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

Abstract

Methods are provided for generating CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ hemogenic endothelial cells (HECs) using chemically defined media that allow for the generation of HECs from early mesodermal progenitor cells in as little as 3 days and HECs from pluripotent stem cells in as little as 5 days. Media, isolated cell populations, and kits are also provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 400,944, filed August 25, 2022. The entire contents of U.S. Provisional Patent Application No. 63 / 400,944 are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Vascular endothelial cells (ECs) are the main type of cell lining blood vessels throughout the body. During embryonic development, a specialized subset of endothelial cells, called hemogenic endothelial cells, acquires the ability to form blood cells and gives rise to hematopoietic stem and progenitor cells (HSPCs). Hematopoietic endothelial cells are a small (1-3% of endothelial cells in distinct tissues) transient population of specialized cells that arise to initiate definitive hematopoiesis (Gritz and Hirschi (2016) Cell. Mol. Life Sci. 73:1547-1567). HSPCs function throughout embryonic development and adult life to generate all blood cells in the body. For an overview of the regulation of hemogenic endothelial cell development and function, see, for example, Wu and Hirschi (2021) Ann. Rev. Physiol. 83:17-37 (Non-Patent Document 2) and Lange et al. (2021) Cell. Mol. Life Sci. 78:4143-4160 (Non-Patent Document 3).

[0003] The earliest methods for generating endothelial cells from pluripotent stem cells devised three different approaches (e.g., as reviewed in Wilson et al. (2014) Stem Cells 32:3037-3045 (Non-Patent Document 4); Yoder (2015) Curr. Opin. Hematol. 22:252-257 (Non-Patent Document 5); Williams and Wu (2019) Arter. Thromb. Vasc. Biol. 39:1317-1329 (Non-Patent Document 6)). In the first approach, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are grown under conditions that induce the self-aggregation of these cells into embryoid bodies (EBs), and then various growth factors are used to promote endothelial lineage differentiation by the EBs. In the second approach, differentiating ESCs or iPSCs are co-cultured with a feeder layer, such as stromal cells (e.g., mouse calvarial 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 culture on plates coated with a protein support, such as Matrigel, gelatin, fibronectin, or similar proteins, under specific conditions with the addition of growth factors or small molecules.

[0004] Activation of canonical Wnt signaling has been reported to promote the differentiation of pluripotent stem cells into hemogenic endothelial cells. Transient treatment of iPSCs with a GSK-3β inhibitor (thereby activating the Wnt signaling pathway) has been shown to induce activation of the CDX / HOX pathway, resulting in hematopoietic posterior mesodermal differentiation of iPSCs (Kitajima et al. (2016) Exp. Hematol. 44:68-74 (Non-Patent Document 7)). A serum-free system using culture with a GSK-3β inhibitor for the differentiation of human pluripotent stem cells into hemogenic endothelial cells has been described (Galat et al. (2017) Stem Cell Res. Therap. 8:67 (Non-Patent Document 8)). A later protocol for generating hemogenic endothelial cells combines treatment with growth factors such as VEGF, FGF2, and BMP4 with Wnt pathway activation (see, e.g., Bruveris et al. (2020) Development 147:dev193037).

[0005] Thus, although 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 culture. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gritz and Hirschi (2016) Cell. Mol. Life Sci. 73:1547-1567 [Non-patent document 2] Wu and Hirschi (2021) Ann. Rev. Physiol. 83:17-37 [Non-patent document 3] Lange et al. (2021) Cell. Mol. Life Sci. 78:4143-4160 [Non-patent document 4] Wilson et al. (2014) Stem Cells 32:3037-3045 [Non-Patent Document 5] Yoder (2015) Curr. Opin. Hematol. 22:252-257 [Non-patent document 6] Williams and Wu (2019) Arter. Thromb. Vasc. Biol. 39:1317-1329 [Non-Patent Document 7] Kitajima et al. (2016) Exp. Hematol. 44:68-74 [Non-patent document 8] Galat et al. (2017) Stem Cell Res. Therap. 8:67 [Non-Patent Document 9] Bruveris et al. (2020) Development 147:dev193037 Summary of the Invention

[0007] The present disclosure provides a method for generating hemogenic endothelial cells (HECs) using a chemically defined medium that enables robust generation of CD31+ CD34+ D143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs from pluripotent stem cells in just five days of culture. The medium contains small molecule agents that stimulate or attenuate specific signaling pathways to promote differentiation along the endothelial cell lineage. Further culture of the resulting hemogenic endothelial cells under lineage-specific cell culture conditions results in short-term and long-term differentiation into hematopoietic stem cells. Furthermore, the use of small molecule agents in the medium allows for precise control of culture components.

[0008] The disclosed methods and compositions utilize a unique cell culture medium that allows for the generation of hemogenic endothelial cells from early mesodermal progenitor cells in just three days. This unique cell culture medium contains certain components previously used in endothelial cell generation, such as VEGFR agonists and FGFR agonists, but also includes novel components and combinations that enable robust hemogenic endothelial cell generation. Early mesodermal progenitor cells can be obtained by culturing pluripotent stem cells under culture conditions that yield early mesodermal progenitor cells, as described herein. Thus, in certain embodiments, the method includes a one-step culture protocol that begins with early mesodermal progenitor cells and develops CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ hemogenic endothelial cells in three days. In other embodiments, the method comprises a two-step culture protocol beginning with differentiation of pluripotent stem cells into early mesodermal progenitor cells for two days in step 1, followed by further differentiation of the early mesodermal progenitor cells into CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ hemogenic endothelial cells for three days in step 2, resulting in a two-step, five-day protocol.

[0009] Accordingly, in one aspect, the present disclosure relates to a method of generating human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs, the method comprising culturing human early mesodermal progenitor cells in a medium comprising a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl 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+ HECs.

[0010] In one embodiment, early mesodermal progenitor cells are cultured in culture medium for 3 days to obtain human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs.

[0011] In one embodiment, the early mesodermal progenitor cells are obtained by culturing human pluripotent stem cells in medium containing a Wnt pathway agonist for two days.

[0012] In another aspect, the present disclosure provides a two-step method for generating human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ hemogenic endothelial cells (ECs), comprising: (a) culturing human pluripotent stem cells in a medium containing a Wnt pathway agonist on days 0 to 2 to generate early mesodermal progenitor cells; and (b) culturing the early mesodermal progenitor cells in a medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl cyclase activator, an actin-binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist on days 2-5 to generate CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs; The present invention relates to a method, comprising:

[0013] In one embodiment, the Wnt pathway agonist used in the culture medium to generate 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 ranging from 3.0 to 9.0 μM. In one embodiment, CHIR99021 is present in the culture at a concentration of 6.0 μM.

[0014] In one embodiment, the VEGFR agonist is VEGF. In one embodiment, VEGF is present in the culture at a concentration ranging from 25 to 75 ng / ml. In one embodiment, VEGF is present in the culture at a concentration of 50 ng / ml.

[0015] In one embodiment, the FGFR agonist is FGF2 or SUN11602. In one embodiment, the FGFR agonist is present in the culture at a concentration ranging from 1 to 20 ng / ml. In one embodiment, the FGFR agonist is FGF2, and the FGF2 is present in the culture at a concentration ranging from 1 to 20 ng / ml. In one embodiment, the FGFR agonist is FGF2, and the FGF2 is present in the culture at a concentration of 10 ng / ml.

