Methods and compositions for generating endothelial cells from pluripotent stem cells

Abstract

Provided is a method for generating CD31+VE-cadherin+KDR+ endothelial cells (ECs) using a chemically defined medium that enables the generation of ECs from early mesoderm progenitor cells in only 3 days and enables the generation of ECs from pluripotent stem cells in only 5 days. Also provided are a medium, an isolated cell population, and a kit.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 391,208, filed on July 21, 2022. The entire content of U.S. Provisional Patent Application No. 63 / 391,208 is incorporated herein by reference.

Background Art

[0002] Background of the Invention Endothelial cells (ECs) are the main type of cells found lining blood vessels, lymphatic vessels, and the heart. Damage to or loss of blood vessels is a major pathophysiological feature of vascular diseases, including ischemic cardiovascular diseases. Despite significant efforts, treatment of such diseases remains difficult. Given the important role of ECs in pathophysiology, an approach focused on ECs or growing blood vessels is biologically reasonable. Thus, the ability to generate endothelial cells from stem cells, such as pluripotent stem cells, is important for both vascular research and vascular regeneration. For example, readily available stem cell-derived ECs would enable the potential use of stem cell-derived ECs in the induction of therapeutic angiogenesis, enhancement of appropriate blood perfusion, or promotion of tissue repair (see, e.g., Losordo et al. (2004) Circulation 109:2692-2697 (Non-Patent Document 1)). For an overview of ECs derived from pluripotent stem cells, see, e.g., Jang et al. (2019) Am. J. Pathol. 189:502-512 (Non-Patent Document 2). Furthermore, iPSC-derived endothelial cells can be used for drug discovery aimed at identifying new anti-angiogenic drugs or safety evaluation in terms of drug-induced vascular toxicity.

[0003] In the earliest approaches to generating ECs from pluripotent stem cells, three different approaches were devised (as outlined, for example, in Wilson et al. (2014) Stem Cells 32:3037-3045 (Non-Patent Document 3); Yoder (2015) Curr. Opin. Hematol. 22:252-257 (Non-Patent Document 4); Williams and Wu (2019) Arter. Thromb. Vasc. Biol. 39:1317-1329 (Non-Patent Document 5)). In the first approach, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are grown under conditions that induce self-aggregation of these cells into embryoid bodies (EBs), and then EC differentiation by the EBs is promoted using various growth factors. In the second approach, differentiating ESCs or iPSCs are co-cultured with a feeder layer, such as stromal cells (e.g., mouse cranial crown mesenchymal OP9 cells or bone marrow stromal cells), to promote differentiation along the EC lineage. In the third approach, ESCs or iPSCs are grown in two-dimensional culture on plates coated with a protein support, such as Matrigel, fibronectin, vitronectin, gelatin, or a similar protein, under specific conditions with the addition of growth factors.

[0004] A chemically-defined culture protocol for generating ECs from human pluripotent stem cells, involving the regulation of classical Wnt signaling, is described (see, e.g., Lian et al. (2014) Stem Cell Reports 3:804-816 (Non-Patent Document 6); Bao et al. (2015) Stem Cell Res. 15:122-129 (Non-Patent Document 7)). For example, albumin-free culture conditions have been reported, in which pluripotent stem cells are cultured in DMEM basal medium supplemented with ascorbic acid and treated with a GSK-3β inhibitor (CHIR99012) that activates Wnt signaling. This protocol has been shown to be sufficient to generate CD34+CD31+ endothelial progenitor cells, but the cell yield was low (e.g., 20-30% CD34+CD31+ cells before sorting).

[0005] Furthermore, the MAPK and PI3K pathways have been linked to the differentiation of ECs from PSCs, and current culture protocols involving treatment with VEGF, FGF2, and BMP4 after GSK-3β inhibitor treatment have been reported. These growth factors are used to induce early vascular progenitor cells from hiPSC-derived mesodermal cells, thereby generating CD31+VE-cadherin+ ECs in 8 days (Harding et al. (2017) Stem Cells 35:909-919 (Non-Patent Document 8)). Cyclic AMP has been reported to enhance the effect of VEGF (Ikuno et al. (2017) PLoS One 12:e0173271 (Non-Patent Document 9)). Subsequently, a chemically-defined protocol also utilized GSK-3β inhibitor treatment and a combination of culture conditions with the addition of VEGF, FGF2, and BMP4, along with DAPT and forskolin (Farkas et al. (2020) Front. Cell. Devel. Biol. 8:309 (Non-Patent Document 10)).

[0006] Thus, although some progress has been made, there is still a need for efficient and robust methods and compositions for generating endothelial cells from pluripotent stem cells in large-scale culture.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

SUMMARY OF THE INVENTION

[0008] The present disclosure provides a method of generating endothelial cells (ECs) using a chemically defined medium that enables robust generation of CD31+ ECs from pluripotent stem cells in as little as 5 days of culture. The medium contains small molecule agonists that stimulate or attenuate specific signaling pathways to promote differentiation along the endothelial cell lineage. The methods of the present disclosure have the advantage of enabling robust endothelial cell generation in a short period of time using reagents that are less expensive than current protocols, including the use of bioreactors, and having a high % endothelial conversion (80 - 90%). Further culturing the resulting ECs in an endothelial cell medium results in cells that exhibit endothelial cell function as measured by a tube formation assay, ac-LDL assay, or nitric oxide production assay. Additionally, the use of small molecule agonists in the medium allows for precise control of the culture components.

