, a WNT inhibitor refers to any inhibitor of a WNT family protein member, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16. In certain embodiments of this method, the term relates to a WNT inhibitor in a differentiation medium. Examples of suitable WNT inhibitors known in this field 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]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), and 2-phenoxybenzoic acid-[(5-methyl-2-furanyl) Examples include methylene hydrazide (PNU74654), 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1), and secreted frizzled-related protein (SFRP1). Furthermore, WNT inhibitors may include antibodies against WNT, dominant-negative variants of WNT, and WNT siRNA, as well as antisense nucleic acids that suppress WNT expression. WNT can also be inhibited using RNA-mediated interference (RNAi).

[0122] BMP pathway inhibitors Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor beta (TGFβ) superfamily. BMPs are thought to constitute a group of morphogenetic signals crucial for regulating the structure of the entire body. The important physiological functions of BMP signaling manifest themselves in numerous roles related to dysregulated BMP signaling in pathological processes.

[0123] BMP pathway inhibitors generally refer to inhibitors of BMP signaling in general, or may include 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]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), and 4-[6-(4-(1-methylethoxy] Examples include oxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347).

[0124] TGFβ pathway inhibitors Transforming growth factor beta (TGFβ) is a secreted protein that regulates proliferation, cell differentiation, and other functions in most cells, and is a type of cytokine that functions in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. TGFβ exists in at least three isoforms called TGFβ1, TGFβ2, and TGFβ3. The TGFβ family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which also includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplesic, and Vg-1.

[0125] TGFβ pathway inhibitors generally include any inhibitor of TGFβ signaling. For example, TGFβ pathway inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imi Dazole-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide hydrate, left-right determinant (Lefty), 3-(6-methyl-2-pyridinyl (A83-01)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide These include (GW788388), 4-[3-(2-pyridinyl))-1H-pyrazole-4-yl]quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine (RepSox).

[0126] MEK inhibitors MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2 and 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-trioxopyrido[4,3-d]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), and 6-(4-bromo-2-fluoroanilino)-7-fluoro-N Examples include -(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).

[0127] bFGF inhibitors Basic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the basement membrane and the extracellular matrix of vascular endothelial cells. Furthermore, bFGF is a common component of human ESC medium, where cells need to remain in an undifferentiated state.

[0128] The bFGF inhibitor generally means an inhibitor of bFGF. Examples of the bFGF inhibitor 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), 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 hydrate (PD166285), N-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD166866), and MK-2206.

[0129] IV. Use of Retinal Pigment Epithelial Cells In certain aspects, provided is a method for producing an RPE or RPE-enriched cell population that can be used for many important research, development, and commercial purposes.

[0130] In some aspects, from the methods disclosed herein, at least about 90% (e.g., at least or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or any range derived therefrom) of RPE cells constitute at least or about 10 6 10 7 10 8 5x10 8 10 9 10 10 (or any range derived therefrom) number of cell populations are generated.

[0131] In certain aspects, the starting cells for the method are at least or about 10 4 10 5, 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 This may involve the use of individual cells or any range of cells derived therefrom. The starting cell population is at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 Seeding can be performed at a density of cells / ml, or any range of densities derived therefrom.

[0132] RPE cells produced by the methods disclosed herein can be used in any method and application currently known in the art for RPE cells. For example, a method for evaluating compounds can be provided, which includes assaying the pharmacological or toxicological properties of a compound on RPE cells. A method for evaluating a compound for its effect on RPE cells can also be provided, which includes a) contacting the RPE cells provided herein with the compound; and b) assaying the effect of the compound on RPE cells.

[0133] A. Screening of test compounds RPE cells can be commercially utilized to screen for factors (solvents, small molecule drugs, peptides, oligonucleotides, etc.) or environmental conditions (culture conditions or manipulations, etc.) that affect the properties of such cells and their diverse progeny. The test compounds may be, for example, compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.

[0134] In one embodiment, the method involves contacting RPE cells with a test drug and determining whether the test drug modulates the activity or function of RPE cells in a population. In some applications, screening assays are used to identify drugs that modulate RPE cell proliferation or alter RPE cell differentiation. Screening assays can be performed in vitro or in vivo. Methods for screening and identifying ophthalmic or RPE drugs include those suitable for high-throughput screening. For example, to identify molecules that may be usable as therapeutic molecules, RPE cells can be placed or set on a culture dish, flask, roller bottle, or plate (e.g., a single multi-well dish, or 8, 16, 32, 64, 96, 384, and 1536-well multi-well plates or dishes) at a predetermined location as needed. Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0135] Other applications of screening systems relate to testing the effects of pharmaceutical compounds on the maintenance or repair of retinal tissue. Such screenings may be performed because compounds designed to have pharmacological effects on cells or other sites may have unintended side effects on cells of this tissue type.

[0136] B. Treatment and transplantation In other embodiments, the use of RPE cells can also be provided to enhance the maintenance and repair of ocular tissue in any condition requiring it, such as retinal degeneration or severe damage.

