Methods for generating hematopoietic stem cells and compositions thereof

Abstract

In various aspects and embodiments, the present disclosure provides methods for generating hematopoietic stem cells (HSCs), as well as compositions comprising the same and methods for treating diseases. The present disclosure provides methods for preparing endothelial cells from pluripotent stem cells by expression (e.g., overexpression) of the E26 transformation-specific variant 2 (ETV2) transcription factor. HSCs are then generated from the endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimuli.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 123,778, filed December 10, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Hematopoietic stem cell (HSC) transplantation (HSCT) involves the infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients with bone marrow or immune system defects, such as in the treatment of blood cancers. HSCs are also useful for generating cancer immunotherapies, including the introduction of genes encoding T cell receptors (TCRs) or chimeric antigen receptors (CARs) against tumor-associated antigens. Specifically, cells that engraft and produce long-term blood cells in a subject can provide a long-term source of targeted anti-cancer effector cells for sustaining remission (Non-Patent Document 1).

[0003] However, ex vivo HSC expansion remains a major obstacle, especially for obtaining HSC products that can engraft effectively after administration to patients (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gschweng et al., Hematopoietic stem cells for cancer immunotherapy, Immunol Rev. 2014 257(1):237-49 [Non-patent document 2] Tajer P, et al., Ex Vivo Expansion of Hematopoietic Stem Cells for Therapeutic Purposes: Lessons from Development and the Niche, Cells 2019 Feb; 8(2): 169 Summary of the Invention

[0005] In various aspects and embodiments, the present invention accomplishes these objectives. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows representative FACS plots of the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] Figure 1 shows that ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (designated CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. DETAILED DESCRIPTION OF THE INVENTION

[0007] In various aspects and embodiments, the present disclosure provides methods for generating hematopoietic stem cells (HSCs), as well as compositions comprising same and methods for treating diseases. HSCs produced in accordance with the present disclosure, in various embodiments, have advantages for establishing hematopoietic function and / or engraftment for long-term production of blood lineages, and / or ex vivo production of cells for immunotherapy.

[0008] In some embodiments, the present disclosure provides a method for generating hematopoietic stem cells (HSCs). The method includes preparing endothelial cells from pluripotent stem cells by expression (e.g., overexpression) of the E26 transformation-specific variant 2 (ETV2) transcription factor. Then, HSCs are generated from the endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation. The nucleotide sequence encoding ETV2 is described in Wang K, et al., "Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA." Sci. Adv. Vol. 6 (2020), the entire contents of which are incorporated herein by reference.

[0009] According to embodiments of the present disclosure, ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0010] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) prepared by reprogramming somatic cells. For example, somatic cells can be reprogrammed by expressing reprogramming factors selected from one or a combination of Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and Klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and Klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and Klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664 (each of which is incorporated by reference in its entirety). In various embodiments, the reprogramming factors are expressed using known viral vector systems, such as lentivirus or Sendai virus systems. Alternatively, reprogramming factors may be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs may be generated by introducing non-integrated episomal plasmids expressing the reprogramming factors, i.e., to generate transgene-free and virus-free iPSCs. Known episomal plasmids with limited replicative capacity, which are therefore lost within a few cell generations, may also be used. In some embodiments, iPSCs are generated from somatic cells, such as, but not limited to, fibroblasts or PBMCs. In some embodiments, iPSCs are derived from T cells, B cells, umbilical cord blood (including CD3+ or CD8+ cells derived from umbilical cord blood), CD34+ cells or CD34-enriched cells, or other primary human tissues. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched) to the recipient. In some embodiments, iPSCs are HLA-modified or HLA-deficient (HLA-null) cells. In these or other embodiments, the iPSCs may be genetically modified to include genes for expression of a chimeric antigen receptor (CAR) or CCR5.

