Methods for generating definitive hematopoietic cells from source cells

Abstract

A method for generating definitive hematopoietic cells from source cells comprising at least one of differentiating iPS cells, cells directly reprogrammed into hematopoietic precursors, cells directly reprogrammed into definitive hematopoietic cells, and adult or neonatal hematopoietic cells from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood, comprising activating the tricarboxylic acid cycle of the source cells using a metabolic regulator. Another method relates to generating primitive hematopoietic cells from source cells comprising at least one of differentiating iPS cells, cells directly reprogrammed into hematopoietic precursors, cells directly reprogrammed into definitive hematopoietic cells, and adult or neonatal hematopoietic cells from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood, comprising inhibiting the tricarboxylic acid cycle of the source cells using a metabolic regulator. Some embodiments relate to metabolic regulators for activation of the tricarboxylic acid cycle of source cells for the production of definitive or primitive hematopoietic cells.

Description

[Technical Field]

[0001] Incorporation by reference to any priority application Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as having been filed with this application are incorporated herein by reference under 37 CFR § 1.57. [Background technology]

[0002] Technical Field 1. A method for generating definitive hematopoietic cells from source cells, wherein the definitive hematopoietic cells comprise at least one of differentiating iPS cells, cells directly reprogrammed into hematopoietic cell precursors, cells directly reprogrammed into definitive hematopoietic cells, and adult or neonatal hematopoietic cells from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood, and wherein the method comprises activating the tricarboxylic acid cycle of the source cells using a metabolic regulator.

[0003] 2. Description of Related Art In the developing embryo, primitive hematopoiesis gives rise to erythrocytes, megakaryocytes, and macrophages in blood islands of the yolk sac (YS) (Palis, J. et al. Development 126, 5073-5084 (1999)). Next, a definitive wave of hematopoiesis produces more mature erythroid-myeloid (Palis, J. et al. Development 126, 5073-5084 (1999)) and lymphoid (Yoder, M. C. et al. Immunity 7, 335-344 (1997) and Boiers, C. et al. Cell Stem Cell 13, 535-548 (2013)) precursors. Around Carnegie stage (CS) 12-13, hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region through the second definitive hematopoietic wave (Medvinsky, A. & Dzierzak, E. Cell 86, 897-906 (1996) and Ivanovs, A. et al. J Exp Med 208, 2417-2427 (2011)). Primitive erythroid cells, erythroid-myeloid progenitors (EMPs), and HSCs are derived from hemogenic endothelial (HE) cells (Lancrin, C. et al. Nature 457, 892-895 (2009); Frame, J. Met al. STEM CELLS 34, 431-444 (2016); and Stefanska, M. et al. Sci Rep 7, 1-10 (2017)) through a process known as the endothelial-to-hematopoietic transition (EHT) (Boisset, J.-C. et al. Nature 464, 116-120 (2010); and Kissa, K. & Herbomel, P. Nature 464, 112-115 (2010)).Studies on the emergence of hematopoiesis during embryonic development have not only described EHT in spatial and temporal context in several animal models (Boisset, J.-C. et al. Nature 464, 116-120 (2010) and Kissa, K. & Herbomel, P. Nature 464, 112-115 (2010)), but also led to a deeper understanding of the growth and transcription factors regulating this process (Chen, M.J. et al. Nature 457, 887-891 (2009); Zhou, F. et al. Nature 533, 487-492 (2016) and Swiers, G. et al. Nat Commun 4, 2924 (2013)). However, the role of metabolites and metabolic pathways in the emergence of hematopoietic cells has not been evaluated during development.

[0004] Increasing evidence indicates that metabolic pathways can control cell fate (Oburoglu, L. et al. Cell Stem Cell 15, 169-184 (2014); Moussaieff, A. et al. Cell Metabolism 21, 392-402 (2015); and Folmes, CD Let al. Cell Metabolism 14, 264-271 (2011)). Specifically, the fate of bone marrow HSCs is regulated by several metabolic pathways. The hypoxic niche of the bone marrow drives HSCs to activate anaerobic glycolysis, the minimal energy-providing pathway, ensuring their quiescence (Takubo, K. et al. Cell Stem Cell 12, 49-61 (2013)). HSC self-renewal and maintenance depend on fatty acid oxidation (Ito, K. et al. Nat Med 18, 1350-1358 (2012)), and differentiating HSCs switch to oxidative phosphorylation (OXPHOS) to meet their energy requirements (Yu, W.-M. et al. Cell Stem Cell 12, 62-74 (2013), and Simsek, T. et al. Cell Stem Cell 7, 380-390 (2010)).

[0005] The EHT process has been extensively modeled in vitro using pluripotent stem cells (PSCs), and the HE intermediates that arise in this context can give rise to both primitive and definitive hematopoietic cells (Garcia-Alegria, E. et al. Stem Cell Reports 11, 1061-1074 (2018)). Much research has focused on obtaining HE with only definitive potential in vitro in efforts to produce functional, transplantable HSCs for therapeutic use (Kennedy, M. et al. Cell Reports 2, 1722-1735 (2012); Sugimura, R. et al. Nature 545, 432-438 (2017); Ng, E. et al. Nature Biotechnology 34, 1168-1179 (2016); and Sturgeon, C. M. et al. Nat Biotech 32, 554-561 (2014)).

[0006] Because EHT involves tight junction dissolution, the acquisition of stem cell-like properties, and leads to widespread transcriptional and phenotypic changes in migrating cells (Zhou, F. et al. Nature 533, 487-492 (2016), Swiers, G. et al. Nat Commun 4, 2924 (2013), and Guibentif, C. et al. Cell Reports 19, 10-19 (2017)), metabolism may contribute to regulating these processes. Previously, in animal models, HSC emergence was shown to be regulated by adenosine signaling and the PKA-CREB pathway (Jing, L. et al. J Exp Med 212, 649-663 (2015), and Kim, P Get al. J Exp Med 212, 633-648 (2015)), which is tightly controlled by ATP levels and availability, indicating altered energy demands during EHT. Glucose metabolism has also been shown to induce HSC emergence in zebrafish (Harris, J. Met al. Blood 121, 2483-2493 (2013)).

[0007] As will be appreciated by those skilled in the art, there is currently a limited availability of appropriately matched hematopoietic cells for transplantation or transfusion procedures, which are necessary in the routine treatment of over 100 blood disorders, malignancies, and other life-threatening conditions. Current sources of hematopoietic cells and hematopoietic stem cells are limited because they typically rely on donations from healthy individuals as part of blood drives (e.g., the Red Cross) and stem cell donor registries for bone marrow, umbilical cord blood, and mobilized peripheral blood. The shortage of these appropriate donor blood products limits the ability to administer necessary therapies; thus, up to 30% of patients requiring hematopoietic stem cell transplants for the treatment of malignancies do not have appropriately matched donors, and because need varies over time and geographic regions, complex infrastructures for the transportation of transfusable blood cells and donation drives from willing donors address the supply shortage. Thus, there is a great need for more robust and reliable systems for obtaining both hematopoietic stem cells and transfusable blood cell products.

[0008] There are also risks associated with using donor-derived products, such as the transmission of infection to the recipient patient, and tissue rejection complications such as graft-versus-host disease, both of which can be life-threatening. Therefore, there is a need to develop alternative sources of these hematopoietic cells that can be perfectly matched to the recipient without the risk of infection transmission and that have virtually unlimited self-renewal capacity. Summary of the Invention

[0009] We determine that metabolic regulation drives HE cells to preferentially adopt a definitive hematopoietic fate. We show that there is a gradual global increase in metabolism during human EHT, fueled by glucose, glutamine, and pyruvate. By analyzing the utilization of these nutrients, we elucidate their role in hematopoietic lineage specification.

[0010] Some aspects relate to methods of generating definitive hematopoietic cells from source cells, the source cells comprising: Differentiating iPS cells, cells directly reprogrammed into hematopoietic cell precursors, cells directly reprogrammed into definitive hematopoietic cells, and adult or neonatal hematopoietic cells from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood; The method involves using a metabolic regulator to activate the tricarboxylic acid cycle in a source cell.

[0011] In some instances, the metabolic regulator inhibits pyruvate dehydrogenase kinase (PDK).

[0012] In some instances, the metabolic regulator activates the pyruvate dehydrogenase complex (PDH).

[0013] In some instances, the metabolic regulator increases pyruvate uptake into mitochondria.

[0014] In some instances, the metabolic regulator accelerates the conversion of pyruvate to acetyl-coenzyme A (Ac-CoA).

[0015] In some examples, the metabolic regulator is dichloroacetate (DCA).

[0016] In some instances, the concentration of dichloroacetate in the culture medium for the source cells is at least 30 μM.

[0017] In some instances, DCA induces definitive hematopoiesis with a lymphoid / myeloid bias.

[0018] In some examples, the metabolic regulator is an LSD1 inhibitor.

[0019] In some examples, the LSD1 inhibitor comprises at least one of GSK2879552 or RO7051790.

[0020] In some instances, LSD1 inhibitors generate definitive hematopoietic cells of the erythroid lineage.

[0021] In some instances, the metabolic regulator increases the production of α-ketoglutarate.

[0022] In some instances, the metabolic regulator is glutamine.

[0023] In some instances, the metabolic regulator results in the generation of CD43+ cells from hemogenic endothelial (HE) source cells.

[0024] In some examples, the method further comprises using a nucleoside triphosphate.

[0025] In some instances, the metabolic regulator is a more potent or more stable equivalent of alpha-ketoglutarate.

[0026] In some examples, the metabolic regulator is dimethyl alpha-ketoglutarate (DMK).

[0027] In some examples, the concentration of dimethyl alpha-ketoglutarate in the culture medium for differentiating iPS cells is at least 17.5 μM.

[0028] In some instances, the metabolic regulator is used in combination with a nucleoside.

[0029] In some instances, the concentration of the nucleoside is at least 0.7 mg / L.

[0030] In some examples, the nucleoside comprises at least one of cytidine, guanosine, uridine, adenosine, and thymidine.

[0031] In some instances, the definitive hematopoietic cells include definitive hematopoietic stem cells.

[0032] In some instances, definitive hematopoietic stem cells have lymphoid and / or bone marrow repopulating potential.

[0033] In some instances, the definitive hematopoietic cells include definitive lymphoid and / or myeloid cells.

[0034] In some examples, the definitive lymphoid cells include at least one of T cells, modified T cells that target tumor cells, B cells, NK cells, and NKT cells.

[0035] In some instances, the definitive hematopoietic cells include mast cells.

[0036] In some instances, the definitive hematopoietic cells include red blood cells suitable for the production of adult hemoglobin.

[0037] In some instances, cells directly reprogrammed into precursors of hematopoietic cells include at least one of mesodermal progenitor cells, hemogenic endothelial cells, and cells undergoing an endothelial to hematopoietic transition.

[0038] In some instances, the adult or neonatal hematopoietic cells comprise hematopoietic stem cells or hematopoietic progenitor cells.

[0039] Some embodiments relate to methods of generating primitive hematopoietic cells from source cells, the source cells comprising at least one of the following: Differentiating iPS cells, cells directly reprogrammed into hematopoietic cell precursors, cells directly reprogrammed into definitive hematopoietic cells, and adult or neonatal hematopoietic cells from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood; The method involves using a metabolic regulator to inhibit the tricarboxylic acid cycle of the source cell.

[0040] In some instances, the metabolic regulator inhibits pyruvate uptake into mitochondria.

[0041] In some instances, the metabolic regulator inhibits the conversion of pyruvate to Ac-CoA.

[0042] In some instances, the metabolic regulator inhibits MPC.

[0043] In some examples, the metabolic regulator is UK5099.

[0044] In some examples, the concentration of UK5099 in the culture medium for the source cells is at least 100 nM.

[0045] In some instances, the metabolic regulator inhibits PDH.

[0046] In some examples, the metabolic regulator is 1-aminoethylphosphinic acid (1-AA).

[0047] In some instances, the concentration of 1-aminoethylphosphinic acid in the culture medium for the source cells is at least 4 μM.

[0048] Some embodiments relate to metabolic regulators for activation of the tricarboxylic acid cycle in source cells for the production of definitive hematopoietic cells.

[0049] Some embodiments relate to metabolic regulators for activation of the tricarboxylic acid cycle in source cells for the production of primitive hematopoietic cells. [Brief explanation of the drawings]

[0050] Those skilled in the art will appreciate that the following figures represent example data and diagrams illustrating the information described below.