[0137] To determine the suitability of a cell composition for therapeutic administration, the cells can first be tested in a suitable animal model. In one aspect, RPE cells are evaluated for their ability to survive and maintain their phenotype in vivo. The cell composition is administered to immunodeficient animals (e.g., nude mice, or animals immunodeficient by chemical or radiation). The tissue is harvested after a period of growth and evaluated for the presence of pluripotent stem cell-derived cells.

[0138] Many animal species are available to test the compatibility of RPE cell compositions. For example, the Royal College of SurgeoN's (RCS) rat is a well-known model for retinal dystrophy (Lund et al., 2006). Furthermore, the compatibility and viability of RPE cells can be determined by transplantation into Matrigel (e.g., subcutaneous or subretinal) of immunodeficient animals such as NOG mice (Kanemura et al., 2014).

[0139] The human RPE cells described herein, or pharmaceutical compositions containing these cells, can be used to manufacture drugs for treating conditions in patients that require them. RPE cells can be cryopreserved in advance. In certain aspects, the disclosed RPE cells are iPSC-derived and therefore can be used to provide “personalized medicine” to patients with eye diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations, differentiate into RPE cells, and form RPE tissue. This RPE tissue can then be used to replace endogenous degenerated RPE cells from the same patient. Alternatively, iPSCs generated from a healthy donor or an HLA homozygous “superdonor” can be used. RPE cells can be treated in vitro with certain factors such as pigment epithelial-derived factor (PEDF), transforming growth factor (TGF) beta, and / or retinoic acid to create an in vivo anti-inflammatory and immunosuppressive environment.

[0140] Various eye conditions can be treated or prevented by introducing RPE cells obtained using the methods disclosed herein. Such conditions include retinal diseases or disorders generally associated with retinal dysfunction or decomposition, retinal injury, and / or loss of retinal pigment epithelium. Treatable conditions include, but are not limited to, degenerative diseases of the retina such as Stargardt macular dystrophy, pigmentary retinitis, macular degeneration (including age-related macular degeneration), glaucoma, and diabetic retinopathy. Other conditions include Lieber congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, gynostosis, total choroidal atrophy, pattern dystrophy, other dystrophies of RPE, and RPE and retinal damage caused by any one of the following: light, laser, inflammatory, infectious, radioactive, neovascular, or traumatic injury. In certain embodiments, a method is provided for treating or preventing conditions characterized by retinal degeneration, comprising administering an effective amount of a composition containing RPE cells to a subject in need. Such a method may involve selecting a subject having 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 a target site in the form of a cell suspension. Alternatively, they may be adhered as a monolayer on a matrix such as a biodegradable polymer, an extracellular matrix or a substrate, or a combination of the above. RPE cells can also be transplanted together with other retinal cells, such as photoreceptors (co-transplantation). In some embodiments, RPE cells are produced from iPSCs derived from the subject to be treated and are therefore autologous. In other embodiments, RPE cells are produced from an MHC-matched donor.

[0141] In some embodiments, RPE cells can be used for autologous RPE grafts for subjects suitable for regenerative medicine. RPE cells can be transplanted in combination with other retinal cells, such as photoreceptors. RPE cells produced by the disclosed method can be transplanted by various techniques well known in the art. For example, methods for performing RPE transplantation are described in U.S. Patents 5,962,027 and 6,045,791, each of which is incorporated herein by reference in whole. According to one embodiment, transplantation is performed by performing a vitrectomy of the pancreas followed by delivery of cells to the subretinal region via a small retinal opening or by direct injection. RPE cells can be introduced to the target site in the form of a cell suspension and may be attached 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, such as retinal cells having photoreceptors (co-transplantation). Thus, compositions comprising RPE cells obtained by the method disclosed herein are provided. In some embodiments, these RPE cells contain nucleic acids encoding tyrosinase enhancers and markers operably linked to a promoter. In other embodiments, the RPE cell also includes a second constitutive promoter operably linked to a nucleic acid encoding a second marker.

[0142] Pharmaceutical compositions of RPE cells prepared by the methods disclosed herein. These compositions contain at least about 1 × 10⁻⁶ cells. 3 RPE cells, approximately 1 × 10⁶ 4 RPE cells, approximately 1 × 10⁶ 5 RPE cells, approximately 1 × 10⁶ 6 RPE cells, approximately 1 × 10⁶ 7 RPE cells, approximately 1 × 10⁶ 8 A single RPE cell, or 1 × 10⁶ 9It may contain RPE cells. In certain embodiments, the composition is a substantially purified (with respect to non-RPE cells) preparation containing differentiated RPE cells produced by the methods disclosed herein. Compositions are also provided that contain a scaffold, such as a polymer carrier, and / or an extracellular matrix, and an effective amount of RPE cells produced by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in vivo. Examples of physiologically acceptable materials include absorbable and / or non-absorbable solid matrix materials such as small intestinal submucosa (SIS), cross-linked or non-cross-linked alginates, hydrophilic colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, fleece, and bioadhesives.

