Method for differentiating pluripotent stem cells into retinal pigment epithelial cells

Abstract

The present invention provides a method of generating retinal pigment epithelium (RPE) cells from pluripotent stem cells (PSCs), as well as methods of using the cells. The present disclosure provides a method of generating RPE cells by differentiation of PSC and includes treating PSC with one or more of a transforming growth factor beta / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a fibroblast growth factor / ERK pathway signaling inhibitor, and a TGF beta family protein. Uses of such RPE cells include methods of treating macular degeneration by administering RPE cells to a subject in need thereof.

Description

[0001] Citations of Related Applications

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 340,736, filed on May 11, 2022. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated herein by reference in its entirety into the disclosure of the present application. Technical Field

[0003] The present invention relates generally to retinal pigment epithelial (RPE) cells, and more particularly to methods of generating RPE cells from pluripotent stem cells (PSCs). Background Art

[0004] Retinal pigment epithelium (RPE) forms a single cell layer of cells below the photoreceptors of the eye. It is essential for the function and survival of these retinal photoreceptors, and is therefore also essential for normal vision. Among other functions, RPE cells also promote the renewal of photoreceptor outer segments (POS, photoreceptor outer segments) by the phagocytosis involving MERTK. By forming tight junctions, the RPE layer also forms the brain-eye barrier separating the inner eye from the blood flow. RPE degeneration or otherwise damaged RPE can cause macular degeneration, which is likely to be treated by in situ transplantation of RPE cells produced from human pluripotent stem cells (hPSC) (including induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC)).

[0005] Different methods of basal culture medium containing various molecules have been designed to promote the differentiation of RPE from PSC. For example, such RPE induction is described, which uses a complex mixture of small molecules and growth factors (including Noggin, Dkk1, IGF1, bFGF, activin A, nicotinamide, SU5402, etc.) in the presence of bovine serum albumin. Some studies use a combination of nicotinamide and activin A or casein kinase / TGFβ / SMAD2 / 3 inhibitors to drive RPE induction (Idelson et al., 2009; Osakada et al., 2009) with limited efficiency. Other combinations and sequences of differentiation-driven molecules are also described, such as using bFGF, SB431542, retinoic acid, Shh and Noggin (Zahabi et al., 2012) to add differentiation-driven molecules on the extracellular matrix of animal origin. Generally, the individual effects produced by this molecule have not yet been determined, and these "mixtures" are not optimized with respect to exact composition, factor concentration, application time, etc.

[0006] RPE cells can also be obtained slowly (within a few weeks) with reduced efficiency by spontaneous differentiation along neural pathways (Plaza Reyes et al., 2016). In fact, it was demonstrated that RPE cells can be obtained by using a matrix based on recombinant human laminin (rhLN-521) in the absence of basic fibroblast growth factor (bFGF) in a xeno-free and defined NutriStem TM Human embryonic stem cells (hESC) can be differentiated into RPE cells by culturing them in hESC XF medium. Under these conditions, hESC RPE differentiated cells were obtained, which had a reduction in the pluripotency-related transcripts OCT3 / 4 and NANOG, as well as a strong expression of the neuroectodermal transcripts sex-determining region Y-box 9 protein (SOX9) and paired box 6 gene (PAX6). This culture strongly supported the formation of pigment structures similar to optical vesicles (OV) as early as 3 weeks of differentiation, with uniform pigmentation obtained at 9 weeks.

[0007] Another alternative approach uses chetonin, an inhibitor of hypoxia-inducible factor (HIF) signaling, as a driver of RPE fate (Maruotti et al., 2015; Sharma et al., 2019).

[0008] The cell mass that existing methods produce is not pure, relies on the molecule (this is a problem for clinical compliance and the immunogenicity of reduction) of animal origin, and may spend several weeks to obtain.This area still needs to develop effective and use convenient culture conditions to produce the method for RPE differentiation of pure cell culture in a short period of time. Summary of the invention

[0009] The present invention is based on the inventive discovery that the combination of a neuroectoderm induction mixture transforming growth factor β (TGF-β) family pathway inhibitory molecules and / or fibroblast growth factor (FGF) / ERK pathway signaling inhibitory molecules and activin A treatment, when used in a clear time sequence and adherent cell culture conditions, produces an induction of RPE cells from PSCs.

[0010] In one embodiment, the present invention provides a method for producing retinal pigment epithelial (RPE) cells, the method comprising: (a) contacting a culture of pluripotent stem cells (PSCs) with a mixture of reagents comprising: (i) one or more of a transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor; and (ii) optionally, a TGFβ family protein; and then (b) culturing the cells of (a) with a TGFβ family protein in the absence of the mixture of reagents of (a)(i).

[0011] In one aspect, the culture of PSC is an adherent monolayer of cells. In one aspect, the cell monolayer is grown in a two-dimensional culture system, such as a culture dish. In one aspect, the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is selected from the group consisting of SB431542, LY3200882, TP0427736HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, galunisertib, vactosertib, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854. In another aspect, the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor is selected from the group consisting of dorsomorphin, PPM1A and LDN-193189. In another aspect, the FGF / ERK pathway signaling inhibitor is selected from the group consisting of PD0325901, PD173074, SU431542, PD161570, PD98059, PD184352, PD198306 and PD334581. In one aspect, the TGFβ protein superfamily member is selected from the group consisting of activin A (activator protein A, Activin A), TGFβ1, TGFβ2 and TGFβ3.

[0012] In one aspect, the mixture of (a) comprises about 0.1-10 μM TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, about 0.1-1 μM BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor and / or about 0.5-5 μM FGF / ERK pathway signaling inhibitor; and at least about 0.1 ng / ml TGFβ family protein. In various aspects, the mixture comprises about 1-5 μM SB431542, about 0.25 μM doxormorphine, about 0.5-1.5 μM PD0325901, and about 25 ng / ml activin A. In one aspect, contacting PSC with TGFβ family protein includes contacting PSC with at least about 0.1 ng / ml TGFβ family protein. In one aspect, contacting PSC with TGFβ family protein includes contacting PSC with at least about 5 ng / ml activin A. In another aspect, contacting the PSC with a TGFβ family protein comprises contacting the PSC with about 25 ng / ml of activin A. In one aspect, the PSC is contacted with the mixture for about 2-7 days. In another aspect, the PSC is contacted with a TGFβ family protein (e.g., activin A) for about 4 weeks.

[0013] In some aspects, the monolayer is cultured on a surface comprising a laminin coating. In various aspects, PSC is a human PSC (hPSC), such as human induced pluripotent stem cells (hiPSC) or human embryonic stem cells (hESC). In one aspect, PSC is contacted with a mixture in the absence of a hypoxia-inducible factor (HIF) pathway regulator. In another aspect, PSC is contacted with a mixture in the absence of nicotinamide. In another aspect, under non-hypoxic conditions, for example, PSC is cultured under normoxic conditions. In another aspect, hPSC is cultured in a system (e.g., a spinner flask) that is not in a three-dimensional culture system.

[0014] In another embodiment, the present invention provides a method for inducing retinal pigment epithelial (RPE) cell differentiation from pluripotent stem cells (PSCs), the method comprising: (a) culturing PSCs under conditions that allow growth as an adherent monolayer in a two-dimensional culture system; (b) contacting the PSCs with a mixture of an agent comprising one or more of a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or an FGF / ERK pathway signaling inhibitor, and optionally a TGFβ superfamily protein for about 2-7 days; and (c) subsequently culturing the cells of (b) with the TGFβ family protein for another about 4 weeks.

[0015] In one aspect, RPE cell differentiation is direct RPE cell differentiation.In another aspect, compared with PSC, the RPE cells differentiated have the expression of the increase of PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1, CLDN19, ATP1B1, NC1, ZO1 and / or TYR.In one aspect, the RPE cells differentiated do not express ECAT11, OCT4, NANOG, SOX2, mir302HT and / or LIN28.

[0016] In another embodiment, the invention provides a method of treating macular degeneration in a subject, the method comprising administering to the subject differentiated retinal pigment epithelial (RPE) cells, wherein the differentiated RPE cells are obtained by one of the methods described herein.

[0017] In one aspect, the RPE cells using differentiation improve photoreceptor (photoreceptor) function and / or survival (survival rate, survival). In some aspects, improving photoreceptor function includes increasing the renewal of photoreceptor outer segment (photoreceptor outer segment), improving the phagocytosis and / or increase, recovery and / or production of cells / cells related to MERTK are tightly connected. In various fields, recovery and / or production of cells / cells are tightly connected to improve or restore brain-eye barrier. In one aspect, the RPE cells using differentiation include in situ injection RPE cells. In another aspect, hPSC is autologous hPSC or allogeneic (allogenic) hPSC.

[0018] In another embodiment, the present invention provides a kit comprising: (a) a neuroectodermal induction cocktail, wherein the neuroectodermal induction cocktail comprises: a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor and / or a FGF / ERK pathway signaling inhibitor; (b) a TGFβ family protein; and (c) instructions for inducing pluripotent stem cells (PSCs) to differentiate into retinal pigment epithelial (RPE) cells.

[0019] In one aspect, the kit further comprises a laminin-coated surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is an illustrative schematic diagram of the method of the present invention.

[0021] Figures 2A-2CShown is the performance of the RPE cell differentiation protocol compared to a reference protocol. Figure 2A are bar graphs showing the expression of RPE-specific markers as obtained by RTqPCR in cells differentiated using the reference protocol or the protocol of the invention. Figure 2B are photographs comparing the pigmentation of cells obtained by the two protocols. Figure 2C is a bar graph showing the expression of RPE specific markers as obtained by RTqPCR in untreated cells, cells treated with Activin A alone and cells differentiated using the protocol of the present invention.

