Method for differentiating stromal cells or pericytes

Abstract

The present disclosure relates to a method for differentiating pluripotent stem cells into stromal cells or pericyte cells. The present disclosure also relates to stromal cells or pericyte cells produced by such a method, organoids containing such stromal cells or pericyte cells, and methods of using the same. The present disclosure also relates to a differentiation medium for use therewith.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,458, filed on July 19, 2022, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to methods for differentiating pluripotent stem cells into stromal cells or pericyte cells. The present disclosure also relates to stromal cells or pericyte cells produced by such methods, organoids containing such stromal cells or pericyte cells, and methods of using the same. The present disclosure also relates to differentiation media for such use.

Background Art

[0003] Pluripotent stem cells (PSCs) are undifferentiated or partially differentiated cells that can differentiate into various other cell types. Induced pluripotent stem cells (iPSCs) are a type of PSC derived from adult somatic cells that have been genetically reprogrammed into an ESC-like state by the expression of genes and factors important for maintaining the properties that define embryonic stem cells (ESCs). iPSCs have gained interest in the medical community in recent years because they address many of the obstacles associated with the use of embryonic stem cells, allowing for the generation of patient-specific PSCs that can be genetically modified, differentiated into somatic lineages, and returned to the same patient as autologous transplants. Yamanaka et al., Cell Stem Cell. 1(1):39-49 (2007); Nishikawa et al., Nat. Rev. Mol. Cell Biol. 9:725 (2008). In addition to genetic disorders, iPSCs can be used for tissue regeneration and disease modeling. Kogut et al., Methods Mol. Biol. 1195:1-12 (2014). PSCs and iPSCs can differentiate into many different cell types, including endothelial cells (ECs). Jang et al., Am. J. Pathol. 189(3):502-512 (2019); Gu et al., Curr. Protoc. Hum. Genet. published online 2018 Jul. 6. doi: 10.1002 / cphg.64.

Summary of the Invention

[0004] The present disclosure provides methods for differentiating pluripotent stem cells (PSCs) into mesenchymal cells or pericyte cells.

[0005] In some embodiments, the method comprises: (i) culturing pluripotent stem cells (PSCs) (e.g., iPSCs) in a basal culture medium comprising bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), glycogen synthase kinase 3 (GSK3) inhibitor, activin A, and Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) culturing the cells of (i) in a basal culture medium comprising VEGF and a transforming growth factor β (TGFβ) inhibitor; and (iii) culturing the cells of (ii) in a basal culture medium comprising TGFβ3 and platelet-derived growth factor (PDGF) to form mesenchymal cells or pericytes.

[0006] In some embodiments, the method further comprises, prior to (iii), culturing the cells of (ii) in a basal culture medium.

[0007] In some embodiments, the method further comprises (iv) culturing the cells of (iii) in a basal culture medium.

[0008] In some embodiments, the cells of (ii) are not passaged prior to (iii).

[0009] In some embodiments, the method comprises: (i) culturing PSCs (e.g., iPSCs) in a basal culture medium comprising BMP4, VEGF, GSK3 inhibitor, and activin A; (ii) culturing the cells of (i) in a basal culture medium comprising VEGF and a TGFβ inhibitor; (iii) culturing the cells of (ii) in a basal culture medium; and (iv) culturing the cells of (iii) in a basal culture medium comprising TGFβ3 and PDGF to form mesenchymal cells.

[0010] In some embodiments, the method further comprises (v) culturing the cells of (iv) in a basal culture medium.

[0011] In some embodiments, the cells of (ii) are not passaged prior to (iii).

[0012] In some embodiments, the culture medium of (i) further comprises a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, Y-27632 is present in the culture medium at a concentration of about 10 μM.

[0013] In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, CHIR99021 is present in the culture medium at a concentration of about 1.5 μM.

[0014] In some embodiments, BMP4 is present in the culture medium at a concentration of about 30 ng / mL.

[0015] In some embodiments, VEGF is present in the culture medium at a concentration of about 50 ng / mL.

[0016] In some embodiments, activin A is present in the culture medium at a concentration of about 25 ng / mL.

[0017] In some embodiments, the TGFβ inhibitor is SB431542. In some embodiments, SB431542 is present in the culture medium at a concentration of about 10 μM.

[0018] In some embodiments, TGFβ3 is present in the culture medium at a concentration of about 2 ng / mL.

[0019] In some embodiments, PDGF is PDGF-BB. In some embodiments, PDGF-BB is present in the culture medium at a concentration of about 4 ng / mL.

[0020] In some embodiments, the stromal cells are pericytes.

[0021] In some embodiments, the iPSCs are not completely confluent at the start of culturing of (i).

[0022] In some embodiments, the iPSCs are about 50% to about 80% confluent at the start of culture (i).

[0023] The present disclosure also provides stromal cells produced by the differentiation methods disclosed herein, pericyte cells produced by the differentiation methods disclosed herein, organoids containing the stromal cells disclosed herein, organoids containing the pericyte cells disclosed herein, and certain methods of using the same.

[0024] The present disclosure also provides a specific differentiation medium. In some embodiments, the differentiation medium comprises a basal culture medium, BMP4, VEGF, a GSK3 inhibitor, activin A, and a ROCK inhibitor. In some embodiments, the differentiation medium comprises a basal culture medium, about 30 ng / mL of BMP4, about 50 ng / mL of VEGF, about 1.5 μM of CHIR99021, about 25 ng / mL of activin A, and about 10 μM of Y-27632.

