Method for differentiating endothelial cells

Abstract

The present invention relates to a method for differentiating pluripotent stem cells into endothelial cells. The present invention also relates to endothelial cells produced by such a method, organoids containing such endothelial cells, and methods of using the same. The present invention also relates to a differentiation medium for use herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 390,445, filed on July 19, 2022, the content of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Field of Disclosure The present disclosure relates to methods for differentiating pluripotent stem cells into endothelial cells. The present disclosure also relates to endothelial cells produced by such methods, organoids containing such endothelial cells, and methods of using the same. The present disclosure also relates to the differentiation medium used therefor.

[0003] Background 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 PSCs obtained from adult somatic cells that have been genetically reprogrammed to an ESC-like state through the expression of genes and factors important for maintaining the defining characteristics of ESCs. iPSCs have recently attracted interest in the medical community because they address many of the obstacles associated with the use of ESCs, enable the generation of patient-specific PSCs, can be genetically modified and differentiated into the somatic lineage, and returned to the same patient as an autologous transplant. 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 diseases, iPSCs can also be used for tissue regeneration and disease modeling. Kogutetal.,MethodsMol.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

Problems to be Solved by the Invention

[0004] Overview The present disclosure provides a method for differentiating pluripotent stem cells (PSCs) into endothelial cells (ECs).

Means for Solving the Problems

[0005] In some embodiments, the method comprises: (i) culturing PSCs (e.g., iPSCs) on a surface coated with collagen IV in a basal culture medium containing a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) culturing the cells of (i) on a surface coated with collagen IV in a basal culture medium containing a glycogen synthase kinase 3 (GSK3) inhibitor; (iii) culturing the cells of (iii) on a surface coated with collagen IV in a basal culture medium containing fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 4 (BMP4) for about 4 days; (iv) culturing the cells of (iii) on a surface coated with collagen IV in a basal culture medium containing FGF2 and VEGF for about 2 days, wherein the culture medium does not contain BMP4; and (v) isolating the cells of (iv) that express CD144 to form endothelial cells.

[0006] In some embodiments, the method further comprises: (vi) culturing the cells of (v) that have expression of CD144 in a basal culture medium containing a transforming growth factor β (TGFβ) inhibitor.

[0007] In some embodiments, the method comprises: (i) culturing PSCs (e.g., iPSCs) on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor; (ii) culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor; (iii) culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4; (iv) isolating the cells of (iii) that have expression of CD144; and (v) culturing the cells of (v) that express CD144 on a collagen I-coated surface in a basal culture medium containing a TGFβ inhibitor to form endothelial cells.

[0008] 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.

[0009] In some embodiments, the culture of (i) is about 1 day.

[0010] In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, CHIR99021 is present in the culture medium at a concentration of about 36 μM.

[0011] In some embodiments, the culture of (ii) is about 1 day.

[0012] In some embodiments, FGF is present in the culture medium at a concentration of about 50 μg / mL.

[0013] In some embodiments, VEGF is present in the culture medium at a concentration of about 50 μg / mL.

[0014] In some embodiments, BMP4 is present in the culture medium at a concentration of about 50 μg / mL.

[0015] In some embodiments, the cells are passaged between (iii) and (iv).

[0016] In some embodiments, the culture of (iii) is from about 4 days to about 6 days.

[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, the culture of (vi) is about 6 days. In some embodiments, the culture of (v) is about 6 days.

[0019] In some embodiments, the separation of (iv) is by immunomagnetic cell separation. In some embodiments, the separation of (v) is by immunomagnetic cell separation.

[0020] In some embodiments, the culture of (i) and / or (ii) is performed under hypoxic conditions.

[0021] The present disclosure also provides endothelial cells produced by the differentiation methods disclosed herein, organoids comprising the endothelial cells disclosed herein, and specific methods of using the same.

[0022] The present disclosure also provides a differentiation medium for use therewith.

Brief Description of the Drawings

[0023] In this specification, some aspects of the present invention are described by way of example only with reference to the accompanying drawings. Here, referring to the drawings in detail, it is emphasized that the details shown are by way of example and for the purpose of an illustrative description of aspects of the present invention.

[0024]

Figure 1

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Figure 2

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Figure 3

[0027] Figure 3B is an exemplary whole-cell image at day 2 of the differentiation protocol described in Example 1.

[0028] Figure 3C is an exemplary whole-cell image at day 5 of the differentiation protocol described in Example 1.

[0029] Figure 3D is an exemplary whole-cell image at day 9 after CD144+ selection of the differentiation protocol described in Example 1.

[0030] Figure 3E is an exemplary whole-cell image at day 13 after CD144+ selection of the differentiation protocol described in Example 1.

[0031] Figure 3F is an example of a whole-cell image on the 17th day after CD144+ selection in the differentiation protocol described in Example 1.

[0032]

Figure 4-1

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Figure 4-2

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Figure 4-3

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Figure 5-1

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Figure 5-2

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Figure 6

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Figure 7

[0039]

Figure 8

Mode for Carrying Out the Invention

[0040] Detailed Description 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 otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. 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.

[0041] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the detailed description and the claims.

[0042] To further define the present disclosure, the following terms and definitions are provided.

[0043] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an") can be used interchangeably herein with the terms "one or more" and "at least one". In certain embodiments, the term "a" or "an" means "single". In other embodiments, the term "a" or "an" includes "two or more" or "plural".

[0044] The term "about" is used herein to mean approximately, roughly, around, or within the range. When the term "about" is used in combination with a numerical range, the range is modified by extending the boundaries above and below the recited numerical values. Generally, the term "about" is used herein to modify a numerical value that is 10 percent above or below the recited value, with the variation being above or below (higher or lower).

[0045] Throughout this disclosure, various aspects of the invention are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose not only each individual numerical value within the range but also all possible sub-ranges. 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 numerical values within the 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 numerical values defining the range and each integer within the defined range.

[0046] Units, prefixes, and symbols are expressed in the form approved by the SI (International System of Units). Numerical ranges include the numerical values that define the range. When a range of values is indicated, it should be understood that each intervening integer value and each fraction between the indicated upper and lower limits of the range are also specifically disclosed, together with each sub-range between such values. The upper and lower limits of any range can be independently included in or excluded from the range, and any range in which either, both, or neither of the limits are included is also included in the disclosed range. Accordingly, ranges shown herein are to be understood as a shorthand for all values within the range including the indicated endpoints. For example, a range from 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.

