Method for differentiating pluripotent stem cells into retinal pigment epithelial cells

Abstract

The present invention provides a method for generating retinal pigment epithelium (RPE) cells from pluripotent stem cells (PSCs), and a method of using such cells. The present disclosure provides a method for generating RPE cells by differentiating PSCs, comprising treating the PSCs with one or more of a transforming growth factor β / 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 TGFβ family protein. Use of such RPE cells includes a method of treating macular degeneration by administering the RPE cells to a subject in need thereof.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 340,736, filed May 11, 2022. The disclosure of this prior application is considered a part of the disclosure of this application and is hereby incorporated by reference in its entirety into the disclosure of this application.

[0002] (Background of the Invention) (Field of the Invention) The present invention generally relates to retinal pigment epithelium (RPE) cells and, more particularly, to methods for generating RPE cells from pluripotent stem cells (PSCs).

Background Art

[0003] (Background Information) The retinal pigment epithelium (RPE) forms a single - cell - layer cell sheet that underlies the eye's photoreceptors. This is essential for the function and survival of these retinal photoreceptors and thus for proper vision. Among several functions, in particular, those RPE cells contribute to the regeneration of photoreceptor outer segments (POS) by phagocytosis involving MERTK. Through the formation of tight junctions, the RPE layer also forms the blood - brain - eye barrier that separates the inside of the eye from the bloodstream. Degenerated or otherwise damaged RPE can give rise to age - related macular degeneration that may be treated by in situ transplantation of RPE cells generated from human pluripotent stem cells (hPSCs) (such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs)).

[0004] Various approaches have been devised to promote RPE differentiation from PSCs, and often, basal media containing various molecules are used. For example, RPE induction has been described using a complex cocktail of small molecules and growth factors, such as Noggin, Dkk1, IGF1, bFGF, activin A, nicotinamide, SU5402, etc., in the presence of bovine serum albumin. Some studies have used combinations of nicotinamide and activin A, or casein kinase / TGFβ / SMAD2 / 3 inhibitors with limited efficiency to drive RPE induction (Idelson et al., 2009; Osakada et al., 2009). Other combinations and orders of adding differentiation-driving molecules to animal-derived extracellular matrices have also been described, for example, using bFGF, SB431542, retinoic acid, Shh, and Noggin (Zahabi et al., 2012). Often, the individual effects exerted by such molecules are not confirmed, and their "cocktails" are not optimized with respect to the exact composition, factor concentrations, timing of administration, etc.

[0005] RPE cells can also be obtained with reduced efficiency slowly (within a few weeks) through spontaneous differentiation along the neural pathway (Plaza Reyes et al., 2016). Indeed, it has been demonstrated that human embryonic stem cells (hESCs) can be differentiated into RPE cells by culturing them in xeno-free synthetic NutriStem™ hESC XF medium in the absence of basic fibroblast growth factor (bFGF) using a recombinant human laminin (rhLN-521)-based matrix. Under such conditions, hESC-RPE differentiated cells were obtained with a decrease in pluripotency-related transcripts OCT3 / 4 and NANOG, along with robust expression of the neuroectodermal transcripts sex-determining region Y box 9 protein (SOX9) and paired box 6 (PAX6). This culture robustly supported the formation of pigmented structures resembling early eye vesicles (OV) at 3 weeks of differentiation, and uniform pigmentation was obtained by week 9.

[0006] 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). Existing methods yield impure cell populations, rely on the use of animal-derived molecules, which are problematic for clinical compliance and reduced immunogenicity, and existing methods can take weeks to obtain. There remains a need in the art to develop methods for efficient RPE differentiation that yield pure cell cultures in a short period using simple culture conditions.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

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Non-Patent Document 3

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0008] (Gist of the Invention) The present invention is based on the important discovery that the combination of a neuroectoderm induction cocktail of a transforming growth factor-β (TGF-β) family pathway inhibitor and / or a fibroblast growth factor (FGF) / ERK pathway signaling inhibitor and activin A treatment results in the induction of RPE cells from PSCs when used in adherent cell culture conditions in a defined chronological order.

[0009] In one embodiment, the present invention provides a method for generating retinal pigment epithelial (RPE) cells, the method comprising: (a) contacting a culture of pluripotent stem cells (PSCs) with a mixture of agents comprising (i) an inhibitor of the transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling, an inhibitor of the bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling, or an inhibitor of the fibroblast growth factor (FGF) / ERK pathway signaling, and (ii) a TGFβ family protein, if desired; and then (b) culturing the cells of (a) in the absence of the mixture of agents of (a)(i) with the TGFβ family protein.

[0010] In one aspect, the culture of the PSCs is an adherent monolayer of cells. In one aspect, the monolayer of cells is grown in a two-dimensional culture system (e.g., a Petri dish). In one aspect, the inhibitor of the TGFβ / SMAD2 / SMAD3 pathway signaling is selected from the group consisting of SB431542, LY3200882, TP0427736 HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, galunisertib, vactosertib, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854. In another aspect, the inhibitor of the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling is selected from the group consisting of dorsomorphin, PPM1A, and LDN-193189. In a further aspect, the inhibitor of the FGF / ERK pathway signaling is selected from the group consisting of PD0325901, PD173074, SU431542, PD161570, PD98059, PD184352, PD198306, and PD334581. In one aspect, the member of the TGFβ protein superfamily is selected from the group consisting of activin A, TGFβ1, TGFβ2, and TGFβ3.

[0011] In one aspect, the mixture of (a) comprises a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor at about 0.1 μM to about 10 μM, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor at about 0.1 μM to about 1 μM, and / or an FGF / ERK pathway signaling inhibitor at about 0.5 μM to about 5 μM; and at least about 0.1 ng / ml of a TGFβ family protein. In various aspects, the mixture comprises about 1 μM to about 5 μM of SB431542, about 0.25 μM of dorsomorphin, about 0.5 μM to about 1.5 μM of PD0325901, and about 25 ng / ml of activin A. In one aspect, the step of contacting the PSC with the TGFβ family protein comprises contacting the PSC with at least about 0.1 ng / ml of a TGFβ family protein. In one aspect, the step of contacting the PSC with the TGFβ family protein comprises contacting the PSC with at least about 5 ng / ml of activin A. In another aspect, the step of contacting the PSC with the TGFβ family protein comprises contacting the PSC with about 25 ng / ml of activin A. In one aspect, the step of contacting the PSC with the above mixture is from about 2 days to about 7 days. In another aspect, the step of contacting the PSC with the TGFβ family protein (e.g., activin A) is about 4 weeks.

[0012] In some aspects, the monolayer is cultured on a surface comprising a laminin coating. In various aspects, the PSC is a human PSC (hPSC) (e.g., a human induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC)). In one aspect, the contact between the PSC and the above mixture is in the absence of a hypoxia-inducible factor (HIF) pathway modulator. In another aspect, the contact between the PSC and the above mixture is in the absence of nicotinamide. In a further aspect, the PSC is cultured under conditions that are not hypoxic conditions (e.g., under normoxic conditions). In a further aspect, the hPSC is cultured in a system that is not in a three-dimensional culture system (e.g., a spinner flask).

[0013] In another embodiment, the present invention provides a method for inducing retinal pigment epithelium (RPE) cell differentiation from pluripotent stem cells (PSCs), the method comprising: (a) culturing the PSCs in a two-dimensional culture system under conditions that allow proliferation as an adherent monolayer; (b) contacting the PSCs with 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 superfamily protein, for about 2 to about 7 days; and (c) then culturing the cells of (b) with the TGFβ family protein for an additional about 4 weeks.

[0014] In one aspect, the RPE cell differentiation is direct RPE cell differentiation. In another aspect, the differentiated RPE cells have increased PMEL17 expression, MITF expression, OTX2 expression, BEST1 expression, RPE65 expression, RLBP1 expression, CLDN19 expression, ATP1B1 expression, NC1 expression, ZO1 expression and / or TYR expression compared to PSCs. In one aspect, the differentiated RPE cells do not express ECAT11, OCT4, NANOG, SOX2, mir302HT and / or LIN28.

[0015] In a further embodiment, the present invention provides a method for treating macular degeneration in a subject, the method comprising administering differentiated retinal pigment epithelium (RPE) cells to the subject, the differentiated RPE cells being obtained by one of the methods described herein.

[0016] In one aspect, administration of differentiated RPE cells increases photoreceptor function and / or photoreceptor survival. In some aspects, the increase in photoreceptor function includes increasing regeneration of photoreceptor outer segments, increasing phagocytosis involving MERTK, and / or increasing, restoring, and / or generating intercellular tight junctions. In various aspects, restoration and / or generation of intercellular tight junctions improves or restores the brain-eye barrier. In one aspect, administration of differentiated RPE cells includes injecting the RPE cells in situ. In another aspect, the hPSCs are autologous hPSCs or allogeneic hPSCs.

[0017] In a further embodiment, the invention provides a kit that comprises: (a) a neuroectoderm induction cocktail comprising a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, and / or an FGF / ERK pathway signaling inhibitor; (b) a TGFβ family protein; and (c) instructions for inducing differentiation of pluripotent stem cells (PSCs) into retinal pigment epithelium (RPE) cells.

[0018] In one aspect, the kit further comprises a laminin-coated surface. BRIEF DESCRIPTION OF THE DRAWINGS

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Figures 9A-D

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Figure 11

Mode for Carrying Out the Invention

[0030] (Detailed Description of the Invention) The present invention is based on the important finding that a combination of a neuroectodermal induction cocktail comprising one or more of a TGFβ family pathway inhibitor and / or an FGF / ERK pathway signal transduction inhibitor and a TGFβ family protein results in the induction of RPE cells from PSCs when used in adherent cell culture conditions in a defined temporal order.

