Method for differentiating endothelial cells

Abstract

The present disclosure relates to methods of differentiating pluripotent stem cells into endothelial cells. The present disclosure also relates to endothelial cells prepared by such methods, organoids comprising such endothelial cells, and methods of use thereof. The present disclosure also relates to differential media for use therein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 390,445, filed on July 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates to methods for differentiating pluripotent stem cells into endothelial cells. The present disclosure also relates to endothelial cells prepared by such methods, organoids comprising such endothelial cells, and methods of using the same. The present disclosure also relates to differentiation medium for use in the above. Background Art

[0003] Pluripotent stem cells (PSCs) are undifferentiated or partially differentiated cells that can differentiate into a variety of other cell types. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells derived from adult somatic cells that are genetically reprogrammed to an embryonic stem cell (ESC)-like state by expressing genes and factors that are important for maintaining the most typical characteristics of embryonic stem cells (ESCs). iPSCs have recently attracted attention in the medical community because they solve many of the obstacles associated with the use of embryonic stem cells and can generate patient-specific PSCs that can be genetically corrected, differentiated into adult cell lineages, 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 being used for genetic diseases, iPSCs can also be used for tissue regeneration and disease modeling. Kogut et al., Methods Mol. Biol. 1195: 1-12 (2014). PSCs and iPSCs can be differentiated into many different cell types, including endothelial cells (EC). 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 endothelial cells (ECs).

[0005] In some aspects, the method comprises: (i) culturing pluripotent stem cells (e.g., iPSCs) on a surface coated with collagen IV in a basal medium containing a Rho-associated coiled-coil protein kinase (ROCK) inhibitor; (ii) culturing the cells in (i) on a surface coated with collagen IV in a basal medium containing a glycogen synthase kinase 3 (GSK3) inhibitor; (iii) culturing the cells in (ii) on a surface coated with collagen IV in a basal 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 in (iii) on a surface coated with collagen IV in a basal medium containing FGF2 and VEGF for about 2 days, wherein the medium does not contain BMP4; and (v) isolating the cells in (iv) having CD144 expression to form endothelial cells.

[0006] In some aspects, the method further comprises: (vi) culturing the cells expressing CD144 described in (v) in a basal medium comprising a transforming growth factor β (TGFβ) inhibitor.

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

[0008] In some aspects, the ROCK inhibitor is Y-27632. In some aspects, Y-27632 is present in the culture medium at a concentration of about 10 μM.

[0009] In some aspects, the culturing in (i) is for about 1 day.

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

[0011] In some aspects, the culturing in (ii) is for about 1 day.

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

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

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

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

[0016] In some aspects, the culturing in (iii) is from about 4 days to about 6 days.

[0017] In some aspects, the TGFβ inhibitor is SB 431542. In some aspects, SB 431542 is present in the culture medium at a concentration of about 10 μM.

[0018] In some aspects, the culturing in (vi) is for about 6 days. In some aspects, the culturing in (v) is for about 6 days.

[0019] In some aspects, the separation in (iv) is performed by immunomagnetic cell separation. In some aspects, the separation in (v) is performed by immunomagnetic cell separation.

[0020] In some aspects, the culturing in (i) and / or (ii) is performed under hypoxic conditions.

[0021] The present disclosure also provides endothelial cells prepared by the differentiation methods disclosed herein, organoids comprising the endothelial cells disclosed herein, and certain methods of use thereof.

[0022] The present disclosure also provides differentiation medium for use in the above. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Some aspects of the present invention are described herein by way of example only and with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that the details shown are by way of example only and for the purpose of illustrative discussion of various aspects of the present invention.

[0024] Figure 1 is a schematic diagram of the differentiation process described in Example 1.

[0025] Figure 2 Flow cytometry data of BJRiPS-EC differentiation described in Example 1 are shown.

[0026] Figure 3 A is an exemplary whole-cell image of day 0 of the differentiation protocol described in Example 1.

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

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

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

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

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

[0032] Figure 4A The progression toward an endothelial cell (EC) phenotype through the SSEA4 flow cytometry results at days 6, 10, 13, 19, and 26 of the differentiation protocol described in Example 1 is shown.

[0033] Figure 4B Progression toward an endothelial cell phenotype by CD140-flow cytometry results at day 6, day 10, day 13, day 19, and day 26 of the differentiation protocol described in Example 1 is shown.

[0034] Figure 4C Progression toward an endothelial cell phenotype by CD90-flow cytometry results at days 6, 10, 13, 19, and 26 of the differentiation protocol described in Example 1 is shown.

[0035] Figure 5 A-5C shows BJRiP (iPSC; Figure 5 A), human pulmonary artery endothelial cells (HPAEC, primary EC; Figure 5 B) and test differentiated cells (iPS-EC, day 13; Figure 5 C) Flow cytometry results of CD140B and CD90 surface markers.

[0036] Figure 5 D-5F shows BJRiP (iPSC; Figure 5 D), HPAEC (primary EC; Figure 5 E ) and test differentiated cells (iPS-EC, day 13; Figure 5 F) Flow cytometry results of CD31 and SSEA-4 surface markers.

[0037] Figure 6 A-6B shows the CD31 ( Figure 6 A) and SSEA-4( Figure 6 B) Expression distribution of surface markers.