[0047] When a value is explicitly recited, values that are approximately the same quantity or amount as the recited value are also understood to be within the scope of the disclosure. When a combination is disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any element of the disclosure is disclosed as having a plurality of alternatives, examples of the disclosure in which each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed by this specification. A plurality of elements of the disclosure can have such exclusions, and all combinations of elements having such exclusions are disclosed by this specification.

[0048] As used herein, the term "and / or" is to be construed as specifically disclosed for each of two particular features or components, regardless of the presence or absence of the other. Thus, when used in a phrase such as "A and / or B" herein, the term "and / or" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, when used in a phrase such as "A, B, and / or C", the term "and / or" is intended to encompass 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).

[0049] It is understood that wherever aspects are described herein in terms of the phrase "comprising", similar aspects are also provided which are described in terms of the phrases "consisting of" and / or "consisting essentially of".

[0050] II. Differentiation Methods The present disclosure relates to a method of differentiating pluripotent stem cells (PSCs, such as iPSCs) into endothelial cells (ECs). By such a method, for example, the viability, yield, and / or differentiation characteristics of endothelial cells are improved.

[0051] As used herein, the terms "differentiate" and "differentiating" refer to the process of inducing or reprogramming young or immature cells (such as pluripotent stem cells) into more mature or specialized cells (such as endothelial 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.

[0052] As used herein, the terms "pluripotent stem cell", "pluripotent stem cell", and "PSC" refer to young or immature cells that can develop into more mature or specialized cells (such as endothelial cells).

[0053] In some embodiments, the 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.

[0054] As used herein, the terms "induced pluripotent stem cell", "induced pluripotent stem cell", and "iPSC" refer to cells generated from differentiated adult, neonatal, or fetal cells that have been induced or reprogrammed into pluripotent stem cells.

[0055] As used herein, the term "endothelial cell", "endothelial cell", or "EC" refers to a cell or group of cells that form a single cell layer lining the inside of blood vessels and regulating the exchange between the blood flow and the surrounding tissues. As used herein, endothelial cells include mature endothelial cells, endothelial progenitor cells, and endothelial precursor cells.

[0056] The endothelial cells generated by the differentiation method provided herein have one or more biochemical, functional, or morphological characteristics of endothelial cells. The biochemical characteristics of endothelial cells include, but are not limited to, the ability to express one or more endothelial cell markers. Endothelial cell markers include, but are not limited to, vascular endothelial (VE) cadherin (CD144), ACE (CD143), BNH9 / BNF13, CD31, CD34, CD54 (ICAM-1), CD62E, CD105, CD146, endocan (ESM-1), endoglin-1, endomucin, eotaxin-3, EPAS1, factor VIII-related antigen, FLI-1, Flk-1 (KDR, VEGFR-2), FLT-1 (VEGFR-1), GATA2, GBP-1, GRO-alpha, HEX, ICAM-2, LMO2, LYVE-1, MRB, nucleolin, PAL-E, RTK, sVCAM-1, TAL1, TEM1, TEM5, TEM7, thrombomodulin (TM, CD141), VCAM-1 (CD106), VEGF, vWF, ZO-1, ESAM, CD102, CD93, CD184, CD304, DLL4, and tight junction proteins (e.g., Claudin5 or ZO-1).

[0057] The functional characteristics of endothelial cells include, but are not limited to, the ability to take up acetylated low-density lipoprotein (ac-LDL); having a barrier function; and the ability to respond to one or more pro-inflammatory stimuli (e.g., TNF, and IL-1) by upregulating the expression of cell adhesion molecules (e.g., CD54 (ICAM-1), CD106, and CD62E).

[0058] The morphological characteristics of endothelial cells include, but are not limited to, the ability to form tubular structures in a three-dimensional matrix, having a flat (or squamous) appearance, and having a large central nucleus.

[0059] The biochemical, functional, and morphological characteristics of endothelial cells can be readily determined by visual inspection or by methods known in the art and described herein.

[0060] In some embodiments, the differentiation methods provided herein include specific cell culture conditions, such as culturing cells in a specific culture medium.

[0061] As used herein, the terms "cell culture", "cell culturing", "culturing", "culture" and "cultured" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. The terms "cell culture medium", "cell culture media", "culture medium" and "media" refer to a cell culture composition containing nutrients that maintain cell viability and support growth and, optionally, differentiation. A cell culture medium includes one or more of salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components such as growth factors and vitamins.

[0062] In some embodiments, the differentiation methods provided herein refer to a cell culture medium (sometimes referred to as a "differentiation medium" or "differentiation media") as a "basal culture medium" supplemented with other components. As used herein, "basal culture medium" or "basal culture media" 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 amino acids, antibiotics, serum, growth factors, etc. Such components are well known in the art and are further described herein.

[0063] In some embodiments, the cell culture medium or basal culture medium of the differentiation methods provided herein "essentially does not contain" or "does not contain" certain components. As used herein, the term "essentially does not contain" 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 of a particular component, or an amount of the particular component that is undetectable. 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 a particular component, or an amount of the particular component that is undetectable. In some embodiments, the culture medium does not contain or essentially does not contain bone morphogenetic protein 4 (BMP4).

[0064] In some embodiments, the differentiation methods of the present disclosure include culturing cells under "normoxic" or "normoxia" conditions. Normoxia conditions typically include culturing cells in vitro at an oxygen level in air of about 15% to 20%.

[0065] In other embodiments, the differentiation methods of the present disclosure include culturing cells under "hypoxic" or "hypoxia" conditions. Hypoxia conditions typically include culturing cells in vitro at an oxygen level of about 10% or less, about 5% or less, or about 1% or less, depending on the cell type. Hypoxia conditions can be created and maintained using a culture apparatus capable of controlling the ambient gas concentration, such as an anaerobic chamber. Unless a hypoxic state is specified, the culture conditions in the present disclosure can be assumed to be normoxic. In some embodiments, the cells of the present disclosure are known in the art and can be cultured under normoxic conditions and then under hypoxic conditions, or vice versa, using the methods further described herein.

[0066] In some embodiments, the differentiation methods provided herein include specific cell culture conditions such as passage of cells in a specific culture medium. As used herein, the terms "passage," "passaged," and "passaging" refer to the act of subculturing and seeding cells at a low density onto one or more cell culture surfaces or vessels when the cells have grown to a desired extent. Passage typically involves detaching the cells by mechanical or enzymatic means (e.g., incubation with Accutase®) prior to seeding. Optionally at a specific cell density. Methods of passaging cells are well known and are further described herein.