[0143] Suitable polymer carriers also include synthetic solutions or natural polymers, as well as porous meshes or sponges formed from polymer solutions. The matrix is, for example, a polymer mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; as well as polysaccharides such as alginic acid and hyaluronic acid polymers. Synthetic polymers include both biodegradable and non-biodegradable polymers. Biodegradable polymers include, for example, polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA) and polylactic acid-glycolic acid (PGLA), polyoltoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetates, and polyvinyl alcohols.

[0144] Polymers capable of forming malleable, ionically or covalently crosslinked hydrogels can be used. Hydrogels are substances formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure that traps water molecules and forms a gel. Examples of materials that can be used to form hydrogels include ionically crosslinked polysaccharides such as alginic acid, polyphosphatidine, and polyacrylates, or block copolymers such as PLURON1CS® or TETRON1CS®, or polyethylene oxide-polypropylene block copolymers that are crosslinked by temperature or H, respectively. Other materials include proteins such as fibrin, and polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0145] The pharmaceutical composition can be packaged in an appropriate container, as needed, along with instructions for use, for a desired purpose such as the reconstitution of RPE cell function to improve diseases or abnormalities of retinal tissue. In some embodiments, RPE cells produced by the disclosed method can be manipulated to form RPEs and used to replace degenerated RPEs in a subject requiring them.

[0146] C. Distribution for commercial, therapeutic, and research purposes. In some embodiments, a reagent system is provided which comprises a set or combination of cells containing an RPE-rich cell population that is present at any time during production, distribution, or use. The cell set comprises any combination of the cell population described herein and undifferentiated pluripotent stem cells or other differentiated cell types, which often have the same genome. Each cell type may be packaged together or in separate containers, at the same time or at different times, within the same facility or in different locations, under the control of the same or different organizations sharing a business relationship.

[0147] The pharmaceutical composition may be packaged in an appropriate container along with instructions for use relating to the desired purpose (e.g., reconstitution of RPE cell function to improve disease or damage of eye tissue), as needed.

[0148] V. Kit In some embodiments, a kit is provided which may contain, for example, one or more culture media and components for producing RPE cells. The reagent system may be packaged in either an aqueous medium or a lyophilized form, as needed. The kit's container means generally includes at least one vial, test tube, flask, bottle, syringe or other container means in which the components are placed, preferably appropriately dispensed. If the kit contains two or more components, the kit generally includes a second, third or other additional container in which the additional components may be placed separately. Also, the vials may contain components in various combinations. The components of the kit may also be supplied as dry powder. If the reagents and / or components are supplied as dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may be supplied in a separate container means. The kit also typically includes means for tightly packaging the kit components for commercial sale. Such containers may include injection or blow-molded plastic containers in which the desired vials are held. The kit may also include instructions in printed or electronic format, such as digital. [Examples]

[0149] VI. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors to function well in the implementation of the present invention and thus constitute a preferred mode for its implementation. However, those skilled in the art will recognize that, in light of this disclosure, many modifications can be made in the particular embodiments disclosed without departing from the spirit and scope of the present invention, and similar or comparable results can be obtained.

[0150] Example 1 - Preparation of a starting pluripotent stem cell population The starting population of RPE cells may be derived from ES cells and pluripotent stem cells such as iPSCs. In exemplary methods, RPE cells are derived from human iPSCs reprogrammed from somatic cells by methods known in the art, such as U.S. Patents No. 8,546,140, ​​8,741,648, 8,691,574, Published U.S. Patent Application No. 20090246875, Published U.S. Patents No. 8,278,104, 9,005,967, 8,058,065, 8,129,187, International Publication No. 2007 / 069666A1, U.S. Patents No. 8,183,038 and 8,268,620, which are incorporated herein by reference. For example, pluripotent stem cells were produced from somatic cells using the nuclear programming factors Oct4, Sox2, c-Myc, and Klf4. In another exemplary method, pluripotent stem cells were produced from somatic cells using nuclear programming factors Oct4, Sox2, Nanog, Lin28, L-Myc, and the SV40 large T antigen.

[0151] iPSCs were grown in a fully defined medium, such as Essential 8™ medium, in plates coated with vitronectin, without a mouse or human feeder layer. Vitronectin was diluted 200-fold with calcium or magnesium-free DPBS, and culture plates were coated with this vitronectin and incubated at room temperature for approximately 1 hour. To prevent unhealthy and / or differentiated cells, iPSCs were divided before they reached confluence and were not permitted to grow any further (Figure 1A).

