Methods and compositions for generating endothelial cells from pluripotent stem cells

Abstract

Methods for producing CD31 + VE-cadherin + KDR + endothelial cells (EC) using chemically defined media are provided, allowing EC production from early mesoderm progenitor cells in a short three days and EC production from pluripotent stem cells in a short five days. Media, isolated cell populations, and kits are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 391,208, filed on July 21, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Endothelial cells (EC) are the main cell types found in blood vessels, lymphatic vessels, and the inner wall of the heart. Damage or loss of blood vessels is the main pathophysiological feature of vascular diseases (including ischemic cardiovascular disease). Despite considerable efforts, the treatment of such diseases remains challenging. In view of the key role of endothelial cells in pathophysiology, methods focusing on EC or vascular growth are biologically meaningful. Therefore, the ability to generate endothelial cells from stem cells (such as pluripotent stem cells) is important for vascular research and angiogenesis. For example, readily available stem cell-derived ECs will allow them to potentially be used to induce therapeutic angiogenesis, enhance proper blood perfusion, or promote tissue repair (see, for example, Losordo et al. (2004) Circulation 109: 2692-2697). For an overview of ECs derived from pluripotent stem cells, see, for example, Jang et al. (2019) Am. J. Pathol. 189: 502-512. In addition, induced pluripotent stem cell (iPSC)-derived endothelial cells can be used in drug discovery for the identification of new anti-angiogenic drugs, or for safety assessment in the context of drug-induced vascular toxicity.

[0004] The earliest methods for generating EC from pluripotent stem cells designed three different methods (e.g., Wilson et al. (2014) Stem Cells 32: 3037-3045; Yoder (2015) Curr. Opin. Hematol. 22: 252-257; Williams and Wu (2019) Arter. Thromb. Vasc. Biol. 39: 1317-1329). In the first method, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) are grown under conditions that induce cells to self-aggregate into embryoid bodies (EBs), and then various growth factors are used to promote EC and EB differentiation. In the second method, differentiated ESCs or iPSCs are co-cultured with feeder layers such as stromal cells (e.g., mouse cranial mesenchymal OP9 cells or bone marrow stromal cells) to promote differentiation along the EC lineage. In the third approach, ESCs or iPSCs are cultured in two dimensions on plates coated with a protein substrate (such as Matrigel, fibronectin, vitronectin, gelatin, or similar proteins) under specific conditions with the addition of growth factors.

[0005] Chemically defined culture protocols for generating ECs from human pluripotent stem cells have been described, involving regulation of canonical Wnt signaling (see, e.g., Lian et al. (2014) Stem Cell Reports 3:804-816; Bao et al. (2015) Stem Cell Res. 15:122-129). 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, although the cell yield is low (e.g., 20-30% CD34+CD31+ cells before sorting).

[0006] In addition, MAPK and PI3K pathways have been implicated in the differentiation of PSCs into ECs, and culture protocols have been reported to treat with VEGF, FGF2, and BMP4 after GSK-3β inhibitor treatment. These growth factors were used to induce early vascular progenitors from human induced pluripotent stem cell (hiPSC)-derived mesodermal cells, resulting in the generation of CD31+VE-cadherin+ECs within eight days (Harding et al. (2017) Stem Cells 35:909-919). Cyclic adenosine monophosphate (Cyclic AMP) has been reported to enhance the effects of VEGF (Ikuno et al. (2017) PLoS One 12:e0173271). Subsequent chemically defined protocols also combined GSK-3β inhibitor treatment with culture conditions utilizing VEGF, FGF2, and BMP4, and added DAPT and forskolin (Farkas et al. (2020) Front. Cell. Devel. Biol. 8:309).

[0007] Thus, while some progress has been made, there remains a need for efficient and robust methods and compositions for generating endothelial cells from pluripotent stem cells in large-scale culture. Summary of the invention

[0008] The present disclosure provides a method for generating endothelial cells (EC) using a chemically defined culture medium that allows for the robust generation of CD31+EC from pluripotent stem cells in just five days of culture. The culture medium contains small molecule agents that agonize or antagonize specific signaling pathways, thereby promoting differentiation along the endothelial cell lineage. The methods of the present disclosure have the following advantages: they have a high % endothelial conversion rate (80-90%) and allow for the generation of robust endothelial cells in a short period of time using reagents that are less expensive than existing solutions, including the use of bioreactors. Further culturing the resulting EC in endothelial cell culture medium results in cells exhibiting endothelial cell function, as measured by tube formation assays, ac-LDL assays, or nitric oxide production assays. In addition, the use of small molecule agents in the culture medium allows for precise control of culture composition.

[0009] The methods and compositions of the present disclosure utilize a unique cell culture medium that allows CD31+ endothelial cells to be generated from early mesodermal progenitor cells in just three days. This unique cell culture medium includes certain components previously used for endothelial cell generation, such as VEGFR agonists and FGFR agonists, but also includes new components and combinations that allow robust endothelial cell generation. As described herein, early mesodermal progenitor cells can be obtained by culturing pluripotent stem cells under culture conditions that generate early mesodermal progenitor cells. Therefore, in certain embodiments, the method includes a single-stage culture scheme, starting with early mesodermal progenitor cells, and generating CD31+ endothelial cells within three days. In other embodiments, the method includes a two-stage culture scheme, first in stage 1, pluripotent stem cells are differentiated into early mesodermal progenitor cells for two days, and then in stage 2, early mesodermal progenitor cells are further differentiated into CD31+ endothelial cells for three days, thereby forming a two-stage, five-day scheme.

[0010] Thus, in one aspect, the present disclosure relates to a method of generating human CD31+ endothelial cells (ECs), the method comprising:

[0011] Human early mesodermal progenitor cells are cultured in a medium containing a VEGFR agonist, a FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist to generate human CD31+ ECs.

[0012] In one embodiment, early mesodermal progenitor cells are cultured in culture medium for 3 days to obtain CD31+ endothelial cells.

[0013] In one embodiment, early mesodermal progenitor cells are obtained by culturing human pluripotent stem cells for two days in a medium comprising a Wnt pathway agonist.