[0051] [Figure 1a] An example of data demonstrating that iPSC-derived cells are consistent with primary human EHT populations. iPSC-derived HE, EHT, and HSC-like cells were sorted, cultured for 1 day, and analyzed by scRNAseq. UMAP visualization of scRNAseq data from HE, EHT, and HSC-like cells is shown, color-coded by sorting phenotype. [Figure 1b]An example of data showing that iPSC-derived cells match primary human EHT populations. The figure includes heat maps showing expression levels of endothelial and hematopoietic genes in HE, EHT, and HSC-like populations. [Figure 1c] An example of data demonstrating that iPSC-derived cells match primary human EHT populations. UMAP showing AEC / Hem cluster cells from Carnegie Developmental Stage (CS) 1333 match HE, EHT, and HSC-like populations in Figure 1a. [Figure 1d] An example of data showing that iPSC-derived cells match primary human EHT populations. Heat maps show the expression levels of endothelial and hematopoietic genes in AEC / Hem cluster cells that map to HE, EHT, and HSC-like populations, as shown in Figure 1c. [Figure 2]Example data showing that glycolysis, oxygen consumption, and mitochondrial activity increase during EHT. (a) Extracellular acidification rate (ECAR) was measured in HE (n = 24), EHT (n = 13), and HSC-like (n = 8) cells. Glycolytic flux was assessed by extracellular flux analysis. Bar graphs show relative levels of the indicated processes ± s.e.m. (from seven (HE, EHT) or three (HSC-like) independent experiments, unpaired t-test). (b) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq based on percent expression (dot size) and average expression level (color intensity). (c) FACS-sorted HE cells were subcultured with or without 2-DG (1 mM). Representative FSC-A / CD43 plots on day 3 of subculture are shown (n = 7, see Extended Data Fig. 3d for bar graph). (d) Representative GPA / CD43 plots on day 3 of subculture and CD45 / CD43 plots on day 6 of subculture are shown (n = 6 and n = 5, respectively; see Extended Data Fig. 3e for bar graphs). (e) CellTrace Violet (CTV) fluorescence on day 3 of subculture was assessed by flow cytometry (representative of n = 4). (f) 2-NBDG uptake was measured by flow cytometry on day 10 for HE, EHT, and HSC-like cells; mean MFI levels ± s.e.m. are shown (n = 4, paired t-test). (g) Oxygen consumption rate (OCR) was measured in HE and EHT cells (n = 7), and oxidative phosphorylation was assessed by extracellular flux analysis. Bar graphs show relative levels ± s.e.m. of the indicated processes (from three independent experiments, unpaired t-test). (h) TMRE fluorescence was measured by flow cytometry on day 10 for HE, EHT, and HSC-like cells with or without 100 μM FCCP treatment, and MFI-MFI FMO levels ± sem compared to HE are shown (n = 5, paired t-test). (i) Basal OCR was measured in HE (n = 6), EHT (n = 5), and HSC-like (n = 4) cells on day 10, and the bar graph shows the mean levels ± sem compared to HE (paired t-test).(j) Live-cell imaging of HE and HSC-like cells stained with TMRE (red) on day 3 of subculture. Representative merged brightfield / TMRE and TMRE images are shown. Scale bar, 100 μm. Bar graphs show the average TMRE staining intensity from all replicate wells across all experiments (HE spindle, n = 9; HE circle, n = 9; HSC-like, n = 6; Kruskal-Wallis test with multiple comparisons). (k) Gene expression levels of TCA cycle enzymes detected by scRNAseq are shown as dot plots based on percent expression (dot size) and average expression level (color intensity). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 3]This is an example of data demonstrating that hematopoietic specification of HE depends on glutamine catabolism. FACS-sorted HE cells were passaged in glutamine-free medium containing the indicated compounds. (a) Representative plots of FSC-A / CD43 and CD34 / CD43 at day 3 of passage are shown (n = 3, see Figure 11, g for bar graph). (b) CellTrace Violet (CTV) fluorescence at day 3 of passage was assessed by flow cytometry (n = 4, see Figure 11, h for bar graph). (c) Representative CTV plots at day 3 for GPA+ (orange) and CD45+ (blue) populations derived from HE cells are shown (n = 6, see Figure 11, i for graph). (d) The percentage of cells expressing GPA or CD45 in the CD43+ population ± sem on day 6 of subculture is shown (control, n = 7; DMK, n = 6; DMK + nucl, n = 5; DMK + nucl + NEAA, n = 3; paired t-test with control; see Extended Data Fig. 5j for plot). (e) Percentage of CD45+CD56+ ± sem cells obtained after 35 days of coculture of 3-day-subcultured HE cells with OP9-DL1 stroma. During the 3-day subculture, HE cells were treated with the indicated compounds (control and DMK, n = 5; -GLN, n = 4; -GLN + nucl and DMK + nucl, n = 3; see Extended Data Fig. 5k for plot). ns, not significant; *p < 0.05; **p < 0.01. [Figure 4]This is an example of data showing that increasing pyruvate flux to mitochondria in HE cells favors definitive hematopoietic fate. (a) Pyruvate is transported into mitochondria via the mitochondrial pyruvate carrier (MPC, inhibitor: UK5099) and converted to acetyl-coA by the pyruvate dehydrogenase complex (PDH, inhibitor: l-AA). Pyruvate dehydrogenase kinase (PDK, inhibitor: DCA) negatively regulates PDH activity. (b) FACS-sorted HE cells were subcultured with or without UK5099 (10 μM) or DCA (3 mM). Representative GPA / CD43 plots (b, d) at subculture day 3 and representative CD45 / CD43 plots (c, e) at subculture day 6 are shown (see Figure 12, a, f, k, and m for corresponding bar graphs). (f) Ratio of CFU-E to CFU-G, M, and GM colonies relative to the control condition obtained from HE cells subcultured for 6 days with the indicated compounds. For percentages, see Extended Data Figure 6r (n = 5, paired t-test). (g) Fold change in expression of HBE1 or HBG1-2 transcripts normalized to KLF1 in CFU obtained from HE cells treated with UK5099 (10 μM) or DCA (3 mM) compared to untreated cells. (h) Percentage of CD45+CD56+ ± sem cells obtained after 35 days of coculture of 3-day-subcultured HE cells with OP9-DL1 stroma. During the 3-day subculture, HE cells were treated with the indicated compounds. (n = 3, one-way ANOVA test; for plots, see Figure 12, u). (ik) Pregnant mice were injected with UK5099 or DCA at E9.5, and fetal livers were analyzed by flow cytometry at E14.5. FL, fetal liver. The levels of LT-HSCs (i), T, and B cells (j) as percentages in fetal livers are shown for control (n = 10), UK5099-treated (n = 14), and DCA-treated (n = 16) conditions (one-way ANOVA). (k) The ratio of BFU-E to CFU-GM colonies obtained from sorted LT-HSCs is shown (see also data in Figure 13, e) (one-way ANOVA).CFU, colony-forming unit; BFU, burst-forming unit; E, erythrocyte; M, macrophage; G, granulocyte. (lp) HE cells cocultured with OP9-DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymus were harvested at week 12. (l) Percentages of human CD4+CD8+ double-positive thymocytes among huCD45+ cells from the thymus are shown ± sem (control, n = 6; DCA, n = 7; unpaired t-test). Percentages of human B cells (m), CLP (n), and CD11b+ myeloid cells (p) among huCD45+ cells from the BM are shown ± sem (control, n = 6; DCA, n = 7; unpaired t-test). (o) Percentage of CD11b+ myeloid cells among huCD45+ cells from PB at week 8 ± sem is shown (control, n = 6; DCA, n = 6; unpaired t-test). ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. [Figure 5] This is an example of data demonstrating how regulation of pyruvate catabolism affects HE commitment at the single-cell level. (a) Control, UK5099-treated, and DCA-treated HE cells were visualized together by UMAP and divided into seven clusters. (b) A heatmap showing scRNAseq data for expressed endothelial or hematopoietic genes in the seven clusters. (c) Clusters 6 (559 cells) and 7 (280 cells) were evaluated individually. Dot plots show the expression levels of the indicated genes as detected by scRNAseq, based on percent expression (dot size) and average expression level (color intensity). (d) Dot plots show the expression levels of the indicated hematopoietic transcription factors in clusters 6 and 7 of HE control, HE+UK5099, and HE+DCA conditions, based on percent expression (dot size) and average expression level (color intensity) as detected by scRNAseq. [Figure 6]Examples of data showing that pyruvate catabolism affects EHT through different mechanisms. (a) FACS-sorted HE cells were subcultured with the indicated compounds. The CD43+GPA+ cell frequencies ± sem on day 3 compared to control are shown (n = 4, one-way ANOVA). (b) HE cells were transduced with shScrambled (shScr) or shLSD1 with or without UK5099 (10 μM) the day after sorting. The CD43+ / GPA+ cell frequencies ± sem on day 3 compared to shScr are shown (n = 3, one-way ANOVA). (c) Acetyl-CoA can be a precursor for ACC-mediated lipid biosynthesis (inhibitor: CP-640186 or CP) or for HMGCR-mediated mevalonate pathway / cholesterol biosynthesis (inhibitor: atorvastatin or Ato). (d) FACS-sorted HE cells were subcultured with CP (5 μM), DCA (3 mM), or both, and CD43+CD45+ cell frequencies ± sem on day 6 compared to control are shown (n = 4, one-way ANOVA). (e) Cholesterol content in HE cells was measured on day 2 of treatment by filipin III staining (n = 3, paired t-test). (f) FACS-sorted HE cells were subcultured with Ato (0.5 μM), DCA (3 mM), or both, and CD43+CD45+ cell frequencies ± sem on day 3 compared to control are shown (n = 3 for control / DCA, n = 2 for Ato / Ato+DCA with two technical replicates, one-way ANOVA). (g) Glycolysis is essential for hematopoietic differentiation of HE cells, and inhibiting pyruvate entry into mitochondria (via UK5099 or shMPC1 / 2) favors a primitive erythroid fate. Increasing pyruvate flux into mitochondria via DCA amplifies acetyl-coA production, which fuels cholesterol biosynthesis and promotes definitive hematopoietic differentiation of HE cells. [Figure 7]Example data includes the generation and characterization of EHT populations of interest. (a) Schematic of the hematopoietic differentiation system. After embryoid body formation, BMP4, activin A, CHIR99021, VEGF, and hematopoietic cytokines were sequentially added to induce HE cell formation and EHT. The cells of interest were sorted on day 8 of the protocol. (b) Sorting strategy for obtaining pure HE, EHT, and HSC-like cell populations. On day 8 of differentiation, a representative plot shows the levels of CD34+ cells after magnetic bead enrichment, separation based on CD43 expression, and further gating on CXCR4-CD73- and CD90+VEcad+ for HE and EHT cells, and CD90+CD38- for HSC-like cells. (c) Pseudochronological analysis of EHT populations following the G0 (c) or S / G2M (d) pathway, and corresponding bar graphs showing population abundance. (e) scCoGAPS mapping of umbilical cord blood CD34+ cells (CB HSC) to the EHT dataset and a violin diagram showing pattern weights. (f-g) scCoGAPS mapping of the EHT dataset to the human CS 13 dorsal aorta dataset (f) and vice versa (g), with plots showing population colocalization. [Figure 8] Example data from the validation of the hematopoietic potential of HE and EHT cells. (a) Sorted HE and EHT cells were subcultured for 6 days (representative of n = 5). The levels of CD43 and CD34 markers (a) and CD43, GPA, and CD45 markers (c) were assessed on days 3 and 6 of subculture. (b) Representative photographs of wells were taken daily during HE and EHT subculture. Scale bar, 100 μm. (d) Globin gene expression in HSC-like cells assessed by scRNAseq. [Figure 9]This is an example of data demonstrating the role of glycolysis in hematopoietic specification. (a) Representative assay data show the extracellular acidification rate (ECAR) measured in HE and EHT cells under basal conditions and after the addition of the indicated compounds. A bar graph is shown in Figure 2a. (b) Dot plots show gene expression levels of glycolytic enzymes in the human CS13 AGM region (data from Zeng et al.) detected by mapping their scRNAseq data onto our dataset (shown in Figure 1c), based on percent expression (dot size) and average expression level (color intensity). (c) Glucose is degraded through glycolysis, and the resulting pyruvate is either converted to lactate or to acetyl-coA for integration into the TCA cycle. 2-Deoxy-D-glucose (2-DG) blocks glycolytic flux. (d) CD43+ cell frequency ± sem on day 3 of subculture compared to control is shown (n = 7, paired t-test). (e) The frequency of CD43+GPA+ cells on subculture day 3 and CD43+CD45+ cells on subculture day 6 compared to control ± sem is shown (n = 6 and n = 5, respectively, paired t-test). (f) CellTrace Violet (CTV) fluorescence on subculture day 3 was assessed by flow cytometry, and median MFI values ​​are shown (n = 4, paired t-test). [Figure 10a] This is an example of data demonstrating increased OXPHOS during EHT, even in the absence of glucose. Representative assay data show the oxygen consumption rate (OCR) measured in HE and EHT cells under basal conditions and after addition of the indicated compounds. A bar graph is shown in Figure 2, g. [Figure 10b] Figure 1 shows an example of data demonstrating increased OXPHOS during EHT even in the absence of glucose. Figure 2 shows a heatmap showing scRNAseq data of OXPHOS-related genes expressed in HE, EHT, and HSC-like populations. [Figure 10c]This is an example of data demonstrating increased OXPHOS during EHT, even in the absence of glucose. This is a heatmap showing scRNAseq data (from Zeng et al.) of OXPHOS-related genes expressed in the human CS 13 AGM region, mapped onto our dataset (shown in Figure 1c). Note that many more OXPHOS-related genes are detectable in this primary cell dataset. [Figure 10d] An example of data demonstrating increased OXPHOS during EHT, even in the absence of glucose. Dot plot showing scRNAseq data for TCA cycle enzymes expressed in the human CS 13 AGM region (data from Zeng et al.) by mapping their scRNAseq data onto our dataset (shown in Figure 1c). [Figure 10e] This is an example of data demonstrating increased OXPHOS during EHT, even in the absence of glucose. OCR was measured in HE and EHT cells (n = 11) in the absence of glucose and after the addition of the indicated compounds. The corresponding bar graphs show the mean levels of OCR ± sem in the absence of glucose and after glucose injection (n = 11, from three independent experiments, unpaired t-test). [Figure 11]This is an example of data demonstrating the contribution of glutamine to different processes for inducing early erythroid and mature hematopoietic lineages. (a) Schematic showing the contribution of glutamine to the TCA cycle. Glutamine is deamidated to glutamate (Glu), which is then converted to α-ketoglutarate (α-KG), an intermediate of the TCA cycle. The conversion of glutamine to glutamate is mediated by the enzyme glutaminase (GLS), which is specifically inhibited by BPTES. (b) Dot plots showing gene expression levels of glutamine transporters detected by scRNAseq based on percent expression (dot size) and average expression level (color intensity). (c and d) FACS-sorted HE cells were subcultured with or without BPTES (25 μM). Representative plots and bar graphs (c) on day 3 of subculture show CD43+GPA+ cell frequency ± s.e.m. (n = 6, paired t-test). Representative plots and bar graphs (d) show CD43+CD45+ cell frequencies ± sem on day 6 of subculture (n = 4, paired t-test). (e and f) FACS-sorted HE cells were subcultured in glutamine-free medium containing the indicated compounds. Representative plots of FSC-A / CD43 (e) on day 3 of subculture are shown. Bar graphs of the percentage of cells expressing CD43 ± sem on day 6 of subculture are depicted (f) (n = 3, paired t-test). (g) Bar graphs of the percentage of CD34-CD43+ cells ± sem in Figure 3, a are shown (n = 3, paired t-test). (h) CTV median MFI ± sem compared to control is shown (n = 5, paired t-test; see corresponding plot in Figure 3, b). (i) CTV median MFI ± sem corresponding to Figure 3, c is shown (n = 6, paired t-test). (j) FACS-sorted HE cells were subcultured in glutamine-free medium containing the indicated compounds. Representative plots for FSC-A / GPA and FSC-A / CD45 at day 3 or 6 of subculture are shown (see Figure 3, d for graph).(e) Plot showing the percentage of CD45+CD56+ cells obtained after 35 days of coculture of 3-day subcultured HE cells with OP9-DL1 stroma. During the 3-day subculture, HE cells were treated with the indicated compounds. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. [Figure 12]Example data showing pyruvate catabolism induces hematopoietic lineage specification. (a) FACS-sorted HE, EHT, or HSC-like cells were passaged with or without UK5099 (10 μM). The CD43+ / GPA+ cell frequency ± sem on passage day 3 compared to control is shown for all populations (HE, n = 5; EHT, n = 6; HSC-like, n = 4; paired t-test). (b) FACS-sorted HE cells were passaged with or without 1-AA (4 mM). The CD43+GPA+ cell frequency ± sem on passage day 3 compared to control is shown (n = 4; paired t-test). (c) The fold change in expression of MPC1 and MPC2 relative to HPRT in shRNA-transduced cells compared to shScrambled (shScr) is shown (n = 3; unpaired t-test). Untr, untransduced. (d) HE cells were transduced with shScrambled (shScr), shMPC1, shMPC2, or both the day after sorting, and the CD43+ / GPA+ cell frequency ± sem on day 3 compared to shScr is shown (n = 4, one-way ANOVA test). Untr, untransduced. (e) FACS-sorted HE cells were stained with CTV, and fluorescence was assessed by flow cytometry for GPA+ cells on day 3 of subculture with or without UK5099 (10 μM). Representative of n = 3. (fg) FACS-sorted HE, EHT, or HSC-like cells were subcultured with or without UK5099 (10 μM). The CD43+ (f) and CD43+CD45+ (g) cell frequencies ± sem on day 6 of passage compared to control for all populations are shown (HE, n = 7; EHT, n = 7; HSC-like, n = 4, paired t-test). (h) FACS-sorted HE cells were passaged with or without 1-AA (4 mM). The CD43+ and CD43+CD45+ cell frequencies ± sem on day 6 of passage compared to control are shown (n = 3, paired t-test). (ij) FACS-sorted HE cells were passaged with or without UK5099 (10 μM) for 3 days. The CTV of HE-derived CD45+ cells (i) and the frequency of HE-derived HSC-like cells ± sem (n = 7, paired t-test) (j) are shown.(kp) FACS-sorted HE and EHT cells were passaged with or without DCA (3 mM). The frequency ± sem of CD43+GPA+ cells on passage day 3 (k, n = 3) and day 6 (l, HE, n = 5, EHT, n = 4) compared to the control is shown (paired t-test). (m) CTV of HE-derived GPA+ cells on passage day 3 is shown (representative of n = 3). (n) The frequency ± sem of CD43+CD45+ cells on passage day 6 compared to the control for both populations is shown (HE, n = 5, EHT, n = 4, paired t-test). (o) CTV of HE-derived CD45+ cells is shown (representative of n = 3). (p) The frequency ± sem of HE-derived HSC-like cells on passage day 6 compared to the control is shown (n = 4, paired t-test). (q) EdU incorporation into HE cells was assessed by flow cytometry after a 24-hour pulse on days 1 and 2 of subculture with or without UK5099 (10 μM) or DCA (3 mM) (n = 3). (rs) Percentage of CFU colony types obtained from HE cells subcultured with the indicated compounds for 3 days (r) (n = 3, two-way ANOVA) or 6 days (s) (n = 5, two-way ANOVA). CFU, colony-forming unit; E, erythroid; M, macrophage; G, granulocyte; GEMM, mixed. (t) EryD and EryP CFU-E obtained from HE cells subcultured with the indicated compounds for 3 days. Scale bar, 100 μm. (u) Fold change in HBA1-2 transcript expression normalized to KLF1 in CFU obtained from HE cells treated with UK5099 (10 μM) or DCA (3 mM) compared to untreated cells. (v) Plot showing the percentage of CD45+CD56+ cells obtained after 35 days of coculture of 3-day subcultured HE cells with OP9-DL1 stroma. During the 3-day subculture, HE cells were treated with the indicated compounds. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. [Figure 13]This is an example of data demonstrating that modulation of pyruvate metabolism affects lineage specification in vivo. (a) Pregnant mice were injected with UK5099 or DCA at E9.5, and fetal livers were analyzed by flow cytometry at E14.5. FL, fetal liver. Percentage levels of HPC-1, HPC-2 (a), and erythroid precursors (b) in fetal liver are shown for control (n = 10), UK5099-treated (n = 14), and DCA-treated (n = 16) conditions (one-way ANOVA). (c) Erythroid differentiation stages by CD71 / Ter119 staining are shown on the control plot. Representative plots showing the percentage of cells at each stage are shown for control, UK5099-treated, or DCA-treated conditions. (d) The gating strategy for sorting LT-HSCs in E14.5 embryos is shown. (e) The percentage of colonies obtained from sorted LT-HSCs is shown for control (n = 4), UK5099-treated (n = 8), and DCA-treated (n = 10) conditions (one-way ANOVA). (f) Irradiated NSG mice were transplanted with DCA-treated HE cells maintained in coculture with OP9-DL1 stroma for 3 days, and human cells in the peripheral blood (PB) were assessed at 4, 8, and 12 weeks. (f) Engraftment levels in the peripheral blood (PB) as a percentage of huCD45+ cells are shown (control, n = 6; DCA, n = 7). (g) Thymuses were harvested 12 weeks post-transplant, and representative plots showing CD4 / CD8-expressing cells are shown for control and DCA-treated conditions. (h) Representative plots and percentages ± sem of CD19+ cells (B cells) among huCD45+ cells from PB at week 8 are shown (control, n = 6; DCA, n = 6; unpaired t-test). (i) Percentages ± sem of human HSCs among huCD45+ cells from BM are shown (control, n = 6; DCA, n = 7; unpaired t-test). ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. [Figure 14](c) 10x10 dot plots showing the percentage of cells belonging to clusters 6 and 7 in each condition. (d) Number of GPA+ clones obtained from single HE cells co-cultured on OP9-DL1 stroma and treated with the indicated compounds for 14 days (n = 6 independent experiments, a total of 552 wells screened for each condition). [Figure 15]Example data showing mechanistic analysis of pyruvate catabolism during EHT. (a) FACS-sorted HE cells were subcultured with or without TSA (60 nM). CD43 MFI levels and representative CD43 histograms at subculture day 3 are shown (n = 4, paired t-test). (b) Dot plots showing gene expression levels of LSD1, GFI1, and GFI1B as detected by scRNAseq and based on percent expression (dot size) and average expression level (color intensity). (c) Fold change in expression of LSD1 relative to HPRT in shRNA-transduced cells compared to shScrambled (shScr) is shown (n = 3, unpaired t-test). Untr, untransduced. (de) FACS-sorted HE cells were subcultured with TCP (300 nM), DCA (3 mM), or both. Day 6 CD43+CD45+ cell frequencies ± sem (d) and day 6 CD43+CD45+CD33+CD11b+ cell frequencies ± sem (e) are shown compared to control (n = 5, one-way ANOVA). (f) Acetate can be directly converted to acetyl-coA by ACSS2 (inhibitor: ACSS2i). Acetyl-coA is the precursor of acetylation marks, which are transferred onto histones via histone acetyltransferase (HAT, inhibitor: C646). (gh) FACS-sorted HE cells were subcultured with ACSS2i (5 μM), DCA (3 mM), or both (n = 5, one-way ANOVA) (g) or C646 (10 μM), DCA (3 mM), or both (n = 3, one-way ANOVA). (h) Day 6 CD43+CD45+ cell frequencies ± sem are shown. (i) FACS-sorted HE cells were subcultured on coverslips for 2 days with or without DCA (3 mM). Staining intensity of H3K9 acetylation and H4K5, 8, 12, and 16 acetylation was assessed by confocal microscopy imaging, and fold changes compared to control are shown (n = 3). (j) Dot plots showing gene expression levels of cholesterol efflux pathway genes detected by scRNAseq based on percent expression (dot size) and average expression level (color intensity). DETAILED DESCRIPTION OF THE INVENTION