[0031] Prior to the description of the present compositions and methods, it should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described, because such compositions, methods, and experimental conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting, because the scope of the present invention is limited only in the appended claims.

[0032] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or procedures of the kind described herein that will be apparent to those of ordinary skill in the art upon reading this disclosure.

[0033] All publications, patents, and patent applications mentioned in this specification are hereby 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.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it is understood that modifications and variations are within the scope of the present disclosure. Preferred methods and materials are described below.

[0035] In one embodiment, the present invention provides a method for generating retinal pigment epithelium (RPE) cells, the method comprising: (a) contacting a culture of pluripotent stem cells (PSCs) with a mixture of agents comprising (i) an inhibitor of transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling, an inhibitor of bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling, or an inhibitor of fibroblast growth factor (FGF) / ERK pathway signaling; and (ii) a TGFβ family protein, if desired; and (b) then culturing the cells of (a) with the TGFβ family protein in the absence of the mixture of agents of (a)(i).

[0036] The pigment epithelium of the retina or retinal pigment epithelium (RPE) is a layer of pigmented cells immediately outside the neurosensory retina, nourishes the retinal photoreceptors, and is firmly attached to the underlying choroid and the overlying retinal photoreceptors. The RPE is composed of a single layer of hexagonal cells (RPE cells) densely packed with pigment granules. When viewed from the outer surface, these cells are smooth and hexagonal in shape. When viewed in cross-section, each cell consists of an outer non-pigmented portion containing a large oval nucleus and an inner pigmented portion extending as a series of parallel screw-like protrusions between the rods, which is particularly applicable when the eye is exposed to light.

[0037] The RPE has several functions, including light absorption, epithelial transport, spatial ion buffering, the visual cycle, phagocytosis, secretion, and immune regulation. RPE cells are responsible for the absorption of scattered light. This role is very important for two main reasons. First, to improve the quality of the visual system. Second, because light is radiation and light is concentrated by the lens onto the cells of the macula, resulting in a strong concentration of photo-oxidative energy. Melanosomes absorb that scattered light and thus reduce its photo-oxidative stress. High perfusion of the retina results in a high partial pressure of oxygen. The combination of light and oxygen results in oxidative stress, and the RPE has many mechanisms to cope with it. RPE cells form the outer blood-retinal barrier, and its epithelium has tight junctions between the sides, accompanied by the isolation of the inner retina from systemic effects. This is important for the highly selective transport of substances for a tightly regulated environment that is an immune privilege of the eye (not only as a barrier but also involving signal transduction processes). The RPE supplies nutrients to photoreceptors, controls ion homeostasis, and removes water and metabolites. Its visual cycle performs the essential task of maintaining visual function and thus needs to be adapted to various visual requirements (e.g., vision in the dark or vision in the light). The photoreceptor outer segment (POS) membrane is constantly exposed to photo-oxidative stress, and those photoreceptor outer segment (POS) membranes constantly experience destruction by that photo-oxidative stress. Those photoreceptor outer segment (POS) membranes are constantly regenerated by shedding their ends, and then the RPE phagocytoses and digests those ends. The RPE is an epithelium that, on one side, interacts closely with photoreceptors but must also be able to interact with cells on the blood side of its epithelium (e.g., endothelial cells or cells of the immune system). To communicate with its neighboring tissues, the RPE is capable of secreting a wide variety of factors and signaling molecules.The RPE secretes ATP, fas-ligand (fas-L), fibroblast growth factors (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), members of the interleukin family, tissue metalloprotease inhibitors (TIMP), and pigment epithelium-derived factor (PEDF). Many of these signaling molecules have important physiopathological roles. The interior of the eye represents an immune-privileged space separated from the bloodstream's immune system. That immune privilege is supported by the RPE in two ways. First, it presents a mechanical and tight barrier that separates the interior space of the eye from the bloodstream. Second, the RPE can communicate with the immune system to suppress the immune response in a healthy eye or, alternatively, activate the immune system in the case of disease.

[0038] Dysfunction of the RPE is associated with RPE diseases that can be called retinal degenerative diseases. Retinal degenerative diseases can result from oxidative stress and inflammation, apoptosis and autophagy, and / or defects in cell polarity and cell interactions.

[0039] The cornea has a transparent structure, and the RPE is exposed to light for a long time and has a rich oxygen supply. As a result, a large amount of reactive oxygen species are easily generated. Furthermore, elevated systemic glucose levels (e.g., in diabetic patients) can promote excessive accumulation of reactive oxygen species (ROS). In degenerative retinopathy, the antioxidant level decreases in cells, that is, the ability of RPE cells to remove reactive oxygen species (ROS) variably decreases, resulting in a large accumulation of POS. Oxidative stress and inflammation cause RPE cell damage, which in turn causes retinal dysfunction and even blindness. Diseased-state RPE shows increased levels of apoptosis, autophagy, and endoplasmic reticulum stress compared to normal cells. RPE cell death via apoptosis and endoplasmic reticulum stress has been observed in age-related macular degeneration (AMD) and other retinal degenerative diseases. The polarity and intercellular junctions of the RPE play an important role at the blood-retinal barrier, maintaining the stability of the internal microenvironment of photoreceptors and supporting the choroidal system. Disrupted cell polarity and intercellular junctions significantly increase the risk of retinal degenerative diseases. The stability of the polarity and intercellular junctions of the RPE is related to the unique basal and apical structures of the retina, and these structures affect phagocytosis and material exchange. Abnormalities in RPE polarity, disruption of the barrier, and abnormalities in retinal stability can contribute to the development of blinding retinal diseases.

[0040] There is no cure for retinal pigment epithelial diseases. Thanks to years of research on retinal diseases, many genes and signaling pathways have been identified as potential targets for gene therapy or other treatment methods. 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 approach for treating RPE diseases.

[0041] 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 factor (GDNF), activin, inhibin, Nodal, Lefty, and Müllerian inhibiting substance (MIS). Ligands of the TGF-β superfamily form dimers that bind to a heterodimeric receptor complex consisting of type I and type II receptor subunits with serine / threonine kinase domains. After ligand binding, the type II receptor phosphorylates and activates the type I receptor to initiate a Smad-dependent signaling cascade that induces or suppresses transcriptional activity. During development, members of the TGF-β family are thought to be required for dorsal-ventral axis patterning, mesoderm induction and mesoderm patterning, limb bud formation, bone and cartilage formation, neuronal differentiation, and the development of various tissues and organs.

[0042] As used herein, the term "pathway signal transduction inhibitor" refers to any molecule capable of inhibiting a target signal transduction pathway. A signal transduction pathway is a series of chemical reactions in which a group of molecules in a cell function together to control cell function (e.g., cell differentiation). A cell receives a signal from its environment when a molecule (e.g., a hormone or growth factor) binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives the signal, it activates another molecule. This process is repeated throughout the entire signal transduction pathway until the last molecule is activated and the cell function is executed. Abnormal activation of a signal transduction pathway or inhibition of a signal transduction pathway can lead to disease or, in the case of pluripotent cells, a change in their pluripotent state and thus differentiation. The term "molecule" includes, but is not limited to, small molecules (including small molecules without 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 small interfering nucleic acids (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triple-stranded forming oligonucleotides, aptamers, and decoys. Bioactive molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antiviral drugs, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triple-stranded forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and their analogs, and small nucleic acid molecules (e.g., small interfering nucleic acids (siNA), small interfering RNAs (siRNA), double-stranded RNAs (dsRNA), microRNAs (miRNA), antagomirs, and small hairpin-type RNA (shRNA) molecules).

[0043] As used herein, the term "TGFβ / SMAD2 / SMAD3 pathway signal transduction inhibitor" refers to any molecule capable of inhibiting the TGFβ / SMAD2 / SMAD3 signal transduction pathway, the term "BMP / SMAD1 / SMAD5 / SMAD8 pathway signal transduction inhibitor" refers to any molecule capable of inhibiting the BMP / SMAD1 / SMAD5 / SMAD8 signal transduction pathway, and the term "FGF / ERK pathway signal transduction inhibitor" refers to any molecule capable of inhibiting the FGF / ERK signal transduction pathway. Signal transduction pathway inhibition is the antonym of signal transduction pathway upregulation. In this process, small molecules referred to as "signal transduction inhibitors" or "pathway signal transduction inhibitors" block the communication between various molecules of the pathway and prevent the molecular signal transduction cascade.

[0044] As used herein, the term "TGFβ superfamily protein" or "TGFβ family protein" refers to any protein or peptide of the transforming growth factor-β (TGF-β) superfamily, 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 in various species (including invertebrates and vertebrates) as members of the TGFβ family and classified into 23 distinct genotypes belonging to the following four major subfamilies: the TGF-β subfamily (including TGFβ1, TGFβ2, and TGFβ3); bone morphogenetic proteins and growth differentiation factors; the activin and inhibin subfamilies; and left-right determinants. Non-limiting examples of TGFβ family proteins for use in the present method include activin A, TGFβ1, TGFβ2, and TGFβ3.

[0045] The method described herein describes cell culture conditions under which human pluripotent stem cells are expanded to give rise to the generation of RPE cells.

[0046] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and maintain their undifferentiated state. In contrast to embryonic stem cells, which can only be isolated from the inner cell mass of a blastocyst, there are three known available sources of adult stem cells: bone marrow (which requires bone drilling), adipose tissue (which is available by liposuction), and blood (from which its stem cells can be extracted among other cells). As used herein, the term "pluripotent stem cell" refers to a cell that can generate all cell types of an organism, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but can only differentiate into cell types of closely related cell families (generally, the cell types of the organ of their origin). Most adult stem cells are multipotent, although a small number of pluripotent adult stem cells can be recovered from the umbilical cord or other tissues. Sources of cells used for retinal cell therapy include stem cells (e.g., embryonic stem cells (ESC), adult stem cells, and induced pluripotent stem cells (iPSC)). Currently, ESC and iPSC are mainly used for differentiation into RPE.

