Production method for hematopoietic stem cells

Abstract

The present invention provides: a production method for hematopoietic stem / precursor cells, said method including a step for culturing hematopoietic stem / progenitor cells in a medium containing a specific substance; cells obtained using said production method; and a use for said cells.

Description

Method for producing hematopoietic stem cells

[0001] The present invention relates to a method for producing hematopoietic stem / progenitor cells, which includes the step of culturing hematopoietic stem / progenitor cells in a culture medium containing a specific substance, the cells obtained by the production method, and the uses of the cells.

[0002] Hematopoietic stem cells are used in cell transplantation therapy for hematopoietic malignancies (leukemia, lymphoma, myeloma) and other blood disorders (e.g., primary immunodeficiency, aplastic anemia, myelodysplasia), with over 5,000 cell transplantation therapies performed annually in Japan. Given the large number of cell transplantation therapies being performed, in vitro amplification of (human) hematopoietic stem cells to be transplanted is also being carried out (Patent Document 1).

[0003] International Publication No. 2005 / 071064

[0004] However, to the best of our knowledge, in vitro amplification of (human) hematopoietic stem cells has been limited, and it has been difficult to significantly increase their number. Therefore, the object of the present invention is to provide a method for producing hematopoietic stem / progenitor cells, which includes the step of culturing hematopoietic stem / progenitor cells in a culture medium containing a specific substance, as well as cells obtained by this method and uses for such cells.

[0005] While conducting research aimed at in vitro amplification of hematopoietic stem / progenitor cells, the inventors accidentally discovered that adding a specific substance to the culture medium significantly amplified hematopoietic stem / progenitor cells. Further investigation into these specific substances revealed that using substances such as protein arginine methyltransferase 5 inhibitors, survivin inhibitors, Toll-like receptor 1 / 2 inhibitors, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, inflammatory cytokine inhibitors, heat shock protein 70 family inhibitors, lysine-specific demethylase 1 inhibitors, histone deacetylase inhibitors, or thalidomide derivatives, either alone or in combination, could stably and significantly amplify hematopoietic stem / progenitor cells. Based on these findings, the inventors conducted further research, ultimately completing the present invention.

[0006] In other words, the present invention is as follows: [1] A method for producing hematopoietic stem cells / progenitor cells, comprising the step of culturing hematopoietic stem / progenitor cells in a medium containing a protein arginine methyltransferase 5 inhibitor. [2] The method according to [1], wherein the medium contains at least one selected from the group consisting of a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide (CFOC), a heat shock protein 70 family inhibitor, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative. [3] The method according to [2], wherein the medium contains a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative. [4] The method according to any one of [1] to [3], wherein the protein arginine methyltransferase 5 inhibitor is GSK591. [5] The method according to any one of [2] to [4], wherein the survivorin inhibitor is YM155, the Toll-like receptor 1 / 2 inhibitor is Cu-CPT22, the inflammatory cytokine inhibitor is JTE607, the heat shock protein 70 family inhibitor is VER155008, the lysine-specific demethylase 1 inhibitor is UM171, the histone deacetylase inhibitor is valproic acid (VPA), and the thalidomide derivative is lenalidomide (LEN). [6] The method according to any one of [1] to [5], wherein the culture is co-cultured with mesenchymal stem cells. [7] The method according to [6], wherein the mesenchymal stem cells are positive for CD271 and SSEA-4. [8] Hematopoietic stem / progenitor cells obtained by the method according to any one of [1] to [7]. [9] A cell transplantation therapy agent comprising the cells described in [8].

[10] The cell transplantation therapy agent according to [9], further comprising mesenchymal stem cells that are positive for CD271 and SSEA-4.

[11] The method according to [6] or [7], hematopoietic stem / progenitor cells obtained by the method according to [6] or [7], a cell transplantation therapy agent comprising hematopoietic stem / progenitor cells obtained by the method according to [6] or [7], or the cell transplantation therapy agent according to

[10] , wherein the mesenchymal stem cells are derived from pluripotent stem cells.

[12] The method according to

[11] , hematopoietic stem / progenitor cells, or a cell transplantation therapy agent, wherein the pluripotent stem cells are induced pluripotent stem cells.

[13] A method for treating or preventing tissue damage or disease, comprising administering or transplanting an effective amount of the cells according to [8] to a target.

[14] The cells according to [8] for use in the treatment or prevention of tissue damage or disease.

[15] Use of the cells according to [8] in the manufacture of a drug for the treatment or prevention of tissue damage or disease.

[16] A method for producing hematopoietic stem cells / progenitor cells, comprising the step of culturing hematopoietic stem / progenitor cells in a medium comprising at least one selected from the group consisting of a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, and a heat shock protein 70 family inhibitor.

[17] The method according to

[16] , wherein at least a protein arginine methyltransferase 5 inhibitor is selected.

[18] The method according to

[16] or

[17] , wherein the culture medium further comprises at least one selected from the group consisting of a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a heat shock protein 70 family inhibitor, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative (excluding the substances selected in

[16] or

[17] ).

[19] The method according to

[18] , wherein the culture medium comprises a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative.

[20] The method according to any one of

[16] to

[19] , wherein the protein arginine methyltransferase 5 inhibitor is GSK591, the survivorin inhibitor is YM155, the Toll-like receptor 1 / 2 inhibitor is Cu-CPT22, the inflammatory cytokine inhibitor is JTE607, the heat shock protein 70 family inhibitor is VER155008, the lysine-specific demethylase 1 inhibitor is UM171, the histone deacetylase inhibitor is valproic acid, and the thalidomide derivative is lenalidomide.

[21] The method according to any one of

[16] to

[20] , wherein the culture is co-cultured with mesenchymal stem cells.

[22] The method according to

[21] , wherein the mesenchymal stem cells are positive for CD271 and SSEA-4.

[23] Hematopoietic stem / progenitor cells obtained by the method according to any one of

[16] to

[22] .

[24] A cell transplantation therapy agent comprising the cells described in

[23] .

[25] The cell transplantation therapy agent according to

[24] , further comprising mesenchymal stem cells that are positive for CD271 and SSEA-4.

[0007] This invention enables the mass production of hematopoietic stem / progenitor cells. This may allow for a stable supply of hematopoietic stem / progenitor cells required for cell transplantation therapy for hematopoietic malignancies (leukemia, lymphoma, myeloma) and other blood disorders (e.g., primary immunodeficiency, aplastic anemia, myelodysplasia).

