Method for culturing cells and composition

Abstract

To provide a culture method for proliferating hematopoietic stem cells or hematopoietic stem progenitor cells while keeping a high ratio of hematopoietic stem cells or hematopoietic stem progenitor cells in the finally cultured cell population during the proliferative culture of the hematopoietic stem cells or hematopoietic stem progenitor cells, and a culture composition for use in these methods.SOLUTION: A cell culture composition comprises cytokines for use in the culture of Lin(-)CD34(+) hematopoietic stem progenitor cells, or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells, wherein the cell culture composition comprises (1) a histone deacetylase inhibitor (HDAC inhibitor), and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3 L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3 L.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a cell culture composition containing an HDAC inhibitor and / or a cytokine for use in culturing hematopoietic stem progenitor cells or hematopoietic stem cells, a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells using the cell culture composition, a method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells using the cell culture composition, and the use of a cell culture composition containing an HDAC inhibitor and / or a cytokine in culturing hematopoietic stem progenitor cells or hematopoietic stem cells.

Background Art

[0002] Stem cells that maintain self-renewal ability and pluripotency over a long period, for example, throughout the life of an organism, can be mass-cultured using their self-renewal ability, and can be differentiated into various cells using their pluripotency. Stem cells having such characteristics are considered to be key cells in the practical application of regenerative medicine. In addition, progenitor cells, which are lineage-committed cells derived from pluripotent stem cells, although differentiated from pluripotent stem cells, still have the ability to differentiate into one or more cell types. Such progenitor cells are also extremely useful in the practical application of regenerative medicine.

[0003] Hematopoietic stem cells (HSC) are mainly present in the bone marrow, are defined as blood cells having self-renewal ability and pluripotency, and are used in hematopoietic stem cell transplantation and the like. Hematopoietic stem progenitor cells or hematopoietic progenitor cells retain the ability to differentiate into one or more of three hematopoietic lineages (lymphocyte lineage, myeloid lineage, or erythroid / megakaryocyte lineage), and are used in regenerative medicine. When these hematopoietic stem cells and hematopoietic stem progenitor cells are proliferated and cultured by culturing, lineage commitment and differentiation and maturation progress in some cells during the culture process, and it is difficult to obtain a sufficient amount of hematopoietic stem cells and hematopoietic stem progenitor cells.

[0004] Therefore, various attempts have been made to proliferate and culture hematopoietic stem cells or hematopoietic stem progenitor cells (Patent Documents 1 to 10, Non-Patent Document 2). However, there is still a need to provide a new culture method for proliferating hematopoietic stem cells or hematopoietic stem progenitor cells while maintaining a high proportion of hematopoietic stem cells or hematopoietic stem progenitor cells contained in the finally cultured cell population, and a new culture composition for use in such culture.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present disclosure provides a culture method for proliferating hematopoietic stem cells or hematopoietic stem progenitor cells, and a culture composition for use in these methods, while maintaining a high proportion of hematopoietic stem cells or hematopoietic stem progenitor cells contained in the finally cultured cell population when proliferating and culturing hematopoietic stem cells or hematopoietic stem progenitor cells.

Means for Solving the Problems

[0008] As a result of intensive research on the above problems, the inventors of the present disclosure unexpectedly found that by using a cell culture composition containing a combination of certain cytokines or a combination of a cytokine and a histone deacetylase inhibitor (HDAC inhibitor), it is possible to promote the proliferation of hematopoietic stem cells or hematopoietic stem progenitor cells while maintaining a high proportion of hematopoietic stem cells or hematopoietic stem progenitor cells contained in the finally cultured cell population when proliferating and culturing hematopoietic stem cells or hematopoietic stem progenitor cells.

[0009] In one aspect, the present disclosure provides a cell culture composition containing an HDAC inhibitor and / or a cytokine for use in the culture of hematopoietic stem progenitor cells or hematopoietic stem cells.

[0010] In one aspect, a step of preparing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells; and a step of culturing the cell population using a cell culture composition containing an HDAC inhibitor and / or a cytokine, are provided, which is a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells.

[0011] In one aspect, a step of preparing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells; and a step of culturing the cell population using a cell culture composition containing an HDAC inhibitor and / or a cytokine, Provided is a method for producing a cell population comprising hematopoietic stem progenitor cells or hematopoietic stem cells, which comprises

[0012] In one aspect, the present disclosure provides the use of a cell culture composition comprising an HDAC inhibitor and / or a cytokine in the culture of hematopoietic stem progenitor cells or hematopoietic stem cells.

[0013] In one aspect, the cytokine used in the present disclosure is a cytokine selected from the group consisting of interleukin 6 (IL-6), interleukin 3 (IL-3), and Fms-related tyrosine kinase 3 ligand (Flt3L).

[0014] More specifically, in one aspect, the present disclosure provides the following. [Item 1] A cell culture composition comprising a cytokine for use in the culture of Lin(-)CD34(+) hematopoietic stem progenitor cells (Lin-CD34+ cells) or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells (Px cells), which (1) a histone deacetylase inhibitor (HDAC inhibitor), and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L The cell culture composition comprising the same. [Item 2] The cell culture composition according to Item 1, comprising an HDAC inhibitor, IL-6, and IL-3 or Flt3L. [Item 3] The cell culture composition according to Item 1, comprising an HDAC inhibitor, IL-6, IL-3, and Flt3L. [Item 4] The cell culture composition according to item 1, wherein the HDAC inhibitor is an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61. [Item 5] Preparing a cell population comprising hematopoietic stem progenitor cells (Lin-CD34+ cells) that are Lin(-)CD34(+) or hematopoietic stem cells (Px cells) that are CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and Culturing the cell population using a cell culture composition containing cytokines, wherein the cell culture composition (1) an HDAC inhibitor and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L A method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, comprising the step of [Item 6] The culture method according to item 5, wherein the cell culture composition contains an HDAC inhibitor, IL-6, and IL-3 or Flt3L. [Item 7] The culture method according to item 5, wherein the cell culture composition contains an HDAC inhibitor, IL-6, and IL-3 and Flt3L. [Item 8] The culture method according to item 5, wherein the HDAC inhibitor is an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61. [Item 9] A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells using the culture method according to item 5. [Item 10] In the culture method according to item 5, (1) One or more cytokines selected from the group consisting of an HDAC inhibitor and IL-6, IL-3, and Flt3L; or (2) Two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L Use of a cell culture composition containing the same. [Advantages of the Invention]

[0015] The present disclosure provides a culture method for proliferating hematopoietic stem cells or hematopoietic stem progenitor cells, and a culture composition for use in these methods, while maintaining a high proportion of hematopoietic stem cells or hematopoietic stem progenitor cells contained in the finally cultured cell population when proliferating and culturing hematopoietic stem cells or hematopoietic stem progenitor cells. [Brief Description of the Drawings]

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

DETAILED DESCRIPTION OF THE INVENTION

[0017] (Definition) In the present disclosure, when a plurality of numerical ranges are shown, a range consisting of any combination of the lower limit value and the upper limit value of those plurality of ranges is also meant in the same way.

[0018] In the present disclosure, "differentiation" has the meaning commonly used in the art, and typically means a process in which a cell with a lower degree of specialization changes into a more specialized cell such as a nerve cell or a muscle cell. "Differentiated" or "differentiating" is a relative term, and a "differentiated cell" or "differentiated cells" means that it has progressed further along the developmental pathway and is more specialized than the cell with which it is being compared.