[0047] In some embodiments, the PSC used in the methods described herein are human PSC, and in some cases, the human PSC are induced pluripotent stem cells (hiPSC) or human embryonic stem cells (hESC).

[0048] By "generating" RPE cells, it is meant that the method provides physical and chemical culture conditions optimized to induce the differentiation of PSC into RPE cells. The differentiation methods described herein result in a population of RPE cells enriched for RPE cells. For example, more than 80%, more than 85%, more than 90%, more than 95%, 96%, 97%, 98% or 99% RPE cells are obtained in a shorter time than methods available in the art and using simpler culture conditions than methods available in the art.

[0049] Physical culture conditions include, but are not limited to, the cell culture environment (e.g., adherent culture or suspension culture, or 2D culture system or 3D culture system), the pH of the culture medium, the gas concentration in the incubator (e.g., CO2 concentration, O2 concentration), and the temperature.

[0050] There are two basic systems for growing cells in culture, either as a monolayer on an artificial substrate (i.e., adherent culture) or floating in the culture medium (suspension culture). The majority of cells derived from vertebrates (except for hematopoietic cell lines and a few others) are anchorage-dependent and must be cultured on a suitable substrate that has been specially treated (i.e., tissue culture-treated) to allow cell attachment and spreading. However, many cell lines can also be adapted to suspension culture.

[0051] In one aspect, the culture of the PSCs is an adherent monolayer of cells. In another aspect, the monolayer of cells is grown in a 2D culture system. In a further aspect, the PSCs are cultured in a system that is not in a 3D culture system.

[0052] In addition to the treatment of the tissue culture surface, cells may require to be grown on a coated surface (i.e., using a coating) to enhance or improve their attachment and / or spreading. "Coating" as an additional surface treatment means all additional modifications made to increase cell adhesion in addition to the standard plasma treatment or corona treatment that is performed by manufacturers on all cell culture plastics. Usually, coatings are performed using proteins or peptides. Various proteins can be used to coat tissue culture-treated dishes, including poly-L-lysine, poly-D-lysine, polyornithine, gelatin, collagen I, collagen IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domain, Matrigel (trademark) (some components of the extracellular matrix to which growth factors etc. are bound), collagen gel, alginate gel, and lactate gel.

[0053] In one aspect, the monolayer is cultured on a surface comprising a laminin coating.

[0054] Physical culture conditions include the gas concentration in the incubator. Incubation of cell cultures is typically carried out in a standard atmosphere containing 15% - 22% oxygen and 5% CO2 for growth and seeding. In various aspects, PSCs are grown in a humidified atmosphere containing a CO2 concentration of about 5% and normal oxygen pressure conditions (non-hypoxic O2 concentration). Although hypoxic culture conditions are generally thought to support the performance of stem cells, in the present method, PSCs are cultured under conditions that are not hypoxic. As used herein, "normal oxygen pressure" conditions refer to culture conditions that include atmospheric O2 concentration (e.g., an O2 concentration of about 15% - 25%). As used herein, hypoxic conditions are characterized by an oxygen concentration that is lower compared to the atmospheric oxygen concentration (approximately 15% - 25% oxygen). In one aspect, hypoxic conditions are characterized by an oxygen concentration of less than about 10%. In another aspect, hypoxic conditions are characterized by an oxygen concentration of about 0.1% - about 10%, about 1% - about 10%, about 1% - about 9%, about 1% - about 8%, about 1% - about 7%, about 1% - about 6%, about 1% - about 5%, about 1% - about 4%, about 1% - about 3%, or about 1% - about 2%. For example, hypoxic conditions include culture conditions having an O2 concentration of 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%, or any intermediate value.

[0055] Examples of chemical culture conditions include, but are not limited to, agents or molecules added to the culture medium to achieve the desired effect (i.e., differentiation of PSCs into RPE cells). The terms "agent" and "molecule" are used interchangeably and include, but are 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).

[0056] In various embodiments, the chemical culture conditions of the methods described herein include 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, in the form of a mixture of agents.

[0057] For example, the mixture of agents includes a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, and a TGFβ family protein. In another example, the mixture of agents includes a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, a FGF / ERK pathway signaling inhibitor, and a TGFβ family protein. In a further example, the mixture of agents includes a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, a FGF / ERK pathway signaling inhibitor, and a TGFβ family protein. Optionally, the mixture includes a FGF / ERK pathway signaling inhibitor and a TGFβ family protein. In an exemplary example herein, the TGFβ family protein is activin A.

[0058] Examples of substances that inhibit TGFβ / SMAD2 / SMAD3 pathway signaling include any molecule that inhibits type I TGFβ receptor (or ALK5) and its analogs 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, galunisertib, vactosertib, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854.

[0059] In one embodiment, the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is SB431542.

[0060] The TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is added to the PSC culture at a concentration in the range of about 0.1 μM to about 10 μM. For example, PSCs are grown in a culture medium containing about 0.1 μM, about 0.3 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM or more. In one embodiment, the mixture contains about 5 μM of the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor. In another embodiment, the mixture contains about 0.1 μM to about 10 μM of SB431542. In some embodiments, the mixture contains about 1 μM to about 5 μM of SB431542. In other embodiments, the mixture contains about 5 μM of SB431542.

[0061] Examples of inhibitors of BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling include any molecule that inhibits the BMP pathway by targeting type I BMP receptors, activin receptor-like kinases (ALK) 2, ALK3, and ALK6. Non-limiting examples of BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors include dorsomorphin, PPM1A, and LDN-193189.

[0062] In one aspect, the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor is dorsomorphin.

[0063] The BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor is added to the PSC culture at a concentration in the range of about 0.1 μM to about 1 μM. For example, PSCs are grown in a culture medium containing about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM or more. In one aspect, the mixture contains about 0.25 μM of the BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor. In another aspect, the mixture contains about 0.1 μM to about 10 μM of dorsomorphin. In some aspects, the mixture contains about 0.25 μM of dorsomorphin.

[0064] Examples of FGF / ERK pathway signaling inhibitors include 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.

[0065] The FGF / ERK pathway signal transduction inhibitor is added to the PSC culture at a concentration in the range of about 0.1 μM to about 5 μM. For example, the PSCs are grown in a culture medium containing about 0.1 μM, about 0.25 μM, about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM or more. In one embodiment, the mixture contains the FGF / ERK pathway signal transduction inhibitor at about 0.5 μM to about 1.5 μM. In another embodiment, the mixture contains PD0325901 at about 0.1 μM to about 5 μM. In some embodiments, the mixture contains PD0325901 at about 0.5 μM to about 1.5 μM.

[0066] 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 culture medium containing at least about 0.1 ng / ml, about 0.2 ng / ml, about 0.3 ng / ml, about 0.4 ng / ml, about 0.5 ng / ml, about 0.75 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml or more of the TGFβ family protein.

[0067] In one embodiment, the member of the TGFβ protein family is selected from the group consisting of activin A, TGFβ1, TGFβ2, and TGFβ3.

[0068] In some embodiments, the TGFβ family protein is activin A.

[0069] Activin A is a member of the TGFβ family of proteins produced by many cell types throughout development. It is a disulfide-bridged homodimer (two β-A chains) that binds to a heterodimeric complex of serine-threonine kinase receptors of type I (Act RI-A and Act RI-B) and type II (Act RII-A and Act RII-B). Activin signals mainly through the SMAD2 / 3 proteins to regulate various functions including cell proliferation, differentiation, wound healing, apoptosis, and metabolism. Activin A maintains the undifferentiated state of human embryonic stem cells and also promotes the differentiation of human embryonic stem cells into definitive endoderm. Activin A is added to PSC cultures at a concentration in the range of about 0.1 ng / ml to about 100 ng / ml. For example, PSCs are grown in culture media containing Activin A at about 0.1 ng / ml, about 0.2 ng / ml, about 0.3 ng / ml, about 0.4 ng / ml, about 0.5 ng / ml, about 0.75 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml or more. In one aspect, the mixture contains at least about 5 ng / ml of Activin A. In some aspects, the mixture contains about 25 ng / ml of Activin A.

[0070] In other aspects, the member of the TGFβ protein family is TGFβ1 or TGFβ3. TGFβ1 or TGFβ3 is added to the PSC culture at a concentration in the range of about 0.1 ng / ml to about 100 ng / ml. For example, the PSC is grown in a culture medium containing TGFβ1 or TGFβ3 at about 0.1 ng / ml, about 0.2 ng / ml, about 0.3 ng / ml, about 0.4 ng / ml, about 0.5 ng / ml, about 0.75 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml or more.

[0071] The methods described herein are further defined by the exclusion of chemical conditions for the culture of PSCs. For example, the method for generating RPE cells involves culturing PSCs in the absence of a hypoxia-inducible factor (HIF) pathway modulator and in the absence of nicotinamide.

[0072] Hypoxia-inducible factor (HIF) is a transcription factor that is activated in response to a decrease in oxygen availability in the cellular environment. Those hypoxia-inducible factors (HIF) affect cell metabolism, cell survival, and angiogenesis to maintain biological homeostasis. As used herein, "HIF pathway modulator" includes any agent that inhibits HIF. Non-limiting examples of HIF inhibitors include adaptaquin, TAT-cyclo-CLLFVY, DMOG, echinomycin, FM19G11, GN44028, IOX2, KC7F2, LW6, PX12, TC-S7009, and VH298.