[0008] Figure 1 shows a schematic diagram of the method for inducing iDP MSCs from pluripotent stem cells. Figure 2 shows the morphological comparison results of BM-DP MSCs and iDP MSCs. Scale bar: 200 μm. Figure 3 shows the comparison results of surface antigen (positive marker and negative marker) expression of BM-DP MSCs and iDP MSCs. Analysis was performed by flow cytometry using antibodies against each surface antigen. Figure 4 shows the comparison results of the support capacity of human hematopoietic stem / progenitor cells by BM-DP MSCs and iDP MSCs. FACS plot (top) of cells harvested after co-culturing human umbilical cord blood-derived hematopoietic stem / progenitor cells with BM-DP MSCs and each iDP MSC for one week, and hematopoietic stem / progenitor cells (CD34 +Graph of the proportion of cells (below). Figure 5 shows the evaluation of substances used in the production method of the present invention in a co-culture system of hematopoietic stem cells (HSCs) and iDP MSCs. In Figure 5, each compound was added at the following concentrations: UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM), both individually (UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC) and in combination (7 compounds: UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC). Figure 6 shows the FACS plot of HSC-derived producing cells after co-culture of hematopoietic stem cells (HSCs) and iDP MSCs. Figure 7 shows the results of dose-dependent studies when VER155008 and JTE607 were added individually after co-culture of hematopoietic stem cells (HSCs) and iDP MSCs. The effect of VER155008 was evaluated by the number of CD34-positive, CD38-negative, CD133-positive, EPCR-positive cells produced from one HSC on day 14 of culture. The effect of JTE607 was evaluated by the number of CD34-positive, CD38-negative, CD133-positive, CD146-positive cells produced from one HSC on day 16 of culture. Figure 8 shows the evaluation of the substances used in the production method of the present invention in a co-culture system of hematopoietic stem cells (HSCs) and iDP MSCs. Specifically, the evaluation of substances is conducted when all seven compounds (UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC) or six compounds (-UM171, -VPA, -LEN, -GSK591, -YM155, -Cu-CPT22, or -CFOC) are added simultaneously, or when one compound (UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, or -CFOC) is removed from the seven compounds. In all cases, the concentrations of the added substances are UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM). Figure 9 shows the evaluation of the substances used in the production method of the present invention in a co-culture system of hematopoietic stem cells (HSCs) and iDP MSCs.Specifically, the evaluation involved all seven compounds (UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC), as well as each compound individually or in all combinations of these four compounds. In all cases, the concentrations of the added substances were UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM). Figure 10 shows the results of transplantation experiments of amplified hematopoietic stem cells (HSCs) after co-culture with iDP MSCs into severely immunodeficient mice (NSG mice; NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) (Jackson Laboratory Japan). HSCs were cultured at a rate of 1 cell / well for 22 days in the presence of UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM). Cells derived from one HSC were harvested, 1 / 4 of the cells were analyzed by flow cytometry, and the remaining 3 / 4 of the cells were transplanted into NSG mice. Four weeks after transplantation, mouse bone marrow was harvested, and the engraftment of human CD45-positive cells in the mouse bone marrow was analyzed by flow cytometry. Engraftment of human hematopoiesis cells was observed in 2 out of 3 transplanted mice.

[0009] 1. Method for Producing Hematopoietic Stem / Progenitor Cells The present invention provides a method for producing hematopoietic stem / progenitor cells (hereinafter sometimes referred to as "the method of production of the present invention"), which includes the step of culturing hematopoietic stem / progenitor cells in a culture medium containing a specific substance (compound). The specific substance is, specifically, at least one selected from the group consisting of a protein arginine methyltransferase 5 (hereinafter sometimes abbreviated as "PRMT5") inhibitor, a survivor inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a heat shock protein 70 family inhibitor, a lysine-specific demethylase 1 (LSD1) inhibitor, a histone deacetylase (HDAC) inhibitor, and a thalidomide derivative. In the method of production of the present invention, the above nine substances may be used in any combination (2, 3, 4, 5, 6, 7, 8, or 9 types). Specifically, the substance is at least one selected from the group consisting of a protein arginine methyltransferase 5 inhibitor, a survivor inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, and a shock protein 70 family inhibitor. In the production method of the present invention, the above six substances may be used in any combination (2, 3, 4, 5, or 6 types).

[0010] In one embodiment, the specific substance used in the production method of the present invention is a protein arginine methyltransferase 5 inhibitor. In one embodiment, the specific substance used in the production method of the present invention is a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a heat shock protein 70 family inhibitor, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative. In another embodiment, the specific substance used in the production method of the present invention is a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, and a shock protein 70 family inhibitor.

[0011] In one embodiment, the specific substances used in the production method of the present invention are a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative. In one embodiment, the specific substances used are a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, and a thalidomide derivative. In one embodiment, the specific substances used in the production method of the present invention are a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, and a histone deacetylase inhibitor. In one embodiment, the inhibitors are a protein arginine methyltransferase 5 inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative.

[0012] In the present invention, the above-mentioned inhibitor may be an expression suppressant or a degradation (promoting) agent. Furthermore, the compounds included in the inhibitors used in the present invention, as described later, may be salts, solvates (e.g., hydrates, etc.), or solvates (e.g., nonhydrates, etc.). The above-mentioned specific substances used in the production method of the present invention may be present in the culture medium throughout the entire culture period, or only for a portion of the period.

[0013] In this specification, "protein arginine methyltransferase 5" refers to a type II arginine methyltransferase that symmetrically methylates the arginine residue of a substrate (protein) twice. In the present invention, the protein arginine methyltransferase 5 inhibitor is not particularly limited in its method or type, as long as it can reduce the symmetric dimethylation of the arginine residue of the substrate (protein). For example, it may be a small molecule, an antibody, or a nucleic acid (e.g., antisense oligonucleotide, siRNA, miRNA, etc.).

[0014] Specifically, examples of protein arginine methyltransferase 5 inhibitors include GSK591 (also known as EPZ015866 or GSK3203591), GSK3235025 (also known as EPZ015666), GSK3326595 (also known as EPZ015938), Onametostat, PRMT5-IN-1, PRMT5-MTA-IN-1, PRMT5-IN-4, and PRMT5-IN -9, PRMT5-IN-14, PRMT5-IN-19, PRMT5-IN-21, PRMT5-IN-23, PRMT5-IN-25, PRMT5-IN-28, PRMT5-IN-30, PRMT5 -IN-33, PRMT5-IN-34, PRMT5-IN-36-d3, PRMT5-IN-39-d3, PRMT5-IN-43, PRMT5-IN-44, MRTX-1719, MRTX-1719 hydrochloride, (S)-MRTX-1719, LLY-283, TNG908, MRTX9768, PF-06939999, DS-437, BRD0639, HLCL-61 hydrochloride, DW14800, etc. In one embodiment, the protein arginine methyltransferase 5 inhibitor is GSK591 (also known as EPZ015866 or GSK3203591) or GSK3235025 (also known as EPZ015666), preferably GSK591 (also known as EPZ015866 or GSK3203591).

[0015] The concentration of the protein arginine methyltransferase 5 inhibitor in the culture medium used in the production method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of GSK591, the final concentration is typically 2 nM to 20 μM, preferably 20 nM to 2 μM, and more preferably 40 nM to 200 nM.

[0016] In this specification, "survivin" refers to a protein belonging to the apoptosis inhibitor (IAP) family, which typically suppresses apoptosis by inhibiting caspase activity. In the present invention, the method and type of survivin inhibitor are not particularly limited as long as they can reduce the apoptotic resistance of survivin, and may include, for example, small molecules, antibodies, or nucleic acids (e.g., antisense oligonucleotides, siRNA, miRNA, etc.). Specifically, examples of survivin inhibitors include YM155 (Sepantronium Bromide), FL118, Flavokawain A, LQZ-7I, etc. In one embodiment, the survivin inhibitor is YM155 (Sepantronium Bromide).

[0017] The concentration of the survivin inhibitor in the culture medium used in the manufacturing method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of YM155, the final concentration is typically 0.5 nM to 5 μM, preferably 5 nM to 500 nM, and more preferably 10 nM to 50 nM.

[0018] In the present invention, the method and type of Toll-like receptor 1 / 2 inhibitor are not particularly limited as long as they reduce signal transduction to at least the MyD88-dependent pathway of the Toll-like receptor signaling pathway, and may be, for example, small molecules, antibodies, or nucleic acids (e.g., antisense oligonucleotides, siRNA, miRNA, etc.). Specifically, examples of Toll-like receptor 1 / 2 inhibitors include Cu-CPT22.

[0019] The concentration of the Toll-like receptor 1 / 2 inhibitor in the culture medium used in the manufacturing method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of Cu-CPT22, the final concentration is typically 20 nM to 200 μM, preferably 200 nM to 20 μM, and more preferably 400 nM to 2 μM.

[0020] In the present invention, an inflammatory cytokine inhibitor is typically one that can reduce the production of at least one inflammatory cytokine from among TNF-α, IL-1β, IL-6, IL-8, and IL-10. In one embodiment, the inflammatory cytokine inhibitor reduces the production of TNF-α, IL-1β, IL-6, IL-8, and IL-10. Specifically, an example of an inflammatory cytokine inhibitor is JTE607.