[0019] In the present disclosure, the term "stem cell" has the meaning commonly used in the art, and typically refers to an undifferentiated cell that has the ability to self-renew at the single cell level and the ability to produce two or more different differentiated cells. That is, stem cells can divide asymmetrically, where one daughter cell retains the stem cell state and the other daughter cell expresses a distinct other specific biological function and phenotype. Alternatively, stem cells can divide symmetrically into two stem cells, and thus, in a cell population containing stem cells, some stem cells are maintained while other cells in the population give rise only to differentiated progeny.

[0020] In the present disclosure, the term "hematopoietic stem cell" has the meaning commonly used in the art, and typically refers to a cell with pluripotency that can ultimately differentiate into all blood cells (such as red blood cells, white blood cells, and platelets). Hematopoietic stem cells can include intermediate stages of differentiation into progenitor cells or blast cells. The terms "progenitor cell" or "blast cell" are used interchangeably in the present disclosure and refer to maturing cells that have reduced differentiation potential but still have the ability to mature into different cells of a specific lineage (e.g., the myeloid or lymphoid lineage).

[0021] In the present disclosure, the term "hematopoietic stem and progenitor cell" or "hematopoietic progenitor cell" has the meaning commonly used in the art, and typically refers to a cell that retains the ability to differentiate into one or more of three hematopoietic lineages (lymphoid lineage, myeloid lineage, or erythroid / megakaryocytic lineage).

[0022] The hematopoietic stem cells and hematopoietic stem progenitor cells of the present disclosure include hematopoietic stem cells and hematopoietic stem progenitor cells derived from various animals. For example, hematopoietic stem cells or hematopoietic stem progenitor cells derived from various mammals, animals belonging to the primate order, animals belonging to the monkey order, or humans can be used in the present disclosure, or the present disclosure can be applied to these cells. In addition, the hematopoietic stem cells and hematopoietic stem progenitor cells of the present disclosure can be isolated from a somatic cell population obtained from a living body, and can also be isolated from a cell population induced to differentiate from ES cells or induced pluripotent stem cells.

[0023] In the present disclosure, "isolated cells" has the meaning commonly used in the art, and typically means cells removed from the organism in which they are originally found or the progeny of such cells. Such cells may be cultured in vitro, for example, in the presence of other cells. Also, such cells or cells that are their progeny may later be introduced into a second organism.

[0024] In the present disclosure, "isolated cell population" has the meaning commonly used in the art, and typically means a population of cells removed and separated from a heterogeneous cell population. In some embodiments, the isolated population may be a substantially pure cell population compared to the heterogeneous cell population from which the cells were isolated or concentrated, or may still be a heterogeneous cell population.

[0025] A cell population being "substantially pure" with respect to a particular cell type has the meaning commonly used in the art. Typically, in such a cell population, at least about 50%, more preferably at least about 60%, 65%, 70%, 75%, 85%, 90%, 92%, 93%, most preferably at least about 95%, 96%, 97%, 98%, 99% of the cells making up the entire cell population are composed of cells of the particular cell type. For example, a "substantially pure" stem cell population means a population of cells containing less than about 50%, more preferably about 40%, 30%, 20%, 15%, 10%, 8%, less than 7%, most preferably about 5%, 4%, 3%, 2% or less than 1% of cells that are not stem cells. Thus, for example, an isolated hematopoietic stem cell in the present disclosure may be a cell population containing at least about 50%, more preferably at least about 60%, 65%, 70%, 75%, 85%, 90%, 92%, 93%, most preferably at least about 95%, 96%, 97%, 98%, 99% of cells identified by the markers specified by the present disclosure.

[0026] In the present disclosure, "enriched" or "enriched for" has the meaning commonly used in the art and typically means increasing the proportion of organisms, cells, substances, data, etc. having a particular property of interest in a population containing a plurality of organisms, cells, substances, data.

[0027] In the present disclosure, "identifying", "isolating", "sorting", "isolating", "purifying" or "screening" has the meaning commonly used in the art and typically refers to selecting a target such as an organism, cell, substance, data, etc. having a particular property of interest from a population containing a large number by a specific operation / evaluation method. The selected target may or may not be physically separated from the population.

[0028] In the present disclosure, "promote proliferation" of hematopoietic stem cells and the like has the meaning commonly used in the art, and typically means shortening the time required for hematopoietic stem cells to undergo cell division, increasing the number of hematopoietic stem cells, or suppressing a decrease in the content ratio of hematopoietic stem cells accompanying proliferation, or maintaining or increasing the content ratio in a cell composition containing hematopoietic stem cells and other cells.

[0029] In the present disclosure, "cytokine" has the meaning commonly used in the art, and typically means a bioactive protein secreted from various cells that has an activating effect or a function-suppressing effect on immune cells. As cytokines, interleukins secreted from leukocytes and having an immunomodulatory effect, chemokines that induce leukocyte migration, interferons that suppress virus and cell proliferation, and the like are known. In one aspect, the cytokine used in the present disclosure is a cytokine isolated or purified from a living body. The species of the cytokine to be used is not particularly limited as long as the effects of the present disclosure are achieved, and can be appropriately selected according to the hematopoietic stem cells or hematopoietic stem progenitor cells to which it is applied. For example, cytokines derived from animals belonging to the primate order, animals belonging to the order of monkeys, or humans, or various mammals such as mice, cats, dogs, pigs, cows, horses, camels, etc. can be used, but are not limited thereto. In one aspect, the cytokine used in the present disclosure is a cytokine produced using cultured cells and the like by utilizing genetic recombination technology. The method for producing cytokines using genetic recombination technology is known, and those skilled in the art can appropriately select the protein expression system such as the species of the cytokine to be produced and the cells to be used according to the purpose. In one aspect, "cytokine" includes cytokine mutants and cytokine analogs described below.

[0030] In the present disclosure, the term "cytokine variant" has the meaning commonly used in the art, and typically refers to a compound in which some of the amino acids constituting the natural cytokine are mutated. The mutation of the amino acid includes, but is not limited to, substitution, deletion, and insertion of amino acids in one or more consecutive or non-consecutive amino acids. When an amino acid is substituted or inserted, the amino acid introduced by the substitution or insertion can be a natural or non-natural amino acid. In one aspect, as the cytokine variant, a peptide composed of only some of the amino acids constituting the natural cytokine can be used.

[0031] In the present disclosure, the term "cytokine mimetic" has the meaning commonly used in the art, and typically refers to a compound that induces or enhances the signal transduction caused by the binding of the cytokine to its receptor. In one aspect, the cytokine mimetic includes a ligand that activates the receptor of the cytokine. In one aspect, the cytokine mimetic includes a fusion protein in which the cytokine or its variant is fused with another compound, a compound in which a translational modification is added to the cytokine or its variant, and a compound having an action of activating the receptor of the cytokine. Such cytokine mimetics may be compounds having a structure of a protein or peptide, nucleic acid, small molecule compound, or a combination thereof.

[0032] When IL-6 is used as the cytokine in the present disclosure, "IL-6" includes natural IL-6, IL-6 variants, and IL-6 mimetics. For example, a compound of CAS No. 339303-87-6 (GP130 receptor agonist-1: MedChemExpress) can be used as the IL-6 mimetic, but is not limited thereto.

[0033] When using IL-3 as a cytokine in the present disclosure, "IL-3" includes natural IL-3, IL-3 variants and IL-3 analogs. For example, SC-55494 (Blood., 1996, Volume 87, Issue 2, pp.581-591, Proc Natl Acad Sci U S A. 1995, Vol 92(9): pp.3779-3783) can be used as an IL-3 analog, but is not limited thereto.

