Serum-free medium suitable for culturing hematopoietic cells such as human hematopoietic stem cells, and culture method

Abstract

The present invention discloses a composition of serum-free medium and a culture method suitable for culturing hematopoietic stem cells.SOLUTION: In accordance with the present invention, there is provided a method of culturing hematopoietic stem cells, the method comprising contacting hematopoietic stem cells with a polyalkylene glycol modified by a copolymer of polyvinyl caprolactam and polyvinyl acetate blocks.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention discloses a serum-free medium (particularly albumin-free) composition suitable for culturing blood cells such as human hematopoietic stem cells, and a culture method. According to the present invention, a method for culturing blood cells such as human hematopoietic stem cells is provided. The method may include contacting blood cells such as human hematopoietic stem cells with polyethylene glycol modified with a linker moiety of a polyvinyl caprolactam block and a polyvinyl acetate block. [Background technology]

[0002] Research is currently being conducted into the proliferation of hematopoietic stem cells using chemically defined media, and it has been demonstrated that mouse hematopoietic stem cells can be cultured using chemically defined media (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Wilkinson et al., Nature, 571:117-121, 2019 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides a composition of a medium suitable for culturing blood cells such as human hematopoietic stem cells (e.g., a serum-free medium, e.g., an albumin-free serum-free medium, or a cytokine-free serum-free medium, e.g., an albumin-free and cytokine-free serum-free medium), and a culture method.

[0005] The present inventors have previously reported that mouse hematopoietic stem cells (sometimes called KSL cells, named after their isolation method) can be expanded in large quantities over long periods of time in serum-free medium containing no albumin by adding polyvinyl alcohol (PVA) (Wilkinson et al., Nature, 571:117-121, 2019). However, no method for expanding human hematopoietic stem cells in serum-free medium containing no albumin has been established. Here, the present inventors have found that human hematopoietic stem cells exhibit significant proliferation while maintaining stemness in the presence of polyethylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, even in serum-free medium containing no albumin. Furthermore, they have found that hematopoietic stem cell proliferation can be maintained even under cytokine-free conditions. Furthermore, under these conditions, they have found that in addition to hematopoietic stem cells, various blood cell types, including erythroblasts, megakaryocytes, and leukocytes such as T cells, B cells, macrophages, and neutrophils, can be expanded. [Means for solving the problem]

[0006] According to the present invention, the following inventions are provided. [1] A method for culturing human hematopoietic stem cells or human blood cells, comprising: Culturing human hematopoietic stem cells or human blood cells in a culture medium, The culture medium is an albumin-free medium (particularly a serum albumin-free medium) containing polyvinyl alcohol, and (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist; The number of human hematopoietic stem cells or human blood cells is maintained or increased by the above culture. method. [2] The method according to [1] above, which comprises obtaining increased human hematopoietic stem cells or human blood cells. [3] The method according to [1] or [2] above, wherein the culture medium contains a PI3K activator but does not contain stem cell factor (SCF). [4] The method according to any one of [1] to [3] above, wherein the culture medium contains a PI3K activator and a TPO receptor agonist. [5] The method according to [4] above, wherein the culture medium does not contain both SCF and TPO. [5A] The method according to [4] or [5] above, wherein the culture medium is a cytokine-free medium. [6] The method according to any one of [1] to [5] above, wherein the culture medium further contains 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (UM171). [7] The method according to [6] above, wherein the culture period is 7 days or longer. [8] A composition that does not contain albumin, Human hematopoietic stem cells or human blood cells, polyvinyl alcohol, (1) a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) PI3K activators and TPO receptor agonists, A composition comprising: [9] The composition according to [8] above, comprising a PI3K activator and a TPO receptor agonist.

[10] The composition according to [8] or [9] above, further comprising 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (UM171).

[11] Human hematopoietic stem cells or human blood cells obtained by the method described in any one of [1] to [7] above.

[12] A composition comprising polyvinyl alcohol and no albumin, (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. Culture medium for human hematopoietic stem cells or human blood cells.

[0007] [1] A method for culturing or producing human hematopoietic stem cells or human blood cells, comprising: Culturing human hematopoietic stem cells or human blood cells in a culture medium, The culture medium comprises polyvinyl alcohol, and (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist; The number of human hematopoietic stem cells or human blood cells is maintained or increased by the above culture. method. [2] The method according to [1] above, wherein the culture medium is a serum-free medium. [3] The method according to [1] above, wherein the culture medium is a chemically defined medium. [4] The method according to any one of [1] to [3] above, wherein the culture medium is substantially free of albumin. [5] The method according to any one of [1] to [4] above, which comprises obtaining increased human hematopoietic stem cells or human blood cells. [6] The method according to any one of [1] to [5] above, wherein the culture medium contains a PI3K activator but does not contain stem cell factor (SCF). [7] The method according to any one of [1] to [6] above, wherein the culture medium contains a PI3K activator and a TPO receptor agonist. [8] The method according to [7] above, wherein the culture medium does not contain both SCF and TPO. [8A] The method according to [7] or [8] above, wherein the culture medium is a cytokine-free medium. [9] The method according to any one of [1] to [8] above, wherein the culture medium further contains 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (UM171).

[10] The method according to [9] above, wherein the culture period is 7 days or longer.

[11] Human hematopoietic stem cells or human blood cells, polyvinyl alcohol as a substitute for albumin, (1) a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) PI3K activators and TPO receptor agonists, A composition comprising:

[12] The composition according to

[11] above, comprising a PI3K activator and a TPO receptor agonist.

[13] The composition according to

[11] or

[12] above, further comprising 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (UM171).

[14] Human hematopoietic stem cells or human blood cells obtained by the method according to any one of [1] to

[10] above.

[15] A composition containing polyvinyl alcohol as a substitute for albumin, and (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. Culture medium for human hematopoietic stem cells or human blood cells.

[16] The culture medium according to

[15] above, which is a serum-free medium.

[17] The medium according to

[15] above, which is a chemically defined medium.

[18] The medium according to any one of

[15] to

[17] above, which does not contain albumin.

[19] The method according to any one of [1] to [8] above, wherein the human cells are human hematopoietic stem cells.

[20] The composition according to any one of

[11] to

[13] above, wherein the human cells are human hematopoietic stem cells.

[21] The method according to any one of [1] to [8] above, wherein the human cells are human blood cells.

[22] The composition according to any one of

[11] to

[13] above, wherein the human cells are human blood cells.

[23] A culture medium containing human cells obtained by the method according to any one of [1] to [8],

[19] and

[21] above.

[24] The medium according to

[23] above, which is a serum-free medium, albumin-free, and cytokine-free.

[0008] The present invention also provides the following inventions. [1B] A method for culturing human cells, comprising: Culturing human cells in a culture medium; The culture medium comprises a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, method. [2B] The method according to [1B] above, which comprises obtaining expanded human cells. [3B] The method according to [1B] or [2B] above, wherein the human cells are human hematopoietic stem cells or human blood cells. [4B] A medium composition for human cell culture, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. [5B] A composition comprising human cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. [6B] The composition according to [4B] or [5B] above, wherein the human cells are human hematopoietic stem cells or human blood cells. [7B] Human cells obtained by the method described in any one of [1B] to [3B] above. [8B] The method according to [3B] above, wherein the human cells are human hematopoietic stem cells. [9B] The composition described in [6B] above, wherein the human cells are human hematopoietic stem cells. [10B] The method according to [3B] above, wherein the human cells are human blood cells. [11B] The composition described in [6B] above, wherein the human cells are human blood cells. [12B] A medium containing human cells obtained by the method according to any one of [1B] to [3B] above. [13B] The medium according to [12B] above, which is a serum-free medium, albumin-free, and cytokine-free. [14B] The method according to any one of [1B] to [3B], [8B], and [10B] above, or the composition according to any one of [4B] to [6B], [9B], and [11B] above, or the culture medium according to