[0009] The methods and compositions of the present disclosure utilize a unique cell culture medium that enables the generation of CD31+ endothelial cells from early mesoderm progenitor cells in as little as 3 days. This unique cell culture medium contains certain components previously used for endothelial cell generation, such as VEGFR agonists and FGFR agonists, but also includes novel components and combinations that enable robust endothelial cell generation. Early mesoderm progenitor cells can be obtained by culturing pluripotent stem cells under culture conditions that give rise to early mesoderm progenitor cells, as described herein. Thus, in certain embodiments, the method comprises a one-step culture protocol that begins with early mesoderm progenitor cells and results in CD31+ endothelial cells in 3 days. In other embodiments, the method comprises a two-step culture protocol that begins with differentiating pluripotent stem cells into early mesoderm progenitor cells for 2 days in step 1, and then further differentiating the early mesoderm progenitor cells into CD31+ endothelial cells for 3 days in step 2, resulting in a two-step 5-day protocol.

[0010] Accordingly, in one aspect, the present disclosure relates to a method for generating human CD31+ endothelial cells (ECs), the method comprising culturing human early mesoderm progenitor cells in a medium comprising a VEGFR agonist, an FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist to generate the human CD31+ ECs.

[0011] In one embodiment, the early mesoderm progenitor cells are cultured in the medium for 3 days to obtain CD31+ endothelial cells.

[0012] In one embodiment, the early mesoderm progenitor cells are obtained by culturing human pluripotent stem cells in a medium containing a Wnt pathway agonist for 2 days.

[0013] In another aspect, the present disclosure relates to a two-step method for generating human CD31+ endothelial cells (ECs), (a) culturing human pluripotent stem cells in a medium containing a Wnt pathway agonist from day 0 to day 2 to generate early mesoderm progenitor cells, and (b) culturing the early mesoderm progenitor cells in a medium containing a VEGFR agonist, an FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist from day 2 to day 5 to generate CD31+ ECs comprising.

[0014] In certain embodiments, the CD31+ ECs produced by the methods of the present disclosure also express at least one marker selected from the group consisting of KDR, vWF, CD34, FL1, VE-cadherin, and CD31. In certain embodiments, the CD31+ ECs express at least two, at least three, at least four, at least five, or all six markers selected from the group consisting of KDR, vWF, FL1, VE-cadherin, CD34, and CD31.

[0015] In one aspect, the Wnt pathway agonist used in the medium for generating initial mesoderm progenitor cells is a GSK-3β inhibitor. In one aspect, the GSK-3β inhibitor is CHIR99021. In one aspect, CHIR99021 is present in the culture at a concentration in the range of 3.0 to 9.0 μM. In one aspect, CHIR99021 is present in the culture at a concentration of 6.0 μM.

[0016] In one aspect, the VEGFR agonist is VEGF. In one aspect, VEGF is present in the culture at a concentration in the range of 10 to 50 ng / ml. In one aspect, VEGF is present in the culture at a concentration of 25 ng / ml.

[0017] In one aspect, the FGFR agonist is FGF2 or SUN11602. In one aspect, the FGFR agonist is present in the culture at a concentration in the range of 1 to 20 ng / ml. In one aspect, the FGFR agonist is FGF2, and FGF2 is present in the culture at a concentration in the range of 1 to 20 ng / ml. In one aspect, the FGFR agonist is FGF2, and FGF2 is present in the culture at a concentration of 10 ng / ml.

[0018] In one aspect, the retinoic acid (RA) pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA), AY 9944 dihydrochloride, ciliobrevin A, cyclopamine, or combinations thereof. In one aspect, the RA pathway agonist is present in the culture at a concentration in the range of 10 to 100 nM. In one aspect, the RA pathway agonist is TTNPB, and TTNPB is present in the culture at a concentration in the range of 25 to 75 nM. In one aspect, the RA pathway agonist is TTNPB, and TTNPB is present in the culture at a concentration of 50 nM.

[0019] In one aspect, the Sonic Hedgehog (SHH) antagonist is selected from the group consisting of Sant-1, cyclopsaponin B1, itraconazole, GANT61, MK4101, HPI-4, bistramide A, jervine, JK184, taladegib, ciliobrevin D, dinapyrazole A, dynarrestin, GANT58, HPI1, IHR1, PF 04449913 maleate, SANT-2, U 18666A, and combinations thereof. In one aspect, the SHH antagonist is present in the culture at a concentration in the range of 10 - 100 nM. In one aspect, the SHH antagonist is Sant-1, and Sant-1 is present in the culture at a concentration in the range of 25 - 75 nM. In one aspect, the SHH antagonist is Sant-1, and Sant-1 is present in the culture at a concentration of 50 nM.

[0020] In one aspect, the heparin or heparin mimetic is selected from the group consisting of heparin, heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof. In one aspect, the heparin or heparin mimetic is present in the culture at a concentration in the range of 10 - 50 ng / ml. In one aspect, heparin is present in the culture at a concentration in the range of 25 - 35 ng / ml. In one aspect, heparin is present in the culture at a concentration of 20 ng / ml.

[0021] In one aspect, the Wnt pathway antagonist is selected from the group consisting of XAV939, ICG-001 (Hosseinib), cabozantinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, tripterinide, KYA1797K, JW55, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, iCRT14, WIF-1, DKK1, isocitrate, lanatoside C, Gigantol, RCM-1, WIKI4, IQ-1, Adavivant, PRI-724, Tegatrabetan, or a combination thereof. In one aspect, the Wnt pathway antagonist is present in the culture at a concentration in the range of 10 to 500 nM. In one aspect, the Wnt pathway antagonist is XAV939, and XAV939 is present in the culture at a concentration in the range of 50 to 150 nM. In one aspect, the Wnt pathway antagonist is XAV939, and XAV939 is present in the culture at a concentration of 100 nM.