[0038] Figure 7 A-7B shows the test differentiated cells of Example 1 ( Figure 7 A) and fibroblasts ( Figure 7 B) Tube formation results.

[0039] Figure 8 A-8B shows the test differentiated cells of Example 1 ( Figure 8 A) and fibroblasts ( Figure 8 B) Exemplary image of Ac-LDL assay results. DETAILED DESCRIPTION I. General Definitions

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions therein) shall prevail. Unless otherwise required by the context, terms in the singular shall include the plural form, and terms in the plural form shall include the singular form. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, just as each individual publication or patent application is expressly 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. These 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 be apparent from the detailed description and claims.

[0042] In order to further clarify the present disclosure, the following terms and definitions are provided.

[0043] The singular forms "a" and "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" are used interchangeably herein. In some aspects, the terms "a" or "an" mean "single". In other aspects, the terms "a" or "an" include "two or more" or "plurality".

[0044] The term "about" as used herein means approximately, roughly, approximately, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to modify a numerical value by floating it within a range of 10% (higher or lower) above or below a numerical value.

[0045] Throughout the disclosure of the present invention, various aspects of the present invention are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of a range should be deemed to have specifically disclosed all possible sub-ranges and single values ​​within the range. For example, a description such as a range of 1 to 6 should be deemed to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this rule applies. The listed numerical ranges include the numbers that define the range and cover each integer within the defined range.

[0046] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. When a numerical range is listed, it should be understood that each intermediate integer value between the upper and lower limits listed in the range and each fractional value thereof, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and any range that includes the upper limit, the lower limit, both or neither is also included in the disclosure of the present invention. Therefore, the ranges listed herein should be understood to be a shorthand representation of all numerical values ​​in the range, including the endpoint values ​​listed. For example, a range of 1 to 10 should be understood to include any number, combination of numbers or subrange in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0047] When a numerical value is explicitly listed, it should be understood that the numerical value of the quantity or amount approximately the same as the listed numerical value is also within the scope of the present disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. On the contrary, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of the invention disclosure is disclosed as having multiple alternatives, examples of the invention disclosure in which each alternative is excluded individually or in any combination with other alternatives are also disclosed; more than one element of the invention disclosure can have such exclusions, and all combinations of elements with such exclusions are also disclosed herein.

[0048] As used herein, the term "and / or" should be considered as a specific disclosure of the presence or absence of the other of the two specified features or components. Thus, the term "and / or" as used in phrases such as "A and / or B" 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 cover the following: 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 should be understood that wherever herein aspects are described with the language "comprising," similar aspects described with "consisting of" and / or "consisting essentially of" are also provided. II. Differentiation Methods

[0050] The present disclosure relates to methods for differentiating pluripotent stem cells (PSCs, such as iPSCs) into endothelial cells (ECs). Such methods achieve, for example, improved endothelial cell viability, yield, and / or differentiation properties.

[0051] As used herein, the term "differentiation" and "differentiating" refers to the process of inducing or reprogramming young or immature cells (e.g., pluripotent stem cells) into more mature or specialized cells (e.g., endothelial cells). In general, differentiation of pluripotent stem cells can be achieved, for example, by changing the culture conditions of the cells (such as changing the stimulatory agent in the culture medium or the physical state of the cells).

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

[0053] In some aspects, 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 aspects, PSCs are from humans. In some aspects, PSCs are from animals. In some aspects, the animals are sheep, pigs, or primates.

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

[0055] As used herein, the term "endothelial cell" or "endothelial cells" or "EC" refers to a cell or a group of cells that form a monolayer of cells that line the inner wall of blood vessels and regulate the exchange of blood flow with surrounding tissues. As used herein, endothelial cells include mature endothelial cells, endothelial progenitor cells, and endothelial precursor cells.

[0056] Endothelial cells generated by the differentiation methods 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, endostatin (Endocan, ESM-1), Endoglyx-1, endothelial mucin (Endomucin), Eotaxin-3, EPAS1, coagulation factor VIII-related antigen, FLI-1, Flk-1 (KDR, VEGFR-2), FLT- 1 (VEGFR-1), GATA2, GBP-1, GRO-α, 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 (such as ClaudiN5 or ZO-1).

[0057] Functional characteristics of endothelial cells include, but are not limited to, the ability to take up acetylated low-density lipoprotein (ac-LDL); to have a barrier function; and 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] Morphological characteristics of endothelial cells include, but are not limited to, the ability to form tubular structures in a three-dimensional matrix, having a flattened (or squamous) appearance, and possessing 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 aspects, the differentiation methods provided herein include certain cell culture conditions, such as culturing cells in certain culture media.

[0061] As used herein, the terms "cell culture", "cell culturing", "culture", "culturing" 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 "culture media" refer to a composition for culturing cells that contains nutrients that maintain cell viability, support proliferation and, optionally, differentiation. A cell culture medium may contain one or more of the following: one or more salts, one or more buffers, one or more amino acids, glucose or other sugars, one or more antibiotics, serum or serum substitutes, and other components such as growth factors, vitamins, and the like.