[0014] In another aspect, the present disclosure relates to a two-stage method for generating human CD31+ endothelial cells (ECs), the method comprising:

[0015] (a) culturing human pluripotent stem cells in a medium containing a Wnt pathway agonist from day 0 to day 2 to generate early mesodermal progenitor cells; and

[0016] (b) On days 2-5, early mesodermal progenitor cells were cultured in a medium containing a VEGFR agonist, a FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist to generate CD31+ ECs.

[0017] In certain embodiments, the CD31+ECs generated 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.

[0018] In one embodiment, the Wnt pathway agonist used in the culture medium for generating early mesodermal progenitor cells is a GSK-3β inhibitor. In one embodiment, the GSK-3β inhibitor is CHIR99021. In one embodiment, CHIR99021 is present in the culture at a concentration of 3.0-9.0 μM. In one embodiment, CHIR99021 is present in the culture at a concentration of 6.0 μM.

[0019] In one embodiment, the VEGFR agonist is VEGF. In one embodiment, VEGF is present in the culture at a concentration of 10-50 ng / ml. In one embodiment, VEGF is present in the culture at a concentration of 25 ng / ml.

[0020] In one embodiment, the FGFR agonist is FGF2 or SUN11602. In one embodiment, the FGFR agonist is present in the culture at a concentration of 1-20 ng / ml. In one embodiment, the FGFR agonist is FGF2, which is present in the culture at a concentration of 1-20 ng / ml. In one embodiment, the FGFR agonist is FGF2, which is present in the culture at a concentration of 10 ng / ml.

[0021] In one embodiment, the retinoic acid (RA) pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, all-trans retinoic acid (ATRA), AY 9944 dihydrochloride, Ciliobrevin A, cyclopamine, or a combination thereof. In one embodiment, the RA pathway agonist is present in the culture at a concentration of 10-100 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture at a concentration of 25-75 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture at a concentration of 50 nM.

[0022] In one embodiment, the sonic hedgehog (SHH) antagonist is selected from the group consisting of Sant-1, saikosaponin B1, itraconazole, GANT61, MK4101, HPI-4, Vismodegib, Jervine, JK184, Taladegib, Ciliobrevin D, Dynapyrazole A, Dynarrestin, GANT58, HPI1, IHR1, PF04449913 maleate, SANT-2, U18666A, and combinations thereof. In one embodiment, the SHH antagonist is present in the culture at a concentration of 10-100 nM. In one embodiment, the SHH antagonist is Sant-1, which is present in the culture at a concentration of 25-75 nM. In one embodiment, the SHH antagonist is Sant-1, which is present in the culture at a concentration of 50 nM.

[0023] In one embodiment, 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 embodiment, heparin or heparin mimetic is present in the culture at a concentration of 10-50 ng / ml. In one embodiment, heparin is present in the culture at a concentration of 25-35 ng / ml. In one embodiment, heparin is present in the culture at a concentration of 20 ng / ml.

[0024] In one embodiment, the Wnt pathway antagonist is selected from the group consisting of XAV939, ICG-001 (Foscenvivint), Capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, Triptonide, KYA1797K, JW55, JW67, JW74, Cardionogen1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, Gigantol, RCM-1, WIKI4, IQ-1, Advavivant, PRI-724, Tegatrabetan or a combination thereof. In one embodiment, the Wnt pathway antagonist is present in the culture at a concentration of 10-500 nM. In one embodiment, the Wnt pathway antagonist is XAV939, which is present in the culture at a concentration of 50-150 nM. In one embodiment, the Wnt pathway antagonist is XAV939, which is present in the culture at a concentration of 100 nM.

[0025] In one embodiment, the pluripotent stem cell is an embryonic stem cell.In one embodiment, the pluripotent stem cell is an induced pluripotent stem cell.

[0026] In another aspect, the present disclosure relates to a culture medium for generating endothelial cells (EC), the culture medium comprising a VEGFR agonist, a 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, 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, the concentration of VEGF is 25ng / ml, the concentration of FGF2 is 10ng / ml, the concentration of TTNBP is 50nM, the concentration of Sant-1 is 50nM, the concentration of heparin is 20ng / ml, and the concentration of XAV939 is 100nM.

[0027] In another aspect, the present disclosure relates to an isolated cell culture of human CD31+ endothelial cells, the culture comprising: human CD31+ endothelial cells cultured in a medium comprising a VEGFR agonist, a 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, antagonists, and concentration ranges are described herein.

[0028] Other features and advantages of the present application will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of a representative culture protocol for generating endothelial cells from early mesodermal progenitors in three days and a two-day pretreatment for generating early mesodermal progenitors from iPSCs.

[0030] Figure 2 The results of an 8-factor experimental model optimized for maximum expression of PECAM1 (CD31) are shown. The upper part of the model shows the prediction of the expression level of 53 pre-selected genes when optimized for PECAM1. The lower part of the model shows the effectors tested in the model and their contribution to the maximum expression of PECAM1. The value column refers to the concentration of each effector required to simulate the model.

[0031] Figure 3 Results of an 8-factor experimental model optimized for maximum expression of PECAM1 (CD31) are shown. Figure 1 shown.

[0032] Figure 4 Results of an 8-factor experimental model optimized for maximum expression of PECAM1 (CD31) are shown. Figure 1 shown.

[0033] Figure 5A-5B A dynamic profile analysis of PECAM1, CD34, CDH5 and FLI1 expression levels relative to the concentrations of the three validated effectors is shown. The positive influence of VEGF, TTNBP and heparin on the expression of the above genes and their factorial contribution are shown by the slope of the plot for each effector.

[0034] Figure 6A-6BA dynamic profile analysis of PECAM1, CD34, CDH5 and FLI1 expression levels relative to the concentrations of the three validated effectors is shown. The positive effects of VEGF on the expression of all of the above genes and their factorial contributions are shown by the slope of the graph for each effector. Positive effects of FGF2 on the expression of PECAM1, FLI1 and CDH5. Positive effects of XAV939 on the expression of PECAM1 and FLI1.

