Methods and compositions for generating hematopoietic endothelial cells from pluripotent stem cells

Abstract

Provided is a method for the production of CD31 + CD34 + CD143 + CD 309 + GATA2 + FLI1 + RUNX1 + vWF + hematopoietic endothelial cells (HEC) using a chemically defined medium, which enables the production of HEC from early mesoderm progenitor cells in a short three days and from pluripotent stem cells in a short five days. A culture medium, an isolated cell population, and a kit are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 400,944, filed on August 25, 2022, the entire contents of which are incorporated herein by reference. Background of the Invention

[0004] Vascular endothelial cells (EC) are the main cell type covering blood vessels throughout the body. During embryonic development, a special subpopulation of vascular endothelial cells, called hemogenic endothelial cells, acquires the potential to form blood cells, thereby producing hematopoietic stem cells and progenitor cells (HSPC). Hemogenic endothelial cells represent a small part (1-3% of endothelial cells in specific tissues), a transient specialized cell population that appears to initiate permanent hematopoiesis (Gritz and Hirschi (2016) Cell. Mol. Life Sci. 73: 1547-1567). The function of HSPC is to generate all blood cells in vivo throughout embryonic development and in adult life. For an overview of the development and functional regulation of hemogenic endothelial cells, see, for example, Wu and Hirschi (2021) Ann. Rev. Physiol. 83: 17-37 and Lange et al. (2021) Cell. Mol. Life Sci. 78: 4143-4160.

[0005] The earliest methodology for generating endothelial cells from pluripotent stem cells designed three different methods (e.g., see review: 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 cell self-aggregation to form embryoid bodies (EBs), and then multiple growth factors are used to promote differentiation of endothelial lineages from EBs. In the second method, differentiated ESCs or iPSCs are co-cultured with feeder layers (such as stromal cells, such as mouse cranial mesenchymal OP9 cells or bone marrow stromal cells) to promote differentiation along the endothelial lineage. In the third approach, ESCs or iPSCs are grown in two-dimensional cultures on plates coated with a protein matrix (such as Matrigel, gelatin, fibronectin, or similar proteins) under specific conditions with the addition of growth factors or small molecules.

[0006] It has been reported that the activation of classical Wnt signaling is involved in promoting the differentiation of hemogenic endothelial cells from pluripotent stem cells. It has been shown that transient treatment of iPSC with GSK-3β inhibitors (thereby activating the Wnt signaling pathway) can trigger the activation of the CDX / HOX pathway, which leads to the differentiation of iPSC to post-hematopoietic mesoderm (Kitajima et al. (2016) Exp. Hematol. 44: 68-74). A serum-free system cultivated with a GSK-3β inhibitor has been described for differentiating hemogenic endothelial cells from human pluripotent stem cells (Galat et al. (2017) Stem Cell Res. Therap. 8: 67). The subsequent scheme for generating hemogenic endothelial cells has combined Wnt pathway activation with the treatment of growth factors (such as VEGF, FGF2 and BMP4) (see, for example, Bruveris et al. (2020) Development 147: dev193037).

[0007] Thus, despite some progress, there remains a need for efficient and robust methods and compositions for generating endothelial cells from pluripotent stem cells in culture. SUMMARY OF THE INVENTION

[0009] The present disclosure provides a method for generating hemogenic endothelial cells (HECs) using a chemically defined culture medium, which can robustly generate CD31+CD34+D143+CD309+GATA2+FLI1+RUNX1+vWF+HECs from pluripotent stem cells within just five days of culture. The culture medium contains small molecule agents that can excite or antagonize specific signal transduction pathways, thereby promoting differentiation along the endothelial cell lineage. The hemogenic endothelial cells thus generated are further cultured under blood lineage-specific cell culture conditions to differentiate into short-term and long-term hematopoietic stem cells. In addition, the use of small molecule agents in the culture medium enables precise control of the culture components.

[0010] The methods and compositions disclosed herein utilize a unique cell culture medium that can generate hemogenic endothelial cells from early mesodermal progenitor cells in just three days. This unique cell culture medium contains some previously used components for endothelial cell generation, such as VEGFR agonists and FGFR agonists, but also contains new components and combinations that can robustly generate hemogenic endothelial cells. Early mesodermal progenitor cells can be obtained by culturing pluripotent stem cells under culture conditions that generate early mesodermal progenitor cells as described herein. Therefore, in certain embodiments, the method comprises a single-stage culture protocol that starts with early mesodermal progenitor cells and generates CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+ hemogenic endothelial cells within three days. In other embodiments, the method comprises a two-stage culture protocol, starting with pluripotent stem cells that are differentiated for two days in a first stage to generate early mesodermal progenitor cells, and then the early mesodermal progenitor cells are further differentiated for three days in a second stage to generate CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+ hemogenic endothelial cells, thereby forming a two-stage, five-day protocol.

[0011] Thus, in one aspect, the present disclosure relates to a method of generating human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HECs, comprising:

[0012] Human early mesodermal progenitor cells are cultured in a culture medium containing a VEGFR agonist, a FGFR agonist, a sonic hedgehog (sSHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist to generate human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC.

[0013] In one embodiment, early mesodermal progenitor cells are cultured in culture medium for three days to obtain human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HECs.

[0014] 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.

[0015] In another aspect, the present disclosure relates to a two-stage method for generating human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+ hemogenic endothelial cells (HECs), the method comprising:

[0016] (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

[0017] (b) From day 2 to day 5, early mesodermal progenitor cells are cultured in a culture medium containing: a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist to generate CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HECs.

[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, the concentration range of CHIR99021 in culture is 3.0-9.0 μM. In one embodiment, the concentration of CHIR99021 in culture is 6.0 μM.

[0019] In one embodiment, the VEGFR agonist is VEGF. In one embodiment, the concentration of VEGF in the culture ranges from 25-75 ng / ml. In one embodiment, the concentration of VEGF in the culture is 50 ng / ml.

[0020] In one embodiment, the FGFR agonist is FGF2 or SUN11602. In one embodiment, the concentration range of the FGFR agonist in culture is 1-20 ng / ml. In one embodiment, the FGFR agonist is FGF2, and its concentration range in culture is 1-20 ng / ml. In one embodiment, the FGFR agonist is FGF2, and its concentration in culture is 10 ng / ml.

[0021] In one embodiment, the sonic hedgehog (SHH) agonist is selected from the group consisting of purmorphamine, SSH, GSA 10, SAG, and combinations thereof. In one embodiment, the SHH agonist is present in the culture at a concentration ranging from 100 to 1000 nM. In one embodiment, the SHH agonist is purmorphamine, and its concentration in the culture ranges from 400 to 600 nM. In one embodiment, the SHH agonist is purmorphamine, and its concentration in the culture is 500 nM.

[0022] In one embodiment, the adenylate cyclase activator is selected from the group consisting of forskolin, NKH 477, PACAP 1-27, PACAP 1-38, adenosine, carbacycline, dopamine, endothelin 1, endothelin 1, L-(–)-epinephrine-(+)-bitartrate, glucagon, isoproterenol hydrochloride, (±)-octopamine hydrochloride, parathyroid hormone 1-34, prostaglandin D2, prostaglandin E1, prostaglandin E2, prostaglandin I2, [Arg 8 ]-Vasopressin, [Lys 8 ]-vasopressin and combinations thereof. In one embodiment, the concentration range of the adenylate cyclase activator in the culture is 0.1-10 μM. In one embodiment, the adenylate cyclase activator is forskolin, and its concentration range in the culture is 0.75-2.5 μM. In one embodiment, the adenylate cyclase activator is forskolin, and its concentration in the culture is 1.0 μM.

[0023] In one embodiment, the actin binding protein is selected from the group consisting of thymosin-β4, HMRef, α-actinin, β-spectrin, dystrophin, utrophin, filamentous protein, and combinations thereof. In one embodiment, the actin binding protein is present in the culture at a concentration range of 0.1-10 μg / ml. In one embodiment, the actin binding protein is thymosin-β4, and its concentration range in the culture is 0.75-2.5 μg / ml. In one embodiment, the actin binding protein is thymosin-β4, and its concentration in the culture is 1 μg / ml.