[0025] Some embodiments of the present invention are described herein with reference to the accompanying drawings, which are for illustrative purposes only. Although the drawings are specifically referred to in detail herein, it should be emphasized that the details shown are for illustrative purposes and for the purpose of explicitly discussing embodiments of the present invention.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0027] I. General Definitions 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 disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0028] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and the claims.

[0029] To further define the present disclosure, the following terms and definitions are provided.

[0030] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single". In other embodiments, the term "a" or "an" includes "two or more" or "plural".

[0031] As used herein, the term "about" is used to mean approximately, roughly, around, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the indicated value. Generally, the term "about" is used herein to modify a numerical value by a variation of up to (higher or lower) 10 percent above and below the indicated value.

[0032] Throughout this disclosure, various aspects of the invention are presented in a range format. It should be understood that the description in range format is for mere convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose all possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. An enumerated numerical range includes the numbers that define the range and each integer within the defined range.

[0033] Units, prefixes, and symbols are expressed in their recognized forms in the International System of Units (SI). A numerical range includes the numbers that define the range. When a range of values is stated, the value of each integer between the values stated as the upper and lower limits of the range, and each fraction thereof, as well as each sub-range between such values, are specifically disclosed. The upper and lower limits of any range can be included in the range independently or excluded from the range, and each range that includes either one of the limits, neither of the limits, or both of the limits is also encompassed within the scope of this disclosure. Thus, the ranges described herein are understood to be a shorthand for all values within that range, including the recited endpoints. For example, the range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0034] When values are explicitly recited, it is understood that values that are substantially equivalent in amount or quantity to the recited values are also within the scope of this disclosure. When combinations are disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, when different elements or groups of elements are disclosed individually, the combination is also disclosed. When any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure where each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein; a plurality of elements of a disclosure can have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0035] As used herein, the term "and / or" is to be interpreted as a specific disclosure of each of two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used in an expression such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0036] It is understood that when an aspect is described in the expression "comprising", other similar aspects described in the words "consisting of" and / or "consisting essentially of" are also provided.

[0037] II. Differentiation Methods This disclosure relates to an improved method for differentiating pluripotent stem cells (PSCs) into stromal cells or pericyte cells. Such a method provides, for example, an improvement in the cell viability, yield, and / or characteristics of the differentiated cells.

[0038] As used herein, the terms "differentiate" and "differentiating" refer to the process of inducing or reprogramming young or immature cells (e.g., pluripotent stem cells) into more mature or specialized cells (mesenchymal cells or pericyte cells). Generally, the differentiation of pluripotent stem cells can be carried out by changing the cell culture conditions, such as by changing the stimulant in the culture medium or the physical state of the cells.

[0039] As used herein, the terms "pluripotent stem cell" and "PSC" refer to young or immature cells that can give rise to more mature or specialized cells (e.g., mesenchymal cells or pericyte cells).

[0040] In some embodiments, PSCs include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells, adult stem cells, or combinations thereof. In some embodiments, the PSCs are of human origin. In some embodiments, the PSCs are of animal origin. In some embodiments, the animal is a sheep, a pig, or a primate.

[0041] As used herein, the terms "induced pluripotent stem cell" and "iPSC" refer to cells that are induced or reprogrammed from differentiated adult, neonatal, or fetal cells to produce pluripotent stem cells.

[0042] As used herein, the term "mesenchymal cell" or "SC" refers to a cell that functions as a connective tissue cell of an organ that supports the function of the parenchymal cells of a particular organ. As used herein, mesenchymal cells include mature mesenchymal cells, stromal progenitor cells, and stromal precursor cells.

[0043] In some embodiments, the stromal cells described herein are pericytes. As used herein, the term "pericyte" refers to fibroblast-like cells that wrap extensive cytoplasmic processes around endothelial cells of arterioles, capillaries, and venules and cover between 22% and 99% of the endothelial cell surface. Pericytes are embedded in the basement membrane of capillaries, where they communicate with endothelial cells by both direct physical contact and paracrine signaling.

[0044] In some embodiments, the differentiation methods provided herein include certain cell culture conditions such as cell culture in a particular culture medium.

[0045] As used herein, the terms "cell culture", "culturing cells", "culture", "culturing", and "cultured" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. The terms "cell culture medium", and "culture medium" refer to compositions for cell culture that contain nutrients that maintain cell viability, support growth, and optionally support differentiation. A cell culture medium can contain one or more of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components such as growth factors, vitamins, etc.

[0046] In some embodiments, the differentiation methods provided herein refer to a cell culture medium as a "basal culture medium" supplemented with other components (sometimes referred to herein as a "differentiation medium"). As used herein, "basal culture medium" refers to a composition containing the minimal elements necessary for the maintenance, growth and / or differentiation of cells in an in vitro environment. Examples of basal culture media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), MEM, Iscove's Modified Dulbecco Medium (IMDM), Glasgow Modified MEM (GMEM), DMEM / F12, Leibovitz L-15, RPMI-1640, CMRL, Ham's F10, and Ham's F12. In some embodiments, the basal culture medium is supplemented with one or more other components such as, for example, amino acids, antibiotics, serum, growth factors. Such components are well known in the art and are further described herein.