[0016] In one embodiment, the sonic hedgehog (SHH) agonist is selected from the group consisting of purmorphamine, SSH, GSA10, 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 the purmorphamine is present in the culture at a concentration ranging from 400 to 600 nM. In one embodiment, the SHH agonist is purmorphamine, and the purmorphamine is present in the culture at a concentration of 500 nM.

[0017] In one embodiment, the adenylyl cyclase activator is forskolin, NKH477, PACAP1-27, PACAP1-38, adenosine, carbacyclin, dopamine, endothelin 1, endothelin 1, L-(-)-epinephrine-(+)-bitartrate, glucagon, isoproterenol HCl, (±)-octopamine HCl, 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 adenylyl cyclase activator is present in the culture at a concentration ranging from 0.1 to 10 μM. In one embodiment, the adenylyl cyclase activator is forskolin, and the forskolin is present in the culture at a concentration ranging from 0.75 to 2.5 μM. In one embodiment, the adenylyl cyclase activator is forskolin, and the forskolin is present in the culture at a concentration of 1.0 μM.

[0018] In one embodiment, the actin-binding protein is selected from the group consisting of thymosin-β4, HMRef, α-actinin, β-spectrin, dystrophin, utrophin, fimbrin, and combinations thereof. In one embodiment, the actin-binding protein is present in the culture at a concentration ranging from 0.1 to 10 μg / ml. In one embodiment, the actin-binding protein is thymosin-β4, and the thymosin-β4 is present in the culture at a concentration ranging from 0.75 to 2.5 μg / ml. In one embodiment, the actin-binding protein is thymosin-β4, and the thymosin-β4 is present in the culture at a concentration of 1 μg / ml.

[0019] 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 BMP pathway agonist is present in the culture at a concentration ranging from 5 to 50 ng / ml. In one embodiment, the BMP pathway agonist is BMP4, and the BMP4 is present in the culture at a concentration ranging from 15 to 30 ng / ml. In one embodiment, the BMP pathway agonist is BMP4, and the BMP4 is present in the culture at a concentration of 20 ng / ml.

[0020] In one embodiment, the Wnt pathway antagonist is XAV939, ICG-001 (fossenvivin), capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptonide, KYA1797K, JW55, JW67, JW7 The Wnt pathway antagonist is selected from the group consisting of 4, cardiogen 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, gigantrol, RCM-1, WIKI4, IQ-1, Adavivant, PRI-724, tegatrabetan, or a combination thereof. In one embodiment, the Wnt pathway antagonist is present in the culture at a concentration ranging from 10 to 500 nM. In one embodiment, the Wnt pathway antagonist is XAV939, and the XAV939 is present in the culture at a concentration ranging from 50 to 150 nM. In one embodiment, the Wnt pathway antagonist is XAV939, and the XAV939 is present in the culture at a concentration of 100 nM.

[0021] 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, or a combination thereof. In one embodiment, the RA pathway agonist is present in the culture at a concentration ranging from 100 to 1000 nM. In one embodiment, the RA pathway agonist is TTNPB, and the TTNPB is present in the culture at a concentration ranging from 400 to 600 nM. In one embodiment, the RA pathway agonist is TTNPB, and the TTNPB is present in the culture at a concentration of 500 nM.

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

[0023] In another aspect, the present disclosure relates to a culture medium for generating HECs, comprising a VEGFR agonist, an FGFR agonist, a Sonic Hedgehog (SHH) agonist, an adenylyl 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 adenylyl 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, VEGF is at a concentration of 50 ng / ml, FGF2 is at a concentration of 10 ng / ml, purmorphamine is at a concentration of 500 nM, forskolin is at a concentration of 1 μM, thymosin-β4 is at a concentration of 1 μg / ml, BMP4 is at a concentration of 20 ng / ml, XAV939 is at a concentration of 100 nM, and retinoic acid is at a concentration of 500 nM.

[0024] In yet another aspect, the present disclosure relates to an isolated cell culture of human hemogenic endothelial cells, comprising human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs cultured in a medium comprising a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl cyclase activator, an actin-binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist.

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

[0026] [Figure 1] FIG. 1 shows a representative culture protocol for generating hemogenic endothelial cells from early mesodermal progenitor cells in 3 days, as well as a schematic diagram of a 2-day pretreatment for generating early mesodermal progenitor cells from iPSCs. [Figure 2] Figure 1 shows results from an HD-DoE model of an eight-factor experiment optimized for maximal GATA2 expression. The top section of the model shows the predicted expression levels of 52 preselected genes when optimized for GATA2. The bottom section of the model shows the effectors tested in the model and their contribution to maximal GATA2 expression. The value columns indicate the required concentration of each effector to mimic the model. [Figure 3] Results from the HD-DoE model of an eight-factor experiment optimized for maximal GATA2 expression are shown. The top and bottom sections are as described in Figure 2. [Figure 4] Results from the HD-DoE model of an eight-factor experiment optimized for maximal GATA2 expression are shown. The top and bottom sections are as described in Figure 2. [Figure 5] Results from the HD-DoE model of an eight-factor experiment optimized for maximal GATA2 expression are shown. The top and bottom sections are as described in Figure 2. [Figure 6] Results from the HD-DoE model of an eight-factor experiment optimized for maximal GATA2 expression are shown. The top and bottom sections are as described in Figure 2. [Figure 7A] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three tested effectors. The effects of VEGF, forskolin, and FGF2 on the expression of these genes and their factor contributions are indicated by the slope of the plot for each effector. [Figure 7B]Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three tested effectors. The effects of VEGF, forskolin, and FGF2 on the expression of these genes and their factor contributions are indicated by the slope of the plot for each effector. [Figure 8A] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three validated effectors. The effects of VEGF, thymosin-β4, and FGF2 on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 8B] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three validated effectors. The effects of VEGF, thymosin-β4, and FGF2 on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 9A] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three validated effectors. The effects of purmorphamine, VEGF, and B27 on the expression of the above genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 9B] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of three validated effectors. The effects of purmorphamine, VEGF, and B27 on the expression of the above genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 10A]Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of four validated effectors. The effects of XAV939, VEGF, FGF2, and L-ascorbic acid on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 10B] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 versus the concentration of four validated effectors. The effects of XAV939, VEGF, FGF2, and L-ascorbic acid on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 11A] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 in response to the concentration of three validated effectors. The effects of retinoic acid, BMP4, and VEGF on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 11B] Dynamic profile analysis of the expression levels of GATA2, PECAM1, CD309 (KDR), CD34, FLI1, TAL1, and CD44 in response to the concentration of three validated effectors. The effects of retinoic acid, BMP4, and VEGF on the expression of these genes and their factorial contributions are indicated by the slope of the plot for each effector. [Figure 12]Figures 12A-12B show flow cytometry staining of iPSC-derived HECs at the end of stage 2. Figure 12A shows cells stained for CD31, CD34, CD61, CD143, and CD309 (KDR). At this stage, cells were positive for all markers expected for HECs. n=5 for CD31, CD34, CD61, and KDR, n=2 for CD143. Figure 12B shows that the medium described herein containing small molecules increases CD31, CD34, and CD61 compared to the known endothelial cell differentiation proteins VEGF and FGF2 alone. [Figure 13] Figure 13A shows a representative plot of the organization of endothelial (blue) and hemogenic endothelial (red) lineage-committing gene sets induced by factor combinations in mesoderm-committed cells. Cells were exposed to stage 1 medium for 2 days before factor addition. The plot was generated using the HDB-unsupervised clustering algorithm. Conditions for the expression of genes enriched in hemogenic endothelial cells, such as GATA2, TAL1, and ETV2, as well as genes expressed by endothelial cells, such as PECAM1 and ERG, were identified. Figure 13B shows photographs of RUNX1-positive HECs differentiated from iPSCs using the stage 2 recipe described herein and RUNX1-negative endothelial cells differentiated using a different optimized stage 2 recipe for endothelial cell differentiation. [Figure 14] Fluorescence imaging of iPSC-derived hemogenic endothelial cells at the end of stage 2. Cells were stained for hemogenic endothelial cell biomarkers, including CD31, FLI1, CD144, CD309 (KDR), vWF, RUNX1, GATA2, and SOX17. At this stage, cells were positive for all markers expected for hemogenic endothelial cells. [Figure 15]Flow cytometry staining of iPSC-derived HECs at the end of stage 2 from two iPSC cell lines (iX Cells CR0000001 and REPROCELL 771-3G) is shown. Cells were stained for HEC markers RUNX1, CD34, CD43, and CD31. Both the 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. [Figure 16] Figure 1 shows the results of bulk RNA-seq used to characterize iPSC-derived HECs. iPSCs were differentiated into lateral plate mesoderm (stage 1) as previously described. Mesoderm cells were then treated with stage 2 medium for 3 days. The heatmap shows the Z-score of transcripts per kilobase million (TPM) from bulk RNA-seq characterization. At the end of stage 2, RNA-seq demonstrated upregulation of stage 2 HEC genes and downregulation of stage 0 and stage 1 pluripotency, primitive streak, and mesoderm genes. TPM values ​​above the average are shown in red, and TPM values ​​below the average are shown in blue. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention Described herein are methods and compositions that enable the robust generation of CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ hemogenic endothelial cells from iPSCs in as few as five days under chemically defined culture conditions using a small-molecule-based approach. Using a high-dimensional design of experiments (HD-DoE) approach, multiple process inputs (e.g., small molecule agonists or antagonists) were simultaneously tested for output responses, such as gene expression. These experiments enabled the identification of chemically defined media containing agonists and / or antagonists of specific signaling pathways sufficient to generate HECs from iPSCs in a very short time. The optimized media was further validated by factor criticality analysis, examining the effect of eliminating individual agonist or antagonist agents. Flow cytometry and immunocytochemistry analyses were used to further confirm the phenotype of cells generated by the differentiation protocol.