[0024] In one embodiment, the BMP pathway agonist is selected from the group consisting of BMP4, BMP2, BMP6, BMP7, GDF6, and combinations thereof. In one embodiment, the concentration range of the BMP pathway agonist in culture is 5-50 ng / ml. In one embodiment, the BMP pathway agonist is BMP4, and its concentration range in culture is 15-30 ng / ml. In one embodiment, the BMP pathway agonist is BMP4, and its concentration in culture is 20 ng / ml.

[0025] In one embodiment, the Wnt pathway antagonist is selected from the group consisting of XAV939, ICG-001 (PRI-724, Foscenvivint), capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797 K, JW55, JW67, JW74, cardionogen-1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanolin C, dendrobium phenol, RCM-1, WIKI4, IQ-1, Advavivant, PRI-724, Tegatrabetan, and combinations thereof. In one embodiment, the Wnt pathway antagonist is cultured at a concentration range of 10-500 nM. In one embodiment, the Wnt pathway antagonist is XAV939, which is cultured at a concentration range of 50-150 nM. In one embodiment, the Wnt pathway antagonist is XAV939, which is cultured at a concentration of 100 nM.

[0026] In one embodiment, the retinoic acid (RA) pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, CD437, Ch55, BMS753, BMS961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid and all-trans retinoic acid (ATRA), AY9944 dihydrochloride, Ciliobrevin A, cyclopamine, and combinations thereof. In one embodiment, the RA pathway agonist is in a culture at a concentration range of 100-1000 nM. In one embodiment, the RA pathway agonist is TTNPB, which is in a culture at a concentration range of 400-600 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in culture at a concentration of 500 nM.

[0027] 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.

[0028] On the other hand, the present disclosure relates to a culture medium for generating HEC, comprising a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylate cyclase activator is forskolin, the actin binding protein is thymosin-β4, the BMP pathway agonist is BMP4, the Wnt pathway antagonist is XAV939, and the RA receptor agonist is retinoic acid. In one embodiment, the concentration of VEGF is 50 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of purmorphamine is 500 nM, the concentration of forskolin is 1 μM, the concentration of thymosin-β4 is 1 μg / ml, the concentration of BMP4 is 20 ng / ml, the concentration of XAV939 is 100 nM and the concentration of retinoic acid is 500 nM.

[0029] In another aspect, the present disclosure relates to a cell culture of isolated human hematuric endothelial cells, comprising: human CD31+CD34+CD143+CD309+GATA2+FLI 1+RUNX1+vWF+HEC cultured in a culture medium comprising a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist.

[0030] 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

[0032] Figure 1 is a schematic diagram of a representative culture protocol for generating hemogenic endothelial cells from early mesodermal progenitors in three days, and a schematic diagram of the two-day pretreatment for generating early mesodermal progenitors from iPSCs.

[0033] Figure 2 Results of an 8-factor experimental HD-DoE model optimized for GATA2 maximum expression are shown. The upper part of the model shows the predictions of the expression levels of the 52 genes pre-selected for GATA2 optimization. The lower part of the model shows the effectors tested in the model and their contribution to GATA2 maximum expression. The value column refers to the concentration of each effector required to simulate the model.

[0034] Figure 3 Results from an 8-factor experimental HD-DoE model optimized for maximal expression of GATA2 are shown. Figure 2 same.

[0035] Figure 4 Results from an 8-factor experimental HD-DoE model optimized for maximal expression of GATA2 are shown. Figure 2 same.

[0036] Figure 5 Results from an 8-factor experimental HD-DoE model optimized for maximal expression of GATA2 are shown. Figure 2 same.

[0037] Figure 6 Results from an 8-factor experimental HD-DoE model optimized for maximal expression of GATA2 are shown. Figure 2 same.

[0038] Figure 7A-7B The dynamic distribution analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1 and CD44 relative to the concentrations of three validated effectors is shown. The effects of VEGF, forskolin and FGF2 on the expression of the above genes and their factorial contributions are shown by the slope of the curve for each effector.

[0039] Figures 8A-8B The dynamic distribution analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1 and CD44 relative to the concentration of three validated effectors is shown. The influence of VEGF, thymosin-β4 and FGF2 on the expression of the above genes and their factorial contribution are shown by the slope of the curve of each effector.

[0040] Figures 9A-9B The dynamic distribution analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1 and CD44 relative to the concentration of three validated effectors is shown. The effects of purmorphamine, VEGF and B27 on the expression of the above genes and their factorial contributions are shown by the slope of the curve for each effector.

[0041] Figures 10A-10B The dynamic distribution analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1 and CD44 relative to the concentrations of four validated effectors is shown. The effects of XAV939, VEGF, FGF2 and L-ascorbic acid on the expression of the above genes and their factorial contributions are shown by the slope of the curve for each effector.

[0042] Figures 11A-11BThe dynamic distribution analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1 and CD44 relative to the concentration of three validated effectors is shown. The effects of retinoic acid, BMP4 and VEGF on the expression of the above genes and their factorial contributions are shown by the slope of the curve for each effector.

[0043] Figures 12A-12B Flow cytometric staining results of iPSC-derived HECs at the end of stage 2 are shown. Fig. 12A Cells stained with CD31, CD34, CD61, CD143 and CD309 (KDR) are shown. At this stage, cells were positive for all expected HEC markers. n=5 for CD31, CD34, CD61 and KDR, and n=2 for CD143. Fig. 12B Depicted are increases in CD31, CD34, and CD61 in culture medium containing small molecules as described herein and compared to culture medium alone with the known endothelial cell differentiation proteins VEGF and FGF2.

[0044] Fig.13A Representative graphs of lineage-determining gene sets induced by factor combinations on mesoderm-committed cells are shown for endothelial (blue) and hemogenic endothelial (red) tissues. Cells were exposed to stage 1 media for 2 days prior to factor addition. The graph was generated using the HDB unsupervised clustering algorithm. Conditions have been identified for the expression of genes enriched in hemogenic endothelial cells, such as GATA2, TAL1, and ETV2, and for genes expressed by endothelial cells, such as PECAM1 and ERG.

[0045] Fig. 13B are photographs showing RUNX1 -positive HECs differentiated from iPSCs using the Stage 2 formulation described herein, and RUNX1 -negative endothelial cells differentiated from iPSCs using a different Stage 2 formulation optimized for endothelial cell differentiation.

[0046] Fig.14 A fluorescent image of iPSC-derived hemogenic endothelial cells at the end of stage 2 is shown. Cells were stained with hemogenic endothelial cell biomarkers including CD31, FLI1, CD144, CD309 (KDR), vWF, RUNX1, GATA2, and SOX17. At this stage, cells were positive for all expected hemogenic endothelial cell markers.

[0047] Fig.15The flow cytometry staining results of iPSC-derived HEC from 2 iPSC cell lines (iX cell CR0000001 and REPROCELL 771-3G) at the end of the second stage are shown. Cells were stained with HEC markers RUNX1, CD34, CD43, and CD31. CR0000001 and 771-3G cell lines were positive for all markers at the expected expression levels. Both cell lines showed similar HEC marker expression levels.

[0048] Fig.16 A large number of RNA-seq results for characterizing iPSC-derived HEC are shown. iPSC is differentiated into the lateral plate mesoderm as described above (Phase 1). Then, the mesodermal cells are treated with Phase 2 culture medium for 3 days. The heat map shows the Z score of transcripts per kilobase (TPM) from a large number of RNA-seq characterizations. At the end of Phase 2, RNA-seq shows the upregulation of Phase 2 HEC genes, and the downregulation of omnipotence, primitive streak and mesoderm genes in Phase 0 and Phase 1. TPM values ​​above the average are shown in red, and those below the average are shown in blue. DETAILED DESCRIPTION OF THE INVENTION

[0050] This article describes methods and compositions that can robustly generate CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+ hemogenic endothelial cells (HECs) from iPSCs in only five days under chemically defined culture conditions using small molecule-based methods. A high-dimensional experimental design (HD-DoE) approach was used to simultaneously test the effects of multiple process inputs (e.g., small molecule agonists or antagonists) on output responses (such as gene expression). These experiments enabled the identification of chemically defined culture media that contained agonists and / or antagonists of specific signaling pathways, which were sufficient to generate HECs 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 eliminating individual agonists or antagonists. Flow cytometry and immunocytochemistry were used to further confirm the phenotypes of cells generated by the differentiation protocol.