[0047] In some embodiments, the differentiation methods provided herein include certain cell culture conditions such as passaging cells in a particular culture medium. As used herein, the terms "passage", "passaged", and "passaging" refer to the act of splitting and seeding cells at a lower concentration onto one or more cell culture surfaces or vessels when the cells have grown to the desired extent. Passaging typically involves detaching the cells by mechanical or enzymatic means (e.g., incubation with Accutane®) at a particular cell density as appropriate prior to seeding. Methods for passaging cells are well known and are further described herein.

[0048] In some embodiments, the culturing and subculturing in the differentiation methods provided herein are performed using one or more substrates coated on the cell culture surface or vessel. Such substrates include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen (e.g., collagen I, collagen IV, or a combination thereof), elastin, osteopontin, thrombospondin, a mixture of naturally occurring cell line-produced matrices such as Matrigel™, and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminal monolayers, or combinations thereof. Methods for coating substrates onto the cell culture surface or vessel are well known and are further described herein.

[0049] Some embodiments of the differentiation methods provided herein include culturing the cells in a basal culture medium containing bone morphogenetic protein 4 (BMP4). BMP4 is known to stimulate the differentiation of upper ectodermal tissues and bone formation in adult animals.

[0050] In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 10 ng / mL to about 50 ng / mL, or any value or range of values therebetween, such as about 20 ng / mL to about 50 ng / mL, about 30 ng / mL to about 50 ng / mL, about 40 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 20 ng / mL to about 40 ng / mL, about 30 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 20 ng / mL to about 30 ng / mL, or about 10 ng / mL to about 20 ng / mL. In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, or about 50 ng / mL. In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 30 ng / mL.

[0051] In other aspects, the basal culture medium of the differentiation methods provided herein does not contain BMP4 or is essentially free of BMP4. As used herein, the term "essentially free of" is known in the art and, when measured by the methods further described herein, refers to a culture medium that contains at least 95%, 96%, 97%, 98%, 99% or 100% less BMP4, or has an amount of BMP4 that cannot be detected, or a culture medium that does not contain BMP4. The terms "do not comprise" and "does not comprise" are known in the art and, when measured by the methods further described herein, refer to a culture medium that does not contain BMP4, or has an amount of BMP4 that cannot be detected.

[0052] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing vascular endothelial growth factor (VEGF). VEGF is a signaling protein that promotes the growth of new blood vessels. VEGF forms part of the mechanism that restores blood supply to cells and tissues deprived of oxygenated blood due to reduced blood circulation.

[0053] In some aspects, VEGF is present in the basal culture medium at a concentration of about 10 ng / mL to about 100 ng / mL, or any value or range of values therebetween, such as about 25 ng / mL to about 100 ng / mL, about 50 ng / mL to about 100 ng / mL, about 75 ng / mL to about 100 ng / mL, about 10 ng / mL to about 75 ng / mL, about 25 ng / mL to about ng / mL, about 50 ng / mL to about 75 ng / mL, about 10 ng / mL to about 50 ng / mL, about 25 ng / mL to about 50 ng / mL, or about 10 ng / mL to about 25 ng / mL. In some aspects, VEGF is present in the basal culture medium at a concentration of about 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL, or about 100 ng / mL. In some aspects, VEGF is present in the basal culture medium at a concentration of about 50 ng / mL.

[0054] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3 is a serine / threonine protein kinase that mediates the addition of phosphate molecules to certain serine and threonine amino acids of cellular substrates (e.g., glycogen synthase). This phosphorylation typically results in inhibition of the substrate. GSK3 is also involved in the control of the cellular response to damaged DNA and in the phosphorylation of Ci in the Wnt signaling and Hedgehog (Hh) pathways, targeting proteolysis to the inactive form.

[0055] As used herein, "GSK3 inhibitor" refers to a compound that inhibits one or more GSK3 enzymes. The family of GSK3 enzymes is well-known and numerous variants have been described (e.g., Schaffer et al., Gene, 302:73-81, 2003). Specific examples of GSK3 inhibitors include, but are not limited to, kenpaullone, 1-azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB415286, SB216763, AR-A014418, lithium, SB 415286, and TDZD-8.Further exemplary GSK3 inhibitors include BIO (2’Z,3’E)-6-bromindirubin-3’-oxime (GSK3 inhibitor IX); BIO-acetoxime (2’Z,3’E)-6-bromindirubin-3’-acetoxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopentadienylruthenium complex (GSK3 inhibitor XV); TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β inhibitor I); 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β inhibitor II); OTDZT 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β inhibitor III); α-4-dibromoacetophenone (GSK3β inhibitor VII); AR-AO 14418 N-(4-methoxybenzyl)-N’-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β inhibitor VIII); 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK3β inhibitor XI); TWS1 19-pyrrolopyrimidine compound (GSK3β inhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form (GSK3β inhibitor XIII); 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β inhibitor VI); AR-AO 144-18; SB216763; and SB415286, but are not limited thereto. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0056] In some embodiments, a GSK3 inhibitor (e.g., CHIR99021) is present in the basal culture medium at a concentration of about 0.5 μM to about 3 μM, or any value or range of values thereof, such as about 0.5 μM to about 1.5 μM, about 0.5 μM to about 1 μM, about 0.5 μM to about 1.5 μM, about 0.5 μM to about 1 μM, or a concentration comprising about 0.5 μM to about 1 μM. In some embodiments, the GSK3 inhibitor (e.g., CHIR99021) is about 0.5 μM, about 1 μM, about 1.5 μM, or about 3 μM. In some embodiments, the GSK3 inhibitor (e.g., CHIR99021) is about 1.5 μM.