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

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

[0030] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSCs) and human embryonic stem cells, such as ES cell lines.Non-limiting examples of artificial iPSCs include 19-11-1, 19-9-7, or 6-9-9 cells (for example, 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 cell markers that can be used to determine that cells are 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. Because the methods of producing HECs of the present disclosure are used to differentiate (maturate) a starting pluripotent stem cell population, in various embodiments, the HEC populations produced by the methods of the present disclosure lack expression of one or more stem cell markers, for example, 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.

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

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

[0033] In some 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 characterizing 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 hematopoietic endothelial cells.

[0034] GATA2 is a transcription factor identified as a master regulator of hematopoiesis and promotes the generation of HECs (Castano, J. et al. (2019) Stem Cell Reports 13:515-529). RUNX1 is a transcription factor governing the emergence of definitive HECs and is widely recognized as an important marker for these cells (Ling, M. et al. (2014) Blood 11:e11-e20). The expression of both GATA2 and RUNX1, key hematopoietic regulators, was simultaneously increased in iPSC-derived HECs (Castano, J. et al. (2019) Stem Cell Reports 13:515-529). SOX17 is a transcription factor identified as a master regulator of the arterial program in hemogenic endothelial cells and is required for hemogenic endothelial cell specification (Jung et al. (2021) Cell Rep. 34:108758). CD143 (ACE) has been shown to characterize 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 of HECs and a regulator of endothelial-hematopoietic transition (Oatley, M. et al. (2020) Nature Communications 11:586). Furthermore, TAL1 is an essential transcription factor for maintaining HSPC pluripotency and can be detected in primitive HSPCs such as HECs (Real, P. et al. (2012) Molecular Therapy 20:1443-1453).

[0035] II. Medium components The disclosed method for generating hemogenic endothelial cells from pluripotent stem cells includes culturing early mesodermal progenitor cells in a medium containing specific agonists and / or antagonists of cell receptors and / or signaling pathways to generate differentiated HECs. Furthermore, early mesodermal progenitor cells can be first obtained from pluripotent stem cells (e.g., ESCs or iPSCs) by culturing the stem cells under defined culture conditions described herein.

[0036] A culture medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl 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 progenitors in just 3 days of culture. Early mesodermal progenitors can be obtained from PSCs by culturing them with a Wnt pathway agonist for 2 days, resulting in a total 5-day protocol for obtaining differentiated HECs from PSCs under defined culture conditions.

[0037] As used herein, an "agonist" of a cell receptor or signaling pathway is intended to refer to an agent that stimulates (upregulates) that cell receptor or signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, for example, by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by using a small molecule agonist that interacts intracellularly with a component of the signaling pathway.

[0038] 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 extracellularly, for example, by using an antagonist that blocks cell surface receptors involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts with a component of the signaling pathway intracellularly.

[0039] The agonist and antagonist used in the chemically defined medium and method of the present disclosure are known in the art and commercially available.They are used in medium at effective concentrations to achieve desired outcome, for example, to produce the cell of interest (for example, early mesodermal progenitor cells or differentiated endothelial cells) that expresses the marker of interest.The non-limiting examples of suitable agonist and antagonist acting substance and effective concentration range are further described below.

[0040] 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 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, and VEGF is present in the culture medium at a concentration within the range of 10-100 ng / ml, 25-75 ng / ml, 40-60 ng / ml, 45-55 ng / ml, or 50 ng / ml.

[0041] Agonists of the FGFR pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the fibroblast growth factor receptor signaling pathway, which is biologically activated by the binding of FGF to FGFR. In one embodiment, the FGFR agonist is FGF2, SUN11602, or a combination thereof. In one embodiment, the FGFR pathway agonist is present in the culture medium at a concentration of 1-20 ng / ml, 5-15 ng / ml, 7.5-12.5 ng / ml, 9-11 ng / ml, or 10 ng / ml. In one embodiment, the FGFR agonist is FGF2 (e.g., recombinant human FGF2). In one embodiment, the FGFR agonist is FGF2, and the FGF2 is present in the culture medium at a concentration of 1-20 ng / ml, 5-15 ng / ml, 7.5-12.5 ng / ml, 9-11 ng / ml, or 10 ng / ml.

[0042] Agonists of the Sonic Hedgehog (SHH) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signaling through the SHH pathway, which biologically involves binding of SHH to the Patched-1 (PTCH1) receptor and transmission 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 SHH pathway agonist is present in the medium at a concentration within the range of 100-1000 nM, 200-800 nM, 250-750 nM, or 450-550 nM, or at a concentration of 500 nM. In one embodiment, the SHH pathway agonist is purmorphamine. In one embodiment, the SHH pathway agonist is purmorphamine, and the purmorphamine is present in the medium at a concentration of 100-1000 nM, 200-800 nM, 250-750 nM, or 500-600 nM. In one embodiment, the SHH pathway agonist is purmorphamine, and the purmorphamine is present in the medium at a concentration of 500 nM.