[0034] When using Flt3L as a cytokine in the present disclosure, "Flt3L" includes natural Flt3L, Flt3L variants and Flt3L analogs. As such an Flt3L analog, GS-3583 (Journal of Clinical Oncology, 2021, Vol.39, no.15, pp.2559-2559) can be used, but is not limited thereto.

[0035] (Cell culture composition containing an HDAC inhibitor and / or a cytokine) In one aspect, the present disclosure provides a cell culture composition containing an HDAC inhibitor and / or a cytokine. The cell culture composition containing an HDAC inhibitor and / or a cytokine of the present disclosure is typically used for culturing hematopoietic stem progenitor cells or hematopoietic stem cells.

[0036] As the HDAC inhibitor contained in the culture composition of the present disclosure, a substance having inhibitory activity against histone deacetylase can be used without particular limitation. Histone deacetylase is an enzyme that regulates chromatin remodeling and is extremely important for epigenetic regulation of various genes. Non-limiting examples of substances that can be used as HDAC inhibitors include trichostatin A (TSA), valproic acid, sodium butyrate, butyryl hydroxamic acid, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61 (see T. Suzuki et al., J. Med. Chem. 2006, 49, 4809-4812, Bozhilov, Y.K at al., Cells (2023), 12, 896. https: / / doi.org / 10.3390 / cells12060896, etc.). In one aspect, it is preferable to use trichostatin A.

[0037] The concentration of the HDAC inhibitor contained in the culture medium composition can be appropriately set by those skilled in the art through preliminary experiments or the like. For example, the concentration of the HDAC inhibitor in the culture medium composition can be set in the range of 0.001 to 0.1 μM, 0.002 to 0.1 μM, 0.003 to 0.1 μM, 0.004 to 0.1 μM, 0.005 to 0.1 μM, 0.006 to 0.1 μM, 0.007 to 0.1 μM, 0.008 to 0.1 μM, 0.009 to 0.1 μM, 0.01 to 0.1 μM, 0.02 to 0.1 μM, 0.03 to 0.1 μM, 0.04 to 0.1 μM, 0.05 to 0.1 μM, 0.06 to 0.1 μM, 0.07 to 0.1 μM, 0.08 to 0.1 μM, 0.09 to 0.1 μM, or 0.001 to 0.09 μM, 0.001 to 0.08 μM, 0.001 to 0.07 μM, 0.001 to 0.06 μM, 0.001 to 0.05 μM, 0.001 to 0.04 μM, 0.001 to 0.03 μM, 0.001 to 0.02 μM, 0.001 to 0.01 μM, 0.001 to 0.009 μM, 0.001 to 0.008 μM, 0.001 to 0.007 μM, 0.001 to 0.006 μM, 0.001 to 0.005 μM, or 0.002 to 0.09 μM, 0.003 to 0.08 μM, 0.004 to 0.07 μM, 0.005 to 0.06 μM, 0.006 to 0.05 μM, 0.007 to 0.04 μM, 0.008 to 0.03 μM, 0.009 to 0.05 μM, 0.01 to 0.04 μM, etc.

[0038] As the cytokine contained in the culture composition of the present disclosure, it is preferable to use a cytokine selected from the group consisting of IL-3, IL-6, Flt3L, and combinations thereof. By using two or more of these cytokines in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0039] In one aspect, the culture composition of the present disclosure contains a cytokine and does not contain an HDAC inhibitor.

[0040] In one aspect, the culture composition of the present disclosure contains a cytokine and an HDAC inhibitor. By using the cytokine and the HDAC inhibitor in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0041] In one aspect, the culture composition of the present disclosure comprises an HDAC inhibitor and IL-6.

[0042] In one aspect, in addition to an HDAC inhibitor and IL-6, the culture composition of the present disclosure comprises IL-3 and / or Flt3L.

[0043] In one aspect, the culture composition of the present disclosure is erythropoietin (EPO) or granulocyte macrophage colony-stimulating factor (GM-CSF); a DNA methylation inhibitor, particularly 5azaD; an inhibitor of TGFβ signaling; an inhibitor of p38 signaling; an activator of classical Wnt signaling; a combination of insulin and transferrin; an aryl hydrocarbon receptor (AHR) modulator compound; an LILRB2 agonist, an antibody against the LILRB2 receptor; TAT-HOXB4; a retinoic acid receptor antagonist, a retinoid X receptor antagonist; an agent that reduces the expression and / or activity of CD38, a vitamin D receptor antagonist, an anti-CD38 antibody, an anti-retinoic acid receptor antibody, an anti-retinoid X receptor antibody, or an anti-vitamin D receptor intracellular antibody; a nicotinamide analog, a benzamide, a nicotinethioamide, a nicotinic acid, an α-amino-3-indolepropionic acid; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), and a combination of insulin, FGF, VEGF, and a Notch agonist or stem cell factor (SCF), IL-3, and thrombopoietin (TPO); a combination of a wnt signaling pathway activator and an FGF signaling pathway activator; or a combination of a Notch agonist and an aryl hydrocarbon receptor (AHR) antagonist; a combination of CAY10433, stemregenin1, and valproic acid; a combination of a TGF-β inhibitor (A83-01), an LSD1 inhibitor, and TSA; or does not include a combination thereof.

[0044] The concentration of the cytokine contained in the medium composition can be appropriately set by those skilled in the art through preliminary experiments or the like. For example, the concentration of the cytokine in the medium composition can be set in the range of 1 ng / mL to 500 ng / mL, 2 ng / mL to 500 ng / mL, 3 ng / mL to 500 ng / mL, 4 ng / mL to 500 ng / mL, 5 ng / mL to 500 ng / mL, 6 ng / mL to 500 ng / mL, 7 ng / mL to 500 ng / mL, 8 ng / mL to 500 ng / mL, 9 ng / mL to 500 ng / mL, 10 ng / mL to 500 ng / mL, 20 ng / mL to 500 ng / mL, 30 ng / mL to 500 ng / mL, 40 ng / mL to 500 ng / mL, 50 ng / mL to 500 ng / mL, 60 ng / mL to 500 ng / mL, 70 ng / mL to 500 ng / mL, 80 ng / mL to 500 ng / mL, 90 ng / mL to 500 ng / mL, 100 ng / mL to 500 ng / mL, or 10 ng / mL to 400 ng / mL, 10 ng / mL to 300 ng / mL, 10 ng / mL to 200 ng / mL, 10 ng / mL to 100 ng / mL, or 20 ng / mL to 400 ng / mL, 30 ng / mL to 300 ng / mL, 40 ng / mL to 200 ng / mL, 50 ng / mL to 100 ng / mL, etc.

[0045] In one aspect, the cell culture composition containing the HDAC inhibitor and cytokine of the present disclosure is used for proliferating hematopoietic stem cells or hematopoietic stem progenitor cells. As the hematopoietic stem cells or hematopoietic stem progenitor cells, hematopoietic stem cells or hematopoietic stem progenitor cells prepared by known methods in the art can be used without particular limitation. In one aspect, the culture composition of the present disclosure can be suitably used for the proliferation of long-term hematopoietic stem cells (WO2022 / 004864) showing a staining pattern of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+). Also, in one aspect, the culture composition of the present disclosure can be suitably used for the proliferation of hematopoietic stem progenitor cells showing a cell surface marker staining pattern of CD34(+), particularly hematopoietic stem progenitor cells showing a cell surface marker staining pattern of Lin(-)CD34(+) (Lin-CD34+ cells). These hematopoietic stem cells or hematopoietic stem progenitor cells can be obtained by using cord blood or a commercially available hematopoietic cell sample as a material and obtaining a cell population in which the target hematopoietic stem cells or hematopoietic stem progenitor cells are concentrated by using a flow cytometer or the like. As a method for obtaining the cell population, those skilled in the art can appropriately use well-known methods.