[0152] To induce RPE cells, iPSCs were dissociated as a single-cell suspension, and aggregates or embryoid bodies were removed. To obtain a single-cell suspension, the cells were washed with DPBS and incubated in a cell dissociation enzyme such as TRYPLE® at 37°C for approximately 10 minutes. The cells were then separated by pipetting using a serological pipette, and the cell suspension was collected in a conical tube. If the cells did not separate by gentle pipetting, the culture was incubated for a longer period, e.g., 2-3 minutes. To recover all cells, the culture vessel was washed with E8® medium at room temperature, and the medium was then added to the tube containing the cell suspension. Furthermore, brevistatin (e.g., 2.5 μM) was added to the E8® medium to ensure high viability of the PSC cells after dissociation into single cells, even if the cells did not adhere to the culture vessel. To recover the cells, they were centrifuged at 400xg for approximately 5 minutes, the supernatant was aspirated, and the cells were resuspended in an appropriate volume of E8® medium.

[0153] To efficiently differentiate RPE cells from single iPSCs, the input density of single iPSC cells is accurately counted using an automated cell counter such as VICELL (trademark), and approximately 1 × 10⁶ cells are obtained at room temperature using E8 (trademark). 5 The cells were diluted to obtain a cell suspension of cells / mL. Once a single-cell suspension of iPSCs was obtained at a known cell density, the cells were seeded into a suitable culture vessel, such as a 6-well plate coated with vitronectin. Cells were seeded at a cell density of approximately 200,000 cells per well and placed in a humidified incubator at 37°C. After approximately 18–24 hours, the medium was aspirated and fresh E8® medium was added to the culture. To ensure proper adhesion to the plate, the cells were cultured in E8® medium for approximately 2 days after seeding.

[0154] Example 2 - Differentiation of iPSCs into RPE cells Single-cell iPSCs seeded at an appropriate cell density were cultured for approximately 2 days as in Example 1, and then cultured in various differentiation media to induce RPE cells. On day 3, E8® medium was aspirated and room temperature retinal induction medium (RIM) (e.g., Table 3) was added. Briefly, RIM contains DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RIM contained WNT pathway inhibitors, BMP pathway inhibitors, TGFβ pathway inhibitors, and insulin growth factor 1 (IGF1). The medium was aspirated daily and fresh RIM was added to the cells. The cells were cultured in RIM for approximately 2–4 days.

[0155] Next, the cells were cultured in retinal differentiation medium (RDM) for approximately 7–14 days. Briefly, RDM (Table 2) contained DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. Furthermore, RDM included a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD325901). The concentrations of the Wnt pathway inhibitor, BMP pathway inhibitor, and TGFβ pathway inhibitor were 10 times higher in RDM than in RIM. The medium was aspirated daily, and room temperature RDM was added to the cells to produce differentiated retinal cells.

[0156] To induce RPE cells, the cells were then cultured in retinal medium (RM) for 7–10 days. The RM contained DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RM contained nicotinamide and activin A. RPE cells were obtained by changing the medium daily in room temperature RM.

[0157] To mature RPE cells, the cells were cultured in RPE maturation medium (RPE-MM) for 5–10 days. RPE-MM (Table 2) contained MEM alpha, fetal bovine serum, N-2 supplement, MEM NEAA, and sodium pyruvate. In addition, RPE-MM contained taurine, hydrocortisone, and 3,3',5-triiodo-L-thyronine (Figure 1C). The medium was changed every other day with RPE-MM at room temperature. The cells were then dissociated with cell dissociation enzymes and re-seed on plates coated with vitronectin. The induced PRE cells can be cryopreserved at this stage in heterogeneous component-free CS10 medium. To continue RPE maturation, the seeded cells were cultured for a further 15 days.

[0158] Example 3 - Maturation of RPE cells To further mature the RPE cells produced in Example 2, the cells were dissociated with a cell-dissociating enzyme such as Triple™ and re-seeded into a special SNAPWELL™ design degradable scaffold assembly in RPE-MM supplemented with a MEK inhibitor, e.g., PD325901, for 1-2 weeks. This yielded a monolayer of differentiated, polarized, and confluent functional RPE cells that could be cryopreserved at this stage using heterogeneous component-free CS10 medium (Figure 1D).

[0159] Mature RPE cells were further developed into a functional RPE cell monolayer that functions as intact RPE tissue by continued culture in RPE-MM supplemented with small molecules such as PGE2 or affidicorin. While not theoretically bound, these primary ciliary inducers suppress the canonical WNT pathway, induce cell cycle departure within cells, and induce apical-basal polarization in the RPE monolayer. Alternatively, RPE maturation can be induced by canonical WNT pathway inhibitors such as IWP2 and endo-IWR1, which induce cell cycle departure in RPE cells to promote RPE maturation. To obtain a mature and functional RPE cell monolayer, the cells were cultured in this medium for a further 2-3 weeks. Thus, the method disclosed hereof provides pluripotent cell-derived mature RPE cells that can be replicated consistently on a large scale for clinical use.