[0052] During embryonic development, hematopoiesis first occurs through primitive and definitive waves, primarily in the yolk sac (YS) and aorta-gonad-mesonephros (AGM) regions, giving rise to different blood lineages (Palis, J. et al. Development 126, 5073-5084 (1999), and Medvinsky, A. & Dzierzak, E. Cell 86, 897-906 (1996)). The first hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells in the AGM through the endothelial-to-hematopoietic transition (EHT) (Boisset, J.-C. et al. Nature 464, 116-120 (2010), and Kissa, K. & Herbomel, P. Nature 464, 112-115 (2010)). In adults, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism (Takubo, K. et al. Cell Stem Cell 12, 49-61 (2013) and Yu, W.-M. et al. Cell Stem Cell 12, 62-74 (2013)). In a specific example disclosed herein, the de novo emergence of blood can be regulated by multiple metabolic pathways that directly induce or regulate hematopoietic specification and lineage commitment in human EHT. EHT can be accompanied by a metabolic switch involving increased glycolysis and oxidative phosphorylation (OXPHOS). Moreover, OXPHOS-fueled glutamine may be essential for hematopoietic emergence and can induce distinct lineage retrievals through its different pathway intermediates. In both in vitro and in vivo settings, manipulating pyruvate utilization toward glycolysis or OXPHOS can differentially bias HE cell commitment toward either a primitive erythroid fate or a definitive fate with lymphoid / myeloid potential, respectively. In certain instances, commitment to primitive or definitive fates in this context may be controlled by different mechanisms. During EHT, metabolism may be a major determinant of hematopoietic specification, lineage commitment, and primitive versus definitive fate decisions. The disclosure provided herein may provide a basis for generating definitive HSCs in vitro using metabolic pathway modulation, thereby providing a valuable source of therapy for, in examples, hematological disorders and malignancies.

[0053] Induced pluripotent stem (iPS) cells are one such ideal source, and perhaps the most feasible, because their functional equivalence to embryonic stem cells allows for unlimited self-renewal potential, and they can be generated from a patient's own somatic cells (e.g., skin cells, or amniotic fluid MSCs, etc.) and thus recognized as autologous. In some instances, the ability to generate hematopoietic stem cells from patient-derived iPS cells allows for the generation of an unlimited supply of human leukocyte antigen (HLA)-matched cells, capable of reconstituting the hematopoietic system of patients with hematological disorders or those undergoing chemotherapy for hematopoietic and some non-hematopoietic solid tumor malignancies. In some instances, depending on the source of somatic cells from which the iPS cells are derived, iPS-derived hematopoietic stem cells may have advantages over conventionally harvested hematopoietic stem cells in terms of: 1) reduced acquired mutations (e.g., when the iPS cells are derived from a neonatal cell source), 2) unlimited proliferation capacity, 3) reduced rejection issues, 4) absence of contaminating cells from the original tumor present, and 5) the ability to correct congenital mutations in patient-derived iPS cell lines using existing gene editing technologies such as Crispr / Cas.

[0054] Moreover, recent advances in the ability to generate T cells specifically engineered to target and destroy malignant cells after their differentiation from iPS cells mean that transplants can be performed with the co-administration of stem cells and antitumor T cells (Trounson et al. Nature Reviews 2016, Vizcardo et al. Cell Stem Cell 2013). As such, in some instances, the ability to generate iPS-derived hematopoietic stem cells provides immediate donor cell demand for many patients, potentially providing an exponential increase in use as surrounding technologies advance. Thus, iPS-derived hematopoietic cells offer a reliable and robust new treatment for patients with the aforementioned life-threatening diseases.

[0055] Furthermore, the ability to generate therapeutically valuable mature or differentiated hematopoietic cells from iPS cells for transfusion into patients represents another, perhaps even greater, aspect of meeting a public need. In some instances, functional red blood cells could be generated en masse for all blood groups to address shortages of transfusion products for patients suffering from blood loss as a result of injury, those requiring transfusions during surgery, or those suffering from various forms of anemia. In addition, other blood cells differentiated from iPS cells may also be useful in the treatment of cancer, such as NK or T cells programmed with antitumor activity.

[0056] The tricarboxylic acid (TCA) cycle, also known as the Krebs or citric acid cycle, is the primary source of energy for cells and is an essential part of aerobic respiration. The cycle utilizes the available chemical energy of acetyl coenzyme A (acetyl-CoA) for the reducing power of nicotinamide adenine dinucleotide (NADH). The TCA cycle is part of the larger glucose metabolism, whereby glucose is oxidized to form pyruvate, which is then oxidized and enters the TCA cycle as acetyl-CoA.

[0057] Differentiating iPS cells function like embryonic stem (ES) cells. Unlike ES cells, iPS cells are more readily available for therapy and research, and their isolation does not carry the same ethical concerns. Because human iPS cells can be derived from the patient themselves, they may be an ideal source for patient-specific therapies. In addition, iPS cells can serve as a useful research tool by providing models of human disease, as well as models of normal development, for use in screening new drugs or studying pathogenesis and toxicology.

[0058] Hematopoietic stem cells (HSCs) are undifferentiated cells whose progeny reconstitute blood cell lineages, such as monocytes / macrophages or T and B lymphocytes, through a process called hematopoiesis. HSCs have the potential for indefinite self-renewal, which explains their purpose in transplantation for the sustained reconstitution of blood cells. B cells are a type of lymphocyte involved in humoral immunity (antibody-mediated immunity).

[0059] Definitive hematopoietic stem cells (HSCs) are responsible for the continuous production of all mature blood cells during an individual's entire adult lifespan. They are clinically important cells in transplant protocols used to treat blood-related diseases. Experimentally, HSCs can result in long-term reconstitution of the entire hematopoietic system in irradiated adult recipients.

[0060] In certain instances, specific metabolic pathway regulators of glycolysis and the TCA cycle (the primary means of energy production in cells) can directly activate transcriptional changes in precursors of hematopoietic cells (cells undergoing the endothelial-to-hematopoietic transition) that enable induced hematopoietic lineage bias and the generation of definitive hematopoietic cells. The ability to generate definitive hematopoietic cells from reprogrammed cells is critical for therapy because only definitive cells give rise to lymphoid blood lineages (NK, B, and T cells), hematopoietic stem cells, and erythroid cells (red blood cells) that express adult hemoglobin. These are cell types currently provided by donors that are already widely used or being developed for use in hematopoietic cell-based therapies, including the millions of red blood cell transfusions that patients receive worldwide each year.