[0046] In one aspect, the present disclosure A cell culture composition containing cytokines for use in the culture of Lin(-)CD34(+) hematopoietic stem progenitor cells (Lin-CD34+ cells) or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells (Px cells), comprising (1) a histone deacetylase inhibitor (HDAC inhibitor) and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L The cell culture composition containing the same is provided.

[0047] In one aspect, the present disclosure A cell culture composition comprising a cytokine for use in culturing hematopoietic stem progenitor cells (Lin−CD34+ cells) that are Lin(−)CD34(+), or hematopoietic stem cells (Px cells) that are CD34(+)CD48(−), CD48(−)c-Kit(+), or CD34(+)CD48(−)c-Kit(+), The cell culture composition comprising a histone deacetylase inhibitor (HDAC inhibitor), IL-6, IL-3, and Flt3L is provided.

[0048] In one aspect, the present disclosure A cell culture composition comprising a cytokine for use in culturing hematopoietic stem progenitor cells (Lin−CD34+ cells) that are Lin(−)CD34(+), or hematopoietic stem cells (Px cells) that are CD34(+)CD48(−), CD48(−)c-Kit(+), or CD34(+)CD48(−)c-Kit(+), The cell culture composition comprising an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61, and IL-6, IL-3, and Flt3L is provided.

[0049] The culture composition of the present disclosure can generally contain components used in cell culture as long as the culture promoting effect on hematopoietic stem progenitor cells or hematopoietic stem cells is not lost. For example, it may contain components such as Penicillin Streptomycin Glutamine, Insulin-Transferrin-Selenium-Ethanolamine, DMSO, bovine serum albumin (BSA), SCF, and TPO. In one aspect, it is preferable to contain BSA.

[0050] The culture composition of the present disclosure is provided in a form that can be used as a culture medium for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, and can also be provided as an additive composition to be added to the culture composition of hematopoietic stem progenitor cells or hematopoietic stem cells. For example, an HDAC inhibitor and / or cytokine may be added to Iscove's MDM (IMDM) medium together with other components to provide a culture medium composition. In another aspect, for example, a composition containing an HDAC inhibitor and / or cytokine may be provided as a composition for adding to a medium composition such as Iscove's MDM (IMDM) medium.

[0051] The culture composition of the present disclosure may be provided in a form containing an HDAC inhibitor and / or cytokine in the same dosage form, or may be provided in a form containing them in separate dosage forms.

[0052] The dosage form of the culture composition of the present disclosure is not particularly limited, and it can be provided in various forms of liquid or solid forms. For example, when provided in a solid form, it can be provided in the form of a gel, powder, tablet, etc. In one aspect, it may be provided in the form of a lyophilized powder. The culture composition of the present disclosure can be formulated together with one or more pharmaceutically acceptable carriers. As the "pharmaceutically acceptable carrier", any non-toxic, inert solid, semi-solid or liquid, filler, diluent, encapsulating material or compounding adjuvant can be used.

[0053] The cell culture composition of the present disclosure has excellent technical effects such as significantly promoting cell proliferation while maintaining the same high self-renewal ability and multi-differentiation ability as hematopoietic stem progenitor cells or hematopoietic stem cells before proliferation.

[0054] When culturing hematopoietic stem progenitor cells or hematopoietic stem cells using the cell culture composition of the present disclosure, for culture conditions other than the composition of the medium, such as culture temperature, atmospheric component composition, presence or absence of binding to a solid layer, culture period, etc., those skilled in the art can appropriately select according to the characteristics of the cells to be cultured. For example, the cells can be cultured by incubating at 37°C, 5% CO 2 in an incubator for about 7 days.

[0055] When culturing hematopoietic stem progenitor cells or hematopoietic stem cells using the cell culture composition of the present disclosure, by culturing these cells for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, a cell growth promoting effect of 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 200-fold can be obtained compared to before culturing. When culturing hematopoietic stem progenitor cells or hematopoietic stem cells using the cell culture composition of the present disclosure, by culturing these cells for the above period, for example, an increase in the number of hematopoietic stem progenitor cells or hematopoietic stem cells, and / or enrichment of hematopoietic stem progenitor cells or hematopoietic stem cells within the cell population is achieved.

[0056] (Method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells) In one aspect, the present disclosure provides a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells. Typically, the present disclosure provides the following culturing method. A step of preparing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells; and A step of culturing the cell population using a cell culture composition containing an HDAC inhibitor and / or a cytokine, A method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, comprising

[0057] In the culturing method of the present disclosure, the step of preparing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells can utilize methods well-known to those skilled in the art. For example, in one aspect, using cord blood or a commercially available hematopoietic cell sample as a material, and fractionating the cell population based on cell surface markers using a flow cytometer or the like, a cell population enriched with the target hematopoietic stem cells or hematopoietic stem progenitor cells can be obtained.

[0058] In the culture method of the present disclosure, the step of culturing the cell population using a cell culture composition containing an HDAC inhibitor and / or a cytokine is typically performed using the culture composition described above (the cell culture composition containing an HDAC inhibitor and / or a cytokine).

[0059] In the culture method of the present disclosure, for culture conditions other than the composition of the culture medium, such as culture temperature, atmospheric component composition, presence or absence of binding to a solid layer, culture period, etc., those skilled in the art can appropriately select according to the characteristics of the cells to be cultured. For example, cells can be cultured by incubating at 37°C, 5% CO 2 for about 7 days in an incubator.

[0060] In one aspect, in the culture method of the present disclosure, it is preferable to use a cytokine selected from the group consisting of IL-3, IL-6, Flt3L, and combinations thereof as the cytokine. In one aspect, the culture method of the present disclosure uses a culture composition containing a cytokine selected from the group consisting of IL-3, IL-6, Flt3L, and combinations thereof. By using two or more of these cytokines in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0061] In one aspect, the culture method of the present disclosure does not use a cytokine and an HDAC inhibitor simultaneously. In one aspect, the culture method of the present disclosure uses a culture composition containing a cytokine and not containing an HDAC inhibitor.

[0062] In one aspect, the culture method of the present disclosure uses a cytokine and an HDAC inhibitor simultaneously. In one aspect, the culture method of the present disclosure uses a culture composition containing a cytokine and an HDAC inhibitor. By using a cytokine and an HDAC inhibitor in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0063] In one aspect, the culture method of the present disclosure uses an HDAC inhibitor and IL-6. In one aspect, the culture method of the present disclosure uses a culture composition containing an HDAC inhibitor and IL-6.

[0064] In one aspect, the culture method of the present disclosure uses IL-3 and / or Flt3L in addition to an HDAC inhibitor and IL-6. In one aspect, the culture method of the present disclosure uses a culture composition containing IL-3 and / or Flt3L in addition to an HDAC inhibitor and IL-6.

