Methods for generating hematopoietic stem cells

Abstract

This invention provides a method for proliferating hematopoietic stem cells (HSCs) using genetic, pharmacological, and mechanical stimulation. [Solution] In various aspects and embodiments, the Disclosure discloses genetic, pharmacological, and mechanical stimulation for converting endothelial cells into hematopoietic endothelial (HE) cells and for converting HE cells into HSCs containing significant levels of long-term (LT)-HSCs. The Disclosure further provides methods for growing HSCs using genetic, pharmacological, and mechanical stimulation.

Description

[Technical Field]

[0001] Claim of priority This application is a U.S. Provisional Patent Application No. 62 / 945,83 filed on December 9, 2019. We claim the benefit of Article 8. The entire contents of the said application are incorporated herein by reference.

[0002] Research funded by the federal government This invention is granted under registration number HL131645 by the National Institutes of Health, USA. This was done with government support. The government has certain rights in this invention. [Background technology]

[0003] Hematopoietic stem cells (HSCs) are formed during embryonic development when specific induced events occur in the mesoderm, along with blood stem cells. They derive in a different region from other cells that are converted into blood progenitor cells. HSCs are called hematopoiesis. In this process, red blood cells, platelets, bone marrow, and lymph (T cells and B cells) cells It can be made to happen.

[0004] HSC transplantation (HSCT) is performed on patients with hematological disorders, bone marrow disorders, metabolic disorders, and immune disorders. It is widely used to treat patients with umbilical cord blood and haplotype-matched stem cells. Despite advances in transplantation, the therapeutic use of HSC transplantation is limited to human leukocyte antigen (HL) A) The difficulty of finding a suitable donor in a timely manner, especially in countries with ethnic minorities. Furthermore, due to the lack of domestic registration for unrelated donors, this is often restricted. People make up 1.6 percent (9.7 million) of the US population, but mixed-race volunteers A represents only 3 percent (21,000 people) of the 7 million registered users, and 60 00 patients still have unsuitable bone marrow. Even when a suitable match is found, the graft... Immunological factors such as GVHD, donor rejection, and high mortality rates associated with the procedure. Comorbidities can endanger the patient's survival. However, these complications can be eliminated by autologous transplantation. Autologous HSCs do not replace all allogeneic HSCs, but especially in hematological malignancies. In this situation, autologous HSCs have a lack of donor availability, as well as widespread malignancy and This includes GVHD in patients with non-malignant hematological, immunological, and metabolic disorders. Overcoming major hurdles in HSCT. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, to generate HSCs for HSCT, including homemade HSCs or pre-made HSCs. It is necessary to do so. [Means for solving the problem]

[0006] This disclosure reveals that changes in the expression or activity of specific endothelial and hematopoietic genes can lead to changes in the production of hematopoiesis from endothelial cells. Hematopoietic stem cell (HSC) formation, for example, self-regeneration, engraftment, and reconstitution of multicellular adult blood cells. The discovery that this induces the formation of a considerable number of long-term (LT)-HSCs is significant. It is also based on part of it.

[0007] cdh5-morphant (cdh5-MO) embryos do not exhibit cardiac output or active blood flow. Furthermore, in the blood vessels, there is a pulsation mediated by the heartbeat. The stretching resulting from the pulsation is controlled by a single piezo device. It activates mechanosensory channels, which then enter the aortic-gonad-mesonephrogenic (AGM) region. This further enhances the expression of Dnmt3b, which in turn enhances the expression of core endothelial and hematopoietic genes. It regulates the expression of its regulatory factors and stimulates the conversion of hematopoietic endothelium to hematopoietic stem cells (HSCs). Intense or piezo-1 pharmacological activation also produces 2-3 times more LT-HSCs. This HSC reconstitutes normal, functional multicellular adult blood through serial transplantation. Morphologically, the hematopoietic stem cells produced in accordance with this disclosure contain a considerable number of LT-HSCs. This shows excellent engraftment and reconstructs functional multicellular adult blood in the recipient. To accomplish.

[0008] In some embodiments, the present invention relates to a population of HSCs including LT-hematopoietic stem cells (HSCs). The present invention provides a method for preparing a substance comprising endothelial and / or hematopoietic endothelial (HE) cells. To provide a cell population, as well as vegfa in endothelial and / or HE cells, h ey2, grp116, gna13, sox17, cdh5, plxnd1, bcl6 Expression of two, three, four, five or more endothelial genes selected from and apln. The method includes reducing or altering the activity of endothelial and / or HE cells. runx1, spi1, cebpa, tal1, gfi1, gata2 and in cells Expression of two, three, four, five or more hematopoietic genes selected from mllt3 This further includes increasing or altering the activity of endothelial genes. By reducing HE cells and increasing the expression or activity of hematopoietic genes, HE cells The formation of cysts or HSCs (including a significant number of LT-HSCs) is stimulated. Endothelial genes Decreased expression or activity, and increased expression or activity of hematopoietic genes, can be caused by, for example, inhibitors or introduction. By administration of genes, episomes, mRNA and their derivatives, and / or (this specification) (As fully described in the book) It can be a direct approach using gene editing methods. Alternatively, such changes in expression or activity may occur, for example, in DNA (cytosine-5). -)-methyltransferase 3 beta (Dnmt3b) and / or GTPase To increase the expression or activity of IMAP family member 6 (Gimap6) Therefore, it may be induced at least partially and indirectly. Furthermore, such gene expression Regulation is at least in part by agonists of mechanosensitive receptors or channels (e.g., P Using an Ezo-1 agonist, or biomechanical stretching by repeated strain on cells This may be done by applying to vegfa, hey 2, grp116, gna13, sox17, cdh5, plxnd1, bcl6, oyo Select at least one, or at least two, three, four, or five from apln. Furthermore, the expression of endothelial genes beyond these is directly or indirectly reduced. In other embodiments, runx1, spi1, cebpa, tal1, gfi1 , at least one or at least two selected from gata2 and mllt3, Three, four, five or more hematopoietic genes are directly or indirectly increased in expression. ru.

[0009] In some embodiments, the expression or activity of endothelial genes and hematopoietic genes is at least Also, under conditions sufficient to stimulate the formation of HE cells or HSCs, the cells By increasing the activity or expression of Dnmt3b and / or Gimap6 in [the specified location] It is regulated in this way. In some embodiments, HE cells are harvested and used for HSC formation. HSCs may be collected and, if necessary, propagated for administration to patients. C may, depending on the circumstances, be a genetic, pharmacological, or mechanical stimulus as described herein. It is propagated using [this method].

[0010] In some embodiments, endothelial cells increase the activity or expression of Dnmt3b to an effective level. Agonists that either enlarge or provide appropriate regulation of endothelial and hematopoietic genes. To be touched. In some embodiments, the agonist is a mechanosensitive receptor or mechanosensitive receptor. It is an agonist of a receptive channel. In some embodiments, the mechanosensible receptor is a pied It is a piezo 1. Typical piezo 1 agonists include Yoda1, Jedi1 and Je di2 is one example. In some embodiments, a piezo agonist (e.g., Yoda1) is used. The effective dose is approximately 0.1 μM to 300 μM, or approximately 0.1 μM to 200 μM. , or approximately 1 μM to approximately 100 μM, or in some embodiments, approximately 10 μM to Approximately 100 μM, or approximately 2.5 μM to approximately 100 μM, or approximately 2.5 μM to approximately 50 μM It is within the range of M.

[0011] Alternative mechanosensitive receptors or mechanosensitive channel agonists (e.g., piezo-1 agonists) The 'nist' can be identified from a chemical library. In these embodiments, Agonists that induce changes in endothelial and hematopoietic gene expression as described in the specification are identified. For example, candidate agonists, upon contact with the candidate compound, affect endothelial cells and / or HE cells. In cells, vegfa, hey2, grp116, gna13, sox17, cdh5, Reduce the expression of endothelial genes selected from plxnd1, bcl6, and apln. Alternatively, altering the activity of runx1, sp in endothelial cells and / or HE cells. Two selected from i1, cebpa, tal1, gfi1, gata2, and mllt3. It increases the expression or alters the activity of one or more hematopoietic genes.

[0012] Sites that directly increase gene activity or expression in endothelial cells and / or HE cells. In total, various approaches can be used. For example, gene mRNA expression can be measured using mR By delivering NA transcripts (including modified mRNA) to cells, or by increasing activity An introduced gene that may have one or more modifications to cause or alter something It can be enlarged by introducing / or episomes. Some implementations Morphologically, gene editing is used to express elements in endothelial cells or HE cells. By introducing gene modifications, for example, promoter strength, ribosome binding, or RNA To increase stability. In some embodiments, gene editing is used to modify gain-of-function mutations. Used for implementation.

[0013] In some embodiments, gene activity or expression is controlled in endothelial cells and / or HE cells. It may be directly reduced in the cell. For example, the expression or activity of endothelial genes may be completely reduced. These include partial gene deletion, RNA silencing, and antisense oligonucleotide inhibition. Introducing and introducing gene modification of the expression element (promoter strength) (including reducing ribosome binding or RNA stability) or endothelial cells and / or by introducing loss-of-function mutations in HE cells, one or more of these It may be reduced.

[0014] In some embodiments, the present invention relates to Gim in endothelial cells and / or HE cells. To increase the activity or expression of ap6, either alone or in combination with Dnmt3b. Includes. To increase Gimap6 activity or expression, Gimap6 mRNA transcription The product, or instead the Gimap6 transgene and / or episome, is introduced into the cell. It is possible to do so, as well as / or the gene of the Gimap6 expression element in cells. Modifications (to increase promoter strength, ribosome binding, or RNA stability) It is also possible to introduce one or more modifications.

[0015] In various embodiments, a population of cells including embryoid bodies, endothelial cells and / or HE cells It is introduced into a bioreactor. In some embodiments, the bioreactor is repeatedly It provides biomechanical stretching to cells in 2D or 3D culture by strain. For example, Biomechanical stretching due to counter-strain may be applied to 2D or 3D culture surfaces. Repeated Biomechanical stretching due to strain is linked to the activity of Dnmt3b and / or Gimap6. This increases expression. For example, nylon membrane (e.g., a culture plate with a soft bottom), P Computer-controlled vacuum connected to a DMS membrane or other biocompatible and biomimetic membrane. Pump systems (e.g., FlexCell® Tension System, C Using the ytostretcher System (or similar), the prescribed control Under controlled repeated strain conditions, circumferential extension is performed in 2D or 3D culture via ex vivo. It can be applied to embryoid bodies, endothelial cells, or HE cells that are in contact with the membrane.

[0016] In various embodiments, HSC conversion involves activation of piezo 1; mechanical extension; Dnmt For 3b, with or without modification by the introduced gene (i.e., introduced gene f Lee), with or without episomal modification, or with or without gene modification Introduction of mRNA without accompanying modification; modification of Gimap6 by the transgene or Without (i.e., transgene-free), with or without episomal modification Introduction of mRNA, with or without gene modification; as described herein. Modification by an introduced gene to one or more hematopoietic genes listed. Not (i.e., transgene-free), with or without episomal modification. , or introduction of mRNA with or without gene modification; and, whole or partial By introducing specific gene deletions, RNA silencing, and antisense oligonucleotide inhibition... By doing so, or by introducing gene modifications to the endothelial genes described herein. It is triggered by one or more selections.

[0017] In some embodiments, endothelial cells and / or HE cells are induced pluripotent stem cells (i Obtained from PSCs, non-hematopoietic stem cells, or somatic cells such as fibroblasts or endothelial cells. or derived therefrom. In some embodiments, endothelial cells and / or HE cells HLA null cells, HLA modified cells, gene-modified cells, viral vector overexpressing cells, From transgene-overexpressing cells and / or transgene-free cells, or from embryoid bodies These are obtained from or derived from genetic induction into endothelial cells and / or HE cells. These are derived from the source cells of an allogeneic donor, or from the subject being treated with HSC. This includes hematopoietic endothelial cells (e.g., Flkl+CD45+ cells, Flkl+CD41+ cells). Cells (or CD31+CD43+ cells) can be obtained in any way (i.e.) Chemical induction, genetic induction, mRNA induction, etc., from autologous or allogeneic cells to hematopoietic endothelial cells. Induction, transgene-free induction, or episome induction). In some embodiments, Dermocytes or HE cells are cells derived from recipient, ready-made bank, or pluripotent donor cells. It is generated from iPSCs fabricated using this method. In some embodiments, it is embryologically flexible. Developmentally plastic endothelial cells are used.

[0018] In various embodiments, HSC prepared by the method described herein A pharmaceutical composition for cell therapy is prepared, comprising a population and a pharmaceutically acceptable vehicle. The pharmaceutical composition contains at least about 10 2 one HSC, or at least about 10 3 H SC, or at least about 10 4 one HSC, or at least about 10 5 Individual HSCs, or at least about 10 6 one HSC, or at least about 10 7 Individual HSCs, or few Kutomo 10 8 It may contain a small amount of HSCs. In various embodiments, the composition contains a small amount of HSCs. At least about 0.1%, or at least about 1%, or at least about 2%, or less Also about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50% are LT-HSCs. For example, in some embodiments, from about 100,000 to about 4×10 6 HSCs per kilogram of recipient body weight (e.g., about 2×10 6 cells / kg) are included in a pharmaceutical composition that is administered .

[0019] In some embodiments, a cell therapy comprising a population of HSCs prepared by the methods described herein is provided. In some embodiments, the cell therapy comprises a pharmaceutically acceptable vehicle. The cell therapy comprises at least about 10 HSCs, or at least about 10 2 HSCs, or at least about 10 3 HSCs, or at least about 10 4 HSCs, or at least about 10 5 HSCs, or at least about 10 6 HSCs, or at least about 1... 7 H SCs, or at least 10 8 HSCs may be included. In various embodiments, at least about 0.1%, or at least about 1%, or at least about 2% of the HSCs in the composition, or at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50% are LT -HSCs. For example, in some embodiments, a pharmaceutical composition comprising from about 100,000 to about 4×10 HSCs per kilogram of recipient body weight (e.g., about 2×10 cells / kg) is administered. The number of HSC cells can be varied based on the age and weight of the patient. 6 HSCs (e.g., about 2×10 6 cells / kg) is included . The number of HSC cells can be varied based on the age and weight of the patient.

[0020] HSCs for transplantation are, in some embodiments, less than approximately 2 months or less than approximately 1 month. Full duration (e.g., about 4 weeks), or less than about 2 weeks, or less than about 1 week, or less than about 6 days or relatively short periods such as less than approximately 5 days, less than approximately 4 days, or less than approximately 3 days. It can be generated in between. In some embodiments, a developmentally flexible endothelium or HE The cells are cultured for 1 to 4 weeks by regulating the activity or expression of endothelial and hematopoietic genes.

[0021] HSC prepared by the method described herein can be administered, for example, by intravenous injection or It is administered to the recipient via intramedullary transplantation. This method is myeloablative, Non-myeloablative or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) pre-treatment This can be done after the treatment regimen.

[0022] The methods described herein are for the treatment of acquired or congenital forms of blood disorders (malignant and For example, treatment of (and non-malignant) bone marrow diseases, metabolic diseases, mitochondrial diseases, and immune diseases. Used to generate a population of HSCs for use in the transplantation protocol. It is possible. In some embodiments, the HSC population is derived from autologous cells, for example, It is generated from iPSCs produced using the target cells. In some embodiments In this context, the HSC population consists of pluripotent donor cells or HLA null hematopoietic endothelial cells or normal cells. It originates from similar cells that produce HSCs.