[0051] Various aspects of the application are described in further detail in the following subsections.

[0052] I. cell

[0053] The starting cell used in the present disclosure is generally a human pluripotent stem cell, which is used to generate early mesodermal progenitor cells, which are subsequently used to obtain hemogenic endothelial cells. As used herein, the term "human pluripotent stem cell" (abbreviated as hPSC) refers to a human stem cell with the ability to be differentiated into a variety of different cell types. As used herein, the term "pluripotent" refers to the ability of a cell to differentiate into the cell type characteristics of all three germ cell layers (endoderm, mesoderm and ectoderm) under different conditions. Pluripotent cells are mainly characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation tests. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is to prove the ability of cells that differentiate into each of the three germ layers.

[0054] For example, human pluripotent stem cells include induced pluripotent stem cells (iPSC) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of inducible iPSCs 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). hPSCs express markers that can be used to identify cells as PSCs. 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 methods of generating HECs disclosed herein are used to differentiate (mature) a starting pluripotent stem cell population, in various embodiments, the HEC population generated by the methods disclosed herein lacks 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.

[0055] As described herein, pluripotent stem cells are placed under culture conditions that induce cell differentiation. 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 to a specific cell lineage. In order to generate hemogenic endothelial cells from pluripotent stem cells, stem cells are first differentiated into mesoderm commitment.

[0056] As used herein, "early mesodermal progenitor cells" refer to cells that are more differentiated than pluripotent stem cells and have been directed to the mesodermal lineage. As described herein, early mesodermal progenitor cells can be obtained from PSC culture by activating Wnt signaling with an agent (such as a GSK-3β inhibitor, such as CHIR99021 for two days).

[0057] 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 used to characterize cells of interest include: Brachyury (T) and KDR as biomarkers for early mesodermal progenitor cells, and CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, and vWF as biomarkers for differentiated hemogenic endothelial cells.

[0058] GATA2 is a transcription factor that has been identified as a master regulator of hematopoiesis and promotes the generation of HEC (Castano, J. et al. (2019) Stem Cell Reports 13: 515-529). RUNX1 is a transcription factor that controls the emergence of defined HECs and is widely considered to be their key marker (Ling, M. et al. (2014) Blood 11: e11-e20). These two master hematopoietic regulators, GATA2 and RUNX1, have shown parallel increased expression in iPSC-derived HECs (Castano, J. et al. (2019) Stem Cell Reports 13: 515-529). SOX17 is a transcription factor that has been identified as a master regulator of the arterial program in the hemogenic endothelium and is essential for the specificity of hemogenic endothelial cells (Jung et al. (2021) Cell Rep. 34: 108758). It has been shown that CD143 (ACE) marks early hematopoietic stem cells (Fadlullah, MZH et al. (2022) Blood 139: 343-346; Jokubaitis, V. et al. (2008) Blood 111: 4055-4063). CD44 has been shown to be a marker for HEC and a regulatory factor for the transition of endothelial cells to hematopoietic cells (Oatley, M. et al. (2020) Nature Communications 11: 586). In addition, TAL1 is a transcription factor that is essential for maintaining the multipotency of hematopoietic stem cells (HSPC) and can be detected in primitive HSPCs (such as HEC) (Real, P. et al. (2012) Molecular Therapy 20: 1443-1453).

[0059] II. Medium composition

[0060] The method for generating hemogenic endothelial cells from pluripotent stem cells disclosed herein comprises: culturing early mesodermal progenitor cells in a culture medium comprising agonists and / or antagonists of specific cell receptors and / or signal transduction pathways to generate differentiated HECs. In addition, early mesodermal progenitor cells can be obtained from pluripotent stem cells (e.g., ESC or iPSC), by culturing stem cells under defined culture conditions as described herein.

[0061] A culture medium containing a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist is sufficient to generate CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+ hemogenic endothelial cells from early mesodermal progenitor cells in just three days of culture. Early mesodermal progenitor cells can be obtained from PSCs by culturing with a Wnt pathway agonist for two days, resulting in a total of five days of protocol for obtaining differentiated HECs from PSCs under defined culture conditions.

[0062] As used herein, an "agonist" of a cell receptor or signaling pathway is intended to refer to an agent that stimulates (upregulates) a cell receptor or signaling pathway. Stimulation of a cell signaling pathway can be initiated from outside the cell, such as by using an agonist that activates a cell surface receptor in the signaling pathway (e.g., an agonist can be a receptor ligand). In addition or alternatively, stimulation of cell signaling can be initiated from within the cell, such as by using a small molecule agonist that interacts with a component of the signaling pathway within the cell.

[0063] As used herein, an "antagonist" of a cell signaling pathway is intended to refer to an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated from outside the cell, such as by using an antagonist that blocks a cell surface receptor in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated from within the cell, such as by using a small molecule antagonist that interacts with a component of the signaling pathway within the cell.

[0064] The agonists and antagonists used in the culture medium and method of chemical composition determination of the present disclosure are known in the art and can be commercially available. Their use concentration in the culture medium is effective to achieve the desired result, such as generating the target cell (such as early mesodermal progenitor cell or differentiated endothelial cell) expressing the target marker. Non-limiting examples of suitable agonist and antagonist reagents and effective concentration ranges are further described below.

[0065] 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 to VEGFR). In one embodiment, the VEGFR agonist is VEGF or an analog thereof, which stimulates the signal through VEGR. In one embodiment, the VEGFR agonist is VEGF (e.g., recombinant human VEGF). In one embodiment, the VEGFR agonist is VEGF, and its concentration in the culture medium ranges from 10-100 ng / ml, 25-75 ng / ml, 40-60 ng / ml, 45-55 ng / ml or a concentration of 50 ng / ml.

[0066] The agonist of FGFR pathway comprises the reagent, molecule, compound or material that can stimulate (upregulate) fibroblast growth factor receptor signaling pathway (it is activated by the combination of FGF and FGFR biologically).In one embodiment, FGFR agonist is FGF2, SUN11602 or its combination.In one embodiment, the concentration range of FGFR pathway agonist in culture medium is 1-20ng / ml, 5-15ng / ml, 7.5-12.5ng / ml, 9-11ng / ml or concentration is 10ng / ml.In one embodiment, FGFR agonist is FGF2 (for example, recombinant human FGF2).In one embodiment, FGFR agonist is FGF2, and its concentration range in culture medium is 1-20ng / ml, 5-15ng / ml, 7.5-12.5ng / ml, 9-11ng / ml or concentration is 10ng / ml.

[0067] Agonists of the sonic hedgehog (SHH) pathway include agents, molecules, compounds or substances that can stimulate (activate) signals through the SHH pathway, which biologically involves the binding of SHH to the Patched-1 (PTCH1) receptor and the conduction through the smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway agonist is selected from the group consisting of purmorphamine, SSH, GSA10, SAG and combinations thereof. In one embodiment, the concentration range of the SHH pathway agonist in the culture medium is 100-1000nM, 200-800nM, 250-750nM or 450-550nM, or a concentration of 500nM. In one embodiment, the SHH pathway agonist is purmorphamine. In one embodiment, the SHH pathway agonist is purmorphamine, and its concentration range in the culture medium is 100-1000nM, 200-800nM, 250-750nM or 500-600nM. In one embodiment, the SHH pathway agonist is purmorphamine, which is present in the culture medium at a concentration of 500 nM.