[0065] In one aspect, the culture method of the present disclosure is erythropoietin (EPO) or granulocyte macrophage colony-stimulating factor (GM-CSF); a DNA methylation inhibitor, particularly 5azaD; an inhibitor of TGFβ signaling; an inhibitor of p38 signaling; an activator of classical Wnt signaling; a combination of insulin and transferrin; an aryl hydrocarbon receptor (AHR) modulator compound; an antibody against LILRB2, the LILRB2 receptor; TAT-HOXB4; a retinoic acid receptor antagonist, a retinoid X receptor antagonist; an agent that reduces the expression and / or activity of CD38, a vitamin D receptor antagonist, an anti-CD38 antibody, an anti-retinoic acid receptor antibody, an anti-retinoid X receptor antibody, or an anti-vitamin D receptor intracellular antibody; a nicotinamide analog, benzamide, nicotinethioamide, nicotinic acid, α-amino-3-indolepropionic acid; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) and a combination of insulin, FGF, VEGF, and Notch or stem cell factor (SCF), IL-3, and thrombopoietin (TPO); a combination of a wnt signaling pathway activator and an FGF signaling pathway activator; or a combination of Notch and an aryl hydrocarbon receptor (AHR) antagonist; a combination of CAY10433, stemregenin1, and valproic acid; a combination of TGF-β inhibitor (A83-01), LSD1 inhibitor, and TSA; or does not use these combinations.

[0066] In one aspect, the culture method of the present disclosure is erythropoietin (EPO) or granulocyte macrophage colony-stimulating factor (GM-CSF); DNA methylation inhibitor, particularly 5azaD; inhibitor of TGFβ signaling; inhibitor of p38 signaling; activator of classical Wnt signaling; combination of insulin and transferrin; aryl hydrocarbon receptor (AHR) modulator compound; antibody against LILRB2, LILRB2 receptor; TAT-HOXB4; retinoic acid receptor antagonist, retinoid X receptor antagonist; agent that decreases the expression and / or activity of CD38, vitamin D receptor antagonist, anti-CD38 antibody, anti-retinoic acid receptor antibody, anti-retinoid X receptor antibody, or anti-vitamin D receptor intracellular antibody; nicotinamide analog, benzamide, nicotinethioamide, nicotinic acid, α-amino-3-indolepropionic acid; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), and combination of insulin, FGF, VEGF, and Notch or stem cell factor (SCF), IL-3, and thrombopoietin (TPO); combination of wnt signaling pathway activator and FGF signaling pathway activator; or combination of Notch and aryl hydrocarbon receptor (AHR) antagonist; combination of CAY10433, stemregenin1 and valproic acid; combination of TGF-β inhibitor (A83-01), LSD1 inhibitor and TSA; or a culture composition not containing these combinations is used.

[0067] In one aspect, the culture method of the present disclosure uses an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61.

[0068] In the production method of the present disclosure, the amount and / or concentration of the HDAC inhibitor and / or cytokine used can be appropriately set by those skilled in the art through preliminary experiments or the like. In one aspect, as the amount and / or concentration of the HDAC inhibitor and / or cytokine used in the production method of the disclosure, the amount / and or concentration exemplified in the above (cell culture composition containing HDAC inhibitor and / or cytokine) can be used.

[0069] In one aspect, the culture method of the present disclosure is used for culturing hematopoietic stem progenitor cells of Lin(-)CD34(+) (Lin-CD34+ cells).

[0070] In one aspect, the culture method of the present disclosure is used for culturing hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+).

[0071] In one aspect, the present disclosure A step of preparing a cell population comprising hematopoietic stem progenitor cells of Lin(-)CD34(+) (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and A step of culturing the cell population using a cell culture composition containing a cytokine, wherein the cell culture composition (1) An HDAC inhibitor and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) Two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L A method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, which comprises the step of containing.

[0072] In one aspect, the present disclosure Preparing a cell population comprising hematopoietic stem progenitor cells (Lin-CD34+ cells) that are Lin(-)CD34(+), or hematopoietic stem cells (Px cells) that are CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and Culturing the cell population using a cell culture composition containing cytokines, wherein the cell culture composition contains an HDAC inhibitor, IL-6, IL-3, and Flt3L, and providing a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells.

[0073] In one aspect, the present disclosure Preparing a cell population comprising hematopoietic stem progenitor cells (Lin-CD34+ cells) that are Lin(-)CD34(+), or hematopoietic stem cells (Px cells) that are CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and Culturing the cell population using a cell culture composition containing cytokines, wherein the cell culture composition contains an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61, and IL-6, IL-3, and Flt3L, and providing a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells.

[0074] The method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells of the present disclosure has excellent technical effects such as significantly promoting the proliferation of hematopoietic stem progenitor cells or hematopoietic stem cells while maintaining a high self-renewal ability and multi-differentiation ability similar to that of hematopoietic stem progenitor cells or hematopoietic stem cells before proliferation.

[0075] In the culture method of the present disclosure, by culturing hematopoietic stem progenitor cells or hematopoietic stem cells for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, a cell growth promoting effect of 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 200-fold can be obtained compared to before culturing. In the culture method of the present disclosure, by culturing hematopoietic stem progenitor cells or hematopoietic stem cells for the above period, for example, an increase in hematopoietic stem progenitor cells or hematopoietic stem cells and / or enrichment of hematopoietic stem progenitor cells or hematopoietic stem cells in the cell population are achieved.

[0076] (Method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells) In one aspect, the present disclosure provides a method for producing hematopoietic stem progenitor cells or hematopoietic stem cells. Typically, the present disclosure uses the above (method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells) to produce hematopoietic stem progenitor cells or hematopoietic stem cells. That is, in one aspect, the present disclosure preparing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells; and culturing the cell population using a cell culture composition containing an HDAC inhibitor and / or a cytokine, A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells, comprising the above steps, is provided.

[0077] The hematopoietic stem progenitor cells or hematopoietic stem cells, the cell culture composition containing an HDAC inhibitor and / or a cytokine, and the culture method used in the production method of the present disclosure may be prepared or carried out according to the description of the above (method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells) or (cell culture composition containing an HDAC inhibitor and / or a cytokine).

[0078] In the production method of the present disclosure, for culture conditions other than the composition of the culture medium, such as culture temperature, atmospheric component composition, presence or absence of binding to a solid layer, culture period, etc., those skilled in the art can appropriately select according to the characteristics of the cells to be cultured. For example, 37°C, 5% CO 2Cells can be cultured by culturing them in an incubator for about 7 days.

[0079] In one aspect, the production method of the present disclosure preferably uses a cytokine selected from the group consisting of IL-3, IL-6, Flt3L, and combinations thereof. In one aspect, the production method of the present disclosure uses a culture composition containing a cytokine selected from the group consisting of IL-3, IL-6, Flt3L, and combinations thereof. By using two or more of these cytokines in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0080] In one aspect, the production method of the present disclosure does not use a cytokine and an HDAC inhibitor simultaneously. In one aspect, the production method of the present disclosure uses a culture composition containing a cytokine and not containing an HDAC inhibitor.

[0081] In one aspect, the production method of the present disclosure uses a cytokine and an HDAC inhibitor simultaneously. In one aspect, the production method of the present disclosure uses a culture composition containing a cytokine and an HDAC inhibitor. By using a cytokine and an HDAC inhibitor in combination, a synergistically excellent cell growth promoting effect can be obtained.

[0082] In one aspect, the production method of the present disclosure uses an HDAC inhibitor and IL-6. In one aspect, the production method of the present disclosure uses a culture composition containing an HDAC inhibitor and IL-6.

[0083] In one aspect, the production method of the present disclosure uses IL-3 and / or Flt3L in addition to an HDAC inhibitor and IL-6. In one aspect, the production method of the present disclosure uses a culture composition containing IL-3 and / or Flt3L in addition to an HDAC inhibitor and IL-6.