[0061] In addition to the above-mentioned metabolic pathway modulation in iPS-derived production of definitive hematopoietic cells, metabolic modulation may also be an important means for generating definitive blood from cell sources other than iPS cells. For example, de novo generation of definitive hematopoietic cells can also be achieved by direct reprogramming of somatic cells into blood progenitor cells, including mesodermal cells and cells undergoing the endothelial-to-hematopoietic transition, in addition to directly reprogrammed blood cells. Metabolic modulation may provide the basis for inducing definitive blood production in all these cases. Furthermore, the self-renewal capacity of already committed definitive blood cells (i.e., from bone marrow, umbilical cord blood, and mobilized peripheral blood, as currently used in hematopoietic stem cell transplantation therapies worldwide today) benefits from metabolic pathway manipulation for the self-renewal and proliferation of therapeutic hematopoietic stem cells or other definitive hematopoietic cells.

[0062] Pyruvate dehydrogenase kinase family members (PDK1, PDK2, PDK3, and PDK4) are serine kinases that catalyze the phosphorylation of the E1α subunit of the pyruvate dehydrogenase complex (PDC). Pyruvate dehydrogenase kinase is activated by ATP, NADH, and acetyl-CoA. It is inhibited by ADP, NAD+, CoA-SH, and pyruvate. Biochemicals that inhibit PDK can be used to induce hematopoietic lineage bias and generate definitive hematopoietic cells. For example, inhibitors of pyruvate dehydrogenase kinase (PDK) include Leelamin HCl, a weak CB1 receptor agonist and PDK inhibitor; quercetin dihydrate, a natural flavonoid antiproliferative kinase inhibitor; sodium dichloroacetate, an inhibitor of mitochondrial pyruvate dehydrogenase kinase; SB 203580 (hydrochloride), a MAPK inhibitor; dichloroacetic acid, a mitochondrial PDK (pyruvate dehydrogenase kinase) inhibitor; PDK1 / Akt / Flt Dual Pathway Inhibitor, a cell-permeable compound that selectively induces apoptosis; BX 795, an inhibitor of PDK1, TBK1, and IKKε SB 203580; a pyridinylimidazole and specific inhibitor that suppresses p38-mediated activation of MK2; KT 5720, a potent, specific, cell-permeable inhibitor of PKA; BX-912, a potent and selective PDK-1 inhibitor that induces apoptosis; GSK 2334470, a potent and selective PDK1 inhibitor that subsequently induces apoptotic cell death; and OSU 03012, a PDK1 inhibitor and inducer of caspase- and p53-independent apoptosis.

[0063] Pyruvate dehydrogenase (PDH) is the first component enzyme of the pyruvate dehydrogenase complex (PDC). The pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA through a process called pyruvate decarboxylation (Swanson conversion). Acetyl-CoA can then be used in the citric acid cycle to carry out cellular respiration. Thus, pyruvate dehydrogenase connects the glycolytic metabolic pathway to the citric acid cycle, releasing energy via NADH. Pyruvate dehydrogenase can be allosterically activated by fructose-1,6-bisphosphate and inhibited by NADH and acetyl-CoA. Phosphorylation of PDH is mediated by pyruvate dehydrogenase kinase. Metabolic regulators that activate the pyruvate dehydrogenase complex (PDH) can be used.

[0064] The PDH inhibitor 1-aminoethylphosphinic acid (1-AA) can be used in the culture medium for source cells, with the concentration of 1-AA preferably being at least 4 μM, but can range from about 0.5 μM to 50 μM, e.g., about: 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM.

[0065] In some instances, metabolic regulators can be used to increase pyruvate uptake into mitochondria. Pyruvate transport across the outer mitochondrial membrane (OMM) occurs via large, nonselective channels, such as voltage-dependent anion channels / porins, which allow passive diffusion (Benz R. Biochim Biophys Acta. 1994;1197:167-196). Voltage-dependent anion channels (VDACs) are the most abundant proteins in the OMM and serve as the primary pathway for metabolite / ion transport between the cytosol and the mitochondrial intermembrane space (IMS). Defects in these channels have been shown to block pyruvate metabolism (Huizing M. et al. Pediatr Res. 1996;39:760-765). Inhibitors of voltage-dependent anion channels / porins can be used to inhibit pyruvate uptake. VDAC phosphorylation by the protein kinases GSK3β, PKA, and protein kinase C epsilon (PKCε) blocks or inhibits VDAC binding to other proteins, such as Bax and tBid, and also regulates VDAC opening. PKA-dependent VDAC phosphorylation and GSK3β-mediated VDAC2 phosphorylation increase VDAC conductance.

[0066] However, the movement of metabolites such as pyruvate through the inner mitochondrial membrane (IMM) can be more restricted than across the OMM. Many metabolites have specific inner mitochondrial membrane transporters that have been identified and studied (Palmieri F. et al. Biochim Biophys Acta. 1996;1275:127-132).

[0067] Metabolic regulators that accelerate the conversion of pyruvate to acetyl-coenzyme A (Ac-CoA) can be used. Dichloroacetate (DCA) inhibits pyruvate dehydrogenase (PDH) kinase, thereby maintaining PDH in an active, dephosphorylated state, thereby promoting pyruvate entry into the Krebs cycle. In cases where the metabolic regulator is dichloroacetate (DCA), the concentration of dichloroacetate in the culture medium for the source cells can be at least about 30 μM and can range from 10 μM to 100 μM, including concentrations of about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM.

[0068] The metabolic regulators used in the methods disclosed herein can inhibit the conversion of pyruvate to Ac-CoA. For example, UK-5099 is a potent inhibitor of the mitochondrial pyruvate carrier (MPC). UK-5099 has an IC of 50 nM. 50 The concentration of UK5099 in the culture medium for source cells can be at least 100 nM, but can range from 10 nM to 1 μM, including approximately 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, and 1 μM.

[0069] Lysine-specific demethylase 1 (LSD1) can be used to treat EHT, especially in the erythroid lineage. Numerous LSD1 inhibitors have been reported, including TCP, ORY-1001, GSK-2879552, IMG-7289, INCB059872, CC-90011, ORY-2001, and RO7051790. One or more of these inhibitors can be used in combination, such as two or more, three or more, four or more, five or more, or six or more combinations.

[0070] Metabolic regulators that increase the production of α-ketoglutarate can be used. For example, L-glutamine is a nutritionally semi-essential amino acid for proper growth in most cells and tissues and plays an important role in determining and protecting normal metabolic processes in cells. With the help of transport systems, extracellular L-glutamine can cross the plasma membrane and be converted to α-ketoglutarate (AKG) through two pathways: glutaminase (GLS) I and II. Different steps in glutamine metabolism (glutamine-AKG axis) can be regulated by several factors (Xiao, D. et al. 2016 Amino Acids 48:2067-2080), making the glutamine-AKG axis a potential target for regulating the generation of definitive hematopoietic cells from source cells by activating the tricarboxylic acid cycle in source cells. Alpha-ketoglutarate is membrane-impermeable, meaning it is usually added to cells in the form of esters such as dimethyl alpha-ketoglutarate (DMKG), trifluoromethylbenzyl alpha-ketoglutarate (TFMKG), and octyl alpha-ketoglutarate (O-KG). Once these compounds cross the plasma membrane, they can be hydrolyzed by esterases to produce alpha-ketoglutarate, which remains trapped within the cell. All three compounds increase intracellular levels of alpha-ketoglutarate. Thus, these compounds are metabolic regulators of alpha-ketoglutarate. In some examples, the concentration of dimethyl alpha-ketoglutarate in culture medium for differentiating iPS cells can be at least about 17.5 μM, but can be between about 10 μM and 100 μM, including concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM.

[0071] The various metabolic regulators disclosed herein can be used in combination with a nucleoside, and the concentration of the nucleoside can be at least 0.7 mg / L, but can be from about 0.1 mg / L to about 10 mg / L, such as about 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L, 31 mg / L, 32 mg / L, 33 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 37 mg / L, 38 mg / L, 39 mg / L, 39 mg / L, 40 mg / L, 41 mg / L, 42 mg / L, 43 mg / L, 44 mg / L, 45 mg / L, 46 mg / L, 47 mg / L, 48 mg / L, 49 mg / L, 50 mg / L, 51 mg / L, 5 6.5mg / L, 7mg / L, 7.5mg / L, 8mg / L, 8.5mg / L, 9mg / L, 9.5mg / L, 10mg / L, and 10.5mg / L. Nucleosides include at least one of cytidine, guanosine, uridine, adenosine, and thymidine, but can include any potential combination, such as two, three, four, or all five nucleosides.

[0072] Example 1 Recapitulation of human EHT and hematopoietic differentiation in vitro In one example of obtaining both primitive and definitive hematopoiesis in culture, two previously mentioned small molecules were combined during human iPSC differentiation (Figure 7, a): CHIR99021, a WNT pathway agonist that supports definitive hematopoiesis (Ng, E. et al. Nature Biotechnology 34, 1168-1179 (2016)), and Activin A, which promotes primitive hematopoiesis (Kennedy, M. et al. Cell Reports 2, 1722-1735 (2012)). After integrating these modifications into a previously described hematopoietic differentiation protocol (Ditadi, A. & Sturgeon, C.M. Methods 101, 65-72 (2016)), we obtained hemogenic endothelial cells (HE), transitioning cells expressing intermediate levels of CD43 (EHT) (Guibentif, C. et al. Cell Reports 19, 10-19 (2017)), and hematopoietic stem-like cells (HSC-like, immunophenotype HSC) (see Figure 7.b for gating strategy). These three populations were transcriptionally characterized using single-cell RNA sequencing (scRNAseq). UMAP visualization placed HE cells distal to HSC-like cells and EHT cells bridged these two populations, confirming the sequential EHT process (Figure 1a). In addition, pseudotime analysis of the dataset was performed, revealing two cell cycle pathways: G0 (Figure 7.c) and S / G2M (Figure 7.d). In both cases, abundant HE cells were observed at the beginning of the trajectory, EHT cells in the middle, and HSC-like cells at the end (Figure 7, c and d, bar graphs). HE cells expressed endothelial markers such as KDR, FLT1, and CDH5, but not hematopoietic markers. In contrast, as previously shown in other EHT systems (Zhou, F. et al. Nature 533, 487-492 (2016); Swiers, G. et al. Nat Commun 4, 2924 (2013); and Guibentif, C. et al. Cell Reports 19, 10-19 (2017)), EHT cells expressed both endothelial and hematopoietic markers, while HSC-like cells expressed only hematopoietic markers such as RUNX1, TAL1, WAS, and SPN (Figure 1b). Isolated cord blood CD34 +We generated a cell dataset and projected it onto the EHT process dataset using the scCoGAPS package, observing that the highest pattern weights were part of pattern 1 and pattern 3, both of which encompass our HSC-like cluster (Figure 7, e), and were associated with cord blood CD34 + We demonstrated that these cells share most transcripts with iPSC-derived HSC-like cells described elsewhere herein. We compared the EHT process data with a recently published scRNA-seq analysis of primary human embryonic cells at Carnegie developmental stage (CS) 13 (Zeng, Y. et al. Cell Res 1-14 (2019)). Of the 99 cells in the arterial endothelial and hematopoietic (AEC / Hem) cluster, 50, 36, and 13 cells mapped to the HE, EHT, and HSC-like populations, respectively (Figure 1c), which clustered similarly to the EHT dataset in Figure 1a. Furthermore, similar to the EHT dataset, AEC / Hem cluster cells mapped to the HE expressed endothelial markers such as KDR, FLT1, and CDH5, while cells mapped to the HSC-like cells expressed hematopoietic markers such as RUNX1, TAL1, WAS, and SPN (Figure 1d). The dataset was mapped to the human CS13 dorsal aorta population dataset from Zeng et al. using the scCoGAPS package. The majority of the HE population (pattern 9) colocalized with the CS13 AEC and EC populations (gray arrows), and a significant portion of the HSC-like population (patterns 7-8) mapped to the CS13 Hem cluster (pink arrow) (Figure 7, f). When reverse mapping of the CS13 data was performed on the dataset, the CS13 EC population mapped near the HE cells (pattern 9, gray arrow), and the CS13 Hem cluster mapped near both EHT and HSC-like cells (pattern 10, green arrows) (Figure 7, g). Thus, in our example, the system successfully captured the human EHT process, and the resulting HE, EHT, and HSC-like populations possess blood-endothelial transcriptional signatures comparable to the cell types that arise in human embryos at CS13.

[0073] Next, we examined the hematopoietic potential of both HE and EHT populations. Both cell types were found to be hematopoietic cells (CD43 + ) (Fig. 8a). On day 6 of subculture, almost all cells (>96%) derived from HE or EHT cells were CD43 + Most of the cells lost CD34 expression (>86%), indicating their maturation. In both cell cultures, spindle-shaped endothelial cells changed their morphology to round hematopoietic cells (Figure 8, b). In both HE- and EHT-derived subcultures, erythrocytes (CD43 + GPA + ) cell populations and non-erythroid panhematopoietic CD43 + CD45 + The cell populations were clearly distinguishable on days 3 and 6, respectively (Fig. 8, c). According to the model described by Kennedy et al. (Kennedy, M. et al. Cell Reports 2, 1722-1735 (2012)), CD43 + GPA + and CD43 + CD45 + The time frame over which cell populations are generated suggests their primitive and definitive nature, respectively. Moreover, the presence of embryonic (HBZ, HBE1), fetal (HBA1, HBA2, HBG1, HBG2), and adult (HBD, HBB) globin upregulation in the subcultured HSC-like cells further supports our ability to obtain both primitive and definitive hematopoietic cells in this setting (Figure 8(d)). Altogether, these results demonstrate that this differentiation system accurately models the human EHT process and that efficient subculturing of the resulting HE cells gives rise to both primitive and definitive hematopoietic populations.