[0068] Adenylate cyclase activators include agents, molecules, compounds or substances that can stimulate (upregulate) the activity of adenylate cyclase (also referred to in the art as adenylate cyclase and adenylate cyclase), which catalyzes the conversion of ATP into cAMP and pyrophosphate. In one embodiment, the adenylate cyclase activator is selected from the group consisting of forskolin, NKH477, PACAP 1-27, PACAP1-38, adenosine, carbacycline, dopamine, endothelin 1, endothelin 1, L-(–)-epinephrine-(+)-bitartrate, glucagon, isoproterenol hydrochloride, (±)-octopamine hydrochloride, parathyroid hormone 1-34, prostaglandin D2, prostaglandin E1, prostaglandin E2, prostaglandin I2, [Arg 8 ]-Vasopressin, [Lys 8 ]-vasopressin and combinations thereof. In one embodiment, the concentration range of the adenylate cyclase activator in the culture medium is 0.1-10 μM, 0.5-5 μM, 0.75-2.5 μM or 0.9-1.1 μM, or the concentration is 1 μM. In one embodiment, the adenylate cyclase activator is forskolin. In one embodiment, the adenylate cyclase activator is forskolin, and the concentration range in the culture medium is 0.1-10 μM, 0.5-5 μM, 0.75-2.5 μM or 0.9-1.1 μM. In one embodiment, the adenylate cyclase activator is forskolin, and the concentration range in the culture medium is 1.0 μM.

[0069] Actin binding protein (also referred to as ABP) is a protein that binds to actin monomers, actin polymers, or both. ABPs known in the art are of various kinds, and non-limiting examples include thymosin-β4, HMRef, α-actinin, β-spectrin, dystrophin, dystrophin-related proteins, and fascin. In one embodiment, the ABP is thymosin. In one embodiment, thymosin is thymosin-β4 or thymosin-α1. In one embodiment, the concentration range of actin binding protein in culture medium is 0.1-10 μg / ml, 0.5-5 μg / ml, 0.75-2.5 μg / ml, or 0.9-1.1 μg / ml, or a concentration of 1 μg / ml. In one embodiment, the actin binding protein is thymosin-β4. In one embodiment, the actin binding protein is thymosin-β4, and its concentration in the culture medium ranges from 0.1-10 μg / ml, 0.5-5 μg / ml, 0.75-2.5 μg / ml, or 0.9-1.1 μg / ml. In one embodiment, the actin binding protein is thymosin-β4, and its concentration in the culture medium is 1 μg / ml.

[0070] Agonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds or substances that can stimulate (activate or upregulate) BMPs (which are biologically activated by the binding of BMPs to BMP receptors). BMP receptors (BMPRs) are activin receptor-like kinases (ALKs) (e.g., type I BMP receptors, including but not limited to ALK2 and ALK3). In one embodiment, the BMP pathway agonist is selected from the group consisting of BMP4, BMP2, BMP6, BMP7, GDF6, and combinations thereof. In one embodiment, the concentration range of the BMP pathway agonist in the culture medium is 5-50 ng / ml, 10-40 ng / ml, 15-30 ng / ml, or 20-25 ng / ml or a concentration of 20 ng / ml. In one embodiment, the BMP pathway agonist is BMP4. In one embodiment, the BMP pathway agonist is BMP4, and its concentration range in the culture medium is 5-50 ng / ml, 10-40 ng / ml, 15-30 ng / ml, or 20-25 ng / ml. In one embodiment, the BMP pathway agonist is BMP4, which is present in the culture medium at a concentration of 20 ng / ml.

[0071] Antagonists of the WNT pathway include agents, molecules, compounds or substances that are capable of inhibiting (downregulating) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of Wnt protein ligands to Frizzled family receptors. 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, triptolide, KYA1797K, JW55, JW 67, JW74, cardiomyogen-1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercetin, lanatoside C, dendrobium, RCM-1, WIKI4, IQ-1, Adavivant, PRI-724, tegatrabectin, and combinations thereof. In one embodiment, the concentration range of the WNT pathway antagonist in the culture medium is 10-500nM, 50-250nM, 50-150nM, 75-125nM or a concentration of 100nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939, and its concentration range in the culture medium is 10-500nM, 50-250nM, 50-150nM or 75-125nM. In one embodiment, the WNT pathway antagonist is XAV939, and its concentration in the culture medium is 100nM.

[0072] Agonists of the retinoic acid (RA) pathway include agents, molecules, compounds or substances that can stimulate the retinoic acid receptor (RAR), which can be activated by all-trans retinoic acid and 9-cis retinoic acid. There are three 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 have been synthesized that are able to activate the retinoic acid pathway. Non-limiting examples of these compounds include TTNPB (agonist of RAR-α, β and γ), AM580 (RAR-α agonist), CD1530 (potent and selective RAR-γ agonist), CD2314 (selective RAR-β agonist), Ch55 (potent RAR agonist), BMS753 (RAR-α selective agonist), tazarotene (receptor selective retinol; binds RAR-β and RAR-γ), isotretinoin (endogenous agonist of retinoic acid receptor; inducer of neural differentiation) and AC261066 (RARβ2 agonist). In certain embodiments, the RA signaling pathway agonist is selected from the group consisting of i) a retinol 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-oxo retinoic acid, and all-trans retinoic acid (ATRA).

[0073] Therefore, in one embodiment, the RA pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, CD437, Ch55, BMS753, BMS961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC 55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid and all-trans retinoic acid (ATRA), AY9944 dihydrochloride, Ciliobrevin A, cyclopamine, and combinations thereof. In one embodiment, the RA pathway agonist is present in the culture medium at a concentration range of 100-1000 nM, 200-800 nM, 250-750 nM, or 400-600 nM, or at a concentration of 500 nM. In one embodiment, the RA pathway agonist is retinoic acid (RA). In one embodiment, the RA pathway agonist is retinoic acid (RA) at a concentration range of 100-1000 nM, 200-800 nM, 250-750 nM or 400-600 nM in the culture medium. In one embodiment, the RA pathway agonist is retinoic acid (RA) at a concentration of 500 nM in the culture medium.

[0074] 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, SB216763, SB415286, LY2090314, 3F8, A1070722, AR-A014418, BIO, BIO-acetoxime, AZD1080, WNT3A, Atepolone, Indirubin-3'-monoxime, 1-azacamperolone, Clairani, TC-G 24, TDZD8, TWS119, NP 031112, AT7519, KY19382, AZD2858, and combinations thereof. In one embodiment, the WNT pathway agonist is present in the culture medium at a concentration 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 embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist is CHIR99021, and its concentration in the culture medium ranges from 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, and its concentration in the culture medium is 6.0 μM (e.g., in stage 1 culture medium for generating early mesodermal progenitor cells).

[0075] III. Culture conditions

[0076] In combination with the chemically defined and optimized culture medium described in Section 2 above, the method of generating hemogenic endothelial cells disclosed herein utilizes standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37°C and 5% CO2. Cells can be cultured in standard culture containers or culture 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, before the differentiation protocol begins, stem cells can be cultured in a dedicated stem cell culture medium (such as Essential8Flex culture medium, Thermo Fisher #A2858501) for at least one day. In a non-limiting exemplary embodiment, stem cells are cultured at 41,666 cells / cm 2 Cells were passaged at a density of 1:1 onto 6-well plates coated with vitronectin (Thermo Fisher #A14700) and cultured in Essential 8Flex medium for one day prior to differentiation.

[0077] When the differentiation protocol is initiated from stem cells, the medium in which the stem cells are being cultured is replaced with a basal differentiation medium supplemented with a Wnt signaling pathway agonist (such as a GSK-3β inhibitor, e.g., CHIR99021) as described in Section 2 above, which is referred to as stage 1 of the differentiation protocol. For example, the basal differentiation medium may comprise a commercially available basal medium supplemented with other standard medium components required to maintain cell viability and growth, but typically without serum (basal differentiation medium is a serum-free medium).

[0078] In one non-limiting exemplary embodiment, the basal differentiation medium in stage 1 of the differentiation protocol is CDM2 medium (e.g. Figure 1 CDM2 medium contains 0.5x IMDM (ThermoFisher #12440046), 0.5x F12 (Thermo Fisher #11765047), 1 mg / ml polyvinyl alcohol (Sigma #p8136), 1% chemically defined lipid concentrate (Thermo Fisher #11905031), 450 μM 1-thioglycerol (Sigma #M6145), 0.7 μg / ml insulin (Sigma #11376497001), and 15 μg / ml transferrin (Sigma #10652202001).

[0079] In one non-limiting exemplary embodiment, the basal differentiation medium (e.g. Figure 1 ) is commercially available RPMI medium containing 2% B-27 supplement, 100 μg / ml L-ascorbic acid, and 1% penicillin / streptomycin.