[0062] In some aspects, the differentiation methods provided herein refer to a cell culture medium (sometimes referred to as "differentiation medium" or "differentiation medium") as a "basal medium" supplemented with other components. As used herein, "basal culture medium" or "basal culture medium" refers to a composition containing the most basic elements required for cells to maintain, grow and / or differentiate in an in vitro environment. Examples of basal culture media include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), MEM, Iskoff's modified Dulbecco's medium (IMDM), Glasgow modified MEM (GMEM), DMEM / F12, Leibovitz L-15, RPMI-1640, CMRL, Ham F10, and Ham F12. In some aspects, the basal medium is supplemented with one or more other components, such as one or more amino acids, one or more antibiotics, serum, one or more growth factors. Such components are well known in the art and are further described herein.

[0063] In some aspects, the cell culture medium or basal medium in the differentiation method provided herein is "substantially free of" a certain component or "does not contain" a certain component. As used herein, the term "substantially free of" refers to a culture medium that is at least 95% free, 96% free, 97% free, 98% free, 99% free or 100% free of a certain component, or has an undetectable amount of a certain component, as measured by methods known in the art and further described herein. The terms "do not comprise" and "does not comprise" refer to a culture medium that does not contain a certain component, or has an undetectable amount of a certain component, as measured by methods known in the art and further described herein. In some aspects, the culture medium does not contain or is substantially free of bone morphogenetic protein 4 (BMP4).

[0064] In some aspects, the differentiation methods of the present disclosure include culturing cells under "normoxic" or "normal oxygen" conditions. Normoxic conditions generally include culturing cells in vitro at oxygen levels in air of about 15%-20%.

[0065] In other aspects, the differentiation methods of the present disclosure include culturing cells under "hypoxic" or "hypoxia" conditions. Hypoxic conditions generally include culturing cells in vitro at oxygen levels of about 10% or less, about 5% or less, or about 1% or less, depending on the cell type. Hypoxic conditions can be created and maintained by using a culture device (e.g., an anaerobic culture chamber) that can control the concentration of ambient gases. Unless specifically indicated as hypoxic conditions, the culture conditions in the present disclosure can be considered to be under normoxic conditions. In some aspects, the cells in the present disclosure can be cultured in normoxic conditions using methods known in the art and further described herein, and then cultured under hypoxic conditions, and vice versa.

[0066] In some aspects, the differentiation methods provided herein include certain cell culture conditions, such as passage of cells in certain culture media. As used herein, the terms "passage," "passaged," and "passaging" refer to the act of subdividing cells at lower concentrations and inoculating them into one or more cell culture surfaces or containers when the cells have proliferated to a desired degree. Passaging generally involves mechanical or enzymatic separation (e.g., at a certain cell density) prior to inoculation. Methods for passaging cells are well known and further described herein.

[0067] In some aspects, the culture and passage in the differentiation methods provided herein are carried out under the condition of coating one or more substrates on the cell culture surface or container. 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, matrix mixtures produced by naturally occurring cell lines (such as Matrigel TM ) and synthetic or artificial surfaces (e.g., polyamine monolayers and carboxyl-terminated monolayers) or combinations thereof. Methods for coating substrates onto cell culture surfaces or containers are well known and are further described herein.

[0068] In some aspects, the differentiation methods provided herein include one or more steps of separating cultured cells with certain biochemical characteristics. As used herein, the terms "separated" and "separating" refer to the process of separating one or more specific cell populations from a heterogeneous mixture of cells. In some aspects, the differentiation methods provided herein include separating cells expressing vascular endothelial (VE)-cadherin (CD144), for example, to form endothelial cell (EC) cultures. As used herein, the term "CD144 expression" includes but is not limited to detectable CD144 expression, CD144 expression comparable to mature endothelial cells or their precursor cells or progenitor cells, or CD144 expression higher than CD144 expression in control cells that do not express CD144 and / or are not endothelial cells.

[0069] Cell separation methods based on a certain biochemical feature 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 separation, microfluidic cell sorting, buoyancy activated cell sorting, aptamer-based cell separation, complement depletion, etc. Affinity separation techniques include, but are not limited to, separation using antibody-coated magnetic beads (e.g., immunomagnetic cell separation), affinity chromatography, cytotoxic agents (e.g., complement and cytotoxins) connected to or used in conjunction with monoclonal antibodies, and "panning" or other convenient techniques using antibodies attached to solid matrices (e.g., culture plates). In some aspects, the cells of the differentiation method of the present invention are separated by immunomagnetic cell separation.

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

[0071] In some aspects, FGF is present in the basal medium at a concentration of about 20 μg / mL to about 100 μg / mL (or any value or range of values ​​thereof), 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 ...70 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 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about In some aspects, FGF is present in basal 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, FGF is present in basal medium at a concentration of about 50 μg / mL.

[0072] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing vascular endothelial growth factor (VEGF). VEGF is a signaling protein that promotes the growth of new blood vessels. VEGF is part of the mechanism for restoring blood supply to cells and tissues when they are deprived of oxygen due to obstructed blood circulation.

[0073] In some aspects, VEGF is present in the basal medium at a concentration of about 20 μg / mL to about 100 μg / mL (or any value or range of values ​​thereof), 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 ... In some aspects, VEGF is present in 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, VEGF is present in a concentration of about 50 μg / mL in the basal medium. In some aspects, VEGF is present in a concentration of about 50 μg / mL in the basal medium.

[0074] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing bone morphogenetic protein 4 (BMP4). In other aspects, the basal medium does not contain or is substantially free of BMP4. BMP4 stimulates the differentiation of overlying ectodermal tissues and is known to stimulate bone formation in adult animals.