[0035] Figure 7A-7B A dynamic profile analysis of PECAM1, CD34, CDH5 and FLI1 expression levels relative to the concentration of two validated effectors is shown. The positive effect of VEGF on the expression of all the above genes and their factorial contribution is shown by the slope of the plot for each effector. Positive effect of SANT1 on PECAM1 and CDH5 expression.

[0036] Figure 8 Shown are flow cytometry staining results of iPSC-derived endothelial cells at the end of stage 2. Cells were stained with CD31, CD34, CD73, KDR, CXCR4, and CD 144. At this stage, cells were positive for all markers expected for endothelial cells.

[0037] Fig. 9 A fluorescent image photograph of iPSC-derived endothelial cells at the end of stage 2 is shown. The cells were stained with endothelial biomarkers including vWF, VE-cadherin (VE-cad), FLI1, KDR, and Ulex oleraceus I-FITC. At this stage, the cells were positive for all markers expected for endothelial cells.

[0038] Figures 10A-10C The figure shows the experimental results of functional verification of iPSC-derived endothelial cells after cryopreservation. Fig. 10A Shown are the results of tube formation assays in Matrigel layers after 3 hours. Fig. 10B Results are shown for acetylated LDL uptake by endothelial cells after 4 hours of incubation, cells were also stained with phalloidin (F-actin in white). Fig. 10C Shown are the results of flow cytometric analysis of cells stained with DAF-FM, comparing undifferentiated iPSCs (red) to iPSC-derived endothelial cells (blue).

[0039] Fig.11 Results of RNA-seq experiments used to characterize iPSC-derived endothelial cells are shown. Analysis was performed on stage 1, stage 2, and frozen stage 2 samples maintained in culture for 4 days. Results are shown as a heat map of log2 fold changes showing differential gene expression in stage 2 cells compared to stage 1 cells (day 2). Blue indicates genes downregulated in endothelial cells, and red indicates genes upregulated in endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0041] The methods and compositions described herein allow KDR+CD31+VE-cadherin+endothelial cells to be robustly generated from iPSCs in just five days using small molecule-based methods under chemically defined culture conditions. The effects of multiple process inputs (e.g., small molecule agonists or antagonists) on output responses (such as gene expression) were tested simultaneously using a high-dimensional experimental design (HD-DoE) approach. These experiments allow identification of chemically defined culture media containing agonists and / or antagonists of specific signaling pathways that are sufficient to generate ECs from iPSCs in a very short period of time. The optimized culture media were further validated by factor criticality analysis, which examined the effects of removing a single agonist or antagonist. The cell phenotypes generated by the differentiation protocol were further confirmed using flow cytometry analysis and immunofluorescence.

[0042] Various aspects of the application are described in further detail in the following subsections.

[0043] I. cell

[0044] The starting cell used in the culture of the present disclosure is generally a human pluripotent stem cell, which is used to generate early mesodermal progenitor cells, which are then used to obtain endothelial cells. As used herein, the term "human pluripotent stem cell" (abbreviated as hPSC) refers to a human stem cell with the ability to differentiate into a variety of different cell types. The term "pluripotent" used herein refers to cells with the ability to differentiate into all three germ cell layers (endoderm, mesoderm and ectoderm) cell type characteristics under different conditions. Pluripotent cells are mainly characterized by their ability to differentiate into all three germ layers, such as using nude mice and teratoma formation assays. Pluripotency can also be demonstrated by the expression of embryonic stem cell (ES) cell markers, although the preferred test of pluripotency is to demonstrate the ability to differentiate into cells of each germ layer in the three germ layers.

[0045] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSC) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSC) include 19-11-1, 19-9-7 or 6-9-9 cells (e.g., as described 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, JA et al. (1998) Science 282: 1145-1147). Human pluripotent stem cells (PSC) express cell markers that can be used to identify whether 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 of generating committed endothelium disclosed herein is used to differentiate (mature) a starting pluripotent stem cell population, the resulting differentiated cells may lack the expression of pluripotent stem cell markers. Therefore, in various embodiments, the endothelial committed cell population generated by the method of the present disclosure lacks the expression of one or more stem cell markers, such as one or more stem cell markers selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2.

[0046] The pluripotent stem cells are placed under culture conditions that induce cell differentiation as described herein. As used herein, the term "differentiation" refers to the development of cells from a more primitive stage to a more mature (i.e., less primitive) cell, typically exhibiting phenotypic characteristics directed toward a specific cell lineage. In order to generate endothelial cells from pluripotent stem cells, the stem cells are first differentiated into a mesoderm-like pattern.

[0047] As used herein, "early mesodermal progenitor cells" refer to cells that are more differentiated than pluripotent stem cells and are directed to the mesodermal lineage. As described herein, early mesodermal progenitor cells can be obtained from PSC by culturing using reagents (such as GSK-3β inhibitors) that activate Wnt signaling (e.g., CHIR99021 culture for two days).

[0048] In embodiments, cells can be identified and characterized based on the expression of one or more biomarkers, such as specific biomarkers for early mesodermal progenitor cells or differentiated endothelial cells. Non-limiting examples of biomarkers whose expression can be assessed in the characterization of target cells include CD31, VE-cadherin, KDR, vWF, FL1 and / or CD34 as biomarkers for differentiated endothelial cells.

[0049] II. Medium components

[0050] The method for generating endothelial cells from pluripotent stem cells disclosed herein comprises culturing early mesodermal progenitor cells in a culture medium comprising specific agonists and / or antagonists of cell receptors and / or signaling pathways to generate differentiated ECs. In addition, early mesodermal progenitor cells can first be obtained from pluripotent stem cells (e.g., ESCs or iPSCs) by culturing stem cells under defined culture conditions as described herein.

[0051] Culture medium containing a VEGFR agonist, a 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 mesodermal progenitors in as little as three days of culture. Early mesodermal progenitors can be derived from PSCs by culturing PSCs with a Wnt pathway agonist for two days, resulting in an overall five-day protocol for obtaining differentiated ECs from PSCs under defined culture conditions.

[0052] As used herein, an "agonist" of a cell receptor or signaling pathway means an agent that stimulates (upregulates) a cell receptor or signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, such as by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., an agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, such as by using a small molecule agonist that interacts intracellularly with a component of the signaling pathway.