[0080] In certain embodiments, the starting pluripotent stem cells adhere to a culture plate, preferably coated with an extracellular matrix material, such as vitronectin. In one embodiment, the stem cells are cultured on a culture surface coated with vitronectin (e.g., a 96-well plate coated with vitronectin).

[0081] The culture medium is usually replaced with fresh culture medium regularly. For example, in one embodiment, the culture medium is replaced every 24 hours.

[0082] In order to generate early mesodermal progenitor cells, starting pluripotent stem cells are cultured in a culture medium containing a GSK-3β inhibitor (e.g., CHIR99021) for a sufficient period of time (generally two days) to achieve cell differentiation and expression of markers associated with directed early mesodermal progenitor cells. This protocol for generating early mesodermal progenitor cells from PSC is referred to herein as "step (a)" or "stage 1".

[0083] In order to generate differentiated hemogenic endothelial cells from early mesodermal progenitor cells, the progenitor cells are cultured in a culture medium for a sufficient period of time (usually three days) to achieve cell differentiation and hemogenic endothelial cell-related markers (e.g., CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+) expression, the culture medium comprises a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist. This scheme of generating hemogenic endothelial cells from early mesodermal progenitor cells is referred to herein as "step (b)" or "Phase 2".

[0084] In various embodiments, early mesodermal progenitor cells are cultured in an optimized culture medium for a sufficient period of time to increase the expression of at least one and preferably multiple hemogenic endothelial cell-related markers. Suitable non-limiting examples of hemogenic EC-related markers include CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, and vWF. In embodiments, cells are cultured for a sufficient period of time to increase the expression levels of at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight hemogenic EC-related markers. In one embodiment, compared to the starting cell population, cells are cultured for a sufficient period of time to increase the expression level of at least one hemogenic EC-related marker by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The expression levels of markers in cultured HECs can be measured by techniques available in the art (e.g., RNAseq analysis and / or flow cytometry).

[0085] Thus, in the first stage method of generating early mesodermal progenitors from PSCs, pluripotent stem cells are cultured in a stage 1 optimized medium from day 0 to day 2, or starting from day 0 and continuing to day 2, or continuing 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.

[0086] Therefore, in the second stage method of generating hemogenic endothelial cells from early mesodermal progenitor cells, the early mesodermal progenitor cells generated in stage 1 are replated and 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.

[0087] The culture medium is usually replaced with fresh culture medium regularly. For example, in certain embodiments, the culture medium is replaced every 24 hours, every 48 hours or every 72 hours.

[0088] IV. use

[0089] The methods and compositions disclosed herein for generating hemogenic endothelial cells provide these cell populations with efficient and robust availability for a variety of purposes. For example, these methods and compositions can be used to study hematopoietic development and differentiation, including to assist in understanding the biology of hematopoietic diseases and conditions. For example, HEC generated using the methods disclosed herein can be further purified using reagents that are combined with surface markers expressed on cells according to established methods in the art.

[0090] The HEC obtained according to the method disclosed herein can be further cultured under blood lineage specific cell culture conditions to differentiate into short-term and long-term hematopoietic stem cells. Therefore, the HEC obtained according to the method disclosed herein provides an opportunity to study the hematopoietic system and its developmental functions. Other uses include 3D bioprinting, drug screening, safety assessment, vascular tissue engineering and disease modeling.

[0091] It is also contemplated that HEC generated according to the methods of the present disclosure or further differentiated hematopoietic lineage cells derived therefrom are used for the treatment of various hematopoietic diseases and disorders, for example, by delivering the cells to a subject suffering from the disease or symptom. Examples of hematopoietic diseases and disorders include, but are not limited to, cancers (such as leukemias and lymphomas), blood diseases, and autoimmune diseases.

[0092] V. Composition

[0093] In other aspects, the present disclosure provides compositions related to methods of generating hemogenic endothelial cells, comprising culture medium and isolated cell cultures.

[0094] On the one hand, the present disclosure provides a culture medium for generating CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC, which comprises a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist. Non-limiting examples of suitable agents and their concentrations include those described in Section 2 above. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylate cyclase activator is forskolin, the actin binding protein is thymosin-β4, the BMP signaling pathway agonist is BMP4, the Wnt pathway antagonist is XAV939, and the RA receptor agonist is retinoic acid. In one embodiment, the concentration of VEGF is 50 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of purmorphamine is 500 nM, the concentration of forskolin is 1 μM, the concentration of thymosin-β4 is 1 μg / ml, the concentration of BMP4 is 20 ng / ml, the concentration of XAV939 is 100 nM and the concentration of retinoic acid is 500 nM.

[0095] On the other hand, the present disclosure provides a cell culture of isolated human hematuric endothelial cells (EC), the culture comprising: human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC cultured in a medium comprising a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist. Non-limiting examples of suitable agents and their concentrations include those described in Section 2 above. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylate cyclase activator is forskolin, the actin binding protein is thymosin-β4, the BMP signaling pathway agonist is BMP4, the Wnt signaling pathway antagonist is XAV939, and the RA receptor agonist is retinoic acid. In one embodiment, the concentration of VEGF is 50 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of purmorphamine is 500 nM, the concentration of forskolin is 1 μM, the concentration of thymosin-β4 is 1 μg / ml, the concentration of BMP4 is 20 ng / ml, the concentration of XAV939 is 100 nM and the concentration of retinoic acid is 500 nM.

[0096] The present application is further illustrated by the following examples, which should not be construed as further limiting.The contents of all figures and references, patents and published patent applications cited in this application are expressly incorporated herein by reference. Example

[0097] Example 1: Development of a culture protocol for generating hemogenic endothelial cells from pluripotent stem cells

[0098] A two-stage formulation for generating hemogenic endothelial cells from pluripotent stem cells was developed, which can direct human iPSCs to hemogenic endothelial cells expressing CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, vWF, and VE-cadherin within 5 days of culture. Hemogenic endothelial cells were generated from pluripotent stem cells by a two-stage protocol, which is schematically shown in Figure 1 middle.

[0099] Briefly, based on an established early differentiation protocol along the endothelial lineage in the art, the starting PSCs are first differentiated into early mesodermal progenitors by culturing the PSCs in a medium containing a GSK-3β inhibitor. In one embodiment, the PSCs are cultured in a medium containing 6 μM CHIR99021 for two days (Day 0 to Day 2).

[0100] Early mesodermal progenitor cells were further used for differentiation along the endothelial lineage, which employed a high-dimensional experimental design (HD-DoE) approach to simultaneously test the effects of multiple process inputs (e.g., small molecule agonists or antagonists) on output responses (e.g., gene expression). Based on the predicted conditions that maximized the expression of hemogenic endothelial cell-enriched genes (e.g., CD31, CD34, CD143, CD309, GATA2, FLI1 and RUNX1, vWF), a complex recipe was developed for generating differentiated hemogenic endothelial cells from early mesodermal progenitor cells, which consisted of 8 reagents, as shown in Table 1 below:

[0101] Table 1 :Hemogenic Endothelial Cell Generation Medium Formulation

[0102] Reagents effect concentration VEGF VEGFR agonists 50ng / mL FGF2 FGFR agonists 10 ng / mL Purmorphamine SHH pathway agonists 500nM Forskolin Adenylate cyclase activator 1μM Thymosin-β4 Actin-binding protein 1μg / ml BMP4 BMP pathway agonists 20ng / ml XAV939 WNT pathway antagonists 100nM Retinoic acid (RA) RA pathway agonists 500nM

[0103] This formulation is referred to in this disclosure as the second-stage formulation for generating hemogenic endothelial cells. The development of the two-stage formulation is described in detail below.