[0021] The concentration of the inflammatory cytokine inhibitor in the culture medium used in the manufacturing method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of JTE607, the final concentration is typically 500 nM to 10 μM, preferably 1 μM to 5 μM.

[0022] In the present invention, the method and type of heat shock protein 70 family inhibitor are not particularly limited as long as they reduce chaperone-mediated autophagy, and may be, for example, small molecules, antibodies, or nucleic acids (e.g., antisense oligonucleotides, siRNA, miRNA, etc.). In one embodiment, the heat shock protein 70 family inhibitor reduces chaperone-mediated autophagy and lowers the level of the complex between heat shock protein 90 and client protein. In another embodiment, the heat shock protein 70 family inhibitor competitively inhibits the binding of ATP to heat shock protein 70. Specifically, examples of heat shock protein 70 family inhibitors include VER155008, Apoptozole, JG98, TRC051384, PES-Cl, MKT-077, etc. In one embodiment, the heat shock protein 70 family inhibitor is VER155008.

[0023] The concentration of the heat shock protein 70 family inhibitor in the culture medium used in the manufacturing method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of VER155008, the final concentration is typically 1 nM to 100 μM, preferably 10 nM to 7.5 μM, and more preferably 100 nM to 1 μM.

[0024] In this specification, "lysine-specific demethylase 1 (LSD1)" refers to an enzyme that demethylates lysine-4 of histone H3 and forms a transcriptional repression complex with histone deacetylase (HDAC). In the present invention, the lysine-specific demethylase 1 inhibitor is not particularly limited in terms of method or type, as long as it can reduce the above-mentioned demethylation, and may be, for example, a small molecule, an antibody, or a nucleic acid (e.g., antisense oligonucleotide, siRNA, miRNA, etc.). Specifically, as a lysine-specific demethylase 1 inhibitor, for example, a pyrimidoindole derivative described in US2015 / 0011543, WO2017 / 022534, WO2021132627 (e.g., UM171 ((1r,4r)-N 1 Examples include -(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine), UMUM729 (Methyl 4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate), RN 1 dihydrochloride (1-(4-methyl-1-piperazinyl)-2-[[(1R*,2S*)-2-[4-phenylmethoxy)phenyl]cyclopropyl]amino]ethanone dihydrochloride), Tranylcypromine, GSK-LSD1, etc. In one embodiment, the lysine-specific demethylase 1 inhibitor is UM171.

[0025] The concentration of the lysine-specific demethylase 1 inhibitor in the culture medium used in the production method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of UM171, the final concentration is typically 0.35 nM to 3.5 μM, preferably 3.5 nM to 350 nM, and more preferably 7 nM to 35 nM.

[0026] In this specification, "histone deacetylase (HDAC)" refers to an enzyme that catalyzes the reaction of removing an acetyl group from the terminal of a histone. In the present invention, the histone deacetylase (HDAC) inhibitor is not particularly limited in its method or type as long as it can reduce the above-mentioned reaction of removing the acetyl group, and may be, for example, a small molecule, an antibody, or a nucleic acid (e.g., antisense oligonucleotide, siRNA, miRNA, etc.). In one embodiment, the histone deacetylase inhibitor used in the production method of the present invention is also a GABA transaminase inhibitor and / or a glycogen synthase kinase-3 (GSK-3) inhibitor. Specifically, examples of histone deacetylase inhibitors include valproic acid, trichostatin A, and sodium butyrate.

[0027] The concentration of the histone deacetylase inhibitor in the culture medium used in the production method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of valproic acid, the final concentration is typically 500 nM to 5 mM, preferably 5 μM to 500 μM, and more preferably 10 μM to 50 μM.

[0028] Examples of thalidomide derivatives include compounds such as those described in US2012071509 (e.g., Lenalidomide). Thalidomide derivatives typically reduce (suppress) the production of inflammatory cytokines. Examples of such inflammatory cytokines include TNF-α, IL-1β, IL-6, and IL-12. In one embodiment, thalidomide derivatives reduce the production of at least TNF-α.

[0029] The concentration of the thalidomide derivative in the culture medium used in the production method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained. For example, in the case of Lenalidomide, the final concentration is typically 0.75 nM to 7.5 μM, preferably 7.5 nM to 750 nM, and more preferably 15 nM to 75 nM.

[0030] The concentration of N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide in the culture medium used in the production method of the present invention is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells is obtained, but is typically 500 nM to 2 μM at the final concentration.

[0031] In this specification, "Hematopoietic Stem / Progenitor Cell (HSPC)" is a general term encompassing hematopoietic stem cells and hematopoietic progenitor cells, and refers to cells that are CD133 positive and have the ability to differentiate into other cells of the hematopoietic lineage. Hematopoietic stem / progenitor cells are typically CD34 positive, but may also be CD34 negative cells that may be present in umbilical cord blood. In addition to the above, hematopoietic stem / progenitor cells may also have properties such as being differentiation antigen negative, CD38 negative, CD45 positive, CD90 positive, GPI-80 (also known as Vanin 2, VNN2, FOAP-4, etc.) positive, and neutrophil marker negative.

[0032] Differentiation antigens are not particularly limited as long as they are known as positive markers for hematopoietic cells, such as leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, or macrophages), erythrocytes, platelets, mast cells, or dendritic cells. Specifically, examples include CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD16, CD19, CD20, CD24, CD33, CD41, CD45RA, CD56, CD66c, CD127, CD235a, etc. The hematopoietic cell marker is preferably at least one selected from the group consisting of CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD16, CD19, CD20, CD24, CD33, CD41, CD45RA, CD56, CD66c, CD127, and CD235a, more preferably at least four, even more preferably at least eight, even more preferably at least twelve, even more preferably at least sixteen, and particularly preferably all eighteen. The differentiation antigen may be a single antigen, or a combination of two or three or more antigens.

[0033] Neutrophil markers are not particularly limited as long as they are known as positive neutrophil markers. Specifically, examples include CD10, CD15s, CD16b, CD17, CD24, CD32, CD35, CD43, CD65, CD65s, CD66a, CD66c, CD66d, CD89, CD92, CD93, CD111, CD112, CD114, CD116, CD123, CD131, CD157, CD170, CD177, CD181, CD281, CD282, CD312, etc. Neutrophil markers may be used individually, or in combination of two or more types.

[0034] Examples of the hematopoietic stem / progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, myeloid progenitor cells, megakaryocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem / progenitor cells can typically be isolated from, for example, bone marrow, peripheral blood, umbilical cord blood, or placenta. Furthermore, the hematopoietic stem / progenitor cells used in this invention may be commercially available cells, or they may be differentiated and induced from pluripotent stem cells (for example, induced pluripotent stem cells derived from the individual to be transplanted).

[0035] The hematopoietic stem / progenitor cells used in the production method of the present invention can be obtained by a method known per se (for example, WO 2015 / 163185 etc.). In one aspect, the hematopoietic stem / progenitor cells are obtained by removing differentiated antigen-positive cells from human umbilical cord blood using the EasySep Human Progenitor Cell Enrichment Kit (manufactured by StemCell Technologies), and staining the remaining differentiated antigen-negative cells with anti-Lineage marker antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD133 antibody and anti-GPI-80 antibody, and then isolating the Linege-negative CD34-positive CD38-negative CD133-positive GPI-80-positive fraction using a cell sorter (FACS AriaIII, BD Biosciences). Alternatively, hematopoietic stem / progenitor cells used in the production method of the present invention may be obtained by inducing differentiation from pluripotent stem cells as described below according to a method known per se (for example, WO 2014 / 200030 etc.).