[0057] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing activin A. Activin A is a dimeric glycoprotein belonging to the transforming growth factor β (TGFβ) family. Activin A regulates several biological functions including hormonal homeostasis, gonadal function, muscle growth, immunity, inflammation, and bone remodeling.

[0058] In some embodiments, activin A is present in the basal culture medium at a concentration of about 5 ng / mL to about 50 ng / mL, or any value or range of values thereof, such as about 10 ng / mL to about 50 ng / mL, about 25 ng / mL to about 50 ng / mL, about 5 ng / mL to about 25 ng / mL, about 10 ng / mL to about 25 ng / mL or a concentration comprising about 5 ng / mL to about 10 ng / mL. In some embodiments, activin A is present in the basal culture medium at a concentration of about 5 ng / mL, about 10 ng / mL, about 25 ng / mL or about 50 ng / mL. In some embodiments, activin A is present in the basal culture medium at a concentration of about 25 ng / mL.

[0059] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that acts as a downstream effector of Rho kinase and has three isoforms (RhoA, RhoB, RhoC). A "ROCK inhibitor" can, for example, reduce the expression and / or the activity of ROCK. Examples of ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides, and small molecules. More specific examples of ROCK inhibitors include, but are not limited to, anti-ROCK antibodies that target ROCK, and dominant negative ROCK variants, siRNA, shRNA, miRNA, and antisense nucleic acids. Examples of other ROCK inhibitors include thiazovivin, Y-27632, fasudil, AR122-86, Y-30141, WF-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, and the ROCK inhibitors disclosed in U.S. Patent No. 8,044,201, which is hereby incorporated by reference in its entirety. In some aspects, the ROCK inhibitor is Y-27632.

[0060] In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 1 μM to about 20 μM, or any value or range of values thereof, such as about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 10 μM to about 15 μM, or about 15 μM to about 20 μM. In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 1 μM, about 5 μM, about 10 μM, about 15 μM, or about 20 μM. In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 10 μM.

[0061] Some aspects of the differentiation methods provided herein involve culturing cells in a basal culture medium containing a TGFβ inhibitor. TGFβ is a highly pleiotropic cytokine that plays important roles in wound healing, angiogenesis, immune regulation, and cancer. As used herein, the term "TGFβ inhibitor" includes, but is not limited to, any inhibitor of TGF signaling in general, or an inhibitor specific for a TGFβ receptor (e.g., ALK5), and may also include antibodies against the TGFβ receptor, dominant negative variants of the TGFβ receptor, as well as siRNA and antisense nucleic acids that suppress the expression of the TGFβ receptor.Examples of TGFβ inhibitors include SB431542, A-83-01 (also known as 3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), 2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388 (-4-[3-(pyridin-2-yl)-1H-pyrazole-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), SMI6, 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide, GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazole-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride), SU5416, lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY2109761; SB-431542; SD-208; SM16; NPC-30345; KI26894; SB-203580; SD-093; ALX-270-448; EW-7195; SB-525334; GN-1233; SKI2162; Gleevec; 3,5,7,2’,4’-pentahydroxyflavone (Morin); activin-M108A; P144; soluble TBR2-Fc; and the pyrimidine derivatives and indolinones reported by Roth et al. in 2010, but are not limited thereto. In some embodiments, the TGFβ inhibitor is SB431542.

[0062] In some embodiments, the TGFβ inhibitor (e.g., SB431542) is present in the basal culture medium at a concentration of about 1 μM to about 20 μM, or any value or range of values thereof, such as a concentration comprising about 5 μM to about 20 μM, about 10 μM to about 20 μM, about 1 μM to about 10 μM, and about 1 μM to about 5 μM. In some embodiments, the TGFβ inhibitor (e.g., SB431542) is present in the basal culture medium at a concentration of about 1 μM, about 5 μM, about 10 μM, or about 20 μM. In some embodiments, the TGFβ inhibitor (e.g., SB431542) is present in the basal culture medium at a concentration of about 10 μM.

[0063] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing transforming growth factor β3 (TGFβ3). TGFβ3 is a cytokine involved in cell differentiation, embryonic development, and morphogenesis. TGFβ3 is thought to regulate molecules involved in cell adhesion and extracellular matrix (ECM) formation during the process of palate development.

[0064] In some embodiments, TGFβ3 is present in the basal culture medium at a concentration of about 0.5 ng / mL to about 5 ng / mL, or any value or range of values thereof, such as a concentration comprising about 1 ng / mL to about 5 ng / mL, about 2 ng / mL to about 5 ng / mL, about 0.5 ng / mL to about 2 ng / mL, about 1 ng / mL to about 2 ng / mL, or about 0.5 ng / mL to about 1 ng / mL. In some embodiments, TGFβ3 is present in the basal culture medium at a concentration of about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, or about 5 ng / mL. In some embodiments, TGFβ3 is present in the basal culture medium at a concentration of about 2 ng / mL.

[0065] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing platelet-derived growth factor (PDGF). PDGF is a growth factor that controls cell growth and division and plays an important role in angiogenesis, the growth of blood vessels from existing vascular tissue, as well as the mitosis and chemotaxis of mesenchymal cells. PDGF is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB). In some aspects, the PDGF is PDGF-BB.