[0043] Activators of adenylyl cyclase include agents, molecules, compounds, or substances that can stimulate (upregulate) the activity of the adenylyl cyclase enzyme (also known in the art as adenyl cyclase and adenylate cyclase), which catalyzes the conversion of ATP to cAMP and pyrophosphate. In one embodiment, the adenylyl cyclase activator is selected from the group consisting of forskolin, NKH477, PACAP1-27, PACAP1-38, adenosine, carbacyclin, dopamine, endothelin 1, endothelin 1, L-(-)-epinephrine-(+)-bitartrate, glucagon, isoproterenol HCl, (±)-octopamine HCl, 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 adenylyl cyclase activator is present in the medium at a concentration within the range of 0.1 to 10 μM, 0.5 to 5 μM, 0.75 to 2.5 μM, or 0.9 to 1.1 μM, or at a concentration of 1 μM. In one embodiment, the adenylyl cyclase activator is forskolin. In one embodiment, the adenylyl cyclase activator is forskolin, and the forskolin is present in the medium at a concentration within the range of 0.1 to 10 μM, 0.5 to 5 μM, 0.75 to 2.5 μM, or 0.9 to 1.1 μM. In one embodiment, the adenylyl cyclase activator is forskolin, and the forskolin is present in the medium at a concentration of 1.0 μM.

[0044] Actin-binding proteins (also known as ABPs) are proteins that bind to actin monomers, actin polymers, or both. Numerous ABPs are known in the art; non-limiting examples of ABPs include thymosin-β4, HMRef, α-actinin, β-spectrin, dystrophin, utrophin, and fimbrin. In one embodiment, the ABP is a thymosin. In one embodiment, the thymosin is thymosin-β4 or thymosin-α1. In one embodiment, the actin-binding protein is present in the medium at a concentration within the range of 0.1-10 μg / ml, 0.5-5 μg / ml, 0.75-2.5 μg / ml, or 0.9-1.1 μg / ml, or at 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 the thymosin-β4 is present in the medium at a concentration of 0.1 to 10 μg / ml, 0.5 to 5 μg / ml, 0.75 to 2.5 μg / ml, or 0.9 to 1.1 μg / ml. In one embodiment, the actin-binding protein is thymosin-β4, and the thymosin-β4 is present in the medium at a concentration of 1 μg / ml.

[0045] BMP (bone morphogenetic protein) pathway agonists include agents, molecules, compounds, or substances that can stimulate (activate or upregulate) the BMP signaling pathway, which is biologically activated by the binding of BMP to a BMP receptor. 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 BMP pathway agonist is present in the culture medium at a concentration within the range of 5-50 ng / ml, 10-40 ng / ml, 15-30 ng / ml, or 20-25 ng / ml, or at 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 the BMP4 is present in the medium at a concentration in the range of 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, and the BMP4 is present in the medium at a concentration of 20 ng / ml.

[0046] WNT pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) 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 (fossenvivint), capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptonide, KYA1797K, JW55, JW67, JW74, cardiogen 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, gigantol, RCM-1, WIKI4, IQ-1, adavivant, PRI-724, tegatrabetan, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the medium at a concentration within the range of 10-500 nM, 50-250 nM, 50-150 nM, 75-125 nM, or at a concentration of 100 nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939, and the XAV939 is present in the medium at a concentration of 10-500 nM, 50-250 nM, 50-150 nM, or 75-125 nM. In one embodiment, the WNT pathway antagonist is XAV939, and the XAV939 is present in the medium at a concentration of 100 nM.

[0047] Agonists of the RA pathway include agents, molecules, compounds, or substances that can stimulate retinoic acid receptors (RARs), which are activated by both all-trans retinoic acid and 9-cis retinoic acid. There are three types of RARs: RAR-α, RAR-β, and RAR-γ, which are encoded by the RARA, RARB, and RARG genes, respectively. Various retinoic acid analogs have been synthesized that can activate the retinoic acid pathway. Non-limiting examples of such compounds include TTNPB (RAR-α, β, and γ agonist), AM580 (RARα agonist), CD1530 (potent and selective RARγ agonist), CD2314 (selective RARβ agonist), Ch55 (potent RAR agonist), BMS753 (RARα-selective agonist), tazarotene (receptor-selective retinoid; binds to RAR-β and -γ), isotretinoin (endogenous agonist for retinoic acid receptors; inducer of neuronal differentiation), and AC261066 (RARβ2 agonist). In some embodiments, the RA signaling pathway agonist is selected from the group consisting of i) retinoid compounds, ii) retinoid X receptor (RXR) agonists, and iii) 25 retinoic acid receptor (RAR) agonists. 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).

[0048] Thus, 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, AC55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA), AY9944 dihydrochloride, ciliobrevin A, cyclopamine, or a combination thereof. In one embodiment, the RA pathway agonist is present in the medium at a concentration within the 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), and the retinoic acid (RA) is present in the medium at a concentration within the range of 100-1000 nM, 200-800 nM, 250-750 nM, or 400-600 nM. In one embodiment, the RA pathway agonist is retinoic acid (RA), and the retinoic acid (RA) is present in the medium at a concentration of 500 nM.

[0049] Agonists of the WNT pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of a Wnt-protein ligand to a Frizzled family receptor. In one 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, alsterpaullone, indirubin-3-oxime, 1-azakempaullone, kenpaullone, TC-G24, TDZD8, TWS119, NP031112, AT7519, KY19382, AZD2858, and combinations thereof. In one embodiment, the WNT pathway agonist is present in the medium at a concentration within the range of 3.0-9.0 μM, 4.0-8.0 μM, 5.0-7.0 μM, 6.5-7.5 μM, or at a concentration of 6.0 μM. In one embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist is CHIR99021 and is present in the medium at a concentration in the range of 3.0-9.0 μM, 4.0-8.0 μM, 5.0-7.0 μM, or 6.5-7.5 μM. In one embodiment, the WNT pathway agonist is CHIR99021 and is present in the medium at a concentration of 6.0 μM (e.g., in Stage 1 medium for generating early mesodermal progenitor cells).

[0050] III. Culture conditions In combination with the chemically defined and optimized medium described in subsection II above, the method of producing hemogenic endothelial cells of the present disclosure uses standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37°C under 5% CO2 conditions. Cells can be cultured in standard culture vessels or plates, such as culture dishes, culture flasks, or 96-well plates. Pluripotent stem cells can be cultured in commercially available media before differentiation. For example, before the differentiation protocol begins, stem cells can be cultured for at least one day in specialized stem cell medium, such as Essential 8 Flex Medium (Thermo Fisher # A2858501). In a non-limiting exemplary embodiment, stem cells can be cultured at a density of 41,666 cells / cm. 2 The cells were passaged at a density onto vitronectin (Thermo Fisher # A14700) coated 6-well plates and cultured in Essential 8 Flex medium for 1 day before differentiation.