[0036] Any undifferentiated cells can be used as the pluripotent stem cells as long as they have the "self-renewal ability" to proliferate while maintaining an undifferentiated state and the "differentiation pluripotency" to differentiate into all three primary germ layers. Examples of such pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer Embryonic stem cell: ntES cells), multipotent germline stem cells ("mGS cells"), embryonic germ cells (EG cells), etc. Preferably, they are iPS cells (more preferably human iPS cells). When the above pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells prepared by destroying the embryo or cells prepared without destroying the embryo. From an ethical perspective, preferably, they are cells prepared without destroying the embryo.

[0037] iPS cells are artificial stem cells derived from somatic cells that possess characteristics nearly identical to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal, and can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. (Takahashi K. and Yamanaka S. (2006) Cell, 126:663-676; Takahashi K. et al. (2007), Cell, 131:861-872; Yu J. et al. (2007), Science, 318:1917-1920; Nakagawa M. et al., Nat. Biotechnol.)26:101-106 (2008); WO 2007 / 069666). When using iPS cells, the iPS cells may be produced from somatic cells by a known method, or already established and stockpiled iPS cells may be used. Reprogramming factors may consist of genes specifically expressed in ES cells, their gene products or non-coding RNA, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNA, or small molecule compounds. Examples of genes included in reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO 2009 / 126655, WO 2009 / 157593, WO 2010 / 009015, WO 2010 / 033906, WO 2010 / 033920, WO 2010 / 042800, WO 2010 / 050626, WO 2010 / 056831, WO 2010 / 068955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat.The combinations described in Biotechnol., 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0038] ES cells are stem cells that possess pluripotency and the ability to proliferate through self-renewal, established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. For methods of establishing and maintaining human and monkey ES cells, see, for example, USP5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. This is described in Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485, etc.Alternatively, ES cells can also be established using only a single blastomere of an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or can be established using embryos that have stopped developing (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0039] ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). That is, ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are ntES (nuclear transfer ES) cells. For the production of ntES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (described above) is used (Kiyoshi Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell can be injected into an enucleated unfertilized egg of a mammal and initialized by culturing for several hours.

[0040] mGS cells are pluripotent stem cells derived from the testes and are the origin of spermatogenesis. Similar to ES cells, these cells can be differentiated into various cell lineages, and possess properties such as the ability to create chimeric mice when transplanted into mouse blastocysts (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004), Cell, 119:1001-1012). They can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and germline stem cells can be obtained by repeatedly passaged under the same culture conditions as ES cells (Takebayashi, Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Suppl.), pp. 41-46, Yodosha (Tokyo, Japan)).

[0041] EG cells are pluripotent cells similar to ES cells, established from primordial germ cells during the embryonic stage. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0042] The origin of the pluripotent stem cells is not particularly limited and may include cells from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus macaques, marmosets, orangutans, and chimpanzees. The preferred origin is human.

[0043] In the present invention, a cell may be a single cell, but typically it is a population of multiple cells. Therefore, unless otherwise specified, "cell" in this specification includes "population of cells." A population of cells may consist of one type of cell, or it may consist of two or more types of cells.

[0044] The hematopoietic stem / progenitor cells used in this invention can be obtained by culturing pluripotent stem cells, as described above, in a culture medium containing a specific cytokine (e.g., IGF2).

[0045] The culture medium used in the manufacturing method of the present invention can typically be prepared using basal media or serum-free media for hematopoietic stem cells. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. Examples of serum-free media for hematopoietic stem cells include StemPro TM -34 SFM (Thermo Fisher Scientific), STEMα.A (Funakoshi), StemSpan TM Examples include XF (STEMCELL Technologies).

[0046] The culture medium may be serum-containing or serum-free. Serum-free medium (SFM) means a medium that does not contain any untreated or unpurified serum. When serum (e.g., fetal bovine serum (FBS), human serum, etc.) is used, its concentration in the medium may be 5-30%, preferably 10-20%. The SFM may or may not contain any serum substitute. Examples of serum substitutes include albumin (e.g., albumin substitutes such as bovine serum albumin, lipid-rich albumin, recombinant albumin, etc.), plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, sodium selenite, 2-mercaptoethanol, ethanolamine, 3'-thioglycerol, or equivalents thereof, etc. Such serum substitutes can be prepared, for example, by the method described in WO 98 / 30679. Alternatively, commercially available products can be used for convenience. Examples of such commercially available substances include Knockout® Serum Replacement (KSR), Chemically-defined Lipid concentrate, Glutamax (Invitorogen), and ITS supplements (e.g., ITS-G, ITS-A, ITS-X, all manufactured by Fujifilm Wako Pure Chemical Industries).

[0047] The culture medium may also contain other known additives. These additives are not particularly limited, but examples include growth factors (e.g., insulin), polyamines (e.g., putrescine), minerals (e.g., sodium selenite), sugars (e.g., glucose), organic acids (e.g., pyruvate, lactic acid), amino acids (e.g., non-essential amino acids (NEAA), L-glutamine), reducing agents (e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin), steroids (e.g., [beta]-estradiol, progesterone), antibiotics (e.g., streptomycin, penicillin, gentamicin), buffers (e.g., HEPES), nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement), and various cytokines (e.g., thrombopoietin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1)). It is preferable that each additive is included within a known concentration range.

[0048] The culture can be carried out by setting appropriate conditions for the survival of hematopoietic stem / progenitor cells based on known methods (e.g., WO 2014 / 200030), for example, by incubation under humid conditions at a CO2 concentration of 5% and 37°C. The culture period is not particularly limited as long as the desired amount of hematopoietic stem / progenitor cells can be obtained, but is typically 1 to 30 days, preferably 4 to 15 days. The culture may be adherent culture or suspension culture. The culture in the production method of the present invention may use the method for producing mesenchymal stem cells used in the present invention, which will be described later.

[0049] Culture may be carried out under feeder-free and / or xeno-free conditions. In the production method of the present invention, the entire process may be carried out under feeder-free and xeno-free conditions. In this specification, "feeder-free" means a culture medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play an auxiliary role in preparing the culture conditions for the cells to be cultured. Also, "xeno-free" means a culture medium or culture conditions that do not contain components of a different biological origin from the biological species of the cells to be cultured.

[0050] The manufacturing method of the present invention can also be described as a large-scale culture of hematopoietic stem / progenitor cells. In this specification, "large-scale culture" means culturing for the purpose of proliferating a desired cell population and increasing the number of cells. The increase in cell number is achieved by the increase in the number of cells due to proliferation exceeding the decrease in the number of cells due to death, and it is not necessary for all cells in the cell population to proliferate. The increase in cell number may be 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 300 times, 500 times, 1000 times, 3000 times, 5000 times, 10000 times or more compared to before the start of the large-scale culture.

[0051] The manufacturing method of the present invention may also involve co-culturing hematopoietic stem / progenitor cells with mesenchymal stem cells (MSCs). Mesenchymal stem cells are found at a low frequency in mesenchymal tissues and are thought to be present in tissues and organs throughout the body, albeit at varying frequencies. Bone marrow mesenchymal stem cells are found within bone marrow stromal cells and are a type of cell that supports hematopoietic cells. In addition to bone marrow, mesenchymal stem cells have been isolated from various tissues such as adipose tissue, placental tissue, umbilical cord tissue, and dental pulp.

[0052] The mesenchymal stem cells (MSCs) used in the manufacturing method of the present invention (also referred to as "bone marrow niche cells") are obtained, for example, by a method comprising: (1) culturing pluripotent stem cells in a medium containing a GSK3β inhibitor to obtain fibroblast-like cells; and (2) culturing the fibroblast-like cells obtained in step (1) in a medium containing fibroblast growth factor and a GSK3β inhibitor to obtain mesenchymal stem cells (hereinafter sometimes referred to as "the method for producing MSCs used in the present invention").