[0074] Glycolysis may fuel different processes during EHT To illustrate the metabolic processes occurring in EHT populations, glycolysis was assessed in HE, EHT, and HSC-like cells. A gradual increase in glycolytic capacity and glycolysis with differentiation was demonstrated (Figure 2a, Figure 9a). Expression of the glycolytic enzymes HK1, PFKFB2, TPI1, GAPDH, PKLR, ENO3, LDHA, and LDHB, assessed by scRNA-seq, also increased during EHT (Figure 2b, Figure 2b). In some cases, increases in most of these glycolytic enzymes during EHT were also observed in human primary cells from CS13 (Zeng, Y. et al. Cell Res 1-14 (2019)), consistent with in vitro results (Figure 9b, Figure 2c).

[0075] To investigate whether glycolytic activity is required during EHT, HE cells were treated with the glucose analog 2-deoxy-D-glucose (2-DG), which blocks glycolysis (Fig. 9c). This treatment resulted in the depletion of CD43 from HE cells on day 3 of subculture. + 2-DG significantly reduced the cell output (Fig. 2c, Fig. 9d). + GPA + Cell populations and CD43 on day 6 + CD45 + The generation of cell populations was significantly impaired, falling to less than 50% of the control (Figure 2, d; Figure 9, e). Interestingly, the proliferation rate of EHT or HSC-like cells, but not HE, was significantly reduced in the presence of 2-DG (Figure 2, e; Figure 9, f). These results indicate that while glycolysis is important for HE cells to induce hematopoietic differentiation, it may also fuel the proliferation of EHT and HSC-like cells at later steps during the EHT process.

[0076] Mitochondrial respiration can gradually increase during the EHT process Along with increased glycolysis and proliferation, HSC-like cells also had increased glucose uptake compared to HE and EHT cells (Figure 2, f). Interestingly, although glycolytic flux was higher in EHT cells compared to HE cells, glucose uptake was comparable in these two cell types. This result prompted us to investigate whether mitochondrial respiration was more active in HE versus EHT cells. Unexpectedly, EHT cells exhibited higher levels of basal respiration, ATP production, and maximal respiration compared to HE cells (Figure 2, g; Figure 10, a). Furthermore, mitochondrial activity, as measured by TMRE staining, was significantly increased in individually analyzed EHT cells compared to HE cells, and we observed even higher rates in HSC-like cells (Figure 2, h). Treatment with FCCP, which depolarizes mitochondria, abolished the TMRE signal in all cell types, indicating that OXPHOS was active in these populations (Figure 2, h). Consistent with TMRE staining, the highest basal respiration rate we detected was in HSC-like cells (Figure 2, i). Using live-cell imaging with confocal microscopy, we compared mitochondrial activity in spindle-shaped HE cells versus their newly formed round hematopoietic progeny in the same wells. TMRE staining intensity measurements showed twofold higher mitochondrial activity in round compared to spindle-shaped cells in HE wells, a value similar to the level detected in HSC-like cells (Figure 2, j). We also observed a gradual increase in the expression of several genes involved in OXPHOS, including subunits of complexes I (a gene called NDUF), II (SDHA), IV (a gene called COX), and V (a gene called ATP5) in HE, EHT, and HSC-like populations by scRNA-seq (Figure 10, b). This result was accompanied by a gradual increase in TCA cycle enzymes in EHT (Figure 2, k). We observed a gradual increase in both OXPHOS-related genes and TCA cycle enzymes during EHT in human primary cells at CS 13 (Zeng, Y. et al. Cell Res 1-14 (2019)), confirming our in vitro findings (Figure 10, c and d).Taken together, these results indicate that TCA cycle activity, mitochondrial respiration, and OXPHOS gradually increase during the EHT process.

[0077] Glutamine may be the limiting step initiating hematopoietic differentiation of HE Even in glucose-free medium, HE and EHT cells had high basal respiration levels (Figure 10, e). Thus, these cells may also rely on other energy sources for mitochondrial respiration. Glutamine can generate α-ketoglutarate (α-KG), an intermediate in the TCA cycle, which subsequently contributes to OXPHOS (Figure 11, a). As shown in the figure, HE, EHT, and HSC-like cells expressed several different glutamine transporters (Figure 11, b). HSC-like cells expressed the highest levels of the SLC1A5 transporter, as previously reported in primary cord blood HSCs (Oburoglu, L. et al. Cell Stem Cell 15, 169-184 (2014)).

[0078] To determine whether glutamine is important for EHT, we blocked the glutaminase (GLS) enzyme, which catalyzes the deamidation of glutamine to glutamate, by treating HE cells with BPTES (Figure 11, a). Day 3 HE-derived CD43 in the presence of BPTES + GPA + Erythroid populations and CD43 on day 6 + CD45 + The sharp decline in population generation is shown in the figures (Fig. 11, c and d). This result indicates that in some instances, glutamine entry into the TCA cycle may be required for hematopoietic differentiation in EHT.

[0079] Glutamine is also involved in several metabolic pathways, including nucleotide and non-essential amino acid (NEAA) synthesis (DeBerardinis, RJ & Cheng, T. Oncogene 29, 313-324 (2009)). Therefore, to better understand its role during EHT, HE cells were cultured in its absence. Glutamine deprivation significantly reduced CD43 expression from HE cells on day 3 of subculture. + The cellular output was abolished (>80% reduction) (Figure 3, a). To rescue this phenotype, glutamine-free culture medium was supplemented with nucleosides, NEAA, or a cell-permeable form of α-KG (dimethyl-ketoglutarate, DMK), all of which are substrates that can be derived from glutamine (DeBerardinis, RJ & Cheng, T. Oncogene 29, 313-324 (2009)). Nucleosides, NEAA, or a combination of both failed to rescue the effects seen in glutamine deprivation (Figure 11, e). However, DMK addition significantly reduced CD43 expression from HE cells. + The combination of DMK / nucleoside or DMK / nucleoside / NEAA rescued up to 60% of the cell output (Fig. 3a, Fig. 11e). Furthermore, the combination of DMK / nucleoside or DMK / nucleoside / NEAA rescued CD43 expression from HE cells. + The percentage of cells further increased and reached the level of the control condition.

[0080] Because pyruvate, another fuel in the TCA cycle, can substitute for glutamine, we treated HE cells with the pyruvate dehydrogenase kinase (PDK) inhibitor, dichloroacetate (DCA), to increase pyruvate dehydrogenase (PDH) activity during glutamine deprivation. Without glutamine, for example, DCA treatment alone reduced the CD43 expression seen in controls. + The cellular level could not be restored (Fig. 11, f).

[0081] In certain instances, CD34 + More mature CD43 that has lost expression +The percentage of cells significantly decreased in the glutamine-free DMK-treated condition compared to the control (Figure 3a, Figure 11g). However, nucleoside addition, alone or together with NEAA, significantly increased the CD43 + CD34 - The percentage of cells recovered to levels observed in controls. Because nucleotides are essential for proliferating cells, in certain instances, the proliferation of differentiating HE cells was dependent on this factor. In some instances, DMK or nucleosides alone were unable to restore the proliferation profile seen in control conditions; indeed, only the addition of both of these factors restored proliferation of HE cells during glutamine deprivation (Figure 3b, Figure 11h). These results suggest that glutamine promotes the proliferation of CD43 from HE. + It may be important for cell biogenesis, showing a role in TCA cycle fueling and in the production of nucleotides to support growth.

[0082] Glutamine differentially maintains hematopoietic populations Previously, it has been shown that erythroid differentiation requires glutamine-fueled nucleotide synthesis (Oburoglu, L. et al. Cell Stem Cell 15, 169-184 (2014)). Therefore, HE cells were stained with a proliferation dye (Cell Trace Violet, CTV) to detect newly formed GPA. + or CD45 + The proliferation status of the cells was evaluated after 3 days. + The cells clustered into dividing cells (low CTV MFI values), but interestingly, CD45 cells derived from HE cells + The cells had little or no division (high CTV MFI values, Fig. 3c, Fig. 11i). On days 3 and 6 of HE subculture without glutamine, DMK alone suppressed CD43 + GPA + However, the DMK / nucleoside or DMK / nucleoside / NEAA combinations failed to rescue the CD43 population to levels seen in controls (Fig. 11, j).+ GPA + As a result, glutamine functioned as both a carbon donor and a nitrogen donor, both of which regulated the CD43 population from HE cells along their proliferation profile (Fig. 3d, Fig. 11j). + GPA + It produces α-KG and nucleotides required for the production of α-KG (Fig. 3, c).

[0083] Interestingly, CD43 in the condition + CD45 + A significant increase in the percentage of HE-derived CD45 cells was restored by DMK or DMK / nucleoside compared to the control (Fig. 3d, Fig. 11j). + Consistent with the finding that cells were slower to initiate proliferation (Fig. 3c), DMK was sufficient for their induction from HE even in the absence of nucleosides. Thus, an increase in the TCA cycle was associated with the CD45 + Favorable for the formation of mature hematopoietic cells. + / nucleoside + Conditions with similar levels of CD43 to controls + GPA + As observed in (Fig. 4, d), this is due to CD43 + CD45 + This ruled out the possibility that cells take over the culture to replace other populations. To understand whether DMK induces the formation of definitive hematopoietic cells, day 3 HE cells were cocultured with OP9-DL1 stroma to induce lymphoid differentiation. Glutamine deprivation or nucleoside supplementation alone during the 3-day subculture prevented HE cells from giving rise to NK cells in the coculture, whereas DMK or DMK / nucleoside supplementation enabled efficient NK cell differentiation (Figure 3, e and Figure 11, k). Thus, glutamine is essential during EHT and contributes to the generation of primitive GPA from HE. + and crucially CD45 + Differentially regulates population growth.

[0084] Modulation of pyruvate can reshape the hematopoietic output from HE. In some instances, HE cells uptake glucose at levels similar to EHT cells, albeit with a lower glycolytic rate (Figure 2f). Therefore, whether pyruvate oxidation is important for the hematopoietic commitment of HE cells was investigated. Pyruvate is taken up by mitochondria via the mitochondrial pyruvate carrier complex (MPC) and converted to acetyl-CoA by the PDH enzyme, which can replenish the TCA cycle (Figure 4a). Pyruvate entry into mitochondria was blocked using a specific MPC inhibitor called UK5099 (Figure 4a). In HE cells, unlike EHT or HSC-like cells, MPC inhibition resulted in the upregulation of CD43 by day 3 of subculture. + GPA + This resulted in a significant increase in cellular output (Figure 4b and Figure 12a). To confirm this result, HE cells were treated with 1-aminoethylphosphinic acid (1-AA), a PDH inhibitor (5) (Figure 4a), which significantly increased GPA compared to controls. + A significant increase in cell output was observed (Fig. 12b). Furthermore, in some instances, both MPC subunits, MPC1 and MPC2, were downregulated using shRNA (Fig. 12c), resulting in CD43 expression on day 3 of subculture. + GPA + A 2.7-fold increase in cell output was observed (Fig. 12, d), confirming the results with UK5099. HE-derived GPA in the presence of UK5099 compared to the control. + No difference in population proliferation was observed (Figure 12, e). These results indicate that the use of glucose for glycolysis can be sufficient to drive erythroid cell formation and that inhibiting pyruvate entry into mitochondria leads to increased differentiation of HE cells toward the erythroid lineage.

[0085] Total CD43 + Although the levels of CD43 cells were unchanged between UK5099-treated and untreated conditions on day 6 (Fig. 12, f), but not HSC-like cells, derived from both HE and EHT cells. + CD45 +A two-fold decrease in the total CD43 population was observed (Fig. 4c, Fig. 12g). + Even though the cellular level was unchanged, HE-derived CD45 + In some instances, UK5099 and UK6099, both derived from HE, resulted in a significant reduction in the CD45 cell population (Fig. 12, h). + These results suggest that blocking pyruvate entry into mitochondria inhibits the expression of CD45 in EHT. + It may be shown to impair differentiation towards a hematopoietic fate.

[0086] In certain instances, the opposite effect can be induced by increasing pyruvate flux into the mitochondria. Using DCA, PDK, which inhibits the PDH complex, is blocked, allowing pyruvate to be converted to acetyl-coA, potentially fueling the TCA cycle (Figure 4, a). + GPA + Although the formation of GPA cells was not significantly altered by DCA on day 3 of HE subculture (Fig. 12, k), a 50% reduction in this population on day 6 was observed in the treated condition (Fig. 4, d; Fig. 12, l). Thus, DCA does not directly block glycolysis and may not affect primitive erythroid differentiation from HE cells. Indeed, the GPA cells derived from HE subcultures + The proliferation of the population was not affected by DCA on day 3 of subculture (Fig. 12, m). On day 6 of subculture with DCA, CD43 cells derived from HE, but not EHT, cells were isolated. + CD45 + An 80% increase in the percentage of cells was observed (Fig. 4e, Fig. 12n). +DCA did not affect cell proliferation or the frequency of HSC-like cells derived from HE (Figure 12, o and p). To completely rule out any effect of UK5099 and DCA on proliferation, EdU incorporation was performed at early time points (days 1 and 2) of subculture, and no difference in proliferation was observed with UK5099 or DCA treatment (Figure 12, q). Collectively, these results indicate that HE cells can preferentially give rise to erythroid cells when pyruvate entry into the TCA cycle is inhibited, whereas increased TCA cycle fuel supply, when pyruvate is driven toward oxidation in mitochondria, promotes the critical CD45 expression of HE cells. + It favors differentiation.

[0087] In an example, after 3 or 6 days of MPC inhibition with UK5099 in HE cells, erythroid colony (CFU-E) formation significantly increased compared to untreated conditions, whereas granulocyte and macrophage colonies (CFU-G, GM, and M) colonies decreased (Figure 12, r and s). In contrast, PDK inhibition with DCA for 3 days had no effect on CFU (Figure 12, r), whereas DCA treatment for 6 days resulted in a decrease in CFU-E and a significant increase in CFU-M colonies (Figure 12, s). The ratio of CFU-E colonies to the sum of CFU-G, CFU-GM, and CFU-M colonies was 20-fold higher in UK5099-treated cells and more than 3-fold lower in DCA-treated cells compared to controls (Figure 4, f). In all conditions, both bright red primitive erythroid (EryP) and brownish definitive erythroid (EryD) colonies were observed (Figure 12, t). However, although UK5099 or DCA had no effect on HBA1-2 (adult globin) expression (Fig. 12, u), a significant increase in HBE1 (embryonic) and HBG1-2 (fetal) globin transcripts in colonies obtained from UK5099-treated HE cells (Fig. 4, g) was observed, confirming that MPC inhibition increases the generation of primitive erythroid cells.