[0160] Example 4 - Cryopreservation of RPE cells To cryopreserve the differentiated RPE cells from Example 2, the culture medium was aspirated and the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS). The cells were then incubated with cell dissociation enzymes, and the cell suspension was pipetted into a conical tube. The cells were centrifuged, the supernatant was aspirated, and the cells were resuspended in room temperature RPE-MM. The cell suspension was then filtered through a STERIFLIP® cell strainer, and the cells were counted. The cells were then centrifuged and transferred to a chilled CryoStor® CS10 at an appropriate density (e.g., 1 × 10⁻⁶). 7 The cells were resuspended in (cells / mL). The cell suspension was divided into pre-labeled cryovials, placed in a freezer container, and stored in a -80°C freezer for 12-24 hours. The vials were then transferred to liquid nitrogen for storage.

[0161] Example 5 - Enrichment of RPE starting cell population by MACS depletion and CD24, CD56 and / or CD90 depletion of contaminated non-RPE cells The RPE cell population obtained in Example 2 or 3 may contain residual contaminated non-RPE cells and immature RPE cells (collectively referred to as "contaminated cells"), both of which can be isolated and removed to obtain a cell population rich in mature RPE cells. Contaminated cells can be removed from the culture by various methodologies, such as magnetically activated cell sorting (MACS®), fluorescence-activated cell sorting (FACS), or single-cell sorting. Using the MACS® methodology, which is known in the field to be able to separate various cell populations based on their surface antigens, contaminated cells were isolated from the more mature target RPE cells.

[0162] Contaminated cells present in a starting population of RPE cells possess specific cell surface markers that can be used to isolate the contaminated cells from the mature RPE cells of interest. For example, CD24, CD56, and / or CD90 are cell surface antigens expressed on pluripotent stem cells and other neuronal cell types (but not limited to these). CD24 is a glycoprotein expressed on the surface of pluripotent stem cells, some B lymphocytes, and differentiating neuroblasts. CD56, or neuronal 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 as well as neurons. The expression of CD24, CD56, and / or CD90 is lost during the process in which stem cells differentiate into many mature cell types, including RPE cells. Therefore, residual contaminated cells can be depleted by removing cells positive for CD24, CD56, and / or CD90.

[0163] To perform the separation technique, it is desirable to dissociate the starting population of RPE cells into single-cell suspensions for the sorting to be performed (e.g., MACS). If the cells have already been cryopreserved, they need to be thawed and reseeded. To obtain single-cell suspensions from cells contained in adherent cultures, the cells were washed (e.g., with DPBS) and a cell dissociation enzyme was added (e.g., Triple®). After incubating the cells at 37°C for about 5 minutes, the neuron clusters were detached by gently tapping the container. The cells were washed twice with DPBS and a cell dissociation enzyme was added (e.g., Triple®). After incubating the cells at 37°C for about 30 minutes, the cell suspension was collected in RPE-MM plating medium and centrifuged at 400xg for 5 minutes. The cell pellet was resuspended in RPE-MM plating medium, and the cell suspension was filtered through a cell strainer (e.g., a 20 μM slit-slip cell strainer) to dissociate the remaining cell clusters. Cell suspensions containing viable cells were counted (e.g., using a Bissell counter) and cell concentrations were examined. The counted cell suspensions provided single-cell suspensions that could be used for sorting or flow cytometry purity assays.

[0164] To remove contaminated cells from the RPE cell starting population, MACS was used to deplete CD24-positive, CD56-positive, and / or CD90-positive cells. After dissociating cells from the RPE cell starting population into single-cell suspensions, the cells were placed in MACS buffer, for example, 1 × 10⁶ cells. 7 The cells were resuspended in MACS buffer at a concentration of cells / mL. An example of MACS buffer is shown in Table 3. Next, the cells were stained with anti-CD24 antibody, anti-CD56 antibody, and / or anti-CD90 antibody (each diluted to 1:500), and incubated 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 label that binds to the secondary antibody (e.g., FITC). After incubation, 20 mL of MACS buffer was added, and the cells were centrifuged at 400xg for 5 minutes. The cell pellet was resuspended in 20 mL of MACS buffer, mixed vigorously, and centrifuged at 400xg for 5 minutes to remove unbound antibodies. The cell pellet was then added to MACS buffer (e.g., 1.11×10⁶). 8The cells were resuspended (at 1 / mL), and microbeads coated with diluted (1:10) secondary antibody (e.g., anti-FITC) were added. The cells were incubated at 4°C for 20 minutes. After incubation, the cells were washed with MACS buffer to remove unbound microbeads, and 1.25 × 10⁶ microbeads were removed. 8 The cells were resuspended in 500 μL of MACS buffer. The cell suspension was transferred to an LD column placed in a strong magnetic field, and cells expressing CD24, CD56, and / or CD90 antigens attached to microbeads remained in the column. The LD column was washed twice with MACS buffer. Unlabeled cells not expressing CD24, CD56, and / or CD90 antigens were eluted and collected. For further characterization and culture, the collected unlabeled cell suspension was centrifuged (400xg for 5 minutes), reseeded in RPE-MM seeding medium, and a certain amount of the cell suspension was used for a flow cytometry purity assay. In this way, MACS cell sorting yielded an RPE-rich cell population depleted of cells positive for CD24, CD56, and / or CD90. The use of this method is not limited to the starting population resulting from the method detailed in Example 2, but can also be used to remove contaminated cells from RPE cell populations produced by other methods (for example, but not limited to, the methods described in U.S. Patent Applications No. 12 / 523,444 and No. 14 / 405,730). [Table 1] The preliminary selection rate of cells positive for the RPE marker represents the proportion of RPE marker-positive cells in the starting population of RPE cells in Example 2. Depleting both CD24-positive and CD56-positive cells enriches RPE cells more than depleting only CD24-positive cells. When all CD24-positive, CD56-positive, and CD90-positive cells were depleted, the purity of RPE cells in the cell population exceeded 99%.