[0067] In some embodiments, culturing and passaging 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 combinations thereof), elastin, osteopontin, thrombospondin, mixtures of natural cell line-forming matrices such as Matrigel®, and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminal monolayers, or combinations thereof. Methods of coating substrates on cell culture surfaces or vessels are well known and are further described herein.

[0068] In some embodiments, the differentiation methods provided herein include one or more steps in which cultured cells having specific biochemical properties are isolated. As used herein, the terms "isolated" and "isolating" refer to the process of isolating one or more specific cell populations from a heterogeneous mixture of cells. In some embodiments, the differentiation methods provided herein include, for example, isolating cells having expression of vascular endothelial (VE) cadherin (CD144) to form a culture of endothelial cells (ECs). As used herein, the term "expression of CD144" includes, but is not limited to, detectable expression of CD144, expression of CD144 comparable to that of mature endothelial cells or their progenitor or ancestral cells, or expression of CD144 that is higher than the expression of CD144 in control cells that do not express CD144 and / or are not endothelial cells.

[0069] Cell separation methods based on specific biochemical properties are well known in the art and include, but are not limited to, affinity separation, fluorescence-activated cell sorting (FACS), density gradient centrifugation, immunodensity cell isolation, microfluidic cell sorting, buoyancy-activated cell sorting, aptamer-based cell isolation, complement depletion, and the like. Techniques for affinity separation include separation using antibody-coated magnetic beads (e.g., immunomagnetic cell separation), affinity chromatography, cytotoxic agents (e.g., complement and cytotoxins) conjugated to monoclonal antibodies or used in combination with monoclonal antibodies, and "panning" by antibodies attached to a solid matrix (e.g., plates), or other convenient techniques. In some embodiments, the cells of the differentiation methods of the invention are separated by immunomagnetic cell separation.

[0070] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing fibroblast growth factor 2 (FGF2). FGF2, also known as basic fibroblast growth factor or FGF-β, is a growth factor and signaling protein encoded by the FGF2 gene. It has a wide range of mitogenic and cell survival activities and is involved in various biological processes including embryonic development, cell proliferation, morphogenesis, tissue repair, tumor growth, and invasion.

[0071] In some aspects, the FGF is present in the basal culture medium at a concentration of about 20 μg / mL to about 100 μg / mL, or, for example, about 40 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 60 μg / mL to about 100 μg / mL, about 80 μg / mL to about 100 μg / mL, about 20 μg / mL to about 80 μg / mL, about 40 μg / mL to about 80 μg / mL, about 50 μg / mL to about 80 μg / mL, about 60 μg / mL to about 80 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 80 μg / mL, about 50 μg / mL to about 60 μg / mL, about 60 μg / mL to about 100 μg / mL, about 60 μg / mL to about 80 μg / mL, or about 80 μg / mL to about 100 μg / mL, or any value or range of values therebetween. In some aspects, the FGF is present in the basal culture medium at a concentration of about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL. In some aspects, the FGF is present in the basal culture medium at a concentration of about 50 μg / mL.

[0072] 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 that are oxygen-starved due to reduced blood circulation.

[0073] In some embodiments, VEGF is present in the basal culture medium at a concentration of about 20 μg / mL to about 100 μg / mL, or, for example, about 40 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 60 μg / mL to about 100 μg / mL, about 80 μg / mL to about 100 μg / mL, about 20 μg / mL to about 80 μg / mL, about 40 μg / mL to about 80 μg / mL, about 50 μg / mL to about 80 μg / mL, about 60 μg / mL to about 80 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 80 μg / mL, about 50 μg / mL to about 60 μg / mL, about 60 μg / mL to about 100 μg / mL, about 60 μg / mL to about 80 μg / mL, or about 80 μg / mL to about 100 μg / mL, or any value or range of values therebetween. In some embodiments, VEGF is present in the basal culture medium at a concentration of about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL. In some embodiments, VEGF is present in the basal culture medium at a concentration of about 50 μg / mL.

[0074] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium that contains bone morphogenetic protein 4 (BMP4). In other embodiments, the basal culture medium does not contain BMP4 or is essentially free of BMP4. BMP4 is known to stimulate the differentiation of overlying ectodermal tissue and to stimulate bone formation in adult animals.

[0075] In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 20 μg / mL to about 100 μg / mL, or any value or range of values including, for example, about 40 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 60 μg / mL to about 100 μg / mL, about 80 μg / mL to about 100 μg / mL, about 20 μg / mL to about 80 μg / mL, about 40 μg / mL to about 80 μg / mL, about 50 μg / mL to about 80 μg / mL, about 60 μg / mL to about 80 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 80 μg / mL, about 50 μg / mL to about 60 μg / mL, about 60 μg / mL to about 100 μg / mL, about 60 μg / mL to about 80 μg / mL, or about 80 μg / mL to about 100 μg / mL. In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL. In some embodiments, BMP4 is present in the basal culture medium at a concentration of about 50 μg / mL.

[0076] In some embodiments, the differentiation method provided herein includes culturing cells in a basal culture medium containing fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF), wherein the basal culture medium does not contain BMP4 or is essentially free of BMP4. In some embodiments, the method includes culturing cells in a basal culture medium containing FGF2 and VEGF for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, wherein the basal culture medium does not contain BMP4 or is essentially free of BMP4. In some embodiments, the culturing is performed on a collagen-coated (e.g., collagen type IV-coated) surface.

[0077] Some aspects of the differentiation methods provided herein involve culturing cells (e.g., PSCs or iPSCs) in a basal culture medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that functions as a downstream effector of Rho kinase and has three isoforms (RhoA, RhoB, and RhoC). A "ROCK inhibitor" can, for example, reduce the expression or 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 and dominant negative ROCK variants that target ROCK, siRNA, shRNA, miRNA, and antisense nucleic acids. Other examples of 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.

[0078] 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 including, for example, 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.

[0079] Some embodiments 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 specific serine and threonine amino acids of cellular substrates (e.g., glycogen synthase). This phosphorylation usually results in inhibition of the substrate. GSK3 is also involved in the control of the cellular response to damaged DNA, Wnt signaling, and the phosphorylation of Ci in the Hedgehog (Hh) pathway, where Ci is targeted for proteolysis to an inactive form.