[0073] In one aspect, the mixture comprises a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor at about 1 μM to about 10 μM, a BMP / SMAD1 / SMAD5 / SMAD8 at about 0.1 μM to about 1 μM, and / or an FGF / ERK pathway signaling inhibitor at about 0.5 μM to about 5 μM; and at least about 5 ng / ml of a TGFβ family protein.

[0074] In some aspects, the mixture comprises about 5 μM of SB431542, about 0.25 μM of dorsomorphin, and / or about 0.5 μM to about 1.5 μM of PD0325901, and about 25 ng / ml of activin A. In one aspect, the mixture comprises about 5 μM of SB431542, about 0.25 μM of dorsomorphin, and about 25 ng / ml of activin A. In another aspect, the mixture comprises about 0.25 μM of dorsomorphin, about 0.5 μM to about 1.5 μM of PD0325901, and about 25 ng / ml of activin A. In yet another aspect, the mixture comprises about 5 μM of SB431542, about 0.25 μM of dorsomorphin, about 0.5 μM to about 1.5 μM of PD0325901, and about 25 ng / ml of activin A.

[0075] In another aspect, contacting the PSC with the TGFβ family protein comprises contacting the PSC with at least about 5 ng / ml of a TGFβ family protein.

[0076] The methods described herein include exposing PSCs to physical and chemical culture conditions, and sequentially exposing such PSCs to such physical and chemical conditions for a defined length of time. In various embodiments, the methods described herein include contacting the PSCs with a mixture of agents for a predetermined length of time and then contacting the PSCs with a TGFβ family protein (e.g., activin A). For example, the contact between the PSCs and the above mixture is for a period ranging from about 2 days to about 7 days. The contact between the PSCs and the above mixture includes contacting the PSCs and the above mixture for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or more days. In one embodiment, the contact between the PSCs and the above mixture is from about 2 days to about 4 days. In various embodiments, the contact between the PSCs and the above mixture is about 2 days.

[0077] The contact between the PSCs and the TGFβ family protein (e.g., activin A) is for a period ranging from about 2 weeks to about 6 weeks. For example, the contact between the PSCs and the TGFβ family protein includes contacting the PSCs and the TGFβ family protein for about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or more weeks. In one embodiment, the contact between the PSCs and the TGFβ family protein is about 4 weeks.

[0078] 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 the PSCs in a two-dimensional culture system under conditions that allow growth as an adherent monolayer; (b) contacting the PSCs with 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, for about 2 days to about 7 days; and (c) then culturing the cells of (b) with the TGFβ family protein for an additional about 4 weeks.

[0079] Cell differentiation is the process by which undifferentiated cells (e.g., stem cells) change from an undifferentiated state to a differentiated state. Usually, the cells change into a more specialized type. Differentiation occurs multiple times during the development of multicellular organisms as they change from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide during tissue repair and normal cell turnover to produce fully differentiated daughter cells. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly regulated modifications in gene expression. A specialized type of differentiation (known as terminal differentiation) is important in some tissues (e.g., the vertebrate nervous system, skeletal muscle, epidermis, and intestine). During terminal differentiation, progenitor cells that were previously capable of cell division permanently exit the cell cycle, disassemble the cell cycle machinery, and often express a defined set of genes (e.g., myosin and actin for muscle cells) that are specific to the cell's final function. Differentiation can continue after terminal differentiation if the cell's capabilities and functions experience further changes. The methods described herein enable the induction of differentiation of PSCs into terminally differentiated RPE cells.

[0080] In one aspect, the method includes (a) culturing PSCs in a two-dimensional culture system (i.e., not in a three-dimensional culture system) under conditions that permit growth as an adherent monolayer; (b) contacting the PSCs with a mixture of agents that includes 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 days to about 7 days; and (c) then culturing the cells from (b) with the TGFβ family protein for an additional about 4 weeks in the absence of an HIF pathway modulator, in the absence of nicotinamide, and under conditions that are not hypoxic conditions.

[0081] In various aspects, the method comprises: (a) culturing PSCs in a two-dimensional culture system (i.e., not in a three-dimensional culture system) under conditions that permit growth as an adherent monolayer; (b) contacting the PSCs with a mixture of agents comprising an inhibitor of TGFβ / SMAD2 / SMAD3 pathway signaling at about 5 μM, an inhibitor of BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling at about 0.25 μM, an inhibitor of FGF / ERK pathway signaling at about 0.5 μM to about 1.5 μM, and activin A at about 25 ng / ml for about 2 days to about 7 days; and (c) thereafter culturing the cells of (b) with activin A at about 25 ng / ml in the absence of an HIF pathway modulator, in the absence of nicotinamide, and under conditions that are not hypoxic conditions for an additional about 4 weeks.

[0082] In one aspect, RPE cell differentiation is direct RPE cell differentiation.

[0083] By "direct" differentiation, it is meant that the methods described herein enable the obtaining of RPE cells from PSCs without the accompaniment of moderately differentiated cells and without the need for intermediate manipulation of those cells. That is, in contrast to indirect methods in which partially differentiated cells (e.g., multipotent cells, or non-terminally differentiated cells) are obtained in an initial process and are then terminally differentiated into RPE, the methods described herein produce RPE cells in a single process.

[0084] Differentiated cells can be characterized based on differences observed compared to the undifferentiated cells from which they were obtained (including changes in cell size, shape, membrane potential, metabolic activity, responsiveness to signals, changes in gene expression, etc.). In one aspect, the characterization of RPE includes analysis of changes in the expression of genes that form a molecular signature specific to RPE cells. Such molecular signatures include, for example, the expression of genes selected from PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1, CLDN19, ATP1B1, NC1, ZO1, and / or TYR.

[0085] PMEL, a melanocyte protein (also called premelanosome protein), is a protein encoded by the PMEL gene or PMEL17 in humans. PMEL is a 100 kDa type I transmembrane glycoprotein that is mainly expressed in pigment cells of the skin and eye. The transmembrane form of PMEL is modified in its secretory pathway by the production of N-linked oligosaccharides and the addition and modification of O-linked sugar chains. It then targets the precursors of melanosomes, the pigment organelles, where it is proteolyzed into several small fragments. Some of these fragments form non-pathological amyloid, and these amyloids aggregate into sheets to form the striated pattern that underlies the melanosome microstructure. The expression of the PMEL gene is regulated by the microphthalmia-associated transcription factor (MITF).

[0086] 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 type transcription factor that is involved in the regulation of lineage-specific pathways in many types of cells, including melanocytes, osteoclasts, and mast cells. The term "lineage-specific" means a gene or trait that is found only in a particular cell type because it is associated with MITF. Thus, MITF can be involved in the rearrangement of signaling cascades, which are particularly required for the survival and physiological functions of their normal cell precursors.

[0087] Homeobox protein OTX2 is a protein encoded by the OTX2 gene in humans. OTX2 is expressed in the brain, ear, nose, and eye, and in the case of mutations, it can lead to significant developmental abnormalities and disorders. Mutations in OTX2 can cause eye disorders, including anophthalmia and microphthalmia. Apart from anophthalmia and microphthalmia, other abnormalities, such as optic nerve hypoplasia, hypoplasia of the optic chiasm, and dysplastic eyes, have also been observed. Other deficits resulting from mutations in the OTX2 gene include pituitary abnormalities and mental retardation. Homeoprotein Otx2 has been identified as a molecular "messenger" that may be required for experience-driven visual plasticity during the critical period. Initially involved in embryonic head formation, Otx2 is re-expressed during the critical period (>P23) in rats and regulates the maturation of parvalbumin-expressing GABAergic interneurons (PV cells), which control the onset of critical period plasticity.

[0088] Bestrophin 1 (Best1) is a protein encoded by the BEST1 gene in humans. The bestrophin protein family includes four evolutionarily related genes (BEST1, BEST2, BEST3, and BEST4) that encode integral membrane proteins. This family was first identified in humans by associating BEST1 mutations with Best vitelliform macular dystrophy (BVMD). Mutations in the BEST1 gene have been identified as the major cause of at least five different degenerative retinal diseases. Bestrophins are an ancient family of structurally conserved proteins that have been identified in almost every organism studied from bacteria to humans. In humans, they function as calcium-activated anion channels, and each of them has a unique tissue distribution throughout the body. Specifically, the BEST1 gene on chromosome 11q13 encodes the bestrophin 1 protein, which has the highest expression in the retina in humans.

[0089] The retinal pigment epithelium-specific 65 kDa protein (also known as retinoid isomerohydrolase) is an enzyme of the vertebrate visual cycle encoded by the RPE65 gene in humans. RPE65 is expressed in the retinal pigment epithelium (RPE), a layer of epithelial cells that nourishes the photoreceptors, and is responsible for the conversion of all-trans-retinyl ester to 11-cis-retinol during phototransduction. Subsequently, 11-cis-retinol is used in the regeneration of visual pigments in the photoreceptors. RPE65 belongs to the carotenoid oxygenase enzyme family. RPE65 is an important enzyme in the vertebrate visual cycle found in the retinal pigment epithelium. It is also found 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 and thus must be reconverted to 11-cis-retinal before it can combine with opsin to form an active visual pigment. RPE65 reverses this photoisomerization by converting all-trans-retinyl ester to 11-cis-retinol. Most commonly, its ester substrate is retinyl palmitate. The other enzymes of the visual cycle complete the reactions necessary to oxidize and esterify all-trans-retinol to retinyl ester (the substrate of RPE65) and to oxidize 11-cis-retinol to 11-cis-retinal (the required photoactive visual pigment component).