[12] or

[13] above, wherein the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block is polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0009] [1C] A method for culturing human cells, comprising: Culturing human cells in a culture medium; The culture medium is a serum albumin-free medium and contains an additive, the additive being selected from the group consisting of polyvinyl alcohol and modified polyalkylene glycol; and (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist; The number of human hematopoietic stem cells or human blood cells is maintained or increased by the above culture. method. [2C] The method according to [1C] above, wherein the human cells are cells selected from the group consisting of human hematopoietic stem cells and human blood cells. [3C] The method according to [1C] above, wherein the human cells are blood cells other than hematopoietic stem cells. [4C] The method according to [3C] above, wherein the additive is polyvinyl alcohol. [5C] The method according to [1C] or [2C] above, wherein the additive is a modified polyalkylene glycol. [6C] The method according to [5C] above, wherein the modified polyalkylene glycol is a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. [7C] The method according to [6C] above, wherein the modified polyalkylene glycol is polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1 shows the results of culturing mouse hematopoietic stem cells (mouse KSL cells) and human hematopoietic stem cells (CD34+CD38- cells) in albumin-free serum-free medium containing polyvinyl alcohol (PVA) in the presence of 10 ng / mL mouse and human tissue factor (SCF) and 100 ng / mL thrombopoietin (TPO), respectively. [Figure 2] Figure 2 shows the level of phosphorylation of downstream signaling factors of SCF and TPO in mouse and human hematopoietic stem cells cultured in the presence of 10 ng / mL tissue factor (SCF) and 100 ng / mL thrombopoietin (TPO) in the absence of albumin and the presence of PVA. The symbol "m" indicates mouse hematopoietic stem cells, and "h" indicates human hematopoietic stem cells. [Figure 3] Figure 3 shows the results of culturing human hematopoietic stem cells in the absence of albumin and the presence of PVA, in the presence of 10 ng / mL human tissue factor (SCF) and 100 ng / mL human thrombopoietin (TPO), and in the presence of AKT activator II (AKTa) or PI3K activator (PI3Ka). [Figure 4] Figure 4 shows the proliferation rates of total cells and CD34+ cells on day 7 of culture of human hematopoietic stem cells in the presence of PVA and 100 ng / mL human thrombopoietin (TPO) in the absence of albumin. Figure 4 compares the SCF-containing condition with the SCF-free condition, and shows that there is no statistically significant difference between the conditions in the total cell count and the CD34+ cell count on day 7 of culture. [Figure 5] Figure 5 shows the change in cell number when human hematopoietic stem cells were cultured in the presence of albumin or PVA, with TPO replaced by various TPO receptor agonists. The symbols "Buz" represent butizamide, "Elt" represent eltrombopag, and "Ava" represent avatrombopag. [Figure 6]FIG. 6 shows the proliferation rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells were cultured in the presence of PVA in the absence of albumin, with various TPO receptor agonists replacing TPO. [Figure 7] Figure 7 shows the total cell count, CD34+ cell count, and GEmM colony count on day 7 after culturing human hematopoietic stem cells in medium containing a PI3K activator and TPO or butizamide (Buty) in the presence of PVA in the absence of albumin. Colony types were determined by microscopically harvesting colonies, preparing cytospin specimens, staining with Giemsa, and microscopically determining colony types. G stands for "granulocyte," E stands for "erythroblast," m stands for "macrophage," and M stands for "megakaryocyte." [Figure 8] Figure 8 shows the proliferation rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator or a TPO receptor agonist, or a combination thereof, in the absence of albumin and the presence of PVA, but not SCF or TPO. [Figure 9] Figure 9 shows the proliferation rate of each cell population in the culture obtained on day 7 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO, in the presence of PVA and in the absence of albumin. [Figure 10] Figure 10 shows the changes in the total number of cells and the number of CD34+ cells on days 7 and 14 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO, in the presence of PVA and in the absence of albumin. [Figure 11] Figure 11 shows the results of gating cells obtained on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator and a TPO receptor agonist but without SCF or TPO in the presence of PVA and in the absence of albumin. The left panel shows the results of flow cytometry using CD34 and CD38 as markers, and the right panel shows the results of flow cytometry using CD41a and CD42b as markers. The photographs in Figure 11 are optical micrographs of the resulting cultures. [Figure 12] Figure 12 shows the results of colony assays of cells obtained on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO in the presence of PVA and in the absence of albumin. Colonies were collected under a microscope, cytospin specimens were prepared, and Giemsa staining was performed to determine the colony type. G indicates a colony containing "granulocytes," E indicates "erythroblasts," m indicates "macrophages," and M indicates "megakaryocytes." [Figure 13] Figure 13 shows the proliferation rates of total cells and CD34+ cells on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator, a TPO receptor agonist, and either or both of SR-1 and UM171, in the absence of albumin and the presence of PVA, but not SCF or TPO. [Figure 14] FIG. 14 shows the proliferation rates of total cells and CD34+ cells, as well as the number of CD41+ cells, on day 14 after culture under each of conditions 1 to 3. [Figure 15] Figure 15 shows the results of flow cytometry of cells in the culture on day 14 after culturing under each of conditions 1 to 3. The top panel shows the results for CD34 (horizontal axis) and CD38 (vertical axis), and the bottom panel shows the results for CD41a (horizontal axis) and CD42b (vertical axis). [Figure 16] FIG. 16 shows the engraftment of human hematopoietic stem cells in the peripheral blood of mice 12 weeks after transplantation of human hematopoietic stem cells cultured under each of conditions 1 to 3 on day 7 into mice. [Figure 17] Figure 17 shows the total cell count, percentage of CD34+ cells, percentage of viable cells, and CD34+ cell count in cultures obtained by separating mononuclear cells from donated umbilical cord blood for 14 days in a medium that does not contain albumin, SCF, or TPO, but does contain PVA, a PI3K activator, and a TPO receptor agonist, in the presence or absence of UM171. [Figure 18]Figure 18 shows the results of an in vitro culture experiment of mouse hematopoietic stem cells in the absence of albumin. The culture medium for mouse hematopoietic stem cells contained the indicated components at a final concentration of 0.1%. More specifically, the above medium was supplemented with the same concentrations of BASF Corifol® P188 bio (188 bio), Corifol™ P 188 Geismar (188 Geismar), Sol+, Corifol™ P 407 Geismar (407 Geismar), Kollidon™ 30 Origin USA (30 USA), Kollidon™ 17 PF (17 PF), Kollidon™ 25 (25), Kollidon™ 90F (90F), and Kollidon™ 12PF (12PF), each at a final concentration of 0.1%. [Figure 19] FIG. 19 shows the results of an in vitro colony formation experiment of mouse hematopoietic stem cells in the presence of PVA or polymer A in a serum-free medium in the absence of albumin. [Figure 20] FIG. 20 shows the positive rate (%) of the stem cell marker CD201 in mouse hematopoietic stem cells cultured in the presence of PVA or polymer A in a serum-free medium in the absence of albumin. [Figure 21] Figure 21 shows a scheme for transplantation of mouse hematopoietic stem cells (HSCs) cultured in serum-free medium without albumin in the presence of PVA or polymer A into irradiated mice (left), and the chimeric rate (%) in the peripheral blood (PB) of the mice 4 weeks after transplantation. The chimeric rate is the percentage of cells derived from the transplanted HSCs among all blood cells. [Figure 22] FIG. 22 shows the CD34-positive cell rate (%) of human hematopoietic stem cells cultured in the presence of PVA or polymer A in a serum-free medium in the absence of albumin. [Figure 23] FIG. 23 shows the change in the number of human hematopoietic stem cells when cultured in the presence of PVA or polymer A in a serum-free medium in the absence of albumin. [Figure 24]24 is a graph showing the degree of phosphorylation of downstream signaling factors of SCF and TPO in mouse and human hematopoietic stem cells cultured in the presence of 10 ng / mL tissue factor (SCF) and 100 ng / mL thrombopoietin (TPO) in the absence of albumin and the presence of PVA. The symbol "m" indicates mouse hematopoietic stem cells, and "h" indicates human hematopoietic stem cells. [Figure 25] Figure 25 shows the results of culturing human hematopoietic stem cells in the absence of albumin and the presence of PVA, in the presence of 10 ng / mL human tissue factor (SCF) and 100 ng / mL human thrombopoietin (TPO), and in the presence of AKT activator II (AKTa) or PI3K activator (PI3Ka). [Figure 26] Figure 26 shows the proliferation rates of total cells and CD34+ cells on day 7 of culture of human hematopoietic stem cells in the presence of PVA and 100 ng / mL human thrombopoietin (TPO) in the absence of albumin. Figure 26 compares the conditions with and without SCF and shows that there is no statistically significant difference between the conditions in the total cell count and the number of CD34+ cells on day 7 of culture. [Figure 27] 27 shows the time course of cell number when human hematopoietic stem cells were cultured in the presence of albumin or PVA, with TPO replaced by various TPO receptor agonists. The symbols "Buz" represent butizamide, "Elt" represent eltrombopag, and "Ava" represent avatrombopag. [Figure 28] FIG. 28 shows the proliferation rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells were cultured in the presence of PVA in the absence of albumin, with various TPO receptor agonists replacing TPO. [Figure 29]Figure 29 shows the total cell count, CD34+ cell count, and GEmM colony count on day 7 after culturing human hematopoietic stem cells in medium containing a PI3K activator and TPO or butizamide (Buty) in the presence of PVA in the absence of albumin. Colony types were determined by microscopically collecting colonies, preparing cytospin specimens, staining with Giemsa, and microscopically determining colony types. G stands for "granulocyte," E stands for "erythroblast," m stands for "macrophage," and M stands for "megakaryocyte." [Figure 30] Figure 30 shows the proliferation rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator or a TPO receptor agonist, or a combination thereof, in the absence of albumin and the presence of PVA, but not SCF or TPO. [Figure 31] Figure 31 shows the proliferation rate of each cell population in the culture obtained on day 7 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO, in the presence of PVA and in the absence of albumin. [Figure 32] Figure 32 shows the changes in the total number of cells and the number of CD34+ cells on days 7 and 14 when human hematopoietic stem cells were cultured in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO, in the presence of PVA and in the absence of albumin. [Figure 33] Figure 33 shows the results of gating cells obtained on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator and a TPO receptor agonist but without SCF and TPO in the presence of PVA and in the absence of albumin. The left panel shows the results of flow cytometry using CD34 and CD38 as markers, and the right panel shows the results of flow cytometry using CD41a and CD42b as markers. The photographs in Figure 33 are optical micrographs of the resulting cultures. [Figure 34]Figure 34 shows the results of colony assays of cells obtained on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator and a TPO receptor agonist but not SCF or TPO in the presence of PVA and in the absence of albumin. Colonies were collected under a microscope, cytospin specimens were prepared, and Giemsa staining was performed to determine the colony type. G indicates a colony containing "granulocytes," E indicates "erythroblasts," m indicates "macrophages," and M indicates "megakaryocytes." [Figure 35] Figure 35 shows the proliferation rates of total cells and CD34+ cells on day 14 after culturing human hematopoietic stem cells in a culture medium containing a PI3K activator, a TPO receptor agonist, and either or both of SR-1 and UM171, in the absence of albumin and the presence of PVA, but not SCF or TPO. [Figure 36] FIG. 36 shows the proliferation rates of total cells and CD34+ cells, as well as the number of CD41+ cells, on day 14 after culture under each of conditions 1 to 3. [Figure 37] Figure 37 shows the results of flow cytometry of cells in the culture on day 14 after culturing under each of conditions 1 to 3. The top graph shows the results for CD34 (horizontal axis) and CD38 (vertical axis), and the bottom graph shows the results for CD41a (horizontal axis) and CD42b (vertical axis). [Figure 38] FIG. 38 shows the engraftment of human hematopoietic stem cells in the peripheral blood of mice 12 weeks after transplantation of human hematopoietic stem cells cultured under each of conditions 1 to 3 on day 7 into mice. [Figure 39] Figure 39 shows the total cell count, percentage of CD34+ cells, percentage of viable cells, and CD34+ cell count in cultures obtained by separating donated umbilical cord blood into mononuclear cells and culturing them for 14 days in a medium that does not contain albumin, SCF, or TPO, but does contain PVA, a PI3K activator, and a TPO receptor agonist, in the presence or absence of UM171. [Figure 40] FIG. 40 shows the proliferation of human T cells in albumin-free and cytokine-free medium in the presence of PVA or SoluPlus™. [Figure 41] Figure 41 shows the results of a differentiation experiment of human hematopoietic stem cells in an albumin-free medium in the presence of PVA or SoluPlus™. It has been shown that hematopoietic stem cells can proliferate and differentiate (particularly proliferate) into a wide range of blood cell lineages. [Figure 42] Figure 42 shows that chronic myeloid leukemia (CML) cells can grow in albumin-free and cytokine-free medium in the presence of PVA or SoluPlus™. In the figure, "IM+" means in the presence of imatinib, and "IM-" means in the absence of imatinib. DETAILED DESCRIPTION OF THE INVENTION

[0011] As used herein, "hematopoietic stem cells" refer to stem cells that can differentiate into blood cells. Hematopoietic stem cells can be collected from bone marrow, umbilical cord, placenta, and peripheral blood. Human hematopoietic stem cells are CD34-positive cells. In humans, hematopoietic stem cells are known to be abundant in the CD34-positive CD38-negative cell fraction. Therefore, human hematopoietic stem cells can be CD34-positive CD38-negative. Hematopoietic stem cells may also be cells obtained by ex vivo differentiation of pluripotent stem cells such as ES cells and iPS cells.