[0011] In some embodiments, iPSCs contain one or more constitutive or inducible suicide genes. In these embodiments, transplanted HSCs can be easily depleted after treatment with HSCs. An exemplary suicide gene is HSV-thymidine kinase (HSV-TK), as described in Liang Q, et al., "Linking a cell-division gene and a suicide gene to define and improve cell therapy safety." Nature Vol. 563: 701-704 (2018). HSV-TK converts ganciclovir into a toxic product, allowing for selective elimination of TK+ cells using this activator. An alternative or additional suicide gene is caspase-9 (Yagyu S. et al., "An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells." Mol. Ther. 2015 23(9):1475-85). See also Wiebking V., et al., Metabolic engineering generates a transgene-free safety switch for cell therapy. Nature Biotechnology Vol. 38: 1441-1450 (2020).

[0012] In these or other embodiments, the iPSCs may contain a reporter gene that allows for detection of HSCs prepared according to the present process, including after administration to a patient. Exemplary reporter genes include positron emission tomography (PET) imaging reporter genes, which allow for whole-body monitoring of, for example, the location, quantity at all locations, survival, and proliferation of therapeutic cells over time. Exemplary PET reporter genes are described, for example, in Yaghoubi S. et al., Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications. Theranostics. 2012; 2(4): 374-391. An exemplary PET reporter gene is PMK2. See Haywood T., et al., Positron emission tomography reporter gene strategy for use in the central nervous system. PNAS 2019 116(23): 11402-11407.

[0013] In various embodiments, iPSCs are differentiated into mesodermal progenitor cells (MPCs), and MPCs are differentiated into endothelial cells (ECs). Differentiation into MPCs is generally due to activation of the Wnt and Noda1 signaling pathways. See Patsch C., et al., Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat. Cell. Biol. 17, 994-1003 (2015). For example, the Wnt and Noda1 signaling pathways can be activated using a glycogen synthase kinase 3 inhibitor (e.g., CHIR99021). See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). ETV2 expression in MPC cells is involved in differentiation into ECs. Heterologous ETV2 can be expressed, for example, by introducing a non-integrated episomal plasmid encoding ETV2 for constitutive or inducible expression and the production of transgene-free ECs. In some embodiments, ETV2 is expressed from mRNA introduced into MPCs. mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing MPCs can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). In some embodiments, viral vectors can be used to express ETV2.

[0014] In some embodiments, iPSCs (e.g., expressing ETV2) can be used to generate embryoid bodies (EBs), which can be used to induce EHT. EB preparation is described, for example, in U.S. Patent Application Publication No. 2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, human iPSC aggregates are grown in a bioreactor, for example, as described in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93.

[0015] In some embodiments, a population of human pluripotent stem cells induced to undergo differentiation (e.g., differentiation into endothelial cells) is enriched, e.g., as described in U.S. Patent No. 9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include inducing differentiation in a population of human pluripotent stem cells (or EBs derived therefrom) and sorting the induced population based on expression of CD34 and / or CD43. + and / or CD43 neg Select the fraction of

[0016] The ECs produced by this process can be converted into hemogenic endothelial cells (HECs) or HSCs using mechanical, biochemical, genetic, and / or pharmacological stimuli. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the endothelial cells (e.g., using mechanical, pharmacological, or genetic means) under conditions sufficient to stimulate the formation of HSCs. See WO 2019 / 236943 (incorporated by reference in its entirety). In some embodiments, the ECs can be converted into hemogenic endothelial (HE) cells using mechanical, genetic, and / or pharmacological stimuli, which can then be optionally converted into HSCs using mechanical, genetic, and / or pharmacological stimuli.

[0017] In some embodiments, the pharmacological stimulation comprises contacting the endothelial cells with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezo1. An exemplary Piezo1 agonist is Yoda1. Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezo1 (Syeda R, Chemical activation of the mechanotransduction channel Piezo1. eLife (2015)). Yoda1 has the following structure: [ka]

[0018] Derivatives of Yodali can be used in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are used in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yodali analogue (Dooku1) which antagonizes Yodali-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759): 2018. Further Piezo1 agonists include Jedali and / or Jedi2, or derivatives thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel." Nature Communications (2018) 9:1300. Jedali and Jedi2 share a 3-methylfuran carboxylate structural motif. Other Piezo1 agonists sharing this motif can be used in accordance with embodiments of the present invention. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 agonist or derivative is in the range of about 0.1 μM to about 500 μM, or about 0.1 μM to about 200 μM, or about 0.1 μM to about 100 μM, or in some embodiments, about 1 μM to about 150 μM, or about 5 μM to about 100 μM, or about 10 μM to about 50 μM, or about 20 μM to about 50 μM.