[0022] In one aspect, the pluripotent stem cells are embryonic stem cells. In one aspect, the pluripotent stem cells are induced pluripotent stem cells.

[0023] In another aspect, the present disclosure relates to a medium for generating endothelial cells (ECs) comprising a VEGFR agonist, an FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist. Suitable agonists and antagonists and concentration ranges are described herein. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the RA pathway agonist is TTNBP, the SHH antagonist is Sant-1, the heparin or heparin mimetic is heparin, and the WNT pathway antagonist is XAV939. In one embodiment, VEGF is at a concentration of 25 ng / ml, FGF2 is at a concentration of 10 ng / ml, TTNBP is at a concentration of 50 nM, Sant-1 is at a concentration of 50 nM, heparin is at a concentration of 20 ng / ml, and XAV939 is at a concentration of 100 nM.

[0024] In another aspect, the present disclosure relates to an isolated cell culture of human CD31+ endothelial cells, the cell culture comprising human CD31+ endothelial cells cultured in a medium comprising a VEGFR agonist, an FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist. Suitable agonists and antagonists and concentration ranges are described herein.

[0025] Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

[0026]

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Mode for Carrying Out the Invention

[0027] Detailed Description of the Invention Methods and compositions are described herein that enable the robust generation of KDR+CD31+VE-cadherin+ endothelial cells from iPSCs in just 5 days under chemically defined culture conditions using a small molecule-based approach. A High-Dimensional Design of Experiments (HD-DoE) approach was used to simultaneously test multiple process inputs (e.g., small molecule agonists or antagonists) for output responses such as gene expression. These experiments made it possible to identify a chemically defined medium containing agonists and / or antagonists of specific signaling pathways sufficient to generate ECs from iPSCs in a very short time. The optimized medium was further validated by factor criticality analysis examining the effect of removing each individual agonist or antagonist agent. Flow cytometry analysis and immunofluorescence were used to further confirm the phenotype of the cells generated by the differentiation protocol.

[0028] Various aspects of the invention are described in further detail in the following subsections.

[0029] I. Cells The starting cells used in the cultures of the present disclosure are typically human pluripotent stem cells, which are used to generate early mesoderm progenitor cells, which are then used to obtain endothelial cells. As used herein, the term “human pluripotent stem cell” (abbreviated as hPSC) refers to human stem cells that have the ability to differentiate into a variety of different cell types. As used herein, the term “pluripotent” refers to cells that have the ability to differentiate, under various conditions, into cell types characteristic of all three germ cell layers (endoderm, mesoderm, and ectoderm). Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mouse and teratoma formation assays. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but the preferred test of pluripotency is demonstration of the ability to differentiate into cells of each of the three germ layers.

[0030] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSCs) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1, 19-9-7, or 6-9-9 cells (as described, for example, in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, J.A. et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to determine that a cell is a PSC. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2. Since the method for generating committed endothelium of the present disclosure is used to differentiate (mature) a starting population of pluripotent stem cells, the resulting differentiated cells may lack expression of pluripotent stem cell markers. Thus, in various embodiments, the endothelial committed cell population produced by the method of the present disclosure lacks expression of one or more stem cell markers selected from the group consisting of one or more stem cell markers, such as TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2.

[0031] Pluripotent stem cells are subjected to culture conditions that induce cell differentiation, as described herein. As used herein, the term "differentiation" refers to the development of a cell from a more primitive stage to a more mature (i.e., less primitive) cell that typically exhibits phenotypic characteristics of commitment to a particular cell lineage. To generate endothelial cells from pluripotent stem cells, first, the stem cells are differentiated into mesoderm commitment.

[0032] As used herein, the term "early mesoderm progenitor cell" refers to a cell that is more differentiated than a pluripotent stem cell and committed to the mesodermal lineage. As described herein, early mesoderm progenitor cells can be obtained from PSCs by culturing them with an agent that activates Wnt signaling, such as a GSK-3β inhibitor (e.g., together with CHIR99021 for 2 days).

[0033] In multiple embodiments, cells can be identified and characterized based on the expression of one or more biomarkers, such as specific biomarkers of early mesoderm progenitor cells or differentiated endothelial cells. Non-limiting examples of biomarkers whose expression can be evaluated in characterizing cells of interest include CD31, VE-cadherin, KDR, vWF, FL1, and / or CD34 as biomarkers of differentiated endothelial cells.

[0034] II. Medium Components The disclosed methods for generating endothelial cells from pluripotent stem cells include culturing early mesoderm progenitor cells in a medium containing specific agonists and / or antagonists of cell receptors and / or signaling pathways to generate differentiated ECs. Additionally, first, early mesoderm progenitor cells can be obtained from pluripotent stem cells (e.g., ESCs or iPSCs) by culturing the stem cells under defined culture conditions as described herein.

[0035] A medium containing a VEGFR agonist, an FGFR agonist, a retinoic acid agonist, a sonic hedgehog antagonist, heparin or a heparin mimetic, and a Wnt pathway antagonist is sufficient to generate CD31+KDR+FL1+VE-cadherin+ endothelial cells from early mesoderm progenitor cells in as little as 3 days of culture. Early mesoderm progenitor cells can be obtained from PSCs by culturing the PSCs with a Wnt pathway agonist for 2 days, so the total protocol for obtaining differentiated ECs from PSCs under defined culture conditions is 5 days.

[0036] As used herein, an "agonist" of a cell receptor or signaling pathway is intended to refer to an agent that stimulates (upregulates) that cell receptor or signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, for example, by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist may be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by using a small molecule agonist that interacts intracellularly with a component of the signaling pathway.

[0037] As used herein, an "antagonist" of a cell signaling pathway is intended to refer to an agent that inhibits (downregulates) the cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts intracellularly with a component of the signaling pathway.