[0080] 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, SB415286, and TDZD-8.Further exemplary GSK3 inhibitors include, but are not limited to, BIO (2’Z,3’E)-6-bromodidehydrovinblastine-3’-oxime (GSK3 inhibitor IX); BIO-acetoxime (2’Z,3’E)-6-bromoindirubin-3’-acetoxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopenta dienylruthenium complex (GSK3 inhibitor XV); TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3 beta inhibitor I); 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 beta inhibitor II); OTDZT 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (GSK3 beta inhibitor III); alpha-4-dibromoacetophenone (GSK3 beta inhibitor VII); AR-AO14418 N-(4-methoxybenzyl)-N’-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3 beta inhibitor VIII); 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK3 beta inhibitor XI); TWS119 - pyrrolopyrimidine compound (GSK3 beta inhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form (GSK3 beta inhibitor XIII); 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3 beta inhibitor VI); AR-AO144-18; SB216763; and SB415286. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0081] In some embodiments, a GSK3 inhibitor (e.g., CHIR99021) is present in the basal culture medium at a concentration of about 10 μM to about 60 μM, or, for example, about 10 μM to about 50 μM, about 10 μM to about 40 μM, about 10 μM to about 30 μM, about 10 μM to about 20 μM, about 20 μM to about 60 μM, about 20 μM to about 50 μM, about 20 μM to about 40 μM, about 20 μM to about 30 μM, about 30 μM to about 60 μM, about 30 μM to about 50 μM, about 30 μM to about 40 μM, about 40 μM to about 60 μM, about 40 μM to about 50 μM, or about 50 μM to about 60 μM, or any value or range of values including those therein. In some embodiments, a GSK3 inhibitor (e.g., CHIR99021) is present in the basal culture medium at a concentration of about 10 μM, about 20 μM, about 30 μM, about 35 μM, about 36 μM, about 40 μM, about 50 μM, or about 60 μM. In some embodiments, a GSK3 inhibitor (e.g., CHIR99021) is present in the basal culture medium at a concentration of about 36 μM.

[0082] In some embodiments, the differentiation method of the present disclosure comprises:

[0083] (i) culturing a PSC (e.g., iPSC) in a basal culture medium containing a ROCK inhibitor;

[0084] (ii) culturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor;

[0085] (iii) culturing the cells of (ii) in a basal culture medium containing FGF2, VEGF, and BMP4; and

[0086] (iv) culturing the cells of (iii) in a basal culture medium containing FGF2 and VEGF, wherein the culture medium does not contain or is essentially free of BMP4.

[0087] In some embodiments, the differentiation method of the present disclosure comprises:

[0088] (i) Culturing PSCs (e.g., iPSCs) in a basal culture medium containing a ROCK inhibitor;

[0089] (ii) Culturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor;

[0090] (iii) Culturing the cells of (ii) in a basal culture medium containing FGF2, VEGF, and BMP4;

[0091] (iv) Culturing the cells of (iii) in a basal culture medium containing FGF2 and VEGF, wherein the culture medium does not contain or is essentially free of BMP4; and

[0092] (v) Separating the cells of (iv) having CD144 expression to form endothelial cells.

[0093] In some embodiments, the culture of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culture of (i) and / or (ii) is for about 1 day. In some embodiments, the cells are passaged between (iii) and (iv).

[0094] In some embodiments, the method further comprises (vi) culturing the cells of (v) having CD144 expression in a basal culture medium containing a transforming growth factor β (TGFβ) inhibitor. In some embodiments, the culture of (vi) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culture of (vi) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0095] TGFβ is a highly pleiotropic cytokine that plays important roles in wound healing, angiogenesis, immune regulation, and cancer. TGFβ inhibitors include, but are not limited to, inhibitors of TGF signaling in general, or inhibitors specific for TGFβ receptors (e.g., ALK5), antibodies that suppress the expression of TGFβ receptors, dominant negative mutants, as well as siRNA and antisense nucleic acids. Examples of TGFβ inhibitors are SB431542, A-83-01 (also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388 (-4-[3-(pyridin-2-yl)-1H-pyrazol-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-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride), SU5416, Reldemizumab (CAT-152); Metelimumab (CAT-192); GC-1008; ID11; AP-12009, AP-11014; LY550410; LY580276; LY364947; LY2109761; SB-431542; SD-208; SM16; NPC-30345; ΚΙ26894; SB-203580; SD-093; ALX-270-448; EW-7195; SB-525334; ΓΝ-1233; SKI2162; Gleevec; 3,5,7,2’,4’-pentahydroxyflavone (morin); Activin-M108A; P144; soluble TBR2-Fc; and pyrimidine derivatives and indolinones reported by Roth et al., 2010, etc., but are not limited thereto. In some embodiments, the TGFβ inhibitor is SB431542.

[0096] 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 including, for example, 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.

[0097] In other embodiments, the differentiation method of the present disclosure includes the following:

[0098] (i) culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0099] (ii) culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0100] (iii) culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4; and

[0101] (iv) culturing the cells of (iii) on a collagen-coated surface in a basal culture medium containing FGF2 and VEGF, wherein the culture medium does not contain BMP4 or is essentially free of BMP4.

[0102] In some embodiments, the differentiation method of the present disclosure includes the following:

[0103] (i) culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0104] (ii) Culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0105] (iii) Culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4;

[0106] (iv) Culturing the cells of (iii) on a collagen-coated surface in a basal culture medium containing FGF2 and VEGF, where the culture medium does not contain or is essentially free of BMP4; and

[0107] (v) Separating the cells of (iv) having CD144 expression to form endothelial cells.

[0108] In some embodiments, the collagen is collagen IV. In some embodiments, the method further comprises (vi) culturing the cells of (v) having CD144 expression in a basal culture medium containing a transforming growth factor β (TGFβ) inhibitor. In some embodiments, the culture of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culture of (i) and / or (ii) is for about 1 day. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culture of (vi) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culture of (vi) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0109] In some embodiments, the differentiation method of the present disclosure comprises:

[0110] (i) Culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0111] (ii) Culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0112] (iii) Culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4 for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days; and

[0113] (iv) Culturing the cells of (iii) on a collagen-coated surface in a basal culture medium containing FGF2 and VEGF for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, wherein the culture medium does not contain BMP4 or is essentially free of BMP4.