[0053] As used herein, an "antagonist" of a cell signaling pathway means an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, such as 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, such as by using a small molecule antagonist that interacts intracellularly with a component of the signaling pathway.

[0054] The agonists and antagonists used in the culture medium and methods determined by the chemical composition of the present disclosure are known in the art and are commercially available. They are used in the culture medium to effectively achieve the desired result, for example, to generate a concentration of the target cell (such as early mesodermal progenitor cells or differentiated endothelial cells) expressing the target marker. The non-limiting examples of suitable agonists, antagonists and effective concentration ranges are further described below.

[0055] 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 combination of VEGF and VEGFR. In one embodiment, the VEGFR agonist is VEGF or its analog that stimulates signaling through VEGR. In one embodiment, the VEGFR agonist is VEGF (e.g., recombinant human VEGF). In one embodiment, the VEGFR agonist is VEGF, which is present in the culture medium at a concentration of 10-50ng / ml, 15-45ng / ml, 20-40ng / ml, 20-30ng / ml or 25ng / ml.

[0056] The agonist of FGFR pathway includes reagents, molecules, compounds or substances that can stimulate (upregulate) fibroblast growth factor receptor signaling pathway, which is biologically activated by the combination of FGF and FGFR. In one embodiment, the FGFR agonist is FGF2, SUN11602 or a combination thereof. In one embodiment, the FGFR pathway agonist is present in culture medium at a concentration of 1-20ng / ml, 5-15ng / ml, 7.5-12.5ng / ml, 9-11ng / ml or 10ng / ml. In one embodiment, the FGFR agonist is FGF2 (for example, recombinant human FGF2). In one embodiment, the FGFR agonist is FGF2, which is present in culture medium at a concentration of 1-20ng / ml, 5-15ng / ml, 7.5-12.5ng / ml, 9-11ng / ml or 10ng / ml.

[0057] Agonists of the RA pathway include agents, molecules, compounds or substances that can stimulate retinoic acid receptors (RARs) activated by all-trans retinoic acid and 9-cis retinoic acid. There are three types of RAR: RAR-α, RAR-β and RAR-γ, encoded by RARA, RARB, and RARG genes, respectively. Different retinoic acid analogs that can activate the retinoic acid pathway have been synthesized. Non-limiting examples of such compounds include TTNPB (RAR-α, β and γ agonists), AM 580 (RARα agonist), CD 1530 (potent and selective RARγ agonist), CD 2314 (selective RARβ agonist), Ch 55 (potent RAR agonist), BMS 753 (RARα-selective agonist), tazarotene (receptor selective retinoid; binding to RAR-β and -γ), isotretinoin (endogenous agonist of retinoic acid receptor; neuronal differentiation inducer), and AC 261066 (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 a specific embodiment, 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-oxo retinoic acid, and all-trans retinoic acid (ATRA).

[0058] Therefore, in one embodiment, the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, all-trans retinoic acid (ATRA), AY 9944 dihydrochloride, Ciliobrevin A, cyclopamine, or a combination thereof. In one embodiment, the RA pathway agonist is present in the culture medium at a concentration of 10-100 nM, 20-80 nM, 25-75 nM, or 40-60 nM, or at a concentration of 50 nM. In one embodiment, the RA pathway agonist is TTNPB. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 10-100 nM, 20-80 nM, 25-75 nM, or 40-60 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 50 nM.

[0059] Antagonists of the SHH (sonic hedgehog) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) signal transduction through the SHH pathway, which is biologically related to the binding of SHH to the Patched-1 (PTCH1) receptor and transduction through the Smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway antagonist is selected from the group consisting of Sant-1, saikosaponin B1, itraconazole, GANT61, MK4101, HPI-4, Vismodegib, Jervine, JK184, Taladegib, Ciliobrevin D, Dynapyrazole A, Dynarrestin, GANT58, HPI1, IHR1, PF 04449913 maleate, SANT-2, U 18666A and combinations thereof. In one embodiment, the SHH pathway antagonist is present in the culture medium at a concentration of 10-100 nM, 20-80 nM, 25-75 nM, or 40-60 nM, or at a concentration of 50 nM. In one embodiment, the SHH pathway antagonist is Sant-1. In one embodiment, the SHH pathway antagonist is Sant-1, which is present in the culture medium at a concentration of 10-100 nM, 20-80 nM, 25-75 nM, or 40-60 nM. In one embodiment, the SHH pathway antagonist is Sant-1, which is present in the culture medium at a concentration of 50 nM.

[0060] Heparin is a glycosaminoglycan anticoagulant known in the art, and heparin mimetics are synthetic and semi-synthetic compounds, which are highly sulfated, structurally different analogs of glycosaminoglycans. In one embodiment, the culture medium comprises heparin or heparin mimetics, such as heparin analogs selected from the group consisting of heparan sulfate, enoxaparin, low molecular weight heparin, AV5026, M402, and combinations thereof. In one embodiment, heparin or heparin mimetics are present in the culture medium at a concentration of 5-50ng / ml, 10-40ng / l, 15-25ng / ml, or 25-35ng / ml, or at a concentration of 20ng / ml. In one embodiment, the culture medium comprises heparin, which is present in the culture medium at a concentration of 5-50ng / ml, 10-40ng / l, 15-25ng / ml, or 25-35ng / ml. In one embodiment, the culture medium comprises heparin, which is present in the culture medium at a concentration of 20ng / ml.

[0061] Antagonists of the WNT pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway antagonist is selected from the following group: XAV939, ICG-001 (Foscenvivint), Capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, Triptonide, KYA1797K, JW55, JW 67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, Gigantol, RCM-1, WIKI4, IQ-1, Advavivant, PRI-724, Tegatrabetan, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the culture medium at a concentration of 10-500nM, 50-250nM, 50-150nM, 75-125nM, or 100nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the culture medium at a concentration of 10-500nM, 50-250nM, 50-150nM, or 75-125nM. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the culture medium at a concentration of 100 nM.