[0075] In some aspects, BMP4 is present in the basal medium at a concentration of about 20 μg / mL to about 100 μg / mL (or any value or range of values ​​thereof), 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 ... In some aspects, BMP4 is present in basal medium with 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, BMP4 is present in basal medium with a concentration of about 50 μg / mL.

[0076] In some respects, the differentiation method provided herein is included in the step of culturing cells in a basal medium containing fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF), wherein the basal medium does not include or is substantially free of BMP4. In some respects, the method is included in the step of culturing cells in a basal 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 medium does not include or is substantially free of BMP4. In some respects, culturing is carried out on a surface coated with collagen (e.g., coated with collagen IV).

[0077] Some aspects of the differentiation methods provided herein include culturing cells (e.g., PSC or iPSC) in a basal medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that acts as a downstream effector of Rho kinase, of which there are three subtypes (RhoA, RhoB, and RhoC). A "ROCK inhibitor" can, for example, reduce ROCK expression and / or ROCK activity. 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, dominant negative ROCK variants, siRNA, shRNA, miRNA, and antisense nucleic acids targeting ROCK. Other examples of ROCK inhibitors include, but are not limited to, thiazovivin, Y-27632, Fasudil, AR122-86, Y-30141, WF-536, HA-1077, hydroxy-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)-cyclohexanecarboxylic acid amide, and ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety. In some aspects, the ROCK inhibitor is Y-27632.

[0078] In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM to about 20 μM (or any value or range thereof) in a basal medium, for example, including 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 aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM, about 5 μM, about 10 μM, about 15 μM or about 20 μM in a basal medium. In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 10 μM in a basal medium.

[0079] Some aspects of the differentiation methods provided herein include culturing cells in a basal 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 cell substrates (e.g., glycogen synthase). This phosphorylation typically results in inhibition of the substrate. GSK3 is also involved in controlling the response of cells to damaged DNA, Wnt signaling, and the phosphorylation of Ci in the Hedgehog (Hh) pathway, targeting it for proteolysis to an inactive form.

[0080] As used herein, "GSK3 inhibitor" refers to a compound that inhibits one or more GSK3 enzymes. The GSK3 enzyme family is well known, and many 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. Other exemplary GSK3 inhibitors include, but are not limited to, BIO(2'Z,3'E)-6-bromoindirubin-3'-oxime (GSK3 inhibitor IX); BIO-acetyloxime (2'Z,3'E)-6-bromoindirubin-3'-acetyloxime (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-AO14418 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); TWS119-pyrrolopyrimidine compound (GSK3β inhibitor XII); L803 H-KEAPP APPQSpP-NH2 or its myristoylated form (GSK3β inhibitor XIII); 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β inhibitor VI); AR-AO144-18; SB216763; and SB415286. In some aspects, the GSK3 inhibitor is CHIR99021.

[0081] In some aspects, the GSK3 inhibitor (e.g., CHIR99021) is present in the basal medium at a concentration of about 10 μM to about 60 μM (or any value or range of values ​​thereof), including, 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. In some aspects, the GSK3 inhibitor (e.g., CHIR99021) is present in 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 the basal medium. In some aspects, the GSK3 inhibitor (e.g., CHIR99021) is present in a concentration of about 36 μM in the basal medium.

[0082] In some aspects, the differentiation methods of the present disclosure include:

[0083] (i) culturing PSCs (eg, iPSCs) in a basal medium comprising a ROCK inhibitor;

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

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

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

[0087] In some aspects, the differentiation methods of the present disclosure include:

[0088] (i) culturing PSCs (eg, iPSCs) in a basal medium comprising a ROCK inhibitor;

[0089] (ii) culturing the cells in (i) in a basal medium comprising a GSK3 inhibitor;

[0090] (iii) culturing the cells in (ii) in a basal medium comprising FGF2, VEGF and BMP4;

[0091] (iv) culturing the cells of (iii) in a basal medium comprising FGF2 and VEGF, wherein the medium does not comprise or is substantially free of BMP4; and

[0092] (v) isolating the cells expressing CD144 in (iv) to form endothelial cells.

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

[0094] In some aspects, the method further comprises (vi) culturing the cells having CD144 expression in (v) in a basal medium containing a transforming growth factor β (TGFβ) inhibitor. In some aspects, the culturing in (vi) is about 3 days to about 9 days, about 4 days to about 8 days, or about 5 days to about 7 days. In some aspects, the culturing in (vi) is 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 an important role in wound healing, angiogenesis, immunoregulation, and cancer. TGFβ inhibitors include, but are not limited to, general TGF signaling inhibitors, or inhibitors specific for TGFβ receptors (e.g., ALK5), which may include antibodies against TGFβ receptors, dominant negative variants against TGFβ receptors, and siRNA and antisense nucleic acids that inhibit TGFβ receptor expression. Examples of TGFβ inhibitors include, but are not limited to, SB431542, A-83-01 (also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-methanthamide), 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-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-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl SU5416, lerdelimumab (CAT-152), metelimumab (CAT-192), GC-1008, ID11, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761, and SB-431542 ; SD-208; SM16; NPC-30345; KI26894; SB-203580; SD-093; ALX-270-448; EW-7195; SB-525334; IN-1233; SKI2162; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (Morin); Activin-M108A; P144; soluble TBR2-Fc and pyrimidine derivatives and indolinone compounds reported in Roth et al., 2010. In some aspects, the TGFβ inhibitor is SB431542.