[0012] As used herein, "blood cells" refer to hematopoietic stem cells and cells derived from hematopoietic stem cells through differentiation. Blood cells are broadly classified into hematopoietic stem cells, hematopoietic progenitor cells, and blood cells, depending on the differentiation stage. Hematopoietic stem cells differentiate into blood cells via hematopoietic progenitor cells. More specifically, hematopoietic stem cells can differentiate into lymphocytes (e.g., T cells, B cells, and NK cells) via lymphoblasts. Hematopoietic stem cells can also differentiate into monocytes via hematopoietic progenitor cells and monoblasts. Hematopoietic stem cells can also differentiate into hematopoietic progenitor cells, myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band cells, and then into neutrophils. Hematopoietic stem cells can also differentiate into granulocytic leukocytes, such as eosinophils or basophils, or macrophages via hematopoietic progenitor cells and myeloblasts. Hematopoietic stem cells can also differentiate into red blood cells via hematopoietic progenitor cells, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and normochromatic erythroblasts. Hematopoietic stem cells can also differentiate into hematopoietic progenitor cells, promegakaryocytes, and megakaryocytes, which can produce platelets. All cells that differentiate from these hematopoietic stem cells are blood cells. Blood cells include cancer cells and non-cancerous cells. Cancer cells include, for example, chronic myeloid leukemia (CML) cells and CML stem cells.

[0013] As used herein, "ex vivo" means outside the body. As used herein, ex vivo is used in contrast to in vivo (inside the living body) and refers to a state in which cells present in a living body are removed from the living body to outside the body. Culturing can be performed ex vivo.

[0014] As used herein, "positive" means that the cell is recognized as expressing the molecule identified by the term immediately preceding it. As used herein, "positive" may be simply expressed as "+".

[0015] In this specification, "polyvinyl alcohol" (PVA) means a polymer of vinyl alcohol. Polyvinyl alcohol can be obtained by saponifying polyvinyl acetate, which is obtained by polymerizing vinyl acetate monomer. The weight-average molecular weight (M W) can be, for example, 1 kDa to 20 kDa, 3 kDa to 17 kDa, 5 kDa to 15 kDa, or 7 kDa to 13 kDa. When PVA is obtained by saponifying polyvinyl acetate using the above method, the saponification rate can be 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more.

[0016] As used herein, "albumin" refers to a protein known as a plasma component. Albumin is said to account for 60% of plasma proteins and is present in large quantities in blood, serum, and plasma. Albumin is thought to be responsible for maintaining the osmotic pressure of blood in vivo and for binding to and transporting biological substances such as fatty acids and hormones. The importance of serum albumin is also known in the maintenance culture of hematopoietic stem cells. Human serum albumin (hereinafter sometimes referred to as "HSA") is human serum albumin and can be, for example, a protein having the amino acid sequence registered in GenBank under registration number AAN17825.1 or a human serum albumin having an amino acid sequence corresponding to this.

[0017] As used herein, the term "agonist" refers to a substance that activates proteins such as receptors and enzymes.

[0018] As used herein, "PI3K" refers to phosphatidylinositol 3-kinase. PI3K is an enzyme that phosphorylates inositol phospholipids, which are components of cells. Phosphatidylinositol 3,4,5-triphosphate (PIP3), which is generated by phosphorylation, phosphorylates Akt (also known as protein kinase B), transmitting the signal downstream. As used herein, "PI3K activator" refers to an agonist of receptor tyrosine kinase and a substance that activates PI3K. PI3K activators may be selective for PI3K.

[0019] As used herein, "TPO" refers to thrombopoietin. TPO is a protein responsible for the differentiation of hematopoietic stem cells into megakaryocytes. TPO is known to be involved in the proper maintenance of hematopoietic stem cells. Human thrombopoietin may be, for example, a protein having the amino acid sequence registered in GenBank under accession number AAB33390.1 or a thrombopoietin having an amino acid sequence corresponding thereto. As used herein, the term "TPO receptor agonist" refers to a substance other than TPO that activates the TPO receptor. Examples of TPO receptor agonists include TPO variants other than TPO, peptides, and compounds that activate the TPO receptor. As used herein, the term "compound" encompasses organic compounds. The TPO receptor agonist may have selectivity for the TPO receptor.

[0020] As used herein, stem cell factor (SCF) refers to a hematopoietic cell growth factor that acts in the early stage of hematopoiesis. Human stem cell factor may be, for example, a protein having the amino acid sequence registered in GenBank under accession number AAA85450.1 or an SCF having an amino acid sequence corresponding thereto.

[0021] As used herein, "culturing" means incubating cells under conditions suitable for their growth or maintenance. In the case of human cells, incubation may preferably be carried out at 37°C and in a 5% CO2 atmosphere. When "culturing" involves growth, it is understood that "culturing" is the production of proliferated cells. As used herein, "culturing" may be carried out in a serum-free medium. As used herein, "culturing" may be carried out in a chemically defined culture medium (or in a synthetic complete medium). A chemically defined culture medium is a serum-free medium.

[0022] As used herein, "culture medium" refers to a medium used for culturing cells. A culture medium can be prepared by adding necessary components to a basal medium. The necessary components can be a pH adjuster, a sugar source such as glucose, antibiotics (e.g., penicillin, streptomycin, etc.), essential amino acids such as glutamine, and culture additives such as insulin, transferrin, selenium (e.g., sodium selenite), and ethanolamine.

[0023] As used herein, "free" means free from concentrations above the detection limit or completely free. For example, a serum-free medium without added serum or albumin is albumin-free and does not contain serum or albumin. "Albumin-free" means that albumin is not contained at concentrations above the detection limit or is not contained. Furthermore, "cytokine-free" means that cytokines are not contained at concentrations above the detection limit or are not contained. The medium may be albumin-free. The medium may be cytokine-free. The medium may be both albumin-free and cytokine-free.

[0024] As used herein, "cytotoxic" refers to the property of reducing cell numbers or killing cells during culture. As used herein, "non-cytotoxic" refers to the property of not reducing cell numbers during culture. Some substances can become cytotoxic at increasing concentrations. In this case, a substance can be defined as non-cytotoxic if its expected effect occurs even when its concentration is reduced to a level that does not cause cytotoxicity. As used herein, "a medium free of cytotoxic agents" or "free of cytotoxic agents" means that the medium does not contain a cytotoxic agent or cytotoxic agent in an amount sufficient to cause cytotoxicity. Therefore, even if a certain agent is cytotoxic at high concentrations, when used at a concentration that does not cause cytotoxicity, the agent is not a cytotoxic agent or a cytotoxic agent.

[0025] The present inventors have previously reported that mouse hematopoietic stem cells (sometimes called KSL cells, named after their isolation method) can be expanded in large quantities over long periods of time in serum-free medium without albumin by adding polyvinyl alcohol (PVA) (Wilkinson et al., Nature, 571:117-121, 2019). However, no method has been established for expanding human hematopoietic stem cells, even in serum-free medium without albumin. Here, the present inventors have found that human hematopoietic stem cells exhibit significant proliferation while maintaining stemness in the presence of polyethylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block. The present inventors also found that human hematopoietic stem cells exhibited weak activation of various signaling pathways, including the PI3K pathway and the Akt pathway, in the presence of PVA, SCF, and TPO, even in serum-free medium without albumin. The present inventors have also found that human hematopoietic stem cells and human blood lineage cells proliferate in albumin-free serum-free medium by adding a PI3K activator in the presence of PVA, SCF, and TPO. The present inventors have further found that a PI3K activator can completely replace SCF in the presence of PVA, even in albumin-free serum-free medium. The present inventors have also found that a TPO receptor agonist can replace TPO in the presence of PVA and a PI3K activator in albumin-free serum-free medium. The present inventors have further found that human hematopoietic stem cells and human blood lineage cells proliferate well in albumin-free serum-free medium in the presence of polyethylene glycol modified with a polyvinyl caprolactam block and a polyvinyl acetate block copolymer, a PI3K activator, and a TPO receptor agonist, without the need for the addition of SCF or TPO to the medium. The present inventors have further found that human hematopoietic stem cells can be maintained and expanded for a long period of time by adding UM171 to the culture medium.

[0026] According to the present invention, 1. A method for culturing human cells, comprising: Culturing human cells in a culture medium; The culture medium is a serum albumin-free medium and contains an additive, the additive being selected from the group consisting of polyvinyl alcohol and modified polyalkylene glycol; and (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist; The number of human hematopoietic stem cells or human blood cells is maintained or increased by the above culture. method is provided.

[0027] The human cells may be cells selected from the group consisting of human hematopoietic stem cells and human blood cells. Alternatively, the human cells may be blood cells other than hematopoietic stem cells. The blood cells may be cells selected from the group consisting of hematopoietic progenitor cells and blood cells. The blood cells may be one or more cells selected from the group consisting of lymphoblasts, lymphocytes (e.g., T cells, B cells, NK cells, and NKT cells), e.g., CD3-positive cells, myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, and granulocytic leukocytes such as neutrophils, myeloblasts, eosinophils, and basophils, macrophages, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, normochromatic erythroblasts, and erythrocytes, as well as promegakaryocytes and megakaryocytes. The human cells may be, for example, one or more cells selected from the group consisting of chronic myeloid leukemia (CML) cells and CML stem cells.

[0028] In some embodiments, the additive may be an additive that can replace albumin. In some embodiments, the additive may be a modified polyalkylene glycol. In some embodiments, the modified polyalkylene glycol may be, for example, a modified polyethylene glycol. The modified polyalkylene glycol or modified polyethylene glycol may preferably be modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0029] In one embodiment, the additive can be polyvinyl alcohol.

[0030] According to the present invention, the addition of PVA or modified polyalkylene glycol can reduce the amount of albumin, such as serum albumin, added to the culture medium, and preferably make the culture medium albumin-free. In one embodiment, the culture medium can be a serum-free medium, for example, an albumin-free serum-free medium.

[0031] According to the present invention, a PI3K activator can replace SCF. Also according to the present invention, a TPO receptor agonist can replace TPO. In this way, according to the present invention, the amount of SCF and TPO added to the culture medium can be reduced, and preferably, the culture medium can be cytokine-free. In one embodiment, the culture medium can be serum-free, for example, a cytokine-free serum-free medium.

[0032] In some embodiments, the culture medium can be supplemented with PVA or modified polyalkylene glycol to reduce the amount of albumin, such as serum albumin, and with a PI3K activator and a TPO receptor agonist to reduce the amount of SCF and TPO. In some embodiments, the culture medium can be albumin-free, cytokine-free, and serum-free.

[0033] The present invention also provides a method for culturing human hematopoietic stem cells or human blood cells, comprising the steps of: Culturing human hematopoietic stem cells or human blood cells in a culture medium, The culture medium contains polyvinyl alcohol, does not contain albumin, and (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. In this embodiment, the culture can increase or maintain the number of human hematopoietic stem cells or human blood cells.

[0034] According to the present invention, there is provided a method for culturing human hematopoietic stem cells or human blood cells in a culture medium, comprising the steps of: contacting human hematopoietic stem cells or human blood cells with polyvinyl alcohol in a culture medium; In a culture medium, human hematopoietic stem cells or human blood cells (1) contacting a phosphatidylinositol 3-kinase (PI3K) activator with one or more selected from the group consisting of thrombopoietin (TPO) and a TPO receptor agonist; (2) contacting a TPO receptor agonist with one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator; or (3) contacting a PI3K activator with a TPO receptor agonist; In this embodiment, the number of human hematopoietic stem cells or human blood cells can be increased or maintained by the culture.