[0019] Alternatively or additionally, the transformation into HE cells or HSCs is by genetic stimulation. For example, Dnmt3b mRNA expression can be increased by transgene-free methods, including but not limited to, delivering a transcript encoding Dnmt3b into cells, introducing a transgene encoding Dnmt3b, or introducing a non-integrated episome into cells. In some embodiments, gene editing is used to introduce genetic modifications into Dnmt3b expression elements in endothelial cells, for example, to increase promoter strength, ribosome binding, RNA stability, or affect RNA splicing.

[0020] In some embodiments, the method includes increasing the activity or expression of Gimap6 in endothelial cells, alone or in combination with Dnmt3b. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cells, or transgene-free approaches can be used, including, but not limited to, introducing an episomal or alternatively, a transgene encoding Gimap6 into the cells. In some embodiments, gene editing is used to introduce genetic modifications (e.g., one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affect RNA splicing) into a Gimap6 expression element in the endothelial cells.

[0021] In embodiments of the present disclosure using mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNA containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can cause translation block, secretion of inflammatory cytokines, and cell death. RNA containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See U.S. Pat. No. 9,181,319 (incorporated herein by reference) for nucleotide modifications specifically for circumventing the innate immune response.

[0022] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing into cells a transgene that can direct desired levels of overexpression (through varying promoter strength or other selection of expression control elements). The transgene can be introduced using a variety of viral vectors or transfection reagents known in the art. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery as described).

[0023] In some embodiments, the expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques to introduce one or more modifications, for example, to increase promoter strength, ribosome binding, or RNA stability. Various editing techniques are known, including CRISPR, zinc finger (ZF), and transcription activator-like effector (TALE). Fusion proteins comprising one or more of these DNA-binding domains and the cleavage domain of Fok1 endonuclease can be used to generate double-strand breaks in desired regions of DNA within cells (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the contents of all of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system known in the art, see e.g., U.S. Patent Nos. 8,697,359, 8,906,616, and 8,999,641, which are incorporated by reference in their entireties.

[0024] In some embodiments, ECs or HECs are converted into HSCs using mechanical stimulation, such as a process involving circumferential stretching in 2D or 3D culture. For example, a cell population containing developmentally plastic endothelial cells or HE cells is introduced into a bioreactor. In some embodiments, the bioreactor provides cyclic strain biomechanical stretching, as described in International Publication No. 2017 / 096215 (incorporated herein by reference in its entirety). The cyclic strain biomechanical stretching increases the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, mechanical means apply stretching forces to cells. For example, cells can be subjected to circumferential stretching ex vivo under defined, controlled cyclic strain conditions using a computer-controlled vacuum pump system (e.g., the FlexCell™ Tension System, Cytostretcher System, etc.) attached to a nylon membrane or similar biocompatible or biomimetic membrane in a flexible-bottom culture plate.

[0025] In some embodiments, the endothelial cells of the HE cells are selected or enriched for cells with a desired cell surface marker phenotype. In some embodiments, the endothelial cells or HE cells are selected or enriched for CD34+ cells. Optionally, the following cell surface markers are selected: CD31 pos , CD144 pos , K.D.R. pos , CD43 neg and CD235 neg The cells are selected or enriched based on one or more of the following:

[0026] In some embodiments, the method comprises, at least in part, inducing hematopoietic differentiation by culturing the endothelial or HE cells with one or more factors selected from insulin-like growth factor-1 (IGF-1), sonic hedgehog (SHH), angiotensin 2, losartan, Flt3, Flt3 ligand, Y27632, FGF, FGF-2 (bFGF), BMP-4, activin A, transferrin, VEGF, DKK, IL-6, IL011, SCF, EPO, IL-3, SB-431542, fibronectin, and vitronectin.