[0090] Retinaldehyde-binding protein 1 (RLBP1), also known as cellular retinaldehyde-binding protein (CRALBP), is a 36 kD water-soluble protein encoded by the RLBP1 gene in humans. This cellular retinaldehyde-binding protein transports 11-cis-retinal (also known as 11-cis-retinaldehyde) as its physiological ligand. It plays an important role as an 11-cis-retinal receptor that promotes the enzymatic isomerization of all-trans-retinal to 11-cis-retinal in the isomerization of rods and cones in the visual cycle. Mutations in RLBP1 are involved in several vision-related diseases. All of these are autosomal recessive (including Bosnian dystrophy, flecked retinopathy, retinitis pigmentosa, Newfoundland rod-cone dystrophy, and flecked fundus). The characteristics of their associated diseases vary with age, severity, and rate of progression. All of these have similar properties such as photoreceptor deterioration and slower dark adaptation, ultimately leading to visual impairment and often complete blindness.

[0091] Claudin 19 is a protein encoded by the CLDN19 gene in humans. It belongs to the group of claudins. Claudin 19 has been associated with magnesium transport. Claudins (e.g., CLDN19) are transmembrane proteins found at tight junctions. Tight junctions form gates that control the passage of ions and molecules across epithelial sheets and the movement of proteins and lipids between the apical and basolateral domains of epithelial cells.

[0092] Sodium / potassium-transporting ATPase subunit beta-1 is an enzyme encoded by the ATP1B1 gene in humans. The protein encoded by this gene belongs to the family of Na+ / K+ ATPase beta-chain proteins and H+ / K+ ATPase beta-chain proteins and belongs to the subfamily of Na+ / K+-ATPase. Na+ / K+-ATPase is an integral membrane protein responsible for establishing and maintaining the electrochemical gradients of Na and K ions across the plasma membrane. These gradients are essential for osmotic control, for the sodium-coupled transport of various organic and inorganic molecules, and for the electrical excitability of nerves and muscles.

[0093] NC1 or non-collagenous 1 (NC1) is a protein domain derived from COL4A1. COL4A1 belongs to the type IV collagen family and contains the following three domains: a short N-terminal domain, a long triple helix 7S domain in the center, and a non-collagenous 1 (NC1) domain at its C-terminus. Its triple helix domain contains interrupted G-X-Y repeats, which are thought to allow flexibility of the domain. Its NC1 domain is composed of two trimer caps (each containing two α1 fragments and one α2 fragment) that form a propeller with six components arranged around an axial tunnel. The interaction between these two caps occurs along a large planar interface and is stabilized by covalent cross-links between its α1 and α2 chains spanning these two caps.

[0094] The tight junction protein 1 (ZO-1), also known as zona occludens-1, is a 220 kD surface membrane protein encoded by the TJP1 gene in humans. It belongs to the family of tight junction proteins (ZO-1, ZO-2, and ZO-3), which are tight junction-associated proteins, among which ZO-1 was first cloned. It serves as a scaffold protein that cross-links tight junction (TJ) strand proteins, which are fibril-like structures within the lipid bilayer, and anchors them to the actin cytoskeleton.

[0095] Tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin. This enzyme is mainly involved in two separate reactions of melanin synthesis, also known as the Raper Mason pathway. First, the hydroxylation of monophenols, and second, the conversion of o-diphenols to the corresponding o-quinones. o-Quinones undergo several reactions to finally 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 found inside the melanosomes synthesized in skin melanocytes. In humans, its tyrosinase enzyme is encoded by the TYR gene. Tyrosinase gene mutations that result in tyrosinase deficiency lead to oculocutaneous albinism type I, a genetic disorder that affects 1 in 20,000 people. When not regulated during melanin synthesis, Tyr activity leads to an increase in melanin synthesis. Reducing tyrosinase activity has been targeted for the improvement or prevention of conditions associated with hyperpigmentation of the skin, such as melasma and senile lentigines.

[0096] In various aspects, differentiated RPE cells have increased expression of PMEL17, MITF, OTX2, BEST1, RPE65, RLBP1, and / or TYR compared to hPSCs.

[0097] In many embodiments, the differentiated RPE cells do not express any pluripotent stem cell markers (i.e., they are terminally differentiated and not pluripotent after the differentiation process). Stem cell markers include genes expressed by stem cells, and examples of stem cell markers include, but are not limited to, ECAT11, OCT4, NANOG, SOX2, mir302HT, and LIN28.

[0098] In one embodiment, the differentiated RPE cells do not express ECAT11, OCT4, NANOG, SOX2, mir302HT, or LIN28.

[0099] In a further embodiment, the present invention provides a method of treating macular degeneration in a subject, the method comprising administering differentiated retinal pigment epithelium (RPE) cells to the subject, the differentiated RPE cells being obtained by one of the methods described herein.

[0100] RPE dysfunction is associated with various retinal degenerative diseases. As used herein, the term "retinal degenerative disease" or "degenerative retinopathy" is intended to refer to any retinopathy consisting of the deterioration of the retina caused by the progressive death of retinal cells. There are several reasons for retinal degeneration, including arterial or venous occlusion, diabetic retinopathy, R.L.F. / R.O.P. (retrolental fibroplasia / retinopathy of prematurity), or disease (usually genetic). These can present in many different ways (e.g., visual impairment, night blindness, retinal detachment, photosensitivity, tunnel vision, and peripheral vision loss to complete loss of vision). Of these retinal degenerative diseases, retinitis pigmentosa (RP) is a very important example. Non-limiting examples of degenerative retinopathies associated with RPE deterioration include macular degeneration (e.g., 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).

[0101] The methods described herein relate in particular to the treatment of age-related macular degeneration (AMD) in a subject, the treatment comprising administering to the subject RPE cells obtained by the methods described herein.

[0102] Age-related macular degeneration or AMD is a disease that affects a person's central vision. It can lead to severe loss of central vision, but people rarely go blind from it. AMD is the most common cause of severe vision loss in people over 50 years old. Only the center of the visual field is affected by this disease. AMD affects central vision and thereby the ability to see details. In AMD, a part of the retina called the macula is damaged. In the advanced stage, people lose the ability to drive, recognize faces, and read small print. In its early stages, AMD may have no signs or symptoms, so people may not suspect that they have AMD. The two main types of age-related macular degeneration have separate causes. The most common type, atrophic AMD (about 80% of people with AMD have this atrophic type), has an unknown exact cause, but both genetic and environmental factors are thought to play a role. This occurs when the light-sensitive cells in the macula are slowly destroyed, usually one eye at a time. Vision loss in this condition is usually slow and progressive. Age-related damage to an important support membrane under the retina is thought to contribute to atrophic age-related macular degeneration. Exudative AMD is the less common type and usually causes more severe vision loss in patients than atrophic AMD. It is the most common cause of severe vision loss. Exudative AMD occurs when abnormal blood vessels begin to grow under the retina. They leak fluid and blood (hence the name exudative AMD) and can create a large blind spot in the center of the visual field. The methods described herein are useful for the treatment of both exudative and atrophic AMD in a subject.

[0103] As used herein, the term "subject" refers to any individual or patient on whom the method is performed. Generally, the subject is human, but as will be understood by those skilled in the art, the subject can be an animal. Thus, other animals (vertebrates such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, chickens, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas)) are included within the definition of subject.

[0104] The term "treatment" is used interchangeably herein with the term "treatment method", and refers to both (1) therapeutic treatments or means that cure, slow, reduce the symptoms of, and / or stop the progression of diagnosed pathological conditions or disorders, and (2) prophylactic / preventive means. Those in need of treatment can include individuals already having a particular medical disorder and those who may ultimately acquire the disorder (i.e., those in need of preventive treatment). The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", "effective amount", etc. refer to the amount of RPE cells that elicits a biological or medical response in a tissue, system, animal or human being sought by a researcher, veterinarian, physician or other clinician. Generally, the response is either a reduction in symptoms in a patient or a desirable biological outcome (e.g., treatment of AMD). Such amount should be sufficient to treat AMD in the subject. The effective amount can be determined as described herein.

[0105] The terms "administration" and / or "administering" should be understood to mean providing a pharmaceutical composition to a subject in need of treatment in a therapeutically effective amount. The route of administration can be enteral, topical or parenteral. Thus, routes of administration include, but are not limited to, intravitreal, subretinal, and any other intraocular administration, as well as injection.

[0106] In one aspect, administration of differentiated RPE cells includes injecting the RPE cells in situ.

[0107] Currently, there is no treatment for atrophic age-related macular degeneration, but vision rehabilitation programs and low vision devices can enhance visual skills, develop new ways to perform activities of daily living, and be used to adapt to life with age-related macular degeneration. The main treatment for exudative AMD is injection of anti-VEGF agents because high VEGF levels in the eye are associated with the formation of abnormal blood vessels that cause much of the damage in exudative AMD. Anti-VEGF agents are used to combat the disease process and reduce the effect of damaging these abnormal leaky blood vessels. They can also effectively stabilize vision in many patients.

[0108] In some aspects, administration of the RPE cells described herein can be combined with one or more additional therapeutic agents. Phrases such as "combination therapy," "in combination with," etc. refer to the simultaneous use of multiple drug therapies or treatments to increase their response. The RPE cells of the present invention can be used, for example, in combination with other drugs or treatments for treating AMD. Specifically, administration of RPE cells to a subject can be in combination with an anti-VEGF agent. Such therapy can be administered before, simultaneously with, or after administration of the RPE cells of the present invention.

[0109] In one aspect, administration of differentiated RPE cells increases photoreceptor function and / or photoreceptor survival.

[0110] As used herein, "photoreceptor" or "photoreceptor cell" refers to a specialized type of neuroepithelial cell found in the retina that is capable of visual light transmission. The critical biological importance of photoreceptors is that they convert light (visible electromagnetic radiation) into signals that can stimulate biological processes. Specifically, photoreceptor proteins in the cell absorb photons, inducing a change in the cell's membrane potential. In the mammalian eye, there are three known types of photoreceptors: rods, cones, and intrinsically photosensitive retinal ganglion cells. The two classical photoreceptors are rods and cones, each of which contributes to the information used by the visual system to form a representation of the visual world (visual field). Rods contribute mainly to night vision (dark adaptation conditions), while cones contribute mainly to daytime vision (light adaptation conditions), but the chemical processes in each that support light transmission are similar. Intrinsically photosensitive retinal ganglion cells are thought not to contribute directly to the visual field but have roles in the entrainment of the circadian rhythm and pupil reflex.