[0066] In some aspects, the PDGF (e.g., PDGF-BB) is present in the basal culture medium at a concentration of about 2 ng / mL to about 8 ng / mL, or any value or range of values therebetween, such as 4 ng / mL to about 8 ng / mL, about 5 ng / mL to about 8 ng / mL, about 2 ng / mL to about 5 ng / mL, about 4 ng / mL to about 5 ng / mL, or about 2 ng / mL to about 4 ng / mL. In some aspects, the PDGF (e.g., PDGF-BB) is about 2 ng / mL, about 4 ng / mL, about 5 ng / mL, or about 8 ng / mL. In some aspects, the PDGF (e.g., PDGF-BB) is about 4 ng / mL.

[0067] In some embodiments, the differentiation method provided herein includes culturing PSCs (e.g., iPSCs) in a basal culture medium containing BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), activin A, and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the basal culture medium contains BMP4 at about 10 ng / mL to about 50 ng / mL, VEGF at about 10 ng / mL to about 100 ng / mL, a GSK3 inhibitor (e.g., CHIR99021) at about 0.5 μM to about 3 μM, activin A at about 5 ng / mL to about 50 ng / mL, and a ROCK inhibitor (e.g., Y-27632) at about 1 μM to about 20 μM. In some embodiments, the basal culture medium contains BMP4 at about 30 ng / mL, VEGF at about 50 ng / mL, a GSK3 inhibitor (e.g., CHIR99021) at about 1.5 μM, activin A at about 25 ng / mL, and a ROCK inhibitor (e.g., Y-27632) at about 10 μM.

[0068] In some embodiments, the differentiation method provided herein includes culturing cells in a basal culture medium containing VEGF and a TGFβ inhibitor (e.g., SB431542). In some embodiments, the basal culture medium contains VEGF at about 10 ng / mL to about 100 ng / mL and a TGFβ inhibitor (e.g., SB431542) at about 1 μM to about 20 μM. In some embodiments, the basal culture medium contains VEGF at about 30 ng / mL and a TGFβ inhibitor (e.g., SB431542) at about 10 μM.

[0069] In some embodiments, the differentiation method provided herein includes culturing cells in a basal culture medium containing TGFβ3 and PDGF (e.g., PDGF-BB). In some embodiments, the basal culture medium contains TGFβ3 at about 1 ng / mL to about 5 ng / mL and PDGF (e.g., PDGF-BB) at about 2 ng / mL to about 8 ng / mL. In some embodiments, the basal culture medium contains TGFβ3 at about 2 ng / mL and PDGF (e.g., PDGF-BB) at about 4 ng / mL.

[0070] In some embodiments, the differentiation method provided herein is as follows: (i) Culturing pluripotent stem cells (e.g., iPSCs) in a basal culture medium containing BMP4, VEGF, a GSK3 inhibitor, activin A, and a ROCK inhibitor; (ii) Culturing the cells of (i) in a basal culture medium containing VEGF and a TGFβ inhibitor; and (iii) Culturing the cells of (ii) in a basal culture medium containing TGFβ3 and platelet-derived growth factor (PDGF) to form mesenchymal cells or pericytes.

[0071] In some embodiments, the method further includes culturing the cells of (ii) in a basal culture medium before (iii). In some embodiments, the method further includes culturing the cells of (iii) in a basal culture medium. In some embodiments, the cells of (ii) are not passaged before (iii).

[0072] In some embodiments, the differentiation method provided herein includes culturing PSCs (e.g., iPSCs) in a basal culture medium containing BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), and activin A. In some embodiments, the basal culture medium contains from about 10 ng / mL to about 50 ng / mL of BMP4, from about 10 ng / mL to about 100 ng / mL of VEGF, from about 0.5 μM to about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), and from about 5 ng / mL to about 50 ng / mL of activin A. In some embodiments, the basal culture medium contains about 30 ng / mL of BMP4, about 50 ng / mL of VEGF, about 1.5 μM of a GSK3 inhibitor (e.g., CHIR99021), and about 25 ng / mL of activin A.

[0073] In some embodiments, the differentiation method provided herein is as follows: (i) Culturing PSCs (e.g., iPSCs) in a basal culture medium containing BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), and activin A; (ii) culturing the cells of (i) in a basal culture medium containing a VEGF and TGFβ inhibitor (e.g., SB431542); (iii) culturing the cells of (ii) in a basal culture medium; and (iv) culturing the cells of (iii) in a basal culture medium containing TGFβ3 and PDGF (e.g., PDGF - BB) to form stromal cells or pericytes.

[0074] In some embodiments, the method further comprises (v) culturing the cells of (iv) in a basal culture medium. In some embodiments, the cells of (ii) are not passaged prior to (iii).

[0075] In some embodiments of any of the methods disclosed herein, at the start of culturing of (i), the PSC (e.g., iPSC) is not fully confluent. In some embodiments of any of the methods disclosed herein, at the start of culturing of (i), the PSC is about 50% - about 80% confluent. In some embodiments of any of the methods disclosed herein, at the start of culturing of (i), the PSC is about 50%, about 60%, about 70%, or about 80% confluent.

[0076] In some embodiments of any of the methods disclosed herein, the stromal cells are pericytes.

[0077] III. Other Embodiments The present disclosure also relates to stromal cells produced by any of the differentiation methods disclosed herein. The present disclosure also relates to pericytes produced by any of the differentiation methods disclosed herein.

[0078] The present disclosure also relates to organoids comprising stromal cells and / or pericytes disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated three-dimensional (3D) cellular organism derived from pluripotent stem cells (e.g., iPSCs) that self-organizes by the dense accumulation of cells in a controlled space. Such organisms can be made to reproduce much of the complexity of an organ, or to represent a selected aspect thereof, for example, to produce only a particular type of cell.