[0027] In various embodiments, the generated HSCs are expanded. For example, HSCs can be expanded according to the methods disclosed in U.S. Patent Nos. 8,168,428, 9,028,811, 10,272,110, and 10,278,990 (incorporated herein by reference in their entireties). For example, in some embodiments, ex vivo expansion of HSCs involves the addition of prostaglandin E2 (PGE2) or a PGE2 derivative or precursor to the culture. In these or other embodiments, ex vivo expansion involves the addition of linoleic acid to the culture. In these or other embodiments, HSC expansion involves the addition of polyvinyl alcohol to the culture. In some embodiments, HSC expansion involves, at least in part, culturing the cells with an aryl hydrocarbon receptor antagonist, such as a substituted imidazopyridine or imidazopyrazine, including those described in U.S. Patent No. 10,457,683 (incorporated herein by reference in its entirety). Other compounds and reagents useful for expanding HSCs are described in Papa L., et al., Distinct Mechanisms Underlying the Ex Vivo Expansion of Human Cord Blood Stem Cells with Different Strategies Currently Used for Allogeneic Transplantation, Blood (2019) 134 (Supplement 1): 4470. Such compounds and reagents can include nicotinamide and valproic acid.

[0028] In various embodiments, hematopoietic stem cells produced according to the present disclosure comprise long-term hematopoietic stem cells (LT-HSCs) that exhibit excellent engraftment and reconstitute functional multilineage adult blood in recipients. In some embodiments, the HSCs comprise Lin- / Scal+ / c-kit+ cells. LT-HSCs self-renew to maintain the stem cell pool or differentiate into short-term HSCs (ST-HSCs) or lineage-restricted progenitor cells, which undergo extensive proliferation and differentiation to produce terminally differentiated, functional hematopoietic cells. While ST-HSCs or multipotent progenitor cells (MPPs) can only sustain hematopoiesis for a short period of time, LT-HSCs persist throughout the life of an organism and can continually replenish the hematopoietic system. In some embodiments of the present disclosure, the HSCs comprise at least about 0.0001% HSCs, or at least about 0.001% LT-HSCs, or at least about 0.01% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 1% LT-HSCs. In some embodiments, a subpopulation of cells (e.g., LT-HSCs) can be isolated or enriched using, for example, a cell sorting approach. For example, in some embodiments, cells are selected or enriched for CD34+ cells. In some embodiments, cells are enriched or sorted based on expression of one or more of CD34, CD45, CD38, CD90, CD49f, and GPI-80. In some embodiments, cells are enriched or sorted for cells with one or more of the following phenotypic markers: CD110+, CD135+, and APLNR+.

[0029] In various embodiments, upon conversion to HSCs, cells that are floating in the culture (eg, non-adherent) are collected.

[0030] In some aspects, the present disclosure provides compositions comprising a population of HSCs produced according to the methods described herein. In various embodiments, a composition for cell therapy is prepared comprising a population of HSCs prepared according to the methods described herein and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprises at least about 10 2 HSCs, or at least about 10 3 HSCs, or at least about 104 HSCs, or at least about 10 5 HSCs, or at least about 10 6 HSCs, or at least about 10 7 HSCs, or at least 10 8 For example, in some embodiments, a pharmaceutical composition containing about 100,000 to about 400,000 (CD34+) HSCs per kg of recipient body weight (e.g., about 200,000 cells per kg) is administered.

[0031] HSCs for therapy or transplantation can, in some embodiments, be generated in a relatively short period of time, for example, less than two months, or less than one month, or less than about two weeks.

[0032] The cell composition may further comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other administration routes, and may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell composition may be provided in unit vials or bags and stored frozen until use. In certain embodiments, the volume of the composition is about 1 fluid ounce to 1 pint.

[0033] HSCs generated using the methods described herein can be administered to a subject (recipient) by, for example, intravenous infusion or intrabone marrow transplantation, which can be performed following myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-kit, anti-CD45, etc.) conditioning regimens.