[0096] In some aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 1 μM to about 20 μM (or any value or range thereof) in a basal medium, for example, including 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 aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 1 μM, about 5 μM, about 10 μM, or about 20 μM in a basal medium. In some aspects, TGFβ inhibitor (e.g., SB431542) is present in a concentration of about 10 μM in a basal medium.

[0097] In other aspects, the differentiation method of the present disclosure comprises:

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

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

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

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

[0102] In some aspects, the differentiation methods of the present disclosure include:

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

[1104] (ii) culturing the cells in (i) on a collagen-coated surface in a basal medium comprising a GSK3 inhibitor;

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

[0106] (iv) culturing the cells of (iii) on the collagen-coated surface in a basal medium comprising FGF2 and VEGF, wherein the medium does not comprise or is substantially free of BMP4; and

[0107] (v) isolating the cells expressing CD144 in (iv) to form endothelial cells.

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

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

[0111] (ii) culturing the cells in (i) on a collagen-coated surface in a basal medium comprising a GSK3 inhibitor;

[0112] (iii) culturing the cells in (ii) on a collagen-coated surface in a basal medium comprising 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 in (iii) on a collagen-coated surface in a basal medium comprising FGF2 and VEGF for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, wherein the medium does not comprise or is substantially free of BMP4.

[0114] In some aspects, the differentiation methods of the present disclosure include:

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

[0116] (ii) culturing the cells in (i) on a collagen-coated surface in a basal medium comprising a GSK3 inhibitor;

[0117] (iii) culturing the cells in (ii) on a collagen-coated surface in a basal medium comprising 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 in (iii) on the collagen-coated surface in a basal medium comprising FGF2 and VEGF for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, wherein the medium does not comprise or is substantially free of BMP4; and

[0119] (v) isolating the cells expressing CD144 in (iv) to form endothelial cells.

[0120] In some aspects, the collagen is collagen IV. In some aspects, the method further includes (vi) culturing cells with CD144 expression in (v) in a basal medium comprising a TGFβ inhibitor. In some aspects, the culture in (i) and / or (ii) is about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In some aspects, the culture in (i) and / or (ii) is about 1 day. In some aspects, cells are passaged between (iii) and (iv). In some aspects, cells are passaged between (iii) and (iv). In some aspects, the culture in (vi) is about 3 days to about 9 days, about 4 days to about 8 days or about 5 days to about 7 days. In some aspects, the culture in (vi) is 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 aspects, the differentiation methods of the present disclosure include:

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

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

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

[0125] (iv) culturing the cells in (iii) on the collagen IV coated surface in a basal medium comprising FGF2 and VEGF for about 2 days, wherein the medium does not comprise or is substantially free of BMP4.

[0126] In some aspects, the differentiation methods of the present disclosure include:

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

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

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

[0130] (iv) culturing the cells in (iii) on the collagen IV coated surface in a basal medium comprising FGF2 and VEGF for about 2 days, wherein the medium does not comprise or is substantially free of BMP4; and

[0131] (v) isolating the cells expressing CD144 in (iv) to form endothelial cells.

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

[0133] On the other hand, the differentiation method of the present disclosure includes the step of culturing cells with CD144 expression in a basal medium comprising a TGFβ inhibitor. In some aspects, the culturing is on a surface coated with collagen. In some aspects, the culturing is on a surface coated with collagen I.

[0134] In some aspects, the differentiation methods of the present disclosure include:

[0135] (i) culturing PSCs (eg, iPSCs) in a basal medium comprising a ROCK inhibitor;

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

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

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

[0139] In some aspects, the differentiation methods of the present disclosure include:

[0140] (i) culturing PSCs (eg, iPSCs) in a basal medium comprising a ROCK inhibitor;

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

[1142] (iii) culturing the cells in (ii) in a basal medium comprising FGF2, VEGF and BMP4;

[0143] (iv) isolating the cells expressing CD144 in (iii); and

[0144] (v) culturing the cells expressing CD144 in (iv) in a basal medium containing a TGFβ inhibitor.

[0145] In some aspects, the culture in (i) and / or (ii) is about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In some aspects, the culture in (i) and / or (ii) is about 1 day. In some aspects, the culture in (iii) is about 1 day to about 10 days or about 4 days to about 6 days. In some aspects, the culture in (iii) is about 4 days, about 5 days or about 6 days. In some aspects, the cell is passaged between (iii) and (iv). In some aspects, the culture in (v) is about 3 days to about 9 days, about 4 days to about 8 days or about 5 days to about 7 days. In some aspects, the culture in (v) is 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 aspects, the differentiation methods of the present disclosure include:

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

[0148] (ii) culturing the cells in (i) on a collagen-coated surface in a basal medium comprising a GSK3 inhibitor;

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

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

[0151] In some aspects, the differentiation methods of the present disclosure include:

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

[0153] (ii) culturing the cells in (i) on a collagen-coated surface in a basal medium comprising a GSK3 inhibitor;