[0022] Figure 3 is a bar graph showing the expression of RPE-specific markers as obtained by RTqPCR in cells differentiated using PSD mixture, without PD, without SB or without DM. PSD mixture: TGFβ / SMAD2 / SMAD3 inhibitor + BMP / SMAD1 / SMAD5 / SMAD8 inhibitor + FGF / ERK inhibitor; without PD: TGFβ / SMAD2 / SMAD3 inhibitor + BMP / SMAD1 / SMAD5 / SMAD8 inhibitor; without SB: BMP / SMAD1 / SMAD5 / SMAD8 inhibitor + FGF / ERK inhibitor; without DM: TGFβ / SMAD2 / SMAD3 inhibitor + FGF / ERK inhibitor.

[0023] Figure 4A-4B are graphs showing the expression of RPE-specific markers in cells differentiated with PD+A or PSD+A. Figure 4A Shown are graphs showing the expression of RPE-specific markers as measured by FACS. Figure 4B is a bar graph showing the expression of RPE-specific markers as measured by RTqPCR. PSD+A: TGFβ / SMAD2 / SMAD3 inhibitor+BMP / SMAD1 / SMAD5 / SMAD8 inhibitor+FGF / ERK inhibitor+Activin A; PD+A: FGF / ERK inhibitor+BMP / SMAD1 / SMAD5 / SMAD8 inhibitor+Activin A.

[0024] Figure 5 is a graph showing the expression of OTX2, MITF, and PMEL in cells during 7 days of PSD+A RPE differentiation.

[0025] Figure 6 are bar graphs showing the expression of RPE-specific markers in cells differentiated with PSD followed by A or PSD+A.

[0026] Figure 7 is a graph showing the titration of activin A treatment.

[0027] Figures 8A-8C is a graph showing the expression of early, mid / late, late REP markers and maturation markers as measured by RTqPCR in cells differentiated using the PSD+A protocol. Fig. 8A is a graph showing the expression of early RPE markers OTX2, MITF, PMEL, and ATP1B1. Figure 8B is a graph showing the expression of the middle / late RPE markers TYR and CLDN19. Figure 8C is a graph showing the expression of late RPE / maturation markers RPE65, RLBP1 and BEST1.

[0028] Figures 9A-9D are photographs showing phase contrast and immunofluorescence labeled RPE cells obtained by the PSD+A protocol. Fig.9A is a phase contrast photograph showing RPE cells. Fig. 9B are photographs showing the expression of BEST1 and ZO1 proteins in RPE cells. Fig. 9C are photographs showing the expression of MITF1 and CRALBP proteins in RPE cells. Fig.9D are photographs showing the expression of CRALBP and ZO1 proteins in RPE cells.

[0029] Figures 10A-10B are photographs showing electron microscopic analysis of iPSC-derived RPE cells generated using the PSD+A protocol. Fig. 10A is a transmission electron microscope photograph showing cells. Fig. 10B is a scanning electron microscope photograph showing cells.

[0030] Fig.11 is a bar graph showing the expression of iPSC markers in iPSC-derived RPE cells as measured by RTqPCR. DETAILED DESCRIPTION

[0031] The present invention is based on the inventive discovery that a neuroectoderm induction mixture comprising one or more of a TGFβ family pathway inhibitory molecule and / or a FGF / ERK pathway signaling inhibitory molecule in combination with a TGFβ family protein produces induction of RPE cells from PSCs when used in a specified time sequence and under adherent cell culture conditions.

[0032] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, as these compositions, methods, conditions may vary. It should also be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting, as the scope of the present invention will only be limited in the appended claims.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0035] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein may be used in the practice or testing of the present invention, it should be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0036] In one embodiment, the present invention provides a method for producing retinal pigment epithelial (RPE) cells, comprising: (a) contacting a culture of pluripotent stem cells (PSCs) with a mixture of reagents comprising: (i) one or more of a transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor; and (ii) optionally, a TGFβ family protein; and (b) subsequently culturing the cells of (a) with a TGFβ family protein in the absence of the mixture of reagents of (a)(i).

[0037] Retinal pigment layer or retinal pigment epithelium (RPE) is the pigment cell layer just outside the sensory neuroretina, which nourishes retinal visual cells, and firmly attaches to the eye choroid below and the retinal visual cells above. RPE is composed of the single layer hexagonal cells (RPE cells) densely filled with pigment particles. When viewed from the outer surface, these cells are smooth and hexagonal in shape. When viewed in cross section, each cell is composed of the non-pigmented part of the outside and the pigmented part of the inside, and the non-pigmented part of the outside comprises a large oval nucleus, and the pigmented part of the inside extends between the rods as a series of straight thread-like (thread-like) projections, especially when eyes are exposed to light.

[0038] RPE has multiple functions, including light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immunoregulation. RPE cells are responsible for absorbing scattered light. This effect is very important, mainly for two reasons, first, in order to improve the quality of the optical system, second, light is radiation, it is gathered on the macula cells by the lens, thereby causing the strong concentration of photo-induced oxidation energy. Melanosomes absorb scattered light, thus weakening photo-induced oxidative stress. High perfusion of the retina produces a high oxygen tension environment. The combination of light and oxygen produces oxidative stress, and RPE has many mechanisms to deal with it. RPE cells form the outer blood-retinal barrier, and epithelial cells have a tight connection between the lateral surfaces, which means that the inner retina is isolated from systemic effects. This is very important for the immune privilege of the eye (not only as a barrier, but also for the signal transduction process), which is a highly selective substance transport in a strictly controlled environment. RPE provides nutrition for photoreceptors, controls ion homeostasis, and eliminates water and metabolites. The visual cycle performs the vital task of maintaining visual function, so it is necessary to adapt to different visual needs, such as vision in the dark or bright. Photoreceptor outer segment (POS) membrane is exposed to continuous light-induced oxidative stress, and they are subjected to its continuous destruction. They are constantly renewed by falling off their ends, and then RPE engulfs and digests. RPE is an epithelial cell, which closely interacts with the photoreceptor of one side, but also must be able to interact with the cells (such as endothelial cells or immune system cells) on the blood side of the epithelial cell. In order to communicate with adjacent tissues, RPE can secrete multiple factors and signal transduction molecules. It secretes ATP, fas-ligand (fas-L), fibroblast growth factor (FGF-1, FGF-2 and FGF-5), transforming growth factor β (TGF-β), insulin-like growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), lens epithelium-derived growth factor (LEDGF), interleukin family members, tissue inhibitors of matrix metalloproteinases (TIMP) and pigment epithelium-derived factor (PEDF). Many of these signal transduction molecules have important physiological-pathological effects. The inner eye represents an immune privileged space that is disconnected from the immune system of the bloodstream. The RPE supports immune privilege in two ways. First, it represents a mechanical and tight barrier that separates the inner space of the eye from the bloodstream. Second, the RPE is able to communicate with the immune system, thereby suppressing immune responses in healthy eyes, or, on the other hand, activating the immune system in the case of disease.

[0039] Dysfunction of the RPE is associated with RPE diseases, which may be referred to as retinal degenerative diseases. Retinal degenerative diseases may be caused by defects in oxidative stress and inflammation, apoptosis and autophagy, and / or cell polarity and interactions.

[0040] Cornea has transparent structure and RPE is exposed to light for a long period of time, has abundant oxygen supply, therefore it is easy to produce a large amount of reactive oxygen. In addition, the rise of systemic glucose level such as in diabetic patients can promote excessive ROS accumulation. In degenerative retinopathy, the antioxidant level in cells is reduced; That is to say, the ability of RPE cells to remove ROS is variably reduced, causing POS to accumulate in large quantities. Oxidative stress and inflammation cause RPE cell damage, which in turn causes retinal dysfunction and even blindness. Compared with normal cells, the RPE of pathology shows increased apoptosis, autophagy and endoplasmic reticulum stress levels. In age-related macular degeneration (AMD) and other retinal degenerative diseases, RPE cell death via apoptosis and endoplasmic reticulum stress has been observed. The polarity and cell connection of RPE play a vital role in the blood-retinal barrier, thereby maintaining the stability of the internal photoreceptor microenvironment and supporting the choroidal system. The destruction of cell polarity and cell connection can significantly increase the risk of retinal degenerative diseases. RPE polarity and cell connection stability are related to the unique base structure and top structure of the retina, which affects phagocytosis and material exchange. Abnormalities in RPE polarity, barrier disruption, and retinal stability may contribute to the pathogenesis of blinding retinal diseases.

[0041] There is no cure for retinal pigment epithelial disease. Due to years of research on retinal diseases, many genes and signal transduction pathways have been identified as potential targets for gene therapy or other therapies. Cell therapy using stem cell-derived RPE and photoreceptors has restored vision in preclinical models of human retinal degenerative diseases. Therefore, stem cell transplantation is an effective method for treating RPE diseases.

[0042] The transforming growth factor β (TGF-β) superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial cell line-derived neurotrophic factors (GDNFs), activins, inhibins, Nodal, Lefty, and Müllerian inhibitory substances (MISs). Ligands of the TGF-β superfamily form dimers that bind to heterodimeric receptor complexes consisting of type I and type II receptor subunits with serine / threonine kinase domains. Upon ligand binding, type II receptors phosphorylate and activate type I receptors, thereby initiating Smad-dependent signaling cascades that induce or inhibit transcriptional activity. During development, members of the TGF-β family are thought to be essential for dorso-ventral patterning, mesoderm induction and patterning, limb bud formation, bone and cartilage formation, neuronal differentiation, and the development of a variety of different tissues and organs.