[0104] First, undifferentiated iPSCs were cultured in CDM2 medium containing 6 μM CHIR99021 for 48 hours (referred to herein as the first phase). The second phase culture medium was subsequently developed. The basis of this is an 8-factor HD-DoE experiment that focuses on the differentiation of cells to the hemogenic endothelial lineage for an additional 3 days after the termination of the first phase treatment. To test the effector, 48 different effector combinations generated using experimental compression design by D-optimality were automatically prepared. These effector combinations were prepared in basal medium and subsequently added to the cells, which were then allowed to differentiate. After 3 days, RNA extraction was performed and gene expression was obtained by using quantitative PCR analysis. The data was standardized and the effector design was modeled using partial least squares regression analysis, so that a gene-specific model was generated, which was adjusted by the model to obtain the maximum Q2 predictive power, explaining the ability of the effector combination and the control of the expression of a single gene alone. Solutions within the test space can then be explored to meet desirability. For the second phase, conditions were created to induce the expression of GATA2, a transcription factor identified as a master regulator of hematopoiesis. GATA2 promotes the generation of HECs and promotes the endothelial to hematopoietic transition (EHT). GATA2 also inhibits cardiac differentiation at the mesoderm stage, which directs cells toward a hematopoietic fate. Optimizing for maximum GATA2 expression resulted in a robust solution. Other genes were also predicted to be highly expressed in this solution, such as CD34, ERG, FLI1, HOPX, KDR, LMO2, and CD31, all of which are highly expressed on hematopoietic endothelial cells, indicating that the cells are committed to this lineage ( Figure 2 ). High levels of KDR (an important receptor for VEGF signaling) were observed in this model. GATA6, PAX6, OTX2, and SOX1 (genes associated with ectoderm and endoderm) were also downregulated. In addition, genes associated with early stages (such as NODAL and EOMES) were also downregulated, indicating that the cells are differentiating. This model was derived from an initial test of eight factors, including: Forskolin, VEGF, FGF2, Albumax, SUN1162, Hydrocortisone, SB341542, and Resveratrol. Three of the effectors: Forskolin, VEGF, and FGF2 showed a positive effect on the expression of the target gene, with factor contributions of 27, 46, and 17, respectively ( Figure 2 ). Factors with low contributions were excluded. Within the specification of achieving about 86% of maximum GATA2 expression, the Cpk value (process capability index) of this complex medium composition was 0.69, corresponding to a risk of failure of 1.9%.

[0105] Next, additional factors were evaluated to potentially increase the complexity of signaling inputs for effective fate control. As previously described, we focused on the expression of GATA2. Optimization for maximal GATA2 expression resulted in a robust solution. In this solution, other genes were also predicted to be abundantly expressed, such as CD34, CD44, ERG, FLI1, KDR, LMO2, TAL1, and vWF, all genes associated with the hemogenic endothelial program, which indicates that the cells are committed to this lineage ( Figure 3 ). Again, high levels of KDR were observed. This model was derived from an initial test of eight factors: CHIR, VEGF, FGF2, TTNBP, AGN194310, Y27632, thymosin-β4, and heparin. Two of these effectors, VEGF and thymosin-β4, showed a positive effect on the expression of the target gene, with factor contributions of 25 and 16, respectively ( Figure 3 ). CHIR, TTNBP, AGN194310, Y27632, and heparin were not included in this formulation because sufficient GATA2 induction was obtained in their absence and they have relatively low factor contributions. Although FGF2 has a low factor contribution in this model, it was included in this formulation because it had a factor contribution of ≥16 in 2 of the 3 models. Within the specification of achieving approximately 87% maximum GATA2 expression, the Cpk value (process capability index) of this complex media composition was 0.7, corresponding to a 2% risk of failure.

[0106] To further refine the recipe for hemogenic endothelial cell differentiation, additional HD-DoEs were performed. This model was derived from an initial test of eight factors: YHHU, DBZ, purmorphamine, SANT1, LPA, Yoda1, VEGF, and B-27. Two of these effectors, purmorphamine and B-27, showed a positive effect on the expression of the target gene, with factor contributions of 20 and 25, respectively. Figure 4 ). Except for VEGF, the remaining factors were not included in this recipe because sufficient GATA2 induction was obtained in their absence and they had relatively low factor contributions. VEGF remained in the recipe because 4 out of 5 models showed a strong positive effect on GATA2 induction. Other genes were also predicted to be highly expressed in this solution, such as CD34, ERG, FLI1, ETV2 and TAL1, all of which are highly expressed on hemogenic endothelial cells, which indicates that the cells are committed to this lineage (see Figure 4 Within the specification of achieving approximately 84% maximal GATA2 expression, this complex medium composition had a Cpk value (process capability index) of 0.7, corresponding to a risk of failure of 1.4%.

[0107] A fourth modeling experiment was conducted to continue improving the formula for hemogenic endothelial cell differentiation. This model was derived from the initial testing of eight factors: VEGF, FGF2, sphingosine 1 phosphate, VH298, Pyrintegrin, erythropoietin, L-ascorbic acid, and XAV939. Four of them, VEGF, FGF2, L-ascorbic acid, and XAV939, showed a positive effect on the expression of the target gene, with factor contributions of 22, 22, 5, and 16, respectively. Figure 5 ). L-ascorbic acid was included in the basal medium as is. None of the remaining factors were included in this formulation, as sufficient GATA2 induction was obtained in their absence and they have relatively low factor contributions. Other genes were also predicted to be highly expressed in this solution, such as CD34, CD44, CDH5, LMO2, MECOM, and TAL1, which are highly expressed on hemogenic endothelial cells, indicating that the cells are committed to this lineage (see Figure 5 ). Within the specification of achieving approximately 82% maximal GATA2 expression, this complex medium composition had a Cpk value (process capability index) of 0.7, corresponding to a risk of failure of 2.2%.

[0108] The fifth and final modeling was performed to finalize the recipe optimized for hemogenic endothelial cell differentiation. This model was derived from the initial testing of eight factors: VPA, SCF, BMP4, EFG, FLT3L, arginine, retinoic acid, and VEGF. Three of them, retinoic acid, BMP4, and VEGF, showed a positive effect on the expression of the target gene, with factor contributions of 22, 31, and 22, respectively. Figure 6 ). None of the remaining factors were included in this recipe, as sufficient GATA2 induction was obtained in their absence and their factor contributions were relatively low. Other genes were also predicted to be highly expressed in this solution, such as CD34, CDH5, ERG, FLI1, LMO2, and CD31, which are highly expressed on hemogenic endothelial cells, indicating that the cells are committed to this lineage ( Figure 6). Within the specification of achieving approximately 81% maximum GATA2 expression, the Cpk value (process capability index) of this complex medium composition was 0.9, corresponding to a risk of failure of 0.51%. Considering all models analyzed, a complex formula was developed for generating differentiated hemogenic endothelial cells from early mesodermal progenitors based on the predicted conditions of maximizing the expression of hemogenic endothelial cell-enriched genes (such as GATA2, LMO2, ERG, CDH5, CD31, FLI1, CD34, TAL1, MECOM, MYB), which consists of 8 effectors as shown in Table 1 above.

[0109] Example 2 :Analysis of the importance of factors in the induction culture conditions of hemogenic endothelial cells

[0110] The various contributing factors for each input of the Phase 2 recipe for inducing hemogenic endothelial cells showed different relative impacts. To assess the impact of each validated factor on endothelial lineage-associated genes, dynamic distribution analysis was used and the expression levels of the target genes were compared when each final factor was removed and kept in the presence of the other factors. Since the expression level of the target gene reveals whether the desired outcome can be achieved, this factor importance analysis reveals the degree of importance of each input effector.

[0111] In this analysis, one final effector was removed while maintaining the other factors to analyze the effect of the factor on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 ( Fig. 7A ). When VEGF was removed, the values ​​for GATA2 decreased from 583 to 236, CD31 from 879 to 2, KDR from 33751 to 16856, CD34 from 2350 to 150, FLI1 from 3971 to 241, TAL1 from 370 to 18, and CD44 from 578 to 349. All changes represent significant decreases in the expression of the desired genes ( Figure 7B When forskolin was removed, the values ​​of GATA2 decreased from 583 to 373, KDR from 33751 to 32839, CD34 from 2350 to 2240, FLI1 from 3971 to 3740, TAL1 from 370 to 318, and CD44 from 578 to 540 ( Figure 7B ). When forskolin was removed, the expression of GATA2 was significantly reduced. When FGF2 was removed, the values ​​of GATA2 decreased from 583 to 458, the values ​​of CD31 decreased from 879 to 505, the values ​​of KDR decreased from 33760 to 23868, the values ​​of FLI1 decreased from 3970 to 3374, and the values ​​of CD44 decreased from 576 to 274 ( Figure 7B). These data indicate that FGF2 is essential for the expression of KDR, CD31, and CD44.