[0062] 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 binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor. In one embodiment, the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetyl oxime, AZD1080, WNT3A, alsterpaullone, indirubin-3-oxime, 1-azakenpaullone, kenpaullone, TC-G 24, TDZD 8, TWS119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof. In one embodiment, the WNT pathway agonist is present in a culture medium at a concentration of 3.0-9.0 μM, 4.0-8.0 μM, 5.0-7.0 μM, 6.5-7.5 μM or 6.0 μM. In one embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist CHIR99021, which is present in a culture medium at a concentration of 3.0-9.0 μM, 4.0-8.0 μM, 5.0-7.0 μM or 6.5-7.5 μM. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in a culture medium (e.g., in a stage 1 culture medium for generating early mesoderm progenitor cells) at a concentration of 6.0 μM.

[0063] III. Culture conditions

[0064] In conjunction with the chemically defined and optimized culture medium described in Section II above, the methods of generating endothelial cells disclosed herein utilize standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37° C., 5% CO2. 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 culture media before differentiation. For example, stem cells can be cultured in a specialized stem cell culture medium, such as Essential 8Flex medium (Thermo Fisher #A2858501), for at least one day before the differentiation protocol is initiated. In a non-limiting exemplary embodiment, stem cells are cultured at 150,000 cells / cm 2Cells were passaged at a density of 1:1 onto vitronectin (Thermo Fisher #A14700) coated 96-well plates and cultured in Essential 8Flex medium for one day prior to differentiation.

[0065] In order to start the differentiation protocol from stem cells, the culture medium for culturing stem cells is replaced with a basic differentiation medium supplemented with a Wnt signaling pathway agonist such as a GSK-3β inhibitor (e.g., CHIR99021), as described in Section II above, referred to herein as Phase 1 of the differentiation protocol. In one embodiment, cells are grown in suspension during differentiation, such as in a vertical wheeled bioreactor. For differentiation, the basic differentiation medium may include, for example, a commercially available matrix supplemented with other standard culture medium components required to maintain cell viability and growth, but typically lacks serum (the basic differentiation medium is a serum-free medium). Non-limiting examples of commercially available basic culture media include IMDM and F-12 culture media. Table 1 shows non-limiting examples of basic differentiation media:

[0066] Table 1: Sample basal differentiation medium

[0067] Compound Final concentration Albumax 0.25％ Ethanolamine 10μM Monothioglycerol 150μM Cholesterol sulfate 200ng / mL(409nM) Trolox 10μM Linolenic acid 4ng / mL(0.01426mM) Oleic acid 10μM insulin 10 μg / mL Holo-transferrin 10 μg / mL Sodium tannate 5ng / mL Vitamin C Phosphate 100 μg / mL IMDM:F12 50:50

[0068] In certain embodiments, the starting pluripotent stem cells adhere 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).

[0069] The culture medium is usually replaced with fresh culture medium regularly. For example, in one embodiment, the culture medium is replaced every 24 hours.

[0070] In order to generate early mesodermal progenitor cells, the starting pluripotent stem cells are cultured in a culture medium comprising a GSK-3β inhibitor (e.g., CHIR99021) for a sufficient time to perform cell differentiation and expression of markers associated with early mesodermal progenitor cells, typically two days. For this stage, cells can be cultured under adherent or suspended conditions. This protocol for generating early mesodermal progenitor cells from PSC is referred to herein as "step (a)" or "stage 1".

[0071] In order to generate differentiated endothelial cells from early mesodermal progenitor cells, the progenitor cells are cultured in a culture medium comprising a VEGFR agonist, a FGFR agonist, a retinoic acid receptor agonist, a sonic hedgehog antagonist, heparin or a heparin mimetic, and a Wnt pathway antagonist, usually under suspension conditions, for a sufficient time to allow cell differentiation and expression of endothelial cell-related markers, usually three days. This protocol for generating endothelial cells from early mesodermal progenitor cells is referred to herein as "step (b)" or "Phase 2."

[0072] In various embodiments, early mesodermal progenitor cells are cultured in an optimized culture medium for a sufficient time to increase at least one, preferably multiple endothelial cell-related markers. Non-limiting examples of suitable EC-related markers include CD31, KDR, FL1, vWF, VE-cadherin, and CD34. In embodiments, cells are cultured for a sufficient time 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, cells are cultured for a sufficient time 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 expression levels of markers in cultured ECs can be measured by techniques available in the art (e.g., qPCR analysis and / or flow cytometry).

[0073] Thus, in the first stage of the method, i.e., generating early mesodermal progenitors from PSCs, pluripotent stem cells are cultured in a medium optimized for stage 1 on days 0-2, or starting on day 0 and continuing to day 2, or for 48 hours (2 days), or at least 36 hours, or at least 40 hours, or at least 44 hours, or at least 48 hours.

[0074] Therefore, in the second stage of the method, i.e., generating endothelial cells from early mesodermal progenitor cells, the early mesodermal progenitor cells generated in stage 1 are further cultured in the medium optimized for stage 2 from day 2 to day 5, or starting from day 2 and continuing to day 5, or starting from day 2 and continuing for 72 hours (3 days), or starting from 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 from day 2 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours or 72 hours.

[0075] The culture medium is usually replaced with fresh culture medium regularly. For example, in certain embodiments, the culture medium is replaced once every 24 hours, or once every 48 hours or once every 72 hours.

[0076] IV. use

[0077] The disclosed methods and compositions for generating endothelial cells allow these cell populations to be used efficiently and robustly for a variety of purposes. For example, these methods and compositions can be used to study the development and differentiation of ECs, including biology, to aid in the understanding of vascular diseases and disorders.

[0078] ECs obtained according to the methods of the present disclosure can be further cultured in standard endothelial cell culture medium, such as for two days or longer, resulting in them 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.

[0079] Therefore, ECs obtained according to the methods of the present disclosure provide an opportunity to study functional endothelial assembly. Other uses include 3D bioprinting, drug screening, safety assessment, organ chips, vascular tissue engineering, and disease modeling.

[0080] The culture methods and compositions disclosed herein have been validated to work on bioreactors, allowing large numbers of endothelial cells to be obtained from pluripotent stem cells, which is important for consistency and low variability.