[0053] The method for producing MSCs used in the present invention may include a step of expanding the culture of mesenchymal stem cells obtained in step (2) (3).

[0054] Step (3) of the method for producing MSCs used in the present invention is not particularly limited as long as it can proliferate mesenchymal stem cells, but is carried out by, for example, culturing them in a mesenchymal stem cell maintenance medium (for example, a basal medium containing serum (for example, 10% FBS) (for example, αMEM medium)).

[0055] Furthermore, the method for producing MSCs used in the present invention may include a step of pre-culturing pluripotent stem cells to adjust the colony size before step (1). Such a step may include, for example, seeding an appropriate number of colonies (e.g., 5 or 6 colonies per well) into a culture vessel and culturing until the median colony size is 40 to 60 cells / colony (especially 50 cells / colony). In this culture step, cells are typically cultured in a pluripotent stem cell maintenance medium (e.g., a basal medium containing bFGF (e.g., NutriStem medium)).

[0056] The method for producing MSCs used in the present invention may include a step of isolating or purifying mesenchymal stem cells after step (2) or (3) above. By performing such a step, the proportion of mesenchymal stem cells in the cell population can be increased. The isolation or purification method can be carried out by a method known to the present day. For example, this can be done by labeling with an antibody against an indicator molecule (e.g., a surface antigen marker such as CD271 or SSEA-4) and purifying using a method using flow cytometry or mass cytometry, magnetic cell separation, or an affinity column immobilized with a desired antigen.

[0057] Examples of GSK3β inhibitors used in the MSC production method of the present invention include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridine-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazole-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CP21R7 (3-(3- Aminophenyl)-4-(1-methyl-1H-indole-3-yl)-pyrrole-2,5-dione), LY2090314 (3-[9-Fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1h-pyrrole-2,5-dione), TDZD-8 (4-benzyl-2-methyl-1,2, 4-Thiasiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yloxy]phenol), Kenpaullone, 1-Azakenpaullone, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4- Examples include (2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18 (1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), CT99021, CT20026, BIO ((2'Z,3'E)-6-bromoindilbine-3'-oxime), BIO-acetoxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, alpha-4-dibromoacetophenone, lithium, etc. Among these, CHIR99021 is preferred. These may be used individually or in combination of two or more.Furthermore, antisense oligonucleotides or siRNAs against GSK3β mRNA, antibodies that bind to GSK3β, and dominant-negative GSK3β variants can also be used as GSK3β inhibitors, and these are either commercially available or can be synthesized according to known methods.

[0058] In the method for producing MSCs used in the present invention, the concentration of the GSK3β inhibitor in the culture medium is appropriately adjusted depending on the type of GSK3β inhibitor added. For example, when CHIR99021 is used, it is typically 0.1 to 1 μM, preferably 0.5 to 1 μM, and more preferably 1 μM. For example, it is 0.01 to 20 μM, preferably 0.1 to 15 μM, and more preferably 0.5 to 10 μM (particularly 3.5 μM).

[0059] The fibroblast growth factors (FGF) used in the MSC production method of the present invention can usually be commercially available. There are 22 types of FGF known in humans, and any that function as a cell growth factor can be used, but bFGF (also called FGF-2) is preferred. Only one type of FGF may be used, or two or more types may be used in combination. The concentration of FGF in the culture medium can be appropriately selected by those skilled in the art depending on the FGF used, but for example, when using bFGF, it is typically 0.1 to 200 ng / ml, preferably 1 to 100 ng / ml, and more preferably 10 to 50 ng / ml (especially 20 ng / ml).

[0060] The culture period in step (1) of the method for producing MSCs used in the present invention is not particularly limited as long as it is the period during which fibroblast-like cells appear, but is typically 3 to 8 days, preferably 4 to 7 days, and more preferably 5 to 6 days.

[0061] The culture period in step (2) of the method for producing MSCs used in the present invention is not particularly limited as long as it is the period during which mesenchymal stem cells appear, but is typically 3 to 8 days, preferably 4 to 7 days, and more preferably 5 to 6 days.

[0062] The culture period in step (3) of the method for producing MSCs used in the present invention is not particularly limited because mesenchymal stem cells are maintained and proliferated by step (3), but is typically 5 to 50 days, preferably 10 to 40 days, and more preferably 15 to 30 days.

[0063] Examples of basal media used in the MSC production method of the present invention include, but are not limited to, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), Eagle MEM medium, αMEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, Hamm medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof.

[0064] The culture medium may be serum-containing or serum-free. In one embodiment, serum-free medium is used in steps (1) and (2) of the method for producing MSCs used in the present invention. In another embodiment, serum-containing medium is used in step (3) of the method for producing MSCs used in the present invention. Serum-free medium (SFM) means a medium that does not contain any untreated or unpurified serum. When serum (e.g., fetal bovine serum (FBS), human serum, etc.) is used, its concentration in the medium may be 5-30%, preferably 10-20%. SFM may or may not contain any serum substitute. Examples of serum substitutes include albumin (e.g., albumin substitutes such as lipid-rich albumin and recombinant albumin, plant starch, dextran and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, sodium selenite, 2-mercaptoethanol, ethanolamine, 3'-thioglycerol, or equivalents thereof, as appropriate. Such serum substitutes can be prepared, for example, by the method described in WO 98 / 30679. Alternatively, commercially available products can be used for greater convenience. Examples of such commercially available products include Knockout® Serum Replacement (KSR), Chemically-defined Lipid concentrated, Glutamax (Invitorogen), and ITS supplements (e.g., ITS-G, ITS-A, ITS-X, all manufactured by Fujifilm Wako Pure Chemical Industries).

[0065] The culture medium may also contain other known additives. These additives are not particularly limited, but examples include growth factors (e.g., insulin), polyamines (e.g., putrescine), minerals (e.g., sodium selenite), sugars (e.g., glucose), organic acids (e.g., pyruvate, lactic acid), amino acids (e.g., non-essential amino acids (NEAAs), L-glutamine), reducing agents (e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin), steroids (e.g., [beta]-estradiol, progesterone), antibiotics (e.g., streptomycin, penicillin, gentamicin), buffers (e.g., HEPES), and nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement). It is preferable that each additive is included within a concentration range known to the present day.

[0066] In one embodiment, the culture medium used in step (1) and / or (2) of the method for producing MSCs used in the present invention contains at least one (preferably all) selected from the group consisting of insulin, transferrin, sodium selenite, and ethanolamine. The concentration of insulin in the culture medium is typically 0.1 to 100 μg / mL, preferably 1 to 50 μg / mL, more preferably 3 to 20 μg / mL (particularly 10 μg / mL). The concentration of transferrin in the culture medium is typically 0.1 to 100 μg / mL, preferably 1 to 50 μg / mL, more preferably 2 to 10 μg / mL (particularly 5.5 μg / mL). The concentration of sodium selenite in the culture medium is typically 0.1 to 100 μg / L, preferably 1 to 50 μg / L, more preferably 2 to 10 μg / L (particularly 6.7 μg / L). The concentration of ethanolamine in the culture medium is typically 0.1 to 100 μg / mL, preferably 0.5 to 50 μg / mL, and more preferably 1 to 5 μg / mL (particularly 2 μg / mL).

[0067] The culture in each step of the MSC production method used in the present invention may be adherent culture or suspension culture. In the case of adherent culture, a culture vessel coated with an extracellular matrix component may be used, or it may be co-cultured with feeder cells. There are no particular limitations on feeder cells, but examples include fibroblasts (mouse fetal fibroblasts (MEF), mouse fibroblasts (STO), etc.). It is preferable that the feeder cells are inactivated by methods known to the present invention, such as irradiation with radiation (gamma rays, etc.) or treatment with anticancer agents (mitomycin C, etc.). Examples of extracellular matrix components include fibrous proteins such as Matrigel (Niwa A, et al. PLoS One.6(7):e22261, 2011), gelatin, collagen, and elastin, glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesion proteins such as fibronectin, vitronectin, and laminin. In one embodiment, in steps (1) and (2) of the method for producing MSCs used in the present invention, an incubator coated with Matrigel is used.