[0023] These and other aspects and embodiments of the present invention are described below in the detailed description of the invention. It is written as follows. [Brief explanation of the drawing]

[0024] [Figure 1A] This image shows slow-speed confocal imaging of cd41:eGFP+ HSCs arising from flk1:mCherry+ endothelial cells in transgenic embryos between 26 and 42 hpf. The data demonstrate that silencing of piezo1 weakens the conversion of endothelial cells to HSCs, while pharmacological activation of piezo1 (Yoda1) stimulates HSC formation in control embryos and restores HSC formation in sih-MO embryos. n=5 per group. *P<0.05 compared to control, P<0.05 compared to sih-MO. [Figure 1B] This is a heatmap of differentially expressed genes in E11.5 AGM cells treated with repeated straining and piezo1 activator (Yoda1), indicating that repeated straining and piezo1 activation result in similar gene expression patterns in AGM during endothelial cell-to-hematopoietic cell conversion. n=3 per group. [Figure 1C] E11.5 This graph shows the hematopoietic colony-forming unit (CFU) assay in AGM cells, demonstrating that piezo1, Yoda1-mediated pharmacological activation stimulates the conversion of endothelial cells to hematopoietic cells. n≧6 per group. *P<0.05 compared to control. Abbreviations: GEMM (granulocyte, erythroid, macrophage, megakaryocyte), GM (granulocyte-macrophage), G (granulocyte), M (macrophage), E (erythroid). [Figure 1D] This graph shows the hematopoietic CFU assay in E11.5 AGM cells, demonstrating that piezo-1, GsMtX4-mediated pharmacological inhibition attenuates the induced effects of repeated strain on endothelial-HSC conversion. n≧6 per group. *P<0.05 compared to control. Abbreviations: GEMM (granulocyte, erythroid, macrophage, megakaryocyte), GM (granulocyte-macrophage), G (granulocyte), M (macrophage), E (erythroid). [Figure 1E]This report presents a hematopoietic CFU assay in E11.5 AGM cells treated with 50 μM Jedi1, 50 μM Jedi2, or 25 μM Yoda1, indicating that Jedi1, Jedi2, or Yoda1-mediated piezo1 activation enhances GEMM formation. N=6 per group. 3.eeAGM cells per sample. *P≦0.05. [Figure 2A] This figure shows the experimental overview (top) and line graph (bottom). The experimental overview (top) shows a diagram representing the serial transplantation of HSCs derived from E11.5 mouse AGM and subsequent treatment with 10% repeated straining or Yoda1 in myeloablative immunodeficient mice. The line graph (bottom) shows the percentage of peripheral blood chimerism occurring in the reconstitution of HSCs derived from E11.5 AGM (donor, 3 embryo equivalents) at 4-week intervals between weeks 8 and 16 in primary transplants (recipients), indicating that repeated straining or piezo-1 pharmacological activation (Yoda1 treatment) of E11.5 AGM stimulates HSC formation. Primary recipients n≧5 per group. *P<0.05 compared to control, P<0.05 compared to chimerism at week 8. 3 embryo equivalents (ee) of AGM donor cells were injected into each recipient. [Figure 2B] This graph shows the percentage of reconstitution of HSCs derived from E11.5 AGM (donor, 3 embryo equivalents) to Mac1+Gr1+ myeloid cells, Cd8+Cd3+ T cells, and B220+Cd19+ B cells in primary transplant recipients at 16 weeks. This indicates that repeated straining of E11.5 AGM or pharmacological activation of piezo1 (Yoda1) stimulates the formation of HSCs to be reconstituted into the blood. Primary recipients with n≧5 per group. [Figure 2C]This line graph shows the percentage of peripheral blood chimerism resulting from the reconstitution of flow-sorted Lin-Sca1+c-Kit+HSPC (n=2000) derived from the primary transplant (donor) at 4-week intervals between weeks 8 and 12 in secondary transplant recipients. This indicates that repeated straining with E11.5 AGM or Yoda1 treatment produces HSCs with continuous engraftment and autoregeneration capabilities. Secondary recipients with n≧5 per group. *P<0.05 compared to control. [Figure 2D] This graph shows the percentage of reconstitution of HSCs derived from the primary transplant (donor) to Mac1+Gr1+ myeloid cells, Cd8+Cd3+ T cells, and B220+Cd19+ B cells in secondary transplant recipients at 12 weeks. This indicates that repeated straining with E11.5 AGM or Yoda1 treatment produces HSCs that can be continuously reconstituted into the blood. Secondary recipients with n≧5 per group. [Figure 3A] This figure shows an overview of the experiment (top) and graphs (bottom). The overview (top) shows the strategies for functional and phenotypic analysis of donor-derived blood lineages in the hematopoietic tissue of primary transplants (recipient mice). The graphs (bottom) show the percentage expression of β-major (adult), εγ (embryonic), and β-H1 (embryonic) hemoglobin in selected Cd71+Ter119+ (donor) erythroid cells derived from bone marrow. The data show that donor HSCs produced after biomechanical stretching of E11.5 AGM or Yoda1 treatment are reconstituted into erythrocytes containing adult hemoglobin. n≧6 per group. [Figure 3B] This graph shows the overnight culture (O / N) of selected Gr1+Mac1+ (donor) neutrophils derived from bone marrow, followed by ELISA-based quantification of myeloperoxidase (MPO) protein. The data demonstrate that donor HSCs were produced to reconstitute functional bone marrow cells exhibiting sufficient MPO levels after biomechanical stretching of E11.5 AGM or Yoda1 treatment. n≧5 per group. [Figure 3C]This graph shows the ELISA analysis of pre-immunization immunoglobulin (Ig) isotypes in the peripheral blood of primary transplant (recipient) mice. The data indicate that the primary transplant produces B cells with a complete immunoglobulin repertoire. n≧6 per group. [Figure 3D] These are images of two gel photographs showing T cell receptor (TCRβ) locus analysis of spleen-selected Cd3+ T cells (donor) (top) or Mac1+ myeloid cells (donor, negative control) (bottom). The data show that after biomechanical stretching of E11.5 AGM or Yoda1 treatment, donor HSCs migrate to the spleen and produce T cells, which are then reconfigured into T cells with sufficient functional recombination mechanisms to rearrange the TCRβ locus, demonstrating T cell receptor β (TCRβ) rearrangement. [Figure 3E] This dot plot shows a delayed-type hypersensitivity assay, demonstrating that primary transplant (recipient) mice reconstituted with donor HSCs derived from biomechanically stretched or Yoda1-treated E11.5 AGMs exhibit a T-cell-mediated immune response. n≧6 per group. *P<0.05 compared to right paw (negative control). [Figure 4A] This is a Venn diagram of upregulated genes in E11.5 AGM cells treated with repeated straining and / or Yoda1, under conditions where genes were upregulated during EC vs. HSC ((1)), EC vs. HEC ((2)), and HEC vs. HSC ((3)). The comparison of Venn diagrams of genes commonly upregulated in the above analyses ((1) vs. (2) vs. (3)) demonstrates that both circumferential stretching and piezo1 activation specifically stimulate the expression of Dnmt3b transcript and Gimap6 transcript during endothelial-to-HSC conversion. [Figure 4B]This is a Venn diagram of upregulated genes in E11.5 AGM cells treated with repeated straining and / or Yoda1, under conditions where genes were upregulated during EC vs. HSC ((1)), EC vs. HEC ((2)), and HEC vs. HSC ((3)). The comparison of Venn diagrams of genes commonly upregulated in the above analyses ((1) vs. (2) vs. (3)) demonstrates that both circumferential stretching and piezo1 activation specifically stimulate the expression of Dnmt3b transcript and Gimap6 transcript during endothelial-to-HSC conversion. [Figure 5A] This figure shows two graphs of Dnmt3b and Dnmt3a protein levels in the nuclear fraction of AGM cells from E11.5 mice treated with repeated straining or Yoda1. The data demonstrate that circumferential stretching or activation of piezo1 specifically stimulates Dnmt3b protein expression levels without affecting Dnmt3a expression. n≧3 per group. *P<0.05 compared to control. [Figure 5B] This graph shows the hematopoietic CFU assay of AGM cells from E11.5 mice treated with repeated straining or Yoda1 in the presence of nanaomycin (Nana). The data indicate that pharmacological inhibition of Dnmt3b weakens endothelial-to-HSC conversion stimulated by circumferential stretching or piezo1 activation. Embryos n≧6 per group. *P<0.05 against control. $P<0.05 against stretching. +P<0.05 against Yoda1. [Figure 5C] This graph shows the results of slow-speed confocal imaging of cd41:eGFP+ HSCs arising from flk1:mCherry+ endothelial cells of transgenic embryos between 26 and 42 hpf. The data demonstrate that silencing of dnmt3bb.1 weakens the endothelial-to-HSC conversion stimulated by piezo1 activation, and that nanaomycin is more specific to Dnmt3b than to Dnmt3a. n≧5 per group. *P<0.05 compared to control. P<0.05 compared to $Yoda1. [Figure 6A]The differential expression of endothelial and hematopoietic genes in E11.5 AGM cells repeatedly stretched or treated with Yoda1 was observed, indicating that repeated straining and piezo1 activation suppress endothelial gene expression while stimulating hematopoietic gene expression during endothelial-to-hematopoietic conversion. [Figure 6B] The qRT-PCR analysis of endothelial and hematopoietic gene expression is shown, demonstrating that repeated straining or piezo1 activation suppresses endothelial gene expression and stimulates hematopoietic gene expression in E11.5 AGM cells. n=5 per group. *P≦0.05 compared to control. [Figure 6C] This study presents a hematopoietic CFU assay of human iPSC-derived MACS-sorted CD34+ cells after Yoda1 treatment, demonstrating that piezo1 pharmacological activation stimulates the formation of pluripotent GEMM progenitor cells and human hematopoiesis. n=6 per group, 20,000 hCD34+ cells per sample. *≤0.05 compared to control. [Figure 6D] Figure 6D shows the percentage of bone marrow chimerism (left) 8-10 weeks after injection (primary transplant) of human PSC (DF19-9-7T)-derived hCD34+ hematopoietic cells into humanized mice, indicating that Yoda1-mediated pharmacological activation of piezo1 enhances the formation of engraftable hCD34+ cells. Figure 6D further shows the percentage of bone marrow rearrangement from human PSC-derived hCD34+ hematopoietic cells (right) to human CD33+ bone marrow cells, human CD3+ T cells, and human CD19+ B cells, indicating that pharmacological activation of piezo1 (Yoda1) stimulates the formation of hCD34+ hematopoietic cells that rearrange multicellular blood. n=8 (control, for primary transplant) and n=8 (Yoda1 treatment, primary transplant). ***P≦0.001. [Figure 6E]Figure 6E shows the percentage of peripheral blood chimerism at 16 weeks after injection of bone marrow cells derived from a primary graft of hCD34+ hematopoietic cells from the DFT19-9-7T hPSC line in humanized mice (secondary transplant) (left), indicating that Yoda1-mediated pharmacological activation of piezo1 enhances the formation of self-regenerating LT-HSCs. Figure 6E further shows the percentage of peripheral blood reconstitution from primary transplant bone marrow-derived human hematopoietic cells to human CD33+ bone marrow cells, human CD3+ T cells, and human CD19+ B cells in secondary transplant (right), indicating that pharmacological activation of piezo1 (Yoda1) stimulates the formation of human LT-HSCs that reconstitute multicellular blood during serial transplantation. N=5 (Yoda1 treatment, secondary transplant). [Figure 7A] FACS analysis demonstrates that repeated stretching promotes conversion from EC to HEC (left) and from HEC to HSPC (center). Analysis of HSPC proliferation by repeated stretching is shown on the right. N=6. **P≦0.001, *P≦0.05. Figure 7A (top) shows the application of 10% repeated stretching to mouse E11.5 AGM-selected EC (CD31+), HEC (CD31+cKit+), and HSPC (cKit+) cells, and subsequent FACS analysis. [Figure 7B] FACS analysis of human embryoid bodies (EBs) at day 8 of hematopoietic differentiation is shown, indicating that Yoda1-mediated piezo1 activation enhances hCD43negCD235negCD144+CD34+HEC formation in controls, but not in piezo1- / -PSCs. Piezo1 loss did not affect HEC formation. N=3 per group. **P≦0.001 *P≦0.05. [Figure 7C] This is a FACS plot of CD34+CD90+HSCs obtained on days 8+7 of hematopoietic differentiation of human PSCs, indicating that Yoda1-mediated piezo1 activation enhances HSC formation from hPSCs. [Modes for carrying out the invention]

[0025] During fetal development, a subset of endothelial cells in the aorta-gonad-mesonephrocyte (AGM) is hematopoietic. These are sexual endothelial cells, which change their fate and ultimately contribute to the development of the fetal liver and bone marrow. This leads to the formation of hematopoietic endothelial cells (HSCs). However, the individuality of the factors that stimulate hematopoietic endothelial cells remains. This is unclear and limits the availability of hematopoietic endothelial cells as a potential source of functional HSCs. Shear stress mediated by blood flow on the intima stimulates the emergence of endothelial properties in HSCs. However, using Cdh5 null zebrafish and mouse models, early Functional HSCs were confirmed to occur despite circulatory arrest. Anderson H, et al. , “Hematopoietic stem cells develop in the absence of endothelial cadherin 5 ex "pression." Blood 2015. These cdh5 models are silenced, functional H Shear stress and / or nitric oxide synthase (NOS) inactivity cause the appearance of SC. As a central point for studying the biomechanical forces of existence, pulse pressure is used in accordance with this disclosure. We investigated further mechanisms by which circumferential extension influences the appearance of HSCs. .

[0026] Attempts to generate HSCs from hematopoietic endothelial cells in the laboratory are... This is partly due to a lack of knowledge about the factors that stimulate the emergence of HSCs. These have largely been unsuccessful. Currently, the circular shape of blood vessels caused by the pulsation from the beating heart is the focus. Circumferential stretching leads to the emergence of functional HSCs from hematopoietic endothelial cells, and functional HSCs It has been confirmed that SCs can eventually engraft and differentiate into definitive cell lines. In addition, Transient receptor potential type cation channel subfamily vanilloid member 4( Activation of the Trpv4 channel is silent heart (tnnt2, sih), i.e. In silenced embryos where heartbeat and blood flow are absent, the formation of HSCs is restored. It is incorporated herein by reference in its entirety by International Publication No. 2017 / 0962. Please refer to pamphlet No. 15.

[0027] This disclosure relates to the biomechanical and / or pharmacological aspects of mechanosensible receptors (e.g., piezo1). This activation enhances the expression of Dnmt3b for hematopoietic stem cell (HSC) formation. This discovery is at least partly based on the endothelial genes of the core set and It regulates the expression of hematopoietic genes and their regulatory factors. This has been demonstrated herein. Uni, cdh5-morphant (cdh5-MO) embryos are associated with cardiac output and active blood flow. In addition, blood vessels have pulsations mediated by the heartbeat. The stretching resulting from the pulsation is due to the piezoelectric effect. 1. Activates a mechanosensitive channel, which further enhances the expression of Dnmt3b in AGM. Enhances the process and stimulates the conversion of endothelium to HSCs. Pulsation stimulation or piezo-1 pharmacology Targeted activation also produces at least three times more LT-HSCs, which is achieved in serial transplantation. The blood is reconstituted into a normal, functional, multicellular adult blood system.