[0114] In some embodiments, the differentiation method of the present disclosure comprises the following:

[0115] (i) Culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0116] (ii) Culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0117] (iii) Culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4 for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days;

[0118] (iv) Culturing the cells of (iii) on a collagen-coated surface in a basal culture medium containing FGF2 and VEGF for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, wherein the culture medium does not contain BMP4 or is essentially free of BMP4; and

[0119] (v) Separating the cells of (iv) having CD144 expression to form endothelial cells.

[0120] In some embodiments, the collagen is collagen IV. In some embodiments, the method further comprises culturing the cells of (v) having CD144 expression in a basal culture medium containing a TGFβ inhibitor. In some embodiments, the culture of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culture of (i) and / or (ii) is for about 1 day. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culture of (vi) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culture of (vi) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0121] In some embodiments, the differentiation method of the present disclosure comprises the following:

[0122] (i) Culturing PSCs (e.g., iPSCs) on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor;

[0123] (ii) Culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0124] (iii) Culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4 for about 4 days; and

[0125] (iv) Culturing the cells of (iii) on a collagen IV-coated surface in a basal culture medium containing FGF2 and VEGF for about 2 days. Here, the culture medium does not contain BMP4 or is essentially free of BMP4.

[0126] In some embodiments, the differentiation method of the present disclosure comprises the following:

[0127] (i) Culturing PSCs (e.g., iPSCs) on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor;

[0128] (ii) Culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0129] (iii) Culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4 for about 4 days;

[0130] (iv) Culturing the cells of (iii) on a collagen IV-coated surface in a basal culture medium containing FGF2 and VEGF for about 2 days, where the culture medium does not contain BMP4 or is essentially free of BMP4; and

[0131] (v) Separating the cells of (iv) having CD144 expression to form endothelial cells.

[0132] In some embodiments, the method further comprises (vi) culturing the cells of (v) having CD144 expression in a basal culture medium containing a TGFβ inhibitor. In some embodiments, the culturing of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culturing of (i) and / or (ii) is for about 1 day. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culturing of (vi) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culturing of (vi) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0133] In another aspect, the differentiation method of the present disclosure includes culturing cells having CD144 expression in a basal culture medium containing a TGFβ inhibitor. In some aspects, the culturing is performed on a collagen-coated surface. In some aspects, the culturing is performed on a surface coated with collagen I.

[0134] In some aspects, the differentiation method of the present disclosure includes the following:

[0135] (i) culturing a PSC (e.g., iPSC) in a basal culture medium containing a ROCK inhibitor;

[0136] (ii) culturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor;

[0137] (iii) culturing the cells of (ii) in a basal culture medium containing FGF2, VEGF, and BMP4; and

[0138] (iv) culturing the cells of (iii) in a basal culture medium containing a TGFβ inhibitor.

[0139] In some aspects, the differentiation method of the present disclosure includes the following:

[0140] (i) culturing a PSC (e.g., iPSC) in a basal culture medium containing a ROCK inhibitor;

[0141] (ii) culturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor;

[0142] (iii) culturing the cells of (ii) in a basal culture medium containing FGF2, VEGF, and BMP4;

[0143] (iv) separating the cells of (iii) having CD144 expression; and

[0144] (v) culturing the cells of (iv) having CD144 expression in a basal culture medium containing a TGFβ inhibitor.

[0145] In some embodiments, the culturing of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culturing of (i) and / or (ii) is for about 1 day. In some embodiments, the culturing of (iii) is for about 1 day to about 10 days, or about 4 days to about 6 days. In some embodiments, the culturing of (iii) is for about 4 days, about 5 days, or about 6 days. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culturing of (v) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culturing of (v) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0146] In some embodiments, the differentiation method of the present disclosure includes the following:

[0147] (i) Culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0148] (ii) Culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0149] (iii) Culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4; and

[0150] (iv) Culturing the cells of (iii) on a collagen-coated surface in a basal culture medium containing a TGFβ inhibitor.

[0151] In some embodiments, the differentiation method of the present disclosure includes the following:

[0152] (i) Culturing PSCs (e.g., iPSCs) on a collagen-coated surface in a basal culture medium containing a ROCK inhibitor;

[0153] (ii) Culturing the cells of (i) on a collagen-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0154] (iii) Culturing the cells of (ii) on a collagen-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4;

[0155] (iv) Separating the cells of (iii) having CD144 expression; and

[0156] (v) Culturing the cells of (iv) having CD144 expression on a collagen-coated surface in a basal culture medium containing a TGFβ inhibitor.

[0157] In some embodiments, the collagen is collagen I or collagen IV. In some embodiments, the collagen of (i) is collagen IV. In some embodiments, the collagen of (ii) is collagen IV. In some embodiments, the collagen of (iii) is collagen IV. In some embodiments, the collagen of (v) is collagen I. In some embodiments, the culturing of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culturing of (i) and / or (ii) is for about 1 day. In some embodiments, the culturing of (iii) is for about 1 day to about 10 days, or about 4 days to about 6 days. In some embodiments, the culturing of (iii) is for about 4 days, about 5 days, or about 6 days. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culturing of (v) is for about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some embodiments, the culturing of (v) is for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0158] In some embodiments, the differentiation method of the present disclosure includes the following:

[0159] (i) Culturing PSCs (e.g., iPSCs) on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor;

[0160] (ii) Culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0161] (iii) Culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4; and

[0162] (iv) Culturing the cells of (iii) on a collagen I-coated surface in a basal culture medium containing a TGFβ inhibitor.

[0163] In some embodiments, the differentiation method of the present disclosure includes the following:

[0164] (i) Culturing PSCs (e.g., iPSCs) on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor;

[0165] (ii) Culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor;

[0166] (iii) Culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4;

[0167] (iv) Separating the cells of (iv) having CD144 expression; and

[0168] (v) Culturing the cells of (v) having CD144 expression on a collagen I-coated surface in a basal culture medium containing a TGFβ inhibitor.