[0038] The agonists and antagonists used in the chemically defined media and methods of the present disclosure are known in the art and are commercially available. These are used in the medium at concentrations effective to achieve a desired outcome, for example, the generation of cells of interest (e.g., early mesoderm progenitor cells or differentiated endothelial cells) that express a marker of interest. Non-limiting examples of suitable agonist and antagonist agents and effective concentration ranges are further described below.

[0039] Agonists of the VEGFR pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the vascular endothelial growth factor receptor signaling pathway, which is biologically activated by the binding of VEGF and VEGFR. In one aspect, the VEGFR agonist is VEGF or an analog thereof that stimulates signal transduction via VEGR. In one aspect, the VEGFR agonist is VEGF (e.g., recombinant human VEGF). In one aspect, the VEGFR agonist is VEGF, and VEGF is present in the medium at a concentration within the range of 10 - 50 ng / ml, 15 - 45 ng / ml, 20 - 40 ng / ml, 20 - 30 ng / ml, or at a concentration of 25 ng / ml.

[0040] Agonists of the FGFR pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the fibroblast growth factor receptor signaling pathway, which is biologically activated by the binding of FGF and FGFR. In one aspect, the FGFR agonist is FGF2, SUN11602, or a combination thereof. In one aspect, the FGFR pathway agonist is present in the medium at a concentration within the range of 1 - 20 ng / ml, 5 - 15 ng / ml, 7.5 - 12.5 ng / ml, 9 - 11 ng / ml, or at a concentration of 10 ng / ml. In one aspect, the FGFR agonist is FGF2 (e.g., recombinant human FGF2). In one aspect, the FGFR agonist is FGF2 present in the medium at a concentration within the range of 1 - 20 ng / ml, 5 - 15 ng / ml, 7.5 - 12.5 ng / ml, 9 - 11 ng / ml, or at a concentration of 10 ng / ml.

[0041] Agonists of the RA pathway include agents, molecules, compounds, or substances that can stimulate retinoic acid receptors (RARs) activated by both all-trans retinoic acid and 9-cis retinoic acid. There are three types of RARs: RAR-α, RAR-β, and RAR-γ, which are encoded by the RARA, RARB, and RARG genes, respectively. A variety of retinoic acid analogs that can activate the retinoic acid pathway have been synthesized. Non-limiting examples of such compounds include TTNPB (an agonist of RAR-α, β, and γ), AM 580 (an RARα agonist), CD 1530 (a potent and selective RARγ agonist), CD 2314 (a selective RARβ agonist), Ch 55 (a potent RAR agonist), BMS 753 (an RARα-selective agonist), tazarotene (a receptor-selective retinoid; binds to RAR-β and -γ), isotretinoin (an endogenous agonist for retinoic acid receptors; an inducer of neuronal differentiation), and AC 261066 (an RARβ2 agonist). In some embodiments, the RA signaling pathway agonist is selected from the group consisting of (i) a retinoid compound, (ii) a retinoid X receptor (RXR) agonist, and (iii) a 25 retinoic acid receptor (RAR) agonist. In certain embodiments, the RA pathway agonist is selected from the group consisting of retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA).

[0042] Accordingly, in one aspect, the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA), AY 9944 dihydrochloride, ciliobrevin A, cyclopamine, or combinations thereof. In one aspect, the RA pathway agonist is present in the medium at a concentration within the range of 10 to 100 nM, 20 to 80 nM, 25 to 75 nM, or 40 to 60 nM, or at a concentration of 50 nM. In one aspect, the RA pathway agonist is TTNPB. In one aspect, the RA pathway agonist is TTNPB and TTNPB is present in the medium at a concentration within the range of 10 to 100 nM, 20 to 80 nM, 25 to 75 nM, or 40 to 60 nM. In one aspect, the RA pathway agonist is TTNPB and TTNPB is present in the medium at a concentration of 50 nM.

[0043] Antagonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signal transduction through the SHH pathway, which biologically involves the binding of SHH to the Patched-1 (PTCH1) receptor and transmission via the Smoothened (SMO) transmembrane protein. In one aspect, the SHH pathway antagonist is selected from the group consisting of Sant-1, sarsasapogenin B1, itraconazole, GANT61, MK4101, HPI-4, vismodegib, jervine, JK184, taladegib, ciliobrevin D, dinapyrazole A, dinarestin, GANT58, HPI1, IHR1, PF 04449913 maleate, SANT-2, U 18666A, and combinations thereof. In one aspect, the SHH pathway antagonist is present in the medium at a concentration within the range of 10 to 100 nM, 20 to 80 nM, 25 to 75 nM, or 40 to 60 nM, or at a concentration of 50 nM. In one aspect, the SHH pathway antagonist is Sant-1. In one aspect, the SHH pathway antagonist is Sant-1 and Sant-1 is present in the medium at a concentration of 10 to 100 nM, 20 to 80 nM, 25 to 75 nM, or 40 to 60 nM. In one aspect, the SHH pathway antagonist is Sant-1 and Sant-1 is present in the medium at a concentration of 50 nM.

[0044] Heparin is a glycosaminoglycan anticoagulant that has long been known in the art, and heparin mimetics are synthetic and semi-synthetic compounds that are highly sulfated and structurally different analogs of glycosaminoglycans. In multiple embodiments, the medium comprises a heparin analog selected from the group consisting of heparin or a heparin mimetic, such as heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof. In one embodiment, the heparin or heparin mimetic is present in the medium at a concentration in the range of 5-50 ng / ml, 10-40 ng / l, 15-25 ng / ml, or 25-35 ng / ml, or at a concentration of 20 ng / ml. In one embodiment, the medium comprises heparin, and the heparin is present in the medium at a concentration in the range of 5-50 ng / ml, 10-40 ng / l, 15-25 ng / ml, or 25-35 ng / ml. In one embodiment, the medium comprises heparin, and the heparin is present in the medium at a concentration of 20 ng / ml.