[0081] ECs generated according to the methods of the present disclosure are also contemplated for use in treating various vascular diseases and conditions, for example by delivering the cells to a subject suffering from a disease or condition, or assembling vascular elements using ex vivo cells and then delivering them to a subject. Examples of vascular diseases and conditions include, but are not limited to, ischemic cardiovascular disease, peripheral vascular disease, and vascular damage caused by infection with SARS-CoV-2. Intended therapeutic uses include use for therapeutic neovascularization, enhancing blood perfusion, and promoting tissue repair.

[0082] V. Composition

[0083] In other aspects, the disclosure provides compositions related to methods of generating endothelial cells, including culture media and isolated cell cultures.

[0084] In one aspect, the present disclosure provides a culture medium for generating CD31+ endothelial cells (EC), which comprises a VEGFR agonist, a 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 reagents and their concentrations include those described in Section 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, the concentration of VEGF is 25ng / ml, the concentration of FGF2 is 10ng / ml, the concentration of TTNBP is 50nM, the concentration of Sant-1 is 50nM, the concentration of heparin is 20ng / ml, and the concentration of XAV939 is 100nM.

[0085] In another aspect, the present disclosure provides an isolated cell culture of human CD31+ endothelial cells (EC), the culture comprising: human CD31+EC cultured in a medium comprising a VEGFR agonist, a 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 Section 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, the concentration of VEGF is 25 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of TTNBP is 50 nM, the concentration of Sant-1 is 50 nM, the concentration of heparin is 20 ng / ml, and the concentration of XAV939 is 100 nM.

[0086] The present application is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Example

[0087] Example 1 :Development of a protocol for generating iPSC-derived endothelial cells

[0088] A two-stage formulation for generating endothelial cells was developed that directed human IPSCs to generate endothelial cells expressing CD31, VE-cadherin, CD34, FLI1, and KDR after 5 days in culture.

[0089] First, undifferentiated iPSCs were grown in CDM2 medium containing 6 μM CHIR99021 for 48 hours (referred to herein as Phase 1). Then Phase 2 medium was designed. The basis is an 8-factor HD-DoE experiment, focusing on the differentiation of cells to the endothelial lineage for an additional 3 days after the termination of Phase 1 treatment. In order to test the effector, 48 different effector combinations generated by D optimality using experimental design compression were automatically prepared. The effector combination was prepared in the basal medium, subsequently added to the cells, and then the cells were differentiated. Three days later, 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 of the effector design to generate a gene-specific model, which explained the ability of the effector to control the expression of a single gene in combination and individually after the model was adjusted for maximum Q2 predictive power. Then, solutions within the test space can be explored to solve the desirability problem. The optimization of the maximum expression of CD31 produced a robust solution. In this solution, other genes such as CD34, ERG, LMO2, FLI1, TAL1, and CDH5 are predicted to be highly expressed as well, all of which are highly expressed on endothelial cells, indicating that the cells are committed to this lineage. In this model, we observed high levels of KDR (an important receptor for VEGF signaling). In the same model, NKX2-5, OTX2, and GBX2 were downregulated (genes associated with other lineages such as heart and brain).

[0090] In addition, genes associated with early stages, such as T, NODAL, and EOMES, were downregulated, indicating that the cells were differentiating. The model was derived from preliminary testing of eight factors including: VEGF, FGF2, TTNBP, AGN194310, Y27632, Thymosin B4, and heparin. Three of the effectors, VEGF, TTNBP, and heparin, showed a positive effect on the expression of target genes, with 39, 21, and 11 factor contributions, respectively ( Figure 2 Within the specification range of achieving approximately 85% of the maximum expression of PECAM, this complex medium composition has a Cpk value (process capability index) of 0.68, corresponding to a risk of failure of 2%.

[0091] Next, we evaluated additional factors that might increase the complexity of signaling inputs to achieve effective fate control. As previously mentioned, we focused on the expression of CD31. Optimization for maximal expression of CD31 produced a robust solution. In this solution, other genes were predicted to be expressed in large quantities, such as CD34, CDH5, vWF, FOXC2, all genes associated with the endothelial program, indicating that the cells are committed to this lineage. The model was derived from preliminary testing of eight factors including: YHHU-3792, DBZ, Pumorphamine, SANT1, LPA, YODA1, VEGF, and B27. Two of the effectors, VEGF and SANT1, showed positive effects on the expression of target genes, with high factor contributions of 32 and 16 factor contributions, respectively ( Figure 3 We did not include B27 (a commonly used additive to enrich basal medium for neural or endothelial cells) in our formulation because it is a complex mixture and expensive, and we obtained adequate CD31 induction using our differentiation factors and basal medium in the absence of B27.

[0092] To further refine the recipe for endothelial cell differentiation, we performed additional HD-DoE experiments. The model was derived from preliminary testing of eight factors including: VEGF, FGF2, Sphingosine 1-phosphate, VH298, Pyrintegrin, Erythropoietin, L-ascorbic acid, and XAV939. Three of the effectors: VEGF, FGF2, and XAV939 showed a positive effect on the expression of target genes, with 42, 16, and 15 factor contributions, respectively ( Figure 4 ). In this solution, other genes such as CD34, CD44, ETV2, MKI67, LMO2, FLI1, ERG, TAL1 and CDH5 were predicted to be expressed in large amounts as well, all of which are highly expressed on endothelial cells, indicating that the cells are committed to this lineage. In this model, we observed high levels of KDR, an important receptor for VEGF signaling. Since FGF2 is critical for the expression of endothelial genes in this model and did not significantly affect the expression of CD31 in the first model, we decided to keep FGF2 in the formulation. Taking into account all models analyzed, an endothelial differentiation complex formulation consisting of 6 effectors was developed based on the predicted conditions that maximized the expression of endothelial enriched genes such as ERG, CDH5, CD31, FLI1, CD34, as shown in Table 2 below:

[0093] Table 2: Validated effectors in endothelial differentiation stage 2 formulations

[0094] Reagents effect concentration VEGF VEGFR agonists 25 ng / mL FGF2 FGFR agonists 10 ng / mL TTNBP RA pathway agonists 50nM Sant1 SHH antagonists 50nM heparin Glycosaminoglycans 20 ng / mL XAV939 WNT pathway antagonists 100nM

[0095] Embodiment 2: Analysis of key factors in endothelial cell induction culture conditions

[0096] The various contributing factors input into each protocol for induced endothelial cell culture indicate the relative impact of the variables. To assess the impact of removing each validated factor on genes associated with the endothelial lineage, a dynamic distribution analysis was used and the expression levels of the target genes were compared in the absence of each finalized factor and in the presence of the other factors. As the expression level of the target gene reveals whether the expected outcome can be achieved, this factor criticality analysis reveals the degree of importance of each input effector.