[0169] In some embodiments, the culture of (i) and / or (ii) is for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In some embodiments, the culture of (i) and / or (ii) is about 1 day. In some embodiments, the culture of (iii) is from about 1 day to about 10 days, or from about 4 days to about 6 days. In some embodiments, the culture of (iii) is about 4 days, about 5 days, or about 6 days. In some embodiments, the cells are passaged between (iii) and (iv). In some embodiments, the culture of (v) is from about 3 days to about 9 days, from about 4 days to about 8 days, or from about 5 days to about 7 days. In some embodiments, the culture of (v) is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0170] III. Other Embodiments The present disclosure also relates to endothelial cells produced by any of the differentiation methods disclosed herein.

[0171] The present disclosure also relates to organoids comprising endothelial cells produced by any of the differentiation methods disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated three-dimensional (3D) cellular organism that self-organizes by aggregating cells in a controlled space and is derived from pluripotent stem cells (e.g., iPSCs). Such organisms can be made to reproduce much of the complexity of an organ or to represent selected aspects of an organ, for example, by generating only specific types of cells. In some embodiments, the organoid is a vascular graft.

[0172] Methods for maintaining differentiated endothelial cells and organoids are well known and include culturing the cells or organoids in the cell culture media described herein and / or cryopreserving them. Methods for producing organoids typically include culturing cells in a three-dimensional (3D) matrix under standard cell culture conditions. 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 natural cell line generation matrices such as Matrigel®, and combinations thereof, such as in the form of a microparticle-filled hydrogel.

[0173] The present disclosure also relates to a method for promoting angiogenesis or vascular development, which includes administering endothelial cells or organoids produced by any of the differentiation methods disclosed herein.

[0174] The present disclosure also relates to a method for treating vasculitis or angiopathy, which includes administering endothelial cells or organoids produced by any of the differentiation methods disclosed herein.

[0175] The present disclosure also relates to a method for treating cardiovascular diseases, which includes administering endothelial cells or organoids produced by any of the differentiation methods disclosed herein. In some embodiments, the cardiovascular disease is coronary artery disease (CAD), arrhythmia, heart failure, heart valve disease, pericardial disease, cardiomyopathy (heart muscle disease), or congenital heart disease.

[0176] The present disclosure also relates to specific differentiation media.

[0177] In some embodiments, the differentiation medium of the present disclosure comprises a basal culture medium, FGF2, VEGF, and BMP4. In some embodiments, the differentiation medium comprises a basal culture medium, from about 20 μg / mL to about 100 μg / mL of FGF2, from about 20 μg / mL to about 100 μg / mL of VEGF, and from about 20 μg / mL to about 100 μg / mL of BMP4. In some embodiments, the differentiation medium comprises a basal culture medium, about 50 μg / mL of FGF, about 50 μg / mL of VEGF, and about 50 μg / mL of BMP4.

[0178] In some embodiments, the differentiation medium of the present disclosure comprises a basal culture medium, FGF2, and VEGF, and the medium does not contain BMP4 or is essentially free of BMP4. In some embodiments, the differentiation medium comprises a basal culture medium, from about 20 μg / mL to about 100 μg / mL of FGF2, and from about 20 μg / mL to about 100 μg / mL of VEGF. In some embodiments, the differentiation medium comprises a basal culture medium, about 50 μg / mL of FGF, and about 50 μg / mL of VEGF.

Examples

[0179] Now, with reference to the above description, refer to the following examples that non - limitatively illustrate some embodiments of the present invention.

[0180] Example 1 Differentiation of Endothelial Cells An experiment was conducted to differentiate endothelial cells from human induced pluripotent stem cells (iPSCs) using the following protocol.

[0181] The following reagents were used:

[0182] Iscove's Modified Dulbecco's Medium (IMDM): Gibco, 12440 - 053

[0183] MEM Non - Essential Amino Acids: Gibco, 11140 - 050

[0184] L - Glutamine: Gibco, 25030 - 081

[0185] Monothioglycerol: Sigma, M1753

[0186] Penicillin / Streptomycin: Gibco, 15140-122

[0187] BIT9500 Serum Substitute: Stem Cell® Technologies, 09500

[0188] Recombinant Human Bone Morphogenetic Protein 4 (BMP4): PeproTech®, 120-05et

[0189] Human Fibroblast Growth Factor (FGF)-Basic: PeproTech®, 100-18b

[0190] Human Vascular Endothelial Growth Factor 165 (VEGF165): PeproTech®, 100-20

[0191] Human Plasma Fibronectin: Life Technologies / Invitrogen, 33016015

[0192] Collagen IV, Mouse: BD Biosciences, 354233 (aliquoted and stored at -70°C)

[0193] mTeSR®1: STEMCELL® Technologies, 05850

[0194] Accutase®: STEMCELL® Technologies, 07920

[0195] Rho-associated Coiled-coil-containing Protein Kinase (ROCK) Inhibitor (Y-27632): Fisher Scientific, 688000

[0196] TrypLE (registered trademark) Express (1x), without phenol red: Life Technologies / Invitrogen, 12604-013

[0197] Human CD31 PeWm59: BD Biosciences, 555446

[0198] CHIR99021 / glycogen synthase kinase (GSK)-3β inhibitor: Stemgent (registered trademark), 04-0004-02

[0199] iPSCs were obtained from the Harvard Stem Cell Institute and maintained in a medium prepared by mixing approximately 400 mL of mTeSR (registered trademark) 1 basal medium and approximately 100 mL of mTeSR (registered trademark) 1 5X supplement. The medium was stored at -20 °C in 4 mL aliquots before use and thawed at room temperature before use. The iPSCs were passaged with Accutase (registered trademark) and seeded at a 1:x ratio every x days onto 10 cm 2 dishes coated with Geltrex (registered trademark).

[0200] Collagen IV (ColIV) was thawed at 4 °C and vigorously vortexed for 10 - 15 seconds to redissolve. Then, ColIV was diluted with ice-cold filtered 0.05 N hydrochloric acid (HCl) to a final stock concentration of 20 μg / ml ColIV in 0.05 N HCl. Approximately 1.5 mL of the diluted ColIV solution was then added to each well of a 6-well plate. The plate was then incubated at 37 °C for approximately 2 hours and washed three times with sterile Dulbecco's phosphate-buffered saline (DPBS) immediately before seeding.

[0201] iPSCs were split by adding approximately 1.5 mL of Accutase (registered trademark) to each well and incubating at 37 °C for 5 - 7 minutes. After centrifugation, the iPSC cell pellet was resuspended by adding 5 mL of warm mTeSR1 (registered trademark) medium containing 10 μM Y-27632 to create a single cell suspension. Then, the iPSCs were seeded at 20,000 cells / cm 2They were seeded onto ColIV-coated plates. The total volume of each well of the 6-well plate was approximately 2 mL. The cells were then incubated at 37°C, 4% O2, and 5% CO2. An exemplary image of the cells at this stage is shown in Fig. 3A.