[0043] As used herein, "pathway signaling inhibitor" refers to any molecule that can inhibit the signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control cell functions, such as cell differentiation. When a molecule (such as a hormone or growth factor) binds to a specific protein receptor on or in a cell, the cell receives a signal from its environment. After the first molecule in the pathway receives a signal, it activates another molecule. The process is repeated through the entire signaling pathway until the last molecule is activated and the cell function is achieved. Abnormal activation of a signaling pathway or inhibition of a signaling pathway may lead to disease, or in the case of pluripotent cells may lead to changes in the pluripotent state, thus leading to differentiation. The term "molecule" includes, but is not limited to, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers (e.g., proteins, peptides, hormones, carbohydrates, or polyamines). Non-limiting examples of polynucleotides include short interfering nucleic acids (siNA), antisense nucleic acid molecules, enzymatic nucleic acid molecules, 2',5'-oligoadenylic acid, triplex forming oligonucleotides, aptamers and decoys. Biologically active molecules include antibodies (e.g., monoclonal antibodies, chimeric antibodies, humanized antibodies, etc.), cholesterol, hormones, antiviral substances, peptides, proteins, chemotherapeutic drugs, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and their analogs, as well as small nucleic acid molecules (such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), antagomirs and short hairpin RNA (shRNA) molecules.

[0044] As used herein, "TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor", "BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor" or "FGF / ERK pathway signaling inhibitor" refers to any molecule that is capable of inhibiting the TGFβ / SMAD2 / SMAD3, BMP / SMAD1 / SMAD5 / SMAD8 or FGF / ERK signaling pathway, respectively. Signaling pathway inhibition is the opposite of signaling pathway upregulation. In this process, small molecules, known as "signal transduction inhibitors" or "pathway signaling inhibitors," block the communication between different molecules of a pathway and interrupt the molecular signaling cascade.

[0045] As used herein, "TGFβ superfamily protein" or "TGFβ family protein" refers to any protein or peptide of the transforming growth factor β (TGF-β) superfamily, which is a large group of structurally related cell regulatory proteins (named after its first member, TGFβ1). TGFβ proteins interact with TGF-β receptors. Since then, many proteins have been described as members of the TGFβ family in various species (including invertebrates as well as vertebrates) and have been classified into 23 different gene types, which belong to four major subfamilies: TGF-β subfamily, including TGFβ1, TGFβ2 and TGFβ3; bone morphogenetic proteins and growth differentiation factors; activin and inhibin subfamilies; and left-right determining factors. Non-limiting examples of TGFβ family proteins used in the present method include activin A, TGFβ1, TGFβ2 and TGFβ3.

[0046] The methods described herein describe cell culture conditions in which human pluripotent stem cells are grown that lead to the generation of RPE cells.

[0047] Stem cell is an undifferentiated cell, which has the ability of self-renewal indefinitely, and is maintained in the undifferentiated state. Contrary to the embryonic stem cell that can only be separated from the internal mass of blastocyst, there are three kinds of known adult stem cells available sources: the bone marrow that needs to be drilled, the adipose tissue and blood (cells and other cells can be extracted from the blood) that can be obtained by liposuction. As used herein, the term "pluripotent stem cell" refers to the cell that can produce all cell types of an organism, i.e., the cell derived from any one of the three germ layers. On the other hand, pluripotent stem cells can be differentiated into a variety of cell types, but can only be differentiated into those cell types of closely related families of cells, usually the cell type of the organ from which they originate. Most adult stem cells are pluripotent, but a small amount of pluripotent adult stem cells can be extracted from umbilical cord or other tissues. The cell source for retinal cell therapy includes stem cells, such as embryonic stem cells (ESC), adult stem cells and induced pluripotent stem cells (iPSC). At present, ESC and iPSC are mainly used in differentiation into RPE.

[0048] In some aspects, the PSC used in the methods described herein is human, and in some cases, the human PSC is an induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC).

[0049] "Produce" RPE cell means that this method provides the physical and chemical culture conditions optimized to induce PSC differentiation into RPE cells.Differentiation method as herein described produces the RPE cell group that is rich in RPE cells.For example, compared with the method available in the art, in a shorter time and using more convenient culture conditions to obtain greater than 80%, greater than 85%, greater than 90%, greater than 95%, 96%, 97%, 98% or 99% RPE cells.

[0050] Physical culture conditions include, but are not limited to, the culture environment of the cells (e.g., adherent culture or suspension culture, or two-dimensional culture or three-dimensional culture system), the pH of the culture medium, the gas concentration in the incubator (e.g., CO2 concentration, O2 concentration), and the temperature.

[0051] There are two basic systems for growing cells in culture: as a monolayer on an artificial substrate (i.e., adherent culture) or floating freely in culture medium (suspension culture). With the exception of hematopoietic cell lines and a few other cell lines, most cells derived from vertebrates are anchorage-dependent and must be cultured on a suitable substrate that has been specially treated to allow the cells to attach and spread (i.e., tissue culture treated). However, many cell lines can also be adapted to suspension culture.

[0052] In one aspect, the culture of PSCs is an adherent monolayer of cells. In another aspect, a monolayer of cells is grown in a two-dimensional culture system. In another aspect, PSCs are cultured in a system that is not in a three-dimensional culture system.

[0053] In addition to treating tissue culture surfaces, cells may need to be grown on coated surfaces to enhance or improve their attachment and / or spreading (i.e., using a coating). "Coating" as an additional surface treatment represents any additional modifications made to increase cell attachment beyond the standard plasma or corona treatment that the manufacturer applies to all cell culture plastics. Typically, coating is accomplished with proteins or peptides. A variety of proteins can be used to coat tissue culture treated culture dishes, including poly-L-lysine, poly-D-lysine, poly-ornithine, gelatin, collagen type I, collagen type IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domains, Matrigel TM (with various components of the extracellular matrix that bind growth factors, etc.), collagen gel, alginate gel, and lactate gel.

[0054] In one aspect, the monolayer is cultured on a surface comprising a laminin coating.

[0055] Physical culture conditions include gas concentrations in an incubator. Cell culture is typically incubated in a normal atmosphere (normal atmosphere) (with 15-22% oxygen and 5% CO2) for expansion and inoculation. In various aspects, PSC is grown in a humidified atmosphere comprising about 5% CO2 concentration and normoxic conditions (non-hypoxic O2 concentration). Although hypoxic culture conditions are generally considered to support the performance of stem cells, in the present method, PSC is cultured under non-hypoxic conditions. As used herein, "normoxic" conditions refer to culture conditions including atmospheric O2 concentrations (e.g., about 15-25% O2 concentrations). As used herein, hypoxic conditions are characterized in that, compared with the oxygen concentration of ambient air (about 15%-25% oxygen), the oxygen concentration is lower. In one aspect, hypoxic conditions are characterized in that the oxygen concentration is less than about 10%. In another aspect, the hypoxic conditions are characterized in that the oxygen concentration is about 0.1% to 10%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2%. For example, hypoxic conditions include culture conditions with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% O2 concentration or any intermediate value.

[0056] Chemical culture conditions include but are not limited to adding to the reagent or molecule of the desired effect (i.e. PSC differentiation into RPE cells) sought subsequently in the culture medium.Terms " reagent " and " molecule " are used interchangeably, include but are not limited to small molecules (including small molecules without optimal cell permeability), lipid, nucleoside, nucleotide, nucleic acid, polynucleotide, oligonucleotide, antibody, toxin, negatively charged polymer and other polymer (such as protein, peptide, hormone, carbohydrate or polyamine).

[0057] In various aspects, the chemical culture conditions of the presently described methods include a mixture of agents comprising one or more of a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a FGF / ERK pathway signaling inhibitor; and, optionally, a TGFβ family protein.

[0058] For example, the mixture of reagents includes TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors, BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors and TGFβ family proteins. In another example, the mixture of reagents includes TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors, BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors, FGF / ERK pathway signaling inhibitors and TGFβ family proteins. In another example, the mixture of reagents includes BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors, FGF / ERK pathway signaling inhibitors and TGFβ family proteins. Optionally, the mixture includes FGF / ERK pathway signaling inhibitors and TGFβ family proteins. In the illustrative example of this article, TGFβ family proteins are activin A.

[0059] TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors include, for example, any molecule that inhibits TGFβ type I receptor (or ALK5) and its relatives (family members) ALK4 and ALK7. Non-limiting examples of TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors include SB431542, LY3200882, TP0427736 HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, Golunisert, Vatosert, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854.

[0060] In one aspect, the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is SB431542.

[0061] The TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is added to the PSC culture at a concentration ranging from about 0.1 μM to 10 μM. For example, the PSC is grown in a culture medium containing about 0.1, 0.3, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM or more. In one aspect, the mixture comprises about 5 μM TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor. In another aspect, the mixture comprises about 0.1-10 μM SB431542. In some aspects, the mixture comprises about 1-5 μM SB431542. In other aspects, the mixture comprises about 5 μM SB431542.

[0062] BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors include, for example, any molecule that inhibits the BMP pathway by targeting type I BMP receptor activin receptor-like kinase (ALK) 2, ALK3, and ALK6. Non-limiting examples of BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors include doxorubicin, PPM1A, and LDN-193189.

[0063] In one aspect, the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor is doxormorphine.

[0064] BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors are added to PSC cultures at a concentration ranging from about 0.1 μM to 1 μM. For example, PSC is grown in a culture medium containing about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μM or more. In one aspect, the mixture comprises about 0.25 μM BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors. In another aspect, the mixture comprises about 0.1-10 μM doxorubicin. In some aspects, the mixture comprises about 0.25 μM doxorubicin.

[0065] FGF / ERK pathway signaling inhibitors include, for example, any molecule that inhibits the MEK1 / 2 signaling pathway. Non-limiting examples of FGF / ERK pathway signaling inhibitors include PD0325901, PD173074, SU431542, PD161570, PD98059, PD184352, PD198306, and PD334581. In one aspect, the FGF / ERK pathway signaling inhibitor is PD0325901.

[0066] The FGF / ERK pathway signaling inhibitor is added to the PSC culture at a concentration ranging from about 0.1 μM to 5 μM. For example, the PSC is grown in a culture medium containing about 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μM or more. In one aspect, the mixture comprises about 0.5-1.5 μM FGF / ERK pathway signaling inhibitor. In another aspect, the mixture comprises about 0.1-5 μM PD0325901. In some aspects, the mixture comprises about 0.5-1.5 μM PD0325901.