[0079] Methods for maintaining differentiated stromal cells, pericytes, and organoids are well known and include culturing the cells or organoids in the cell culture media described herein, and / or cryopreservation. Methods for making organoids typically include culturing the cells in a 3D matrix. Suitable 3D matrices include, but are not limited to, polymers (natural or synthetic), ceramics, or composite materials. The 3D matrix can be in the form of a hydrogel, a porous 3D scaffold, a rapid prototyping scaffold, a foam, a sponge, a mesh, microparticles, a fibrous network, a mixture of naturally occurring cell line-derived matrices such as Matrigel™, and combinations thereof, for example, a hydrogel filled with microparticles.

[0080] The present disclosure also relates to methods for promoting angiogenesis or vascular development, including administration of stromal cells, pericytes, or organoids made by any of the differentiation methods disclosed herein.

[0081] The present disclosure also relates to methods for treating vasculitis or vascular disorders, including administration of stromal cells, pericytes, or organoids made by any of the differentiation methods disclosed herein.

[0082] The present disclosure also relates to a method of treating cardiovascular diseases, including administration of stromal cells, pericytes, or organoids produced by any of the differentiation methods disclosed herein. In some embodiments, the cardiovascular disease is coronary artery disease (CAD), cardiac arrhythmia, heart failure, heart valve disease, pericardial disease, cardiomyopathy (heart muscle disease), or congenital heart disease.

[0083] The present disclosure also relates to a specific differentiation medium.

[0084] In some embodiments, the present disclosure provides a differentiation medium comprising a basal culture medium, BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), activin A, and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation medium comprises a basal culture medium, about 10 ng / mL to about 50 ng / mL of BMP4, about 10 ng / mL to about 100 ng / mL of VEGF, about 0.5 μM to about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), about 5 ng / mL to about 50 ng / mL of activin A, and about 1 μM to about 20 μM of a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation medium comprises a basal culture medium, about 30 ng / mL of BMP4, about 50 ng / mL of VEGF, about 1.5 μM of a GSK3 inhibitor (e.g., CHIR99021), about 25 ng / mL of activin A, and about 10 μM of a ROCK inhibitor (e.g., Y-27632).

[0085] In some embodiments, the present disclosure provides a differentiation medium comprising a basal culture medium, VEGF, and a TGFβ inhibitor (e.g., SB431542). In some embodiments, the differentiation medium comprises a basal culture medium, about 10 ng / mL to about 100 ng / mL of VEGF, and about 1 μM to about 20 μM of a TGFβ inhibitor (e.g., SB431542). In some embodiments, the differentiation medium comprises a basal culture medium, about 30 ng / mL of VEGF, and about 10 μM of a TGFβ inhibitor (e.g., SB431542).

[0086] In some embodiments, the present disclosure provides a differentiation medium comprising a basal culture medium, TGFβ3, and PDGF (e.g., PDGF-BB). In some embodiments, the differentiation medium comprises a basal culture medium, from about 1 ng / mL to about 5 ng / mL of TGFβ3, and from about 2 ng / mL to about 8 ng / mL of PDGF (e.g., PDGF-BB). In some embodiments, the differentiation medium comprises a basal culture medium, about 2 ng / mL of TGFβ3, and about 4 ng / mL of PDGF (e.g., PDGF-BB).

[0087] In some embodiments, the present disclosure provides a differentiation medium comprising a basal culture medium, BMP4, VEGF, a GSK3 inhibitor (e.g., CHIR99021), and activin A. In some embodiments, the differentiation medium comprises a basal culture medium, from about 10 ng / mL to about 50 ng / mL of BMP4, from about 10 ng / mL to about 100 ng / mL of VEGF, from about 0.5 μM to about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), and from about 5 ng / mL to about 50 ng / mL of activin A. In some embodiments, the differentiation medium comprises a basal culture medium, about 30 ng / mL of BMP4, about 50 ng / mL of VEGF, about 1.5 μM of a GSK3 inhibitor (e.g., CHIR99021), and about 25 ng / mL of activin A.

Example

[0088] [Example 1]

[0089] Now, with the above description, reference is made to the following examples, which illustrate some embodiments of the invention in a non-limiting manner.

[0090] Differentiation of induced pluripotent stem cells into mesenchymal cells An experiment to differentiate mesenchymal cells from human induced pluripotent stem cells (iPSCs) (BJRiPS iPSCs provided by Jintang Du) was conducted using the following protocol.

[0091] - Day 1: The day before the experiment started, Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning®, product number 354230) was placed on ice and thawed overnight.

[0092] Day 0: On the initial seeding day of iPSCs, 120 μL of Matrigel® GFR was added to 12 mL of Dulbecco's Modified Eagle Medium (DMEM) / F12 medium (Thermo Fisher, product number 11320033) and mixed. 2 mL of this mixture was added to each well of a 6-well plate and incubated at room temperature (RT) for at least 1 hour.

[0093] For the initial seeding, an iPSC cell culture plate that was approximately 70% confluent was used. The medium was removed from the plate, and the iPSCs were washed once with phosphate-buffered saline (PBS) (Gibco, product number 12604-013). Then, 1.5 mL of Accutase® (Biolegend, product number 423201) was added to the plate and incubated at 37°C for 7 minutes. Subsequently, the cells were solubilized with Accutase®, passed through a strainer to collect cell clumps, and introduced into mTESR™ medium (STEMCELL™ Technologies Inc.) to quench Accutase®. This cell solution was counted using the MoxiCyte Viability Kit and the Moxi Flow Instrument (ORFLO®).