[0202] Next, a series of differentiation experiments were performed (Fig. 1). Plate A was grown under hypoxic conditions and differentiated in a medium containing VEGF, BMP4, and FGF2 for 6 days. Plate B was grown under normoxic conditions for the first 24 hours and then differentiated in a medium containing VEGF, BMP4, and FGF2 for 7 days. Plate C was grown under hypoxic conditions and cultured in a medium containing VEGF, BMP4, and FGF2 for 4 days. The cells were then split and grown in a medium containing VEGF, BMP4, and FGF2 or a medium containing VEGF and FGF2 for 3 days. Plate D was grown under normoxic conditions for the first 24 hours and then grown under hypoxic conditions and in a medium containing VEGF, BMP4, and FGF2 for 4 days. The cells were then split and grown in a medium containing VEGF, BMP4, and FGF2 or a medium containing VEGF and FGF2 for 3 days. Under all conditions, single-cell seeding and 24-hour exposure to CHIR99021 were performed. Human pulmonary artery endothelial cells (HPAEC) were used as a positive control.

[0203] Even when the cells were split on day 4, based on standard EC markers (CD31 / CD144), there was no adverse effect on the differentiation of iPSCs into endothelial cells (EC). However, when split on day 4, an unexpectedly slightly matured EC phenotype of CD73 / CD105 was generated, and the cell viability was increased during harvesting and flow staining. The results of flow staining are shown in Fig. 2, and a summary of cell viability and yield is shown in Table 1. Furthermore, when the cells were split on day 4, the cell number increased by at least 3-fold without sacrificing %CD144 for downstream magnetic separation. Based on this data, the conditions of Plate C were used for the next experiment.

Table 1

[0204] Day 1 of differentiation from iPSC to EC One day after seeding iPSCs on collagen IV (ColIV)-coated plates, mTeSR1® containing 36 μM CHIR99021 was added to the existing medium in each well to a final concentration of 12 μM. The cells were then incubated at 37 °C, 4% O2, and 5% CO2. During and after differentiation, whole-well imaging of the cells was performed using the Incucyte® live-cell analysis system.

[0205] Days 2 - 5 of differentiation from iPSC to EC The cell medium was replenished daily using EC medium containing basal differentiation medium (BD medium), recombinant human bone morphogenetic protein 4 (BMP4), human basic fibroblast growth factor (bFGF), and human vascular endothelial growth factor 165 (VEGF165). BD medium consisted of 400 mL of IMDM, 100 mL of BIT9500 serum replacement, 5 mL of non-essential amino acids (Thermo Fisher Scientific 11-140-050), 450 μM of monothioglycerol, 2 mM of GlutaMAX® (Thermo Fisher Scientific), and 100 μg / mL of Primocin® (InvivoGen). The cells were cultured in an incubator at 37 °C, 4% O2, 5% CO2. Exemplary images of the cells at this stage are shown in FIGS. 3B and 3C.

[0206] ECs are characterized by the expression of surface protein markers platelet endothelial cell adhesion molecule (PECAM / CD31) and vascular endothelial cadherin (VEcadherin / CD144). ECs also express von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (Flk-1 / VEGFR-2 / KDR), vascular endothelial growth factor receptor 1 (Flt-1 / VEGFR1), and endothelial nitric oxide synthase (eNOS). In vitro functions of ECs include uptake of low-density lipoprotein (LDL) and lectin binding.

[0207] Day 6 of differentiation from iPSC to EC - Preparation of cells for CD144+ magnetic selection Using this protocol, the success of EC differentiation from iPSCs was determined by the selection of CD144+ cells. Cells were harvested and washed three times with DPBS. An appropriate amount of TrypLE™ was added and the cells were incubated at room temperature for 5 - 7 minutes. The detached cells were collected and pelleted at 200 x g for 5 minutes. The cells were then counted and used for flow cytometry analysis. Approximately 4E6 cells were transferred to a 15 mL centrifuge and flow cytometry analysis of endothelial protein expression (CD144, CD31), mesenchymal progenitor cells (CD140b), and stem cell marker expression (SSEA4) was performed. The results of this analysis are shown in FIGS. 4A - 4C and FIGS. 5A - 5F.

[0208] 250,000 cells per flow cytometry tube / sample were used for analysis. The remaining cells were centrifuged at 200 x g for 5 minutes. The cells were then resuspended in sterile filtered running buffer containing PBS (Gibco), 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Gibco), and 0.5% bovine serum albumin (BSA) (Miltenyi catalog number 130 - 091 - 376) at a ratio of 10E7 cells / 80 μL. Next, CD144 microbeads (Miltenyi catalog number 130 - 097 - 857) were added to the running buffer suspension at a ratio of 20 μL per 10E7 cells, mixed, and incubated at 4°C for 15 minutes. The cells were then washed with 2 mL of running buffer per 10E7 cells and pelleted at 300 x g for 5 minutes. The pellet was then resuspended in running buffer at a ratio of 25E6 cells / mL. The cells were then run on a Miltenyi Biotec AutoMACS™ Pro system and CD144+ cells were selected according to the Miltenyi AutoMACS™ Pro system CD144 microbead protocol. The results of this analysis are shown in FIGS. 6A - 6B.

[0209] Days 7 to 9 / 10 of iPSC-EC differentiation: From step E4 to step P0 The medium was carefully removed and warm EC growth medium [EGM2 medium (Lonza CC-3162), 10 μM SB431542 (Reprocell 4001010), 100 μg / mL Primocin (registered trademark) (InvivoGen ant-pm-2), and supplemented with 16.2% FBS (VWR 76294-180)] was added. The cells were cultured in a normoxic incubator at 37 °C, 20% O2, 5% CO2. The medium was removed and replaced approximately every 48 hours.

[0210] Day 9 / 10 of iPSC-EC differentiation: Collection of iPSC-ECP0 cells Approximately 24 hours after the previous medium change, the cells were first rinsed three times with DPBS and then harvested. Accutase (registered trademark) was added and the cells were incubated at 37 °C for 5 minutes. Once the cells were detached, they were transferred to a centrifuge tube. The cell culture vessel was rinsed twice with BD medium and this was added to the centrifuge tube.