[0067] The TGFβ family protein is added to the PSC culture at a concentration of at least about 0.1 ng / ml. For example, the PSCs are grown in a medium comprising at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ng / ml or more of the TGFβ family protein.

[0068] In one aspect, the TGFβ protein family member is selected from the group consisting of activin A, TGFβ1, TGFβ2, and TGFβ3.

[0069] In some aspects, the TGFβ family protein is activin A.

[0070] Activin A is a member of the protein TGFβ family produced by many cell types throughout development. It is a disulfide-bonded homodimer (two β-A chains), which binds to the heteropolymeric complex of type I (Act-RI-A and Act-RI-B) and type II (ActRII-A and ActRII-B) serine-threonine kinase receptors. Activin mainly sends signals through SMAD2 / 3 proteins to regulate multiple functions, including cell proliferation, differentiation, wound healing, apoptosis and metabolism. Activin A maintains the undifferentiated state of human embryonic stem cells and promotes the differentiation of human embryonic stem cells into a determined endoderm. Activin A is added to PSC cultures at a concentration ranging from about 0.1-100 ng / ml. For example, PSCs are grown in a medium comprising about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ng / ml, or more activin A. In one aspect, the mixture comprises at least about 5 ng / ml activin A. In some aspects, the mixture comprises about 25 ng / ml activin A.

[0071] In other aspects, the TGFβ protein family member is TGFβ1 or TGFβ3. TGFβ1 or TGFβ3 is added to the PSC culture at a concentration ranging from about 0.1-100 ng / ml. For example, PSCs are grown in a culture medium comprising about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ng / ml or more TGFβ1 and TGFβ3.

[0072] The methods described herein are further defined by excluding chemical conditions for culturing PSCs.For example, the present method for generating RPE cells comprises culturing PSCs in the absence of a hypoxia inducible factor (HIF) pathway modulator and in the absence of nicotinamide.

[0073] Hypoxia inducible factor (HIF) is a transcription factor that is activated in response to reduced oxygen availability in the cell environment. They affect cell metabolism, cell survival and angiogenesis to maintain biological homeostasis. As used herein, "HIF pathway regulators" include any agent that inhibits HIF. Non-limiting examples of HIF inhibitors include adaptaquin, TAT-ring-CLLFVY, DMOG, echinomycin, FM19G11, GN 44028, IOX 2, KC7F2, LW 6, PX 12, TC-S7009 and VH 298.

[0074] In one aspect, the mixture comprises about 1-10 μM TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, about 0.1-1 μM BMP / SMAD1 / SMAD5 / SMAD8 and / or about 0.5-5 μM FGF / ERK pathway signaling inhibitor; and at least about 5 ng / ml TGFβ family protein.

[0075] In some aspects, the mixture comprises about 5 μM SB431542, about 0.25 μM doxorphine and / or about 0.5-1.5 μM PD0325901, and about 25 ng / ml activin A. In one aspect, the mixture comprises about 5 μM SB431542, about 0.25 μM doxorphine, and about 25 ng / ml activin A. In another aspect, the mixture comprises about 0.25 μM doxorphine, about 0.5-1.5 μM PD0325901, and about 25 ng / ml activin A. In yet another aspect, the mixture comprises about 5 μM SB431542, about 0.25 μM doxorphine, about 0.5-1.5 μM PD0325901, and about 25 ng / ml activin A.

[0076] In another aspect, contacting the PSC with a TGFβ family protein comprises contacting the PSC with at least about 5 ng / ml TGFβ family protein.

[0077] The methods described herein include physical and chemical culture conditions, and continuous exposure of PSC to such physical and chemical conditions for a defined duration. In various aspects, the methods described herein include contacting PSC with a mixture of reagents for a given duration, and then contacting PSC with a TGFβ family protein (e.g., activin A). For example, the PSC is contacted with the mixture for a period of about 2 to 7 days. Contacting PSC with the mixture includes contacting PSC with the mixture for about 2, 3, 4, 5, 6, 7 days or more. In one aspect, PSC is contacted with the mixture for about 2-4 days. In various aspects, PSC is contacted with the mixture for about 2 days.

[0078] The PSC is contacted with a TGFβ family protein (e.g., activin A) for a period of time ranging from about 2 to 6 weeks. For example, contacting the PSC with a TGFβ family protein includes contacting the PSC with a TGFβ family protein for about 2, 3, 4, 5, 6 weeks or more. In one aspect, the PSC is contacted with a TGFβ family protein for about 4 weeks.

[0079] In another embodiment, the present invention provides a method for inducing retinal pigment epithelial (RPE) cell differentiation from pluripotent stem cells (PSCs), the method comprising: (a) culturing PSCs under conditions that allow growth as an adherent monolayer in a two-dimensional culture system; (b) contacting the PSCs with a mixture of an agent comprising one or more of a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or an FGF / ERK pathway signaling inhibitor, and optionally a TGFβ superfamily protein for about 2-7 days; and (c) subsequently culturing the cells of (b) with the TGFβ family protein for another about 4 weeks.

[0080] Cell differentiation refers to the process in which undifferentiated cells (e.g., stem cells) are transformed from an undifferentiated state into a differentiated state. Typically, cells are transformed into more specialized types. During the development of multicellular organisms, differentiation occurs many times because it is transformed from a simple fertilized egg into a complex system of tissues and cell types. As adult stem cells divide and produce fully differentiated daughter cells during tissue repair and during normal cell conversion, differentiation continues in adulthood. Differentiation significantly changes the size, shape, membrane potential, metabolic activity and responsiveness to signals of cells. These changes are mainly due to the highly controlled modification in gene expression. A special type of differentiation referred to as terminal differentiation is important in some tissues (e.g., vertebrate nervous system, striated muscle, epidermis and intestinal tract). During terminal differentiation, the precursor cells that were previously able to carry out cell division permanently leave the cell cycle, cancel the cell cycle mechanism, and often express a series of gene features of the final function of the cell (e.g., myosin and actin of muscle cells). If the capacity (ability, capacity) and function of the cell change further, differentiation may continue to occur after terminal differentiation. Methods described herein allow inducing PSC to differentiate into terminally differentiated RPE cells.

[0081] In one aspect, the method comprises (a) culturing PSCs under conditions that allow growth as an adherent monolayer in a two-dimensional culture system (i.e., not in a three-dimensional culture system); (b) contacting the PSCs with a mixture of an agent comprising one or more of a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a FGF / ERK pathway signaling inhibitor; and optionally a TGFβ family protein, for about 2-7 days; and (c) subsequently culturing the cells of (b) with the TGFβ family protein under conditions that serve as non-hypoxic conditions, in the absence of a HIF pathway modulator, in the absence of nicotinamide, for an additional about 4 weeks.

[0082] In various aspects, the method comprises (a) culturing PSCs under conditions that permit growth as an adherent monolayer in a two-dimensional culture system (i.e., not in a three-dimensional culture system); (b) contacting the PSCs with a mixture of an agent comprising about 5 μM TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, about 0.25 μM BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, about 0.5-1.5 μM FGF / ERK pathway signaling inhibitor, and about 25 ng / ml activin A for about 2-7 days; and (c) subsequently culturing the cells of (b) with about 25 ng / ml activin A under conditions that serve as non-hypoxic conditions, in the absence of a HIF pathway modulator, in the absence of nicotinamide, for an additional about 4 weeks.

[0083] In one aspect, the RPE cell differentiation is direct RPE cell differentiation.

[0084] "directly" differentiation means that the method described herein allows to obtain RPE cells from PSC, rather than the cells of intermediate differentiation, and does not need to carry out intermediate operations to cells. That is, contrary to indirect methods, the indirect methods obtain partially differentiated cells (for example, pluripotent cells or non-terminal differentiated cells) in the first process, and then make their terminal differentiation into RPE, and the method described herein produces RPE cells in a separate process.

[0085] Differentiated cells can be based on the observed differences compared to the undifferentiated cells obtained therefrom, including changes in cell size, shape, membrane potential, metabolic activity, responsiveness to signals, gene expression, etc. to characterize. In one aspect, RPE characterizes the changes in gene expression including analyzing the molecular signature characteristic of RPE cells. Such molecular markers include, for example, the expression of genes selected from PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1, CLDN19, ATP1B1, NC1, ZO1 and / or TYR.

[0086] Melanocyte protein PMEL, also known as premelanosome protein, is a protein encoded by PMEL gene or PMEL17 in humans. PMEL is a 100kDa type I transmembrane glycoprotein, which is mainly expressed in pigment cells of the skin and eyes. The transmembrane form of PMEL is modified by the elaboration of N-linked oligosaccharides and the addition and modification of O-linked oligosaccharides in the secretory pathway. Then, it is targeted to the precursor of pigment organelles (i.e., melanosomes), where it is processed into multiple small fragments by proteolysis. Some of these fragments form non-pathological amyloid proteins, which assemble into sheets and form striped patterns below the ultrastructure of melanosomes. The expression of PMEL gene is regulated by microphthalmia-related transcription factor (MITF).

[0087] Microphthalmia-associated transcription factor, also known as class E basic helix-loop-helix protein 32 or bHLHe32, is a protein encoded by the MITF gene in humans. MITF is a basic helix-loop-helix leucine zipper transcription factor that is involved in lineage-specific pathway regulation of multiple types of cells, including melanocytes, osteoclasts, and mast cells. The term "lineage specificity," as it relates to MITF, means a gene or trait that is present only in a certain cell type. Thus, MITF may be involved in the rewiring of signaling cascades specifically required for the survival and physiological function of their normal cell precursors.

[0088] Homeobox protein OTX2 is a protein encoded by the OTX2 gene in humans. OTX2 is expressed in the brain, ears, nose, and eyes, and in the case of mutations; it may lead to significant developmental abnormalities and disorders. OTX2 mutations can lead to eye conditions, including anophthalmia and microphthalmia. In addition to anophthalmia and microphthalmia, other abnormalities such as optic nerve hypoplasia, optic chiasm hypoplasia, and optic glomeruli dysplasia (abnormal eye development) have been observed. Other defects that occur due to mutations in the OTX2 gene include pituitary abnormalities and mental retardation. The homeobox protein Otx2 has been identified as a possible molecular "messenger" that is necessary for experience-driven visual plasticity during the critical period. Otx2 is initially involved in embryonic head formation, is re-expressed during the critical period (>P23) in rats, and regulates the maturation of GABAergic interneurons (PV cells) expressing parvalbumin that control the onset of critical period plasticity.