[0094] Thereafter, the cells were seeded at 200,000 cells per well in warmed mTEST™ medium supplemented with 10 μM of Y-27632 (STEMCELL™ Technologies Inc.) and incubated overnight.

[0095] Day 1: The medium was removed from the culture plate together with non-adherent iPSCs, and 2 mL of fresh warmed mTESR™ supplemented with 10 μM of Y-27632 per well was added to the cells. The cells were imaged across the entire well using the Incucyte® Live-Cell Analysis System (Sartorius). An exemplary image is shown in FIG. 1A.

[0096] Day 2: The medium was removed from the culture plate together with non-adherent iPSCs, and 2 mL of fresh warmed mTESR™ supplemented with 10 μM of Y-27632 per well was added to the cells. The cells were imaged across the entire well using the Incucyte® Live-Cell Analysis System. An exemplary image is shown in FIG. 1B.

[0097] Day 3: Mesoderm differentiation. The medium was removed from the nearly confluent culture plate, and replaced with 2 mL of filtered mesoderm induction medium containing APEL™ 2 medium (STEMCELL™ Technologies Inc.) supplemented with 25 ng / mL of activin A (Peprotech, product number AF-120-14E), 30 ng / mL of bone morphogenetic protein 4 (BMP4) (Peprotech AF-120-05ET), 50 ng / mL of vascular endothelial growth factor (VEGF), 1.5 μM of CHIR99021 (Biovision, product number 1748-5), and 10 μM of Y-27632 per well. The cells were imaged across the entire well using the IncuCyte® Live-Cell Analysis System and incubated for approximately 4 days. Exemplary images at day 4 are shown in FIG. 1C and at day 5 are shown in FIG. 1D.

[0098] Day 7: Vascular Differentiation. The medium was removed from the culture plates and replaced with 2 mL of filtered vascular induction medium containing APEL™ 2 medium supplemented with 50 ng / mL of VEGF and 10 μM of SB431542 (Tocris, product number 3211) per well. Cells were imaged across the well using the Incucyte® Live-Cell Analysis System and incubated for approximately 4 days with one change of the vascular differentiation medium. Exemplary images at day 7, day 9, and day 10 are shown in FIGS. 1E, 1F, and 1G, respectively.

[0099] Day 11: Cell Proliferation. The medium was removed, and the cells were harvested, washed once with PBS, resuspended by adding 1.5 mL of Accutase®, and incubated at 37 °C for 7 minutes. The Accutase® was quenched with APEL™ 2 medium, and the cell clumps were removed by passing through a strainer. The cells were then counted using a Moxi Flow Instrument (ORFLO®) and seeded at 800,000 cells per plate onto gelatin-coated T-75 flasks (Sigma, product number G1394; Falcon, product number 353136) in 15 mL of complete EGM™-2 medium (EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™, Lonza, product numbers CC-3156 and CC-4176). The cells were grown until approximately 90% confluent and fed with fresh complete EGM™-2 medium every 3 days. An exemplary image at day 17 is shown in FIG. 1H.

[0100] Mesenchymal / Pericyte Differentiation: Differentiation was initiated by removing EGM™-2 medium and adding 15 mL of Pericyte Growth Media (PromoCell) supplemented with 2 ng / mL of transforming growth factor-β3 (TGF-β3) (Peprotech, catalog number 100-36E) and 4 ng / mL of platelet-derived growth factor-BB (PDGF-BB) (Peprotech, catalog number 100-14B). Cells were incubated for 3 days. Exemplary images of the resulting test differentiated cells are shown in FIGS. 1I and 1J.

[0101] Next, the test differentiated cells were verified by determining the expression levels of various mesenchymal and control markers using flow cytometry. A summary of the results of this verification showing the relative expression levels is presented in Table 1. The results of the test differentiated cells are shown together with the results of control endothelial cells, pericytes, fibroblasts, and smooth muscle cells.

[0102]

Table 1

[0103] The test differentiated cells were then further characterized by a tube formation assay using the following protocol. The test differentiated cells were co-cultured with human pulmonary artery endothelial cells (HPAEC) in the presence or absence of 1 μM imatinib mesylate to inhibit angiogenesis, and tube formation was observed. Specifically, the wells of a 96-well plate (Costar, catalog number 387) were coated with 30 μL of Matrigel® GFR. The plate was centrifuged at 1,500 revolutions per minute (RPM) for 1 minute and incubated at 30 °C for 30 minutes. The cells were then detached from the culture plate, counted, and resuspended in DMEM / F12 medium such that 30 μL of medium was available per well. 30,000 HPAEC cells and 5,000 differentiated cells were added to each well.

[0104] The green labeling of HPAECs was performed using Nuclight Green Lentivirus (Essen / Sartorius #4475). 100 μL of transfection reagent was added to 5 mL of medium and mixed. To another 5 mL of medium, 10 μL of the required lentivirus was added and mixed. Next, both 5 mL preparations were added together, mixed, and added to plates of HPAECs or pericytes that were approximately 70% confluent. The plates were grown overnight in an incubator.

[0105] The medium and lentivirus preparation were removed, replaced with fresh medium, and the plates were incubated overnight. The plates were incubated for one week with daily medium changes using 1 μg of puromycin in the medium.