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

[0155] (iv) isolating the cells expressing CD144 in (iii); and

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

[0157] In some aspects, the collagen is collagen I or collagen IV. In some aspects, the collagen in (i) is collagen IV. In some aspects, the collagen in (ii) is collagen IV. In some aspects, the collagen in (iii) is collagen IV. In some aspects, the collagen in (v) is collagen I. In some aspects, the culture in (i) and / or (ii) is about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In some aspects, the culture in (i) and / or (ii) is about 1 day. In some aspects, the culture duration in (iii) is about 1 day to about 10 days or about 4 days to about 6 days. In some aspects, the culture duration in (iii) is about 4 days, about 5 days or about 6 days. In some aspects, cells are passaged between (iii) and (iv). In some aspects, the culture in (v) is about 3 days to about 9 days, about 4 days to about 8 days or about 5 days to about 7 days. In some aspects, the culturing in (v) is 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 aspects, the differentiation methods of the present disclosure include:

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

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

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

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

[0163] In some aspects, the differentiation methods of the present disclosure include:

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

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

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

[0167] (iv) isolating the cells expressing CD144 in (iv); and

[0168] (v) Cells expressing CD144 in (v) were cultured on a collagen I-coated surface in basal medium containing a TGFβ inhibitor.

[0169] In some aspects, the culture in (i) and / or (ii) is about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In some aspects, the culture in (i) and / or (ii) is about 1 day. In some aspects, the culture in (iii) is about 1 day to about 10 days or about 4 days to about 6 days. In some aspects, the culture in (iii) is about 4 days, about 5 days or about 6 days. In some aspects, the cell is passaged between (iii) and (iv). In some aspects, the culture in (v) is about 3 days to about 9 days, about 4 days to about 8 days or about 5 days to about 7 days. In some aspects, the culture in (v) is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days or about 9 days. III. Other aspects

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

[0171] The present disclosure also relates to an organoid comprising endothelial cells prepared by any differentiation method disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated three-dimensional (3D) cell structure derived from pluripotent stem cells (e.g., iPSCs), which is self-organized by dense aggregation of cells in a controlled space. Such structures can be constructed to replicate most of the complexity of an organ, or to express specific aspects thereof, for example, only producing certain types of cells. In some aspects, the organoid is a vascular graft.

[0172] Methods for maintaining differentiated endothelial cells and organoids are well known, including culturing the cells or organoids in a cell culture medium as described herein and / or cryopreservation. Methods for preparing organoids generally 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, a microparticle, a fibrous network, a matrix mixture produced by a naturally occurring cell line (e.g., Matrigel TM ) and combinations thereof, such as microparticle-loaded hydrogels.

[0173] The present disclosure also relates to methods of promoting neovascularization or vascular development comprising administering endothelial cells or organoids prepared by any of the differentiation methods disclosed herein.

[0174] The present disclosure also relates to methods of treating vasculitis or vascular disease comprising administering endothelial cells or organoids prepared by any of the differentiation methods disclosed herein.

[0175] The present disclosure also relates to a method for treating cardiovascular disease, the method comprising administering endothelial cells or organoids prepared by any differentiation method disclosed herein. In some aspects, the cardiovascular disease is coronary artery disease (CAD), arrhythmia, heart failure, valvular heart disease, pericardial disease, cardiomyopathy (myocardial disease) or congenital heart disease.

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

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

[0178] In some aspects, the differentiation medium of the present disclosure comprises a basal medium, FGF2 and VEGF, wherein the medium does not comprise or is substantially free of BMP4. In some aspects, the differentiation medium comprises a basal medium, about 20 μg / mL to about 100 μg / mL FGF2 and about 20 μg / mL to about 100 μg / mL VEGF. In some aspects, the differentiation medium comprises a basal medium, about 50 μg / mL FGF2 and about 50 μg / mL VEGF. Example

[0179] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Example 1 Endothelial cell differentiation

[0180] An experiment was performed to differentiate human induced pluripotent stem cells (iPSCs) into endothelial cells using the following protocol.

[0181] The following reagents were used:

[0182] Iskoff'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] Pen / Strep: Gibco, 15140 -122.

[0187] BIT9500 Serum Replacement: StemCell TM Technologies, 09500.

[0188] Recombinant human bone morphogenetic protein 4 (BMP4): 120-05et.

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

[0190] Human vascular endothelial growth factor 165 (VEGF165): 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 TM 1: STEMCELL TM Technologies, 05850.

[0194] Accutase TM :STEMCELL TM Technologies, 07920.

[0195] Rho-associated coiled-coil kinase (ROCK) inhibitor (Y-27632): Fisher Scientific, 688000.

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

[0197] Human CD31 Pe Wm59: BD Biosciences, 555446.

[0198] CHIR99021 / Glycogen synthase kinase (GSK)-3β inhibitor: Stemgent TM , 04-0004-02.

[0199] iPSCs were obtained from the Harvard Stem Cell Institute and maintained in approximately 400 mL of mTeSR TM 1 basal medium and about 100 mL of mTeSR TM15X supplement set. The medium was stored in 4 mL aliquots at -20°C and thawed at room temperature before use. iPSCs were grown using Accutase TM Subculture, inoculate to the coating at a ratio of 1:x every x days 10cm 2 On the culture dish.