[0068] The incubators used for culture are not particularly limited, but examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles.

[0069] The culture temperature is not particularly limited, but is about 30 to about 40°C, preferably about 37°C, and the culture is carried out in the presence of CO2-containing air, with a CO2 concentration of preferably about 2 to 5%.

[0070] In this specification, “mesenchymal stem cells (MSCs)” means stem cells that have a fibroblast-like morphology and are positive for the surface antigen markers CD73, CD90, and CD105, and such MSCs typically have the ability to differentiate into one or more mesenchymal cells (e.g., osteoblasts, chondrocytes, adipocytes, etc.). In addition to the above characteristics of MSCs, the MSCs used in the production method of the present invention may also have the following characteristics (A) and (B): (A) They are derived from pluripotent stem cells. (B) They are positive for the surface antigen markers CD271 and SSEA-4.

[0071] Furthermore, the MSCs used in the manufacturing method of the present invention may also have at least one (preferably all) of the following characteristics (C) to (E): (C) Possesses hematopoietic stem / progenitor cell supporting ability. (D) Possesses at least one (preferably all) of the surface antigen markers selected from CD29, CD44, and HLA-ABC. (E) Possesses at least one (preferably all) of the surface antigen markers selected from CD31, CD34, CD41, CD45, and HLA-DR.

[0072] In this specification, "positive for a surface antigen marker" (or "expression of a surface antigen marker") is used to mean "production of the mRNA-encoded protein." Therefore, if the production of the mRNA-encoded protein is detected at a level above the background by cell sorting, the surface antigen marker can be said to be expressed. On the other hand, if the protein production is not detected by the same method (cell sorting) (i.e., below the detection limit) or is at a background level, the surface antigen marker can be said to be negative.

[0073] In this specification, "having hematopoietic stem / progenitor cell supporting ability" means that when co-cultured with hematopoietic stem / progenitor cells, the differentiation of hematopoietic stem / progenitor cells into other cells is inhibited (in other words, hematopoietic stem / progenitor cells are maintained). MSCs can be evaluated as having hematopoietic stem / progenitor cell supporting ability if, when cultured in co-culture with hematopoietic stem / progenitor cells, the proportion of hematopoietic stem / progenitor cells in the cell population is higher compared to when hematopoietic stem / progenitor cells are cultured for the same period in the absence of MSCs.

[0074] In the production method of the present invention, hematopoietic stem / progenitor cells obtained by culturing hematopoietic stem / progenitor cells in a medium containing a specific substance may be further isolated or purified using indicators such as CD34 negativity, CD34 positivity, differentiation antigen negativity, CD38 negativity, CD45 positivity, CD90 positivity, CD49f positivity, GPI-80 (also known as Vanin 2, VNN2, FOAP-4, etc.) positivity, and neutrophil marker negativity, and cell quality evaluation may also be performed. Such isolation, purification, and quality evaluation can be performed by known methods such as flow cytometry, mass cytometry, magnetic cell separation, or affinity columns immobilized with the desired antigen (e.g., WO 2014 / 200030, WO 2015 / 163185, etc.).

[0075] 2. The hematopoietic stem / progenitor cells obtained by the production method of the present invention (hereinafter sometimes referred to as "the hematopoietic stem / progenitor cells of the present invention") can be suitably used in cell transplantation therapy. Accordingly, in another embodiment of the present invention, a cell transplantation therapy agent (hereinafter sometimes referred to as "the cell transplantation therapy agent of the present invention") containing the hematopoietic stem / progenitor cells of the present invention is provided. Furthermore, a method for treating or preventing tissue damage (including defects) or disease by administering or transplanting an effective amount of the hematopoietic stem / progenitor cells of the present invention into a target mammal (e.g., human, mouse, rat, monkey, cattle, horse, pig, dog, etc.) is also provided. It is clearly encompassed. Furthermore, "treatment of tissue damage" also includes the regeneration of damaged tissue. Examples of such diseases include hematopoietic malignancies (leukemia, lymphoma, myeloma) and other blood disorders (e.g., primary immunodeficiency, aplastic anemia, myelodysplasia). As described above, the MSCs used in the manufacturing method of the present invention have hematopoietic stem / progenitor cell supporting ability. Therefore, when transplanting hematopoietic stem / progenitor cells into a living organism, co-transplanting the mesenchymal cells of the present invention can be expected to improve the survival rate of hematopoietic stem / progenitor cells. Accordingly, the cell transplantation agent of the present invention can also use the MSCs used in the manufacturing method of the present invention as a co-transplantation agent.

[0076] When using the hematopoietic stem / progenitor cells of the present invention in cell transplantation therapy, it is desirable to use cells isolated from the bone marrow, peripheral blood, umbilical cord blood, placenta, etc., of an individual whose HLA genotype is identical or substantially identical to that of the recipient individual, or cells derived from iPS cells established from somatic cells whose HLA genotype is identical or substantially identical to that of the recipient individual, from the viewpoint of preventing rejection. Here, "substantially identical" means that the HLA genotype matches to the extent that the immune response to the transplanted cells can be suppressed by immunosuppressants. For example, this includes somatic cells having an HLA type in which the three gene loci of HLA-A, HLA-B, and HLA-DR, or four gene loci including HLA-C, match. If sufficient cells cannot be obtained due to age, constitution, etc., it is also possible to transplant them while avoiding rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0077] The hematopoietic stem / progenitor cells of the present invention are manufactured as parenteral formulations such as injections, suspensions, and intravenous infusions by mixing them with a pharmaceutically acceptable carrier according to conventional methods. Accordingly, in one embodiment, a method for manufacturing a cell transplantation therapy agent is also provided, which includes a step of manufacturing the hematopoietic stem / progenitor cells of the present invention. Such a manufacturing method may include a step of preparing the hematopoietic stem / progenitor cells of the present invention. Furthermore, it may also include a step of preserving the hematopoietic stem / progenitor cells of the present invention.

[0078] Examples of pharmaceutically acceptable carriers that may be included in the parenteral formulation include aqueous solutions for injection such as physiological saline, glucose, and other isotonic solutions containing adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The cell transplantation therapy agent of the present invention may also be formulated with, for example, buffering agents (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. When the cell transplantation therapy agent of the present invention is formulated as an aqueous suspension, for example, about 1 × 10⁶ of the above aqueous solution may be added. 6 ~Approx. 1×10 8The cells should be suspended to a concentration of cells / ml. Furthermore, the dosage or transplantation amount and the number of administrations or transplants of the hematopoietic stem / progenitor cells or cell transplantation therapy agent of the present invention can be appropriately determined depending on the age, weight, symptoms, etc., of the mammal to be administered the therapy.