[0106] Differentiated cells were stained with 0.35 μM Nuclight Rapid Red (Essen Bioscience) in PBS at 37 °C for 20 minutes. Subsequently, the dye was quenched with medium and the cells were dispersed. For the tube formation assay, after gelation, 30 μL of the cell suspension was carefully placed on top. The plates were loaded into the Incucyte® Live-Cell Analysis System and images were taken every hour.

[0107] As shown in Figure 2, the test differentiated cells developed tube formation and co-localized with HPAECs, but the control cells treated with imatinib mesylate did not.

[0108] Figures 3A - 3C show exemplary images of tube formation at the 3-hour time point in co-cultures of HPAECs and test differentiated cells (Figure 3A), HPAECs and test differentiated cells treated with imatinib mesylate (Figure 3B), and HPAECs and placental pericytes (Figure 3C). These results indicate that the test differentiated cells are binding to HPAEC tubes in a manner similar to primary cultured pericytes.

[0109] Furthermore, based on the results of flow cytometry and the stability of tube formation and the duration of structure, the test differentiated cells that grew the longest in the growth medium had the best characteristics.

[0110] All publications, patents, and patent applications mentioned in this application are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Further, any citation or identification of a reference in this application shall not be construed as an admission that such reference is available as prior art with respect to the present invention. Where section headings are used, they shall not necessarily be construed as limiting.

Claims

**Claim 1** A method for differentiating pluripotent stem cells into mesenchymal cells, comprising the following steps: (i) culturing pluripotent stem cells in a basal culture medium containing bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), glycogen synthase kinase 3 (GSK3) inhibitor, activin A, and Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) culturing the cells of (i) in a basal culture medium containing VEGF and transforming growth factor β (TGFβ) inhibitor; and (iii) culturing the cells of (ii) in a basal culture medium containing TGFβ3 and platelet-derived growth factor (PDGF) to form mesenchymal cells. **Claim 2** The method according to claim 1, further comprising culturing the cells of (ii) in a basal culture medium before (iii). **Claim 3** The method according to claim 1 or 2, further comprising culturing the cells of (iii) in a basal culture medium. **Claim 4** The method according to any one of claims 1 to 3, wherein the cells of (ii) are not passaged before (iii). **Claim 5** A method for differentiating pluripotent stem cells into mesenchymal cells, comprising the following steps: (i) culturing pluripotent stem cells in a basal culture medium containing BMP4, VEGF, GSK3 inhibitor, and activin A; (ii) culturing the cells of (i) in a basal culture medium containing VEGF and TGFβ inhibitor; (iii) culturing the cells of (ii) in a basal culture medium; and (iv) culturing the cells of (iii) in a basal culture medium containing TGFβ3 and PDGF to form mesenchymal cells. **Claim 6** The method according to claim 5, further comprising culturing the cells of (iv) in a basal culture medium. **Claim 7** The method according to claim 5 or 6, wherein the cells of (ii) are not passaged before (iii). **Claim 8** The method according to any one of claims 5 to 7, wherein the culture medium of (i) further contains a ROCK inhibitor. **Claim 9** The method according to any one of claims 1 to 8, wherein the ROCK inhibitor is Y-27632. **Claim 10** The method according to claim 9, wherein Y-27632 is present in the culture medium at a concentration of about 10 μM. **Claim 11** The method according to any one of claims 1 to 10, wherein the GSK3 inhibitor is CHIR99021. **Claim 12** The method according to claim 11, wherein CHIR99021 is present in the culture medium at a concentration of about 1.5 μM.

13. The method according to any one of claims 1 to 12, wherein BMP4 is present in the culture medium at a concentration of about 30 ng / mL.

14. The method according to any one of claims 1 to 13, wherein VEGF is present in the culture medium at a concentration of about 50 ng / mL.

15. The method according to any one of claims 1 to 14, wherein activin A is present in the culture medium at a concentration of about 25 ng / mL.

16. The method according to any one of claims 1 to 15, wherein the TGFβ inhibitor is SB431542.

17. The method according to claim 16, wherein SB431542 is present in the culture medium at a concentration of about 10 μM.

18. The method according to any one of claims 1 to 17, wherein TGFβ3 is present in the culture medium at a concentration of about 2 ng / mL.

19. The method according to any one of claims 1 to 18, wherein PDGF is PDGF-BB.

20. The method according to claim 19, wherein PDGF-BB is present in the culture medium at a concentration of about 4 ng / mL.

21. The method according to any one of claims 1 to 20, wherein the stromal cells are pericytes.

22. The method according to any one of claims 1 to 21, wherein the pluripotent stem cells are not completely confluent at the start of the culture (i).

23. The method according to any one of claims 1 to 22, wherein the pluripotent stem cells are about 50% to about 80% confluent at the start of the culture (i).

24. The method according to any one of claims 1 to 23, wherein the pluripotent stem cells are embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), embryonic germ cells, or adult stem cells.

25. Stromal cells produced by the method according to any one of claims 1 to 24.

26. Pericytes produced by the method according to any one of claims 1 to 24.

27. An organoid comprising the stromal cells according to claim 25.

28. An organoid comprising the pericytes according to claim 26.

29. A differentiation medium comprising a basal culture medium, BMP4, VEGF, a GSK3 inhibitor, activin A, and a ROCK inhibitor.

30. A differentiation medium containing a basal culture medium, about 30 ng / mL of BMP4, about 50 ng / mL of VEGF, about 1.5 μM of CHIR99021, about 25 ng / mL of activin A, and about 10 μM of Y-27632.

Images