[0028] Therefore, the results of this disclosure show how the biomechanical force mediated by the heartbeat affects the mechanical sensation. By activating receptive channels and epigenetic mechanisms, cell fate can be altered. It has been demonstrated that it stimulates cell turnover and stem cell formation. Development, proliferation, and Maintaining stem cell properties is crucial for HSC transplantation and cell therapy to treat hematological and bone marrow disorders. This disclosure is the primary objective in developing LT-HSCs through genetic and pharmacological means. Provides a scientific target. In various embodiments, this disclosure provides endothelial cells to hematopoietic endothelial (HE) cells. To convert into cells, and HE cells, including HSCs containing significant levels of LT-HSCs. This disclosure provides genetic, pharmacological, and mechanical stimuli for conversion to HSC. This provides a method for increasing HSCs using genetic, pharmacological, and mechanical stimulation.

[0029] In one embodiment, the present invention provides a method for preparing a population of HSCs including LT-HSCs. In certain embodiments, the method involves a cell population comprising endothelial cells and / or HE cells. To provide vegfa in endothelial cells and / or HE cells, hey 2, grp116, gna13, sox17, cdh5, plxnd1, bcl6, oyo It reduces the expression of two or more endothelial genes selected from apln, This involves altering the activity. The method further involves endothelial cells and / or HE cells. runx1, spi1, cebpa, tal1, gfi1, gata2 and mllt Increase the expression or activity of two or more hematopoietic genes selected from 3. This includes altering the process to stimulate the formation of HSCs, including LT-HSCs.

[0030] In some embodiments, the method involves vegfa in endothelial cells and / or HE cells. , hey2, grp116, gna13, sox17, cdh5, plxnd1, bcl 6. Expression of 3, 5 or more endothelial genes selected from apln. To reduce or alter the activity of endothelial cells and / or HE cells runx1, spi1, cebpa, tal1, gfi1, gata2, and m Increase the expression of three, five, or more hematopoietic genes selected from LLT3. , or including altering the activity. For example, in some embodiments, the method involves endothelial cells vegfa, hey2, grp116, gna13 in vesicles and / or HE cells To reduce the expression of cdh5 and plxnd1, or to alter their activity, In addition, runx1, spi1, cebpa, ta in endothelial cells and / or HE cells This includes increasing the expression of l1 and gata2 or altering their activity.

[0031] In some embodiments, the development of at least one, two, three, or five hematopoietic genes The current or activity is directly increased. When used herein, gene expression is Or activity is when the nucleic acid encoding a functional copy of the gene is introduced into the cell, or If an endogenous gene has been modified to increase its expression or related activity, then "directly It is increased "in a specific way". For example, activity or expression is increased by introducing coding mRNA, coding Introducing a transgene or episome, or introducing gene modification of an expression element. and using an approach that is selected independently of introducing gain-of-function mutations. This can be directly increased. In some embodiments, increased expression or activity of hematopoietic genes The full set of genes directly increases expression or activity.

[0032] According to various embodiments of this disclosure that use the expression of factors using episomes, One embodiment involves introducing a non-integrated episomal plasmid that expresses a desired element, for example, into a generative plasmid. This may include introducing them to create gene-free and virus-free cell populations. Replication The ability is limited and therefore lost in several cell generations, known epidemiological A plasmid may be used.

[0033] In some embodiments, the expression of at least one, two, three, or five endothelial genes. Or the activity is directly reduced. When used herein, gene expression and Activity is related to the introduction of nucleic acids or pharmacological inhibitors into cells, or their expression or associated effects. If an endogenous gene is modified to reduce its activity, the activity is directly reduced. For example, Activity or expression is affected by complete or partial gene deletion, RNA silencing, antiseptic Introducing oligonucleotide inhibition, pharmacological inhibition, and the legacy of the expression element Independent of introducing gene modifications or loss-of-function mutations, selection was performed. It can be directly reduced using an approach that may be used in some embodiments. The full set of endothelial genes whose expression or activation is reduced in morphology is expressed or The activity is directly reduced.

[0034] In some embodiments, one or more endothelial genes and one or more hematopoietic genes Gene expression or activity is, for example, under conditions sufficient to stimulate HSC formation (expression) DNA (cytosine-5-) in endothelial cells (including periods of higher levels and expression) - Methyltransferase 3 beta (Dnmt3b) and / or GTPase I By increasing the activity or expression of MAP family member 6 (Gimap6) It can be indirectly adjusted.

[0035] Dnmt3b (DNA (cytosine-5-)-methyltransferase 3 beta) It is a DNA methyltransferase. Dnmt3b is mainly localized in the nucleus, and its expression is... It is developmentally regulated. Gimap6 is part of the immune-associated protein (GIMAP) family. It is a member of the GTPase family. The GIMAP protein has a GTP-binding motif and It contains a coiled coil motif.

[0036] In some embodiments, endothelial cells, or HE cells or HS cells according to various embodiments. C is a mechanosensitive receptor or mechanism that increases the activity or expression of Dnmt3b in an effective amount. It is brought into contact with a mechanosensory channel agonist, thereby indirectly affecting endothelial genes and To regulate the expression level or alter the activity of hematopoietic genes. In some embodiments, The mechanosensitive receptor is piezo1. A typical piezo1 agonist is Yoda1. Other typical piezo agonists include Jedi1 and Jedi2. It is possible.

[0037] Yoda1(2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3, 4-thiadiazole-2-yl]-pyrazine is a mechanosensitive ion channel called Piezo This is a small molecule agonist developed for Zo-1. Syeda R, "Chemical activation of The mechanotransduction channel Piezo1. eLife (2015). Yoda1 has the following structure. It holds.

[0038] [ka]

[0039] Derivatives of Yoda1 can be used in various embodiments. For example, 2, Derivatives containing a 6-dichlorophenyl core are used in some embodiments. Typical A typical agonist is Evans EL, et al., Yoda1 analogue (Dooku1), which antagonizes Yo da1-evoked activation of Piezo1 and aortic relaxation, British J. of Pharmacolog Disclosed in y 175(1744-1759): 2018. Jedi1 and Jedi2 are Wang Y., et al., “A lever-like transduction pathway for long-distance chemical- and “mechano-gating of the mechanosensitive Piezo1 channel”, Nature Communications ( It is described in 2018)9:1300. Jedi1 and Jedi2 are methyl 3-carboxylate. It has a furan structure motif. Other piezo agonists that share this motif are from this development It can be used according to the explicit embodiment.

[0040] In some embodiments, a piezo agonist (e.g., Yoda1, Jedi1 or The effective dose of Jedi2 is approximately 0.1 μM to approximately 500 μM, or approximately 0.1 μM to approximately 300 μM, or approximately 0.1 μM to approximately 200 μM, or approximately 0.1 μM to approximately 100 μM M, or in some embodiments, about 1 μM to about 300 μM, about 1 μM to about 20 0 μM, approximately 1 μM to approximately 100 μM, approximately 1 μM to approximately 50 μM, or approximately 10 μM to approximately 100 μM, or in the range of approximately 10 μM to approximately 100 μM, or approximately 10 μM to approximately 50 μM It is located within the enclosure.

[0041] Alternative agonists, including piezo1, can be identified in chemical libraries. This chemical library binds to piezo1 or other mechanosensible receptors or channels. It may contain compounds that activate and / or activate it. The library is Yoda1 It may also contain derivatives of which may include 2,6-dichlorophenyl core or It may have a chemical mimic of it. The library contains Jedi1 and / or Jedi2. It may also contain derivatives, which may include francoa (for example) This may include methylfuran 3-carboxylate, or its derivatives or chemical mimics. The library reacts in endothelial cells and / or HE cells upon contact with candidate compounds. This specification reduces the expression or activity of endothelial genes, and affects endothelial cells and / Alternatively, in HE cells, increasing the expression or activity of hematopoietic cells as described herein. Compounds that induce this change can be screened. Changes in expression or activity can be observed in control cells, i.e., This can be determined by comparing it with cells that have not been in contact with the candidate compound. In some embodiments And, cells that have come into contact with Yoda1, Jedi2, and / or Jedi2, are genetically modified. It can be used as a positive control for expression regulation.

[0042] In these embodiments, the present invention relates to a method for producing hematopoietic stem cells (HSCs). : A panel of chemical compounds is brought into contact with endothelial cells and / or hematopoietic endothelial cells, and the chemicalization Dnmt3b or Gimap6 induced by compound;vegfa, hey2, gr p116, gna13, sox17, cdh5, plxnd1, bcl6, and apln at least two (or at least three or at least five); and runx1 , spi1, cebpa, tal1, gfi1, gata2 and mllt3 at least It also determines changes in the expression levels of two (or at least three or at least five) genes. The method provides the following gene expression changes: Dnmt3b and / or G Increased expression of imap6; vegfa, hey2, grp116, gna13, sox1 Decreased expression of three or more of the following: 7, cdh5, plxnd1, bcl6, and apln. ; and runx1, spi1, cebpa, tal1, gfi1, gata2 and m Compounds that induce one or more increased expression of two or more LLT3 proteins are then Selected. The selected compound is then used (for example, in a bioreactor) in the endothelial cells. It may induce the conversion of cells and / or hematopoietic endothelial cells into HSCs. The resulting HSCs are viable. It is a self-regenerating HSC that can attach and reconstruct multicellular blood. In some embodiments In this context, the selected compound is vegfa in endothelial and / or HE cells, hey 2, reduce the expression of grp116, gna13, cdh5 and plxnd1; endothelium runx1, spi1, cebpa, tal1 and ga in HE cells It increases TA2 expression.

[0043] In some embodiments, the activity or expression of Dnmt3b is controlled by endothelial cells or HE cells. It can be directly increased in Dnmt3b. For example, Dnmt3b mRNA expression can be increased in Dnm By delivering a transcript encoding t3b to cells, or by encoding Dnmt3b By introducing a transgene, or, limited to the introduction of episomes into cells... One or more nucleotide modifiers that do not increase or alter activity. By a transgene-free method that may have embellishments (or modifications of the encoded amino acids) This can be increased. In some embodiments, gene editing is performed to modify genes. It is used to introduce Dnmt3b into the Dnmt3b expression element in endothelial cells, for example, to promote It increases the strength of the ribosome, ribosome binding, and RNA stability, or RNA splicing. It affects the Dnmt3b gene. In some embodiments, gain-of-function mutations affect the Dnmt3b gene. It will be introduced to [location / platform].

[0044] In some embodiments, the present invention relates to the activity or expression of Gimap6 in endothelial cells. This can be used alone or with Dnmt3b and / during repeated straining or activation of piezo 1. This includes increasing the activity of Gimap6 in combination with other modified genes. Alternatively, to increase expression, the transcript of the mRNA encoding Gimap6 is introduced into the cells. It can be introduced, and a gene-free approach can also be used, to This includes, but is not limited to, the introduction of episomes into cells, or, instead, increasing activity. One or more nucleotide modifications (or coding) to cause or change something. Using a transgene encoding Gimap6, which may have the amino acid modifications described above. It is possible. In some embodiments, gene editing modifies genes in endothelial cells. Introduce into imap6 expression element (promoter strength, ribosome binding, RNA To increase stability or to influence RNA splicing, one or more It is used for purposes such as modifying the number. In some embodiments, gain-of-function mutations are used for Gi It is introduced into the map6 gene.

[0045] In some embodiments, mRNA and / or episomes (e.g., Dnmt3b) are used. or one or more hematopoiesis that code for Gimap6 or as described herein. Genes (which encode genes) can be synthesized, for example, by direct chemical synthesis or in vitro transcription. It is produced synthetically and introduced into endothelial cells. Known chemical modifications are used in the cells. This can avoid the innate immune response. For example, synthetic RNs containing only reference nucleotides. A can bind to pattern recognition receptors and trigger a strong immune response in cells. This can lead to translation blockade, secretion of inflammatory cytokines, and cell death. This can cause RNA containing certain non-standard nucleotides to be affected by the innate immune system. It can avoid detection and be translated into protein with high efficiency. In particular, it can avoid innate immune responses. With regard to nucleotide modifications for this purpose, the United States is incorporated herein by reference. See Specification No. 9,181,319. mRNA is obtained by known methods from HSC It can be introduced into cells once or periodically during production.

[0046] In some embodiments, Dnmt3b and / or Gimap6 and / or this The expression of one or more hematopoietic genes described in the specification is overexpressed to a desired level. Transgenes that can lead to (various promoter strengths, or other selections of expression regulatory elements) It is increased by introducing it into cells (with selection). The introduced gene is in this technology Introduction using various viral vectors or transfection reagents known to be used. It is possible to do so. In some embodiments, Dnmt3b and / or Gimap6 and / or hematopoietic gene expression by transgene-free methods (e.g., episome delivery) It increases due to ).

[0047] In some embodiments, gene expression or activity is controlled using gene editing techniques, for example. For example, changing promoter strength, ribosome binding, RNA stability, or RNA splicing. Adjustments are made to introduce one or more modifications to make changes. Various editing techniques are known. It is used in CRISPR, zinc finger (ZF) and transcription activator-like effectors. —(TALEN) is one example. One or more of these DNA-binding domains and F Using a fusion protein having an okl endonuclease cleavage domain, Double-strand breaks can be induced in desired regions of DNA (for example, these All of the above is incorporated herein by reference in U.S. Patent Application Publication No. 2012 / 0 Specifications No. 064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent 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 / 03010 Specification No. 73, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8, U.S. Patent Nos. 450, 471, U.S. Patent Nos. 8,440, 431, U.S. Patent Nos. 8,44 See Patent No. 0,432 and U.S. Patent Application Publication No. 2013 / 0122581. (Please refer to the following). In some embodiments, gene editing is known in the art. This is done using a CRISPR-coupled Cas system. For example, by reference the whole The following is incorporated herein: U.S. Patent No. 8,697,359, U.S. Patent No. 8,9 See U.S. Patent No. 06,616 and U.S. Patent No. 8,999,641.

[0048] In various embodiments, a population of cells (embryonic) containing developmentally flexible endothelial cells or HE cells A substance (including, but not limited to, a form) is introduced into the bioreactor. Some implementations In this context, the bioreactor is an internationally recognized instrument, which is incorporated herein by reference in its entirety. As stated in the brochure publication No. 2017 / 096215, It provides biomechanical extension due to strain. Biomechanical extension due to repeated strain is Dn This increases the activity or expression of mt3b and / or Gimap6, which then further enhances the present specification. The expression of endothelial genes described in this document is reduced, and the hematopoietic genes described in this specification are reduced. This increases the expression of [the function]. In these embodiments, mechanical means apply tensile force in 2D or 3D It is applied to cultured cells. For example, a computer-controlled vacuum pump system (e.g., F lexCell(TM) Tension System, Cytostretcher The system (or similar) uses nylon membranes, PDMS, etc., as the culture surface. It can be connected to a membrane or other biocompatible or biomimetic membrane. Then, using the system, Under specified and controlled repeated strain conditions, circumferential extension is ex vivo in 2D or It can be used to infuse cells into 3D culture.

[0049] In some embodiments, biomechanical stretching due to repeated strain is performed on endothelial cells and / Or it reduces the expression or activity of endothelial genes in HE cells, and endothelial cells and / or This increases the expression or activity of hematopoietic genes in HE cells, stimulating the formation of HSCs. .

[0050] In various embodiments, HSC conversion involves the activation of piezo1, mechanical extension, and mRNA Introduction, modification of Dnmt3b by mRNA and transgene, transgene-free Modification (e.g., by episomes), or introduction of gene modification, and / or Gi For map6, mRNA, modification by transgenes, and transgene-free (for example, E By at least one means selected from modification (by bisomes) or introduction of gene modification This is induced. In various embodiments, in endothelial cells or HE cells, according to this specification At least one hematopoietic gene described in [the text] is directly increased in expression or activity. , and / or at least one endothelial gene described herein is expressed or Activity is directly reduced.