[0045] WNT pathway antagonists include agents, molecules, compounds, or substances that can inhibit (down-regulate) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of Wnt-protein ligands to Frizzled family receptors. In one aspect, the WNT pathway antagonist is selected from the group consisting of XAV939, ICG-001 (Hosseinibivin), Capmatinib (INCB28060), Endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, Triptonide, KYA1797K, JW55, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, iCRT14, WIF-1, DKK1, Isoquercitrin, Lanatoside C, Gigantol, RCM-1, WIKI4, IQ-1, Adavivint, PRI-724, Tegatrabetan, and combinations thereof. In one aspect, the WNT pathway antagonist is present in the medium at a concentration in the range of 10 - 500 nM, 50 - 250 nM, 50 - 150 nM, 75 - 125 nM, or at a concentration of 100 nM. In one aspect, the WNT pathway antagonist is XAV939. In one aspect, the WNT pathway antagonist is XAV939 and XAV939 is present in the medium at a concentration of 10 - 500 nM, 50 - 250 nM, 50 - 150 nM, or 75 - 125 nM. In one aspect, the WNT pathway antagonist is XAV939 and XAV939 is present in the medium at a concentration of 100 nM.

[0046] Agonists of the WNT pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of a Wnt-protein ligand to a Frizzled family receptor. In one aspect, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor. In one aspect, the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB216763, SB415286, LY2090314, 3F8, A1070722, AR-A014418, BIO, BIO-acetoxime, AZD1080, WNT3A, alsterpaullone, indirubin-3-oxime, 1-azakenpaullone, kenpaullone, TC-G24, TDZD8, TWS119, NP031112, AT7519, KY19382, AZD2858, and combinations thereof. In one aspect, the WNT pathway agonist is present in the medium at a concentration within the range of 3.0 - 9.0 μM, 4.0 - 8.0 μM, 5.0 - 7.0 μM, 6.5 - 7.5 μM, or at a concentration of 6.0 μM. In one aspect, the WNT pathway agonist is CHIR99021. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium at a concentration within the range of 3.0 - 9.0 μM, 4.0 - 8.0 μM, 5.0 - 7.0 μM, or 6.5 - 7.5 μM. In one aspect, the WNT pathway agonist is CHIR99021 and CHIR99021 is present in the medium at a concentration of 6.0 μM (e.g., in stage 1 medium for generating early mesoderm progenitor cells).

[0047] III. Culture Conditions The method for generating the endothelial cells of the present disclosure, in combination with the chemically defined and optimized medium described in Subsection II above, utilizes standard culture conditions established in the art for cell culture. For example, the cells can be cultured at 37 °C under 5% CO2 conditions. The cells can be cultured in standard culture containers or plates such as culture dishes, culture flasks, or 96-well plates. Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, prior to the initiation of the differentiation protocol, the stem cells can be cultured in a specialized stem cell medium such as Essential 8 Flex Medium (Thermo Fisher # A2858501) for at least one day. In a non-limiting exemplary embodiment, the stem cells are seeded at a density of 150,000 cells / cm2 on a vitronectin (Thermo Fisher # A14700) coated 96-well plate and cultured in Essential 8 Flex Medium for one day prior to differentiation.

[0048] To initiate the differentiation protocol from the stem cells, the medium containing the cultured stem cells is replaced with a basal differentiation medium supplemented with a Wnt signaling pathway agonist, such as the GSK-3β inhibitor (e.g., CHIR99021) described above in Subsection II. This is referred to herein as Stage 1 of the differentiation protocol. In one embodiment, the cells are grown in suspension during the differentiation process, for example, in a vertical wheel bioreactor. For differentiation, the basal differentiation medium may contain, for example, additional standard medium components necessary to maintain cell viability and growth, and typically may include a commercially available base lacking serum (the basal differentiation medium is a serum-free medium). Non-limiting examples of commercially available basal media include IMDM and F-12 medium. Non-limiting examples of the basal differentiation medium are shown in Table 1.

[0049] (Table 1) Basal Differentiation Medium of Samples TIFF2025523194000001.tif65128

[0050] In certain embodiments, the starting pluripotent stem cells are attached to a plate, preferably a plate coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0051] The medium is typically changed regularly to fresh medium. For example, in one embodiment, the medium is changed every 24 hours.

[0052] To generate early mesoderm progenitor cells, the starting pluripotent stem cells are typically cultured for 2 days in a medium containing a GSK-3β inhibitor (e.g., CHIR99021) for a time sufficient for cell differentiation and expression of markers associated with committed early mesoderm progenitor cells. For this stage, the cells may be cultured under adherent conditions or under suspension conditions. This protocol for generating early mesoderm progenitor cells from PSCs is referred to herein as "step (a)" or "stage 1".

[0053] To generate differentiated endothelial cells from the early mesoderm progenitor cells, the progenitor cells are typically cultured for 3 days under suspension conditions in a medium containing a VEGFR agonist, an FGFR agonist, a retinoic acid receptor agonist, a sonic hedgehog antagonist, heparin or a heparin mimetic, and a Wnt pathway antagonist for a time sufficient for cell differentiation and expression of endothelial cell-associated markers. This protocol for generating endothelial cells from early mesoderm progenitor cells is referred to herein as "step (b)" or "stage 2".