[0084] In one aspect, the production method of the present disclosure is erythropoietin (EPO) or granulocyte macrophage colony-stimulating factor (GM-CSF); a DNA methylation inhibitor, particularly 5azaD; an inhibitor of TGFβ signaling; an inhibitor of p38 signaling; an activator of classical Wnt signaling; a combination of insulin and transferrin; an aryl hydrocarbon receptor (AHR) modulator compound; an antibody against LILRB2, the LILRB2 receptor; TAT-HOXB4; a retinoic acid receptor antagonist, a retinoid X receptor antagonist; an agent that decreases the expression and / or activity of CD38, a vitamin D receptor antagonist, an anti-CD38 antibody, an anti-retinoic acid receptor antibody, an anti-retinoid X receptor antibody, or an anti-vitamin D receptor intracellular antibody; a nicotinamide analog, benzamide, nicotinethioamide, nicotinic acid, α-amino-3-indolepropionic acid; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), and a combination of insulin, FGF, VEGF, and Notch or stem cell factor (SCF), IL-3, and thrombopoietin (TPO); a combination of a wnt signaling pathway activator and an FGF signaling pathway activator; or a combination of Notch and an aryl hydrocarbon receptor (AHR) antagonist; a combination of CAY10433, stemregenin1, and valproic acid; a combination of a TGF-β inhibitor (A83-01), an LSD1 inhibitor, and TSA; or does not use a combination thereof.

[0085] In one aspect, the production method of the present disclosure uses a culture composition that does not contain erythropoietin (EPO) or granulocyte macrophage colony-stimulating factor (GM-CSF); a DNA methylation inhibitor, particularly 5azaD; an inhibitor of TGFβ signaling; an inhibitor of p38 signaling; an activator of classical Wnt signaling; a combination of insulin and transferrin; an aryl hydrocarbon receptor (AHR) modulator compound; an antibody against LILRB2, the LILRB2 receptor; TAT-HOXB4; a retinoic acid receptor antagonist, a retinoid X receptor antagonist; an agent that decreases the expression and / or activity of CD38, a vitamin D receptor antagonist, an anti-CD38 antibody, an anti-retinoic acid receptor antibody, an anti-retinoid X receptor antibody, or an anti-vitamin D receptor intracellular antibody; a nicotinamide analog, benzamide, nicotinethioamide, nicotinic acid, α-amino-3-indolepropionic acid; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), and a combination of insulin, FGF, VEGF, and Notch or stem cell factor (SCF), IL-3, and thrombopoietin (TPO); a combination of a wnt signaling pathway activator and an FGF signaling pathway activator; or a combination of Notch and an aryl hydrocarbon receptor (AHR) antagonist; a combination of CAY10433, stemregenin1, and valproic acid; a combination of a TGF-β inhibitor (A83-01), an LSD1 inhibitor, and TSA; or a combination thereof.

[0086] In one aspect, the production method of the present disclosure uses an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and the HDAC6 inhibitor l61.

[0087] In the production method of the present disclosure, the amount and / or concentration of the HDAC inhibitor and / or cytokine used can be appropriately set by those skilled in the art through preliminary experiments or the like. In one aspect, as the amount and / or concentration of the HDAC inhibitor and / or cytokine used in the production method of the disclosure, the amount / or concentration exemplified in the above (cell culture composition containing HDAC inhibitor and / or cytokine) can be used.

[0088] In one aspect, the production method of the present disclosure is used for the production of a cell population containing hematopoietic stem progenitor cells of Lin(-)CD34(+) (Lin-CD34+ cells).

[0089] In one aspect, the production method of the present disclosure is used for the production of a cell population containing hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+).

[0090] In one aspect, the present disclosure A step of preparing a cell population containing hematopoietic stem progenitor cells of Lin(-)CD34(+) (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and A step of culturing the cell population using a cell culture composition containing a cytokine, wherein the cell culture composition (1) An HDAC inhibitor and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) Two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells, comprising the step of containing, is provided.

[0091] In one aspect, the present disclosure A step of preparing a cell population containing hematopoietic stem progenitor cells (Lin-CD34+ cells) of Lin(-)CD34(+) or hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and A step of culturing the cell population using a cell culture composition containing cytokines, wherein the cell culture composition contains an HDAC inhibitor, IL-6, IL-3, and Flt3L, A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells is provided.

[0092] In one aspect, the present disclosure A step of preparing a cell population containing hematopoietic stem progenitor cells (Lin-CD34+ cells) of Lin(-)CD34(+) or hematopoietic stem cells (Px cells) of CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+); and A step of culturing the cell population using a cell culture composition containing cytokines, wherein the cell culture composition contains an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61, and IL-6, IL-3, and Flt3L, A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells is provided.

[0093] The method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells of the present disclosure has excellent technical effects such as providing a cell population that maintains a high self-renewal ability and multi-differentiation ability similar to that of hematopoietic stem progenitor cells or hematopoietic stem cells before proliferation.

[0094] In the method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells of the present disclosure, by culturing the hematopoietic stem progenitor cells or hematopoietic stem cells for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, a cell growth promoting effect of 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 200-fold can be obtained compared to before culturing. In the method for producing a cell population of the present disclosure, by culturing the hematopoietic stem progenitor cells or hematopoietic stem cells for the above-mentioned period, for example, an increase in the hematopoietic stem progenitor cells or hematopoietic stem cells and / or enrichment of the hematopoietic stem progenitor cells or hematopoietic stem cells in the cell population is achieved.

[0095] (Use of a cell culture composition containing an HDAC inhibitor and / or a cytokine) In one aspect, the present disclosure provides the use of a cell culture composition containing an HDAC inhibitor and / or a cytokine in a method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, or in a method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells.

[0096] In the use of the present disclosure, the method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells is typically the method described in the above (method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells). The method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells is typically the method described in the above (method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells). In the use of the present disclosure, the cell culture composition containing an HDAC inhibitor and / or a cytokine is typically the composition described in the above (cell culture composition containing an HDAC inhibitor and / or a cytokine).

[0097] In one aspect, the present disclosure In a method for culturing a cell population containing Lin(-)CD34(+) hematopoietic stem progenitor cells (Lin-CD34+ cells) or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells (Px cells), or in a method for producing a cell population containing the above hematopoietic stem progenitor cells or hematopoietic stem cells (1) An HDAC inhibitor and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) Two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L The use of a cell culture composition comprising the same is provided.

[0098] In one aspect, the present disclosure A method for culturing a cell population comprising Lin(-)CD34(+) hematopoietic stem progenitor cells (Lin-CD34+ cells) or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells (Px cells), or in a method for producing a cell population comprising the hematopoietic stem progenitor cells or hematopoietic stem cells, The use of a cell culture composition comprising an HDAC inhibitor, IL-6, IL-3, and Flt3L is provided.

[0099] In one aspect, the present disclosure A method for culturing a cell population comprising Lin(-)CD34(+) hematopoietic stem progenitor cells (Lin-CD34+ cells) or CD34(+)CD48(-), CD48(-)c-Kit(+), or CD34(+)CD48(-)c-Kit(+) hematopoietic stem cells (Px cells), or in a method for producing a cell population comprising the hematopoietic stem progenitor cells or hematopoietic stem cells, The use of a cell culture composition comprising an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61, and IL-6, IL-3, and Flt3L is provided.