[0112] In another model, the effects of VEGF, FGF2, and thymosin-β4 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 were evaluated ( Fig. 8A ). Similarly, VEGF was essential for increasing the expression of all analyzed hemogenic endothelial genes. When FGF2 was removed, the values ​​for CD31 decreased from 807 to 677, the values ​​for KDR decreased from 40630 to 29902, and the values ​​for CD44 decreased from 748 to 626 ( Figure 8B ). In the absence of FGF2, KDR expression was significantly reduced. When thymosin-β4 was removed, GATA2 values ​​decreased from 633 to 437, KDR values ​​decreased from 40660 to 37788, and CD44 values ​​decreased from 748 to 626 ( Figure 8B ). The addition of thymosin-β4 is necessary for the upregulation of GATA2 and therefore it is used in the Phase 2 formulation.

[0113] In another model, the effects of purmorphamine, a sonic hedgehog (SHH) activator, on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 were evaluated ( Fig.9A ). B-27 was retained in the model for this dynamic distribution analysis because it was a component of the basal medium used in the validation experiments. When purmorphamine was removed, GATA2 levels decreased from 275 to 105, KDR levels decreased from 13612 to 7614, CD34 levels decreased from 3230 to 2369, FLI1 levels decreased from 3571 to 2324, and TAL1 levels decreased from 1008 to 428. In the absence of purmorphamine, the expression of GATA2, KDR, FLI1, and TAL1 was significantly reduced ( Fig. 9B When VEGF was removed, CD31 levels decreased from 869 to 262, KDR levels decreased from 7518 to 6209, CD34 levels decreased from 2341 to 868, and FLI1 levels decreased from 2292 to 974. In the absence of VEGF, the expression of CD31, KDR, CD34, and FLI1 was significantly reduced ( Fig. 9B The model revealed that B-27 acted synergistically with each factor, increasing the expression of all genes except CD44 and KDR ( Figures 9A-9BThis model suggests that the SHH pathway is a key regulator of the hemogenic endothelial program. Addition of SANT1 (a SHH antagonist) reduced the levels of all target genes, most notably: KDR from 13469 to 12504, CD34 from 3159 to 1995, and TAL1 from 1010 to 498 ( Fig.9A ). This model suggests that the SHH pathway acts as a regulator of endothelial cell phenotype by acting as a lineage-determining pathway. The data suggest that the SHH pathway is a key regulator of endothelial differentiation, as inhibition of the SHH pathway with SANT1 commits cells to the endothelial lineage, and activation of the pathway with purmorphamine commits cells to the hemogenic endothelial lineage ( Fig.13A Since RUNX1 is enriched on hemogenic endothelial cells, we compared RUNX1 expression in cells cultured with our hemogenic endothelial formula with that in cells cultured with an endothelial formula containing SANT1. Fig. 13B RUNX1 was shown to be expressed only on hemogenic endothelial cells and not on endothelial cells. Activation of the SHH pathway is essential for HEC differentiation, so purmorphamine is included in the Phase 2 formula.

[0114] In another model, the effects of XAV939 and FGF2 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 were evaluated ( Fig. 10A ). When performing this dynamic distribution analysis, L-ascorbic acid was retained in the model because it is present in the basal medium and increases the expression of key genes ( Fig. 10B ). Similarly, VEGF was essential for the expression of all hemogenic endothelial genes analyzed. When XAV939 was removed, GATA2 levels decreased from 992 to 560, CD31 levels decreased from 6619 to 2181, KDR levels decreased from 36067 to 31179, CD34 levels decreased from 7050 to 5740, FLI1 levels decreased from 13094 to 7237, TAL1 levels decreased from 1265 to 982, and CD44 decreased from 838 to 799. In the absence of XAV939, the expression of GATA2, CD31, CD34, and FLI1 was significantly reduced ( Fig. 10B When FGF2 was removed, GATA2 levels decreased from 992 to 397, CD31 levels from 6619 to 2002, KDR levels from 36067 to 22308, CD34 levels from 7050 to 6550, FLI1 levels from 13094 to 5406, TAL1 levels from 1265 to 866, and CD44 levels from 838 to 466 ( Fig. 10B ). In the absence of FGF2, the expression of GATA2, CD31, KDR, FLI1, and CD44 was significantly reduced ( Fig. 10B). This model, along with the previous model, revealed that FGF2 exerted a powerful synergistic effect with each factor, which helped to increase gene expression and was therefore selected as the Phase 2 formula.

[0115] In the final model, the effects of retinoic acid and BMP4 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 were evaluated ( Fig.11A ). Similarly, VEGF was essential for the expression of all hemogenic endothelial genes analyzed. When retinoic acid was removed, GATA2 levels decreased from 519 to 281, CD31 levels from 768 to 486, CD34 levels from 3971 to 2902, FLI1 levels from 3250 to 2936, and TAL1 levels from 490 to 235 ( Fig. 11B ). In the absence of retinoic acid, the expression of GATA2, CD31, and TAL1 was significantly reduced. When BMP4 was removed, GATA2 levels decreased from 519 to 338, CD31 levels from 768 to 682, KDR levels from 17667 to 10025, CD34 levels from 3971 to 3455, FLI1 levels from 3250 to 2213, and TAL1 levels from 490 to 422 ( Fig. 11B ). In the absence of FGF2, the expression of GATA2, KDR, FLI1 and CD44 was significantly reduced ( Fig. 10B ).

[0116] To obtain additional supporting evidence for the phase 2 formulation, an experiment was performed in the presence or absence of effector factors in the phase 2 formulation. In this experiment, only VEGF2 and FGF2 (two factors commonly used for endothelial differentiation) were used for comparison with the complete phase 2 formulation. Fig. 12B The increase in CD31, CD34 and CD61 expression levels after the addition of small molecules was demonstrated. CD61 has been shown to be a marker for HEC (Huang, K. et al. (2016) Stem Cell Reports 7: 854-868), and is significantly upregulated after the addition of phase 2 effectors to the formula. The combination of phase 2 effectors with VEGF and FGF guides cell differentiation into HEC. The complete phase 2 formula ingredients can be found in Table 1.

[0117] Example 3 : Flow cytometric analysis and immunocytochemistry of stem cell-derived hemogenic endothelial cells expressing hemogenic endothelial cell markers

[0118] To further validate the formulation developed in Example 1, iPSCs were cultured in stage 1 medium for 2 days, then the cells were replated and cultured in stage 2 medium for 3 days, and the expression of hemogenic endothelial cell markers was assessed using flow cytometry and immunohistochemistry analysis. The basal differentiation medium used in this experiment was RPMI medium containing 2% B-27 supplement, 100 μg / ml L-ascorbic acid, and 1% penicillin / streptomycin. Flow cytometric analysis confirmed the efficiency of the stage 2 formulation in promoting the conversion of iPSCs into hemogenic endothelial cells ( Figures 12A-12B 83% of cells were positive for CD31, 61% of cells were positive for CD34, 6% of cells were positive for CD61, and 95% of cells were positive for CD309 (KDR) ( Fig. 12A ). 63% of cells were positive for CD143 ( Fig. 12A In addition, immunofluorescence staining showed strong staining of various hemogenic endothelial markers such as CD31, FLI1, RUNX1, GATA2, vWF and CD309 (KDR) as well as SOX17 ( Fig.14 ).

[0119] Example 4 : Flow cytometric analysis of stem cell-derived hemogenic endothelial cells expressing hemogenic endothelial cell markers from two isolated iPSC lines

[0120] To further validate the development formula described in Example 1 in another cell line, iPSCs from two cell lines (iXCellsCR0000001 and REPROCELL771-3G) were cultured in stage 1 medium for 2 days, and then the cells were replated and cultured in stage 2 medium for 3 days, and flow cytometry analysis was used to evaluate the expression of hemogenic endothelial cell markers. The basal differentiation medium used in this experiment was RPMI medium containing 2% B-27 supplement, 100 μg / ml L-ascorbic acid, and 1% penicillin / streptomycin. Flow cytometric analysis confirmed the efficiency of the stage 2 formula in promoting the conversion of iPSCs into hemogenic endothelial cells in two iPSC cell lines. Fig.15 It was shown that in iX cells CR0000001 and REPROCELL771-3G, 22% and 49% of the cells expressed RUNX1, 87% and 75% of the cells expressed CD34, 3% and 10% of the cells expressed CD43, and 97% and 99% of the cells expressed CD31, respectively.