[0035] In the present invention, the PI3K activator used does not have cytotoxicity to human hematopoietic stem cells or human blood cells in the absence of albumin and the presence of PVA, i.e., the PI3K activator used in the present invention is non-cytotoxic. Furthermore, the TPO receptor agonists used in the present invention are non-cytotoxic to human hematopoietic stem cells or human blood cells in the absence of albumin and the presence of PVA. Those skilled in the art can appropriately confirm whether a PI3K activator is non-cytotoxic by culture tests of human hematopoietic stem cells or human blood cells. Culture tests of human hematopoietic stem cells or human blood cells can be conducted using IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 100 ng / mL TPO, and 0.1% PVA. Those skilled in the art can appropriately confirm whether a TPO receptor agonist is non-cytotoxic by culture tests of human hematopoietic stem cells or human blood cells. Here, culture tests of human hematopoietic stem cells or human blood cells can be confirmed using IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 10 ng / mL SCF, and 0.1% PVA.

[0036] In one aspect of the present invention, the culture medium used in the culture method of the present invention comprises: (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. The culture medium may be for human hematopoietic stem cells or human blood cells.

[0037] In one aspect of the present invention, the culture medium used in the culture method of the present invention comprises: comprising polyvinyl alcohol or modified polyalkylene glycol, (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. The culture medium may be for human hematopoietic stem cells or human blood cells.

[0038] In one aspect of the present invention, the culture medium used in the culture method of the present invention comprises: Albumin (especially serum albumin)-free, (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. The culture medium may be for human hematopoietic stem cells or human blood cells.

[0039] In one aspect of the present invention, the culture medium used in the culture method of the present invention comprises: Contains polyvinyl alcohol, does not contain albumin, and (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. The culture medium may be for human hematopoietic stem cells or human blood cells.

[0040] The culture medium of the present invention may contain an effective amount of a non-cytotoxic PI3K activator and an effective amount of PVA for growing human hematopoietic stem cells or human blood lineage cells in the absence of albumin. The culture medium of the present invention may further contain either or both of SCF and TPO. The culture medium of the present invention may contain an effective amount of a non-cytotoxic TPO receptor agonist instead of TPO. The culture medium of the present invention may contain an effective amount of a non-cytotoxic PI3K activator, an effective amount of PVA, either or both of TPO and a non-cytotoxic TPO receptor agonist in effective amounts, and UM171.

[0041] According to the present invention, the method for culturing human hematopoietic stem cells can be a method for producing megakaryocytic lineage cells from human hematopoietic stem cells. In this embodiment, the culture medium used in the culture method can be a culture medium containing a PI3K activator and a TPO receptor agonist, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide. In this specific embodiment, the culture medium can be free of UM171.

[0042] In one embodiment of the present invention, the culture method of the present invention may further comprise culturing in the presence of 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (hereinafter also referred to as "UM171"). In one embodiment of the present invention, the culture medium used in the culture method of the present invention may further comprise UM171. This may make it easier for human hematopoietic stem cells to maintain their properties as human hematopoietic stem cells, or may suppress differentiation into megakaryocyte-lineage cells (e.g., megakaryocyte progenitor cells and megakaryocytes).

[0043] In one embodiment of the present invention, the culture method of the present invention does not include culturing in the presence of 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (hereinafter also referred to as "UM171"). In one embodiment of the present invention, the culture medium used in the culture method of the present invention further does not contain UM171. This makes it easier for human hematopoietic stem cells to differentiate into megakaryocyte-lineage cells (e.g., megakaryocyte progenitor cells and megakaryocytes).

[0044] The present invention also provides a method for culturing human hematopoietic stem cells or human blood cells, comprising the steps of: Culturing human hematopoietic stem cells or human blood cells in a culture medium, The culture medium comprises polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block; In this method, the culture medium for human hematopoietic stem cells or human blood lineage cells can be albumin-free. In this method, the culture medium (1) comprising one or more selected from the group consisting of a phosphatidylinositol 3-kinase (PI3K) activator and thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) The culture medium may further contain a PI3K activator and a TPO receptor agonist. In this embodiment, the number of human hematopoietic stem cells or human blood cells can be increased or maintained by the culture medium.

[0045] According to the present invention, there is provided a method for culturing human hematopoietic stem cells or human blood cells in a culture medium, comprising the steps of: A method is provided which comprises contacting human hematopoietic stem cells or human blood cells with polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block in a culture medium. In this method, the culture medium for human hematopoietic stem cells or human blood cells can be albumin-free. This method comprises: In a culture medium, human hematopoietic stem cells or human blood cells (1) contacting a phosphatidylinositol 3-kinase (PI3K) activator with one or more selected from the group consisting of thrombopoietin (TPO) and a TPO receptor agonist; (2) contacting a TPO receptor agonist with one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator; or (3) contacting a PI3K activator with a TPO receptor agonist; In this embodiment, the number of human hematopoietic stem cells or human blood cells can be increased or maintained by the culture.

[0046] The present inventors have demonstrated that, in the absence of albumin but in the presence of PVA, some compounds, including some TPO receptor agonists, can exhibit cytotoxicity against human hematopoietic stem cells or human blood cells. Therefore, according to the present invention, the PI3K activator is non-cytotoxic to human hematopoietic stem cells or human blood cells in the absence of albumin and in the presence of PVA. In the present invention, the TPO receptor agonist is non-cytotoxic to human hematopoietic stem cells or human blood cells in the absence of albumin and the presence of PVA. Whether a PI3K activator is non-cytotoxic can be appropriately confirmed by those skilled in the art through a culture test of human hematopoietic stem cells or human blood cells. Here, the culture test of human hematopoietic stem cells or human blood cells can be confirmed using IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 100 ng / mL TPO, and 0.1% PVA. Furthermore, whether a TPO receptor agonist is non-cytotoxic can be appropriately confirmed by those skilled in the art through a culture test of human hematopoietic stem cells or human blood cells. Here, culture tests of human hematopoietic stem cells or human blood cells can be confirmed using IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 10 ng / mL SCF, and 0.1% PVA.

[0047] In one embodiment of the present invention, the PI3K activator may be a peptide represented by the amino acid sequence RQIKIWFQNRRMKWKKSDGGYMDMS in which Y is phosphorylated (also referred to as "740Y-P").

[0048] In one embodiment of the present invention, the TPO receptor agonist can be 3-[4-[[[4-[2-methoxy-3-(1-tert-butyl-2-oxapentan-1-yl)phenyl]thiazol-2-yl]amino]carbonyl]-2,6-dichlorophenyl]-2-methylpropenoic acid (hereinafter also referred to as "butizamide").

[0049] In one embodiment of the invention, the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide.

[0050] In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a chemically defined medium. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is an albumin-free, cytokine-free, or albumin-free and cytokine-free medium (preferably a serum-free medium, more preferably a chemically defined medium). In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and an albumin-free medium. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and a cytokine-free medium. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and an albumin-free and cytokine-free medium. In one embodiment of the present invention, the culture medium used in the culture method of the present invention may contain a PI3K activator and a TPO receptor agonist. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium), an albumin-free and cytokine-free medium, and comprises polyvinyl alcohol, a PI3K activator, and a TPO receptor agonist. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and comprises polyethylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and may comprise polyethylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks, a PI3K activator instead of SCF, and a TPO receptor agonist instead of TPO.In one embodiment of the present invention, the culture medium used in the culture method of the present invention is an albumin-free, cytokine-free, serum-free medium (preferably a chemically defined medium) and may contain polyethylene glycol modified with a copolymer of polyvinyl caprolactam and polyvinyl acetate blocks, a PI3K activator, and a TPO receptor agonist. In one embodiment of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium (preferably a chemically defined medium) and may contain polyethylene glycol modified with a copolymer of polyvinyl caprolactam and polyvinyl acetate blocks, a PI3K activator instead of SCF, a TPO receptor agonist instead of TPO, and 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine (UM171). In one embodiment of the present invention, the culture medium used in the culture method of the present invention is an albumin-free, cytokine-free, serum-free medium (preferably a chemically defined medium), and may contain polyethylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, a PI3K activator, a TPO receptor agonist, and UM171. As used herein, the expression "containing B instead of A" means that the culture medium does not contain A but contains B.

[0051] In one aspect of the present invention, a medium for culturing human hematopoietic stem cells or human blood cells comprises: It comprises polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and (1) comprising a PI3K activator and one or more selected from the group consisting of TPO and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of SCF and PI3K activators and a TPO receptor agonist; or (3) A compound comprising a PI3K activator and a TPO receptor agonist. The medium may be a culture medium for human hematopoietic stem cells or human blood cells. In this embodiment, the medium may not contain albumin. The medium for culturing human hematopoietic stem cells or human blood cells may further contain UM171. The medium for culturing human hematopoietic stem cells or human blood cells may be used in the culturing method of the present invention. The medium for culturing human hematopoietic stem cells or human blood cells of the present invention is capable of proliferating human hematopoietic stem cells or human blood cells. The medium for culturing human hematopoietic stem cells or human blood cells of the present invention is capable of maintaining the stemness of human hematopoietic stem cells. The medium for culturing human hematopoietic stem cells of the present invention is capable of proliferating human hematopoietic stem cells while maintaining their stemness.

[0052] As the culture medium, one suitable for culturing human hematopoietic stem cells or human blood cells can be used as appropriate. The basal media used include S-clone SF-3 medium, F12 medium, StemSpan (Stem Cell Technologies), STEMα (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium (Invitrogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitrogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM / F-12 medium (Invitrogen), Stemline hematopoietic stem cell growth medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), and MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen and others), MEM medium (Gibco Invitrogen and others), MEMα (Gibco Invitrogen and others), DMEM medium (Gibco Invitrogen and others), IMDM medium (Gibco Invitrogen and others), PRMI1640 medium (Gibco Invitrogen and others), Ham's F-12 medium (Gibco and others), RD medium, and the like can be used. The culture medium includes a basal medium. The culture medium may contain, for example, one or more or all of insulin, transferrin (apo), sodium selenite, and ethanolamine. The culture medium may also contain HEPES, sodium pyruvate, vitamins, amino acids, heparin, heparan sulfate, chondroitin sulfate, and the like. The culture medium may also contain antibiotics (e.g., penicillin and streptomycin). The culture medium may contain glutamine.The culture medium may contain, for example, insulin, transferrin (apo), sodium selenite, ethanolamine, and antibiotics, and may further contain HEPES.

[0053] The culture medium of the present invention may comprise a polyalkylene glycol (eg, polyethylene glycol) modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0054] In all aspects of the present invention, the polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may have the following chemical formula:

[0055] [ka] {In the formula, n is a natural number between 5 and 50, m is a natural number between 10 and 100, and l is a natural number between 10 and 200.}

[0056] In one embodiment, n can be a natural number between 5 and 20, or between 10 and 20, m can be a natural number between 20 and 50, or between 30 and 40, and l can be a natural number between 30 and 100, or between 50 and 60.

[0057] The modified polyalkylene glycol may be a compound in which the ethylene glycol unit is replaced with an alkylene glycol unit. The alkylene may be an alkylene having 1 to 10 carbon atoms, for example, 1 to 5 carbon atoms, and preferably 2 or 3 carbon atoms.