[0051] To initiate a differentiation protocol from stem cells, the medium containing the cultured stem cells is exchanged for a basal differentiation medium supplemented with a Wnt signaling pathway agonist, e.g., a GSK-3β inhibitor (e.g., CHIR99021) as described above in subsection II. This is referred to herein as step 1 of the differentiation protocol. The basal differentiation medium may, for example, comprise a commercially available base supplemented with additional standard media components necessary to maintain cell survival and proliferation, but typically lacks serum (basal differentiation medium is a serum-free medium).

[0052] In a non-limiting exemplary embodiment, the basal differentiation medium for stage 1 of the differentiation protocol is CDM2 medium supplemented with 1% penicillin / streptomycin (as shown in FIG. 1 ). CDM2 medium contains 0.5x IMDM (Thermo Fisher #12440046), 0.5x F12 (Thermo Fisher #11765047), 1 mg / ml poly(vinyl 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).

[0053] In a non-limiting exemplary embodiment, the basal differentiation medium for stage 2 of the differentiation protocol is commercially available RPMI medium containing 2% B-27 supplement, 100 μg / ml L-ascorbic acid, and 1% penicillin / streptomycin (as shown in FIG. 1).

[0054] In certain embodiments, the starting pluripotent stem cells are attached to a plate, preferably a plate coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0055] The medium is typically replaced with fresh medium periodically, for example, in one embodiment, the medium is replaced every 24 hours.

[0056] To generate early mesodermal progenitor cells, starting pluripotent stem cells are cultured in medium containing a GSK-3β inhibitor (e.g., CHIR99021) for a period of time sufficient for cell differentiation and expression of committed early mesodermal progenitor-associated markers, typically for two days. This protocol for generating early mesodermal progenitor cells from PSCs is referred to herein as "step (a)" or "stage 1."

[0057] To generate differentiated hemogenic endothelial cells from early mesodermal progenitor cells, progenitor cells are cultured in medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl cyclase activator, an actin-binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist for a period sufficient for cell differentiation and expression of hemogenic endothelial cell-associated markers (e.g., CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+), typically for 3 days. This protocol for generating hemogenic endothelial cells from early mesodermal progenitor cells is referred to herein as "step (b)" or "step 2."

[0058] In various embodiments, early mesodermal progenitor cells are cultured in optimized medium for a time sufficient to increase the expression of at least one, and preferably multiple, hemogenic endothelial cell-associated markers. Non-limiting examples of suitable hemogenic EC-associated markers include CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, and vWF. In several embodiments, the cells are cultured for a time sufficient to increase the expression level of at least two, at least three, at least four, or at least five, at least six, at least seven, or at least eight hemogenic EC-associated markers. In one embodiment, the cells are cultured for a time sufficient to increase the expression level of at least one hemogenic EC-associated marker by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the starting cell population. The level of marker expression in cultured HECs can be measured by techniques available in the art (e.g., RNAseq analysis and / or flow cytometry).

[0059] Thus, in the first step of the method for generating early mesodermal progenitor cells from PSCs, pluripotent stem cells are cultured in the medium optimized for step 1 from day 0 to 2, or starting on day 0 and continuing through day 2, or for 48 hours (2 days), or for at least 36 hours, or at least 40 hours, or at least 44 hours, or for at least 48 hours.

[0060] Thus, in the second step of the method for generating hemogenic endothelial cells from early mesodermal progenitor cells, on days 2 to 5, or starting on day 2 and continuing through day 5, or starting on day 2 and continuing for 72 hours (3 days), or starting on day 2 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 2 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours, starting on day 2 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0061] The medium is typically replaced with fresh medium periodically, for example, in certain embodiments, the medium is replaced every 24 hours, or every 48 hours, or every 72 hours.

[0062] IV. use The disclosed methods and compositions for generating hemogenic endothelial cells allow for efficient and robust access to these cell populations for a variety of uses. For example, the methods and compositions can be used in studying hematopoietic development and differentiation, including biology, to aid in understanding hematopoietic diseases and disorders. For example, HECs generated using the disclosed methods can be further purified according to established methods in the art using agents that bind to surface markers expressed on the cells.

[0063] The HECs obtained according to the disclosed method can be further cultured under lineage-specific culture conditions to differentiate into short-term and long-term hematopoietic stem cells. Therefore, the HECs obtained according to the disclosed method provide an opportunity to investigate functional aspects of the hematopoietic system and its development. Other uses include 3D bioprinting, drug screening, safety evaluation, vascular tissue engineering, and disease modeling.

[0064] HECs produced according to the methods of the present disclosure, or further differentiated hematopoietic lineage cells derived therefrom, are also contemplated for use in treating various hematopoietic diseases and disorders, for example, by delivering the cells to subjects with such diseases and disorders, including, but not limited to, cancers such as leukemia and lymphoma, blood disorders, and autoimmune disorders.

[0065] V. composition In other aspects, the present disclosure provides compositions related to methods of producing hemogenic endothelial cells, including media and isolated cell cultures.

[0066] In one aspect, the present disclosure provides a medium for generating CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs, comprising a VEGFR agonist, an FGFR agonist, a Sonic hedgehog (SHH) agonist, an adenylyl 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 concentrations thereof include those described in subsection II above. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylyl 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 VEGF is at a concentration of 50 ng / ml, the FGF2 is at a concentration of 10 ng / ml, the purmorphamine is at a concentration of 500 nM, the forskolin is at a concentration of 1 μM, the thymosin-β4 is at a concentration of 1 μg / ml, the BMP4 is at a concentration of 20 ng / ml, the XAV939 is at a concentration of 100 nM, and the retinoic acid is at a concentration of 500 nM.

[0067] In another aspect, the present disclosure provides an isolated cell culture of human hemogenic endothelial cells (ECs), comprising human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs cultured in a medium containing a VEGFR agonist, an FGFR agonist, a Sonic Hedgehog (SHH) agonist, an adenylyl 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 concentrations thereof include those described in subsection II above. In one embodiment, the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylyl 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 VEGF is at a concentration of 50 ng / ml, the FGF2 is at a concentration of 10 ng / ml, the purmorphamine is at a concentration of 500 nM, the forskolin is at a concentration of 1 μM, the thymosin-β4 is at a concentration of 1 μg / ml, the BMP4 is at a concentration of 20 ng / ml, the XAV939 is at a concentration of 100 nM, and the retinoic acid is at a concentration of 500 nM.

[0068] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The figures, as well as the contents of all references, patents, and published patent applications cited throughout this application, are hereby expressly incorporated by reference. [Example]

[0069] Example 1 : Development of a culture protocol for generating hemogenic endothelial cells from pluripotent stem cells We developed a two-step recipe for generating hemogenic endothelial cells that can induce human iPSCs to become hemogenic endothelial cells expressing CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, vWF, and VE-cadherin after 5 days in culture. HECs were generated from pluripotent stem cells using a two-step protocol as shown schematically in Figure 1.

[0070] Briefly, PSCs were first differentiated into early mesodermal progenitor cells by culturing the starting PSCs in medium containing a GSK-3β inhibitor, based on established protocols in the art for early differentiation along the endothelial lineage. In one embodiment, PSCs are cultured for 2 days (days 0-2) in medium containing 6 μM CHIR99021.