[0097] In this analysis, the final identified effector was removed while the other factors were retained for analysis of the effect of the factors on the expression levels of CD31, CD34, FLi1, and CDH5 ( Figure 5A When VEGF was removed, the values ​​for CD31 dropped from 2000 to 340, the values ​​for CD34 changed from 6500 to 1000, the values ​​for CDH5 dropped from 10000 to 0, and the values ​​for FLI1 dropped from 4900 to 1035. All changes indicated a significant loss of expected gene expression ( Figure 5B When TTNBP was removed, the values ​​of CD31 decreased from 2000 to 1077, CD34 decreased from 6500 to 4300, CDH5 decreased from 10000 to 2600, and Fli1 decreased from 2883 to 1703. TTNBP removal had no significant effect on FLI1 expression ( Figure 5B When heparin was removed, the values ​​of CD31 decreased from 2000 to 1500, CD34 decreased from 6500 to 4800, CDH5 decreased from 10000 to 5800, and Fli1 decreased from 2883 to 1703. Heparin removal had no significant effect on FLI1 expression ( Figure 5B ).

[0098] In another model, the effects of XAV939 and FGF2 on the expression levels of CD31, CD34, FLi1, and CDH5 were evaluated ( Fig. 6A ). Likewise, VEGF was essential for the expression of all endothelial genes analyzed. When FGF2 was removed, the values ​​for CD31 dropped from 6500 to 1990, the values ​​for CD34 did not change significantly, the values ​​for CDH5 dropped from 19000 to 5000, and the values ​​for FLI1 dropped from 1300 to 500 ( Figure 6B ). Removal of XAV939 decreased CD31 levels from 6500 to 2225, CD34 levels from 7180 to 5836, and FLI1 from 13400 to 7300.

[0099] In another model, we evaluated the effect of the sonic hedgehog (SHH) inhibitor SANT1 on the expression levels of CD31, CD34, FLi1, and CDH5 ( Fig. 7A ). When performing this dynamic distribution analysis, L-ascorbic acid was retained in the model because it was present in the basal medium. When SANT1 was removed, CD31 levels changed from 808 to 563, and CDH5 levels changed from 2928 to 2340 ( Figure 7B ). Furthermore, the model shows how the SHH pathway is a key regulator of the endothelial program. Adding the SHH agonist pumorphamine significantly reduced CD31 levels from 800 to 400, consistent with evidence that SANT1 is a good effector to include in a phase 2 formulation.

[0100] Example 3 Flow cytometric analysis and immunocytochemistry of stem cell-derived endothelial cells expressing endothelial cell markers

[0101] To further validate the formulation developed in Example 1, iPSCs were grown in stage 1 medium for 2 days and then in stage 2 medium for 3 days in a vertical wheel bioreactor and evaluated for expression of endothelial markers using flow cytometry and immunohistochemistry analysis. The basal differentiation medium used for this experiment is shown in Table 1. Flow cytometric analysis confirmed the efficiency of the stage 2 formulation in promoting the conversion of iPSCs into 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. In addition, immunofluorescence staining confirmed the homogeneous and robust staining of various endothelial markers such as VE-cadherin, KDR, vWF, and FLI1. Immunofluorescence staining showed that the cells were in a proliferative state, as shown by KI67 staining. Finally, staining with Ulex oleraceus agglutinin I (UEA), a lectin that has been identified as a robust marker for endothelial cells, confirmed the differentiation into an endothelial state ( Fig. 9 ).

[0102] Example 4 Functional validation of iPSC-derived endothelial cells

[0103] In order to evaluate the function of iPSC-derived endothelial cells, three endothelial cell assays known in the art were performed: tube formation assay in the matrigel layer, acetylated LDL uptake, and measurement of nitric oxide by using a probe called DAF-FM. The cells used for these assays were cryopreserved cells after stage 2. Cells were thawed in the basal medium shown in Table 1, and 25ng / mL of VEGF and 5μM SB431542 were added. All assays were performed in the presence of the same medium. Acetylated LDL and DAF-FM assays were performed after the cells recovered from the thawing process (72-96h). Tube formation assays were performed after the cells were passaged once.

[0104] For the tube formation assay, 50K cells (in 100 μL of culture medium) were seeded on a layer of growth factor-reduced Matrigel in a 96-well plate. Three hours later, photographs were taken to determine the formation of tube structures. Fig. 10A As shown, it is demonstrated that endothelial cells form tubes.

[0105] As background for the acetylated LDL assay, LDL containing unmodified apolipoproteins is used to study normal cholesterol delivery and internalization. If the lysine residues of the apolipoprotein of LDL are acetylated, the LDL complex no longer binds to the LDL receptor but is taken up by endothelial cells and microglia that have "scavenger" receptors specific for this modified form. Fig. 10B The results shown indicate that our iPSC-derived endothelial cells have the ability to take up ac-LDL after 4 h of incubation.

[0106] 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. DAF-FM staining showed that 97% of endothelial cells were labeled, indicating that they were producing nitric oxide. Fig. 10C As shown, iPSC-derived endothelial cells produced significantly more nitric oxide than undifferentiated iPSCs (mean fluorescence intensity, MFI=26539 vs. MFI of undifferentiated cells: 8582).

[0107] Embodiment 5: RNA-Seq analysis to characterize iPSC-derived endothelial cells

[0108] Bulk transcriptome sequencing (RNA-seq) analysis was used to characterize iPSC-derived endothelial cells. Stage 1, stage 2, and cryopreserved stage 2 samples maintained in culture for 4 days were analyzed. Differential gene expression analysis was performed by comparing stage 1 samples with stage 2 samples. Fig.11As shown, transcriptome analysis revealed an early endothelial cell population expressing venous (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.