[0089] Bestrophin-1 (Best1) is a protein encoded by the BEST1 gene in humans. The Bestrophin protein family contains four evolutionarily related genes (BEST1, BEST2, BEST3 and BEST4) that encode integral membrane proteins. The family was first identified in humans by linking BEST1 mutations to Best vitelliform macular dystrophy (BVMD). Mutations in the BEST1 gene have been identified as the main cause of at least five different degenerative retinal diseases. Bestrophin is an ancient family of structurally conserved proteins that have been identified in almost every organism studied, from bacteria to humans. In humans, they serve as calcium-activated anion channels, each with a unique tissue distribution throughout the body. Specifically, the BEST1 gene on chromosome 11q13 encodes the Bestrophin-1 protein in humans, which is most highly expressed in the retina.

[0090] The retinal pigment epithelium-specific 65kDa protein, also known as retinoid isomerohydrolase, is an enzyme of the vertebrate visual cycle, which is encoded by the RPE65 gene in humans. RPE65 is expressed in the retinal pigment epithelium (RPE, an epithelial cell layer that nourishes photoreceptor cells) and is responsible for converting all trans-retinyl esters to 11-cis retinol during the phototransduction process. 11-cis retinol is then used for visual pigment regeneration in photoreceptor cells. RPE65 belongs to the carotenoid oxygenase family of enzymes. RPE65 is a key enzyme in the vertebrate visual cycle present in the retinal pigment epithelium. It is also present in rods and cones. The photoisomerization of 11-cis retinal to all-trans retinal initiates the phototransduction pathway through which the brain detects light. All-trans retinol is not photoactive, so it must be reconverted into 11-cis retinal before it can be recombined with opsin to form active visual pigments. RPE65 reverses photoisomerization by converting all-trans retinyl esters to 11-cis retinol. Most commonly, the ester substrate is retinyl palmitate. Other enzymes of the visual cycle complete the reactions required to oxidize and esterify all-trans retinol to retinyl esters (substrates for RPE65) and to oxidize 11-cis retinol to 11-cis retinol (the desired photoactive visual pigment component).

[0091] Retinal binding protein 1 (RLBP1), also known as cellular retinal binding protein (CRALBP), is a 36 kD water-soluble protein that is encoded by the RLBP1 gene in humans. Cellular retinal binding protein transports 11-cis-retinal (also known as 11-cis-retinaldehyde) as its physiological ligand. It plays a crucial role as the 11-cis-retinal receptor, which promotes the enzymatic isomerization of all 11-trans retinal to 11-cis retinal in the isomerization of rods and cones in the visual cycle. Mutations in RLBP1 involve a variety of vision-related diseases. All of these are autosomal recessive and include Bothnia dystrophy, retinitis punctata albescens, retinitis pigmentosa, Newfoundland rod-cone dystrophy, and fundus albipunctatus. The features of the associated diseases vary by age, severity, and rate of progression. All share similar characteristics, such as deterioration of photoreceptors and slower dark adaptation, which ultimately lead to impaired vision, often resulting in complete blindness.

[0092] Claudin-19 is a protein encoded by the CLDN19 gene in humans. It belongs to the Claudin group. ClAudin-19 is involved in magnesium transport. Claudin, such as CLDN19, is a transmembrane protein present at tight junctions. Tight junctions form a barrier that controls the movement of ions and molecules across the epithelial layer, as well as the movement of proteins and lipids between the apical and basolateral domains of epithelial cells.

[0093] Sodium / potassium transporting ATPase subunit beta-1 is an enzyme that is encoded by the ATP1B1 gene in humans. The protein encoded by this gene belongs to the beta chain protein family of Na+ / K+ and H+ / K+ ATPases and to the subfamily of Na+ / K+-ATPases. Na+ / K+-ATPases are integral membrane proteins responsible for establishing and maintaining electrochemical gradients of Na and K ions across the plasma membrane. These gradients are essential for osmoregulation, sodium-coupled transport of various organic and inorganic molecules, and for electrical excitability of nerves and muscles.

[0094] NC1 or non-collagenous 1 (NC1) is a protein domain of COL4A1. COL4A1 belongs to the type IV collagen family and contains three domains: a short N-terminal domain, a long triple-helical 7S domain at its center, and a non-collagenous 1 (NC1) domain at its C-terminus. The triple-helical domain contains interrupted GXY repeats, which are suspected to allow flexibility of the domain. The NC1 domain consists of two trimeric caps, each containing two α1 segments and one α2 segment, which form a six-fold propeller arranged around an axial channel. The interaction between the two caps occurs along a large planar interface and is stabilized by covalent cross-links between the α1 and α2 chains that run through the two caps.

[0095] Zonula occludens-1, also known as tight junction protein-1, is a 220 kD peripheral membrane protein encoded by the TJP1 gene in humans. It belongs to the family of zona occludens (ZO-1, ZO-2 and ZO-3), which are tight junction-associated proteins, and among them ZO-1 was the first to be cloned. It has the function of a scaffold protein that crosslinks and anchors tight junction (TJ) tethers (fibrillar structures within the lipid bilayer) to the actin cytoskeleton.

[0096] Tyrosinase is an oxidase that is the rate-limiting enzyme for controlling melanin production. The enzyme is mainly involved in two different melanin synthesis reactions (also known as the Raper Mason pathway). First, the hydroxylation of monophenols, and secondly, the conversion of ortho-diphenols to the corresponding ortho-quinones. Ortho-quinones undergo multiple reactions to ultimately form melanin. Tyrosinase is a copper-containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation. It is present in the melanin bodies synthesized in skin melanocytes. In humans, tyrosinase is encoded by the TYR gene. Mutations in the tyrosinase gene result in impaired tyrosinase production, leading to type I oculocutaneous albinism (a hereditary disorder that affects one in every 20,000 people). If uncontrolled during melanin synthesis, Tyr activity can lead to increased melanin synthesis. Reducing tyrosinase activity has been directed to improve or prevent conditions associated with hyperpigmentation of the skin, such as dark spots and age spots.

[0097] In various aspects, the differentiated RPE cells have increased expression of PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1 and / or TYR compared to hPSCs.

[0098] In many aspects, the RPE cells of differentiation do not express any pluripotent stem cell markers (i.e., they are not pluripotent after the differentiation process, but terminally differentiated). Stem cell markers include genes expressed by stem cells, including but not limited to ECAT11, OCT4, NANOG, SOX2, mir302HT and LIN28.

[0099] In one aspect, the differentiated RPE cells do not express ECAT11, OCT4, NANOG, SOX2, mir302HT, nor LIN28.

[0100] In another embodiment, the present invention provides a method of treating macular degeneration in a subject, comprising administering to the subject differentiated retinal pigment epithelial (RPE) cells, wherein the differentiated RPE cells are obtained by one of the methods described herein.

[0101] The dysfunction of RPE is relevant with various retinal degenerative diseases.As used herein, term " retinal degenerative diseases " or " degenerative retinopathy " means any retinal disease, and it comprises the retinal deterioration caused by the progressive death of retinal cells.Retinal degeneration has multiple reasons, including arterial or venous occlusion, diabetic retinopathy, RLF / ROP (retrocrystalline fibroplasia / retinopathy of prematurity) or disease (usually hereditary).These may exist in a variety of different ways, such as visual impairment, night blindness, retinal detachment, light sensitivity, tunnel vision, peripheral vision loss to complete loss of vision.In retinal degenerative diseases, retinitis pigmentosa (RP, retinitis pigmentosa) is a very important example. Non-limiting examples of degenerative retinal diseases associated with RPE deterioration include macular degeneration (such as age-related macular degeneration (AMD)), retinitis pigmentosa, diabetic retinopathy, and Gardner syndrome (characterized by FAP (familial adenomatous polyposis), bone and soft tissue tumors, retinal pigment epithelial hypertrophy, and impacted teeth).

[0102] The methods described herein are particularly directed to the treatment of age-related macular degeneration (AMD) in a subject, comprising administering to the subject RPE cells obtained by the methods described herein.

[0103] Age-related macular degeneration, or AMD, is a disease that affects a person's central vision. It can cause severe loss of central vision, but people rarely go blind from it. AMD is the most common cause of severe vision loss in people aged 50 and older. Only the center of vision is affected by this disease. AMD affects central vision, and having AMD affects the ability to see fine details. In AMD, a part of the retina called the macula is damaged. In later stages, people lose their ability to drive, see faces clearly, and read small print. In its early stages, AMD may not have any signs or symptoms, so people may not suspect they have it. The two main types of age-related macular degeneration have different causes. The exact cause of dry AMD, the most common type (about 80% of people with AMD have dry AMD), is unknown, but both genetic and environmental factors are thought to play a role. This happens when the light-sensitive cells in the macula slowly fail, usually one eye at a time. In this case, vision loss is usually slow and gradual. It is believed that age-related damage to the important supporting membrane under the retina causes dry AMD. Wet AMD is the less common type and usually causes patients to suffer more severe vision loss than dry AMD. This is the most common cause of severe vision loss. Wet AMD occurs when abnormal blood vessels begin to grow under the retina. They leak fluid and blood, so they are called wet AMD and may cause a large blind spot in the center of the visual field. The methods described herein can be used to treat wet AMD and dry AMD in a subject.

[0104] The term "subject" as used herein refers to any individual or patient who performs the subject method. Typically, the subject is a human, but as will be appreciated by those skilled in the art, the subject may be an animal. Thus, other animals, including vertebrates such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, chickens, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas) are included in the definition of a subject.