[0121] Example 5 RNA-seq analysis to characterize iPSC-derived hemogenic endothelial cells

[0122] Using bulk RNA-seq analysis to characterize iPSC-derived hemogenic endothelial cells ( Fig.16 ). iPSCs were differentiated into lateral plate mesoderm as described previously. Then, mesodermal cells were treated with stage 2 medium for 3 days. Fig.16 As shown, S0 cells express pluripotency markers such as NANOG, SOX2, and POUF51. S1 cells express genes associated with primitive streak and mesoderm such as TBXT, MIXL1, and FOXF1. Finally, extensive RNAseq validation confirmed the expression of blood and endothelial transcripts in S2 cells. The blood-related transcription factors expressed were: RUNX1, GATA2, GATA3, FLI1, GFI1, MEIS2, MECOM, and SPI1. The endothelial transcripts expressed included: SOX17, SOX7, SOX18, KDR, PECAM1, ERG, ETS1, and CDH5.

[0123] Equivalent

[0124] 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 invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for generating human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vW F+HEC, comprising: Human early mesodermal progenitor cells are cultured in a culture medium containing a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist to generate human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC. 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 medium containing a Wnt pathway agonist for two days.

4. A method for generating human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vW F+HEC, comprising: (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 (b) From day 2 to day 5, culturing the early mesodermal progenitor cells in a culture medium containing a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist to generate human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC.

5. The method of claim 3 or 4, wherein the Wnt pathway agonist is a GSK-3β inhibitor. The method according to claim 5 , wherein the GSK-3β inhibitor is CHIR99021.

7. The method according to claim 6, wherein the concentration of CHIR99021 in the culture ranges from 3.0 to 9.0 μM.

8. The method according to claim 7, wherein the concentration of CHIR99021 in the culture is 6.0 μM.

9. The method of any one of claims 1-8, wherein the VEGFR agonist is VEGF.

10. The method of claim 9, wherein the concentration of VEGF in the culture is in the range of 25-75 ng / ml.

11. The method of claim 9, wherein the concentration of VEGF in the culture is 50 ng / ml.

12. The method of any one of claims 1-8, wherein the FGFR agonist is FGF2 or SUN11602.

13. The method of claim 12, wherein the concentration of the FGFR agonist in the culture ranges from 1-20 ng / ml.

14. The method of claim 12, wherein the FGFR agonist is FGF2 at a concentration ranging from 1-20 ng / ml in culture.

15. The method of claim 12, wherein the FGFR agonist is FGF2, and its concentration in culture is 10 ng / ml.

16. The method of any one of claims 1-8, wherein the Sonic Hedgehog (SHH) agonist is selected from the group consisting of purmorphamine, SSH, GSA10, SAG, and combinations thereof.

17. The method of claim 16, wherein the concentration of the SHH agonist in the culture ranges from 100-1000 nM.

18. The method of claim 16, wherein the SHH agonist is purmorphamine, and its concentration in the culture ranges from 400-600 nM.

19. The method of claim 16, wherein the SHH agonist is purmorphamine, and its concentration in culture is 500 nM.

20. The method of any one of claims 1-8, wherein the adenylate cyclase activator is selected from the group consisting of forskolin, NKH477, PACAP 1-27, PACAP 1-38, adenosine, carbacycline, dopamine, endothelin 1, endothelin 1, L-(–)-epinephrine-(+)-bitartrate, glucagon, isoproterenol hydrochloride, (±)-octopamine hydrochloride, parathyroid hormone 1-34, prostaglandin D2, prostaglandin E1, prostaglandin E2, prostaglandin I2, [Arg 8 ]-Vasopressin, [Lys 8 ]-Vasopressin and combinations thereof.

21. The method according to claim 20, wherein the concentration of the adenylate cyclase activator in the culture ranges from 0.1 to 10 μM.

22. The method of claim 20, wherein the adenylate cyclase activator is forskolin, and its concentration in the culture ranges from 0.75 to 2.5 μM.

23. The method of claim 20, wherein the adenylate cyclase activator is forskolin, and its concentration in culture is 1.0 μM.

24. The method of any one of claims 1-8, wherein the actin binding protein is selected from the group consisting of thymosin-β4, HMRef, α-actinin, β-spectrin, dystrophin, dystrophin, fascin, and combinations thereof.

25. The method of claim 24, wherein the concentration of the actin binding protein in the culture ranges from 0.1 to 10 μg / ml.

26. The method of claim 24, wherein the actin binding protein is thymosin-β4, and its concentration in the culture ranges from 0.75 to 2.5 μg / ml.

27. The method of claim 24, wherein the actin binding protein is thymosin-β4, and its concentration in culture is 1 μg / ml.

28. The method of any one of claims 1-8, wherein the BMP pathway agonist is selected from the group consisting of BMP4, BMP2, BMP6, BMP7, GDF6, and combinations thereof.

29. The method of claim 28, wherein the concentration of the BMP pathway agonist in culture ranges from 5-50 ng / ml.

30. The method of claim 28, wherein the BMP pathway agonist is BMP4 at a concentration in the culture ranging from 15-30 ng / ml.

31. The method of claim 28, wherein the BMP pathway agonist is BMP4 at a concentration of 20 ng / ml in culture.

32. The method of any one of claims 1-8, 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, JW67, JW74, cardiomyogen 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, dendrobium, RCM-1, WIKI4, IQ-1, Advavivant, PRI-724, tegatrabectin, and combinations thereof.

33. The method of claim 32, wherein the Wnt pathway antagonist is present in culture at a concentration ranging from 10-500 nM.

34. The method of claim 32, wherein the Wnt pathway antagonist is XAV939, and its concentration in culture ranges from 50-150 nM.

35. The method of claim 32, wherein the Wnt pathway antagonist is XAV939, and its concentration in culture is 100 nM.

36. The method of any one of claims 1-8, wherein the retinoic acid (RA) pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, CD437, Ch55, BMS753, BMS961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid and all-trans retinoic acid (ATRA), AY9944 dihydrochloride, Ciliobrevin A, cyclopamine, and combinations thereof.

37. The method of claim 36, wherein the RA pathway agonist is present in culture at a concentration ranging from 100-1000 nM.

38. The method of claim 36, wherein the RA pathway agonist is retinoic acid at a concentration in the culture ranging from 400-600 nM.

39. The method of claim 36, wherein the RA pathway agonist is retinoic acid, which is present in culture at a concentration of 500 nM.

40. The method of any one of claims 3-39, wherein the pluripotent stem cells are embryonic stem cells.

41. The method of any one of claims 3-39, wherein the pluripotent stem cells are induced pluripotent stem cells.

42. A culture medium for generating HECs, comprising a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist.

43. The culture medium of claim 42, wherein the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylate cyclase activator is forskolin, the actin binding protein is thymosin-β4, the BMP pathway agonist is BMP4, the Wnt pathway antagonist is XAV939, and the RA receptor agonist is retinoic acid.

44. The culture medium of claim 43, wherein the concentration of VEGF is 50 ng / ml, the concentration of FGF2 is 10 ng / ml, the concentration of purmorphamine is 500 nM, the concentration of forskolin is 1 μM, the concentration of thymosin-β4 is 1 μg / ml, the concentration of BMP4 is 20 ng / ml, the concentration of XAV939 is 100 nM, and the concentration of retinoic acid is 500 nM.

45. A cell culture of isolated human hematurgical endothelial cells, the culture comprising: human CD31+CD34+CD143+CD309+GATA2+FLI1+RUNX1+vWF+HEC cultured in a culture medium comprising a VEGFR agonist, a FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylate cyclase activator, an actin binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist.

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