[0079] The cell transplantation therapy agent of the present invention is provided in a state of cryopreservation under conditions normally used for cell cryopreservation, and can be thawed and used at the time of use. In this case, it may further contain serum or a substitute thereof, an organic solvent (e.g., DMSO), etc. In this case, the concentration of serum or a substitute thereof is not particularly limited but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0080] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0081] Example 1: Induction of Mesenchymal Stem Cells iPS cells were maintained in a 6-well plate coated with Matrigel until the colony size reached approximately 50 cells / colony. NutriStem medium (2 ml / well) containing 10 ng / mL bFGF was used as the culture medium. The entire volume of the maintained iPS cell medium was replaced with DMEM / F12 medium containing 1% (v / v) ITS ITS-X (100X) (Fujifilm Wako Pure Chemical Industries) and 3.5 μM CHIR99021 to initiate differentiation induction (Day 0). Culture was continued in the same medium until Day 5 or 6. During this time, the medium was changed daily (at this point, infiltration of cells with fibroblast-like morphology from the cell colonies was observed). Next, the cells were detached from each well and subcultured into gelatin-coated T75 culture flasks. DMEM / F12 medium (10 ml / flask) containing 1% (v / v) ITS-X (100X), 20 ng / ml bFGF, and 3.5 μM CHIR99021 was used as the culture medium. The following day, the medium was replaced with a medium of the same composition to remove dead cells, and the culture was continued. During this process, the medium was replaced every 2-3 days. When the cells became confluent on the 11th or 12th day of culture, 1 / 10 of the medium containing the cells was subcultured into a gelatin-coated T75 culture flask. αMEM medium containing 10% FBS was used as the culture medium. The remaining 9 / 10 of the medium was cryopreserved. Mesenchymal stem cells (iDP MSCs) obtained by continuing the culture until the 30th day of culture were used in the following experiments (images of the cells taken with a phase-contrast microscope are shown in Figure 2).

[0082] For comparison, human bone marrow-derived DP MSCs (BM-DP MSCs) were established using the method described in Matsuoka Y et al., Stem Cells. 33(5):1554-65, 2015. Briefly, human mononuclear cells (MNCs) were isolated from bone marrow samples of healthy donors using density gradient centrifugation, further enriched using the EasySep Human Progenitor Cell Enrichment Kit (STEMCELL Technologies), and lineage-positive cells were depleted by treatment with RoboSep (STEMCELL Technologies). Subsequently, Lin - BM cells were stained with antibodies against 11 types of lin-specific antibodies (CD2, CD16, CD24, and CD235a (DAKO), CD3, CD41, and CD66c (Beckman Coulter), as well as CD4 and CD14 (eBioscience), CD19, and CD56 (BD Biosciences)), anti-CD45 antibody (BioLegend), anti-SSEA-4 antibody (R&D Systems), and 7-aminoactinomycin D (7-AAD) (Beckman Coulter). CD271 and SSEA-4 co-positive cells were sorted using FACSAria (BD Biosciences) or FACSAria III (BD Biosciences). The harvested cells were cultured in α-MEM (Nacalai Tesque) containing 10% fetal bovine serum (BioWest) in a T-75 culture flask (BD Biosciences) under fully humidified atmosphere at 37°C and 5% CO2. Antibody information is shown in Table 1.

[0083] Example 2: Surface Antigen Analysis To analyze the expression of surface markers in iDP MSCs, cells were stained with anti-CD29, anti-CD31, anti-CD34, anti-CD41, anti-CD44, anti-CD45, anti-CD73, anti-CD90, anti-CD105, anti-CD271, anti-SSEA-4, anti-HLA-ABC, and anti-HLA-DR antibodies conjugated with FITC, PE, APC, or PB. As a negative control, samples stained with isotype IgG antibodies conjugated with FITC, PE, APC, or PB were prepared. The stained cells were analyzed using FACSCantoII (BD Biosciences). Antibody information is shown in Table 1.

[0084]

[0085] The results are shown in Figures 2 and 3. These results reveal that iDP MSCs exhibit similar morphology and surface antigen expression patterns to BM-DP MSCs.

[0086] Example 3: Evaluation of the support capacity of human hematopoietic stem / progenitor cells The support capacity of iDP MSCs for human hematopoietic stem / progenitor cells was evaluated by co-culturing them with human umbilical cord blood-derived hematopoietic stem cells. Human umbilical cord blood-derived hematopoietic cells were isolated by the method described in Sumide K et al., Nature Communications. 9(1):2202, 2018. Briefly, human mononuclear cells (MNCs) were isolated from umbilical cord blood samples from healthy donors using density gradient centrifugation, and further enriched using the EasySep Human Progenitor Cell Enrichment Kit (STEMCELL Technologies) to deplete lineage-positive cells. Then, Lin -The cells were stained with antibodies against 18 types of lineage-specific antibodies (CD2, CD16, CD24 and CD235a (DAKO), CD3, CD7, CD10, CD11b, CD20 and CD41 (Beckman Coulter), and CD4, CD14, CD33, and CD127 (eBioscience), CD19 and CD56 (BD Biosciences), CD45RA (SouthernBiotech), CD66b (BioLegend)), an anti-CD45 antibody (BioLegend), an anti-CD34 antibody (BD Biosciences), an anti-CD38 antibody (BD BioLegend), an anti-CD133 / 1 antibody (Miltenyi Biotec), an anti-GPI-80 antibody (MBL), and 7-aminoactinomycin D (7-AAD) (Beckman Coulter). Using FACSAria (BD Biosciences) or FACSAria III (BD Biosciences), Lin - CD34 + CD38 - CD133 + GPI-80 + fraction cells were sorted. The antibody information is shown in Table 1. The cells recovered by this method are hereinafter referred to as CD34 + HCS. The recovered CD34 + HSCs (hematopoietic stem cells) were seeded at 1,000 cells per well on iDP MSCs or DP MSCs seeded as feeder cells in a 24-well plate (BD Biosciences). Then, in a fully humidified atmosphere at 37°C, 5% CO2, and 5% O2, 1% ITS-X (100X) (FUJIFILM Wako Pure Chemical Corporation), 10 ng / mL thrombopoietin (PeproTech), and 50 ng / mL stem cell factor (PeproTech) were added and cultured in StemPro-34 SFM (Thermo Fisher Scientific) for 7 days. Thereafter, the cells were recovered by pipetting, stained with an anti-CD45 antibody, an anti-CD34 antibody, and 7-AAD, and then the proportion of the CD45 + CD34 + fraction in the 7-AAD-negative fraction was evaluated using FACSCanto II.

[0087] The results are shown in Figure 4. From Figure 4, it became clear that iDP MSCs exhibit an expression pattern that shows a similar level of supportive capacity for human hematopoietic stem / progenitor cells as BM-DP MSCs.

[0088] Example 4: Evaluation of substances used in the production method of the present invention in a co-culture system. The effects of the substances used in the production method of the present invention shown in Table 2 were evaluated by adding them to a system in which iDP MSCs obtained in Example 1 and human hematopoietic stem / progenitor cells obtained by the method described below were co-cultured.

[0089]

[0090] Human hematopoietic stem cells were isolated according to a known method (WO 2015 / 163185). Specifically, differentiation antigen-positive cells were removed from human umbilical cord blood using the EasySep Human Progenitor Cell Enrichment Kit (StemCell Technologies). The remaining differentiation antigen-negative cells were stained with anti-Lineage marker antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD133 antibody, and anti-GPI-80 antibody. The Lineage-negative, CD34-positive, CD38-negative, CD133-positive, and GPI-80-positive fraction was then isolated using a cell sorter (FACS AriaIII, BD Biosciences) and used as hematopoietic stem cells. The obtained hematopoietic stem cells were co-cultured with iDP MSCs obtained in Example 1.

[0091] 600 hematopoietic stem cells were cultured for 11 days in a medium containing equal amounts of RPMI1640 and D-MEM, supplemented with cytokines (thrombopoietin, stem cell factor, and stromal cell-derived factor-1 (SDF-1)) and bovine serum albumin, with the addition of the listed small molecular weight compounds (UM171 (35 nM), Valproic acid (VPA) (50 μM), Lenalidomide (LEN) (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide (CFOC) (500 nM), and all seven of these compounds (at the same concentrations as for individual compounds)). Dimethyl sulfoxide (DMSO) was added to the control group.

[0092] Subsequently, the cells were harvested and stained with anti-CD34 antibody, anti-CD38 antibody, anti-CD133 antibody, and anti-CD90 antibody. The absolute number of CD34-positive, CD38-negative, CD133-positive, and CD90-positive cells was evaluated using a flow cytometer (Attune NxT, Thermo Fisher Scientific). Using this value, the number of CD34-positive, CD38-negative, CD133-positive, and CD90-positive cells produced from one pre-culture HSC was calculated.