[0100] By using the present disclosure, hematopoietic stem progenitor cells or hematopoietic stem cells are cultured for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, and a cell growth promoting effect of 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 200-fold can be obtained as compared with before culturing. By using the present disclosure, hematopoietic stem progenitor cells or hematopoietic stem cells are cultured for the above-mentioned period, for example, and an increase in hematopoietic stem progenitor cells or hematopoietic stem cells and / or enrichment of hematopoietic stem progenitor cells or hematopoietic stem cells in the cell population are achieved.

Example

[0101] The present disclosure will be described in more detail below using examples. However, these examples are illustrative for explanation purposes, and the present disclosure is not limited to these examples.

[0102] (Materials and Methods) 1. Human CD34-positive cells Human CD34-positive cells derived from umbilical cord blood were obtained by processing human umbilical cord blood obtained from a collaborating hospital or commercially available human umbilical cord blood-derived CD34-positive cells (Lonza, Swiss). When processing umbilical cord blood, red blood cells were removed from the umbilical cord blood to roughly purify and prepare CD34-positive cells. In some samples, after the red blood cell removal treatment, they were cryopreserved at around -80°C. When using the cryopreserved stock, after thawing according to the recommended method of Lonza, CD34-positive cells were roughly purified. When obtaining the target cell fraction such as hematopoietic stem cells, for the roughly purified CD34-positive cells, cells of the target fraction were seeded by a cell sorter after surface marker staining. When using commercially available human umbilical cord blood-derived CD34-positive cells, the human umbilical cord blood-derived CD34-positive cells stored in liquid nitrogen after purchase were thawed according to the recommended method of Lonza, and cells of the target cell fraction were seeded by a cell sorter after surface marker staining. In any method, the main culture was performed after seeding by a cell sorter without performing recovery culture after freeze-thawing.

[0103] 2. Cell culture medium The following components were added to Iscove's Modified Dulbecco's Medium (IMDM) to prepare the culture medium. TIFF2025081152000001.tif84156PSG and ITS-X were added to the stock solution of the product to a final concentration of 1%. When adding BSA at a final concentration of 1%, a protein solution product in which BSA was dissolved at a concentration of 30% in PBS was used as the stock solution and added to the culture medium to a final concentration of 1%. TSA and UM171 were dissolved in dimethyl sulfoxide (DMSO) and prepared to a final concentration of 0.1% under all cell culture conditions. DMSO is considered to be replaceable with ethanol.

[0104] 3. Surface marker staining and seeding of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells) Using a cell sorter (BD FACS Aria III, Dickinson and Company, U.S.A.), hematopoietic stem progenitor cells (Lin-CD34+ cells) were seeded in the preparation medium for cell culture. Prior to that, they were stained with a fluorescent molecule-conjugated antibody against the surface marker. (Surface marker staining) Each surface marker of CD34, c-Kit, CD48, and Lineage (maturation factor) was stained. The surface markers used for Lineage were CD11b, CD14, CD19, CD20, CD235ab, CD3, CD4, CD56, and CD8a. Propidium iodide (PI) was used as the dead cell marker. The recognition antigens used as each surface marker and the fluorescent molecules used for staining the recognition antigens are shown in the following table. TIFF2025081152000002.tif77130Also, streptavidin conjugated with the fluorescent molecule: APC / Cy7 and Propidium iodide (PI) were used for staining.

[0105] (Seeding into the medium) Using a cell sorter, hematopoietic stem progenitor cells (Lin-CD34+ cells) were seeded into the medium. Specifically, 200 μL of the prepared medium was placed in each well of a 96-well microplate, and hematopoietic stem progenitor cells (Lin-CD34+ cells) were seeded into each well at 300 cells / well using a cell sorter. The gating strategy used to obtain hematopoietic stem progenitor cells (Lin-CD34+ cells) is shown in Fig. 1. Hematopoietic stem cells (Px cells) are also included in the fraction of hematopoietic stem progenitor cells (Lin-CD34+ cells).

[0106] (Cell culture) The cells seeded with the cell sorter were cultured in an incubator at 37 °C and 5% CO 2 for 7 days according to a conventional method.

[0107] 5. Measurement and analysis after cell culture The cells after culture were stained according to the above 2. (Surface marker staining). Subsequently, using a FACS apparatus (BD FACS Canto, Becton, Dickinson and Company, U.S.A.), the culture product was measured. Using software for FACS analysis (FlowJo, Becton, Dickinson and Company, U.S.A.), as shown in the gating strategy of Fig. 1, the number and ratio of live cells, hematopoietic stem progenitor cells (Lin-CD34+ cells), and hematopoietic stem cells (Px cells) were determined.

[0108] 6. Colony-forming unit (CFU) assay According to the above 3. (Surface marker staining and seeding of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells)), hematopoietic stem progenitor cells (Lin-CD34+ cells) before culture and after culture in each prepared medium were seeded into the medium for CFU assay. 1 cell / well, 120 wells per condition were seeded. After seeding, the cells were cultured in an incubator at 37 °C and 5% CO 2 for about 14 days, and then each well was imaged using a microscope with an imaging function. Based on the image data of each imaged well, the presence or absence of colony formation and the type of formed colonies were determined.

[0109] The generated colonies were determined to be three types of colonies (CFU-GEMM, CFU-GM, BFU-E) and unclassifiable (Unclassifiable). The medium for the CFU assay was prepared as follows. Add 50 mL of Iscove’s MDM with 2% FBS to 100 mL of MethoCult H4435 Enriched and stir well to prepare a mixed medium. Dispense 40 μL of the prepared mixed medium into a 96-well microplate.

[0110] Example 1: Cell proliferation promoting effect on hematopoietic stem progenitor cells or hematopoietic stem cells The effects of each medium component on the cell proliferation promotion of hematopoietic stem progenitor cells and hematopoietic stem cells were examined. Specifically, the test media 1 to 10 as follows and 2. (Cell culture medium) described in (Materials and methods) were prepared. Cells were seeded in the prepared test media by 3. (Surface marker staining and seeding of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells)). Then, they were cultured for 7 days using test media 1 to 10 containing BSA (1%), UM171 (0.035 μM), TSA (0.01 μM), and cytokines (IL-3, Flt3L, or IL-6 at 50 ng / mL or 100 ng / mL).

[0111] TIFF2025081152000003.tif55154

[0112] After culturing, the cell numbers and ratios of viable cells, hematopoietic stem progenitor cells (Lin-CD34+ cells), or hematopoietic stem cells (Px cells) were determined according to 5. (Measurement and analysis after cell culture).

[0113] As a result of measuring hematopoietic stem progenitor cells (Lin-CD34+ cells), in the medium without BSA (test medium 1), the hematopoietic stem progenitor cells (Lin-CD34+ cells) hardly proliferated. Among the media containing BSA, in the medium containing UM171 as a positive control (test medium 2), promotion of cell proliferation was obtained. On the other hand, in the medium containing BSA and TSA (test medium 3), cell proliferation decreased compared to the medium containing only BSA (test medium 1). In the media containing IL-3 and Flt3L at 50 ng / mL each (test medium 5) and the medium containing IL-3 and Flt3L at 100 ng / mL each (test medium 7), significantly higher promotion of cell proliferation was obtained compared to the medium containing only BSA (test medium 1). In the media containing IL-6 at 50 ng / mL (test medium 6) or 100 ng / mL in addition to IL-3 and Flt3L (test medium 8), further higher promotion of cell proliferation was obtained by the addition of IL-6. In the media containing 0.01 μM TSA in addition to IL-3, Flt3L, and IL-6 (test medium 9: IL-3, Flt3L, and IL-6 at 50 ng / mL each, test medium 10: IL-3, Flt3L, and IL-6 at 100 ng / mL each), further enhancement of the cell proliferation promoting effect was obtained by the addition of TSA. In particular, when test medium 10 was used, an approximately 40-fold enhancement of the cell proliferation promoting effect was obtained compared to before culture, and a synergistic enhancing effect was obtained by the combination of the HDAC inhibitor and the cytokine (Figure 2).