[0111] There are significant functional differences between rods and cones. Rods are extremely sensitive and can be induced 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 greater number of photons) to generate signals. In humans, there are three separate types of cone cells, distinguished by their response patterns to light of various wavelengths. Those three types of cone cells respond (robustly) to short-wavelength, medium-wavelength, and long-wavelength light and can thus be called S-cones, M-cones, and L-cones, respectively.

[0112] The human retina contains approximately 120 million rod cells and 6 million cone cells. The number and ratio of rods and cones vary between species depending on whether the animal is primarily diurnal or nocturnal. In the human visual system, in addition to the photosensitive rods and cones, there are approximately 2.4 to 3 million ganglion cells, 1% to 2% of which are photosensitive. The axons of the ganglion cells form the two optic nerves. The photoreceptors are typically arranged in an irregular but approximately hexagonal lattice (known as the retinal mosaic).

[0113] Rod photoreceptors and cone photoreceptors are found in the outermost layer of the retina; both have the same basic structure. The closest to the visual field (farthest from the brain) is the axon terminal, which releases a neurotransmitter called glutamate to bipolar cells. Further away is the cell body, which contains the cell's organelles. Even further away is the inner segment, a specialized part of the cell filled with the cell's mitochondria. The main function of the inner segment is to provide ATP (energy) to the sodium-potassium pump. Finally, the closest to the brain (farthest from the visual field) is the outer segment, the light-absorbing part of the photoreceptor. The outer segment is actually a modified cilium containing discs filled with opsin (a molecule that absorbs photons) and voltage-gated sodium channels.

[0114] Opsin, a membrane photoreceptor protein, contains a pigment molecule called retinal. In rod cells, these together are called rhodopsin. In cone cells, there are various types of opsins that combine with retinal to form a pigment called photopsin. The three different classes of photopsin in cones respond to various ranges of light frequencies, which is the discrimination that enables the visual system to calculate color. The function of the photoreceptor is to convert the light information of photons into information in a form that can be communicated to the nervous system and is suitable for the organism to easily use: this conversion is called signal transduction.

[0115] By "increasing the function and / or survival of photoreceptors", it is meant that administration of RPE cells increases the function and / or survival of any type of photoreceptor, including rods, S-cones, M-cones, L-cones, and intrinsically photosensitive retinal ganglion cells.

[0116] In some embodiments, increasing the function of photoreceptors includes increasing the regeneration of photoreceptor outer segments, increasing phagocytosis involving MERTK, and / or increasing, restoring, and / or generating intercellular tight junctions.

[0117] In various embodiments, restoring and / or generating intercellular tight junctions improves or restores the blood-eye barrier or the brain-eye barrier.

[0118] The "blood-ocular barrier" or "brain-eye barrier" is a barrier formed by the endothelium of the capillaries of the retina and of the iris, ciliary epithelium, and retinal pigment epithelium. It is a physical barrier between the local blood vessels and most parts of the eye itself, preventing many substances, including drugs, from moving across it. Inflammation can disrupt this barrier, allowing drugs and large molecules to enter the eye. When the inflammation subsides, the barrier usually returns to normal. It consists of the blood-aqueous barrier, which includes the ciliary epithelium and the capillaries of the iris, and the blood-retinal barrier, which includes the tight junctions between the non-fenestrated capillaries of the retinal circulation and the retinal epithelial cells. 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. The blood-retinal barrier prevents the passage of large molecules from the choroidal capillaries into the retina and is formed by the endothelium of the retinal blood vessels and the retinal pigment epithelium.

[0119] In another embodiment, the PSC is an hPSC. In some embodiments, the hPSC is an autologous hPSC or an allogeneic hPSC.

[0120] hPSCs can be obtained from a subject in need of treatment (i.e., autologous hPSCs). Autologous hPSCs have the advantage of virtually eliminating the chance of graft-versus-host disease rejection because there is no risk of incompatibility and thus no risk of any problems associated with incompatibility. If the hPSCs cannot be obtained from the subject itself, the hPSCs can be obtained from an allogeneic non-autologous donor that is histocompatible with the subject. Allogeneic hPSCs offer the advantage of being prepared / stored in advance for immediate use if needed. However, the risks associated with the lack of histocompatibility between the donor and the recipient, and the risk of rejection of those cells (e.g., acute or chronic graft-versus-host disease) remain.

[0121] In a further embodiment, the present invention provides a kit comprising: (a) a neuroectoderm induction cocktail comprising a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor, a BMP / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitor, and / or an FGF / ERK pathway signaling inhibitor; (b) a TGFβ family protein; and (c) instructions for inducing differentiation of pluripotent stem cells (PSCs) into retinal pigment epithelial (RPE) cells.

[0122] In one aspect, the kit further comprises a laminin-coated surface.

[0123] Presented below are examples discussing methods involving the differentiation of PSCs into RPE cells contemplated for the applications discussed above. The following examples are provided to further illustrate embodiments of the present invention but are not intended to limit the scope of the present invention. Those examples are typical of what can be used, but other procedures, methodologies, or techniques known to those skilled in the art can alternatively be used.

Examples

[0124] (Example 1) (Design of a retinal pigment epithelial cell differentiation protocol) In a stepwise iterative development process, a novel combination of factors promoting highly efficient and excellent RPE differentiation was identified using a simple and GMP-compliant workflow. The rationale is the combination of a neuroectoderm induction cocktail in a defined temporal order and activin A treatment (see Figure 1 for an exemplary example of the method of the present invention). Briefly, a combination of two or three pathway inhibitors, including an FGF / ERK signaling inhibitor (e.g., PD0325901 ("PD")), a TGFβ / SMAD2 / 3 signaling inhibitor (e.g., SB431542 ("SB")), and a BMP / SMAD1 / 5 / 8 inhibitor (e.g., dorsomorphin ("DM")), was used to stimulate iPSCs towards the neuroectodermal lineage. Inhibiting both SMAD pathways for an extended period with or without additional FGF / ERK signaling inhibition is known to result in a neuronal fate (not RPE), and RPE is not a neuron. Treatment of iPS cells with the above neuroinductive cocktail does not significantly induce RPE beyond background, even over a shorter time. Activin A is known to have a positive signaling effect on RPE differentiation in vivo. However, activin A is an important ligand that promotes the self-renewal of hPSCs rather than differentiation.

[0125] However, it was discovered that their limited use on hPSCs, when combined with activin A, stimulated hPSCs towards differentiation into RPE cells.

[0126] As shown in Figure 1, in an exemplary method, highly efficient RPE induction (i.e., high purity in a shorter period) occurs under adherent culture conditions with a combination of activin A treatment and SMAD / FGF pathway inhibition.

[0127] (Example 2) (Materials and Methods) (Maintenance of hiPSCs)

[0128] Before starting differentiation, hiPSCs were maintained with iMatrix-511 in iPS Brew medium and passaged using EDTA dissociation for conventional culture.

[0129] Thereafter, the cells were grown to 90% - 100% confluence on 6-well plates pre-coated with 4× iMatrix-511, i.e., 12 μl of iMatrix-511 instead of 3 μl per 6-well, before starting. Full confluence at the start point yielded better differentiation results than starting from semiconfluent cultures. However, this experiment can be carried out under semiconfluent starting conditions. The laminin coating for performance improvement in the initial differentiation stage seemed to have a beneficial effect on morphology and pigmentation, but perhaps more importantly, it was after reseeding RPE cells at a later stage. A coating of 12 μl per 6-well seemed to be optimal for RPE maintenance.

[0130] One week before differentiation, the desired number of 6-well plates were coated with 125 μl of iMatrix-511 in 2 ml of PBS per 6-well at 37 °C for 1 hour or at 2 °C - 8 °C overnight. The hiPSCs were dissociated in their new culture wells using approximately a quarter of the EDTA digestion time or TrypLE Select supplemented with 10 μM Rocki to better control cell density and uniformity, aiming for 90% - 100% confluence. During this time, the cells were appropriately nourished (including 6 ml - 8 ml per well medium change).

[0131] (Differentiation of hiPSCs)

[0132] (Day 0): Confirmed that the overall undifferentiated and dense hiPSC morphology was close to 100% confluence. If not, supply another 4 ml of pre-warmed iPS Brew medium to those cells and start differentiation the next day instead. Prepared and / or pre-warmed KSR medium (4 ml per 6 wells was required), PD0325901 (PD) aliquot / SB431542 (SB) aliquot / Dorsomorphin (DM) aliquot, and Activin A. After vigorously stirring the culture plate to completely remove the iPS Brew medium by immersion, all reagents were thawed and thoroughly mixed, and then 18 μM of PD (1:1000 from 1 mM stock), 59 μM of SB10 (1:2000 from 10 mM stock), 0.2511 μM of DM (1:2000 from 0.5 mM stock), and 2512 ng / ml of Activin A13 (1:400 from 10 μg / ml stock) were added to 4 ml of KSR medium per confluent hiPSC culture well.

[0133] (Day 1 and Day 2): Prepared the medium as described above, replaced the old medium on the culture wells with 4 ml of fresh differentiation medium per well, then stirred vigorously and aspirated the medium completely.

[0134] (Day 3): Prepared KSR medium containing only Activin A, replaced the old medium on the culture wells with 4 ml of fresh differentiation medium containing only Activin A (ActA only) per well, then stirred vigorously and aspirated the medium completely.