[0051] Endothelial cells or HE cells are associated with blood disorders, bone marrow disorders, metabolic disorders, or immune disorders. It can be obtained from or derived from the subject. In some embodiments, the subject They do not have blood malignancies. The HSC population can be administered to recipients. The source cells for autologous HSC transplantation are iPS cells, endothelial cells and / or HE cells. It likely originates from the word "recipient."

[0052] In some embodiments, endothelial cells and / or HE cells are induced pluripotent stem cells (i PSCs), non-hematopoietic stem cells, or somatic cells including, but not limited to, fibroblasts and endothelial cells. Obtained from or derived from. In some embodiments, endothelial cells or HE cells. Cells are derived from HLA null cells, HLA modified cells, and / or transgene-free cells. or obtained from or derived from the genetic induction of HE cells from endothelial cells. Hematopoietic endothelial cells (e.g., Flkl+CD45+ cells, Flkl+CD41+ cells, or CD31+CD43+ cells are treated, for example, with allogeneic donor or HSCs. They can be obtained from elephant source cells in any manner. For example, HE cells can be obtained from autologous cells or Chemical induction, genetic induction, and gene-free induction from allogeneic cells to hematopoietic endothelial cells, Alternatively, it can be obtained by induction by episomes. In some embodiments, HE cells Cells can be obtained from recipient cells, or from HLA-modified cells, or from HLA-nucleated cells. It is derived from iPSCs created from cells that are a type of cell. In some embodiments, HE cells are used. Cells are obtained from or derived from the target cells of a pluripotent donor. Methods for preparing skin cells are known in the art, and these methods include: This includes generation from human pluripotent stem cells. The whole is incorporated herein by reference. International Publication No. 2017 / 096215 and U.S. Patent Application Publication No. 201 Please refer to specification number 9 / 0119643. Also, see Ditadi et al., Nature Cell Biol. 17(5) 580-591 (2015), Sugimura et al., Nature 2017; 545(7655):432-438, Nakajima -Takagi et al., Blood. 2013; 121(3):447-458, Zambidis et al., Blood. 2008 Nov 1; 112(9):3601-14, and Park et al, Cytometry A. 2013 Jan; 83(1): 114-126 (efficient A blood-endothelial differentiation method based on human embryoid bodies (hEBs) for hiPSC differentiation; Choi et al., Cell Rep. 2012 Sep 27; 2(3): 553-567 (hPSCs in co-culture with OP9) differentiation);Sandler et al, 2014 July 17; 511(17509):312-318(from endothelial cells to hematopoietic cells) See also Sluvkin, Blood 2013 122:4035-4046. In the application morphology, the number of HE cells required to initiate HSC production is at least approximately 10 2 pieces cells, about 10 3 Individual cells, about 10 4 Individual cells, about 10 5 Individual cells, about 10 6 Individual cells, about 10 7 pieces cells, or at least 10 8 It is an individual cell. In some embodiments, according to this disclosure The hematopoietic stem cells produced show excellent engraftment in the recipient, forming a functional multicellular system. Includes long-term hematopoietic stem cells (LT-HSCs) for reconstituting human blood. In some embodiments, HSCs include CD34+ cells.

[0053] In some embodiments, pluripotent stem cells are produced by reprogramming somatic cells. These are induced pluripotent stem cells (iPSCs) prepared using the following method. For example, somatic cells are Sox2, Oc Reprogramming selected from t3 / 4, c-Myc, Nanog, Lin28 and klf4 It can be reprogrammed by the expression of ramming factors. The programming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28. The reprogramming factors are Sox2, O These are ct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs include, for example, the US Japanese Patent No. 10,676,165, U.S. Patent No. 9,580,689, and These are described in U.S. Patent No. 9,376,664, and these are by reference. The whole is incorporated herein. In various embodiments, the reprogramming factor is Well-known viral vector systems, such as lentiviruses or Sendai virus cells. It is expressed using a stem. Alternatively, a reprogramming factor is a reprogramming factor It can be expressed by introducing the mRNA encoding it into somatic cells. Furthermore, iP SC expresses non-integrated episomal plasmids that express reprogramming factors, i.e. By introducing it, for the creation of transgene-free and virus-free iPSCs... It can be produced by [method]. Its replication ability is limited, and therefore it lasts for several cell generations. Known episomal plasmids that are lost may be used. In this context, iPSCs are cells such as fibroblasts or PBMCs (but are not limited to these). It is generated from cells. In various embodiments, iPSCs are autologous or identical to the recipient. They are heterogeneous (e.g., HLA-matched). In some embodiments, iPSCs are HLA-modified particles. These are cells or HLA null cells.

[0054] In various embodiments, the fabricated HSCs are propagated. For example, HSCs are used in the United States. U.S. Patent No. 8,168,428, U.S. Patent No. 9,028,811, U.S. Disclosed in U.S. Patent No. 10,272,110 and U.S. Patent No. 10,278,990. They may be propagated according to the methods described herein, and these may be propagated in their entirety by reference as described herein. It is incorporated into. For example, in some embodiments, ex vivo proliferation of HSCs is pros Taglandin E2 (PGE2) or a PGE2 derivative is used.

[0055] In various embodiments, HSC prepared by the method described herein A pharmaceutical composition for cell therapy is prepared, comprising a population and a pharmaceutically acceptable vehicle. The pharmaceutical composition contains at least about 10 2 one HSC, or at least about 10 3 H SC, or at least about 10 4 one HSC, or at least about 10 5 Individual HSCs, or at least about 10 6 one HSC, or at least about 10 7 Individual HSCs, or a few At least about 10 8 It may contain individual HSCs. In some embodiments, a subpopulation of cells (e.g.) For example, LT-HSCs can be isolated or concentrated using cell sorting approaches. In various embodiments, at least about 0.1% of the HSC in the composition, or at least Approximately 0.5%, or at least about 1%, or at least about 2%, or at least about 3% %, or at least about 5%, or at least about 10%, or at least about 20%, Or at least about 30%, or at least about 40%, or at least about 50% is L It is T-HSC. In various embodiments, the composition is about 2 to about 25% LT-HSC. It contains C, and in some embodiments it contains about 5% to about 25%. For example, some actual In the application method, approximately 100,000 to approximately 4 × 10 per kilogram of recipient body weight. 6 individual (CD34+)HSC (for example, approximately 2 × 10) 6 A pharmaceutical composition containing individual cells / kg is administered. In some embodiments, the pharmaceutical composition is at least about 10 3 individual, at least about 1 0 4 one, or at least about 10 5 Contains 1 LT-HSC cell.

[0056] HSCs for treatment or transplantation are, in some embodiments, administered for less than two months, or one month. Less than a month, or less than approximately two weeks, or less than approximately one week, or less than approximately six days, or approximately Generated over a relatively short period, such as less than 5 days, or less than approximately 4 days, or less than approximately 3 days. In some embodiments, Dnmt3b and / or Gimap6 can be used. Endothelial cells with increased activity or expression of [substance name] are cultured for 1 to 4 weeks.

[0057] Cell composition, a pharmaceutically acceptable carrier suitable for intravenous injection or other routes of administration or It may further contain a vehicle and may also contain a suitable cryoprotective substance. A typical carrier is DM The cell composition is SO (for example, about 10% DMSO). The cell composition is supplied in individual vials or bags. It may be stored frozen until use. In a particular embodiment, the volume of the composition This is approximately 1 fluid ounce to 1 pint.

[0058] HSC produced using the methods described herein can be, for example, administered intravenously. Alternatively, it is administered to the recipient via intra-bone marrow transplantation. This method is myeloablative. , non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) It can be performed after a pretreatment regimen.

[0059] The methods described herein are applicable, for example, to blood disorders (malignant and non-malignant), bone marrow. Used in transplantation protocols for treating diseases, metabolic disorders, and immune disorders. It can be used to generate a population of HSCs for some embodiment, HSC populations are autologous cells or pluripotent donor cells or HLA-modified cells or HLA-nucleated cells. It is derived from cells. In other words, the HSC population is prepared from the recipient cells. or donor cells (for example, pluripotent donor cells, HLA-compatible cells, HLA-modified cells, or HLA null cells) prepared from developmentally flexible endothelial cells or iPSCs. It is generated from E cells. In some embodiments, self-derived cells are used, and the The target diseases for Piento include multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, and acute myeloid leukemia. Neuroblastoma, germ cell tumor, autoimmune disorder (systemic lupus erythematosus (SLE), systemic Treatment using autologous HSC transplantation for sclerosis, myelodysplastic syndrome, amyloidosis, or other conditions. It has a state which is selected from other possible states. In some embodiments, it originates from itself. Cells that produce (for example, HSCs are generated from recipient target cells) are used, The patient does not have a blood malignancy.

[0060] In some embodiments, the recipient is acute myeloid leukemia, acute lymphoblastic disease Leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndrome, Multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, aplastic anemia, pure red blood cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia Severe combined immunodeficiency (SCID), Wiscott-Aldrich syndrome, hemophagocytic phosphorus Pa histiocytosis, congenital metabolic disorders, epidermolysis bullosa, severe congenital neutropenia, Schwachmann's disease. Diamond syndrome, Diamond-Blackfan anemia, Pearson syndrome, and leukemia It has a condition selected from bulbar adhesion disorders. Cells derived from other species or pluripotent donor cells or HLA-modified cells or HLA-null cells , used to generate HE cells. For example, HSCs are used in the donor subject, i.e., recipient It is generated from cells of a target other than the target. In some embodiments, the donor target is blood type and are matched to the recipient based on human leukocyte antigen (HLA) typing. .

[0061] Where used herein, the term "approximately" means ±10% of the number it is associated with. ru.

[0062] These and other embodiments of the present invention will be described below in non-limiting examples. [Examples]

[0063] During secondary hematopoiesis, the first set of HSCs develop hematopoietic endothelial cells in the AGM during fetal development. They arise from cells. Therefore, endothelial cells and / or hematopoietic endothelial cells are part of the AGM microenvironment. If the repertoire of endogenous and exogenous factors present is established, clinical use It can be a source for the development or proliferation of HSCs.

[0064] Once formed, AGM-derived HSCs migrate to the fetal liver and bone marrow, where they mature. It undergoes asymmetric division into late-stage (LT) and short-term (ST) HSCs. LT HSCs are asymmetric While preserving the HSC pool by causing further splitting, ST-HSCs are, Symmetric division supports the dynamic demand for blood production. Seven transcription factors (ERG, HOXA5, Induction of HOXA9, HOXA10, LCOR, RUNX1 and SPI1 (Sugimura, R, et al., Nature, 2017) and angiocline cytokines derived from the vascular niche ( FGRS (Fosb, Gfi1) with TGFβ, CXCR7, CXCR4, and BMP Induction of the transcription factors Runx1 and Spi1 (Lis, R. et al., Nature, 2017) is internal Enhances the conversion from endothelial cells to hematopoiesis. However, these approaches target endothelial cells or hematopoiesis It does not confer the function and characteristics of LT-HSC to endothelial cells. Furthermore, the multiplicity of these transcription factors Because it is associated with hematopoietic malignancies, when decontrolled, the embedded vector or Studies based on overexpression using a transgene-free approach show gene-to-quantity effects. This does not enable analysis of HLA. Furthermore, HSCs proliferated ex vivo are HLA-compatible. This does not eliminate the need to find a suitable healthy donor HSC. Therefore, long-term hematopoietic development To develop autologous or off-the-shelf reservoirs for LT-HSCs from hematopoietic endothelial cells Analyzing extracellular or non-integrated factors that contribute to the formation of novel LT-HSCs is important. It is essential.

[0065] The process of endothelial cell fate conversion to HSCs is accompanied by a progressive degeneration of the hematopoietic program, characterized by an initial loss of endothelial potential. During EHT, epigenetic mechanisms that confer long-term silencing of endothelial genes may exist. EZH1 actively suppresses the final hematopoietic program during the primitive hematopoietic period (Vo, LT, et al., Nature, 2018). On the other hand, ISWI chromatin remodeling controls both primitive and definitive hematopoiesis (Huang HT, et al., Nat. Cell. Biol., 2013). Furthermore, although Dnmt3b controls c-myb expression for HSPC maintenance (Gore AV, et al., Elife, 2016), the role of Dnmt3b in endothelial gene silencing or new LT-HSC formation is unknown. Which mechanisms can continuously alter the endothelial epigenetic landscape to support the formation of LT-HSCs is unknown. As disclosed herein, the present disclosure demonstrates how biomechanical stretch

[0066] mediated by heartbeat and / or pulsation and / or pharmacological activation of the Piezo1 mechanosensitive pathway affects the expression of core genes and erases the endothelial epigenetic landscape for the formation of HSCs (including LT-HSCs). Furthermore, a bioreactor that mimics a pulsatile state was developed, and Piezo1 was identified as a pharmacological target to stimulate LT-HSC formation and scale up. Pulsations mediated by heartbeat stimulate the conversion of endothelial cells to HSCs.

[0067] Heartbeat-mediated pulsation stimulates the conversion of endothelial cells to HSCs. Screening of unbiased zebrafish ethylnitrosourea (ENU) mutagenesis The zebrafish mutant of cadherin-5 (cdh5, ve-cdh) malbec(bw209 mlb ) were obtained. Malbec embryos and CDH5-mol Phantom (MO) embryos exhibit normal primary and secondary hematopoiesis despite circulatory failure. .

[0068] Biomechanical forces independent of blood flow and shear stress stimulate the conversion of endothelium to hemostatic cells (HSCs). To identify the cause, we analyzed the cardiac function, anatomy, and vascularity of CDH5-deficient embryos. Ta.

[0069] Microangiography involves injecting fluorescent dextran beads into the atria of a two-chambered heart in a zebrafish embryo. First, the procedure was performed by irradiation, followed by tracking with dextran beads in the circulation. In control embryos, Kistran beads enter the systemic circulation via the atrioventricular (AV) valve and ventricle, but These beads were captured in the atria of cdh5-morphant embryos.

[0070] To study the structure of the heart, the heart was examined in control embryos and in embryos where CDH5 was silenced. The cells were isolated, and immunohistochemistry was performed on the intima (GFP) and cardiomyocytes (MF20). The atria (A), atrioventricular (AV) valves, ventricles (V), and outflow tract (OT) were all affected by 5-mol of cdh. While the phantom valve was formed, the AV valve was found to be elongated and distorted.

[0071] To investigate why the circulation of cdh5 weakened in embryos where cdh5 was silenced, c Vascular structure, as well as blood circulation, heart rate, and cardiac output in embryos where dh5 was silenced. The volume and cardiac tamponade were analyzed.

[0072] Endometrial integrity was analyzed in mlb×kdr:dsRED embryos. Arterial and venous... The structure was found to be intact in cdh5-deficient embryos.

[0073] The temporal development of the heart, heartbeat, blood vessels, blood circulation, and HSC formation are observed in zebrafish. It is conserved in mice and humans. During zebrafish development, the heart receives The sperm begins to pulsate approximately 23 hours after birth (hpf), and blood circulation begins at around 24-26 hpf. Therefore, definitive HSCs are identified from hematopoietic endothelial cells in the AGM region between 30 and 48 hpf. To occur.

[0074] To analyze intravascular circulation before and after the heart begins to beat, control embryos and CDH5 were used. Slow-speed cofocal imaging of silenced lcr:eGFP×flk1:mCherry embryos Point imaging was performed.