[0071] Using a high-dimensional experimental design (HD-DoE) approach, we used early mesodermal progenitor cells to differentiate along the endothelial lineage, with the aim of simultaneously testing multiple process inputs (e.g., small molecule agonists or antagonists) for output responses such as gene expression. Based on predicted conditions that maximize the expression of genes enriched in hemogenic endothelial cells, such as CD31, CD34, CD143, CD309, GATA2, FLI1, RUNX1, and vWF, we developed a complex recipe for generating differentiated hemogenic endothelial cells from early mesodermal progenitor cells, consisting of eight agents as shown in Table 1 below.

[0072] Table 1. Recipe for medium for producing hemogenic endothelial cells TIFF2025528897000001.tif67161

[0073] This recipe is referred to herein as the Stage 2 recipe for generating hemogenic endothelial cells.

[0074] The development of the two-step recipe is described in detail below.

[0075] First, undifferentiated iPSCs were grown in CDM2 medium containing 6 μM CHIR99021 for 48 hours (referred to herein as Phase 1). Phase 2 medium was then manipulated. This was based on an eight-factor HD-DoE experiment focused on differentiating cells into the hemogenic endothelial lineage for an additional three days after Phase 1 treatment was completed. To test effectors, 48 ​​different effector combinations, obtained using D-optimality design of experiment compression, were robotically prepared. Effector combinations were prepared in basal medium and then added to cells, which were then differentiated. After three days, RNA was extracted and gene expression was obtained using quantitative PCR analysis. Data were normalized and the effector design was modeled using partial least squares regression analysis, resulting in the creation of gene-specific models. These gene-specific models described the effector's ability to combinatorially and individually regulate the expression of individual genes after model tuning for maximum Q2 predictive power. Solutions within the tested space could then be explored to address desirability. For stage 2, we created conditions that induce the expression of GATA2, a transcription factor identified as a master regulator of hematopoiesis. GATA2 promotes the generation of HECs and facilitates endothelial-hematopoietic transition (EHT). GATA2 also suppresses cardiac differentiation at the mesoderm stage, committing cells to a hematopoietic fate. Optimizing for maximal GATA2 expression yielded a robust solution. Other genes predicted to be abundantly expressed in this solution included CD34, ERG FLI1, HOPX, KDR, LMO2, and CD31. All genes are highly expressed on hemogenic endothelial cells, suggesting cell commitment to this lineage (Figure 2). High levels of KDR, a receptor important for VEGF signaling, were observed in this model. Furthermore, GATA6, PAX6, OTX2, and SOX1 (genes associated with ectoderm and endoderm) were downregulated. Furthermore, genes associated with earlier stages, such as NODAL and EOMES, were downregulated, indicating that the cells were undergoing differentiation.This model was derived from an initial test of eight factors, including forskolin, VEGF, FGF2, Albumax, SUN1162, hydrocortisone, SB341542, and resveratrol. Three of these effectors: forskolin, VEGF, and FGF2, showed a positive impact on the expression of the gene of interest, with factor contributions of 27, 46, and 17, respectively (Figure 2). Factors with small contributions were excluded. Within the specification range for achieving approximately 86% maximal expression of GATA2, the Cpk value (process capability index) of this complex medium composition was 0.69, with a corresponding risk of failure of 1.9%.

[0076] Next, we evaluated additional factors that likely increase the complexity of signaling inputs to achieve effective fate control. As before, we focused on GATA2 expression. Optimizing for maximum GATA2 expression yielded a robust solution. This solution predicted abundant expression of other genes, including CD34, CD44, ERG, FLI1, KDR, LMO2, TAL1, and vWF. All genes are associated with the hemogenic endothelial program, suggesting that cells are committed to this lineage (Figure 3). High levels of KDR were again observed. This model was derived from an initial test of eight factors, including CHIR, VEGF, FGF2, TTNBP, AGN194310, Y27632, thymosin-β4, and heparin. Two of these effectors, VEGF and thymosin-β4, showed positive effects on the expression of genes of interest, with high factor contributions of 25 and 16, respectively (Figure 3). This recipe did not include CHIR, TTNBP, AGN194310, Y27632, or heparin because sufficient GATA2 induction was obtained without them and their factor contributions were relatively small. Although the factor contribution of FGF2 was small in this model, it was included in the recipe because its factor contribution was ≥16 in two of the three models. Within the specification range for achieving approximately 87% maximal expression of GATA2, this complex medium composition had a Cpk value (process capability index) of 0.7, corresponding to a 2% risk of failure.

[0077] To further refine the recipe for hemogenic endothelial cell differentiation, we conducted additional HD-DoE experiments. This model was derived from an initial test of eight factors, including 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 genes of interest, with a factor contribution of 20 and 25, respectively (Figure 4). All remaining factors except VEGF were not included in the recipe because sufficient GATA2 induction was obtained without them and their factor contribution was relatively small. VEGF was retained in the recipe because four of the five models showed a strong positive effect toward GATA2 induction. Other genes, such as CD34, ERG, FLI1, ETV2, and TAL1, were also predicted to be abundantly expressed in this solution. All genes are highly expressed on hemogenic endothelial cells, suggesting cell commitment to this lineage (Figure 4). Within the specification range to obtain approximately 84% maximum expression of GATA2, the Cpk value (process capability index) of this complex medium composition was 0.7, with a corresponding risk of failure of 1.4%.

[0078] To continue improving the recipe for hemogenic endothelial cell differentiation, we conducted a fourth modeling experiment. This model was derived from an initial test of eight factors, including VEGF, FGF2, sphingosine-1-phosphate, VH298, pyrintegrin, erythropoietin, L-ascorbic acid, and XAV939. Four of these effectors: VEGF, FGF2, L-ascorbic acid, and XAV939, showed a positive effect on the expression of genes of interest, with factor contributions of 22, 22, 5, and 16, respectively (Figure 5). L-ascorbic acid was included because it is present in the basal medium. All remaining factors were not included in this recipe because sufficient GATA2 induction was obtained without them and their factor contributions were relatively small. Other genes, such as CD34, CD44, CDH5, LMO2, MECOM, and TAL1, were also predicted to be abundantly expressed in this solution. All genes were highly expressed on hemogenic endothelial cells, suggesting cell commitment to this lineage (Figure 5). Within the specification range for achieving approximately 82% maximal expression of GATA2, the Cpk value (process capability index) of this complex medium composition was 0.7, with a corresponding risk of failure of 2.2%.

[0079] To complete the optimization of the recipe for hemogenic endothelial cell differentiation, we performed a fifth and final model. This model was derived from an initial test of eight factors, including VPA, SCF, BMP4, EFG, FLT3L, arginine, retinoic acid, and VEGF. Three of these effectors—retinoic acid, BMP4, and VEGF—positively affected the expression of genes of interest, with factor contributions of 22, 31, and 22, respectively (Figure 6). The remaining factors were not included in this recipe because sufficient GATA2 induction was obtained without them and their factor contributions were relatively small. This solution predicted abundant expression of other genes, including CD34, CDH5, ERG, FLI1, LMO2, and CD31. All genes are highly expressed on hemogenic endothelial cells, suggesting cell commitment to this lineage (Figure 6). Within the specification range for achieving approximately 81% maximum expression of GATA2, the Cpk value (process capability index) of this complex medium composition was 0.9, with a corresponding risk of failure of 0.51%. Based on the predicted conditions for maximizing the expression of genes abundant in hemogenic endothelial cells, such as GATA2, LMO2, ERG, CDH5, CD31, FLI1, CD34, TAL1, MECOM, and MYB, and taking into account all the analyzed models, a complex recipe for hemogenic endothelial differentiation was developed, consisting of eight effectors as shown in Table 1 above.