[0165] Example 6 - Flow cytometry purity assay for characterization of RPE-enriched cell populations Before and after MACS sorting, RPE cells were characterized using a panel of relevant markers including BEST1, CRALBP, TYRP1, PMEL17, MAP2, NES, and MITF (e.g., pre-sorting and post-sorting). Flow cytometry purity assays were performed to obtain measured percentages of cells positive for each marker before and after MACS-based removal of CD24-positive, CD56-positive, and / or CD90-positive cells (Table 1) (Figures 2 and 3).

[0166] Flow cytometry purity assays were performed to determine the percentage of RPE cells obtained by the selection method described herein. A fixed amount of cell suspension (2 × 10⁶ cells per sample in a 5 mL FACS tube) was collected from the MACS assay. 6 The cells were centrifuged at 400 × g for 3 minutes. The cell pellet was resuspended in 1 mL of staining solution (e.g., Live-Dead red staining solution) 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 solution. 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, discarding the supernatant. The cell pellet was resuspended in 2 mL of wash buffer and incubated at 1 × 10⁶. 6The cells were prepared as a cell / mL suspension, and 200 μL of the cell suspension was transferred to a FACS tube. 2 mL of palm buffer was added to each tube, and the cells were centrifuged at 400 × g for 3 minutes. The primary antibody for the RPE-specific marker was diluted in palm 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 palm buffer. The secondary antibody solution was added to each tube, and the cells were incubated in the dark at room temperature for 1–2 hours. After incubation, the cells were washed twice with palm buffer, centrifuged (400 × g for 3 minutes), and resuspended in 100 μL of wash buffer for flow cytometry analysis. Flow cytometry analysis was performed by methods known to those skilled in the art, as described in U.S. Patent No. 8,682,810 (incorporated herein by reference) and Herzenberg et al., 2006, et al., and the percentage of positive cells was obtained for each marker tested (Table 1). Flow cytometry purity assays showed that MACS sorting, which depletes CD24, CD56, and / or CD90-positive contaminated cells, yielded a population rich in RPE cells (95–99%) compared to the percentage in the starting cell population determined by the BEST1 marker (78.6%).

[0167] Example 7 - Another method for differentiating RPE cells With respect to the methods described in Examples 2 and 3, including 1 μM PD0325901 in the culture medium within a specific time frame from day 2 after iPSC seeding until the end of the differentiation process (including culture after MACS) may improve the maturity and purity (meaning a reduction in contaminating cells) of the resulting RPE population. In the RPE processes described herein, it has been shown that including 1 μM PD0325901 in RDM and RPE-MM (approximately 42–50 days) improves both the purity and maturity of the RPE population.

[0168] Example 8 - Another method for differentiating RPE cells With respect to the methods described in Examples 2 and 3, reducing the percentage of fetal bovine serum in RPE-MM and RPE-MM plate media from 5% to 0.5-1% may improve the purity of the RPE population (meaning a reduction in contaminated cells) and the resulting maturity of the RPE population.

[0169] Example 9 - Functionality of mature RPE cells To analyze mature RPE cells produced from PGE2 treatment, immunostaining of the RPE monolayer was performed, and ZO1 staining and transmission electron microscopy of iPSC-RPE cells confirmed the hexagonal structure of tight junctions (Figures 4A-4C) (Figure 4D). This staining showed that PGE2-treated RPE cells exhibited decreased beta-catenin and increased RPE65. Treatment with IWP2 + endo-IWR1 or IWP2 also resulted in decreased beta-catenin (Figure 5A) and increased RPE65 (Figure 5C). The IWP2 + endo-IWR1 combination was found to be more effective than IWP2 alone or endo-IWR1 alone. Therefore, mature RPE cells are produced by treatment with PGE2, IWP2, or IWP2 + endo-IWR1.

[0170] To measure the barrier function of RPE cells generated by the method of the present invention, transepithelial potential (TEP) is used to measure the ion gradient across the entire monolayer generated by energy-driven ion pumps that control intercellular flow, and transepithelial electrical resistance (TER) is used to measure the resistance of materials through the paracellular space, mainly through the ultrastructure of tight junctions (Figure 7A).