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

[0201] By adding approximately 1.5 mL of Accutase TM The iPSCs were isolated by incubation at 37°C for 5-7 minutes. After centrifugation, 5 mL of warm mTeSR1 containing 10 μM Y-27632 was added. TM Resuspend the iPSC pellet in culture medium to form a single-cell suspension. Then, iPSCs were plated at 20,000 cells / cm 2 The total volume of each well in the 6-well plate was approximately 2 mL. The cells were then incubated at 37°C, 4% O2 and 5% CO2. Example images of cells at this stage are shown in Figure 3 As shown in A.

[0202] Then a series of differentiation experiments were performed ( Figure 1). Plate A was grown under hypoxia and differentiated for 6 days in medium containing VEGF, BMP4, and FGF2. Plate B was first cultured under normoxia for 24 hours and then differentiated for 7 days in medium containing VEGF, BMP4, and FGF2. Plate C was grown under hypoxia and cultured for 4 days in medium containing VEGF, BMP4, and FGF2. The cells were then separated and cultured for 3 days in medium containing VEGF, BMP4, and FGF2 or medium containing VEGF and FGF2. Plate D was first grown under normoxia for 24 hours and then grown for 4 days in hypoxia and medium containing VEGF, BMP4, and FGF2. The cells were then separated and grown for 3 days in medium containing VEGF, BMP4, and FGF2 or medium containing VEGF and FGF2. All conditions underwent single cell seeding and 24 hours of CHIR99021 exposure. Human pulmonary artery endothelial cells (HPAEC) were used as a positive control.

[0203] Based on typical EC markers (CD31 / CD144), isolating cells at day 4 did not negatively affect the differentiation of iPSCs into endothelial cells (EC). However, isolation at day 4 unexpectedly resulted in a slightly more mature CD73 / CD105 EC phenotype and higher cell viability at the time of harvest and flow staining. The results of flow staining are shown in Figure 2. Figure 2 A summary of cell viability and yield is shown in Table 1. In addition, isolating cells on day 4 resulted in at least a 3-fold increase in cell number without sacrificing %CD144 during downstream magnetic separation. Based on these data, the culture conditions of plate C were used for subsequent experiments. Table 1: Cell viability and productivity sample % Survival Yield at harvest Tablet A 78.15％ 4.50E+06 cells Tablet B 81.70％ 4.86E+06 cells Flat C-BMP4 93.75％ 16E+06 cells Plate C - No BMP4 93.90％ 14.7E+06 cells Flat D-BMP4 91.65％ 13.9E+06 cells Plate D - No BMP4 89.15％ 13.1E+06 cells Day 1 of iPSC to EC differentiation

[0204] One day after iPSCs were seeded onto collagen IV (Col IV)-coated culture plates, mTeSR1 containing 36 μM CHIR99021 was added to the existing culture medium in each well. TM to a final concentration of 12 μM. The cells were then incubated at 37°C, 4% O2, and 5% CO2. Live cell analysis system images cells throughout the well. Day 2-5 of iPSC differentiation into EC

[0205] The cell culture medium was refilled daily with 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 IMDM, 100 mL BIT9500 serum replacement, 5 mL non-essential amino acids (Thermo Fisher Scientific, 11-140-050), 450 μM monothioglycerol, 2 mM GlutaMAX TM (Thermo Fisher Scientific) and 100 μg / mL (InvivoGen). The cells were cultured in an incubator at 37°C, 4% O2 and 5% CO2. An exemplary image of the cells at this stage is shown in Figure 3 B and Figure 3 As shown in C.

[0206] ECs are characterized by the expression of the surface protein markers platelet endothelial cell adhesion molecule (PECAM / CD31) and vascular endothelial cadherin (VE-Cadherin / 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). EC functional abilities in vitro include uptake of low-density lipoprotein (LDL) and lectin binding. Day 6 of iPSC to EC Differentiation - Preparation of Cells for CD144 + Magnetic selection

[0207] Select CD144 + The cells were used to determine the success of differentiation from iPSC to EC using this protocol. The cells were collected and rinsed 3 times with DPBS. An appropriate volume of TrypLE TM , and then incubate the cells at room temperature for 5-7 minutes. The detached cells were collected and pelleted at 200xg for 5 minutes. The cells were then counted and used for flow cytometric analysis. Approximately 4E6 cells were transferred to a 15 mL centrifuge tube for flow cytometric analysis of endothelial protein expression (CD144, CD31), mesenchymal progenitor cells (CD140b), and stem cell marker expression (SSEA4). The results of this analysis are shown in Figures 4A-4C as well as Figure 5 In A-5F.

[0208] 250,000 cells were used for analysis per flow cytometry tube / sample. The remaining cells were centrifuged at 200xg for 5 minutes. The cells were then resuspended in running buffer at 10E7 cells / 80 μL of 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 cat#130-091-376)). CD144 microbeads (Miltenyi cat#130-097-857) were then added and 10×10 7 20 μL of each cell suspension was mixed with the running buffer and then incubated at 4°C for 15 minutes. 7 The cells were washed with 2 mL of running buffer per 10 cells and pelleted at 300 x g for 5 minutes. The pellet was resuspended in running buffer at 25E6 cells / mL. The cells were then centrifuged at Miltenyi Biotec Pro system based on Miltenyi Biotec Pro System CD144 Bead Program Run for Selection of CD144 + The results of this analysis are shown in Figure 6 A-6B. Day 7-9 / 10 of iPSC-EC differentiation: E4 step to P0 step