[0093] The results are shown in Figure 5. From Figure 5, it was revealed that UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC all had an amplification effect on hematopoietic stem / progenitor cells. In particular, it was revealed that GSK591 and YM155 had high amplification effects. Furthermore, it was revealed that using all seven of the above substances resulted in a remarkable amplification effect.

[0094] Example 5: FACS plot of HSC-derived producible cells after co-culture. Cells cultured in Example 4 were harvested on day 12 and analyzed by flow cytometry. Specifically, when developing with forward scatter and side scatter, a gate was set for the hematopoietic cell population, and the viable cell population negative for 7-Amino-Actinomycin D (7-AAD) was analyzed. Subsequently, the CD34-positive, CD38-negative fraction was gated, and development was performed with CD133 and CD90. Finally, the CD34-positive, CD38-negative, CD133-positive, CD90-positive cells were gated, and their proportions were evaluated.

[0095] The results are shown in Figure 6. Figure 6 also clearly shows that UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC all have an amplification effect on hematopoietic stem / progenitor cells. Furthermore, it was revealed that GSK591 and YM155, in particular, exhibit high amplification effects. It was also found that using all seven of the above substances together produced a remarkable amplification effect.

[0096] Example 6: FACS plot of HSC-derived cells after co-culture. HSCs were cultured under the same conditions as in Example 4, and dose-dependent studies were performed when VER155008 and JTE607 were added individually. Cells were harvested on day 14 of co-culture and stained with anti-CD34 antibody, anti-CD38 antibody, anti-CD133 antibody and anti-EPCR antibody or anti-CD34 antibody, anti-CD38 antibody, anti-CD133 antibody and anti-CD146 antibody. The absolute number of CD34-positive, CD38-negative, CD133-positive, EPCR-positive, or CD34-positive, CD38-negative, CD133-positive, CD146-positive cells produced from one HSC was evaluated using a flow cytometer.

[0097] The results are shown in Figure 7. From Figure 7, it is clear that VER155008 and JTE607 also have an amplification effect on hematopoietic stem / progenitor cells.

[0098] Example 7: Evaluation of Substances Used in the Production Method of the Present Invention in a Co-Culture System The effects of the substances used in the production method of the present invention shown in Tables 21-4 and 7-9 were evaluated under the same conditions (culture period of 12 days) and methods as in Example 4. Specifically, the effects of simultaneously adding all seven compounds (UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM)) or six compounds (-UM171, -VPA, -LEN, -GSK591, -YM155, -Cu-CPT22, or -CFOC, respectively) were evaluated.

[0099] The results are shown in Figure 8. Under these concentration conditions, the removal of GSK591 reduced the cell amplification efficiency in the combination of the seven compounds, indicating that it plays a major role in the mixture of the seven compounds (Figure 8). Furthermore, it was understood that any combination of the six compounds used in the production method of the present invention can exhibit a favorable effect compared to the control (Figure 8).

[0100] Example 8: Evaluation of Substances Used in the Production Method of the Present Invention in a Co-Culture System 3 The effects of the substances used in the production method of the present invention shown in Tables 21-4 and 7-9 were evaluated under the same conditions (culture period of 13 days) and methods as in Example 4. Specifically, the effects of all seven compounds (UM171, VPA, LEN, GSK591, YM155, Cu-CPT22, and CFOC), as well as the effects of each compound individually or in all combinations of these four compounds, were evaluated.

[0101] The results are shown in Figure 9. The combination of GSK591 and CFOC was shown to achieve an amplification effect of about two-thirds that of the combination with seven compounds, even with only two compounds (Figure 9). Furthermore, it was understood that all combinations of the one or two to four compounds used in the example of the substance used in the production method of the present invention can exhibit a favorable effect compared to the control (Figure 9).

[0102] Example 9: Transplantation Experiment of Amplified HSCs into Severely Immunodeficient Mice Transplantation experiments of amplified human umbilical cord blood-derived hematopoietic stem cells (HSCs) obtained by the same method as in the above example was performed in severely immunodeficient mice (NSG mice; NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ, 6 weeks old) (Jackson Laboratory Japan) after co-culture of HSCs with iDP MSCs was performed. HSCs were cultured for 22 days at 1 cell / well in the presence of UM171 (35 nM), VPA (50 μM), LEN (75 nM), GSK591 (200 nM), YM155 (50 nM), Cu-CPT22 (1 μM), and CFOC (500 nM). Cells derived from one HSC were harvested, 1 / 4 of the cells were analyzed using a flow cytometer (Attune NxT flow cytometer, Thermo Fisher Scientific), and the remaining 3 / 4 of the cells were transplanted into NSG mice. Four weeks after transplantation, mouse bone marrow was collected, and the engraftment of human CD45-positive cells in the mouse bone marrow was analyzed using a flow cytometer.

[0103] The results are shown in Figure 10. Of the three NSG mice transplanted with HSCs co-cultured using the substance used in the manufacturing method of the present invention, engraftment of human blood cells was observed in two mice (Figure 10). Therefore, it was understood that hematopoietic stem / progenitor cells produced using the substance used in the manufacturing method of the present invention can be adequately used as transplantation therapy or as a transplantation therapy agent.

[0104] This invention makes it possible to mass-produce hematopoietic stem / progenitor cells. Hematopoietic stem / progenitor cells are necessary for cell transplantation therapy for hematopoietic malignancies (leukemia, lymphoma, myeloma) and other blood disorders (e.g., primary immunodeficiency, aplastic anemia, myelodysplasia), and are therefore extremely useful, especially in the medical field.

[0105] This application is based on Japanese Patent Application No. 2024-186937 (filing date: October 23, 2024), the contents of which are fully incorporated herein.

Claims

1. A method for producing hematopoietic stem cells / progenitor cells, comprising the step of culturing hematopoietic stem / progenitor cells in a medium containing at least one selected from the group consisting of a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, and a heat shock protein 70 family inhibitor.

2. The method according to claim 1, wherein at least one protein arginine methyltransferase 5 inhibitor is selected.

3. The method according to claim 1 or 2, wherein the culture medium further comprises at least one selected from the group consisting of a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, an inflammatory cytokine inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a heat shock protein 70 family inhibitor, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative (excluding the substance selected in claim 1 or 2).

4. The method according to claim 3, wherein the culture medium comprises a protein arginine methyltransferase 5 inhibitor, a survivorin inhibitor, a Toll-like receptor 1 / 2 inhibitor, N-cyclopropyl-5-(furan-2-yl)-1,2-oxazole-3-carboxamide, a lysine-specific demethylase 1 inhibitor, a histone deacetylase inhibitor, and a thalidomide derivative.

5. The method according to claim 1 or 2, wherein the protein arginine methyltransferase 5 inhibitor is GSK591, the survivorin inhibitor is YM155, the Toll-like receptor 1 / 2 inhibitor is Cu-CPT22, the inflammatory cytokine inhibitor is JTE607, the heat shock protein 70 family inhibitor is VER155008, the lysine-specific demethylase 1 inhibitor is UM171, the histone deacetylase inhibitor is valproic acid, and the thalidomide derivative is lenalidomide.

6. The method according to claim 1 or 2, wherein the culture is a co-culture with mesenchymal stem cells.

7. The method according to claim 6, wherein the mesenchymal stem cells are positive for CD271 and SSEA-4.

8. Hematopoietic stem / progenitor cells obtained by the method described in claim 1 or 2.

9. A cell transplantation therapy agent comprising the cells described in claim 8.

10. The cell transplantation therapy agent according to claim 9, further comprising mesenchymal stem cells that are positive for CD271 and SSEA-4.

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