[0135] (Day 4): Prepared fresh KSR + Activin A (ActA) differentiation medium and supplied 8 ml per well.

[0136] (Second week): Supplied 4 ml per 6 wells to the cells daily and 8 ml during weekends.

[0137] (Weeks 3 and 4): During the 3rd and 4th weeks of differentiation, no further small molecules were applied. Only activin A was added to the basal medium (either KSR medium or B27 medium). Activin A was added daily to the pre-warmed basal medium, replacing the old medium on the cells (thoroughly mixed at 1:100).

[0138] (Day 29 (Week 5)): Newly coated dishes were prepared by coating 12-well (5 μl) format or 6-well format (12 μl iM) with 4×iMatrix-511 in PBS at 37 °C for 1 hour. KSR medium was prepared and pre-warmed for the splitting procedure. The differentiated cells were washed once (4 ml) or twice (2 × 2 ml) with PBS, and then 1 ml of pre-warmed TrypLE Select was added. The cells were dissociated by pipetting up and down several times using a 1 ml pipette, and the result was checked under a microscope. The cell suspension was transferred to a 15 ml tube and centrifuged at 300 g for 2 minutes. A portion of those cells was transferred to a 1.5 ml tube for downstream FACS and / or RT-qPCR analysis. The cells were resuspended in an appropriate amount of KSR medium, and the cell titer was determined using a Neubauer chamber. 400,000 cells per cm 2 were seeded in 6-well format (4 ml of KSR medium containing activin A24 or 4 ml of KSR medium without activin A24) or 12-well format (2 ml of medium).

[0139] (Day 31): The cells were supplied with KSR medium (6-well format: 4 ml, 12-well format: 2 ml).

[0140] (Day 33): The cells were supplied with KSR medium (6-well format: 8 ml, 12-well format: 4 ml).

[0141] (Week 6): The procedure of Week 5 was repeated. Depending on the state of the culture (morphology, results of in-process control), the cells were either passaged again (for further purification or expansion) or left to mature in KSR medium without activin A.

[0142] Since required for a specific characterization assay, RPE cells after Px1 (xn = passage number after the first differentiation) were re-seeded onto transwells. Briefly, 24-well transwells with a surface area of 0.33 cm 2 were coated with 0.5 μl of iMatrix-511 per well. Depending on the RPE maturation stage (Px1 or after Px2, e.g., with a long incubation exceeding several weeks), the cells were re-seeded with 1× TrypLE29 or 10× TrypLE29. After PBS washing, the cells were digested for 10 minutes, the results were checked under a microscope, then gently pipetted with a 1 ml pipette, and then re-checked under a microscope. If the majority of the cells were single cells, digestion was stopped by adding 3 volumes of KSR medium, the cells were transferred to a 15 ml tube, and centrifuged at 300 g for 2 minutes. Otherwise, the incubation time was lengthened and the results were checked as described above. The pelleted cells were resuspended in an appropriate volume of KSR medium, the cell titer was determined using a Neubauer chamber, and 100,000 cells per well (total internal volume: 0.5 ml, total external volume: 1 ml) were seeded. The medium was replaced twice a week until the time of analysis.

[0143] (Analysis by RT-qPCR)

[0144] Samples analyzed by RT-qPCR were cells lysed directly from culture wells using RA1 buffer from an RNA isolation kit. For example, the culture medium after vigorous agitation was removed by immersion, and then 600 μl of buffer RA1 was added to the culture wells, and the samples were lysed and homogenized by pipetting up and down quickly using a 1 ml pipette. 350 μl was transferred to a 1.5 ml tube or directly onto a filtration column, following the kit instructions. Alternatively, a sharp plastic scraper was used to scrape a representative fraction of the cells (e.g., 25% of a 6-well) from the running culture, and then its floating aggregates were pipetted into a 1.5 ml tube. The tube was centrifuged briefly, for example, in a mini centrifuge, and the supernatant was completely removed (ideally, there was no liquid remaining on top of the cell pellet). The cells were then quickly resuspended in 350 μl of buffer RA1. After adding buffer RA1, the sample was homogenized immediately. As a third option, cells were collected for further purposes (e.g., reseeding or parallel FACS analysis). In this case, the cells were collected by generating a cell suspension, and the appropriate fraction was pipetted into a 1.5 ml tube for RNA isolation. These cells were centrifuged in a mini centrifuge or tabletop centrifuge and used as described above. RNA was isolated using an elution volume of 40 μl according to the manufacturer's instructions. RNA concentration was measured using a NanoDrop that monitors the A260 / 280 ratio and A260 / 230 ratio. RNA was always kept cold. The cDNA synthesis reaction was set up in a PCR strip and incubated at 42 °C for 1 hour in a PCR machine: 18.5 μl of diluted RNA, the same amount (e.g., μg or 500 ng) for all samples 5 μl of 5× RT buffer (as part of the master mix) 0.5 μl of dNTP mix (as part of the master mix) 0.5 μl of oligo-dT primer (as part of the master mix) 0.5 μl of M-MLV reverse transcriptase (as part of the master mix).

[0145] The cDNA reactants were diluted by adding 150 μl of water each. qPCR was set up using the following gene-specific primers according to the experimental-specific template: 10 μl of SYBR Green mix 3 μl of gene-specific primer working mix 7 μl of diluted cDNA.

[0146] qPCR was performed according to the corresponding GMP SOP and the data were analyzed based on the experimental-specific template.

[0147] (Analysis by FACS)

[0148] An aliquot of FACS buffer was prepared or thawed to set up the primary antibody solution and the secondary antibody solution (200 μl per one sample and antibody): MITF1 antibody 1:150 > A11001 secondary antibody 1:750 - 1:1000 BEST1 antibody 1:150 > A11001 secondary antibody 1:750 - 1:1000 PMEL17 antibody 1:50 > A21206 secondary antibody 1:750 - 1:1000 ZO1 antibody 1:250 > A21206 secondary antibody 1:750 - 1:1000.

[0149] Samples of only the secondary antibody served as negative controls. Cells were collected for RPE dissociation to yield a single cell suspension. After cell counting, a defined number of cells were transferred to the required number of 1.5 ml tubes (in the range of 0.5 - 1 Mio cells per staining). The cells were centrifuged at 400 g for 1 minute, the supernatant was discarded, the pellet was washed with 180 μl - 200 μl of PBS, a pellet was generated again, and the supernatant was discarded. The pellet was resuspended in 400 μl each of 2% formaldehyde / PBS, pipetted up and down several times quickly, and incubated at room temperature (RT) for about 10 minutes. The tubes were centrifuged at 400 g for 1 minute, the supernatant was completely removed by dipping, the cells were resuspended in 180 μl - 200 μl of FACS buffer, the tubes were left standing for 1 - 2 minutes for blocking, centrifuged again, and the supernatant was discarded. Thereafter, the cells were resuspended in the primary antibody solution and incubated at room temperature (RT) for 15 minutes, the tubes were gently flicked at least once during incubation, centrifuged as described above, and the supernatant was removed by dipping. The cells were washed as described above using 180 μl - 200 μl of FACS buffer. The cells were resuspended in the appropriate secondary antibody solution and incubated at room temperature (RT) for 15 minutes, the tubes were gently flicked at least once during incubation, centrifuged as described above, and the supernatant was removed by dipping. The cells were washed and centrifuged with 180 μl - 200 μl of FACS buffer as described above, each cell pellet was resuspended in 300 μl of PBS, and analyzed by flow cytometry using an appropriate template. The percentage of positive cells was based on hierarchical gating with respect to (i) cells, (ii) single cells, and (iii) an encircled positive cloud in the fluorescence intensity against the forward scatter plot.

[0150] (Example 3) (Characterization of Retinal Pigment Epithelial Cells Differentiated from Human Pluripotent Stem Cells) Human induced pluripotent stem cells (hiPSCs) were differentiated into RPE according to the protocol described in the exemplary method shown in Example 2 and FIG. 1.

[0151] The protocol using the PD0325901 (PD, or P) aliquot / SB431542 (SB, or S) aliquot / dorsomorphin (DM, or D) aliquot, as well as activin A (A) described in this specification is referred to as the PSD+A protocol. This PSD+A protocol was first compared with the procedure described by Maruotti et al. (2015). As shown in FIGS. 2A-2C, this PSD+A protocol resulted in RPE cells expressing high expression levels of BEST1, RPE65, RLBP1, TYR, PMEL17, MITF, and OTX2 at the 4-week time point when measured by RTqPCR, compared to REF cells obtained using the reference protocol (see FIG. 2A). As shown in FIG. 2B, this corresponded to RPE cells having a pigmentation phenotype that was significantly different from the cells obtained by the reference protocol. FIG. 2C further shows that treatment with A alone was not sufficient to induce this RPE phenotype and that PSD+A was the optimal protocol, indicating the importance of treatment with these small molecules.

[0152] As shown in FIG. 3, RPE gene expression was quantified by RTqPCR at week 4 after initial induction with PSD, SD (without PD), PD (without SB), or PS (without DM). Removal of DM showed the most drastic decrease in RPE-specific marker expression compared to the standard PSD protocol. Thus, this removal of DM may be considered essential, while removal of PD and SB may be tolerated over the long term.

[0153] As shown in FIGS. 4A-4B, the long-term equivalence of PSD+A induction and PD+A induction after reseeding of cells treated for 4 weeks was evaluated. iPS cells were induced with a two-factor small molecule cocktail (PD) or a three-factor cocktail (PSD), and RPE-specific marker expression was evaluated after the first 4 weeks of differentiation. FIG. 4A shows the levels of protein expression measured by flow cytometry, and FIG. 4B shows the levels of gene expression measured by RTqPCR. The results showed that induction with those three pathway inhibitors was preferred, but that RPE cells induced with two factors showed equivalent RPE-specific marker expression, and thus could be equivalent.