[0209] Carefully remove the culture medium and add warm EC expansion medium [EGM2 medium (Lonza CC-3162) supplemented with 10 μM SB431542 (Reprocell 4001010), 100 μg / mL (InvivoGen ant-pm-2) and 16.2% FBS (VWR, 76294-180)]. The cells were cultured in a normoxic incubator at 37°C, 20% O2 and 5% CO2. The culture medium was removed and replaced approximately every 48 hours. Day 9 / 10 of iPSC-EC differentiation: Harvesting of iPSC-EC P0 cells

[0210] Approximately 24 hours after the previous media change, cells were harvested by first washing the cells 3 times with DPBS. Accutase TM , and incubate the cells at 37°C for 5 minutes. After detachment, the cells were transferred to a centrifuge tube. The cell culture container was rinsed twice with BD medium, and BD medium was added to the centrifuge tube.

[0211] The medium containing the cells was then filtered using a 30 μM filter and centrifuged at 200 x g for 5 minutes at 8°C, maximum acceleration and deceleration to pellet the cells. The cell pellet was resuspended in 5 mL of EC expansion medium. Total cell counts, cells / ml, and percent viability were then determined for quality control testing.

[0212] Cells were imaged every 12 hours starting at day 0 and were shown to have a cobblestone morphology upon terminal differentiation. Exemplary images of cells at day 9, day 13, and day 17 are shown in Figure 3 In D-3F.

[0213] The differentiated cells were further characterized using a tube formation assay using the following protocol. Test differentiated cells were co-cultured with or without 1 μM imatinib mesylate to inhibit angiogenesis and then observed for tube formation. Growth factor reduced (GFR) basement membrane matrix The wells of a 96-well plate (Costar, Product No. 387) were coated. The culture plate was rotated at 1500 revolutions per minute (RPM) for 1 minute and then incubated at 30°C for 30 minutes. The cells were then removed from the culture plate, counted and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) / F12 medium so that 30 μL of medium was available per well. Each well received 30,000 cells to be tested for differentiation. For the tube formation assay, after gelation, 30 μL of the cell suspension was carefully placed on top. The culture plate was placed In the live cell analysis system, images were taken every hour. An exemplary image of the resulting differentiated cells is shown in FIG. Figure 7 As shown in A-7B, the differentiated cells were found to rearrange into tubular structures resembling angiogenic sprouts, and the angiogenic ability was confirmed.

[0214] The differentiated cells were further characterized using the acetylated low-density lipoprotein (AC-LDL) assay. This assay is based on the principle that "scavenger" receptors on endothelial cells can bind and take up Ac-LDL. The test differentiated cells were collected, 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 culture medium was replaced with 100μL (+ / -) 10μg / mL Ac-LDL and a 1:2000 dilution of the nuclear stain NucLight Fast Red (non-interfering, membrane-permeable). The cells were incubated at 37°C and subsequently washed with PBS. Then Cells were imaged every 30 minutes at 20X magnification on S3. Exemplary images of the resulting differentiated cells are shown in Figure 8 A-8B. The data showed that iPSC-derived ECs (CD144 + BJRIP38) internalizes Ac-LDL via receptor-mediated endocytosis and has significant Ac-LDL uptake.

[0215] All publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was expressly and individually indicated to be incorporated herein by reference. In addition, the citation or identification of any reference in this application should not be construed as an admission that such reference constitutes prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily 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 medium containing a Rho-associated coiled-coil kinase (ROCK) inhibitor; (ii) culturing the cells in (i) on a collagen IV-coated surface in a basal medium containing a glycogen synthase kinase 3 (GSK3) inhibitor; (iii) culturing the cells in (ii) on a collagen IV-coated surface in a basal medium comprising fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP4) for about 4 days; (iv) culturing the cells in (iii) on the collagen IV coated surface in a basal medium comprising FGF2 and VEGF for about 2 days, wherein the medium does not comprise or is substantially free of BMP4; and (v) isolating the cells expressing CD144 in (iv) to form endothelial cells.

2. The method of claim 1, further comprising: (vi) culturing the cells expressing CD144 described in (v) in a basal medium containing a transforming growth factor β (TGFβ) inhibitor.

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 medium containing a ROCK inhibitor; (ii) culturing the cells in (i) on a collagen IV-coated surface in a basal medium containing a GSK3 inhibitor; (iii) culturing the cells in (ii) on a collagen IV-coated surface in a basal medium comprising FGF2, VEGF and BMP4; (iv) isolating the cells expressing CD144 in (iii); and (v) culturing the cells expressing CD144 described in (iv) on a collagen I-coated surface in a basal medium containing a TGFβ inhibitor to form endothelial cells.

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

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

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

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

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

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

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

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

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

13. The method of claim 1, wherein the cells are passaged between (iii) and (IV).

14. The method of claim 3, wherein the culturing in (iii) is from about 4 days to about 6 days.

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

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

17. The method of claim 1, wherein the culturing in (vi) is for about 6 days.

18. The method of claim 3, wherein the culturing in (v) is for about 6 days.

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

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

21. The method of any one of claims 1-20, wherein the pluripotent stem cell (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 prepared by the method according to any one of claims 1 to 21.

23. An organoid comprising the endothelial cells of claim 22.

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