[0075] Even after the heart has started beating, lcr:eGFP + Red blood cells, CDH5, silensin It was found to have accumulated in the blood vessels of the embryo after it had started beating. Despite the formation of a morphan, active circulation is absent in the cdh5-morphant. This demonstrates that...

[0076] To investigate cardiac function in embryos where CDH5 has been silenced, electrophysiological... Evaluation and cardiac ultrasound were performed. Heart rates in cdh5-MO embryos were comparable to those of controls. Although it was present, stroke volume was almost nonexistent in the cdh5-MO embryo. Therefore, cardiac output (= stroke volume × heart rate) is decreased in cdh5-MO embryos. This was confirmed.

[0077] cdh5-MO embryos had pericardial fluid retention in the cardiac cavity that could be caused by the backflow of blood from the heart and was present. The accumulation of body fluid in the pericardial cavity reduced ventricular filling, and subsequently, the hemodynamics deteriorated . To investigate whether cardiac tamponade was a factor in the accumulation of body fluid in the pericardial cavity, the cardiac cavity of cdh5-MO embryos was punctured like pericardiocentesis, and then the pericardial fluid was aspirated to reduce the fluid pressure applied to the heart. However, the lack of cardiac output in cdh5-morphant hearts could not be restored .

[0078] Heartbeat was normal in cdh5-morphants, but the cardiac output of cdh5-morphants was impaired due to structural defects of the heart, and as a result, blood accumulated in the pericardial cavity . Since cdh5-MO embryos have normal hematopoiesis, it was hypothesized that the biomechanical forces derived from heartbeat affect HSC formation in the absence of active circulation [[ID=2१]] .

[0079] cdh5-MO embryos have a beating heart but no active circulation, yet cdh5-MO embryos have HSC formation in the aortic endothelium of their blood vessels. When the AGM of control zebrafish embryos was enlarged, different pulsations of the blood vessels were confirmed . To distinguish the presence of pulsations within the blood vessels, which are independent of circulating blood cells, and what is presumably blood flow, the pulsation frequency of the blood vessels was compared with the pulsation frequency of circulating blood cells and the movement caused by blood flow . Specifically, low-speed imaging confocal imaging of a double transgenic system with circulating lcr:eGFP in blood vessels and red blood cells, and of both blood vessels and specifically, flk1:mCherry + circulating lcr:eGFP + in blood vessels with red blood cells was used, as well as of both blood vessels and ​Fourier analysis was performed on signals from circulating blood cells. The frequency spectrum of blood vessels was analyzed separately. It was found to have a peak. Therefore, intravascular pulsation and blood flow coexist, Their existence and properties are independent of each other.

[0080] To investigate the temporal, spatial, and functional presence of pulsation in AGM at 36 hpf Furthermore, light sheet microscopy of the vascular region in control zebrafish embryos and subsequent ho... A sievert analysis was performed. The data showed that AGM was flk1:eGFP. + r produced from endothelial cells unx1:mCherry + Endothelial cells observed in low-speed confocal imaging with HSPC At 36 hpf, the time and location for the conversion of cells to hematopoietic cells, there is a distinct pulsation. This further supports the existence of frequency. In summary, the AGM region is pulsating. It was discovered that pulsation in AGM occurs simultaneously with the conversion of endothelial cells to hematopoietic cells.

[0081] Blood vessels generate circumferential wall stress and endothelial shear stress, and heartbeats contribute to blood pressure. and is under constant mechanical load from blood flow. Blood flow imparts shear stress to endothelial cells and blood vessels. It induces dilation, but the pulsation mediated by the heartbeat is circular for both endothelial cells and smooth muscle cells. This causes circumferential stretching and mechanical expansion.

[0082] HSC-derived cdh5-MO embryos exhibit blood flow-mediated and shear stress-mediated NOS activation. To analyze whether it is mediated or dependent on it, HSPC expression was subjected to NOS inhibition. Analysis was performed on control embryos and cdh5-MO embryos treated with the agent L-NAME. S inhibition weakens HSPC formation in control embryos, but in cdh5-MO embryos, HSP It was demonstrated that it does not affect C formation. Therefore, cdh5-MO embryos derived from HSCs. This does not depend on NOS activation.

[0083] In summary, pulsations mediated by heartbeats stimulate the formation of HSCs from the endothelium, which is independent of circulation. To become agitated.

[0084] Stretching activates piezo 1 for HSC formation. Biomechanical forces stimulate changes in cell shape and fate, thus influencing the pulsation of hematopoietic endothelial cells. We hypothesized that intervening periodic stretching stimulates HSC formation.

[0085] To test the function of pulsation in HSC formation from endothelium, E11.5 mouse embryos We developed a bioreactor that can repeatedly apply strain to AGM cells collected from the cells. Figure 2A, top panel). The assay for analyzing hematopoietic colony formation and flow is performed using a 10% repeat cycle. Rebound strain enhances the formation of pluripotent hematopoietic progenitor cells, and this formation is linked to stretch-activated receptors (S We demonstrated that AR is attenuated by all GdCl3-mediated pharmacological inhibition. GdCl3 also increases HSPC expression in zebrafish embryos, and in sih-MO embryos. I weakened it to the bell level.

[0086] SAR Family Members have four subcategories: K1-Family Member, It also has piezo channels, TRP channels, and DEG / ENaC channels. Tissue expression and computer analysis revealed piezo1 and T in endothelial and hematopoietic tissues. We demonstrated rpv4 and therefore investigated their roles in the conversion from endothelium to HSCs. .

[0087] Loss of function analysis and pharmacological inhibition of trpv4 and piezo1 are used to analyze HSPC markers. Expression and conversion from endothelium to HSCs were eliminated (Figure 1A). Conversely, trpv4 or pie were eliminated. Pharmacological activation of Zo1 enhances the expression of HSPC markers in control embryos, and in sih embryos. HSPC expression was restored in [location]. In temporal and spatial analyses, trpv4 was 36 hpf was not detected in the AGM region of zebrafish embryos, while piezo1 was detected. In E11.5 AGM, it co-localizes with Cd31 (endothelium) and c-Kit (hematopoiesis). He was.

[0088] To confirm the molecular mechanism underlying stretch-mediated HSC formation, repeated straining All transcendences of AGM treated with either a piezo-1 pharmacological activator Lipome analysis was performed. Repeated strain and activation of piezo1 were found to be similar gene signatures. It was found that this caused char (Figure 1B).

[0089] Pharmacological activation of piezo1 further enhanced the formation of pluripotent hematopoietic progenitor cells (Figure 1). C) On the other hand, pharmacological inhibition of piezo1 is associated with repeated strain-mediated HSPC formation. The induction was weakened (Figure 1D). Overall, biomechanical extension mediated by repeated strain is Activating Ezo1 stimulates the conversion of endothelium to HSCs.

[0090] Similar results were obtained with the piezo agonists Yoda1, Jedi1, and Jedi2. In particular, as shown in Figure 1E, 50 μM Jedi1 and 50 μM Jedi2 were obtained. Alternatively, hematopoietic C in E11.5 AGM cells treated with 25 μM Yoda1. The FU assay determines whether Jedi1, Jedi2, or Yoda1-mediated piezo1 activation occurs. We demonstrated that it enhances MM formation.

[0091] Biomechanical stretching or activation of piezo1 produces LT-HSCs. Repeated strain or activation of piezo 1 produces long-term self-regenerating HSCs (LT-HSCs). To analyze whether this occurs, a serial transplant assay was performed. Repeated strain or piezo 1 Primary grafts of AGM treated with an activator exhibit higher engraftment and normal multicellularity. The reconstruction is shown (Figures 2A and 2B). Also, repeated strain or the activation of piezo 1 The bone marrow of primary recipients who received AGM treated with a graft agent was two to three times larger in volume. Lin - Sca1 + c-Kit + Cd48 - Cd150 + HSC (i.e., LT-H SC) was shown. Selected L from primary recipient to immunocompromised secondary recipients. in - Sca1 + c-Kit + HSPC transplantation also results in higher engraftment and normal multicellularity. This resulted in a system reconstruction (Figures 2C, 2D). Therefore, repeated strain and / or It is expected that both activation of piezo1 will produce a larger amount of normal LT-HSCs. To test this hypothesis, a limiting dilution assay was performed using stepwise amounts of Lin - Sca1 + c-Kit + This was performed by transplanting HSPCs into immunodeficient tertiary recipients. Analysis of tertiary transplants showed that repeated straining produced 2 to 3 times more LT-HSCs. This was demonstrated.

[0092] To determine whether AGM-HSC (donor) engrafts and reconstitutes into normal adult blood, The molecular and functional characteristics of the reconstructed blood system are then subjected to control, repeated strain, or In primary recipients who have received an AGM treated with a piezo 1 activator, Analysis was performed. Analysis of donor-derived erythroid cells in the bone marrow was performed in the presence of Bcl11a. Cd71 + / Ter119 + Expression of, and adult globinmer using embryonic globin. Enhanced Kerr expression was observed (Figure 3A). Donor-derived bone marrow cells in bone marrow and serum. Further analysis of a sufficient amount of Gr1 + / Mac1 + Bone marrow cells, and their myelope Luoxidase (MPO) production was shown (Figure 3B). Next, lymph nodes, thymus, and spleen In this context, donor-derived chimerism, Mac1 + Bone marrow cells, Cd19 + B cells, and C d4 + / Cd8 + T cell analysis shows that progenitor cells derived from donor HSCs circulate and enter the hematopoietic niche. We demonstrated that colonies can be formed and adult blood systems can be reconstructed using blood derived from primary transplants. Qing's analysis revealed that these are IgG1, IgG2a, IgG2b, IgA, and IgM. It was also found that the body produced a normal repertoire of pre-immunization immunoglobulins (Ig). (Figure 3C). Spleen donor-derived Cd3 + T cell selection is performed using Mac1 cells derived from a spleen donor. + bone We demonstrated the rearrangement of T cell receptor β (TCRβ), which is absent in myeloid cells (negative control) (Figure). (3D). To analyze the functional characteristics of T cells in primary transplants, delayed-type hypersensitivity reactions were observed. Sei sensitizes the primary transplant with sheep red blood cell injections, thereby achieving antigen specificity in the soles of the feet. This demonstrated the successful recruitment of functional T cells (Figure 3E). Therefore, AGM or hematopoiesis Repeated straining or piezo-1 activation of endothelial cells produces HSCs, and these HSCs are formed The transplanted cells engrafted in the blood niche, reconstructing functional multicellular adult blood cells.

[0093] Biomechanical stretching and activation of piezo1 are necessary for the conversion of endothelium to HSCs using Dnmt3. Adjust b upwards. Since AGM is a heterogeneous tissue, how does the activation of piezo 1, which is mediated by stretching, affect HSC? It was unclear whether this stimulated a change in the fate of aortic endothelial cells. E10.5 AGM selection type Differential gene expression signatures were obtained from endothelial cells, hematopoietic endothelial cells, and HSCs. In the context of endothelial cells derived from AGM, hematopoietic endothelial cells, and HSCs, the status of AGM Hierarchical classes of gene signatures induced by repeated strain or piezo1 activation Tarling further explores overexpressed biological processes, molecular pathways, and gene expression clusters. This document provides a quantitative overview of these gene ontology (GO) terms. Upward-regulated, repeated stretching and / or piezo 1 activation during the transition from skin to HSC. Venn diagram analysis of genes involved in sexualization revealed that Dnmt3b is involved in the endothelial mechanism necessary for HSC formation. This was identified as a potential candidate mechanism involved in the silencing of (Figure 4). In addition, G imap6 is also a potential candidate involved in silencing the endothelial mechanism necessary for HSC formation. The mechanism was identified.

[0094] To validate bioinformatics and computer analysis, E11.5 A Temporal and spatial protein expression of Dnmt3b in GM was analyzed. Immunohistochemistry The scientific assay showed that Dnmt3b was Cd31 + Endothelium and c-Kit + Co-localizes with hematopoietic cells This demonstrated that Dnmt3b can stimulate the conversion of endothelium to HSCs. It was assumed.

[0095] Dnmt3b and Dnmt3a are highly homologous and play a role in the maintenance or differentiation of HSCs. Although they have functions different from others, these latent processes in the conversion of endothelium to HSCs in AGM Its role was unknown. Analysis of gene signature and tissue expression in AGM All involvement of Dnmt3a in HSC formation was ruled out. Dnmt3b and Dnm To distinguish the distinct or overlapping hematopoietic roles of t3a, Dnmt3b and Dn The level of mt3a protein was measured in AGMs treated with repeated strain treatment or Yoda1 treatment. Analysis in the nuclear fraction of cells revealed that repeated strain or activation of piezo1 is E1 1.5 Stimulating the expression of Dnmt3b protein in AGM cells, Dnmt3a It was revealed that it does not cause agitation (Figure 5A).

[0096] Vascular pulsation in the absence of blood flow stimulates HSC formation via Dnmt3b activation. To analyze whether this was the case, the expression of HSPC markers was analyzed using a Dnmt3b inhibitor. Measurements were taken in cdh5-MO embryos treated with naomycin. Pharmacological results of Dnmt3b Inhibition reduced the expression of HSPC markers in control embryos and cdh5-MO embryos.

[0097] Next, the experiment in this embodiment showed that biomechanical extension or activation of piezo 1 was Dnmt3b We analyzed whether it stimulated the conversion of endothelial cells to hematopoietic cells through the activation of Dnm. Inhibition of T3b is mediated by biomechanical extension or piezo 1 in pluripotent hematopoietic progenitor cell formation. The activation of [the cell name] mediated induction (Figure 5B), and the conversion of endothelial cells to hematopoietic cells (Figure 5B) It was found to have weakened. Nanaomycin treatment returns hematopoietic cells to phenotypic endothelial cells, but this Endothelial cells like these were not functional. Whole-mount HSPC markers in sit u Hybridization, and treatment with Yoda1 or Yoda1 Treatment without accompanying, treatment with nanaomycin, or injection with dnmt3b-MO Slow-speed imaging of the transition from endothelium to HSCs in zebrafish embryos is further Furthermore, inhibition or loss of dnmt3b weakens the increase in piezo1 activation-mediated HSC formation. This was confirmed (Figure 5C). In summary, the activation of piezo 1 mediated by pulsation is AG We enhanced Dnmt3b expression in M ​​to stimulate the conversion of endothelial cells to hemoglobin cells (HSCs).

[0098] To determine the role of Dnmt3b in EHT, the inventors used the whole transcript Endothelial and hematopoietic gene expression levels were analyzed using 2D tome analysis. The inventors used 2D Repeated stretching or Yoda1 treatment of AGM samples results in endothelial genes (Vegfa, Apl Expression of n, Hey2, Gpr116, Bcl6, Gna13, Cdh5, Plxnd1) It reduces hematopoietic genes (Sca1, Tal1, Flt3, Spi1, Gata2, Ce We found that it increases the expression of bpa (Figure 6A). To further strengthen our findings... Therefore, the inventors further independently measured the transcription levels of endothelial and hematopoietic genes. They found that repeated strain-induced—or piezo1 activation-mediated—Dnmt3b overexpression is Both involve endothelial gene silencing during EHT (Vegfa, Hey2, Gpr116) , Gna13) and higher expression of hematopoietic genes (Runx1, Spi1, Cebpa, We found that this resulted in Tal1, Gfi1) (Figure 6B). In summary, pulsation-mediated Piezo1 activation enhances Dnmt3b expression, repressing endothelial genes and releasing HSCs from the endothelium. This stimulates the conversion to the piezo1-mediated mechanosensitivity mechanism in human hematopoiesis. To analyze the preserved roles, the inventors constructed RUNX1c:tdTomat Using the directional differentiation of human induced pluripotent stem cells (iPSCs) into hematopoietic endothelial cells, Eel hematopoietic endothelial cells were treated with Yoda1. The inventors of the present invention have shown that Yoda1-mediated We have discovered that piezo1 activation stimulates the conversion of human endothelium to hematopoiesis. Furthermore, Yoda1-mediated piezo1 activation enhanced DNMT3B expression, but DN MT3A expression was not enhanced, but endothelial genes (VEGFA, HEY2, GPR116, GNA) were not. 13. Silence the hematopoietic genes (CDH5, PLXND1) and RUNX1, SPI 1. Induces the expression of CEBPA, TAL1, and GATA2; thereby promoting pluripotent hematopoietic precancerous growth. We found that this leads to the formation of progenitor cells and an increase in human hematopoiesis (Figure 6C). Therefore, Pulsation-mediated piezo1 activation is observed in zebrafish, mouse, and human model systems. It stimulates the conversion from endothelium to hematopoiesis in the endothelium.