[0054] In various embodiments, the early mesoderm progenitor cells are cultured in an optimized medium for a time sufficient to increase the expression of at least one, preferably a plurality of endothelial cell-related markers. Non-limiting examples of suitable EC-related markers include CD31, KDR, FL1, vWF, VE-cadherin, and CD34. In multiple embodiments, the cells are cultured for a time sufficient to increase the expression levels of at least two, at least three, at least four, or at least five EC-related markers. In one embodiment, the cells are cultured for a time sufficient to increase the expression level of at least one EC-related marker by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the starting cell population. The level of marker expression in the cultured ECs can be measured by techniques available in the art (e.g., qPCR analysis and / or flow cytometry).

[0055] Accordingly, in the first stage of the method of making early mesoderm progenitor cells from PSCs, on days 0 to 2, or starting on day 0 and continuing through day 2, or over 48 hours (2 days), or for at least 36 hours, or at least 40 hours, or at least 44 hours, or at least 48 hours, the pluripotent stem cells are cultured in a medium optimized for stage 1.

[0056] Accordingly, in the second stage of the method of making endothelial cells from early mesoderm progenitor cells, on days 2 to 5, or starting on day 2 and continuing through day 5, or starting on day 2 and continuing for 72 hours (3 days), or starting on day 2 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 2 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours, the early mesoderm progenitor cells prepared in stage 1 are further cultured in a medium optimized for stage 2.

[0057] The medium is typically replaced regularly with fresh medium. For example, in certain embodiments, the medium is replaced every 24 hours, or every 48 hours, or every 72 hours.

[0058] IV. Use The methods and compositions of the present disclosure for generating endothelial cells enable the efficient and robust obtaining of these cell populations for various uses. For example, the methods and compositions can be used in the study of EC development and differentiation, including biology, to aid in the understanding of vascular diseases and disorders.

[0059] ECs obtained according to the methods of the present disclosure can be further cultured in a standard endothelial cell medium, for example, for 2 days or more, thereby becoming functional endothelial cells as measured by standard assays demonstrating endothelial cell function. Non-limiting examples of standard assays demonstrating endothelial cell function include tube formation assays, ac-LDL assays, and / or nitric oxide production assays.

[0060] Thus, ECs obtained according to the methods of the present disclosure provide an opportunity to examine functional endothelial aggregates. Other uses include those for 3D-bioprinting, drug screening, safety evaluation, organ on a chip, vascular tissue engineering, and disease modeling.

[0061] The culture methods and compositions of the present disclosure have been verified to work well in bioreactors, and thus, a large number of endothelial cells can be obtained from pluripotent stem cells. This is important for consistency and low variability.

[0062] ECs produced according to the methods of the present disclosure are also intended for use in the treatment of various vascular diseases and disorders, for example, by delivering the cells to a subject having the various vascular diseases and disorders, or for ex vivo use of the cells for assembling vascular elements and then delivering them to a subject. Examples of vascular diseases and disorders include, but are not limited to, ischemic cardiovascular diseases, peripheral vascular diseases, and vascular damage resulting from SARS-CoV-2 infection. Intended therapeutic uses include therapeutic uses for therapeutic angiogenesis, enhancing blood perfusion, and promoting tissue repair.

[0063] V. Compositions In another aspect, the present disclosure provides compositions related to methods of making endothelial cells, including media and isolated cell cultures.

[0064] In one aspect, the present disclosure provides a medium for making CD31+ endothelial cells (ECs) comprising a VEGFR agonist, an FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist. Non-limiting examples of suitable agents and their concentrations include those described in Subsection II above. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the RA pathway agonist is TTNBP, the SHH antagonist is Sant-1, the heparin or heparin mimetic is heparin, and the WNT pathway antagonist is XAV939. In one embodiment, VEGF is at a concentration of 25 ng / ml, FGF2 is at a concentration of 10 ng / ml, TTNBP is at a concentration of 50 nM, Sant-1 is at a concentration of 50 nM, heparin is at a concentration of 20 ng / ml, and XAV939 is at a concentration of 100 nM.

[0065] In another aspect, the present disclosure provides a cell culture of isolated human CD31+ endothelial cells (ECs) cultured in a medium comprising a VEGFR agonist, an FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist. Non-limiting examples of suitable agents and their concentrations include those described in Subsection II above. In one aspect, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the RA pathway agonist is TTNBP, the SHH antagonist is Sant-1, the heparin or heparin mimetic is heparin, and the WNT pathway antagonist is XAV939. In one aspect, VEGF is at a concentration of 25 ng / ml, FGF2 is at a concentration of 10 ng / ml, TTNBP is at a concentration of 50 nM, Sant-1 is at a concentration of 50 nM, heparin is at a concentration of 20 ng / ml, and XAV939 is at a concentration of 100 nM.

[0066] The present invention is further illustrated by the following examples. The following examples should not be construed as further limiting. The contents of the drawings and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

Examples

[0067] Example 1 : Protocol Development for Generating iPSC-Derived Endothelial Cells A two-step recipe for generating endothelial cells was developed that can direct human iPSCs to endothelial cells that express CD31, VE-cadherin, CD34, FLI1, and KDR after 5 days in culture.