[0105] The term "treatment" can be used interchangeably with the term "therapeutic method" in this article, and refers to the following two: 1) cure, slow down, alleviate the pathological condition diagnosed or the symptom of illness and / or prevent the therapeutic treatment (disposal) or measures of the progress of the pathological condition diagnosed or illness, and 2) and prevention / preventative measures. The people who need treatment can include the individuality of having suffered from a specific medical condition and those individuals (that is, those individuals who need preventive measures) that may eventually suffer from illness. The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose" and "effective dose" etc. refer to the amount of the RPE cells of the biological or medical response (response) of the tissue, system, animal or people sought by researchers, veterinarians, doctors or other clinicians. Generally, reaction is either the improvement of patient symptoms, or the biological result (for example, treatment AMD) of expectation. This amount should be enough to treat the AMD of experimenter. Effective dose can be determined as described herein.

[0106] The terms "administration" and / or "administering" are understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration may be enteral, topical or parenteral. Thus, routes of administration include, but are not limited to, intraorbital, intravitreal, subretinal and any other ocular administration, as well as infusion.

[0107] In one aspect, administering the differentiated RPE cells comprises injecting the RPE cells in situ.

[0108] Currently, there is no cure for dry AMD, but vision rehabilitation programs and low vision devices can be used to build visual skills, develop new ways to perform daily living activities, and adjust to living with AMD. The main treatment for wet AMD is injections of anti-VEGF agents, because high levels of VEGF in the eye are associated with the formation of abnormal blood vessels that cause most of the damage in wet AMD. Anti-VEGF agents are used to fight disease progression and reduce the damaging effects of these leaky abnormal blood vessels. They are also effective in stabilizing vision in many patients.

[0109] In some aspects, the use of RPE cells as described herein can be combined with one or more other therapeutic agents. The wording "therapy combination", "combined with " etc. refers to the use of more than one medicine or treatment to increase reaction simultaneously. RPE cells of the present invention can be used in combination with other medicines or treatments for the treatment of AMD, for example. Specifically, RPE cells can be combined with anti-VEGF agents to the experimenter. Such therapy can be used before, simultaneously or afterwards using RPE cells of the present invention.

[0110] In one aspect, administration of differentiated RPE cells increases photoreceptor function and / or survival.

[0111] As used herein, "photoreceptor" or "photoreceptor cell" refers to a special (specific) type of neuroepithelial cell present in the retina that is capable of visual phototransduction. The great biological importance of photoreceptors is that they convert light (visible electromagnetic radiation) into signals that can stimulate biological processes. Specifically, photoreceptor proteins in cells absorb photons, thereby inducing changes in the cell membrane potential. There are three known types of photoreceptor cells in the mammalian eye: rods, cones, and intrinsically photosensitive retinal ganglion cells. The two classical photoreceptor cells are rods and cones, each of which contributes information used by the visual system to form a diagram of the visual world (i.e., vision). Rods contribute primarily to night vision (scotopic conditions), while cones contribute primarily to daytime vision (photopic conditions), but the chemical processes supporting phototransduction in each cell are similar. Intrinsically photosensitive retinal ganglion cells are not thought to contribute directly to vision, but play a role in the regulation (entrainment) of circadian rhythms and pupil reflexes.

[0112] There are major functional differences between rods and cones. Rods are extremely sensitive and can be triggered by a single photon. At very low light levels, the visual experience is based solely on rod signals. Cones require significantly brighter light (i.e., a larger number of photons) to generate signals. In humans, there are three different types of cones, distinguished by their response patterns to different wavelengths of light. The three types of cones respond (roughly) to short, medium, and long wavelengths of light, so they can be called S-cones, M-cones, and L-cones, respectively.

[0113] The human retina contains about 120 million rods and 6 million cones. The number and ratio of rods to cones varies from species to species, depending on whether the animal is primarily diurnal or nocturnal. In the human visual system, in addition to the light-sensitive rods and cones, there are about 2.4 to 3 million ganglion cells, of which 1% to 2% are light-sensitive. The axons of the ganglion cells form the two optic nerves. The photoreceptor cells are usually arranged in an irregular but approximately hexagonal grid, known as a retinal mosaic.

[0114] Rod and cone photoreceptors are found in the outermost layer of the retina; they all have the same basic structure. Closest to the field of vision (and farthest from the brain) are the axon terminals, which release a neurotransmitter called glutamate to the bipolar cells. Further back is the cell body, which contains the cell's organelles. Still further back is the inner segment, which is a specialized part of the cell filled with mitochondria. The primary function of the inner segment is to provide ATP (energy) for the sodium-potassium pump. Finally, closest to the brain (and farthest from the field of vision) is the outer segment, which is the part of the photoreceptor that absorbs light. The outer segment is actually a modified cilium that contains a disk filled with opsins, molecules that absorb photons, and voltage-gated sodium channels.

[0115] The membrane photoreceptor proteins opsins contain a pigment molecule called retinal. In rod cells, these are together called rhodopsin. In cone cells, there are different types of opsins, which combine with retinal to form pigments called photopsins. The three different classes of photopsins in cone cells respond to different ranges of light frequencies (allowing the visual system to calculate the differentiation of colors). The function of the photoreceptor cells is to convert the light information of the photons into a form of information that can be communicated to the nervous system and easily used by the organism: this conversion is called signal transduction.

[0116] By "increasing photoreceptor function and / or survival" it is meant that administration of RPE cells increases the function and / or survival of any type of photoreceptors, including rods, S-cones, M-cones, L-cones, and intrinsically photosensitive retinal ganglion cells.

[0117] In some aspects, increasing photoreceptor function comprises increasing photoreceptor outer segment turnover, increasing phagocytosis involving MERTK, and / or increasing, restoring, and / or generating cell / cell tight junctions.

[0118] In various aspects, restoring and / or generating cell / cell tight junctions improves or restores the brain-eye barrier.

[0119] The "blood-eye barrier" or "brain-eye barrier" is a barrier formed by the endothelium of the capillaries of the retina and iris, the ciliary epithelium, and the retinal pigment epithelium. It is a physical barrier between the local blood vessels and most of the eye itself, preventing many substances including drugs from traveling through it. Inflammation can break this barrier, allowing drugs and macromolecules to penetrate into the eye. As inflammation subsides, the barrier usually recovers. It consists of a blood-aqueous barrier including the capillaries of the ciliary epithelium and iris and a blood-retinal barrier consisting of tight connections between the non-porous capillaries and retinal epithelial cells of the retinal circulation. The blood-aqueous barrier is formed by the non-pigmented ciliary epithelial cells of the ciliary body and the endothelial cells of the blood vessels in the iris. If formed by the endothelium of the retinal blood vessels and the retinal pigment epithelium, the blood-retinal barrier prevents macromolecules from passing through the choroidal capillaries and entering the retina.

[0120] In another aspect, the PSC is an hPSC. In some aspects, the hPSC is an autologous hPSC or an allogeneic hPSC.

[0121] hPSC, i.e., autologous hPSC, can be obtained from a subject in need of treatment. The advantage of autologous hPSC is that almost all possibilities of graft-versus-host disease rejection are eliminated, because there is no risk of incompatibility, and therefore there is no risk of any problems associated with incompatibility. When hPSC cannot be obtained from the subject itself, hPSC can be obtained from an allogeneic non-autologous donor that has been matched with the subject for tissue compatibility. Allogeneic hPSC has the advantage of pre-preparation / storage to be used as soon as possible when necessary. However, there are still risks associated with lack of tissue compatibility between the donor and the recipient, as well as the risk of cell rejection (e.g., acute or chronic graft-versus-host disease).

[0122] In another embodiment, the present invention provides a kit comprising: (a) a neuroectoderm induction mixture, which comprises: a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor and / or a FGF / ERK pathway signaling inhibitor; (b) a TGFβ family protein; and (c) instructions for inducing pluripotent stem cells (PSCs) to differentiate into retinal pigment epithelial (RPE) cells.

[0123] In one aspect, the kit further comprises a laminin-coated surface.

[0124] The embodiment of the method that the discussion inducing PSC to differentiate into RPE cell that is expected to be used for the application discussed is presented below.Following examples are provided for further illustrating embodiments of the present invention, but are not intended to limit the scope of the present invention.Although they are those typical procedures, methods or techniques that can be used, other programs, methods or techniques known to those skilled in the art can be used alternatively.

[0125] Example

[0126] Example 1

[0127] Design of retinal pigment epithelial cells

[0128] Differentiation scheme

[0129] In a stepwise iterative development process, a novel combination of factors that efficiently promotes superior RPE differentiation was identified using a simple and GMP-compliant workflow. The basic principle is to combine a small molecule neuroectoderm induction cocktail with activin A treatment in a defined temporal sequence (see for an illustrative example of the method of the invention). Figure 1 ). In short, the iPSC of neuroectodermal pedigree is initiated using a combination of two or three pathway inhibitors, including FGF / ERK signaling inhibitors (e.g., PD0325901, "PD" or analogs), TGFβ / SMAD2 / 3 signaling inhibitors (e.g., SB431542, "SB") and BMP / SMAD1 / 5 / 8 inhibitors (e.g., doxormorphine, "DM" or analogs). It is known that long-term inhibition of two SMAD pathways with or without additional FGF / ERK signaling inhibition can lead to neuronal fate (not RPE), and RPE is not a neuronal cell. iPS cells are processed with a neural induction mixture, even if a shorter time period is continued, RPE will not be significantly induced relative to background. It is known that activin A has a positive signaling effect on RPE differentiation in vivo. However, activin A is a key ligand that promotes hPSC self-renewal rather than differentiation.

[0130] However, it was discovered that, when combined with Activin A, they can be used to confine hPSCs, triggering their differentiation into RPE cells.

[0131] like Figure 1 What is shown, in illustrative methods, is that the combination of Activin A treatment and SMAD / FGF pathway inhibition results in highly efficient RPE induction (ie, high purity, in a short time) under adherent culture conditions.