[0099] Furthermore, as shown in Figure 6D, Yoda1-mediated pharmacological activation of piezo1 is engraftable. It enhances the formation of functional human CD34+ cells, and such piezo-1 pharmacological activation is multifacial. It stimulates the formation of human CD34+ hematopoietic cells that reconstitute cytoplasmic blood. Furthermore, it is a piezo-1 drug. Physical activation leads to the formation of self-regenerating LT-HSCs that reconstitute multicellular blood cells during serial transplantation. Enhance. See Figure 6E.

[0100] Repeated extension and piezo agonist are used for conversion from EC to HSC, for example from EC to It promotes conversion to HE cells and conversion from HE cells to HSCs. Figure 7A (top) shows Mau S E11.5 AGM Selection EC (CD31 + ), HEC (CD31 + cKit + ) and HSPC(cKit + ) Perform FACS analysis after repeated 10% spread on the cells. The test is shown. As shown in Figure 7A (bottom), repeated stretching is converted from EC to HE cells. (Left) Promoting the transition from HEC to HSPC (center). The impact on HSPC is Shown on the right.

[0101] Figure 7B shows FACS analysis of human germ cells (EBs) on day 8 of hematopoietic differentiation. This is because Yoda1-mediated piezo1 activation is present in the control, while hCD43 neg C D235 neg CD144 + CD34 + It enhances HE cell formation, but piezo1 - / - P This indicates that enhancement does not occur in SCs. Yoda1-mediated piezoelectric 1 activation occurs in hPSCs. This enhances HSC formation. Figure 7C shows the use of Yoda1-mediated piezoelectric 1 activation. This shows a twofold improvement in the number of HSCs produced from hPSCs.

[0102] Production of HSCs from HE cells generated from human iPSCs Differentiation of embryoid bodies and hematopoietic endothelial cells (Sugimura et al. 2017, Ditadi et al. 2015) This was done as described in ( ). In short, the hiPSC colony was set to 0.0 Dissociate with 5% trypsin for 5 minutes at 37°C, wash with PBS + 2% FBS, and then L- Glutamine (2 mM), penicillin / streptomycin (10 ng / ml), ascorbic acid Vinic acid (1 mM), human holo-transferrin (150 μg / ml, Sigma T0) 665), Monothioglycerol (MTG, 0.4 mM), BMP4 (10 ng / ml) , and StemPro-34 (Invitro) with Y-27632 (10 μM) added. The cells were resuspended in gen (10639-011). 5 million cells were placed in a 10 cm dish. Seeds were sown in Ezsphere (Asahi Glass Co., Ltd.) to form spheroids. 1 On day [number], bFGF (5 ng / ml) and BMP4 (10 ng / ml) were added to the culture medium. On the second day, the culture medium was changed to SB431542 (6 μM), CHIR99021 (3 μM), b StemPro supplemented with FGF (5 ng / ml) and BMP4 (10 ng / ml) The medium was changed to -34. On the third day, the culture medium was changed to VEGF (15 ng / ml) and bFGF (1 The medium was replaced with StemPro-34 with 0 ng / ml added. On day 6, the medium was changed to bF GF (5 ng / ml), VEGF (15 ng / ml), Interleukin (IL)-6 ( 10ng / ml), IGF-1 (25ng / ml), IL-11 (5ng / ml), SC Replace with StemPro-34 supplemented with F (50 ng / ml) and EPO (2 IU). The cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. All cytokines were purchased from Peprotech.

[0103] CD34 + To isolate the cells, the embryoid body (obtained on day 8) was treated with 0.05% trypsin. It then dissociates, is filtered through a 70μm strainer, and is CD34 + Cells are bisected by CD34 magnetic They were isolated by cellulose staining and then passed through an LS column (Miltenyi). All bacteria The samples were tested by FACS, and their purity was verified by a panel. The following antibodies were used: Using: CD34-PEcy7 (Clone 581, Biolegend), FL K1-PE (CLONE #89106, BD), and 4',6-diamidino-2-fe Nilindole (DAPI).

[0104] Isolated CD34 + Cells were treated with Y-27632 (10 μM) and TPO (30 ng / ml). ), IL-3 (10ng / ml), SCF (50ng / ml), IL-6 (10ng / m l), IL-11 (5ng / ml), IGF-1 (25ng / ml), VEGF (5ng ( / ml), bFGF (5ng / ml), BMP4 (10ng / ml), and FLT3 ( (Ferrel et al 2) resuspended in StemPro-34 medium containing 10 ng / ml 015). The cells were coated with a thin layer of Matrigel at a cell density of 50,000 cells per well. Seeds were sown in a 4-well plate. One day after sowing, Yoda1 (6.25 to 100 μM) was used. The substance was added to the culture medium. After 7 days, the suspension cells were collected and FACS analysis was performed. In the analysis, cells were treated with CD34-PEcy7 (Clone 581, Biolegend) All were stained with CD45-APC (clone 2D1, Biolegend). I purchased the tokine from Peprotech.

[0105] conclusion The development, proliferation, and maintenance of long-term HSCs are highly significant in stem cell biology and hematopoiesis. This was what was needed. Confocal, light-sea low-speed photography in zebrafish. Based on the analysis of the scalable violins and Fourier transform, scalable violins stimulate pulsations within blood vessels. Not only was the actor established, but the activation of piezo1 transformed endothelial cells into LT-HSCs. It was identified as a pharmacological target for replacement. This study aims to determine if it can engraft during serial transplantation. To develop LT-HSCs that can self-regenerate and be reconstituted into functional adult blood cells with a multicellular system. This provides a novel, gene-free approach to achieve this.

[0106] Pulsations mediated by heartbeats cause circumferential stretching in both endothelial cells and smooth muscle cells. This caused mechanical expansion. However, piezo 1 in E11.5 AGM It is co-expressed between endothelial cells and hematopoietic cells, but not in vascular smooth muscle cells. This suggests that the hematopoietic role of biomechanical stretching and piezo1 activation is related to AGM-endothelium. This suggested that the findings were specific to each cell.

[0107] Biomechanical stretching of blood vessels involves piezo1, Trpv4, K1-family members, and The DEG / ENaC channel was activated. Activation of both piezo1 and Trpv4 was observed. This stimulated the conversion of endothelial cells to hematopoietic cells. However, only piezo1 inhibition was mediated by the stretching process. This weakens the hematopoietic effect, and this means that piezo1 and Trpv4 play a partially superfluous role. This suggested that it was possible.

[0108] Activation of Dmnt3b is linked to the endothelial mechanism, which enhances the self-regeneration capacity of HSCs and multicellular regeneration. The conferral of structural ability was eliminated. Inhibition of Dnmt3b reverts hematopoietic cells back to phenotypic endothelial cells, These cells lacked functional endothelial characteristics. This indicates that the transfer from endothelial cells to hematopoietic cells is not possible. This suggests that the temporal and spatial roles of Dnmt3b in the transition were irreversible. Biomechanical stretching or activation of piezo1 does not affect Dnmt3a expression. This enhanced the temporal and spatial expression of Dnmt3b. The data showed the occurrence and fractional The distinction between the hematopoietic role of Dnmt3b and the leukemic role of Dnmt3a during the evolutionary process. It was proven.

[0109] The findings disclosed herein relate to how biomechanical forces alter the fate of cells. By stimulating and triggering epigenetic mechanisms, stem cells gain the ability to regenerate themselves. This study demonstrates that it can be conferred. This research also examines pluripotent stem cells (PSCs) or donor cells. A platform for inducing LT-HSCs from endothelial cells or hematopoietic endothelial cells derived from cells. The form is provided. The objective is to develop a universally compatible HSC, but in this specification The disclosed bio-inspired bioreactors are free of universally adaptable transgenes. - Source cells have benign and malignant hematological disorders, metabolic disorders, immune disorders, and bone marrow disorders. This provides a foothold that can be used to treat patients.

[0110] material and method All procedures were performed in animal experiments at Brigham and Women's Hospital and Boston Children's Hospital. It was approved by the committee.

[0111] The mice Cd45.2 (C57BL6 / J) and Cd45.1 (SJL) are Jackson. The zebrafish morpholino was purchased from the research institute, and the zebrafish morpholino was purchased from GeneTools. Microangiography involves injecting a fluorescently labeled dextran dye into the atria of a zebrafish heart. This was done by recording the passage using live imaging. Zebraf Immunostaining of AGM in fish hearts and mice was analyzed using an inverted fluorescence microscope. The cardiac tamponade, heart rate, and pulsation frequency in zebrafish embryos were clearly observed. Zebrafish were analyzed using field imaging or slow-motion confocal microscopy. The movement of red blood cells within blood vessels and the conversion of endothelium to HSCs in transgenic embryos, Analysis was performed using slow-speed confocal imaging.

[0112] The pulsating vascular condition was detected in vitro using the Flexcell FX-4000 device. I was excited. To analyze the role of pharmacological targets in regulating the conversion from endothelium to HSCs, AGM or whole mouse embryos derived from mouse embryos are subjected to biomechanical stretching, chemicals, or drugs. They were exposed ex vivo. Next, a hematopoietic colony formation assay was performed using mouse AGM. This was performed by incubating the cells in StemCell M3434 medium for 7 days. In SJL mice irradiated with a lethal dose, serial transplantation of AGM-derived HSCs was performed. The frequency of stem cells during biomechanical extension was analyzed using a limiting dilution assay. Primary transplantation To characterize the properties of AGM-HSC-derived blood cells in the body, chimerism and restructuring The percentage of the composition was measured using FACS, and the globin transcript was quantitatively reverse transcribed. The amount of myeloperoxidase was analyzed using enzyme PCR, and PicoKine EL The ISA kit was used to measure TCR-β rearrangement at the TCR-β locus. Analysis using PCR to detect pre-immune IG, Thermo-Fisher Mous The Ig Isotyping kit was used for analysis, and the delayed-type hypersensitivity reaction was detected. The feet of mice before sensitization with diRBC (Rockland Immunochemicals) The analysis was performed by injecting the substance into the sole.

[0113] RNA sequencing analysis is performed, followed by repeated straining or treatment with pharmacological modifiers. Gene expression patterns were measured in mouse AGM. A computer algorithm was used. Using this method, hierarchical clustering of differentially expressed genes is performed, and these genes that appear excessively are identified. Physical processes and pathways were measured. Gene expression clusters of differentially expressed genes were analyzed. Next, we compared the average expression levels of these genes across different cell populations. Then, we compared the up-gene and down-gene expression levels. Construct a Venn diagram comparison of genes to show the endothelium mediated by repeated strain or pharmacological regulators. We analyzed key candidates for the conversion from HSC to Dnmt3b protein and D The expression of nmt3a protein was controlled in the nuclear fraction of mouse AGM cells using EqiQuick. Analysis was performed using an assay kit. Unless otherwise stated, data are mean ± standard deviation. It is expressed as follows. Statistical analysis is performed using paired or unpaired Student's t-tests. Therefore, the study was conducted. Significance was set at P<0.05.

[0114] animal In the experiment, wild-type AB, Casper, and transgenic zebrafish strains were used. Synthetic lcr:eGFP, flk1:mCherry, flk1:eGFP, cd41:eG FP was used. Embryos were used up to 4 days after fertilization. The experiment was conducted at Jackson Laboratory C Using d45.2(C57BL6 / J) mice and Cd45.1(SJL) mice .

[0115] Morpholino Obtain a morpholino antisense oligo (Gene Tools, the following sequence), It was injected into cell-stage Casper zebrafish embryos. The injected embryos and the injected... The embryos were incubated in E3 medium at 28°C until fixation was performed.

[0116] [Table 1]

[0117] Chemical treatment of embryos Zebrafish embryos were treated with the following chemical modifiers in E3 fish medium:1 00µM L-NAME (Fisher Scientific), 50µM digitox Sigenin (Sigma), 25-50 μM Yoda1 (Cayman Chemica l) 1 μM nanaomycin (Nana, Fisher Scientific), 1 00 μM gadolinium chloride (GdCl3, Sigma), 5-10 μM 4α-phorb 12,13-Didecanate (4Apdd, Sigma), or GSK205 (10 uM).

[0118] Microangiography Fluorescently labeled dextran beads were injected into the atria of control embryos and cdh5-MO embryos. Real-time bright-field video using a Nikkon SMZ1500 stereomicroscope Captured.

[0119] Heart rate and cardiac output Images of a live zebrafish heart were taken using a built-in incandescent light with a 5x objective lens. Axioplan (Zeiss) upright microscope with lenses, and a 512×480 pixel microscope. FastCam-PCI high-speed digital camera with a grayscale image sensor The images were obtained using a Photron camera. Images were obtained at 250 frames per second, and 10 88 frames (8 cardiac cycles) were obtained for each condition using custom software. Heart rate was determined from a series of image files (implemented in MATLAB). The long and short axes of the chamber are defined in ImageJ for both the diastolic and systolic phases of each video. The cardiac chamber volume is measured manually using [a specific tool / method] and then calculated using standard geometric assumptions. It was estimated that for at least 10 embryos per dose of morpholino, cardiac output was measured during diastolic period. It was measured by subtracting the systolic ventricular volume from the total ventricular volume and multiplying the result by the heart rate. (Shin et al., 2010).

[0120] Analysis of periodicity Zebrafish Casper embryos with 0.8% low tricaine (Sigma) It was embedded in melting point agarose and placed in a Petri dish. Next, NIS Elements (Co., Ltd.) Using a Nikon SMZ1500 stereo microscope equipped with Nikon software, Real-time bright-field video of pulsating blood vessels in the GM region was captured. The video was used... The pulsation frequency within the blood vessels was then quantified.

[0121] Bright-field live imaging To perform bright-field live imaging, zebrafish Casper embryos were used. The substance was embedded in 0.8% low-melting-point agarose containing lycaine (Sigma) and placed in a Petri dish. Nikon SM with NIS Elements (Nikon Corporation) software Capture real-time brightfield video and still images using the Z1500 stereo microscope. did.

[0122] Confocal microscopy cd41:eGFP was crossed with flk1:mCherry zebrafish, and flk1 :mCherry was crossed with lcr:eGFP zebrafish, and Morpholino was produced from these The transgenic embryo was injected. The transgenic embryo was placed on low-melting-point agarose. Using a spinning disk confocal microscope, flk1 30 to 42 hpf + From the endodermis The resulting cd41:eGFP + We performed slow-speed confocal imaging of HSCs. eGFP + The relative movement of red blood cells, flk1:mCherry + The analysis was based on the condition of the endothelium. The present inventors perform image analysis using Imaris (Bitplane) software. .