[0068] First, undifferentiated iPSCs were grown for 48 hours in CDM2 medium containing 6 μM CHIR99021 (referred to herein as stage 1). Subsequently, the stage 2 medium was manipulated. The basis for this was an 8-factor HD-DoE experiment focused on further differentiating the cells into the endothelial lineage for an additional 3 days after the stage 1 treatment was completed. To test the effectors, 48 different effector combinations obtained using experimental design compression by D-optimality were prepared by a robot. The effector combinations were prepared by dissolving them in the basal medium, then added to the cells, and then the cells were differentiated. After 3 days, RNA extraction was performed and gene expression was obtained using quantitative PCR analysis. The data were normalized and modeled using partial least squares regression analysis against the effector design, resulting in the creation of gene-specific models. These gene-specific models explained the ability of the effectors to control the expression of each gene, both combinatorially and individually, after model tuning for maximum Q2 predictive power. Subsequently, solutions within the tested space were explored to address desirability. An optimal solution was obtained for maximizing CD31 expression. With this solution, other genes such as CD34, ERG, LMO2, FLI1, TAL1, and CDH5 were also predicted to be highly expressed. All genes were highly expressed on endothelial cells. This suggests cell commitment to this lineage. In this model, the inventors observed high levels of KDR, a receptor important for VEGF signaling. In the same model, NKX2-5, OTX2, and GBX2 (genes related to other lineages such as the heart and brain) were downregulated.

[0069] Furthermore, genes associated with early stages such as T, NODAL, and EOMES were downregulated. From this, it can be seen that the cells are in the process of differentiation. This model was derived from a first - test of eight factors including VEGF, FGF2, TTNBP, AGN194310, Y27632, thymosin B4, and heparin. Among these three effectors: VEGF, TTNBP, and heparin showed positive effects on the expression of genes of interest with factor contributions of 39, 21, and 11 respectively (Figure 2). Within the range of the standard to obtain about 85% maximum expression of PECAM, the Cpk value (process capability index) of this complex medium composition was 0.68, and the corresponding risk rate of failure was 2%.

[0070] Next, the inventors evaluated additional factors that presumably increase the complexity of the signaling input in order to obtain effective fate management. As before, the inventors focused on CD31 expression. An optimized solution for maximum CD31 expression resulted in a robust solution. With this solution, other genes such as CD34, CDH5, vWF, FOXC2 were predicted to be abundantly expressed. All genes are related to the endothelial program. From this, cell commitment to this lineage is suggested. This model was derived from a first - test of eight factors including YHHU - 3792, DBZ, Pumorphamine, SANT1, LPA, YODA1, VEGF, and B27. Among these two effectors: VEGF and SANT1 showed positive effects on the expression of genes of interest with high factor contributions of 32 and 16 factor contributions respectively (Figure 3). Since B27 (an additive commonly used to enrich the basal medium for neurons or endothelial cells) is a complex mixture and expensive, the inventors did not include B27 in their recipe. The inventors obtained sufficient CD31 induction without using their differentiation factors together with their basal medium and using their differentiation factors. DBZ and LPA had low contribution factors and thus were not used in the recipe.

[0071] To further improve the recipe for endothelial cell differentiation, the inventors conducted additional HD-DoE experiments. This model was derived from an initial screen of eight factors including VEGF, FGF2, sphingosine 1-phosphate, VH298, Pyrintegrin, Erythropoetin, L-ascorbic acid, and XAV939. Three of these effectors: VEGF, FGF2, and XAV939, each showed a positive effect on the expression of genes of interest with factor contributions of 42, 16, and 15, respectively (Figure 4). With this solution, other genes such as CD34, CD44, ETV2, MKI67, LMO2, FLI1, ERG, TAL1, and CDH5 were also predicted to be abundantly expressed. All genes were highly expressed on endothelial cells, suggesting cell commitment to this lineage. In this model, the inventors observed high levels of KDR, a receptor important for VEGF signaling. Since FGF2 was important for endothelial gene expression in this model and did not significantly affect CD31 expression in the first model, the inventors decided to include FGF2 in the recipe. Based on the predicted conditions that maximize the expression of genes abundant in endothelium such as ERG, CDH5, CD31, FLI1, CD34, etc., considering all the analyzed models, a complex recipe for endothelial differentiation composed of six effectors was developed as shown in Table 2 below.

[0072] (Table 2) Validated effectors for the endothelial differentiation stage 2 recipe TIFF2025523194000002.tif52140

[0073] Example 2 : Factor importance analysis of culture conditions for inducing endothelial cells Variable-related effects were suggested from various contributing factors for each protocol input for the culture to induce endothelial cells. To evaluate the effect of removing each validated factor on genes related to the endothelial lineage, dynamic profile analysis was used to compare the expression levels of genes of interest in the absence of each final determined factor while leaving other factors intact. This factor importance analysis revealed the degree of importance of each input effector, as it became clear whether a desirable outcome was achievable from the expression levels of the genes of interest.

[0074] In this analysis, the effect of factors on the expression levels of CD31, CD34, FLi1, and CDH5 was analyzed by removing the final determined effector while securing other factors (Figure 5A). When VEGF was removed, the value of CD31 decreased from 2000 to 340, the value of CD34 changed from 6500 to 1000, the value of CDH5 decreased from 10000 to 0, and the value of FLI1 decreased from 4900 to 1035. All changes represent a significant loss of expression of the desired genes (Figure 5B). When TTNBP was removed, the value of CD31 decreased from 2000 to 1077, the value of CD34 decreased from 6500 to 4300, the value of CDH5 decreased from 10000 to 2600, and the value of Fli1 decreased from 2883 to 1703. Removal of TTNBP did not significantly affect FLI1 expression (Figure 5B). When heparin was removed, the value of CD31 decreased from 2000 to 1500, the value of CD34 decreased from 6500 to 4800, the value of CDH5 decreased from 10000 to 5800, and the value of Fli1 decreased from 2883 to 1703. Removal of heparin did not significantly affect FLI1 expression (Figure 5B).