[0114] In the same experiment, as a result of measuring hematopoietic stem cells (Px cells), it was confirmed that for hematopoietic stem cells (Px cells) as well, a synergistic enhancement of the cell proliferation promoting effect was obtained by using the cytokine and TSA in combination. In particular, in the media containing 0.01 μM TSA (test medium 9: IL-3, Flt3L, and IL-6 at 50 ng / mL each, test medium 10: IL-3, Flt3L, and IL-6 at 100 ng / mL each), further promotion of cell proliferation was obtained by the addition of TSA. In particular, when test medium 10 was used, an approximately 110-fold enhancement of the cell proliferation promoting effect was obtained compared to before culture, and a synergistic enhancing effect was obtained by the combination of the HDAC inhibitor and the cytokine (Figure 3).

[0115] To more thoroughly examine the effect of IL-6 addition, hematopoietic stem progenitor cells (Lin-CD34+ cells) were seeded in conditions containing BSA (1%), TSA (0.01 μM), and cytokines (IL-3, Flt3L, and IL-6) at concentrations of 50 ng / mL or 100 ng / mL and cultured for 7 days.

[0116] As a result, it was confirmed that in both cases of concentrations of 50 ng / mL or 100 ng / mL, the addition of IL-6 enhanced the cell proliferation promoting effect on hematopoietic stem progenitor cells (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) (Figure 4).

[0117] Example 2: Effect by combination of cytokines The effects of various cytokines alone or in combination on the promotion of cell proliferation of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells) were examined. Specifically, hematopoietic stem progenitor cells (Lin-CD34+ cells) were cultured for 7 days using test media 1 to 10 containing BSA (1%), UM171 (0.035 μM), and cytokines (IL-3, Flt3L, or IL-6 at 100 ng / mL) as in Example 1, and the cell numbers and ratios of the cultured hematopoietic stem progenitor cells (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) were determined.

[0118] TIFF2025081152000004.tif49155

[0119] As a result of measuring hematopoietic stem progenitor cells (Lin-CD34+ cells), in test media containing IL-3, Flt3L, and IL-6 alone (test media 4 to 6), a significant enhancement of the cell growth promoting effect was obtained only in test medium 4 containing IL-3. On the other hand, in test media containing any two of IL-3, Flt3L, and IL-6 (test media 7 to 9) or test medium containing all of these (test medium 10), a synergistic enhancement of the cell growth promoting effect was obtained. In particular, when test medium 10 was used, an enhancement of the cell growth promoting effect about 10 times that before culture was obtained, and a synergistic enhancement effect was obtained by the combination of the HDAC inhibitor and the cytokine (left in Fig. 5).

[0120] As a result of measuring hematopoietic stem cells (Px cells) in the same experiment, an enhancement of the cell growth promoting effect by the combined use of cytokines was similarly obtained for the hematopoietic stem cell (Px cell) population. In particular, when test medium 10 was used, an enhancement of the cell growth promoting effect about 30 times that before culture was obtained, and a synergistic enhancement effect was obtained by the combination of the HDAC inhibitor and the cytokine (right in Fig. 5).

[0121] Example 3: Effect by addition of HDAC inhibitor As shown in Example 1, the addition of the HDAC inhibitor further enhanced the growth promoting effect of cytokines on hematopoietic stem progenitor cells and hematopoietic stem cells, and the effect was further examined in more detail (Figs. 6 to 7). As a result, when TSA was added as the HDAC inhibitor, while increasing the cell numbers of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells), the proportion of hematopoietic stem progenitor cells (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) in the cell population after culture was the same as or increased compared to the start of culture.

[0122] On the other hand, when only BSA and cytokines were added without adding TSA, although the cell numbers of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells) increased, the proportion of hematopoietic stem progenitor cells (Lin-CD34+ cells) or hematopoietic stem cells (Px cells) present in the cell population obtained after proliferation decreased compared to the control (addition of BSA only: test medium 2).

[0123] Example 4: Maintenance of self-renewal ability and multi-differentiation ability of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells) The self-renewal ability and multi-differentiation ability of hematopoietic stem progenitor cells (Lin-CD34+ cells) and hematopoietic stem cells (Px cells) that had proliferated by the addition of cytokines and TSA were evaluated by the CFU assay. The method was to compare the colony-forming ability of hematopoietic stem progenitor cells (Lin-CD34+ cells) cultured in the following culture media with that of hematopoietic stem progenitor cells (Lin-CD34+ cells) before culture according to 6. (Colony Forming Unit (CFU) assay). TIFF2025081152000005.tif47149

[0124] As a result, the determination ratio of CFU-GEMM was generally equal to or higher than that before culture. The addition of cytokines and TSA can enhance the cell proliferation promoting effect while maintaining the self-renewal ability and multi-differentiation ability (Figure 8).

Claims

**Claim 1** A cell culture composition comprising a cytokine for use in culturing hematopoietic stem progenitor cells of Lin(−)CD34(+), or hematopoietic stem cells of CD34(+)CD48(−), CD48(−)c-Kit(+), or CD34(+)CD48(−)c-Kit(+), (1) a histone deacetylase inhibitor (HDAC inhibitor), and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L The cell culture composition comprising the same. **Claim 2** The cell culture composition according to claim 1, comprising an HDAC inhibitor, IL-6, and IL-3 or Flt3L. **Claim 3** The cell culture composition according to claim 1, comprising an HDAC inhibitor, IL-6, IL-3, and Flt3L. **Claim 4** The cell culture composition according to claim 1, wherein the HDAC inhibitor is an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MGCd-0103, DLS3, SAHA, and HDAC6 inhibitor l61. **Claim 5** A step of preparing a cell population comprising hematopoietic stem progenitor cells of Lin(−)CD34(+) or hematopoietic stem cells of CD34(+)CD48(−), CD48(−)c-Kit(+), or CD34(+)CD48(−)c-Kit(+); and A step of culturing the cell population using a cell culture composition comprising a cytokine, wherein the cell culture composition (1) an HDAC inhibitor, and one or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L A method for culturing hematopoietic stem progenitor cells or hematopoietic stem cells, comprising the step of comprising the same. **Claim 6** The culturing method according to claim 5, wherein the cell culture composition comprises an HDAC inhibitor, IL-6, and IL-3 or Flt3L. **Claim 7** The culturing method according to claim 5, wherein the cell culture composition comprises an HDAC inhibitor, IL-6, IL-3, and Flt3L. **Claim 8** The culturing method according to claim 5, wherein the HDAC inhibitor is an HDAC inhibitor selected from the group consisting of trichostatin A (TSA), valproic acid, butyryl hydroxamic acid, sodium butyrate, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, MCD-0103, DLS3, SAHA, and HDAC6 inhibitor l61.

9. A method for producing a cell population containing hematopoietic stem progenitor cells or hematopoietic stem cells, using the culturing method according to claim 5.

10. In the culturing method according to claim 5, (1) one or more cytokines selected from the group consisting of an HDAC inhibitor and IL-6, IL-3, and Flt3L; or (2) two or more cytokines selected from the group consisting of IL-6, IL-3, and Flt3L Use of a cell culture composition containing the same.

Images