[0058] The culture medium of the present invention may contain an effective amount of a non-cytotoxic PI3K activator and an effective amount of a polyalkylene glycol (e.g., polyethylene glycol) modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block for growing human hematopoietic stem cells or human blood lineage cells in the presence of polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. Here, the medium may be albumin-free. The culture medium of the present invention may further contain either or both of SCF and TPO. The culture medium of the present invention may contain an effective amount of a non-cytotoxic TPO receptor agonist instead of TPO. The culture medium of the present invention may contain an effective amount of a non-cytotoxic PI3K activator, an effective amount of PVA, either or both of effective amounts of TPO and a non-cytotoxic TPO receptor agonist, and UM171.

[0059] According to the present invention, the method for culturing human hematopoietic stem cells can be a method for producing megakaryocytic lineage cells from human hematopoietic stem cells. In this embodiment, the culture medium used in the culture method can be a culture medium containing a PI3K activator and a TPO receptor agonist, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide. In this specific embodiment, the culture medium can be free of UM171.

[0060] In one embodiment of the present invention, the culture method of the present invention may further comprise culturing in the presence of UM171. In one embodiment of the present invention, the culture medium used in the culture method of the present invention may further comprise UM171.

[0061] In the present invention, human hematopoietic stem cells or human blood cells can be cultured under conditions suitable for the proliferation of human hematopoietic stem cells or the human blood cells. The present invention may further comprise isolating human hematopoietic stem cells or human blood cells from the culture. Isolation of human hematopoietic stem cells can be performed by methods known to those skilled in the art, for example, by flow cytometry using a hematopoietic stem cell marker. Human hematopoietic stem cells may be obtained as human hematopoietic stem cells, or may be further differentiated into other cells before use. When differentiating human hematopoietic stem cells into other cells, the cells to be differentiated can be cultured under conditions suitable for the differentiation of the other cells. Isolation of human blood cells can also be performed by methods known to those skilled in the art, for example, by flow cytometry using a marker for the human blood cells.

[0062] According to the present invention, human hematopoietic stem cells or human blood cells obtained by the culture method of the present invention are provided. Human hematopoietic stem cells obtained by the culture method of the present invention can be purified using CD34 and preferably CD38 as markers. Human hematopoietic stem cells obtained by the culture method of the present invention have a better engraftment rate after transplantation into a recipient than human hematopoietic stem cells obtained by conventional methods. Thus, according to the present invention, human hematopoietic stem cells obtained by the culture method of the present invention are provided that have an improved engraftment rate after transplantation into a recipient compared to before culture.

[0063] In the culture method of the present invention, the culture can be performed in the presence of fibronectin. In the culture method of the present invention, the culture can be performed under conditions that allow contact between hematopoietic stem cells and fibronectin. In the culture method of the present invention, the culture is preferably performed, for example, by coating the inside (e.g., bottom) of a culture vessel with fibronectin.

[0064] The albumin-free medium used in the culture method of the present invention contains less than 0.1 (w / v)%, less than 0.05 (w / v)%, less than 0.01 (w / v)%, less than 0.005 (w / v)%, less than 0.001 (w / v)%, less than 0.0005 (w / v)%, or less than 0.0001 (w / v)%, or is completely free of serum albumin.

[0065] The medium used in the culture method of the present invention may contain recombinant TPO. The recombinant TPO may be, for example, a mammalian recombinant TPO, or may be recombinant human TPO. In one embodiment of the present invention, the TPO concentration is 20 to 200 ng / mL, more preferably 30 to 150 ng / mL, and even more preferably 40 to 150 ng / mL, and can be, for example, 100 ng / mL.

[0066] The medium used in the culture method of the present invention may further contain recombinant SCF. The recombinant SCF may be, for example, mammalian recombinant SCF or recombinant human SCF. In one embodiment of the present invention, the SCF concentration is 1 to 200 ng / mL, more preferably 1 to 150 ng / mL, even more preferably 1 to 100 ng / mL, for example, 1 to 50 ng / mL, even more preferably 1 to 30 ng / mL, even more preferably 1 to 20 ng / mL, for example, 5 to 15 ng / mL.

[0067] The medium used in the culture method of the present invention may contain recombinant TPO and recombinant SCF. In this embodiment, the TPO concentration is 20 to 200 ng / mL, more preferably 30 to 150 ng / mL, even more preferably 40 to 150 ng / mL, and can be, for example, 100 ng / mL, and the SCF concentration is 1 to 200 ng / mL, more preferably 1 to 150 ng / mL, even more preferably 1 to 100 ng / mL, for example, 1 to 50 ng / mL, even more preferably 1 to 30 ng / mL, and even more preferably 1 to 20 ng / mL, and can be, for example, 5 to 15 ng / mL. In a preferred embodiment, the medium used in the culture method of the present invention may contain 40 to 150 ng / mL of recombinant TPO and 1 to 50 ng / mL of recombinant SCF. In a preferred embodiment, the culture medium used in the culture method of the present invention has a TPO concentration higher than the SCF concentration, which may be, for example, any concentration within the above concentration range, and may be 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more higher.

[0068] The culture medium used in the culture method of the present invention may further contain Flt3L (particularly, human Flt3L). FLT3 is a cell surface receptor known as FMS-related tyrosine kinase 3 (also known as CD135). FLT3 is expressed on the surface of, for example, hematopoietic stem cells. FLT3L is a ligand for FLT3 and is involved in, for example, the normal development of hematopoietic stem cells and hematopoietic progenitor cells. The culture medium used in the culture method of the present invention may further contain IL-3 and / or GM-CSF in addition to Flt3L. Interleukin-3 (IL-3) is a hematopoietic growth factor that plays an important role in the proliferation and survival of myeloid progenitor cells. GM-CSF is a granulocyte-monocyte colony-stimulating factor and a cytokine that promotes the differentiation of hematopoietic stem cells. GM-CSF can act cooperatively with IL-3 and IL-5 to differentiate hematopoietic stem cells into myeloid progenitor cells. For example, GM-CSF can differentiate hematopoietic stem cells into, for example, early erythroid progenitor cells (BFU-E), colony-forming cells of granulocytes and monocytes (CFU-GM), colony-forming cells of eosinophils (CFU-Eo), and colony-forming cells of basophils (CFU-Ba). GM-CSF differentiates CFU-GM into neutrophils and monocytes, and CFU-Eo into eosinophils. CFU-Ba can be differentiated into basophils by IL-3 or IL-5. Therefore, the culture medium used in the culture method of the present invention may contain Flt3L and may further contain IL-3 and / or GM-CSF. Furthermore, the culture method of the present invention can be performed under conditions suitable for the proliferation and / or differentiation of each blood cell lineage. Such culture conditions are well known as culture conditions for albumin-containing media and can be similarly applied to the culture method of the present invention using a medium with reduced or no albumin.

[0069] The culture medium used in the culture method of the present invention may contain a PI3K activator instead of SCF and / or a TPO receptor agonist instead of TPO. The culture medium used in the culture method of the present invention may contain a PI3K activator instead of SCF and a TPO receptor agonist instead of TPO. The culture medium used in the culture method of the present invention may be a cytokine-free medium and contain a PI3K activator and a TPO receptor agonist. The culture medium used in the culture method of the present invention may contain a PI3K activator instead of SCF, a TPO receptor agonist instead of TPO, and further contain UM171. The culture medium used in the culture method of the present invention may be a cytokine-free medium and contain a PI3K activator, a TPO receptor agonist, and UM171.

[0070] The method for culturing human hematopoietic stem cells of the present invention may further comprise expanding the hematopoietic stem cells under conditions sufficient for the maintenance and / or proliferation of the hematopoietic stem cells. In this embodiment, for example, the method may comprise expanding the hematopoietic stem cells by 10-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 400-fold or more, or 500-fold or more from the start of the culture.

[0071] Conditions sufficient for the maintenance and / or proliferation of human hematopoietic stem cells may be, for example, conditions for culturing in the above-mentioned medium. Preferably, conditions sufficient for the maintenance and / or proliferation of human hematopoietic stem cells may be, for example, conditions in the presence of fibronectin, or conditions that allow contact between human hematopoietic stem cells and fibronectin.

[0072] The method for culturing human blood cells of the present invention may further comprise expanding the human blood cells under conditions sufficient for the maintenance and / or proliferation of the human blood cells. In this embodiment, for example, the method may comprise expanding the human blood cells by 10-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 400-fold or more, or 500-fold or more from the start of culture.

[0073] The culture method of the present invention comprises: Recovering expanded human hematopoietic stem cells or human blood cells from the culture medium The collected human hematopoietic stem cells or human blood cells may further be enriched or isolated. Enrichment or isolation of human hematopoietic stem cells or human blood cells can be performed using a cell surface marker. Examples of cell surface markers that can be used for enrichment or isolation of human hematopoietic stem cells include CD34 and CD38. Enrichment or isolation of hematopoietic stem cells or human blood cells can be performed using a cell sorter.

[0074] According to the present invention, there is provided a method for producing human hematopoietic stem cells or human blood cells ex vivo, comprising the steps of: Methods are provided that include the culture methods of the present invention. The production methods of the present invention can obtain functional human hematopoietic stem cells or human blood lineage cells. Here, "functional" means that human hematopoietic stem cell transplantation can restore the hematopoietic system in the human individual that has received the transplant (recipient). [Example]

[0075] Reference Example 1: Hematopoietic stem cell proliferation test using albumin-free culture medium In this reference example, we tested the proliferation of mouse hematopoietic stem cells (KSL) and human hematopoietic stem cells (CD34+CD38-) in an albumin-free culture medium. The culture medium was a serum-free medium containing polyvinyl alcohol (PVA) and not containing albumin, as described by Wilkinson et al., Nature, 571:117-121, 2019.

[0076] Specifically, for the culture of mouse hematopoietic stem cells, the culture medium was F12 medium containing 1% insulin-transferrin-selenium-ethanolamine (ITSX), 10 mM HEPES, 1% penicillin-streptomycin-glutamine, 100 ng / mL mouse thrombopoietin (mTPO), and 10 ng / mL mouse stem cell factor (mSCF). PVA was added to the medium at a final concentration of 0.1%. For the culture of human hematopoietic stem cells, the culture medium was IMDM medium containing 1% insulin-transferrin-selenium-ethanolamine (ITSX), 25 mM HEPES, 1% penicillin-streptomycin-glutamine, 100 ng / mL human thrombopoietin (hTPO), and 10 ng / mL human stem cell factor (hSCF). PVA was added to the medium at a final concentration of 0.1%. Unless otherwise specified, all media used in the following examples are IMDM medium containing 1% insulin-transferrin-selenium-ethanolamine (ITSX), 25 mM HEPES, 1% penicillin-streptomycin-glutamine, and PVA (hereinafter also referred to as "common medium"). Therefore, hereafter, the medium will be described by focusing on the components added to the common medium. For example, the above medium contains TPO and SCF in addition to the common medium, so for convenience, it will be referred to as TPO+SCF medium. When the concentration of each factor is also indicated, it will be written as SCF10+TPO100, or it may be abbreviated as S10+T100.