[0211] Next, the medium containing the cells was filtered through a 30 μM filter, the cells were pelleted by centrifugation at 200 x g for 5 minutes with maximum acceleration and maximum deceleration at 8 °C. The cell pellet was resuspended in 5 mL of EC growth medium. Then, the total cell number, cells / ml, and viability were measured for use in quality control tests.

[0212] The cells were imaged every 12 hours from day 0 and were demonstrated to take on a cobblestone-like morphology at final differentiation. Exemplary images of the cells on days 9, 13, and 17 are shown in Figures 3D - 3F.

[0213] The differentiated cells were further characterized in a tube formation assay using the following protocol. The test differentiated cells were co-cultured with or without 1 μM imatinib mesylate to inhibit angiogenesis, and tube formation was observed. Specifically, the wells of a 96-well plate (Costar, product number 387) were coated with 30 μL of Matrigel® Growth Factor Reduced (GFR) basement membrane matrix (Corning®). The plate was rotated at 1,500 revolutions per minute (RPM) for 1 minute and incubated at 30 °C for 30 minutes. The cells were then removed from the culture plate, counted, and resuspended in Dulbecco's Modified Eagle Medium (DMEM) / F12 medium such that 30 μL of medium was available per well. 30,000 test differentiated cells were placed in each well. In the tube formation assay, after gelation, 30 μL of the cell suspension was carefully placed on top. The plate was loaded into an Incucyte® live cell analysis system and images were taken every hour. Exemplary images of the resulting differentiated cells are shown in FIGS. 7A-7B. The differentiated cells were reorganized into tubular structures resembling angiogenesis sprouts, confirming their angiogenic capacity.

[0214] The differentiated cells were further characterized using an acetylated low-density lipoprotein (Ac-LDL) assay. This assay is based on the principle that "scavenger" receptors on endothelial cells can bind to and take up Ac-LDL. The test differentiated cells were harvested, counted, and seeded at 5,000 - 10,000 cells per well in a 96-well plate coated with collagen I. Human umbilical vein endothelial cells (HUVEC) and fibroblasts were used as positive and negative controls, respectively. The cells were then incubated overnight at 37°C. The medium was replaced with 100 μL (+ / -) 10 μg / mL Ac-LDL and a 1:2000 dilution of the nuclear stain NucLight Rapid Red (non-disruptive, cell-permeable). After incubating the cells at 37°C, they were washed with PBS. The cells were then imaged every 30 minutes at 20x magnification with an Incucyte® S3. Exemplary images of the resulting differentiated cells are shown in FIGS. 8A - 8B. The data suggest that iPSC-derived ECs (CD144+ BJRIP38) internalize Ac-LDL via receptor-mediated endocytosis and exhibit significant Ac-LDL turnover.

[0215] 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 to the present invention. To the extent that section headings are used, they should not necessarily be construed as limiting.

Claims

1. A method for differentiating pluripotent stem cells into endothelial cells, comprising: (i) culturing pluripotent stem cells on a collagen IV-coated surface in a basal culture medium containing a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a glycogen synthase kinase 3 (GSK3) inhibitor; (iii) culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 4 (BMP4) for about 4 days; (iv) culturing the cells of (iii) on a collagen IV-coated surface for about 2 days in a basal culture medium containing FGF2 and VEGF, wherein the culture medium does not contain BMP4 or is essentially free of BMP4; and (v) isolating the cells of (iv) having CD144 expression to form endothelial cells A method comprising the above steps.

2. (vi) culturing the cells of (v) having CD144 expression in a basal culture medium containing a transforming growth factor β (TGFβ) inhibitor The method according to claim 1, further comprising the above step.

3. A method for differentiating pluripotent stem cells into endothelial cells, comprising: (i) culturing pluripotent stem cells on a collagen IV-coated surface in a basal culture medium containing a ROCK inhibitor; (ii) culturing the cells of (i) on a collagen IV-coated surface in a basal culture medium containing a GSK3 inhibitor; (iii) culturing the cells of (ii) on a collagen IV-coated surface in a basal culture medium containing FGF2, VEGF, and BMP4; (iv) isolating the cells of (iii) having CD144 expression; and (v) culturing the cells of (iv) having CD144 expression on a collagen I-coated surface in a basal culture medium containing a TGFβ inhibitor to form endothelial cells A method comprising the above steps.

4. The method according to any one of claims 1 to 3, wherein the ROCK inhibitor is Y-27632.

5. The method according to claim 4, wherein Y-27632 is present in the culture medium at a concentration of about 10 μM.

6. The method according to any one of claims 1 to 5, wherein the culture in (i) is for about 1 day.

7. The method according to any one of claims 1 to 6, wherein the GSK3 inhibitor is CHIR99021.

8. The method according to claim 7, wherein CHIR99021 is present in the culture medium at a concentration of about 36 μM.

9. The method according to any one of claims 1 to 8, wherein the culture in (ii) is for about 1 day.

10. The method according to any one of claims 1 to 9, wherein FGF is present in the culture medium at a concentration of about 50 μg / mL.

11. The method according to any one of claims 1 to 10, wherein VEGF is present in the culture medium at a concentration of about 50 μg / mL.

12. The method according to any one of claims 1 to 11, wherein BMP4 is present in the culture medium at a concentration of about 50 μg / mL.

13. The method according to claim 1, wherein the cells are passaged between (iii) and (iv).

14. The method according to claim 3, wherein the culture in (iii) is for about 4 days to about 6 days.

15. The method according to any one of claims 1 to 14, wherein the TGFβ inhibitor is SB431542.

16. The method according to claim 15, wherein SB431542 is present in the culture medium at a concentration of about 10 μM.

17. The method according to claim 1, wherein the culture in (vi) is for about 6 days.

18. The method according to claim 3, wherein the culture in (v) is for about 6 days.

19. The method according to any one of claims 1 to 18, wherein the separation is by immunomagnetic cell separation.

20. The method according to any one of claims 1 to 19, wherein the culture in (i) and / or (ii) is performed under hypoxic conditions.

21. The method according to any one of claims 1 to 20, wherein the PSC is an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), an embryonic germ cell, or an adult stem cell.

22. An endothelial cell produced by the method according to any one of claims 1 to 21.

23. An organoid comprising the endothelial cell according to claim 22.

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