[0123] Hall mount in situ hybridization Hall mount in situ hybridization, as previously described. I went.

[0124] Heart tamponade The pericardium is punctured using a microinjection needle, and 48hpf of CDH5-MO is administered. The injected zebrafish embryos released the fluid that makes up the area around their hearts.

[0125] immunostaining E10.5 chimeric mouse embryos were collected, embedded in paraffin blocks, and cross-sections were prepared. And the primary antibodies are piezo1 (rabbit anti-mouse IgG, Abcam), Cd31 (donkey Anti-mouse IgG, R&D Systems), c-Kit (Rabbit anti-mouse IgG, R& D Systems), or Dnmt3b (donkey anti-mouse IgG, Abcam), and The 4,6-diamidino-2-phenylindole (DAPI) antibody, as well as the secondary antibody, Alexa Fluor 488 (Donkey Anti-Rabbit IgG, Fisher Scient ific) and Alexa Fluor 647 (donkey anti-goat IgG, Abcam) Immunostaining was performed to detect the expression of these molecules in the E10.5 AGM region.

[0126] flk1 (GFP), mf2 (mCherry), and DAPI (violet) Expression was isolated from control embryos and zebrafish embryos in which cdh5-MO was silenced. Measurements were taken in the heart.

[0127] AGM explant E11.5 AGM was collected from C57BL6 / J Cd45.2 mouse embryos, and 3 cells were collected. A single-cell suspension of embryo equivalents is cultured in a BioFlex 6-well culture plate (FlexCel). The cells were seeded in each well of (l). The present inventors subjected the cells to repeated straining overnight (Flexcel). l(Registered Trademark) FX-4000(Trademark) Tension System), and / or These are chemical modifiers (2-100 μM Yoda1, 1 μM Nanaomycin, 100 μM Gdcl3, 1 μM GsMTx4, 5-20 μM 4αPDD, 10 μM GSK20 The cells were cultured after applying the treatment described in 5). Next, the collected cells were used for transplantation and fluorescence activation. Cell sorting (FACS) analysis and colony-forming unit (CFU) assay were performed.

[0128] Ex vivo incubation of embryos with medication E11.5 mouse embryos were mated with time-mated pregnant females. The sample was taken from the uterus and treated with FBS, 1 mM glucose, and 1% penicillin-streptomycin. , and / or selected chemical modifiers (2-100 μM Yoda1, 1 μM Nana) Contains omycin, 5-20 μM 4αPDD, or 10 μM GSK205. It was placed in a sterile glass vial. The inventors of this invention placed the glass vial in a roller device (≒30 Rotating at rpm, constant gas supply (21% O2, 5% CO2, remainder N2), and 3 Exvivo incubator (BTC Engineer) consisting of a constant temperature of 7°C It was placed in (ng, Cambridge, UK). After 24 hours, the AGM was collected and FAC Hematopoietic cell formation was analyzed using the S assay and CFU assay.

[0129] transplant In primary transplantation, untreated or treated (repeated straining or 25 μM Yoda) 1) Three embryonic equivalents of AGM and spleen helper cells (approximately 500,000 cells per mouse) were used to test for lethality. In mice with Cd45.1 (SJL) that were irradiated (fractionated dose 10.5 cGy), the treatment was administered via retroorbital injection. Therefore, it was injected. In secondary and tertiary transplants, bone marrow was isolated from the transplanted mice. Legs, arms, pelvic bones, spine, sternum). Bone marrow is Ficol gradient (Histopaque (Registered Trademark) Loaded onto (Standard)-1083, Sigma-Aldrich), and finely ground from the buffy coat. The cells contain biotin conjugate type antibodies and streptavidin microbeads (M It was incubated with iltenyi Biotec. Next, it was incubated with cell line negative (Lin - ) Cells were isolated using a MACS LS column (Miltenyi Biotec), and donor C Lin of d45.2 - Sca1 + c-Kit + (LSK) cells, MoFlo Beck The samples were sorted using a Mann Coulter sorter. The sorted Cd45.2 LSK samples were then processed. The cells were mixed with Cd45.1 spleen helper cells (approximately 500,000 per mouse) and irradiated. The drug was administered to SJL mice with a fractional dose of 10.5 cGy (Cd45.1) via retroorbital injection. I planted it.

[0130] Surviving recipients were counted as responses to the limiting dilution assay: The interval 1 / (stem cell frequency) was calculated using ELDA according to a Poisson distribution.

[0131] CFU assay and FACS assay In the CFU assay, cells from AGM explants or ex vivo cells are used in MethoCult In GF M3434 medium (StemCell Technologies), plate The cells were cultured. Seven days after seeding, the present inventors cultured granulocytes, erythrocytes, macrophages, and megakaryocytes (G EMM), granulocyte-macrophage (GM), granulocyte (G), macrophage (M), and We analyzed their ability to form erythrocyte (E) colonies.

[0132] Explant and ex vivo AGM cells were converted to Sca1-Pacific-Blue(E 13-161.7, Biolegend) and Flk1-APC-Cy7(Avas The cells were stained with 12α1 (BD). The transplanted mouse blood was stained with the following antibody cocktail. Ta: Cd45.2-Pacific-Blue (104, Biolegend), Cd4 5.1-FITC (A20, Biolegend), Cd3-PE (145-2C11, Biolegend), Cd8-PE(53-6.7, Biolegend), Mac1 -APC (M1 / 70, Biolegend), Gr1-APC (108412, Bio legend), Cd19-APC-CY7(6D5, Biolegend), B220 -APC-CY7(RA3-6B2, Biolegend).

[0133] Cells from the bone marrow, spleen, thymus, and lymph nodes of mice transplanted with E11.5 AGM cells. The following antibody panel was used to stain the bone marrow LT-HSC: Cd45.2-FITC(104 , Biolegend), Ter119-Biotin (TER-119 BD), Gr1- Biotin (RB6-8C5, BD), Cd5-Biotin (53-7.3, BD), Cd8 α-Biotin (53-6.7, BD), B220-Biotin (RA3-6B2, BD), Streptavidin-Pacific Blue (eBioscience), Sca1-PE -CY7 (D7, eBioscience), cKit-APC (2B8, eBiosc ience), Cd48-APC-CY7(HM48-1, BD), Cd150-PE- CY5 (TC15-12F12.2, Biolegend). Erythroid differentiation in bone marrow. RI-RV:Cd45.2-Pacific-Blue(104, Biolegend) , Cd45.1-FITC(A20, Biolegend), Ter119-APC(T ER-119, Biolegend), Cd71-PE (R17217, eBiosci ence). Bone marrow granulocytes: Cd45.2-Pacific-Blue (104, Biol egend), Cd45.1-FITC(A20, Biolegend), Gr1-PE (RB6-8C5, BD), Mac1-APC (M1 / 70, Biolegend). spleen T cells in the thymus, lymph nodes, and lymph nodes: Cd45.2-Pacific-Blue(104, Biolegend), Cd45.1-FITC(A20, Biolegend), Cd 8-PE (53-6.7, Biolegend), Cd4-APC (RM4-5, eBi (oscience). Bone marrow cells and B cells of the spleen, thymus, and lymph nodes: Cd45. 2-Pacific-Blue (104, Biolegend), Cd45.1-FIT C(A20, Biolegend), Cd19-APC-CY7(6D5, Bioleg (end), Mac1-APC (M1 / 70, Biolegend). The present inventors have created all FACS analysis was performed using a BD Fortessa cytometer. The present inventors are concerned with hematopoietic organs. The biopsy was performed 16 weeks after the transplant.

[0134] Quantitative reverse transcriptase-polymerase chain reaction analysis (qRT-PCR) Using FACS, pre-erythrocytes obtained from undissolved bone marrow isolated from AGM-transplanted mice were used. Carbohydrate (Cd45.2 + Ter119 + , Cd71 + ) was selected. All RNA was selected. Isolation was performed using the easy Minikit (QIAGEN), and cDNA synthesis was carried out using Sup This was done using erscript III (Invitrogen). Quantitative real-time analysis. Immuno PCR was performed using the MX3000P machine with SYBR Green (QuantaBio) markings. Performed using the primers described (Sankaran et al., 2009). The inventors normalized the expression to that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (Ochida et al., 2010).

[0135] Expression of myeloperoxidase (MPO) Neutrophils (Cd45.2 + , Gr1 + , Mac1 + ) were FACS sorted from the isolated bone marrow of 16-week-old primary transplanted mice and cultured overnight (500,000 cells / mL) in IMDM with 10% FBS in 24-well plates. The supernatant was collected, and the MPO concentration was measured using the Mouse MPO / Myeloperoxidase PicoKine™ ELISA Kit ( ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0138] Detection of pre-immune Ig Serum was isolated from 16-week-old primary transplanted mice, and the isotype of pre-immune Ig was quantified using a Mouse Ig Isotyping Kit (Thermo Fisher).

[0139] Delayed-type hypersensitivity Transplanted mice were sensitized via subcutaneous injection (lumbar region) and intradermal injection (right plantar) with sheep red blood <000158६>cells (sRBC, 10 9 cells / mL, 50 μL per site, Rockland Immu nochemicals). Six days after sensitization, pre-sensitized mice were challenged with 2 ×10 9 RBC / mL in the left plantar and an equal volume of PBS in the right plantar (as a control). Forty-eight hours after challenge, the thickness of the plantar was measured with a microcaliper. The present inventors normalized the percent change on day 6 with the thickness of each plantar before challenge.

[0140] Expression of DNA methyltransferase Nuclear extracts from AGM explants were harvested using an EpiQuik Nuclear Extracti on kit (Epigentek Group Inc.). Levels of Dnm t3b protein and Dnmt3a protein were analyzed using a colorimetric EpiQuik a<00ϰ1597>ssay kit (Epigentek Group Inc.) according to the manufacturer's instructions. The concentrations of Dnmt3b and Dnmt3a were related to 1 μg of nuclear extract protein.

[0141] RNAseq analysis and computational analysis Total RNA from E11.5 mouse AGM explant cultures was isolated using an RNAeasy MiniKi ​Isolation using t(QIAGEN) (control conditions, stretching conditions, Yoda1 conditions, and 4αPD (Condition D). The cDNA library of this invention is available from BGI Americas Corp. Fabricated by ration and processed lane by HiSeq4000 (Illumina) Sequenced with 8 samples per sample. The present inventors' sequencing The read fragments are processed by Genomic Short-Read Nucleotide Using the Alignment program (version 2012-07-20), The data was mapped to the reference genome GRCm38 (ENSEMBL release 69). Using ESeq2 and DEXSeq, we obtained differential expression (FDR=0.1) and Variations in exon use were investigated. Gene expression clusters of differentially expressed genes were analyzed. Next, we compared the average expression levels of these genes across different cell populations. Then, we compared the up-gene and down-gene expression levels. By comparing Venn diagrams of genes, we can determine whether repeated strain or pharmacological regulatory substances are involved in the endothelium. We analyzed key candidates for conversion to HSC. Specifically, we have developed R(R Deve gplots package (Wa Hierarchical clustering was performed using bootstrap analysis (Merners et al., 2017). In GO analysis, the present inventors observed changes in the expression of their inventions in GO categories or pathways. The excessive occurrence of genes was tested using Fisher's exact test, and Bonfe The Roni method was used to correct for multiple tests. The inventors statistically determined that a p-value of 0.001 is statistically significant. As a minimum significant enrichment, as previously described, GO Ta A room enrichment analysis was performed.

[0142] statistical analysis Unless otherwise specified, data is expressed as mean ± standard error of the mean (mean ± SEM). The statistical analysis was performed using paired and unpaired Student's t-tests. The significance level was set at P<0.05.

Claims

1. A method for preparing a population of hematopoietic stem cells (HSCs), including long-term (LT) HSCs, To provide a population containing endothelial cells and / or hematopoietic endothelial (HE) cells, In the endothelial cells and / or HE cells, vegfa, hey2, grp116, g Selected from na13, sox17, cdh5, plxnd1, bcl6 and apln Reducing the expression or activity of two or more endothelial genes, runx1, spi1, cebpa, ta in the endothelial cells and / or HE cells Two or more hematopoiesis selected from L1, GF1, GATA2, and MLT3 To increase gene expression or activity, thereby stimulating the formation of HSCs, including LT-HSCs. thing The method, including the method described above.

2. In the endothelial cells and / or HE cells, vegfa, hey2, grp116, Select from gna13, sox17, cdh5, plxnd1, bcl6 and apln To reduce the expression or activity of three or more endothelial genes, and the aforementioned runx1, spi1, cebpa, tal1 in endothelial cells and / or HE cells , three or more hematopoietic genes selected from gfi1, gata2 and mllt3 To increase the expression or activity of the offspring, thereby stimulating the formation and, if applicable, proliferation of the HSCs. The method according to claim 1, including the following:

3. In the endothelial cells and / or HE cells, vegfa, hey2, grp116, To reduce the expression or activity of gna13, cdh5 and plxnd1, and runx1, spi1, cebpa, ta in the endothelial cells and / or HE cells By increasing the expression or activity of l1 and gata2, the formation of the HSC The method according to claim 1, further comprising stimulating proliferation in the case thereof.

4. The increase in the activity or expression of the hematopoietic gene is due to the coding mRNA or mRNA derivative. Introducing a coding gene or episome, and expressing an element To introduce gene modifications to the hematopoietic gene, or to introduce gain-of-function mutations to the hematopoietic gene The method according to any one of claims 1 to 3, comprising one or more of the following:

5. The aforementioned decrease in endothelial gene expression or activity is due to complete or partial gene deletion, RNA saturation, etc. Introducing erasing, antisense oligonucleotide inhibition, and pharmacological inhibition. Introducing gene modifications to the expression element, or introducing loss-of-function mutations into the endothelial gene. The method according to any one of claims 1 to 4, which includes one or more of introducing natural mutations. Law.

6. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, by increasing the expression or activity of Dnmt3b to an effective level and duration The method according to any one of claims 1 to 3.

7. The increased expression or activity of Dnmt3b may affect the encoding mRNA or mRNA derivative. Introducing, introducing a coding gene or episome, and the Dnmt One of the methods involves introducing gene modification of the expression element or gain-of-function mutation into the 3b gene. The method according to claim 6, comprising one or more.

8. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, by increasing the expression or activity of Gimap6 to an effective level and duration The method according to any one of claims 1 to 3.

9. The increased expression or activity of Gimap6 may affect the encoding mRNA or mRNA derivative. Introducing, introducing a coding gene or episome, and Gimap6 One method involves introducing gene modification or gain-of-function mutations into a gene to modify the expression element. The method according to claim 8, which includes a plurality of or

10. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, the endothelial cells or HE cells are subjected to repeated 2D or 3D strain, effective concentration and This is done by bringing the piezo 1 agonist, or a combination thereof, into contact with the period. The method described in any one of the requests 1 to 3.

11. The aforementioned piezo agonist is Yoda1, Jedi1, and / or Jedi2. The method according to claim 10.

12. The effective amount of the aforementioned piezo-agonist is in the range of 0.1 to 500 μM, or 0.1 to The method according to claim 11, wherein the concentration is in the range of 100 μM.

13. The agonist of Piezo 1, upon contact with the candidate compound, the endothelial cells and / or vegfa, hey2, grp116, gna13, sox17, cd in HE cells Expression or activation of endothelial genes selected from h5, plxnd1, bcl6, and apln Decreased sex, and runx1, spi1, in the endothelial cells and / or HE cells Two selected from cebpa, tal1, gfi1, gata2 and mllt3 Based on the increased expression or activity of hematopoietic genes, in the chemical library The method according to claim 10, as identified.