[0088] In a specific example, to understand whether DCA induces the formation of definitive hematopoietic cells, lymphoid differentiation was induced in HE cells at day 3 in OP9-DL1 stromal coculture. UK5099 treatment impaired NK cell formation, whereas DCA treatment significantly increased NK cell differentiation compared to untreated HE cells (Figure 4h, Figure 12v). Overall, in this example, these results confirm the flow cytometry data and demonstrate that UK5099 can increase primitive erythropoiesis, while DCA favors myeloid / lymphoid differentiation from HE at later stages.

[0089] To validate these findings in an in vivo setting, pregnant mice were injected with UK5099 or DCA at embryonic day (E) 9.5 to affect the hemogenic endothelium, which gives rise to definitive hematopoiesis (both the second and third waves) occurring at E9-9.5 and E10.5, rather than primitive hematopoiesis, which occurs at E7-7.25 (Palis, J. et al. Development 126, 5073-5084 (1999) and Medvinsky, A. & Dzierzak, E. Cell 86, 897-906 (1996)). In some instances, blood lineage output in the embryo was assessed by characterizing the cellular composition of the fetal liver (FL) at E14.5, when the FL is the primary site of hematopoiesis. In some instances, the frequency of phenotypical long-term HSCs (LT-HSCs) was unaffected by UK5099 or DCA (Figure 4, i), confirming in vitro findings (Figure 12, j and p). Hematopoietic progenitor cells (HPC)-1, a restricted precursor with lymphoid / myeloid potential and HPC-2, which give rise to primarily megakaryocytic progeny, were significantly increased in embryos from DCA-injected mice compared to control and UK5099-injected conditions (Figure 13, a). Consistent with this, both T and B cell levels were increased in DCA versus control and UK5099-injected embryos (Figure 4, j), with increased CD45 expression in DCA. +This supported the in vitro results demonstrating definitive output. In some cases, DCA treatment led to a significant reduction in stage 0, 4, and 5 erythrocyte populations in the FL, with no significant differences in stages 1, 2, and 3 compared to control and UK5099 conditions (Figure 13, b and c). In some cases, this profile indicated impairment in definitive erythroid cell production (a reduction in stage 0), and as previously described (Fraser, ST et al., Blood 109, 343-352 (2007) and Isern, J. PNAS 105, 6662-6667 (2008)), primitive erythrocytes formed before injection were in late maturation stages in the FL (stages 1, 2, and 3) or had exited the FL into the circulation (a reduction in stages 4 and 5).

[0090] Furthermore, in our example, LT-HSCs from DCA-treated embryos sorted according to the gating strategy shown in Figure 13, d, produced significantly more CFU-GM colonies and fewer BFU-E colonies (Figure 13, e), with an 80% reduction in the BFU-E to CFU-GM ratio (Figure 4, k), compared with control and UK5099-treated conditions. No significant effect of UK5099 on in vivo EHT and hematopoiesis (Figure 4, i), confirming that MPC inhibition primarily affects the primitive hematopoietic wave. Thus, similar to in vitro results, PDK inhibition by DCA increases the frequency of lymphoid / myeloid cells at the expense of mature erythroid cells in vivo.

[0091] In a specific example, to assess the definitive hematopoietic potential of iPS-derived cells, 3-day DCA-treated HE cells co-cultured with OP9-DL1 stroma were intravenously injected into irradiated NSG mice. Engraftment levels comparable to previous studies were obtained (Rahman, N. et al. Nat Commun 8, 1-12 (2017)), with approximately 1% human CD45 expression in the peripheral blood (PB) at 8 weeks. +At week 8, significantly more human B cells were detected in the PB of NSG mice injected with DCA-treated cells (Fig. 13, g), whereas myeloid cell levels were similar to the untreated condition (Fig. 12, h). At week 12, similar levels of human HSCs (CD34 + CD38 - CD90 + CD49f + CD45RA - Although a significant increase in the common lymphoid precursor (CLP) population was detected in the DCA-treated condition (Fig. 4, m), a significant increase in the common lymphoid precursor (CLP) population was detected in the DCA-treated condition (Fig. 4, n). Consistent with this result, significantly more human B cells were observed in the BM of NSG mice injected with DCA-treated HE cells (Fig. 4, n), with no difference in the levels of myeloid cells (Fig. 4, o). Furthermore, significantly more CD4 + CD8 + DP thymocytes were detected (Fig. 4p, Fig. 13i). Collectively, these results indicate that increasing pyruvate flux to mitochondria with DCA drives HE cells toward definitive hematopoiesis and preferentially lymphoid fates in vivo.

[0092] Pyruvate fate can direct hematopoietic lineage commitment of HE cells at the single-cell level In a specific example, to analyze the molecular effects of pyruvate manipulation, the transcriptome profiles of HE cells were assessed at the single-cell level in control and UK5099- or DCA-treated cells at the early time point of treatment (day 2). First, all conditions were grouped together and cells were separated into seven clusters (Figure 5A). The majority of HE cells expressed endothelial markers, including ENG, CDH5, PROCR, and ANGPT2 (Figure 1C, a), and their expression was primarily restricted to clusters 1–5 (Figure 5B). In contrast, cells in clusters 6 and 7 expressed hematopoietic genes, including RUNX1, GATA2, MYB, and SPN (Figure 5B and Figure 1C, b). Thus, this time point may capture the commitment of HE cells to hematopoietic cell lineages that occurs within clusters 6 and 7.

[0093] In an example focusing on isolated clusters 6 and 7 (Figure 5c), we found that the early erythropoiesis regulator RYK (Tusi, BK et al. Nature 555, 54-60 (2018)) and the erythroid-specific KLF3 were already expressed at high levels in cluster 6, whereas other erythroid markers such as TAL1, GATA2, ZFPM1, KLF1, NFE2, ANK1, and HBQ1 were more highly expressed in cluster 7 (erythroid markers, Figure 5c). The early lymphoid cell fate regulators POU2F2 (B cells) and GATA3 (T cells) and myeloid markers SWAP70 and IRF8 were expressed at higher levels in cluster 6, whereas T lymphocyte BCL11B, myelomonocytic CSF1R, CEBPE, and megakaryocyte PF4 were most highly expressed in cluster 7 (lymphoid / myeloid markers, Figure 5c). Thus, cluster 6 cells expressed early regulators of specific lineages, whereas cluster 7 cells began to express transcription factors characteristic of more mature hematopoietic cells. Also, in cluster 7, the percentage of cells expressing erythroid transcription factors was over 75%, whereas cells expressing lymphoid or myeloid markers were significantly higher than those expressing GPA from HE, respectively. + and CD45 + Depending on the early and late appearance of cells, they represented less than 20% of the total (Fig. 5, c).

[0094] In some instances, the percentage of cells in cluster 6 was consistent across conditions, but there were 38% more UK5099-treated HE cells and 35% fewer DCA-treated HE cells in cluster 7 compared to controls (Figure 14, c). This result indicates that pyruvate modulation may not affect early hematopoietic commitment (cluster 6). However, it appears to have an effect on lineage commitment (cluster 7).

[0095] In particular, in clusters 6 and 7, the average expression levels of the erythroid lineage genes RYK, KLF3, TAL1, GATA2, ZFPM1, KLF1, NFE2, ANK1, and HBQ1 were higher in UK5099-treated HE cells compared to untreated HE cells, whereas these factors were nearly absent in DCA-treated HE cells (left dot plot, Figure 5D). In contrast, DCA-treated HE cells expressed higher levels of the lymphoid / myeloid transcription factors SWAP70, POU2F2, GATA3, CSF1R, PF4, BCL11B, CEBPE, and IRF8 compared to control and UK5099-treated HE cells (right dot plot, Figure 5D).

[0096] In some instances to further assess the effect of pyruvate manipulation on the single cell level, single HE cells were sorted onto OP9-DL1 stroma and analyzed using GPA + Clones were scored on day 14. From a total of 552 single cells per condition, 9 GPAs in the control + 12 GPAs in UK5099 treatment conditions compared to clones + clone, 7 GPA in DCA treatment condition + Clones were detected (Figure 14, d). This result may confirm the preferential commitment of HE cells to the erythroid lineage in the presence of UK5099. Taken together, these results may indicate that, at the early stage of HE differentiation, modulation of pyruvate utilization directly affects the expression of lineage-specific transcription factors, inducing the lineage commitment of HE cells.

[0097] Primitive erythroid commitment during MPC inhibition can depend on LSD1 Previous studies have shown that lysine-specific demethylase 1 (LSD1) may be important for EHT and especially for the erythroid lineage (Takeuchi, M. et al. PNAS 112, 13922-13927 (2015) and Thambyrajah, R. et al. Nat Cell Biol 18, 21-32 (2016)). During EHT, LSD1 acts in concert with HDAC1 / 2 (Thambyrajah, R. et al. Stem Cell Reports 10, 1369-1383 (2018)) and GFI1 / GFI1B (Thambyrajah, R. et al. Nat Cell Biol 18, 21-32 (2016)) to induce epigenetic changes. In some instances, CD43 + We demonstrated that HDACs may be important for EHT using an HDAC1 / 2 inhibitor (trichostatin A, TSA), which impairs the emergence of hematopoietic cells (Figure 15, a). Furthermore, we observed that LSD1, GFI1, and GFI1B were expressed at higher levels in UK5099-treated cells compared to DCA-treated cells (Figure 15, b), indicating that lineage specification through pyruvate catabolism may be LSD1-dependent. Under conditions in which LSD1 was blocked with tranylcypromine (TCP) or downregulated by shRNA (Figure 15, c), CD43 expression was significantly reduced at day 3 after UK5099 treatment of HE cells. + GPA + No increase in cell frequency was detected (Figure 6, a and b). On the other hand, TCP-treated HE cells expressed more CD43 cells on day 6, similar to DCA treatment. + CD45 + Although DCA specifically increased myeloid differentiation (Fig. 15, d), TCP, unlike DCA, specifically increased myeloid differentiation (Fig. 15, e), as previously described in the literature (Schenk, T. et al. Nat Med 18, 605-611 (2012)). Thus, mechanistically, induction of primitive erythropoiesis through MPC inhibition may depend on epigenetic regulation by LSD1 in HE cells.

[0098] DCA-dependent definitive hematopoiesis can be promoted by cholesterol metabolism In some instances, dichloroacetate can be used directly as a precursor to the acetylation mark, where acetate is converted to acetyl-coA by ACSS2 and transferred to histones via histone acetyltransferases (HATs) (Figure 15, f). Inhibiting ACSS2 significantly reduced CD43 expression on day 6 of HE subculture. + CD45 + This did not perturb the DCA effect on HE cells (Fig. 15g), indicating that DCA is not directly converted to acetyl-coA. Also, blocking HAT with C646 alone had no effect on HE cells, whereas C646 + DCA treatment significantly reduced CD43 expression compared to DCA alone. + CD45 + DCA increased the proliferation of CD45 cells by 2-fold (Fig. 15h). Blocking HAT did not inhibit the DCA effect, and no changes in the global acetylation of H3K9 or H4 were observed (Fig. 15i). Thus, inhibiting HAT together with enhancing PDH activity may promote acetyl-coA availability for other metabolic processes, potentially increasing the proliferation of CD45. + Acetyl-coA is a precursor for both lipid biosynthesis (via ACC) and the mevalonate pathway (via HMGCR) that produces cholesterol (Figure 6c). Blocking ACC with CP-640186 (CP) had the same effect as DCA, and combined treatment with both CP and DCA significantly increased CD43 cell proliferation at day 6 compared to DCA alone. + CD45 + DCA treatment further increased the frequency of HE cells (Figure 6, d). Thus, preventing lipid biosynthesis may increase the availability of acetyl-coA for cholesterol production. Indeed, in DCA-treated HE cells, an 8% increase in cholesterol content (Figure 6, e) was detected, along with higher levels of cholesterol efflux genes on day 2 (Figure 6, j). Surprisingly, treating HE cells with DCA in combination with atorvastatin (Ato), an inhibitor of the mevalonate pathway, abolished the effect of DCA (Figure 6, f). Taken together, these results suggest that DCA promotes cholesterol biosynthesis, which favors definitive hematopoietic commitment in HE cells (Figure 6, g).

[0099] As explained above, during EHT, migrating cells may undergo fundamental changes in energy use and metabolism, with a concomitant increase in glycolysis and TCA cycle / OXPHOS. The disclosures and results presented herein demonstrate for the first time that glutamine is critical to the EHT process and plays distinct roles in the specification of primitive erythroid and definitive hematopoietic cells. Meanwhile, in some instances, glucose may play a role in both glycolysis and the TCA cycle. Blocking its use in 2-DG may impair hematopoietic differentiation of HE cells. It has been shown that in quiescent HSCs, glycolysis is regulated by hypoxia through the stabilization of hypoxia-inducible factor-1α (HIF-1α) (Takubo, K. et al. Cell Stem Cell 7, 391-402 (2010)). The transition from HE to HSC has also been shown to be regulated by HIF-1α (Harris, J. Met al. Blood 121, 2483-2493 (2013) and Imanirad, P. et al. Stem Cell Research 12, 24-35 (2014)). Thus, in some instances, HIF-1α-dependent glycolytic induction may be important for EHT.

[0100] As shown herein and explained above, glycolysis is sufficient to provide energy for primitive hematopoiesis. Indeed, in early embryonic stages, oxygen is not systemically available, and glycolysis is the preferred pathway for producing energy (Gardner, DK et al. Semin Reprod Med 18, 205-218 (2000)). In embryonic development, primitive erythroid cells have been shown to undergo high rates of glycolysis to fuel their rapid proliferation (Baron, MH et al. Blood 119, 4828-4837 (2012)). Similarly, in the setting described herein, GPA derived from HE + The cells were CD45 +MPCs proliferate faster than HSCs and depend on glutamine to provide nucleotides for this process. Similarly, the important role of glutamine in supplying nucleotides for erythroid differentiation has previously been described in the context of HSCs obtained from umbilical cord blood (Oburoglu, L. et al. Cell Stem Cell 15, 169-184 (2014)). Blocking MPC can redirect HE commitment toward primitive erythropoiesis at a very early stage of EHT, as indicated by an increased frequency of committed cells at the single-cell level and higher levels of erythroid factors and embryonic / fetal-specific globins in this condition.