[0109] Thus, RNA-seq characterization of endothelial cells confirmed upregulation of endothelial genes and downregulation of pluripotency, primitive streak, and mesoderm genes. Overall, upregulation of expected genes and downregulation of stem cell markers confirmed conversion of iPSCs to endothelial cells within 5 days.

[0110] Equivalent

[0111] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A method for generating human CD31+ endothelial cells (ECs), comprising culturing human early mesodermal progenitor cells in a medium containing a VEGFR agonist, a FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist to generate human CD31+ ECs. 2 . The method according to claim 1 , wherein the early mesodermal progenitor cells are cultured in the culture medium for three days. 3 . The method according to claim 1 , wherein the early mesodermal progenitor cells are obtained by culturing human pluripotent stem cells in a culture medium containing a Wnt pathway agonist for two days.

4. A method for generating human CD31+ endothelial cells (EC), comprising: (a) From day 0 to day 2, human pluripotent stem cells are cultured in a medium containing a Wnt pathway agonist to generate early mesodermal progenitor cells; as well as (b) on day 2 to day 5, culturing the early mesodermal progenitor cells in a medium containing a VEGFR agonist, a FGFR agonist, a retinoic acid pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist to generate CD31+ECs. 5 . The method according to claim 4 , wherein the EC further expresses at least one marker selected from the group consisting of KDR, vWF, FL1, VE-cadherin and CD34. The method of claim 4 , wherein the Wnt pathway agonist is a GSK-3β inhibitor.

7. The method of claim 6, wherein the GSK-3β inhibitor is CHIR99021.

8. The method of claim 7, wherein CHIR99021 is present in the culture at a concentration of 3.0-9.0 μM.

9. The method of claim 8, wherein CHIR99021 is present in the culture at a concentration of 6.0 μM.

10. The method of claim 4, wherein the VEGFR agonist is VEGF.

11. The method of claim 10, wherein the VEGF is present in the culture at a concentration of 10-50 ng / ml.

12. The method of claim 10, wherein the VEGF is present in the culture at a concentration of 25 ng / ml.

13. The method of claim 4, wherein the FGFR agonist is FGF2 or SUN11602.

14. The method of claim 13, wherein the FGFR agonist is present in the culture at a concentration of 1-20 ng / ml.

15. The method of claim 13, wherein the FGFR agonist is FGF2, which is present in the culture at a concentration of 1-20 ng / ml.

16. The method of claim 13, wherein the FGFR agonist is FGF2, which is present in the culture at a concentration of 10 ng / ml.

17. The method of claim 4, wherein the retinoic acid (RA) pathway agonist is selected from the group consisting of TTNPB, AM580, 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-oxo retinoic acid, all-trans retinoic acid (ATRA), AY 9944 dihydrochloride, Ciliobrevin A, cyclopamine, or a combination thereof.

18. The method of claim 17, wherein the RA pathway agonist is present in the culture at a concentration of 10-100 nM.

19. The method of claim 17, wherein the RA pathway agonist is TTNPB, which is present in the culture at a concentration of 25-75 nM.

20. The method of claim 17, wherein the RA pathway agonist is TTNPB, which is present in the culture at a concentration of 50 nM.

21. The method of claim 4, wherein the sonic hedgehog (SHH) antagonist is selected from the group consisting of Sant-1, saikosaponin B1, itraconazole, GANT61, MK4101, HPI-4, vismodegib, allicerrine, JK184, taladegib, Ciliobrevin D, Dynapyrazole A, Dynarrestin, GANT58, HPI1, IHR1, PF 04449913 maleate, SANT-2, U 18666A, and combinations thereof.

22. The method of claim 21, wherein the SHH antagonist is present in the culture at a concentration of 10-100 nM.

23. The method of claim 21, wherein the SHH antagonist is Sant-1, which is present in the culture at a concentration of 25-75 nM.

24. The method of claim 21, wherein the SHH antagonist is Sant-1, which is present in the culture at a concentration of 50 nM.

25. The method of claim 4, wherein 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.

26. The method of claim 25, wherein the heparin or heparin mimetic is present in the culture at a concentration of 10-50 ng / ml.

27. The method of claim 25, wherein the heparin is present in the culture at a concentration of 25-35 ng / ml.

28. The method of claim 25, wherein the heparin is present in the culture at a concentration of 20 ng / ml.

29. The method of claim 4, wherein the Wnt pathway antagonist is selected from the group consisting of XAV939, ICG-001 (Foscenvivint), capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW 67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, dendrobium, RCM-1, WIKI4, IQ-1, Advavivant, PRI-724, Tegatrabetan, or a combination thereof.

30. The method of claim 29, wherein the Wnt pathway antagonist is present in the culture at a concentration of 10-500 nM.

31. The method of claim 29, wherein the Wnt pathway antagonist is XAV939, which is present in the culture at a concentration of 50-150 nM.

32. The method of claim 29, wherein the Wnt pathway antagonist is XAV939, which is present in culture at a concentration of 100 nM.

33. The method of claim 4, wherein the pluripotent stem cells are embryonic stem cells.

34. The method of claim 4, wherein the pluripotent stem cells are induced pluripotent stem cells.

35. A culture medium for generating endothelial cells (ECs), comprising a VEGFR agonist, a FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist.

36. The culture medium of claim 35, wherein 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.

37. The culture medium of claim 36, wherein the concentration of VEGF is 25 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of TTNBP is 50 nM, the concentration of Sant-1 is 50 nM, the concentration of heparin is 20 ng / ml, and the concentration of XAV939 is 100 nM.

38. An isolated cell culture of human CD31+ endothelial cells, the culture comprising: human CD31+ endothelial cells cultured in a medium comprising a VEGFR agonist, a FGFR agonist, a retinoic acid (RA) pathway agonist, a sonic hedgehog (SHH) antagonist, heparin or a heparin mimetic, and a WNT pathway antagonist.

Images