[0034] The methods described herein can be used to generate populations of HSCs for use in transplantation protocols to treat, for example, hematological disorders (malignant and non-malignant), bone marrow disorders, metabolic disorders, and immune disorders. In some embodiments, the HSC population is derived from autologous cells, or universally-compatible donor cells, or HLA-modified or HLA-deficient cells. In some embodiments, the HSC population is derived from gene-edited cells containing one or more transgenes (e.g., CCR5, TCR, or CAR). That is, the HSC population is generated from iPSCs prepared from the recipient subject's cells or prepared from donor cells (e.g., universal donor cells, HLA-matched cells, HLA-modified cells, or HLA-deficient cells) and optionally gene-edited.

[0035] In some embodiments, the recipient subject suffers from a condition selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, a myeloproliferative disorder, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), acquired immunodeficiency syndrome (AIDS), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

[0036] In yet other embodiments, HSCs produced in accordance with the present disclosure are used to produce immune cells for immunotherapy, such as T cells (e.g., xenogeneic TCR-expressing T cells, chimeric antigen receptor T cells (CAR-T cells), regulatory T cells, cytotoxic T cells, γδ T cells, αβ T cells, etc.), NK cells (including CAR-NK cells), or B cells (including CAR-B cells).

[0037] In other embodiments, HSCs produced in accordance with the present disclosure are used to generate macrophages to treat, for example, transplant tolerance, autoimmune disorders, or fibrosis.

[0038] In some embodiments, HSCs produced in accordance with the present disclosure are used to produce red blood cells that can be used, for example, to treat hemoglobinopathies. In yet other embodiments, HSCs produced in accordance with the present disclosure are used to produce platelets for the direct or indirect treatment of bleeding disorders. In some embodiments, HSCs are used to prepare blood products for the treatment of co-morbidities associated with hematological, bone marrow, immune, metabolic, or mitochondrial disorders.

[0039] Methods for differentiating into desired cell lineages are known in the art.

[0040] As used herein, the term "about" means plus or minus 10% of the relevant numerical value. [Example]

[0041] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency Induced pluripotent stem cells (iPSCs) were transduced with an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSCs retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a representative FACS plot of the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences.

[0042] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies, followed by differentiation into hemogenic endothelial cells (Strugeon et al., 2014). - CD34 in the population +and CD31 + As evidenced by marker expression, overexpression of ETV2 suggests that it enhances the formation of hemogenic endothelial cells (Figure 2). Specifically, Figure 2 shows a representative flow cytometry analysis of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), and relative quantification demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells.

[0043] Furthermore, the results showed that ETV2-OE was associated with CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows a representative flow cytometry analysis of CD34+ cells, and the relative quantification demonstrates that ETV2-OE enhances CD34+ cell formation.

[0044] Taken together, these data indicate that ETV2 overexpression in iPSCs promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation without affecting their pluripotent properties.

Claims

1. 1. A method for generating hematopoietic stem cells (HSCs), comprising: preparing endothelial cells (ECs) from pluripotent stem cells by expression of E26 transformation specific variant 2 (ETV2) transcription factor; converting the endothelial cells into hemogenic endothelial cells and converting the hemogenic endothelial cells into hematopoietic stem cells; A method comprising:

2. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs) prepared by reprogramming somatic cells.

3. 3. The method of claim 2, wherein the somatic cells are reprogrammed by expression of reprogramming factors selected from one or more of Sox2, Oct3 / 4, c-Myc, Nanog, Lin28 and Klf4.

4. The method of claim 2 or 3, wherein the iPSCs are HLA-modified or HLA-deficient cells.

5. The method of any one of claims 2 to 4, wherein the iPSCs are genetically modified.

6. The method of any one of claims 2 to 5, wherein the iPSCs comprise a constitutive or inducible suicide gene.

7. The method of any one of claims 2 to 6, wherein the iPSCs comprise a reporter gene.

8. The method of any one of claims 1 to 7, wherein ETV2 is expressed from a non-integrated episome.

9. The method of any one of claims 2 to 7, wherein ETV2 is expressed from mRNA introduced into the iPSCs.

10. The method of claim 9, wherein ETV2 mRNA is introduced into mesodermal progenitor cells (MPCs) prepared from the iPSCs.