[0132] Example 2

[0133] Materials and methods

[0134] Maintenance of hiPSCs

[0135] Prior to initiating differentiation, hiPSCs were maintained on iMatrix-511 in iPS Brew medium and passaged using EDTA lysis as per routine culture.

[0136] The cells were then grown to 90-100% confluence on 4×iMatrix-511, i.e., 6 wells coated with 12 μl instead of 3 μl Matrix-511 per 6 wells, before starting. Complete confluence at the beginning produced better differentiation results than starting from a semi-confluent culture. However, experiments can be performed under semi-confluent starting conditions. Enhancing the laminin coating appears to have a positive effect on morphology and pigmentation during the initial differentiation phase, but may be more important at a later stage after the RPE cells are re-plated. 12 μl per 6-well coating appears to be optimal for RPE maintenance.

[0137] One week before differentiation, coat the required number of 6 wells with 125 μl iMatrix-511 per 6 wells in 2 ml PBS (each at 37 ° C for 1 h, or overnight at 2-8 ° C). HiPSCs were lysed (dissociated) using an EDTA digestion time of ~4 min, or, to better control cell density and uniformity, TrypLESelect supplemented with 10 μM Rocki was used in new culture wells, aiming to reach 90-100% confluence. At the same time, the cells were fed when appropriate, including 6-8 ml of medium replacement per well.

[0138] Differentiation of hiPSCs

[0139] Day 0: Confirm that the hiPSC morphology is generally undifferentiated and compact and close to 100% confluence. Otherwise, feed the cells again with 4 ml pre-warmed iPS Brew medium and start differentiation on the second day instead. Prepare and / or pre-warm KSR medium (4 ml per 6 wells), PD0325901 (PD) / SB431542 (SB) / Doxorubicin (DM) aliquots, and Activin A. After thawing and thoroughly mixing all reagents, 18 μM PD (1:1000 from 1 mM stock), 59 μM SB10 (1:2000 from 10 mM stock), 0.2511 μM DM (1:2000 from 0.5 mM stock), and 2512 ng / ml Activin A13 (1:400 from 10 μg / ml stock) were added to 4 ml KSR medium per confluent hiPSC culture well after vigorously agitating the culture plate and completely aspirating the iPS Brew medium.

[0140] Day 1 and Day 2: Prepare medium as described above and replace the old medium on the wells with 4 ml of fresh differentiation medium per well after vigorous agitation and complete aspiration of the medium.

[0141] Day 3: Prepare KSR medium containing only Activin A and replace the old medium on the culture wells with 4 ml of fresh differentiation medium containing only ActA per well after vigorous stirring and complete aspiration of the medium.

[0142] Day 4: Prepare fresh KSR+ActA (Activin A) differentiation medium and feed 8 ml per well.

[0143] Week 2: Feed cells with 4 ml / 6 wells daily and 8 ml during weekends.

[0144] Week 3 and Week 4: During the third and fourth weeks of differentiation, no small molecules were applied. Only Activin A was added to the basal medium (KSR or B27 medium). Activin A was added to the pre-warmed basal medium every day and the old medium was replaced on the cells (1:100 mixed thoroughly).

[0145] Day 29 (Week 5): Prepare freshly coated culture dishes by coating 12-well (5 μl) or 6-well format (12 μl iM) with 4×iMatrix-511 in PBS for 1 h at 37°C. Prepare KSR medium and preheat for the lysis procedure. Wash the differentiated cells once (4 ml) or twice (2×2 ml) with PBS, then add 1 ml of preheated TrypLE Se-lec. Use a 1 ml pipette to separate the cells by pipetting up and down several times and check the results under a microscope. Transfer the cell suspension to a 15 ml tube and centrifuge at 300g for 2 min. Transfer a portion of the cells to a 1.5 ml tube for downstream FACS and / or RT-qPCR analysis. Resuspend the cells in an appropriate amount of KSR medium and determine the cell titer using a Neubauer chamber. Plate each cm in a 6-well (4 ml KSR medium with or without activin A24) or 12-well format (2 ml medium). 2 400,000 cells were plated.

[0146] Day 31: Feed cells with KSR medium (6-well format: 4 ml, 12-well format: 2 ml).

[0147] Day 33: Feed cells with KSR medium (6-well format: 8 ml, 12-well format: 4 ml).

[0148] Week 6: Repeat the procedure of week 5. Depending on the culture status (morphology, results of in-process control), cells are either passaged again (for further cleaning or expansion) or left to mature in KSR medium without Activin A.

[0149] RPE cells at Px1 or higher (xn = passage number after initial differentiation) were re-plated onto transwells as required for the specific characterization assay. 2 24-well transwells of surface area are coated with 0.5 μl iMatrix-511 per well. According to the RPE maturation stage (Px1 or Px2 or higher, for example, long-term incubation for several weeks), cells are re-plated with 1x or 10x TrypLE29. After PBS washing, the cells are digested for 10min, and the results are checked under a microscope, and then gently pipetted with a 1ml pipette, and then re-checked under a microscope. If the vast majority of cells are single cells, digestion is stopped by adding 3 times the volume of KSR culture medium, and the cells are transferred to a 15ml tube and centrifuged at 300g for 2min. Otherwise, the incubation time is extended, and the results are checked as described above. The precipitated cells are resuspended in an appropriate volume of KSR culture medium, and the cell titer is determined using a Neubauer chamber, 100,000 cells per well are plated-total internal volume: 0.5ml, total external volume: 1ml. The culture medium is replaced twice a week until the analysis time point.

[0150] Analysis by RT-qPCR

[0151] The sample to be analyzed by RT-qPCR is a cell dissolved directly from the culture well using the RA1 buffer from the RNA isolation kit. For example, the culture medium is sucked off after vigorous stirring, and then 600 μl of buffer RA1 is added to the culture well, and the sample is dissolved and homogenized by quickly pipetting up and down with a 1 ml pipette. 350 μl is transferred to a 1.5 ml tube or directly to a filter column and carried out according to the instructions for use of the kit. Alternatively, a representative cell fraction (proportional part, fraction, fraction) (e.g., 25% of 6 wells) is scraped out from the flowing culture using a sharp plastic scraper, and the floating mass is then moved into a 1.5 ml tube. For example, the tube is briefly spun on a microcentrifuge to completely remove the supernatant (ideally, no liquid remains on top of the cell pellet). The cells are then quickly resuspended in 350 μl of buffer RA1. After adding buffer RA1, the sample is immediately homogenized. As a third option, cells are harvested for additional purposes, such as re-plating or parallel FACS analysis. In this case, cells are harvested by creating a cell suspension and appropriate fractions are transferred into 1.5 ml tubes for RNA isolation. Cells are centrifuged in a mini or benchtop centrifuge and used as described above. RNA is isolated according to the manufacturer's instructions using an elution volume of 40 μl. RNA concentration is measured on a NAnoDrop monitoring the A260 / 280 and A260 / 230 ratios. RNA is always kept cold. Assemble the cDNA synthesis reaction in a PCR strip and incubate in a PCR machine at 42°C for 1 h:

[0152] 18.5 μl diluted RNA, same amount for all samples, e.g. μg or 500 ng

[0153] 5 μl 5× RT buffer (as part of the master mix)

[0154] 0.5 μl dNTP mix (as part of the master mix)

[0155] 0.5 μl oligo-dT primer (as part of the master mix)

[0156] 0.5 μl M-MLV reverse transcriptase (as part of the master mix)

[0157] Dilute the cDNA reactions by adding 150 μl of water each. Assemble qPCR according to the experiment-specific template using gene-specific primers:

[0158] 10 μl SYBR Green mix

[0159] 3 μl gene-specific primer working mix

[0160] 7 μl diluted cDNA

[0161] Run qPCR according to the appropriate GMP SOPs and analyze the data based on the experiment-specific template.

[0162] Analysis by FACS

[0163] Prepare or thaw aliquots of FACS buffer to assemble primary and secondary antibody solutions (200 μl per sample and antibody):

[0164] MITF1 antibody 1:150>A11001 secondary antibody 1:750-1:1000

[0165] BEST1 antibody 1:150>A11001 secondary antibody 1:750-1:1000

[0166] PMEL17 antibody 1:50>A21206 secondary antibody 1:750-1:1000

[0167] ZO1 antibody 1:250>A21206 secondary antibody 1:750-1:1000

[0168] Secondary antibody sample only is used as negative control.Cells are harvested to lyse RPE, thereby producing single cell suspension.After cell counting, a limited number of cells are transferred to the desired number of 1.5ml tubes (each staining range is 0.5-1Mio cells).Cells are centrifuged at 400g for 1min, supernatant is discarded, precipitate is washed with 180-200μl PBS, precipitate is produced again, supernatant is discarded.Precipitate is resuspended in 400μl 2% formaldehyde / PBS respectively, pipetted up and down (blowing) several times quickly, incubated at room temperature for about 10min.Pipe is centrifuged at 400g for 1min, supernatant is completely sucked off (soak off), cells are resuspended in 180-200μl FACS buffer, pipet is left to stand for 1-2min with block, centrifuged again, supernatant is discarded. The cells were then resuspended in the primary antibody solution, incubated at room temperature for 15 min, the tube was flicked at least once during the incubation period, centrifuged as described above, and the supernatant was removed. The cells were washed as described above using 180-200 μl FACS buffer. The cells were resuspended in the appropriate secondary antibody solution, incubated at room temperature for 15 min, the tube was flicked at least once during the incubation period, centrifuged as described above, and the supernatant was removed. The cells were washed and centrifuged with 180-200 μl FACS buffer as described above, each cell pellet was resuspended in 300 μl PBS, and analyzed on a flow cytometer using a suitable template. The percentage of positive cells was based on the following hierarchical gating: (i) cells, (ii) single cells, and (iii) surrounding positive clouds in the fluorescence intensity and forward scatter plots.