[0080] Example 2 : Factor criticality analysis of culture conditions for inducing hemogenic endothelial cells For the stage 2 culture recipe for inducing hemogenic endothelial cells, various contributing factors for each protocol input suggested variable related effects. To assess the impact of eliminating each tested factor on genes associated with the endothelial lineage, dynamic profile analysis was used to compare the expression levels of genes of interest in the absence of each tested factor with the presence of other factors. This factor criticality analysis revealed the degree of importance of each input effector, as the expression levels of genes of interest revealed whether the desired outcome was achievable.

[0081] In this analysis, we analyzed the effect of factors on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 by removing the final effector while retaining other factors (Figure 7A). Upon VEGF removal, GATA2 levels decreased from 583 to 236, CD31 levels changed from 879 to 2, KDR levels decreased from 33,751 to 16,856, CD34 levels decreased from 2,350 to 150, FLI1 levels decreased from 3,971 to 241, TAL1 levels decreased from 370 to 18, and CD44 levels decreased from 578 to 349. All changes represented a significant loss of expression of desired genes (Figure 7B). Upon removal of forskolin, GATA2 expression decreased from 583 to 373, KDR expression decreased from 33751 to 32839, CD34 expression decreased from 2350 to 2240, FLI1 expression decreased from 3971 to 3740, TAL1 expression decreased from 370 to 318, and CD44 expression decreased from 578 to 540 (Figure 7B). Upon removal of forskolin, GATA2 expression decreased significantly. Upon removal of FGF2, GATA2 expression decreased from 583 to 458, CD31 expression decreased from 879 to 505, KDR expression decreased from 33760 to 23868, FLI1 expression decreased from 3970 to 3374, and CD44 expression decreased from 576 to 274 (Figure 7B). This data indicates that FGF2 is crucial for KDR, CD31, and CD44 expression.

[0082] In another model, we evaluated the effects of VEGF, FGF2, and thymosin-β4 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 (Figure 8A). Again, VEGF was important for the increased expression of all hemogenic endothelial genes analyzed. Upon removal of FGF2, CD31 levels decreased from 807 to 677, KDR levels decreased from 40,630 to 29,902, and CD44 levels decreased from 748 to 626 (Figure 8B). In the absence of FGF2, KDR expression was significantly lost. Upon removal of thymosin-β4, GATA2 levels decreased from 633 to 437, KDR levels decreased from 40,660 to 37,788, and CD44 levels decreased from 748 to 626 (Figure 8B). GATA2 upregulation requires the addition of thymosin-β4 and was therefore used in the stage 2 recipe.

[0083] In another model, we evaluated the effect of purmorphamine, a sonic hedgehog (SHH) activator, on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 (Figure 9A). For this dynamic profile analysis, we retained B-27 in the model because it was present in the basal medium for validation experiments. Upon removal of purmorphamine, 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, GATA2, KDR, FLI1, and TAL1 expression were significantly reduced (Figure 9B). Upon VEGF removal, 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, expression of CD31, KDR, CD34, and FLI1 was significantly lost (Figure 9B). This model reveals that B-27, together with other factors, plays a synergistic role in contributing to the increased expression of all genes except CD44 and KDR (Figures 9A-9B). This model indicates that the SHH pathway is a crucial regulator of the hemogenic endothelial program. Addition of the SHH antagonist SANT1 reduced the levels of all genes of interest, most importantly, KDR (from 13,469 to 12,504), CD34 (from 3,159 to 1,995), and TAL1 (from 1,010 to 498) (Figure 9A). This model indicates that the SHH pathway is a regulator of endothelial cell phenotype by acting as a lineage commitment pathway. Our data indicate that the SHH pathway is a critical regulator of endothelial differentiation, as inhibition of the SHH pathway with SANT1 commits cells to the endothelial lineage, whereas activation of this pathway with purmorphamine commits cells to the hemogenic endothelial lineage (Figure 13A).Because RUNX1 is abundant on hemogenic endothelial cells, we compared RUNX1 expression on cells cultured using our hemogenic endothelial recipe with that on cells cultured using the endothelial recipe containing SANT1. Figure 13B shows that RUNX1 expression is only present on hemogenic endothelial cells, not on endothelial cells. Activation of the SHH pathway is crucial for HEC differentiation; therefore, purmorphamine was included in the stage 2 recipe.

[0084] In another model, we evaluated the effects of XAV939 and FGF2 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 (Figure 10A). When performing this dynamic profile analysis, we retained L-ascorbic acid in the model because it was present in the basal medium and increased the expression of critical genes (Figure 10B). Again, VEGF was critical for the expression of all hemogenic endothelial genes analyzed. Upon removal of XAV939, 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 levels decreased from 838 to 799. In the absence of XAV939, the expression of GATA2, CD31, CD34, and FLI1 was significantly lost (Fig. 10B). Upon FGF2 removal, GATA2 levels decreased from 992 to 397, CD31 levels decreased from 6619 to 2002, KDR levels decreased from 36067 to 22308, CD34 levels decreased from 7050 to 6550, FLI1 levels decreased from 13094 to 5406, TAL1 levels decreased from 1265 to 866, and CD44 levels decreased from 838 to 466 (Figure 10B). In the absence of FGF2, expression of GATA2, CD31, KDR, FLI1, and CD44 was significantly lost (Figure 10B). Based on previous and current models, this model revealed that FGF2 plays a robust and synergistic role in combination with other factors to contribute to increased gene expression and was therefore selected for the stage 2 recipe.

[0085] In the final model, we evaluated the effects of retinoic acid and BMP4 on the expression levels of GATA2, CD31, KDR, CD34, FLI1, TAL1, and CD44 (Figure 11A). Again, VEGF was crucial for the expression of all analyzed hemogenic endothelial genes. Upon removal of retinoic acid, GATA2 levels decreased from 519 to 281, CD31 levels decreased from 768 to 486, CD34 levels decreased from 3971 to 2902, FLI1 levels decreased from 3250 to 2936, and TAL1 levels decreased from 490 to 235 (Figure 11B). In the absence of retinoic acid, GATA2, CD31, and TAL1 expression were significantly lost. Upon BMP4 removal, GATA2 levels decreased from 519 to 338, CD31 levels decreased from 768 to 682, KDR levels decreased from 17667 to 10025, CD34 levels decreased from 3971 to 3455, FLI1 levels decreased from 3250 to 2213, and TAL1 levels decreased from 490 to 422 (Figure 11B). In the absence of FGF2, the expression of GATA2, KDR, FLI1, and CD44 was significantly lost (Figure 10B).

[0086] To further support the Stage 2 recipe, we performed experiments with the Stage 2 recipe in the presence and absence of effectors. In this experiment, we compared only VEGF2 and FGF2 (two common factors used in endothelial differentiation) against the complete Stage 2 recipe. Figure 12B shows that the addition of small molecules increased the expression of CD31, CD34, and CD61. CD61 has been shown to be a marker for HECs (Huang, K. et al. (2016) Stem Cell Reports 7:854-868) and is significantly upregulated when Stage 2 effectors are added to the recipe. The combination of Stage 2 effectors with VEGF and FGF directs cells to differentiate into HECs. The complete composition of this Stage 2 recipe can be found in Table 1.