11. The method of any one of claims 2 to 10, wherein the iPSCs are differentiated into mesodermal progenitor cells (MPCs) and the MPCs are differentiated into endothelial cells (ECs).

12. The method according to claim 11, wherein ECs are differentiated into hemogenic endothelial cells (HECs) by addition of a Piezo1 agonist.

13. CD34 ｐｏｓ and optionally CD31 ｐｏｓ , CD144 ｐｏｓ , K.D.R. ｐｏｓ , CD235 ｎｅｇ and CD43 ｎｅｇ The method of any one of claims 1 to 12, further comprising selecting or enriching for cells having a cell surface phenotype selected from one or more of:

14. The method according to any one of claims 1 to 13, wherein HECs are differentiated into HSCs by adding a Piezo1 agonist.

15. 15. The method of any one of claims 1 to 14, wherein inducing hematopoietic differentiation comprises or further comprises culturing the endothelial or HE cells with one or more of insulin-like growth factor-1 (IGF-1), sonic hedgehog (SHH), angiotensin 2, losartan, Flt3, Flt3 ligand, Y27632, FGF, FGF-2 (bFGF), BMP-4, activin A, transferrin, VEGF, DKK, IL-6, IL011, SCF, EPO, TPO, IL-3, SB-431542, fibronectin, and vitronectin.

16. The method of any one of claims 1 to 15, wherein the ECs are converted to HECs or HSCs by a process comprising increasing expression or activity of Dnmt3b.

17. The HSCs are optionally treated with linoleic acid, PGE ２ , P.G.E. ２ Derivatives and / or PGE ２ The method according to any one of claims 1 to 16, wherein the progenitor(s) are used for proliferation.

18. The method of any one of claims 1 to 17, wherein the HSCs comprise long-term HSCs (LT-HSCs).

19. CD34 ＋ 19. The method of claim 18, further comprising selecting or enriching the cells, optionally sorting or enriching the cells based on positive or negative expression of one or more of CD45, CD38, CD90, CD49f and GPI-80.

20. 20. The method of claim 18, comprising harvesting cells in suspension in the culture.

21. A composition comprising a hematopoietic stem cell (HSC) population produced according to the method of any one of claims 1 to 20.

22. 22. The composition of claim 21 for treating a disease or condition in a recipient.

23. 23. The composition of claim 22, wherein the disease or condition is a blood disease, a bone marrow disease, a metabolic disease, an immune disease or a mitochondrial disease.

24. 24. The composition of claim 23, wherein the disease or condition is selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), acquired immune deficiency syndrome (AIDS), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

25. 25. The composition of claim 23 or 24, wherein the HSCs are derived from allogeneic or universally matched donor cells, HLA-modified cells or HLA-deficient cells, and are optionally genetically modified.

26. The composition of any one of claims 23 to 25, wherein the HSCs are derived from cells of the recipient.

27. A method for preparing a cell population for immunotherapy, comprising differentiating HSCs produced according to the method of any one of claims 1 to 20 into an immune cell lineage.

28. 28. The method of claim 27, wherein the immune cell lineage is a T cell, a B cell, or a NK cell.

29. 29. The method of claim 28, wherein the immune cell lineage is a regulatory T cell, a cytotoxic T cell, a γδ T cell, or an αβ T cell.

30. 30. The method of claim 29, further comprising expressing a heterologous TCR.

31. 31. The method of claim 29 or 30, further comprising expressing a chimeric antigen receptor (CAR).

32. 21. A method for preparing a cell population for treating a hemoglobinopathy, comprising differentiating HSCs produced according to the method of any one of claims 1 to 20 into red blood cells.

33. 21. A method for preparing a cell population for treating transplant tolerance, an autoimmune disorder or a fibrotic disease, comprising differentiating HSCs produced according to the method of any one of claims 1 to 20 into macrophages.

34. 21. A method for preparing a cell population for directly or indirectly treating a bleeding disorder, comprising differentiating HSCs produced according to the method of any one of claims 1 to 20 into platelets.

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