[0154] The timing of inhibition of those three pathways was evaluated to assess how long those cells needed to be contacted with those three small molecules to induce RPE cell differentiation. As shown in FIG. 5, the RTqPCR analysis showed that 1-day treatment or complete removal (activin A alone) was insufficient to induce RPE cell fate, but that more than 2 days (up to at least 7 days) was optimal and that little difference was observed among the various exposure times.

[0155] Therefore, it was evaluated when treatment with activin A should be started and what concentration should be used for optimal RPE induction. As shown in FIG. 6, changes from the standard protocol, which is the form of starting activin A treatment after signal transduction inhibition, resulted in decreased RPE gene expression, suggesting the importance of treating those cells simultaneously with the above small molecule inhibitors and activin A to obtain optimal RPE cell induction. As shown in FIG. 7, titration of activin A revealed that concentrations of activin A lower than 20 ng / ml or complete removal of activin A resulted in inferior RPE induction, but that there was no complete absence of RPE induction.

[0156] The time course of RPE markers was assayed, and the expression levels of early markers, mid / late markers, late markers, and mature markers were evaluated up to 10 weeks after RPE induction. As shown in FIGS. 8A-8C, PSD+A induced early RPE markers (FIG. 8A) / mid / late RPE markers (FIG. 8B), late / mature (FIG. 8C) RPE markers at various differentiation stages after 7 days of induction.

[0157] RPE cells were also characterized by immunofluorescence and using light microscopy. As shown in FIG. 9A, RPE cells showed a typical pigmented cobblestone morphology in phase contrast light, and it was found that RPE cells expressed BEST1 (FIG. 9B), ZO1 (FIGS. 9B and 9D), MITF1 (FIG. 9C), and CRALBP (FIGS. 9C and 9D), which are RPE markers.

[0158] RPE cells were further analyzed using electron microscopy as shown in FIGS. 10A-10B. Transmission electron microscopy analysis (FIG. 10A) and scanning electron microscopy analysis (FIG. 10B) of RPE cells generated using the above PSD+A protocol showed typical RPE characteristics (e.g., epithelial polarity with the nucleus at the basal side, melanosomes at the apical side, and microvilli facing the apical cell surface).

[0159] Finally, the terminal differentiation of those cells was assayed by evaluating whether the cells had any residual expression of any stem cell markers. As shown in FIG. 11, RPE cells obtained by the above PSD+A protocol were assayed for the expression of ECAT11, OCT4, NANOG, SOX2, mir-302 HT, and LIN28. The expression levels were compared with the expression levels in iPSCs. As shown in FIG. 11, RPE cells showed no expression of any stem cell markers.

[0160] (References) Almedawar, S., Vafia, K., Schreiter, S., Neumann, K., Khattak, S., Kurth, T., Ader, M., Karl, M.O., Tsang, S.H., and Tanaka, E.M. (2020). MERTK-Dependent Ensheathment of Photoreceptor Outer Segments by Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium. Stem Cell Reports 14, 374-389. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280. da Cruz, L., Fynes, K., Georgiadis, O., Kerby, J., Luo, Y.H., Ahmado, A., Vernon, A., Daniels, J.T., Nommiste, B., Hasan, S.M., et al. (2018). Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. Nat Biotechnol 36, 328-337. Fuhrmann, S., Levine, E.M., and Reh, T.A. (2000). Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127, 4599-4609. Greber, B., Coulon, P., Zhang, M., Moritz, S., Frank, S., Muller-Molina, A.J., Arauzo-Bravo, M.J., Han, D.W., Pape, H.C., and Scholer, H.R. (2011). FGF signalling inhibits neural induction in human embryonic stem cells. EMBO J 30, 4874-4884. Greber, B., Lehrach, H., and Adjaye, J. (2008). Control of early fate decisions in human ES cells by distinct states of TGFbeta pathway activity. Stem Cells Dev 17, 1065-1077. Greber, B., Wu, G., Bernemann, C., Joo, J.Y., Han, D.W., Ko, K., Tapia, N., Sabour, D., Sterneckert, J., Tesar, P., et al. (2010). Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6, 215-226. Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5, 396-408. Mandai, M., Kurimoto, Y., and Takahashi, M. (2017). Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration. N Engl J Med 377, 792-793. Maruotti, J., Sripathi, S.R., Bharti, K., Fuller, J., Wahlin, K.J., Ranganathan, V., Sluch, V.M., Berlinicke, C.A., Davis, J., Kim, C., et al. (2015). Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells. Proc Natl Acad Sci U S A 112, 10950-10955. 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 from human pluripotent stem cells by small-molecule induction. J Cell Sci 122, 3169-3179. Plaza Reyes, A., Petrus-Reurer, S., Antonsson, L., Stenfelt, S., Bartuma, H., Panula, S., Mader, T., Douagi, I., Andre, H., Hovatta, O., et al. (2016). Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model. Stem Cell Reports 6, 9-17. Rao, J., and Greber, B. (2017). Concise Review: Signaling Control of Early Fate Decisions Around the Human Pluripotent Stem Cell State. Stem Cells 35, 277-283. Sharma, R., Khristov, V., Rising, A., Jha, B.S., Dejene, R., Hotaling, N., Li, Y., Stoddard, J., Stankewicz, C., Wan, Q., et al. (2019). Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sci Transl Med 11. Xu, R.H., Sampsell-Barron, T.L., Gu, F., Root, S., Peck, R.M., Pan, G., Yu, J., Antosiewicz-Bourget, J., Tian, S., Stewart, R., et al. (2008). NANOG is a direct target of TGFbeta / activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3, 196-206. Zahabi, A., Shahbazi, E., Ahmadieh, H., Hassani, S.N., Totonchi, M., Taei, A., Masoudi, N., Ebrahimi, M., Aghdami, N., Seifinejad, A., et al. (2012). A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev 21, 2262-2272. Zhu, Y., Carido, M., Meinhardt, A., Kurth, T., Karl, M.O., Ader, M., and Tanaka, E.M. (2013). Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium. PLoS One 8, e54552.

[0161] Although the present invention has been described with reference to the above embodiments, it is understood that modifications and variations are included 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, (a) Cultures of pluripotent stem cells (PSCs) (i) one or more of the following: transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitors, bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors, or fibroblast growth factor (FGF) / ERK pathway signaling inhibitors; (ii) TGFβ family proteins as needed A step of contacting a mixture of drugs containing; and (b) The step of subsequently culturing the cells of (a) with the TGFβ family protein in the absence of the drug mixture of (a)(i) to generate RPE cells. Methods that include...

2. The method according to claim 1, wherein the culture of the PSC is a cell-adherent monolayer.

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

4. The method according to claim 1, wherein the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is selected from the group consisting of SB431542, LY3200882, TP0427736 HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, garnicertive, bactocertive, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854.

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

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

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

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

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

11. The method according to claim 1, wherein the contact between the PSC and the TGFβ family protein in (b) comprises contacting the PSC with about 25 ng / ml of activin A.

12. The method according to claim 1, wherein the contact between the PSC and the mixture of (a) is for about 2 to 7 days.

13. The method according to claim 1, wherein the contact between the PSC and the TGFβ family protein in (b) is approximately 4 weeks.

14. The method according to claim 3, wherein the monolayer is cultured on a surface including a laminin coating.

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

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

17. The method according to claim 1, wherein the contact between the PSC and the mixture occurs in the absence of a hypoxia-inducible factor (HIF) pathway modulator.

18. The method according to claim 1, wherein the contact between the PSC and the mixture occurs in the absence of nicotinamide.

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

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

21. A method for inducing differentiation of retinal pigment epithelial (RPE) cells from pluripotent stem cells (PSCs), (a) A step of culturing PSCs in a two-dimensional culture system under conditions that enable growth as an adhesive monolayer; (b) A step of contacting the PSC with a mixture of drugs comprising one or more of the transforming growth factor β (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitors, bone morphogenetic protein (BMP) / SMAD1 / SMAD5 / SMAD8 pathway signaling inhibitors, or fibroblast growth factor (FGF) / ERK pathway signaling inhibitors, and optionally TGFβ family proteins, for about 2 to about 7 days; and (c) Subsequently, the cells from (b) are cultured with the TGFβ family protein for approximately four weeks to induce differentiation of RPE cells from PSCs. Methods that include...

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

23. The method according to claim 21, wherein 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 according to claim 21, wherein differentiated RPE cells do not express ECAT11, OCT4, NANOG, SOX2, mir302HT, or LIN28.

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

26. A composition for use in a method for treating macular degeneration in a subject, comprising differentiated retinal pigment epithelial (RPE) cells, the method comprising the step of administering the composition to the subject, A composition characterized in that differentiated RPE cells are obtained by the method described in claim 1 or 21.

27. The composition according to claim 26, wherein the step of administering the composition increases the function and / or survival of the photoreceptor.

28. The composition according to claim 27, wherein the increased function of the photoreceptor includes increasing the regeneration of the photoreceptor outer segment, increasing MERTK-mediated phagocytosis, and / or increasing, restoring, and / or generating intercellular tight junctions.

29. The composition according to claim 28, wherein the restoration and / or formation of intercellular tight junctions improves or restores the brain-ophthalmological barrier.

30. The composition according to claim 26, wherein the step of administering the composition includes injecting RPE cells in situ.

31. The composition according to claim 26, wherein the PSC is human PSC (hPSC).

32. The composition according to claim 31, wherein the hPSC is self-hPSC or homogeneous hPSC.

33. (a) a neuroectoderm induction cocktail 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; (c) Instructions for inducing pluripotent stem cell (PSC) differentiation into retinal pigment epithelial (RPE) cells and A kit that includes this.

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

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