[0087] Example 3: Flow cytometric analysis and immunocytochemistry of stem cell-derived hemogenic endothelial cells expressing hemogenic endothelial cell markers To further validate the recipe developed in Example 1, iPSCs were grown in stage 1 medium for 2 days, then replated and grown in stage 2 medium for 3 days. The expression of hemogenic endothelial cell markers was assessed using flow cytometry and immunohistochemistry. 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 cytometry analysis confirmed the efficiency of the stage 2 recipe in promoting the conversion of iPSCs to hemogenic endothelial cells (Figures 12A-12B). 83% of the cells were CD31 positive, 61% of the cells were CD34 positive, 6% of the cells were CD61 positive, and 95% of the cells were CD309 (KDR) positive (Figure 12A). 63% of the cells were CD143 positive (Figure 12B). Furthermore, immunofluorescence staining showed robust staining of various hemogenic endothelial cell markers, such as CD31, FLI1, RUNX1, GATA2, vWF, and CD309 (KDR), and SOX17 (Figure 14).

[0088] Example 4 Flow cytometry analysis of stem cell-derived hemogenic endothelial cells expressing hemogenic endothelial cell markers from two separate iPSC cell lines To further validate the developed recipe described in Example 1 with other cell lines, iPSCs derived from two cell lines (iX Cells CR0000001 and REPROCELL 771-3G) were grown in Stage 1 medium for 2 days, then replated and grown in Stage 2 medium for 3 days, and the expression of hemogenic endothelial cell markers was assessed using flow cytometry 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 cytometry analysis confirmed the efficiency of the Stage 2 recipe in promoting the conversion of iPSCs to hemogenic endothelial cells in both iPSC cell lines. Figure 15 shows 22% vs. 49% RUNX1-expressing cells, 87% vs. 75% CD34-expressing cells, 3% vs. 10% CD43-expressing cells, and 97% vs. 99% CD31-expressing cells in iX Cells CR0000001 vs. REPROCELL 771-3G, respectively.

[0089] Example 5 : RNA-seq analysis to characterize iPSC-derived hemogenic endothelial cells We characterized iPSC-derived hemogenic endothelial cells using bulk RNA-seq analysis (Figure 16). iPSCs were differentiated into lateral plate mesoderm as previously described. Mesodermal cells were then treated with stage 2 medium for 3 days. As shown in Figure 16, S0 cells express pluripotency markers such as NANOG, SOX2, and POUF51. S1 cells express genes associated with the primitive streak and mesoderm, such as TBXT, MIXL1, and FOXF1. Finally, bulk RNA-seq analysis 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.

[0090] equivalent 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 scope of the following claims.

Claims

1. 1. A method for producing human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs, comprising: The method comprises culturing human early mesodermal progenitor cells in a medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl cyclase activator, an actin-binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist to produce human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs.

2. The method of claim 1, wherein the early mesodermal progenitor cells are cultured in the medium for 3 days.

3. 2. The method of 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 2 days.

4. 1. A method for producing human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs, 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) culturing the early mesodermal progenitor cells in a medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl cyclase activator, an actin-binding protein, a BMP pathway agonist, a Wnt pathway antagonist, and a retinoic acid (RA) receptor agonist on days 2-5 to generate human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF hematopoietic ECs. The method comprises:

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

6. 6. The method of claim 5, wherein the GSK-3β inhibitor is CHIR99021.

7. 7. The method of claim 6, wherein CHIR99021 is present in the culture at a concentration ranging from 3.0 to 9.0 μM.

8. 8. The method of claim 7, wherein CHIR99021 is present in the culture at a concentration of 6.0 μM.

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

10. 10. The method of claim 9, wherein VEGF is present in the culture at a concentration ranging from 25 to 75 ng / ml.

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

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

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

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

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

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

17. 17. The method of claim 16, wherein the SHH agonist is present in the culture at a concentration ranging from 100 to 1000 nM.

18. 17. The method of claim 16, wherein the SHH agonist is purmorphamine, and the purmorphamine is present in the culture at a concentration ranging from 400 to 600 nM.

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

20. The adenylyl cyclase activator is selected from the group consisting of forskolin, NKH477, PACAP1-27, PACAP1-38, adenosine, carbacyclin, dopamine, endothelin 1, endothelin 1, L-(-)-epinephrine-(+)-bitartrate, glucagon, isoproterenol HCl, (±)-octopamine HCl, parathyroid hormone 1-34, and prostaglandin D 2 , prostaglandin E 1 , prostaglandin E 2 , prostaglandin I 2 , [Arg 8 ]-vasopressin, [Lys 8 9. The method of any one of claims 1 to 8, wherein the vasopressin is selected from the group consisting of vasopressin, vasopressin, vasopressin-1, vasopressin-2, vasopressin-3, vasopressin-4, vasopressin-5, vasopressin-6, vasopressin-7, vasopressin-8, vasopressin-9, vasopressin-10, vasopress

21. 21. The method of claim 20, wherein the adenylyl cyclase activator is present in the culture at a concentration ranging from 0.1 to 10 μM.

22. 21. The method of claim 20, wherein the adenylyl cyclase activator is forskolin, and the forskolin is present in the culture at a concentration ranging from 0.75 to 2.5 μM.

23. 21. The method of claim 20, wherein the adenylyl cyclase activator is forskolin and the forskolin is present in the culture at a concentration of 1.0 μM.

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

25. 25. The method of claim 24, wherein the actin-binding protein is present in the culture at a concentration ranging from 0.1 to 10 μg / ml.

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

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

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

29. 29. The method of claim 28, wherein the BMP pathway agonist is present in the culture at a concentration ranging from 5 to 50 ng / ml.

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

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

32. The Wnt pathway antagonist is selected from the group consisting of XAV939, ICG-001 (fossenvivin), capmatinib (INCB28060), endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptonide, KYA1797K, JW55, JW67, JW74, and cardioplegia. The method of any one of claims 1 to 8, wherein the therapeutic agent is selected from the group consisting of iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, gigantrol, RCM-1, WIKI4, IQ-1, adavivant, PRI-724, tegatrabetan, or a combination thereof.

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

34. 33. The method of claim 32, wherein the Wnt pathway antagonist is XAV939, and the XAV939 is present in the culture at a concentration ranging from 50 to 150 nM.

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

36. 9. The method of any one of claims 1 to 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, or a combination thereof.

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

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

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

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

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

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

43. 43. The medium of claim 42, wherein the VEGFR agonist is VEGF, the FGFR agonist is FGF2, the SHH agonist is purmorphamine, the adenylyl 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. 44. The medium of claim 43, wherein VEGF is at a concentration of 50 ng / ml, FGF2 is at a concentration of 10 ng / ml, purmorphamine is at a concentration of 500 nM, forskolin is at a concentration of 1 μM, thymosin-β4 is at a concentration of 1 μg / ml, BMP4 is at a concentration of 20 ng / ml, XAV939 is at a concentration of 100 nM, and retinoic acid is at a concentration of 500 nM.

45. 1. An isolated cell culture of human hemogenic endothelial cells, comprising: The cell culture comprises human CD31+ CD34+ CD143+ CD309+ GATA2+ FLI1+ RUNX1+ vWF+ HECs cultured in a medium containing a VEGFR agonist, an FGFR agonist, a sonic hedgehog (SHH) agonist, an adenylyl 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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