[0075] In another model, the effects of XAV939 and FGF2 on the expression levels of CD31, CD34, FLi1, and CDH5 were evaluated (Figure 6A). Again, VEGF was important for the expression of all endothelial genes analyzed. When FGF2 was removed, the value of CD31 decreased from 6500 to 1990, the value of CD34 did not change significantly, the value of CDH5 decreased from 19000 to 5000, and the value of FLI1 decreased from 1300 to 500 (Figure 6B). Removal of XAV939 decreased the CD31 level from 6500 to 2225, the CD34 level decreased from 7180 to 5836, and FLI1 decreased from 13400 to 7300.

[0076] In another model, the inventors evaluated the effect of SANT1, a sonic hedgehog (SHH) inhibitor, on the expression levels of CD31, CD34, FLi1, and CDH5 (Figure 7A). Since L-ascorbic acid was present in the basal medium during this kinetic profile analysis, L-ascorbic acid was ensured in this model. When SANT1 was removed, the CD31 level changed from 808 to 563 and the CDH5 level changed from 2928 to 2340 (Figure 7B). Furthermore, this model demonstrated that the SHH pathway is an important regulator of the endothelial program. Addition of purmorphamine, an SHH agonist, significantly decreased the CD31 level from 800 to 400. This provides evidence that SANT1 is a good effector included in the stage 2 recipe.

[0077] Example 3 : Flow cytometry analysis and immunocytochemistry of stem cell-derived endothelial cells expressing endothelial cell markers To further validate the recipe developed in Example 1, iPSCs were placed in a vertical wheel bioreactor and grown in stage 1 medium for 2 days and then in stage 2 medium for 3 days. Flow cytometry and immunohistochemical analysis were used to evaluate endothelial marker expression. The basic differentiation medium used in this experiment is shown in Table 1. Flow cytometry analysis confirmed the efficiency of the stage 2 recipe to promote the conversion of iPSCs to endothelial cells (Figure 8). More than 80% of the cells were positive for CD31, CD144, and KDR. Endothelial cells also expressed CD34, CXCR4, and CD73, but at lower levels. Furthermore, immunofluorescence staining confirmed the homogeneity and robust staining of various endothelial markers such as VE-cadherin, KDR, vWF, and FLI1. From immunofluorescence staining, it was found that the cells were in a proliferative state as shown by KI67 staining. Finally, differentiation into an endothelial state was confirmed by staining with Ulex Europaeus Agglutinin I (UEA), a lectin established as a robust marker for endothelial cells (Figure 9).

[0078] Example 4 :Functional verification of iPSC-derived endothelial cells To evaluate the function of iPSC-derived endothelial cells, three endothelial cell assays known in the art were performed: tube formation assay within a Matrigel layer, acetylated LDL uptake, and nitric oxide measurement using a probe called DAF-FM. The cells used for these assays were cryopreserved cells after stage 2. The cells were thawed in the basic medium shown in Table 1 supplemented with 25 ng / mL of VEGF and 5 μM SB431542. All assays were performed in the presence of the same medium. The acetylated LDL and DAF-FM assays were performed after recovering the cells from the thawing process (72 - 96 hours). The tube formation assay was performed after the cells were passaged once.

[0079] For the tube formation assay, 50K cells (in 100 uL of medium) were plated on the surface of a layer of growth factor-reduced Matrigel in a 96-well plate. After 3 hours, photographs were taken to identify the formation of tube structures. The results are shown in Figure 10A. Figure 10A demonstrates tube formation by endothelial cells.

[0080] As background for the acetylated LDL assay, LDL containing unmodified apoprotein is used to study normal cholesterol delivery and internalization. If the lysine residues of the apoprotein of LDL are acetylated, the LDL complex no longer binds to the LDL receptor and is taken up by endothelial cells and microglia cells that have "scavenger" receptors specific for its modified form. The results shown in Figure 10B demonstrate that our iPSC-derived endothelial cells had the ability to take up ac-LDL after 4 hours of incubation.

[0081] As background for the DAF-FM assay, DAF-FM is non-fluorescent until it reacts with nitric oxide to form a fluorescent compound called benzotriazole. From DAF-FM staining, it was found that 97% of our endothelial cells were labeled. This indicates that our endothelial cells are producing nitric oxide. As shown in Figure 10C, when compared to undifferentiated iPSCs, iPSC-derived endothelial cells produce significantly more nitric oxide than undifferentiated cells (mean fluorescence intensity MFI = 26539 compared to MFI = 8582 for undifferentiated cells).

[0082] Example 5 :RNA-Seq analysis for characterizing iPSC-derived endothelial cells iPSC-derived endothelial cells were characterized using bulk RNA-seq analysis. Samples at stage 1, stage 2 maintained in culture for 4 days, and cryopreserved stage 2 samples were analyzed. Differential gene expression analysis was performed by comparing stage 1 samples with stage 2 samples. As shown in Figure 11, transcriptome analysis revealed an initial population of endothelial cells that expressed both venous markers (NR2F2 and EPHB4) and arterial markers (GJA4 and NRP1). Expression of tight junction proteins (TJP1, PECAM1, CDLN5ESAM), SOX transcription factors (SOX7, SOX17, and SOX18), and many NOTCH family members (HEY1, DLL4, JAG2, NOTCH1, NOTCH4) was observed. iPSC-derived endothelial cells expressed receptor tyrosine kinases for VEGF such as KDR and FLT1. Expression of endothelial-specific ETS transcription factors (ETS1, ERG, FLI1) was also found.

[0083] Therefore, RNA-seq characterization of endothelial cells confirmed upregulation of endothelial genes and downregulation of pluripotency, primitive streak, and mesoderm genes. Overall, upregulation of desirable genes and downregulation of stem cell markers support the conversion of iPSCs to endothelial cells within 5 days.

[0084] Equivalents One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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