[0077] Mouse bone marrow KSL cells were used as hematopoietic stem cells. Specifically, mouse bone marrow cells were isolated from the tibia, femur, and pelvis and stained with APC-c-KIT antibody. c-KIT+ cells were enriched using anti-APC magnetic beads and an LS column (Miltenyi Biotec). The c-KIT-enriched cells were then stained with a lineage antibody cocktail (biotinylated CD4, CD8, CD45R, TER119, LY-6G / LY-6C, and CD127) before staining with anti-CD34, anti-c-KIT, anti-SCA1, and streptavidin-APC / eFluor 780 for 90 minutes. The cell population was then purified by sorting directly into wells containing medium using a FACS Aria II (BD) with propidium iodide as a death stain.

[0078] Human bone marrow CD34+CD38- cells were used as human hematopoietic stem cells. Specifically, commercially available human bone marrow CD34-positive cells (Lonza 2C-101) were purchased.

[0079] The results are shown in Figure 1. Wilkinson et al. (2019) reported that mouse hematopoietic stem cells were unable to maintain long-term proliferation in serum-free medium without albumin, even in the presence of SCF and TPO. However, Wilkinson et al. (2019) demonstrated that adding PVA to the medium enabled long-term proliferation of mouse hematopoietic stem cells in serum-free medium without albumin. As shown in Figure 1, mouse hematopoietic stem cells proliferated well in serum-free medium containing PVA but without albumin, whereas human hematopoietic stem cells did not proliferate well in this medium.

[0080] Reference Example 2: Differences in signal transduction between mouse and human hematopoietic stem cells We analyzed the signaling pathways of mouse and human hematopoietic stem cells in the presence of SCF and TPO.

[0081] Mouse and human hematopoietic stem cells were cultured in the presence of 10 ng / mL tissue factor (SCF) and 100 ng / mL thrombopoietin (TPO) in the absence of albumin but the presence of PVA. The phosphorylation levels of downstream signaling factors of SCF and TPO in these hematopoietic stem cells were analyzed by phosphorylation immunostaining. The phosphorylation status of each downstream factor of SCF and TPO signaling was detected using antibodies specific for the phosphorylated forms of each factor. Specifically, the cells were stimulated with cytokines and then exposed to phosphoantibodies against representative signaling molecules, such as Akt, PI3K, and Stat5. The fluorescence intensity of the antibodies reacted with the cells was quantitatively analyzed by fluorescence microscopy.

[0082] As shown in Figure 2, several of the seven factors examined were found to have different phosphorylation states between mouse and human hematopoietic stem cells. Among these, PI3K and Akt were found to be highly phosphorylated in mouse hematopoietic stem cells 24 hours after the addition of SCF and TPO, whereas in human hematopoietic stem cells, the phosphorylated forms were barely observed or were lower than those in mouse hematopoietic stem cells.

[0083] Therefore, human hematopoietic stem cells were cultured in a culture medium supplemented with 0.3 μM AKTa (Sigma-Aldrich, product number 123871) or 20 μM PI3Ka. Cells were counted after 3, 5, and 7 days of culture, and the ratio of the cell number at each time point to the cell number on the first day of culture was calculated. In the following examples, 740Y-P (Tocris, product number 1983) was used as PI3Ka. As shown in Figure 3, human hematopoietic stem cells showed clear proliferation in the presence of PI3Ka. On the other hand, AKTa did not exhibit any proliferative effect on human hematopoietic stem cells under the experimental conditions.

[0084] Next, we investigated whether PI3Ka completely replaces SCF in the culture of human hematopoietic stem cells. After 7 days of culture, human hematopoietic stem cells were cultured in the above-mentioned culture medium for human hematopoietic stem cells, supplemented with 20 μM PI3Ka (S10 + PI3Ka20 + T100) and in the above-mentioned culture medium supplemented with 20 μM PI3Ka but omitting SCF (PI3Ka20 + T100). The total cell count and the number of CD34+ cells isolated by cell sorting using anti-CD34 antibodies were determined. The results are shown in Figure 4. As shown in Figure 4, when PI3Ka was added, there was no significant difference in the total cell count or the number of CD34+ cells, regardless of the presence or absence of SCF.

[0085] Next, we investigated whether TPO could be replaced by a TPO receptor agonist in the culture of human hematopoietic stem cells. Known TPO receptor agonists include butizamide, eltrombopag, and avatrombopag. Human hematopoietic stem cells were cultured in the above-mentioned culture medium without TPO, supplemented with 0.1 μM butizamide, 3 μg / mL eltrombopag, or 3 μM avatrombopag in the presence of 0.1% recombinant human serum albumin (Albumin Biosciences) or PVA. Mpl32D cells were used as human hematopoietic stem cells in this experiment. The results are shown in Figure 5. As shown in Figure 5, in the presence of albumin, butizamide, eltrombopag, and avatrombopag were able to support the proliferation of human hematopoietic stem cells in the absence of TPO. In contrast, in the presence of PVA (absence of albumin), butizamide had a significant cell proliferation effect on human hematopoietic stem cells in the absence of TPO, but the effect was small with avatrombopag, and almost no effect was observed with eltrombopag.

[0086] In addition, purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood was used for the culture experiments. CD34+ cells were isolated using microbeads. 24-well plates were cultured at 0.2–1.0 × 10 per well. 5Cells were aliquoted, and TPO was removed from the culture medium for human hematopoietic stem cells and replaced with one of the above TPO receptor agonists (hereinafter sometimes referred to as "TPOago"). The ratio of cell number on day 7 to that on day 1 of culture was calculated. The results are shown in Figure 6. As shown in Figure 6, human hematopoietic stem cells showed significant cell proliferation only in the presence of butizamide in the absence of albumin and in the presence of PVA. In the absence of albumin and in the presence of PVA, hematopoietic stem cells underwent cell death in the presence of abatrombopag or eltrombopag. However, these TPO receptor agonists are highly safe compounds used in vivo to treat patients undergoing surgical treatment for liver cirrhosis and patients with aplastic anemia. The results of this example demonstrate that some TPO receptor agonists can exhibit cytotoxicity against human hematopoietic stem cells in the absence of albumin and in the presence of PVA. This also suggests that some TPO receptor agonists are cytotoxic to human hematopoietic stem cells, and that in order to culture human hematopoietic stem cells, it is sufficient to use one that is not cytotoxic to human hematopoietic stem cells.

[0087] Next, we investigated whether SCF and TPO could be replaced by PI3Ka and a TPO receptor agonist in the presence of PVA in the absence of albumin. In the following examples, butizamide was used as the TPO receptor agonist. CD34+ cells were isolated from purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood using microbeads as described above and used in the culture experiments. 0.2 to 1.0 × 10 cells per well were placed in a 24-well plate. 5Cells were aliquoted and cultured in a medium containing 20 μM PI3Ka substituted for SCF (PI3Ka 20 μM + TPO 100 μM) or 20 μM PI3Ka and 0.1 μM butizamide substituted for TPO (PI3Ka 20 μM + TPO 0.1 μM). After 7 days, the total cell count was determined, and the CD34+ cell count was determined by flow cytometry. The proliferation rate was then calculated compared to the initial count. The results are shown in Figure 7. As shown in Figure 7, butizamide completely replaced TPO. CD34+ cells were sorted before and after culture using a cell sorter, and 100 cells were seeded onto Methocult H4415 for colony assay. After 2 weeks, colonies were picked, cytospun, stained with Giemsa, and characterized under a microscope. The number of GEmM colonies per 50 CD34+ cells was counted, and the increase in the number of colonies compared to before culture was calculated. This showed that butizamide could completely replace TPO in terms of GEmM colony formation ability.

[0088] Next, we cultured human hematopoietic stem cells in medium containing 20 μM PI3Ka and / or 0.1 μM TPOago in culture media lacking SCF and TPO. On day 7 of culture, the total cell numbers and the CD34+ cell counts were counted by flow cytometry, and the proliferation rate relative to the initial time point was calculated. As shown in Figure 8, while PI3Ka alone or butizamide alone did not significantly increase the cell number, the presence of both PI3Ka and TPOago significantly increased the cell number.

[0089] Next, we investigated which cell populations were more likely to expand in a medium in which SCF and TPO were replaced with PI3Ka and TPOago. Fresh cord blood samples were used to separate CD34+ cells using microbeads, as described above. Cells were fractionated using a cell sorter using anti-CD34-PE-Cy7 antibody (BD Biosciences, Product No. 348791), anti-CD38-V450 antibody (BD Biosciences, Product No. 646851), anti-CD133-PE antibody (Miltenyi Biotec, Product No. 130-080-801), anti-CD45RA-APC antibody (BioLegend, Product No. 304112), and anti-CD49f-PE antibody (BioLegend, Product No. 313611). The CD34+ fraction, CD34+CD38-CD133+ fraction, and CD34+CD38-CD45RA-CD49f+ fraction, which have been reported as purification markers for human umbilical cord blood-derived hematopoietic stem cells, were counted by flow cytometry on day 7 after the initiation of culture, and the proliferation rate relative to the initiation of culture was calculated. The results are shown in Figure 9. As shown in Figure 9, significant cell proliferation was observed in all cell fractions, but cell proliferation was particularly pronounced in the CD34+CD38-CD133+ fraction and the CD34+CD38-CD45RA-CD49f+ fraction, especially the CD34+CD38-CD45RA-CD49f+ fraction.

[0090] Reference Example 3: Long-term culture experiment of human hematopoietic stem cells Human hematopoietic stem cells were cultured in the same culture medium as above, but containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO. The total cell count and CD34+ cell count were determined in the same manner as above on days 7 and 14 after the start of culture. The results are shown in Figure 10. As shown in Figure 10, the total cell count increased with the passage of culture days, whereas the CD34+ cell count was lower on day 14 than on day 7.

[0091] Observation of the cultured cells under an optical microscope revealed the presence of giant cells on day 14. To confirm the possibility that these giant cells were megakaryocytes or megakaryocyte progenitor cells, the cells were fractionated using a flow cytometer with anti-CD41a-FITC antibody (BD Pharmingen, product number 555466) and anti-CD42b antibody (BD Pharmingen, product number 555473). As shown in Figure 11, the majority of cells on day 14 after the start of culture were CD41a+CD42b+ cells. However, as shown in Figure 11, CD34+CD38- cells proliferated even under these conditions.

[0092] In addition, 100 CD34+ cells from the culture obtained on day 14 after the start of culture were seeded onto Methocult H4415 and subjected to colony assay. After two weeks, colonies were collected and cytospin specimens were prepared. The specimens were then stained with Giemsa and the colony types were determined under a microscope. The results are shown in Figure 12. Regarding colony types, G indicates a colony containing "granulocytes," E indicates a colony containing "erythroblasts," M indicates a colony containing "macrophages," and M indicates a colony containing "megakaryocytes." As shown in Figure 12, most of the cell colonies contained megakaryocytes.