[0101] In some instances, the results presented herein and above may elucidate the role of the TCA cycle and OXPHOS in specifying definitive hematopoietic identity. Fueling the TCA cycle with DMK during glutamine deprivation or DCA treatment may enhance the definitive CD45 expression of HE cells. + This can lead to increased differentiation into lineages. PDK inhibition with DCA does not affect primitive erythroid cell formation, but can induce definitive hematopoiesis with a lymphoid / myeloid bias, as shown herein both in vitro and in vivo. DCA treatment of HE cells can lead to increased lymphoid reconstitution in NSG mice. The results presented herein are consistent with previous findings in Pdk2 / Pdk4 double knockout mice, which were shown to be anemic but retained normal frequencies of T, B, and myeloid populations (Takubo, K. et al. Cell Stem Cell 12, 49-61 (2013)). In an example, the results herein demonstrate that DCA inhibits CD45 by fueling cholesterol biosynthesis. +These results support elegant studies in zebrafish showing that Srebp2-dependent regulation of cholesterol biosynthesis is essential for HSC emergence (Gu, Q. et al. Science 363, 1085-1088 (2019)). As shown here, direct metabolic changes in HE cells, namely increased acetyl-CoA content, can promote cholesterol metabolism and regulate definitive hematopoietic output.

[0102] It has previously been reported that different EHT cell subsets, or pre-HSCs, express different lineage tendencies (Zhou, F. et al. Nature 533, 487-492 (2016) and Guibentif, C. et al. Cell Reports 19, 10-19 (2017)). In specific examples, such as those shown herein, metabolism can reshape the fate of HE cells, indicating that lineage tendencies can be determined at the HE level. Consistent with the results herein, a recent study combining scRNAseq with lentiviral lineage tracing revealed that cell fate biases emerge at a much earlier stage during hematopoietic development than previously described using conventional methods (Weinreb, C. et al. Science. 2020 Feb 14;367(6479)). Furthermore, mouse HSCs have been shown to exhibit lymphoid or myeloid lineage bias due to epigenetic priming established prior to their formation (Yu, VW C et al. Cell 167, 1310-1322. e17 (2016)). Indeed, linking epigenetic changes to metabolism is an emerging field that coordinates metabolic modifications with transcriptional regulation of cellular processes. Thus, as shown by example herein, erythroid fate induction by MPC inhibition may depend on the epigenetic factor LSD1.

[0103] The examples and results herein suggest that the lineage predisposition of the primitive and definitive hematopoietic waves is shaped by nutrient availability in the YS and AGM niches. Due to the lack of oxygen during early embryogenesis, the primitive hematopoietic wave may rely on glycolysis to form erythroid cells expressing embryonic globin, which has a high affinity for oxygen (Figure 6, g). This allows for efficient distribution of oxygen to newly forming tissues and promotes the use of OXPHOS, which may initiate the emergence of the definitive hematopoietic wave (Figure 6, g).

[0104] As described herein, in examples, using metabolic determinants to induce definitive HSC development in vitro from PSCs can provide a method for producing transplantable cells capable of reconstituting the hematopoietic system in patients with hematological malignancies and disorders.

[0105] hiPSC culture, hematopoietic differentiation, and cell isolation methods Those skilled in the art will understand that the methods and materials described below and elsewhere herein are merely examples, and that such examples may be performed using different combinations of methods and materials. Furthermore, elements of the methods and materials described herein may be optional. The RB9-CB1 human iPSC line was co-cultured with mouse embryonic fibroblasts (MEFs, Millipore), passaged every 6 days, and treated as previously described to form embryoid bodies (EBs) (Guibentif, C. et al. Cell Reports 19, 10-19 (2017)). The differentiation protocol used in this study has been previously described (Ditadi, A. & Sturgeon, C.M. Methods 101, 65-72 (2016)), with minor modifications made to induce both primitive and definitive hematopoiesis, as shown below and in Figure 7, a). Newly formed EBs were first kept in SFD medium supplemented with 1 ng / ml activin A (days 0–2) and 3 μM CHIR99021 (day 2 only). On day 3, the medium was switched to "Day 3-SP34" medium supplemented with 1 ng / ml activin A (day 3 only) and 3 μM CHIR99021 (day 3 only) by day 6. On day 6, the medium was replaced by "Day 6-SP34" medium by day 8. In some experiments shown, to obtain higher yields of HSC-like cells, EBs were kept until day 10, in this case, EBs were placed in Matrigel (8 μg / cm). 2 EBs were seeded on day 8 onto dishes coated with TryPLE Express (Corning) and maintained until day 10. Medium was changed daily except for days 5 and 7. On days 8 or 10 (as indicated), EBs were singularized by 5–6 rounds of 5-minute incubation with TryPLE Express (Thermo Fisher Scientific). CD34 + Cells were selected using a human CD34 MicroBead kit (Miltenyi Biotec) and analyzed according to the aforementioned markers: CD34-FITC, CD73-PE, VECad-PerCPCy5.5, CD38-PC7, CD184-APC, CD45-AF700, CD43-APCH7, GPA-eF450, CD90-BV605, and HE (CD34+ CD43 - CXCR4 - CD73 - CD90 + VECad + ), EHT(CD34 + CD43 int CXCR4 - CD73 - CD90 + VECad + ), and HSC-like (CD34 + CD43 + CD90 + CD38 - ) Cells were stained with the viability marker 7AAD for sorting (Guibentif, C. et al. Cell Reports 19, 10-19 (2017); Harris, J. Met al. Blood 121, 2483-2493 (2013); and Schenk, T. et al. Nat Med 18, 605-611 (2012)).

[0106] HE, EHT, and HSC-like subcultures Sorted HE (40,000), EHT (30,000), and HSC-like (5-20,000) cells were plated on Matrigel (16 μg / cm ) in HE medium ( 30 ) with 1% penicillin-streptomycin. 2Cells were seeded onto 96-well flat-bottom plates coated with PEG-1000 (Corning) and maintained overnight in a humidified incubator at 37°C, 5% CO2, and 4% O2. The following day (day 0), wells were washed twice with PBS, and fresh HE medium was added along with glutamine-free medium containing 2-DG (1 mM), UK5099 (10 μM), DCA (3 mM), BPTES (25 μM), TSA (60 nM), TCP (300 nM), ACSS2i (5 μM), C646 (10 μM), CP-640186 (5 μM), atorvastatin (0.5 μM), or DMK (1.75 mM), nucleosides (1X), or NEAA (1X), where indicated. The medium was changed and drugs were added every 2 days, and the cells were kept in a humidified incubator at 37°C, 5% CO , and 20% O for 6–7 days. Photographs were taken using an Olympus IX70 microscope equipped with a CellSens DP72 camera and CellSens Standard 1.6 software (Olympus).

[0107] Extracellular flux analysis For comparisons between HE, EHT, and HSC-like cells, day 10 FACS-sorted cells (≥40,000) were seeded directly onto Seahorse XF96 cell culture microplate wells coated with CellTak (0.56 μg / well) in two to four replicates, and extracellular flux was immediately assessed with a Seahorse XF96 analyzer. For comparisons between HE and EHT cells, day 8 FACS-sorted cells (≥40,000) were seeded onto Matrigel (16 μg / cm) in three to four replicates. 2 Cells were seeded onto Seahorse XF96 cell culture microplate wells coated with PEG-400 (Corning), and extracellular flux was assessed 2 days after seeding using a Seahorse XF96 analyzer. To assess glycolytic flux, ECAR was measured in XF medium containing 2 mM glutamine under basal conditions (after 1 hour of glucose starvation according to the manufacturer's instructions) and after the addition of 25 mM glucose, 4 μM oligomycin, and 50 mM 2-DG, and data were normalized to cell number. Glycolytic capacity (ECAR) was calculated as オリゴマイシン -ECAR2-DG ) and glycolysis (ECAR グルコース -ECAR 2-DG The levels of basal respiration (OCR) were calculated. To assess oxidative phosphorylation, OCR was measured in XF medium containing 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate under basal conditions and after the addition of 4 μM oligomycin, 2 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 μM rotenone / 40 μM antimycin A, and data were normalized to cell number. Basal respiration (OCR) 基礎 -OCR ロテノン / アンチマイシンA ), ATP production (OCR 基礎 -OCR オリゴマイシン ), and maximum respiration (OCR FCCP -OCR ロテノン / アンチマイシンA ) levels were calculated.

[0108] Flow cytometry analysis On days 3 and 6 of subculture, cells were harvested after a 2-minute incubation with StemPro Accutase Cell Dissociation Reagent at 37°C and stained with CD34-FITC, CD14-PE, CD33-PC7, CD11b-APC, CD45-AF700, CD43-APCH7, GPA-eF450, CD90-BV605, and the viability marker 7AAD. Fluorescence was measured on a BD LSRII. To measure mitochondrial activity, cells were incubated with tetramethylrhodamine ethyl ester (TMRE, 20 nM) for 30 minutes at 37°C. A negative control was incubated with 100 μM FCCP for 30 minutes at 37°C prior to TMRE staining. Fluorescence was measured on a BD FACSARIA III, and MFI levels (MFI FMO) were calculated. To measure glucose uptake, cells were incubated with 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 30 minutes at 37°C, and fluorescence was measured on a BD FACSARIA III. To measure proliferation, cells were treated with the CellTrace Violet (CTV) kit according to the manufacturer's instructions (10 minutes of incubation), and fluorescence was measured on a BD LSRFortessa. To measure EdU incorporation, HE cells were assessed on day 1 or 2 of subculture after a 24-hour EdU pulse using the Click-iT EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher Scientific, C10424) according to the manufacturer's instructions. Flow cytometry output was analyzed in FlowJo software using initial gating on SSC-A / FSC-A, FSC-H / FSC-A, SSC-H / SSC-A, and 7-AAD to exclude doublets and dead cells in all experiments.

[0109] Colony-forming unit assay Subcultured HE cells were treated with StemPro Accutase Cell Dissociation Reagent at 37°C for 2 minutes, and the dissociated cells were resuspended in 3 ml of Methocult H4230 (STEMCELL Technologies, France) (prepared according to the manufacturer's instructions with 20 ml of Iscove's Modified Dulbecco's Medium containing 2.5 μg of hSCF, 5 μg of GM-CSF, 2.5 μg of IL-3, and 500 U of EPO). Each mixture was divided into two wells of a non-tissue-culture-treated 6-well plate. After 12 days of incubation at 37°C in a humidified incubator at 5% CO2 and 20% O2, colonies were morphologically identified and scored. For globin analysis, colonies in the Methocult wells were harvested with PBS, thoroughly washed, and frozen in RLT buffer containing β-mercaptoethanol. After RNA extraction and RT (Qiagen), gene expression was assessed by q-PCR using Taqman probes. The Taqman probes used in this study are HBA1 / 2 (Hs00361191_g1), HBE1 (Hs00362216_m1), HBG2 / 1 (Hs00361131_g1), and KLF1 (Hs00610592_m1).

[0110] Lymphocyte differentiation assay against OP9-DL1 stroma Day 3 subcultured HE cells, cultured in glutamine-free medium containing UK5099 (10 μM), DCA (3 mM), or DMK (1.75 mM), nucleosides (1X), or NEAA (1X), as indicated, were harvested after a 2-minute incubation with StemPro Accutase Cell Dissociation Reagent at 37°C and plated onto 80% confluent OP9-DL1 stroma. Cells were cultured in OP9 medium containing SCF (10 ng / ml), FLT3-L (10 ng / ml), IL-2 (5 ng / ml), IL-7 (5 ng / ml for the first 15 days only), and IL-15 (10 ng / ml) as previously described (Renoux, VM et al., Immunity 43, 394-407 (2015)), and passaged weekly onto fresh OP9-DL1 stroma. On day 35 of co-culture, cells were analyzed on a BD LSRFortessa.

[0111] Single-cell RNAseq library preparation and sequencing Sorted HE, EHT, and HSC-like cells, as well as magnetically selected (Miltenyi Biotec) cord blood CD34 + Cells were cultured on Matrigel (16 μg / cm ) in HE medium (Ditadi, A. & Sturgeon, CM Methods 101, 65-72 (2016)) with 1% penicillin-streptomycin. 2Cells were seeded onto 96-well flat-bottom plates coated with PEG-1000 (Corning) and kept overnight in a humidified incubator at 37°C, 5% CO2, and 4% O2. The following day (day 0), wells were washed twice with PBS, and fresh HE medium was added along with UK5099 (10 μM) or DCA (3 mM), where indicated. On days 1 and 2 (as indicated), cells were washed twice with PBS 0.04% UltraPure BSA and harvested after a 2-minute incubation with StemPro Accutase Cell Dissociation Reagent at 37°C. Cells were spun down, resuspended in PBS 0.04% UltraPure BSA, and counted (yields of 8,000–18,000 cells). Library preparation was performed according to the Chromium Single Cell 3' Reagent Kit v3 instructions (10x Genomics). Sequencing was performed on an Illumina NOVASeq 6000 with run parameters recommended by 10x Genomics (28-8-0-91) and a final pooled library loading concentration of 300 pM. Human umbilical cord blood samples were collected from Skane University Hospital (Lund and Malmo) and Helsingborg Hospital with informed consent in accordance with guidelines approved by the local ethical committee.