14. A population including embryoid bodies, endothelial cells, hematopoietic endothelial (HE) cells, or a combination thereof, is called a bioliary. The method according to any one of claims 1 to 13, which includes providing to an actor.

15. The bioreactor undergoes biomechanical extension due to repeated strain, and maintains an effective concentration and duration. The method according to claim 14, which provides an agonist for the intermediate piezo 1 or a combination thereof.

16. The biomechanical stretching due to repeated strain is performed on the endothelial cells and / or HE cells. okeru vegfa, hey2, grp116, gna13, sox17, cdh5, pl Development of three or more endothelial genes selected from xnd1, bcl6, and apln It reduces the activity of runx1,s in the endothelial cells and / or HE cells. Selected from pi1, cebpa, tal1, gfi1, gata2, and mllt3. To increase the expression or activity of two or more hematopoietic genes, the formation of the HSC and The method according to claim 15, wherein proliferation is stimulated in some cases.

17. The aforementioned HSCs engraft in the hematopoietic niche and reconstruct functional multicellular adult blood. The method described in any one of the requests 1 to 16.

18. HE cells are induced pluripotent stem cells (iPSCs), non-hematopoietic stem cells, somatic cells, or endothelial cells. The method according to any one of claims 1 to 17, obtained from there.

19. The hematopoietic stem cells consist of at least 1% long-term hematopoietic stem cells (LT-HSCs) or at least The method according to any one of claims 1 to 18, further comprising 5% LT-HSC.

20. The hematopoietic stem cells include at least 0.1% long-term hematopoietic stem cells (LT-HSCs). The method described in item 19.

21. The endothelial cells and / or HE cells are HLA-modified cells or HLA-null cells, and Derived from transgene-free cells, gene-modified cells, or transgene-overexpressing cells, In some cases, iPS cells or somatic cells are derived by genetic or chemical induction. The method described in any one of the requests 1 to 20.

22. The source cells are obtained from or derived from a pre-existing library of target cells, as described above. The method according to any one of claims 1 to 21, wherein the subject is, in some cases, a universally compatible donor. 。

23. The aforementioned source cells are associated with hematological diseases, bone marrow diseases, lysosomal storage diseases, mitochondrial diseases, and metabolic disorders. Claim 22, obtained from or derived from a subject having a disease or immune disorder Method of description.

24. The method according to claim 23, wherein the subject does not have a blood or non-blood malignant tumor.

25. Claim further comprising recovering the HSCs and optionally propagating them. The method described in any one of items 1 to 24.

26. The aforementioned HSC population is administered to the recipient, and the recipient may be a donor. The method according to claim 25.

27. At least about 10 ２ The method according to claim 26, wherein an HSC is administered.

28. At least about 10 ３ The method according to claim 26, wherein an HSC is administered.

29. At least about 10 ４ The method according to claim 26, wherein an HSC is administered.

30. At least about 10 ５ The method according to claim 26, wherein an HSC is administered.

31. A method for converting a cell population into hematopoietic endothelial (HE) cells, Provide a population containing embryoid bodies or endothelial cells, In the aforementioned cells, vegfa, hey2, grp116, gna13, sox17, cd One or more endothelial genes selected from h5, plxnd1, bcl6, and apln To reduce the expression or activity of the child, and runx1, spi1, in the cell Two selected from cebpa, tal1, gfi1, gata2 and mllt3 This involves increasing the expression or activity of hematopoietic genes beyond a certain point. Applying repeated 2D or 3D strain, and The cells are brought into contact with the agonist of piezo 1 at an effective concentration and for an effective period of time. Provides one or more genetic, pharmacological, and / or mechanical stimuli selected from the following: The cells are then converted into HE cells. The method, including the method described above.

32. The increase in the activity or expression of the hematopoietic gene is due to the coding mRNA or mRNA derivative. Introducing a coding gene or episome, expressing an element Introducing gene modifications, and introducing gain-of-function mutations into the hematopoietic genes. The method according to claim 31, comprising one or more of the above.

33. The aforementioned decrease in endothelial gene expression or activity is due to complete or partial gene deletion, RNA saturation, etc. Elimination, antisense oligonucleotide inhibition, and pharmacological inhibition are introduced. Introducing gene modifications to the existing element, and introducing loss-of-function mutations into the endothelial gene. The method according to claim 31, comprising one or more of introducing the following.

34. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, by increasing the expression or activity of Dnmt3b to an effective level and duration A method according to claim 33 or 33.

35. The increased expression or activity of Dnmt3b may affect the encoding mRNA or mRNA derivative. To introduce, to introduce a coding gene or episome, to introduce an expression element Introducing genetic modifications and incorporating gain-of-function mutations into the Dnmt3b gene The method according to claim 34, comprising one or more of the above.

36. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, by increasing the expression or activity of Gimap6 to an effective level and duration, The method according to any one of claims 32 to 35, which is carried out as follows.

37. The increased expression or activity of Gimap6 may affect the encoding mRNA or mRNA derivative. To introduce, to introduce a coding gene or episome, to introduce an expression element Introducing genetic modifications and introducing gain-of-function mutations into the Gimap6 gene The method according to claim 36, comprising one or more of the above.

38. The cells are divided into an effective amount selected from Yoda1, Jedi1 and / or Jedi2. The method according to claim 31, wherein the piezo agonist is brought into contact with the piezo 1 agonist.

39. The effective amount of the aforementioned piezo-agonist is in the range of 0.1 to 500 μM, or 0.1 to The method according to claim 38, wherein the concentration is in the range of 100 μM.

40. The process includes supplying the population to a bioreactor, and the bioreactor repeatedly The method according to any one of claims 31 to 39, which provides biomechanical extension due to strain. Law.

41. Depending on the circumstances, the aforementioned genetic, pharmacological, and / or mechanical stimulation may be applied. The method according to claim 40, wherein the HE cells are recovered or converted into HSCs. 。

42. The aforementioned HE cells engraft in the hematopoietic niche and reconstruct functional multicellular adult blood. The method according to claim 41, which is converted to HSC.

43. Claims 31-42, wherein the embryoid body or endothelial cells are derived from induced pluripotent stem cells (iPSCs). The method described in any one of the items.

44. The method according to any one of claims 31 to 42, wherein the endothelial cells are derived from non-hematopoietic stem cells. Law.

45. The HE cells are converted into hematopoietic stem cells, including long-term hematopoietic stem cells (LT-HSCs), claim. The method described in any one of items 31 to 44.

46. Claims 31-45, wherein the cell population is derived from HLA-modified cells or HLA-null cells. The method described in any one of the items.

47. The cell population is a transgene-free cell population, as described in any one of claims 31 to 45. The method.

48. This includes applying the aforementioned genetic, pharmacological, and / or mechanical stimuli to the HSC cells. Any of claims 31 to 47 further comprises increasing the HSCs in the process The method described in any one of the items.

49. The HSC is administered to the recipient, and the recipient may be a donor. A method according to claim 48.

50. A method for proliferating a population of hematopoietic stem cells (HSCs), We provide an HSC group, In the aforementioned cells, vegfa, hey2, grp116, gna13, sox17, cd One or more endothelial genes selected from h5, plxnd1, bcl6, and apln To reduce the expression or activity of the child, and runx1, spi1, in the cell Two selected from cebpa, tal1, gfi1, gata2 and mllt3 This involves increasing the expression or activity of hematopoietic genes beyond a certain point. Applying repeated 2D or 3D strain, and The cells are brought into contact with the agonist of piezo 1 at an effective concentration and for an effective period of time. Provides one or more genetic, pharmacological, and / or mechanical stimuli selected from the following: to increase HSC The method, including the method described above.

51. The increase in the activity or expression of the hematopoietic gene is due to the coding mRNA or mRNA derivative. Introducing a coding gene or episome, expressing an element Introducing gene modifications, and introducing gain-of-function mutations into the hematopoietic genes. The method according to claim 50, comprising one or more of the above.

52. The aforementioned decrease in endothelial gene expression or activity is due to complete or partial gene deletion, RNA saturation, etc. Elimination, antisense oligonucleotide inhibition, and pharmacological inhibition are introduced. Introducing gene modifications to the current element, and introducing a 53-loss type mutation into the endothelial gene. The method according to claim 51, comprising one or more of introducing the following.

53. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes By increasing the expression or activity of Dnmt3b to an effective level and duration, The method according to claim 51 or 52, which is carried out by...

54. The increased expression or activity of Dnmt3b may affect the encoding mRNA or mRNA derivative. To introduce, to introduce a coding gene or episome, to introduce an expression element Introducing genetic modifications and incorporating gain-of-function mutations into the Dnmt3b gene The method according to claim 53, comprising one or more of the above.

55. The aforementioned decrease in the expression or activity of endothelial genes and the aforementioned increase in the expression or activity of hematopoietic genes However, by increasing the expression or activity of Gimap6 to an effective level and duration, The method according to any one of claims 50 to 54, which is carried out as follows.

56. The increased expression or activity of Gimap6 may affect the encoding mRNA or mRNA derivative. To introduce, to introduce a coding gene or episome, to introduce an expression element Introducing genetic modifications and introducing gain-of-function mutations into the Gimap6 gene The method according to claim 55, comprising one or more of the above.

57. The cells are divided into an effective amount selected from Yoda1, Jedi1 and / or Jedi2. The method according to claim 50, wherein the piezo agonist is brought into contact with the piezo 1 agonist.

58. The effective amount of the aforementioned piezo-agonist is in the range of 0.1 to 500 μM, or 0.1 to The method according to claim 57, wherein the concentration is in the range of 100 μM.

59. The process includes supplying the population to a bioreactor, and the bioreactor repeatedly The method according to any one of claims 50 to 58, which provides biomechanical extension due to strain. 。

60. The aforementioned HSCs engraft in the hematopoietic niche and reconstruct functional multicellular adult blood. The method described in item 59.

61. The HSC is converted from endothelial cells or HE cells, as per any of claims 50 to 60. The method described in item 1.

62. The HSC includes long-term hematopoietic stem cells (LT-HSC), any one of claims 50 to 61. The method described in section [section number].

63. Claims 50-6, wherein the HSC population is derived from HLA-modified cells or HLA-null cells. The method described in any one of item 2.

64. The method according to claim 63, wherein the cell population is transgene-free cells.

65. A population of HSCs prepared by the method described in any one of claims 1 to 64, and A pharmaceutical composition containing a pharmaceutically acceptable vehicle.

66. at least 10 4 The pharmaceutical composition according to claim 65, comprising 1 LT-HSC cell.

67. A method for treating subjects requiring hematopoietic stem cell therapy or transplantation, wherein a therapeutically effective amount of Hematopoietic stem cells (HSCs) prepared by the method described in any one of claims 1 to 64 Administering the above to the subject, or administering the pharmaceutical composition according to claim 65 or 66 to the subject A method, including administration to an elephant.

68. The aforementioned subjects include malignant or non-malignant forms of hematological disorders, bone marrow disorders, and lysosomal storage disorders. The method according to claim 67, wherein the patient has a mitochondrial disease, a metabolic disease, or an immune disease. 。

69. The aforementioned subjects include acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, and myeloproliferative disease. Sexual disorders, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin's disease, neuroblastoma Claim 67, having a condition selected from cystic tumor, germ cell tumor, or amyloidosis. Or the method described in 68.

70. The aforementioned subjects have autoimmune disorders such as systemic lupus erythematosus (SLE) or systemic sclerosis. Harmful, aplastic anemia, pure red blood cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia Thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiscotch To-Aldrich syndrome, hemophagocytic lymphohistiocytosis, congenital metabolic disorders, epidermolysis bullosa, severe Congenital neutropenia, Schwachmann-Diamond syndrome, Diamond Black Claims have a condition selected from Fan anemia, Pearson syndrome, and leukocyte adhesion disorder. The method described in paragraph 69.

71. A method for producing hematopoietic stem cells (HSCs), A panel of chemical compounds is brought into contact with embryoid bodies, endothelial cells and / or hematopoietic endothelial cells, and the Dnmt3b or Gimap6; vegfa, hey2, which are induced by chemical compounds. ,grp116,gna13,sox17,cdh5,plxnd1,bcl6, and At least two of apln; and runx1, spi1, cebpa, tal1, gf To determine changes in the expression levels of at least two of i1, gata2, and mllt3. ； The following changes in gene expression: Increased expression of Dnmt3b and / or Gimap6, vegfa, hey2, grp116, gna13, sox17, cdh5, plxnd 1. Decreased expression of two or more bcl6 and apln; and runx1, spi1, cebpa, tal1, gfi1, gata2, and mllt3 Increased expression of two or more of the following Selecting compounds that induce; and The conversion of endothelial cells and / or hematopoietic endothelial cells to HSCs by the selected compound Induced by contact with cortical cells and / or hematopoietic endothelial cells, thereby producing To create self-regenerating HSCs that can reconstitute adult multicellular blood. The method, including the method described above.

72. The selected compound is found to affect vegfa in the endothelial cells and / or HE cells. hey2, grp116, gna13, sox17, cdh5, plxnd1, bcl6 and reduces the expression of five or more endothelial genes selected from apln, and the runx1, spi1, cebpa, tal1 in endothelial cells and / or HE cells Five or more hematopoietic genes selected from gfi1, gata2, and mllt3 To increase the expression of the offspring, the formation of the HE cells, HSCs or combinations thereof and, if applicable, The method according to claim 71, wherein growth is stimulated.

73. The selected compound is found to affect vegfa in the endothelial cells and / or HE cells. It reduces the expression of hey2, grp116, gna13, cdh5 and plxnd1. runx1, spi1, cebpa, ta in the endothelial cells and / or HE cells The method according to claim 72, which increases the expression of l1 and gata2.

74. The selected compound increases the expression of Dnmt3b, according to claims 71 to 73. The method described in any one of the items.

75. The selected compound is a piezo-agonist, any one of claims 71 to 73. The method described in section [section number].

76. The selected compound is a derivative of Yoda1, Jedi1 and / or Jedi2. The method according to claim 74.

77. Embryoid bodies, endothelial cells, or HE cells are used to create induced pluripotent stem cells (iPSCs), non-hematopoietic stem cells, The method according to any one of claims 71 to 76, obtained from somatic cells or endothelial cells.

78. The obtained hematopoietic stem cells contain at least 0.1% long-term hematopoietic stem cells (LT-HSCs). The method according to any one of claims 71 to 76, including the method according to any one of claims 71 to 76.

79. The obtained hematopoietic stem cells constitute at least about 1% or at least about 10% of long-term hematopoietic stem cells. The method according to claim 78, comprising cells (LT-HSC).

80. Claim 78, wherein the obtained hematopoietic stem cells contain approximately 2% to approximately 25% LT-HSCs. Method of description.

81. The endothelial cells and / or HE cells are HLA-modified cells or HLA-null cells, introduced Derived from gene-overexpressing cells and / or transgene-free cells, and in some cases iPS cells. Derived by genetic or chemical induction of cells or somatic cells, according to claims 71 to 80. The method described in either of the above terms.

82. Source cells are obtained from or derived from a subject, and the subject may be a patient. , a suitable or unsuitable donor, or a universally suitable donor, any of claims 71 to 81 The method described in item 1.

83. The method according to any one of claims 71 to 82, further comprising recovering the HSC. Law.

84. Produced in accordance with this disclosure, at least about 10 ３ one or at least about 10 ４ Individual LT- A composition for cell therapy containing HSC cells.

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