[0169] Example 3

[0170] Characterization of retinal pigment epithelial cells differentiated from human pluripotent stem cells

[0171] According to the protocol described in Example 2 and Figure 1 An illustrative method is shown for differentiating human induced pluripotent stem cells (hiPSC) into RPE.

[0172] The regimen described herein using aliquots of PD0325901 (PD or P) / SB431542 (SB or S) / dosomorphine (DM or D) and activin A (A) is referred to as the PSD+A regimen. The PSD+A regimen was initially compared to the procedure described by Maruotti et al. (2015). Figures 2A-2C As shown, at the 4 week time point, the PSD+A protocol produced RPE cells expressing higher levels of BEST1, RPE65, RLBP1, TYR, PMEL17, MITF, and OTX2 as measured by RTqPCR compared to REF cells obtained using the reference protocol (see Figure 2A).like Figure 2B As shown, this corresponds to RPE cells having a pigmentation phenotype that is clearly different from that of cells obtained by the reference protocol. Figure 2C It was further shown that treatment with A alone was not sufficient to induce the RPE phenotype, and PSD+A was the optimal regimen, indicating the importance of small molecule treatment.

[0173] like Figure 3 As shown, RPE gene expression was quantified by RTqPCR 4 weeks after initial induction with PSD, SD (no PD), PD (no SB), or PS (no DM). DM withdrawal showed the most severe decrease in RPE-specific marker expression compared to the standard PSD regimen and can therefore be considered essential, while PD and SB withdrawal can be tolerated in the long term.

[0174] like Figure 4A-4B As shown, the long-term equivalence of PSD+A and PD+A induction after re-plating of cells treated for 4 weeks was assessed. iPS cells were induced with a 2-factor small molecule cocktail (PD) or a 3-factor cocktail (PSD) and RPE-specific marker expression was assessed after the first 4 weeks of differentiation. Figure 4A Shown are protein expression levels as measured by flow cytometry, Figure 4B Gene expression levels as measured by RTqPCR are shown. The results indicate that although induction with a triple pathway inhibitor is preferred, 2-factor induced RPE cells exhibit equivalent expression of RPE-specific markers and therefore may be equivalent.

[0175] The timing of triple pathway inhibition was evaluated to assess how long cells needed to be exposed to the three small molecules to induce RPE cell differentiation. Figure 5 As shown, RTqPCR analysis indicated that 1 day of treatment or complete withdrawal (activin A alone) was insufficient to induce RPE cell fate, but 2 days or more (at least up to 7 days) was optimal, with few differences observed between different exposure times.

[0176] Therefore, we evaluated when activin A treatment should be initiated for optimal RPE induction and which concentration should be used. Figure 6 As shown, unlike the standard protocol, a form of activin A treatment that began after signaling inhibition produced reduced RPE gene expression, indicating the importance of treating cells simultaneously with small molecule inhibitors and activin A for optimal RPE cell induction. Figure 7 As shown, titration of activin A revealed that concentrations below 20 ng / ml or complete withdrawal of activin resulted in poor, but not complete absence of, RPE induction.

[0177] The time course of RPE markers was assessed to evaluate the expression levels of early, mid / late, late, and mature markers after RPE induction for up to 10 weeks. Figures 8A-8C As shown, after several days of induction, PSD+A induced early ( Fig. 8A ), mid / late stage( Figure 8B ), Late / Mature( Figure 8C )RPE markers.

[0178] RPE cells were also characterized by immunofluorescence and light microscopy. Fig.9A As shown, RPE cells displayed a typical pigmented cobblestone morphology under phase contrast light and were found to express the RPE marker BEST1 ( Fig. 9B )、ZO1( Fig. 9B and Fig.9D )、MITF1( Fig. 9C ) and CRALBP( Fig. 9C and Fig.9D ).

[0179] RPE cells were further analyzed using electron microscopy, as Figures 10A-10B The transmission of RPE cells generated by the PSD+A protocol ( Fig. 10A ) and Scan( Fig. 10B ) Electron microscopic analysis showed typical RPE features such as epithelial polarity, with nuclei located at the basal side and melanosomes and microvilli towards the apical side and apical cell surface, respectively.

[0180] Finally, the cells were evaluated for terminal differentiation by assessing whether they had residual expression of any stem cell markers. Fig.11 As shown, the expression of ECAT11, OCT4, NANOG, SOX2, mir-302HT, and LIN28 in RPE cells obtained by the PSD+A protocol was evaluated. The expression levels were compared with those in iPSCs. Fig.11 As shown, RPE cells did not express any stem cell markers.

[0181] References

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[0192] Osakada,F.,Jin,Z.B.,Hirami,Y.,Ikeda,H.,Danjyo,T.,Watanabe,K.,Sasai,Y.,and Takahashi,M.(2009).In vitro differentiation of retinal cells fromhuman pluripotent stem cells by small-molecule induction.J Cell Sci 122,3169-3179.

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[0199] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the appended claims.

Claims

1. A method for generating retinal pigment epithelial (RPE) cells, comprising: (a) contacting a culture of pluripotent stem cells (PSCs) with a mixture comprising: (i) one or more of a transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor; and (ii) optionally a TGFβ family protein; and (b) subsequently culturing the cells of (a) with the TGFβ family protein in the absence of the mixture of agents of (a)(i), Thus, RPE cells are generated.

2. The method of claim 1, wherein the culture of PSCs is an adherent monolayer of cells. The method according to claim 2 , wherein the monolayer of the cells is grown in a two-dimensional culture system.

4. The method of claim 1, wherein the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is selected from the group consisting of SB431542, LY3200882, TP0427736HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, gorunisert, vatosert, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854.

5. The method of claim 1, wherein the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor is selected from the group consisting of doxormorphine, PPM1A, and LDN-193189.

6. The method of claim 1, wherein the FGF / ERK pathway signaling inhibitor is selected from the group consisting of PD0325901, PD173074, SU431542, PD161570, PD98059, PD184352, PD198306, and PD334581.

7. The method of claim 1, wherein the TGFβ family protein is selected from the group consisting of activin A, TGFβ1, TGFβ2, and TGFβ3.

8. The method of claim 1, wherein the mixture of (a) comprises about 0.1-10 μM TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, about 0.1-1 μM BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor and / or about 0.5-5 μM FGF / ERK pathway signaling inhibitor; and at least about 0.1 ng / ml of a TGFβ family protein.

9. The method of claim 1, wherein the mixture comprises about 1-5 μM SB431542, about 0.25 μM dorsomorphine, about 0.5-1.5 μM PD0325901, and about 25 ng / ml activin A.

10. The method of claim 1, wherein contacting the PSC with the TGFβ family protein in (b) comprises contacting the PSC with at least about 0.1 ng / ml TGFβ family protein.

11. The method of claim 1, wherein contacting the PSC with the TGFβ family protein in (b) comprises contacting the PSC with about 25 ng / ml Activin A.

12. The method of claim 1, wherein the PSC is contacted with the mixture of (a) for about 2-7 days.

13. The method of claim 1, wherein the PSCs are contacted with the TGFβ family protein in (b) for about 4 weeks.

14. The method of claim 3, wherein the monolayer is cultured on a surface comprising a laminin coating.

15. The method of claim 1, wherein the PSC is a human PSC (hPSC).

16. The method of claim 14, wherein the hPSC is a human induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC).

17. The method of claim 1, wherein the PSCs are contacted with the mixture in the absence of a hypoxia inducible factor (HIF) pathway modulator.

18. The method of claim 1, wherein the PSC is contacted with the mixture in the absence of nicotinamide.

19. The method of claim 1, wherein the PSCs are cultured under conditions other than hypoxic conditions.

20. The method of claim 1, wherein the PSCs are cultured in a system that is not a three-dimensional culture system.

21. A method for inducing retinal pigment epithelial (RPE) cell differentiation from pluripotent stem cells (PSCs), comprising: (a) PSCs were cultured in a 2D culture system under conditions that allowed growth as an adherent monolayer; (b) contacting the PSC with a mixture of agents for about 2-7 days, wherein the mixture of agents comprises: one or more of a transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor, and optionally a TGFβ family protein; and (c) subsequently culturing the cells of (b) with the TGFβ family protein for about 4 more weeks, Thereby inducing RPE cell differentiation from PSCs.

22. according to the method for claim 21, wherein RPE cell differentiation is direct RPE cell differentiation.

23. The method of claim 21, wherein the differentiated RPE cells have increased expression of PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1, CLDN19, ATP1B1, NC1, ZO1 and / or TYR compared to hPSCs.

24. The method of claim 21, wherein the differentiated RPE cells do not express ECAT11, OCT4, NANOG, SOX2, mir302HT or LIN28.

25. The method of claim 21, wherein the PSC is a human PSC (hPSC).

26. A method of treating macular degeneration in a subject, comprising administering to the subject differentiated retinal pigment epithelial (RPE) cells, The differentiated RPE cells are obtained by the method of claim 1 or 21.

27. according to the method for claim 26, wherein using the RPE cell of differentiation improves photoreceptor function and / or survival.

28. The method of claim 27, wherein increasing photoreceptor function comprises: increasing the turnover of photoreceptor outer segments, enhancing phagocytosis involving MERTK, and / or increasing, restoring and / or generating cell / cell tight junctions.

29. The method of claim 28, wherein restoring and / or generating cell / cell tight junctions improves or restores the brain-eye barrier.

30. The method of claim 26, wherein administering differentiated RPE cells comprises orthotopic injection of RPE cells.

31. The method of claim 26, wherein the PSC is a human PSC (hPSC).

32. The method of claim 31, wherein the hPSCs are autologous hPSCs or allogeneic hPSCs.

33. A kit comprising: (a) a neuroectoderm induction mixture comprising: a transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor, a bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, and / or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor; (b) TGFβ family proteins; and (c) Instructions for use in inducing pluripotent stem cells (PSCs) to differentiate into retinal pigment epithelial (RPE) cells.

34. The kit of claim 33, further comprising a laminin-coated surface.

Images