[0093] Reference Example 4: Examination of conditions more suitable for long-term culture of human hematopoietic stem cells We attempted to culture human hematopoietic stem cells for a long period in the presence of compounds that can proliferate human hematopoietic stem cells.

[0094] We prepared the above-mentioned culture medium for human hematopoietic stem cells, containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO (PI3Ka 20 μM + TPOago 0.1 μM), and the above-mentioned medium supplemented with either SR-1 (500 nM) or UM171 (35 nM) (+SR-1, +UM171, and +SR-1 + UM171). CD34+ cells were isolated from purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood using microbeads as described above and used for the culture experiments. 0.2–1.0 × 10 cells were cultured per well in a 24-well plate. 5 The cells were dispensed and cultured as CD34+ cells in the prepared medium. On day 14 after the start of culture, the total number of cells and CD34+ cells were counted as described above, and the proliferation rate from before the start of culture was calculated. The results are shown in Figure 13. As shown in Figure 13, the total number of cells and the number of CD34+ cells increased in the culture medium further containing UM171, and the proliferation rate of CD34+ cells was significantly increased in the medium containing UM171 compared to the medium without UM171. In contrast, SR-1 killed the cells under these experimental conditions.

[0095] Furthermore, CD34+ cells were cultured under three conditions: Condition 1: the same culture medium as above for human hematopoietic stem cells, but containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO (PI3Ka 20 μM + TPOago 0.1 μM), for 14 days; Condition 2: culture in medium without butizamide (PI3Ka 20 μM) from Day 7 onwards (Day 7 - No Buty); and Condition 3: culture medium (PI3Ka 20 μM + TPOago 0.1 μM) with the addition of UM171 (+UM171) for 14 days. The total number of cells and the rate of increase in CD34+ cell count were also measured using a flow cytometer. The results are shown in Figure 14. As shown in Figure 14, under condition 2, in which cells were cultured in a medium lacking butizamide on day 7 after the start of culture, the number of CD34+ cells increased and the number of CD41+ cells decreased compared to condition 1. As shown in Figure 14, under condition 3, in which cells were cultured in a medium further containing UM171, the rate of increase in CD34+ cells significantly increased and the number of CD41+ cells significantly decreased compared to conditions 1 and 2. These results demonstrate that, in serum-free medium conditions in the presence of PVA, PI3Ka, and TPOago, UM171 proliferates human hematopoietic stem cells and suppresses their differentiation into megakaryocyte progenitor cells and megakaryocytes (see Figure 15).

[0096] Reference Example 5: Transplantation experiment of CD34+ cells after culture Human CD34+ cells cultured for 7 days under each of conditions 1 to 3 were transplanted into irradiated NOG mice to confirm cell engraftment. Specifically, 1 × 10 human CD34+ cells were transplanted into NOG mice irradiated with 1.5 Gy of gamma rays. 4Cells were transplanted. Twelve weeks after transplantation, peripheral blood was collected from the NOG mice, and the cellular components in the peripheral blood were analyzed using a flow cytometer. The results are shown in Figure 16. As shown in Figure 16, under conditions 1 and 2, the hematopoietic stem cell engraftment rates were 14.9% and 11.1%, respectively, when pre-culture CD34+ cells were transplanted, but increased to 66.6% and 54.9%, respectively, when cultured CD34+ cells were transplanted. Furthermore, under condition 3, the hematopoietic stem cell engraftment rate was 17% when pre-culture CD34+ cells were transplanted, but increased to 67% when cultured CD34+ cells were transplanted.

[0097] These results demonstrate that in serum-free medium without albumin and in the presence of PVA, PI3K activators and TPO receptor agonists effectively proliferate human hematopoietic stem cells, and that the expanded human CD34+ cells maintain their hematopoietic stem cell properties and improve engraftment rates after transplantation into other individuals. Furthermore, when human hematopoietic stem cells are cultured for long periods under these conditions, some differentiate into megakaryocytes, thereby producing megakaryocytes, while others tend to proliferate as CD34+ cells. The addition of UM171 to these cells also improved the proliferation rate of CD34+ cells and suppressed their differentiation into megakaryocytes.

[0098] Next, human mononuclear cells were isolated from fresh human umbilical cord blood. The cells obtained under conditions 1 and 3 (5 × 10 5 The total cell count, CD34+ cell count, percentage of CD34+ cells among total cells, and cell viability were determined 7 and 14 days after the start of culture. The results are shown in Figure 17. As shown in Figure 17, under all conditions, the total cell count of mononuclear cells obtained from human umbilical cord blood decreased with culture. On the other hand, the percentage of CD34+ cells among total cells was significantly higher under Condition 3 than under Condition 1. Cell viability was also significantly higher under Condition 3 than under Condition 1. Furthermore, the number of CD34+ cells showed a significant increase under Condition 3, whereas under Condition 1, the cell count was maintained, but no increase in cell count was observed.

[0099] Example 1: Hematopoietic stem cell proliferation test using albumin-free culture medium In this example, we tested the proliferation of mouse hematopoietic stem cells (KSL) and human hematopoietic stem cells (CD34+CD38-) in an albumin-free culture medium. The culture medium was the albumin-free serum-free medium described by Wilkinson et al., Nature, 571:117-121, 2019, but contained various polymers instead of polyvinyl alcohol (PVA).

[0100] Specifically, for the culture of mouse hematopoietic stem cells, the culture medium was Ham's F12 medium containing 1% insulin-transferrin-selenium-ethanolamine (ITSX), 10 mM HEPES, 1% penicillin-streptomycin-glutamine, 100 ng / mL mouse thrombopoietin (mTPO), and 10 ng / mL mouse stem cell factor (mSCF). For the culture of human hematopoietic stem cells, the culture medium was IMDM medium containing 1% insulin-transferrin-selenium-ethanolamine (ITSX), 25 mM HEPES, 1% penicillin-streptomycin-glutamine, 100 ng / mL human thrombopoietin (hTPO), and 10 ng / mL human stem cell factor (hSCF). In the following examples, unless otherwise specified, the above culture medium was used for the culture of hematopoietic stem cells.

[0101] The polymers added to the medium were as follows: polyvinylpyrrolidone K12, povidone K17, povidone, polyoxyethylene polyoxypropylene glycol, and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (hereinafter referred to as "Polymer A"). More specifically, the same concentrations of BASF Corifol™ P188 bio (188 bio), Corifol™ P 188 Geismar (188 Geismar), Sol+, Corifol™ P 407 Geismar (407 Geismar), Kollidon™ 30 Origin USA (30 USA), Kollidon™ 17 PF (17 PF), Kollidon™ 25 (25), Kollidon™ 90F (90F), and Kollidon™ 12PF (12PF) were added to the medium at a final concentration of 0.1%.

[0102] We used the CD34-CD150+ KSL cell fraction from mouse bone marrow as the hematopoietic stem cell. Specifically, mouse bone marrow cells were isolated from the tibia, femur, and pelvis and stained with APC-c-KIT antibody. c-KIT+ cells were enriched using anti-APC magnetic beads and an LS column (Miltenyi Biotec). The c-KIT-enriched cells were then stained with a lineage antibody cocktail (biotinylated CD4, CD8, CD45R, TER119, LY-6G / LY-6C, and CD127) before staining with anti-CD34, anti-c-KIT, anti-SCA1, and streptavidin-APC / eFluor 780 for 90 minutes. The cell population was then purified by sorting directly into wells containing medium using a FACS Aria II (BD) with propidium iodide as a death stain.

[0103] Human bone marrow CD34+CD38- cells were used as human hematopoietic stem cells. Specifically, commercially available human bone marrow CD34-positive cells (Lonza 2C-101) were purchased.

[0104] Mouse hematopoietic stem cells were cultured in each medium for one week, and the number of cells after culture was counted.

[0105] The results are shown in Figure 18. As shown in Figure 18, mouse hematopoietic stem cells proliferated well in a serum-free medium containing PVA but not containing albumin, but did not proliferate in the presence of other compounds tested in place of PVA, and proliferated only in the presence of Compound A.

[0106] Soluplus™ was a compound having the following structure: [ka] {wherein n is 13, m is 30, and l is 57.}

[0107] In Wilkinson et al., 2019, mouse hematopoietic stem cells were unable to maintain long-term proliferation in serum-free medium without albumin, even in the presence of SCF and TPO. However, in Wilkinson et al., 2019, Figure 2b showed that adding PVA to the medium enabled long-term proliferation of mouse hematopoietic stem cells in serum-free medium without albumin.

[0108] This demonstrated that mouse hematopoietic stem cells proliferate well in the presence of PVA or polymer A even in serum-free medium without albumin.

[0109] Mouse hematopoietic stem cells were sorted at a rate of 50 cells / well and cultured in the presence of 0.1% PVA or polymer A with SCF and TPO. The results are shown in Figure 19. As shown in Figure 19, mouse hematopoietic stem cells successfully formed colonies in the presence of PVA or polymer A, even in serum-free medium without albumin. This demonstrated that hematopoietic stem cells exhibit cell division ability in the presence of PVA and polymer A.

[0110] The percentage of CD201-positive cells among the expanded cells was determined using alkaline phosphatase-labeled anti-mouse CD201 antibody (eBio1560). As shown in Figure 20, mouse hematopoietic stem cells cultured in the presence of polymer A had a higher percentage of CD201-positive cells than those cultured in the presence of PVA. CD201 is known as a marker for hematopoietic stem cells, and the high percentage of CD201-positive cells suggests that stemness was better maintained by expansion.

[0111] Furthermore, mouse hematopoietic stem cells were cultured for one week in the presence of polymer A or PVA at a final concentration of 0.1% and then transplanted into irradiated mice. Four weeks after transplantation, peripheral blood samples from the transplanted irradiated mice were analyzed to determine the chimeric rate of blood cells derived from the transplanted hematopoietic stem cells. As a result, hematopoietic stem cells cultured in the presence of polymer A showed a chimeric rate (contribution rate) equivalent to that of hematopoietic stem cells cultured in PVA. This indicates that the bone marrow reconstitution capacity of hematopoietic stem cells cultured in the presence of polymer A was equivalent to that of hematopoietic stem cells cultured in the presence of PVA.

[0112] Example 2: Differences in signal transduction between mouse and human hematopoietic stem cells We analyzed the signaling pathways of mouse and human hematopoietic stem cells in the presence of SCF and TPO.

[0113] Mouse and human hematopoietic stem cells were cultured in the presence of 10 ng / mL tissue factor (SCF) and 100 ng / mL thrombopoietin (TPO) in the absence of albumin but the presence of PVA. The phosphorylation levels of downstream signaling factors of SCF and TPO in these hematopoietic stem cells were analyzed by phosphorylation immunostaining. The phosphorylation status of each downstream factor of SCF and TPO signaling was detected using antibodies specific for the phosphorylated forms of each factor. Specifically, the cells were stimulated with cytokines and then exposed to phosphoantibodies against representative signaling molecules, such as Akt, PI3K, and Stat5. The fluorescence intensity of the antibodies reacted with the cells was quantitatively analyzed by fluorescence microscopy.