[0171] Functional analysis of mature RPE cells treated with IWP2 or endo-IWR1 was also performed. Comparison of TEP and TER measurements between RPE cells treated with PGE2 or IWP2 + endo-IWR1 and untreated RPE cells showed that the functionality of treated mature RPE cells was improved (Figures 7C-7E).

[0172] Next, we tested whether increasing the PGE2 concentration in the RPE-MM+PGE2 medium from 50 μM to 100 μM improved both the purity of the RPE population (i.e., reduction in contaminated cells) and the resulting maturity of the RPE population. To determine the maturity and functionality of 50 μM and 100 μM PGE2-treated cultures, barrier function was measured in terms of transepithelial electrical resistance (TER) (Figure 7F), comparing the resistance of substances passing through the paracellular space, as described in Example 9. To measure the percentage of pure RPE cells obtained after treating iPSC-derived RPE cultures with 50 μM or 100 μM in RPE-MM+PGE2 medium, flow cytometry purity assays were performed for RPE-specific markers, as described in Example 6 (Figure 7G). The results showed that higher concentrations of primary ciliary inducer PGE2 promoted both the purity and maturity of the RPE population during the process of iPSC-derived RPE differentiation.

[0173] Example 10 - Reproducibility of the RPE differentiation method To test the reproducibility of the RPE differentiation process, the differentiation of three iPSC lines into RPE cells was performed by multiple operators (Figure 6A). The average purity of the obtained RPE cells was analyzed by measuring the RPE marker retinal aldehyde-binding protein 1 (Craplbp) by flow cytometry (Table 2). The RPE differentiation process was found to be highly reproducible even when the starting cell populations and operators were different. Furthermore, reproducibility was confirmed by RPE differentiation from different starting cell lines, including 3D1, AMD1B, BEST1L, BEST3A, BEST8A, AMD donor 3D, and HLA cell line A (Figure 6B). HLA cell line A (21525.102) is an iPSC line produced from donors homozygous for HLA-A*01 and HLA-B*08, providing beneficial similarity for 11.38% of the US population. Furthermore, we have successfully produced RPEs using this process from iPSC lines derived from HLA cell line C(21526.101), which is homozygous for HLA-A*03 and HLA-B*07, potentially providing beneficial similarity for 7.63% of the US population. The aforementioned HLA cell lines A(21525.102) and C(21526.101), which are homozygous for HLA-A and HLA-B, are the property of Cellular Dynamics International Inc. In addition, we further confirmed reproducibility by differentiating RPEs 109 times from 28 iPSC cell lines derived from 13 donors and measuring the percentage of Cralbp-positive cells before and after purification (Figure 6C-D). Although the percentage of Cralbp-positive cells after RPE differentiation varied, MACS purification consistently yielded purity of over 95%, and in most cases, results close to 100% purity. Therefore, this method of RPE differentiation is clearly more useful than any other method for producing RPE cells from embryoid bodies, as it provides more consistent and reproducible results, even when there is a wide variety of donor genotypes or when the procedure is performed by different operators. [Table 2]

[0174] Example 11 - Materials and Methods Table 3 shows the materials used in Examples 1 to 10. [Table 3] JPEG2023162277000004.jpg193170JPEG2023162277000005.jpg229170JPEG202 3162277000006.jpg202170JPEG2023162277000007.jpg206170JPEG20231622770 00008.jpg237170JPEG2023162277000009.jpg234170JPEG2023162277000010.j pg245170JPEG2023162277000011.jpg237170JPEG2023162277000012.jpg197170

[0175] The 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 can be filtered and stored at 4°C for up to 4 weeks.

[0176] Palm buffer for flow cytometry was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium-free). 1 gram of saponin was added and thoroughly mixed. The buffer can be filtered and stored at 4°C for up to 4 weeks.

[0177] The live-dead red stain for flow cytometry was prepared by diluting the live-dead stain 1000-fold with DPBS (i.e., calcium and magnesium-free). 1 × 10⁶ samples were assayed. 6 One mL of staining solution was prepared for each individual cell. The staining solution was freshly prepared before use.

[0178] The fixation buffer for flow cytometry was prepared by adding 1 mL of 36.5% formaldehyde to 8.1 mL of DPBS (i.e., calcium and magnesium-free). 1 × 10⁶ samples were assayed. 6 One mL of staining solution was prepared for each individual cell. The buffer was freshly prepared before use.