[0112] Single-cell RNAseq analysis Data were processed and analyzed using Seurat v3.1.0, allowing cells to have up to 20% mitochondrial reads before log-normalization, and the "vst" method was used to find the top 500 variable genes. Cell cycle scores were calculated, and data were scaled by regression on the difference between mitochondrial content and S and G2M scores. Principal components were calculated before calculating UMAP. Pseudo-time trajectories describing two developmental pathways were identified in our EHT dataset using Slingshot (Street, K. et al. BMC Genomics 19, 477 (2018)), along which cells were ordered. Cells were then binned along each trajectory, and the cell type composition of each bin was calculated as a percentage. Cord blood CD34 +Cells were mapped onto our data and labeled using scCoGAPS (Stein-O'Brien, G. Lett. et al. Cell Syst 8, 395-411.e8 (2019)). CS13 data from Zeng et al. (Zeng, Y. et al. Cell Res 1-14 (2019)) were read and processed to create a UMAP, from which cells designated "AEC" and "Hem" were identified. These 99 cells were mapped onto our data and labeled using SCMAP (Kiselev, V. et al. Nat Methods 15, 359-362 (2018)). Our EHT data were mapped onto the data from Zeng et al. (Zeng, Y. et al. Cell Res 1-14 (2019)) using scCoGAPS, and vice versa. Ten patterns were identified in each dataset and then projected onto each other using projectR. Each cell was assigned to the group that achieved the highest weight. A correlation between cell type and pattern was calculated by forming a contingency table. Correspondence analysis was performed using the ca package in R. The FindAllMarkers function was used to find differentially expressed genes. The cell counts for the day 1 samples were as follows: HE = 1451, EHT = 1523, HSC-like = 732. The cell counts for the day 2 samples were as follows: HE ctrl = 1195, HE+UK5099 = 718, HE+DCA = 2309. All evaluated endothelial and hematopoietic genes have been previously used in several publications to validate the EHT process (Zhou, F. et al. Nature 533, 487-492 (2016); Swiers, G. et al. Nat Commun 4, 2924 (2013); Ng, E. et al. Nature Biotechnology 34, 1168-1179 (2016); and Guibentif, C. et al. Cell Reports 19, 10-19 (2017)). For gene expression analysis, gene sets for glycolysis, oxidative phosphorylation, glutamine transport, and cholesterol efflux were downloaded from the Molecular Signatures Database (MSigDB).

[0113] shRNA-mediated downregulation A short hairpin sequence recognizing the gene of interest was cloned into the GFP-expressing pRRL-SFFV vector, embedded in a microRNA context for minimal toxicity, as previously described (Fellmann, C. et al., Cell Reports 5, 1704-1713 (2013)). Each lentivirus batch was produced in two T175 flasks of HEK 293T cells by cotransfecting 22 μg of pMD2.G, 15 μg of pRSV-Rev, 30 μg of pMDLg / pRRE, and 75 μg of shRNA vector using 2.5 M CaCl2. The medium was changed 16 h posttransfection, and the virus was harvested 48 h posttransfection, pelleted at 20,000 × g for 2 h at 4°C, resuspended in 100 μl of DMEM, aliquoted, and stored at -80°C. The downregulation efficiency of each shRNA was measured using umbilical cord blood CD34 cells. + Three days after lentiviral transduction of HSPCs, sorted GFP + This was measured by assessing the corresponding gene expression in the cells by qPCR. HE cells were transduced the day after sorting by adding lentiviral particles directly to the culture medium.

[0114] In vivo compound injection and mouse hematopoietic evaluation Pregnant female C57Bl / 6xB6.SJL mice were intraperitoneally injected with UK5099 (4 mg / kg), DCA (200 mg / kg), or PBS (control) at E9.5. Embryos were harvested at E14.5, individually weighed, and processed. Fetal livers were dissected and homogenized in 800 μL of ice-cold PBS supplemented with 2% fetal bovine serum (FBS). FL cells were washed with PBS containing 2% FBS. For differentiated lineage panels, cells were stained with B220 and CD19 (B cell markers)-PE, CD3e-APC, Ter119-PeCy7, and CD71-FITC and analyzed on a BD FACSARIA III. For the HSC panel, samples were first treated with ammonium chloride solution (STEMCELL Technologies, France) to lyse red blood cells, washed twice with ice-cold PBS containing 2% FBS, stained with CD3e, B220, Ter119, Gr1 (lineage)-PeCy5, c-Kit-Efluor780, Sca1-BV421, CD48-FITC, CD150-BV605, and 7-AAD (for dead cell exclusion), and analyzed on a BD FACSARIA III. Flow cytometry output was analyzed using FlowJo software with initial gating on SSC-A / FSC-A and FSC-H / FSC-A to exclude doublets. For seeding of CFU assays, 100 LT-HSCs were sorted (gating strategy shown in Figure 13, d) and resuspended in 3.0 mL of Methocult M3434 (STEMCELL Technologies, France). Each mixture was divided into two wells of a non-tissue culture treated 6-well plate. After 14 days of incubation in a humidified incubator at 37°C, 5% CO2, 20% O2, colonies were morphologically identified and scored.

[0115] NSG mouse transplantation Sorted human HE cells (350,000) were mixed with OP9-DL1 stroma (60,000) and cultured in HE medium on Matrigel (16 μg / cm 2 The cells were subcultured for 3 days on 12-well plates coated with DCA (3 mM) or without DCA (3 mM). 30100,000–150,000 cells from control or DCA samples were transfected into sublethally irradiated (300 cGy) 8-week-old female NOD / Cg-Prkdc mice. scid Il2rg tm1Wjl NSG mice (The Jackson Laboratory) were transplanted with 20,000 whole bone marrow support cells from C57Bl / 6.SJL mice (CD45.1+ / CD45.2+, in-house bred). Cells were transplanted in a single-cell solution in 250 μL of PBS containing 2% FBS via intravenous tail vein injection. The drinking water of transplanted NSG mice was supplemented with ciprofloxacin (125 mg / L, HEXAL) for 3 weeks after transplantation to prevent infection. Mice were housed in a controlled environment with a 12-hour light / dark cycle and free access to food and water. Experiments and animal care were performed in accordance with the Lund University Animal Ethical Committee.

[0116] Peripheral blood analysis after transplantation in NSG mice Peripheral blood (PB) was collected from the tail vein into EDTA-coated microvette tubes (Sarstedt, catalog no. 20.1341.100). The peripheral blood was lysed for mature red blood cells (STEMCELL technologies) in ammonium chloride solution for 10 minutes at room temperature, washed and stained for cell surface antibodies for 45 minutes at 4°C, washed, filtered, and then subjected to flow cytometry analysis on a FACS AriaIII (BD). Flow cytometry output was analyzed using FlowJo software with initial gating on SSC-A / FSC-A and FSC-H / FSC-A for doublet exclusion, DAPI for dead cell exclusion, and huCD45 / muCD45.1 for mouse cell exclusion.

[0117] Bone marrow analysis after transplantation in NSG mice Bone marrow was analyzed at the 12-week transplantation endpoint. Mice were euthanized by spinal dislocation, followed by dissection of both the left and right femurs, tibias, and ilium. Bone marrow was harvested by grinding with a pestle and mortar, and cells were collected in 20 mL of ice-cold PBS containing 2% FBS, filtered, and washed (350 x g, 5 min). Bone marrow cells were lysed for red blood cells (ammonium chloride solution, STEMCELL technologies) for 10 min at room temperature, washed and stained for cell surface antibodies for 45 min at 4°C, washed, filtered, and then subjected to FACS analysis on a FACS AriaIII (BD). Flow cytometry output was analyzed using FlowJo software with initial gating on SSC-A / FSC-A and FSC-H / FSC-A for doublet exclusion, DAPI or 7AAD for dead cell exclusion, and huCD45 / muCD45.1 for mouse cell exclusion.

[0118] Thymus analysis after transplantation in NSG mice Whole thymi were harvested at the 12-week transplantation endpoint. Thymocytes were mechanically dissociated from connective tissue in the thymus by pipetting up and down in PBS containing 2% FBS, followed by filtration through a 50 μm sterile filter. Red blood cell contamination was removed by lysing the sample in ammonium chloride solution (STEMCELL Technology) for 10 minutes at room temperature. The sample was washed and then spun down. The thymocyte pellet was resuspended in FACS buffer and stained with cell surface antibodies for 45 minutes at 4°C before FACS analysis on a FACS AriaIII (BD), washed, and filtered. Flow cytometry output was analyzed using FlowJo software with initial gating on SSC-A / FSC-A and FSC-H / FSC-A for doublet exclusion, DAPI for dead cell exclusion, and huCD45 / muCD45.1 for mouse cell exclusion.

[0119] Confocal microscopy imaging and quantification For TMRE staining, on day 3 of subculture, half of the culture medium was removed and cells were stained with 20 nM TMRE (Thermo Fisher Scientific, T669) by directly adding a 2x concentrated solution to the culture medium. After 20 min of incubation at 37 °C, the wells were carefully washed with PBS, and fresh HE medium was added. During acquisition, cells were maintained in a humidified incubator at 37 °C, 5% CO2, and 20% O2. For immunocytochemistry, HE cells (seeded on coverslips) on day 2 of subculture were washed twice with PBS, fixed with 4% PFA for 15 min at RT, and washed three times with PBS. For filipin staining, fixed cells were incubated with 100 μg / ml filipin III (Sigma-Aldrich, F4767) for 1 h, washed three times with PBS, rinsed with distilled water, and mounted with PVA / DABCO. For H3K9 and H4 acetylation staining, fixed cells were permeabilized and blocked with PBS + 0.25% Triton X-100 + 5% normal donkey serum (blocking solution) for 1 h at RT, followed by overnight incubation at 4°C with primary antibodies diluted in blocking solution. Cells were then washed 2 x 5 min with PBS + 0.25% Triton X-100 (TPBS) and 5 min with blocking solution, followed by 2 h of incubation with secondary antibodies diluted in blocking solution at RT. Cells were then washed twice with PBS containing 1 μg / ml Hoechst for 5 min, rinsed with distilled water, and mounted with PVA:DABCO. Images were obtained with a 10x (TMRE) or 20x (filipin and acetylation) objective on a Zeiss LSM 780 confocal microscope using Zen software and a 1.5x (TMRE) or 0.6x (filipin and acetylation) zoom. Acquisition settings were the same for all images in each experiment, and the same number of stacks were taken. Intensity quantification was performed using Fiji software as follows: For TMRE, ROIs were selected for five spindle-shaped and five round cells (randomly selected) using the brightfield channel, and the average intensity of each ROI was calculated over the total Z-stack in the TMRE channel. For filipin and acetylation, total Z-stacks were obtained over the filipin channel, and the average intensity was calculated.A total of two to three independent experiments with two to three replicate wells were quantified. For each replicate well, four to six images were acquired.

[0120] statistical analysis The significance of differences between conditions was calculated using paired / unpaired t-tests, one-way / two-way analysis of variance (ANOVA) tests, or Kruskal-Wallis tests for multiple comparisons in GraphPad Prism 6 software, as indicated. p-values ​​are indicated in the figures with the following abbreviations: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0121] While this description sets forth specific details of various embodiments, it will be understood that the description is illustrative only and should not be construed as limiting in any way. Moreover, various applications of such embodiments and modifications thereto that may occur to those skilled in the art are also encompassed by the general concepts described herein. Each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present invention, provided that the features included in such combinations are not mutually inconsistent. All figures, tables, and appendices, as well as patents, applications, and publications referenced above, are incorporated herein by reference.

[0122] Some embodiments are described in conjunction with the accompanying drawings. However, it should be understood that the figures are not drawn to scale. Distances, angles, and the like are merely illustrative and do not necessarily bear a precise relationship to the actual dimensions and layout of the illustrated devices. Components may be added, removed, and / or rearranged. Furthermore, any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed herein in connection with various embodiments can be used in all other embodiments described herein. In addition, it will be recognized that any method described herein may be implemented using any apparatus suitable for performing the recited steps.

[0123] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. It should be understood that not all such advantages may necessarily be achieved in accordance with any particular embodiment. Thus, for example, one skilled in the art will recognize that the present disclosure may be embodied or implemented in a way that achieves one advantage or group of advantages taught herein without necessarily achieving other advantages that may be taught or suggested herein.

[0124] While these inventions have been disclosed in the context of certain preferred embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the invention, as well as obvious modifications and equivalents thereof. In addition, while several variations of the invention have been shown and described in detail, other modifications, which are within the scope of these inventions, will be readily apparent to those skilled in the art based on this disclosure. It is also contemplated that various combinations or subcombinations of specific features and aspects of the embodiments may be made and still fall within the scope of the invention. It should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form varying modes of the disclosed invention(s). Furthermore, the actions of the disclosed processes and methods may be modified in any manner, including rearranging actions and / or inserting additional actions and / or deleting actions. Thus, it is intended that the scope of at least some of the inventions disclosed herein should not be limited by the specific disclosed embodiments described above. The limitations in the claims should be interpreted broadly based on the language used in the claims and not limited to the examples set forth herein or during prosecution of the application, which examples should be construed as non-exclusive.

Claims

1. 1. A method for generating definitive hematopoietic cells, comprising: providing a plurality of source cells selected from the group consisting of differentiating iPS cells, cells directly reprogrammed into hematopoietic cell precursors, and adult or neonatal hematopoietic cells derived from bone marrow, umbilical cord blood, placenta, or mobilized peripheral blood, wherein the adult or neonatal hematopoietic cells are hematopoietic stem cells or hematopoietic progenitor cells; and treating the source cells with dichloroacetate (DCA); Including, The method is not performed on or within the human body.

2. 2. The method of claim 1, wherein the concentration of dichloroacetate is at least 30 μM.

3. The method of claim 1 or 2, wherein the definitive hematopoietic cells comprise definitive hematopoietic stem cells.

4. 4. The method of claim 3, wherein the definitive hematopoietic stem cells have lymphoid and / or bone marrow repopulating potential.

5. The method of any one of claims 1 to 4, wherein the definitive hematopoietic cells comprise definitive lymphoid and / or myeloid cells.

6. 6. The method of claim 5, wherein the definitive lymphoid cells comprise cells selected from the group consisting of T cells, modified T cells that target tumor cells, B cells, NK cells, and NKT cells.

7. The method of any one of claims 1 to 6, wherein the definitive hematopoietic cells comprise mast cells.

8. The method of any one of claims 1 to 7, wherein the definitive hematopoietic cells comprise red blood cells suitable for the production of adult hemoglobin.

9. 9. The method of any one of claims 1 to 8, wherein the cells directly reprogrammed into precursors of hematopoietic cells comprise cells selected from the group consisting of mesodermal progenitor cells, hemogenic endothelial cells, and cells undergoing an endothelial to hematopoietic transition.

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