[0179] All methods disclosed and claimed herein can be prepared and performed without excessive experimentation in light of this disclosure. While the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to these methods and processes or the order of steps in the described methods without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that the same or similar results can be achieved by substituting certain chemically and physiologically relevant agents with those described herein. All such similar substitutions and modifications that are 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. References The following references are incorporated herein by reference to the extent that they provide illustrative procedures or other details that supplement those described herein. Amit et al., Developmental Biology (Dev.Bio.), 227:271-278, 2000. Buschholz et al., Stem Cells, 27:2427-2434, 2009. Billun et al., Nature, 450(7169):497-502, 2007. Hirami et al., Neuroscience Letters, 48:126-131, 2009. Kanemura et al., PLoS One, 9, 2014. Ludwig et al., Nature Biotechnology, 24:185-187, 2006b Ludwig et al., Nature Methods, 3:637-646, 2006a International Publication No. 2007 / 069666A1 International Publication No. 2014 / 121077 Smith, "Origins and Properties of Mouse Embryonic Stem Cells," 2000. Strauss et al., *Physiological Reviews*, 85:845-881, 2005. Takahashi et al., Cell, 126, 663-676, 2006 Takahashi et al., Cell, 131, 861-872, 2007. Thomson and Marschel, Latest Findings in Developmental Biology (Curr.Top.Dev.Biol.), 38:133-165, 1998. Thomson and Odoriko, Trends Biotechnol., 18(2):53-57, 2000. Thomson et al., Proceedings of the National Academy of Sciences, 92:7844-7848, 1995. U.S. Patent No. 8,546,140 U.S. Patent Application No. 2002 / 0076747 U.S. Patent Application No. 2009 / 0246875 U.S. Patent Application No. 2010 / 0210014 U.S. Patent Application No. 2012 / 0196360 U.S. Patent Application No. 2012 / 0276636 U.S. Patent No. 5,843,780 U.S. Patent No. 6,103,470 U.S. Patent No. 6,200,806 U.S. Patent No. 6,416,998 U.S. Patent No. 6,833,269 U.S. Patent No. 7,029,913 U.S. Patent No. 7,442,548 U.S. Patent No. 7,598,364 U.S. Patent No. 7,682,828 U.S. Patent No. 7,989,425 U.S. Patent No. 8,058,065 U.S. Patent No. 8,071,369 U.S. Patent No. 8,129,187 U.S. Patent No. 8,268,620 U.S. Patent No. 8,278,620 U.S. Patent No. 8,546,140 U.S. Patent No. 8,741,648 Published U.S. Patent Application No. 2003 / 0211603 Published U.S. Patent Application No. 2010 / 0003757 Shu et al., Nature Biotechnology, 19:971-974, 2001. In et al., Cell, 115:281-292, 2003 You et al., Science, 318:1917-1920, 2007

Claims

1. A population enriched in retinal pigment epithelial (RPE) cells, in which cells positive for CD56 are removed compared to a non-enriched population.

2. The population described in claim 1, obtained by a method comprising the step of enriching a starting cell population containing RPE cells prepared from human pluripotent stem cells for RPE cells by removing cells positive for CD56, thereby resulting in an RPE-enriched cell population in which RPE cells are enriched compared to the starting cell population.

3. The population described in claim 2, wherein the enrichment level of RPE cells in the RPE enriched cell population is determined by the method.

4. The population described in claim 3, wherein the enrichment level is determined through the use of TYRP1 and PMEL17.

5. A population described in any one of claims 2 to 4, wherein the RPE-enriched cell population is enriched in RPE cells compared to the starting cell population as measured by BEST1 sorting.

6. A population described in any one of claims 2 to 5, wherein the enrichment step is performed without genetically manipulating the starting cell population.

7. A population described in any one of claims 1 to 6, wherein at least 95% of the RPE-enriched cell population are RPE cells.

8. A population described in any one of claims 1 to 7, wherein at least 99% of the RPE-enriched cell population are RPE cells.

9. A population described in any one of claims 1 to 8, wherein the RPE-enriched cell population is essentially pure RPE cells.

10. A population according to any one of claims 1 to 9, wherein CD56-positive cells have been removed by magnetic bead-based sorting or fluorescence-based sorting.

11. A population described in any one of claims 1 to 10, in which cells positive for CD56 have been removed using an antibody or aptamer that recognizes CD56.

12. A population described in any one of claims 1 to 11, wherein the RPE-enriched cell population is not genetically modified.

13. A population described in any one of claims 1 to 12, wherein RPE cells are enriched by removing cells positive for CD56 and cells positive for CD90 from a starting cell population containing RPE cells prepared from human pluripotent stem cells.

14. A population described in any one of claims 1 to 13, wherein RPE cells are enriched by removing cells positive for CD56 and cells positive for CD24 from a starting cell population containing RPE cells prepared from human pluripotent stem cells.

15. A population described in any one of claims 1 to 14, wherein RPE cells are enriched from a starting cell population containing RPE cells prepared from human pluripotent stem cells by removing cells positive for CD56, cells positive for CD90, and cells positive for CD24.

16. The population of any one of claims 1 to 15, wherein the RPE cells are derived from human iPSCs, and the human iPSCs do not contain exogenous genetic elements.

17. The population of claim 16, wherein the exogenous genetic elements include retroviral and lentiviral vector elements.

Images