[0114] As a result, as shown in Figure 24, among the seven factors examined, several factors were found to have different phosphorylation states between mouse and human hematopoietic stem cells. Among these, PI3K and Akt were found in high phosphorylated forms in mouse hematopoietic stem cells 24 hours after the addition of SCF and TPO, whereas in human hematopoietic stem cells, the phosphorylated forms were hardly observed or were present in amounts lower than those in mouse hematopoietic stem cells.

[0115] Therefore, human hematopoietic stem cells were cultured in a culture medium supplemented with 0.3 μM AKTa (Sigma-Aldrich, Product No. 123871) or 20 μM PI3Ka. Cells were counted after 3, 5, and 7 days of culture, and the ratio of the cell number at each time point to the cell number on the first day of culture was calculated. In the following examples, 740Y-P (Tocris, Product No. 1983) was used as PI3Ka. As shown in Figure 25, human hematopoietic stem cells showed clear proliferation in the presence of PI3Ka. On the other hand, AKTa did not have any proliferative effect on human hematopoietic stem cells under the experimental conditions.

[0116] Next, we investigated whether PI3Ka completely replaces SCF in the culture of human hematopoietic stem cells. Human hematopoietic stem cells were cultured for 7 days in the above-mentioned culture medium for human hematopoietic stem cells, supplemented with 20 μM PI3Ka (S10 + PI3Ka20 + T100) and in the above-mentioned culture medium supplemented with 20 μM PI3Ka without SCF (PI3Ka20 + T100). The total cell count and the number of CD34+ cells isolated by cell sorting using anti-CD34 antibodies were determined. The results are shown in Figure 26. As shown in Figure 26, when PI3Ka was added, there was no significant difference in the total cell count or the number of CD34+ cells, regardless of the presence or absence of SCF.

[0117] Next, we investigated whether TPO could be replaced by a TPO receptor agonist in the culture of human hematopoietic stem cells. Known TPO receptor agonists include butizamide, eltrombopag, and avatrombopag. Human hematopoietic stem cells were cultured in the above-mentioned culture medium without TPO, supplemented with 0.1 μM butizamide, 3 μg / mL eltrombopag, or 3 μM avatrombopag in the presence of 0.1% recombinant human serum albumin (Albumin Biosciences) or PVA. Mpl32D cells were used as human hematopoietic stem cells in this experiment. The results are shown in Figure 27. As shown in Figure 27, in the presence of albumin, butizamide, eltrombopag, and avatrombopag were able to support the proliferation of human hematopoietic stem cells in the absence of TPO. In contrast, in the presence of PVA (absence of albumin), butizamide had a significant cell proliferation effect on human hematopoietic stem cells in the absence of TPO, but the effect was small with avatrombopag, and almost no effect was observed with eltrombopag.

[0118] In addition, purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood was used for the culture experiments. CD34+ cells were isolated using microbeads. 24-well plates were cultured at 0.2–1.0 × 10 per well. 5Cells were aliquoted, and TPO was removed from the culture medium for human hematopoietic stem cells and replaced with one of the above TPO receptor agonists (hereinafter sometimes referred to as "TPOago"). The ratio of cell number on day 7 to that on day 1 of culture was calculated. The results are shown in Figure 28. As shown in Figure 28, human hematopoietic stem cells showed significant cell proliferation only in the presence of butizamide in the absence of albumin and in the presence of PVA. In the absence of albumin and in the presence of PVA, hematopoietic stem cells underwent cell death in the presence of abatrombopag or eltrombopag. However, these TPO receptor agonists are highly safe compounds used in vivo to treat patients undergoing surgical treatment for liver cirrhosis and patients with aplastic anemia. The results of this example demonstrate that some TPO receptor agonists can exhibit cytotoxicity against human hematopoietic stem cells in the absence of albumin and in the presence of PVA. This also suggests that some TPO receptor agonists are cytotoxic to human hematopoietic stem cells, and that in order to culture human hematopoietic stem cells, it is sufficient to use one that is not cytotoxic to human hematopoietic stem cells.

[0119] Next, we investigated whether SCF and TPO could be replaced by PI3Ka and a TPO receptor agonist in the presence of PVA in the absence of albumin. In the following examples, butizamide was used as the TPO receptor agonist. CD34+ cells were isolated from purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood using microbeads as described above and used in the culture experiments. 0.2 to 1.0 × 10 cells per well were placed in a 24-well plate. 5Cells were aliquoted and cultured in a medium containing 20 μM PI3Ka substituted for SCF (PI3Ka 20 μM + TPO 100) or 20 μM PI3Ka and 0.1 μM butizamide substituted for TPO (PI3Ka 20 μM + TPO ago 0.1 μM). After 7 days, the total cell count was determined, and the CD34+ cell count was determined by flow cytometry. The proliferation rate was then calculated relative to the initial count. The results are shown in Figure 29. As shown in Figure 29, butizamide completely replaced TPO. CD34+ cells were sorted before and after culture using a cell sorter, and 100 cells were seeded onto Methocult H4415 for colony assay. After 2 weeks, colonies were picked, cytospun, stained with Giemsa, and microscopically characterized for colony type. The number of GEmM colonies per 50 CD34+ cells was counted, and the increase in the number of colonies compared to before culture was calculated. This showed that butizamide could completely replace TPO in terms of GEmM colony formation ability.

[0120] Next, we cultured human hematopoietic stem cells in culture medium lacking SCF and TPO, supplemented with either 20 μM PI3Ka or 0.1 μM TPOago. On day 7 of culture, the total cell numbers and the CD34+ cell counts were counted by flow cytometry, and the proliferation rate relative to the initial cell count was calculated. As shown in Figure 30, while no cell proliferation was observed with PI3Ka or butizamide alone, the presence of both PI3Ka and TPOago significantly increased the cell number.

[0121] Next, we investigated which cell populations were more likely to expand in a medium in which SCF and TPO were replaced with PI3Ka and TPOago. Fresh cord blood samples were used to separate CD34+ cells using microbeads, as described above. Cells were fractionated using a cell sorter using anti-CD34-PE-Cy7 antibody (BD Biosciences, Product No. 348791), anti-CD38-V450 antibody (BD Biosciences, Product No. 646851), anti-CD133-PE antibody (Miltenyi Biotec, Product No. 130-080-801), anti-CD45RA-APC antibody (BioLegend, Product No. 304112), and anti-CD49f-PE antibody (BioLegend, Product No. 313611). The CD34+ fraction, CD34+CD38-CD133+ fraction, and CD34+CD38-CD45RA-CD49f+ fraction, which have been reported as purification markers for human umbilical cord blood-derived hematopoietic stem cells, were counted by flow cytometry on day 7 after the initiation of culture, and the proliferation rate relative to the initiation of culture was calculated. The results are shown in Figure 31. As shown in Figure 31, significant cell proliferation was observed in all cell fractions, but cell proliferation was particularly pronounced in the CD34+CD38-CD133+ fraction and the CD34+CD38-CD45RA-CD49f+ fraction, especially the CD34+CD38-CD45RA-CD49f+ fraction.

[0122] Example 3: Long-term culture experiment of human hematopoietic stem cells Human hematopoietic stem cells were cultured in the same culture medium as above, but containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO. The total cell count and CD34+ cell count were determined in the same manner as above on days 7 and 14 after the start of culture. The results are shown in Figure 32. As shown in Figure 32, the total cell count increased with the passage of culture days, whereas the CD34+ cell count was lower on day 14 than on day 7.

[0123] Observation of the cultured cells under an optical microscope revealed the presence of giant cells on day 14. To confirm the possibility that these giant cells were megakaryocytes or megakaryocyte progenitor cells, the cells were fractionated using a flow cytometer with anti-CD41a-FITC antibody (BD Pharmingen, product number 555466) and anti-CD42b antibody (BD Pharmingen, product number 555473). As shown in Figure 33, the majority of cells on day 14 after the start of culture were CD41a+CD42b+ cells. However, as shown in Figure 33, CD34+CD38- cells proliferated even under these conditions.

[0124] In addition, 100 CD34+ cells from the culture obtained on day 14 after the initiation of culture were seeded onto Methocult H4415 and subjected to colony assay. After two weeks, colonies were collected and cytospin specimens were prepared. The specimens were then stained with Giemsa and the colony types were determined under a microscope. The results are shown in Figure 34. Regarding colony types, G indicates a colony containing "granulocytes," E indicates a colony containing "erythroblasts," M indicates a colony containing "macrophages," and M indicates a colony containing "megakaryocytes." As shown in Figure 34, most of the cell colonies contained megakaryocytes.

[0125] Example 4: Examination of conditions more suitable for long-term culture of human hematopoietic stem cells We attempted to culture human hematopoietic stem cells for a long period in the presence of compounds that can proliferate human hematopoietic stem cells.

[0126] We prepared the above-mentioned culture medium for human hematopoietic stem cells, containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO (PI3Ka 20 μM + TPOago 0.1 μM), and the above-mentioned medium supplemented with either SR-1 (500 nM) or UM171 (35 nM) (+SR-1, +UM171, and +SR-1 + UM171). CD34+ cells were isolated from purchased human bone marrow CD34+ cells (sold by Lonza, product number 2C-101) or donated fresh umbilical cord blood using microbeads as described above and used for the culture experiments. 0.2–1.0 × 10 cells were cultured per well in a 24-well plate. 5 The cells were dispensed and cultured as CD34+ cells in the prepared medium. On day 14 after the start of culture, the total number of cells and CD34+ cells were counted as described above, and the proliferation rate from before the start of culture was calculated. The results are shown in Figure 35. As shown in Figure 35, the total number of cells and the number of CD34+ cells increased in the culture medium further containing UM171, and the proliferation rate of CD34+ cells was significantly increased in the medium containing UM171 compared to the medium without UM171. In contrast, SR-1 killed the cells under these experimental conditions.

[0127] Furthermore, CD34+ cells were cultured under three conditions: Condition 1: 14 days of culture in the same culture medium as above, but containing 20 μM PI3Ka and 0.1 μM TPOago instead of SCF and TPO (PI3Ka 20 μM + TPOago 0.1 μM); Condition 2: 14 days of culture in medium without butizamide (PI3Ka 20 μM) from Day 7 onward (Day 7 - No Buty); and Condition 3: 14 days of culture in medium (PI3Ka 20 μM + TPOago 0.1 μM) plus UM171 (+UM171) (PI3Ka 20 μM + TPOago 0.1 μM). The total number of cells and the rate of increase in CD34+ cell count were also measured using a flow cytometer. The results are shown in Figure 36. As shown in Figure 36, under condition 2, in which cells were cultured in a medium lacking butizamide on day 7 after the start of culture, the number of CD34+ cells increased and the number of CD41+ cells decreased compared to condition 1. As shown in Figure 36, under condition 3, in which cells were cultured in a medium further supplemented with UM171, the rate of increase in CD34+ cells significantly increased and the number of CD41+ cells significantly decreased compared to conditions 1 and 2. These results demonstrate that, in serum-free medium conditions in the presence of PVA, PI3Ka, and TPOago, UM171 proliferates human hematopoietic stem cells and suppresses their differentiation into megakaryocyte progenitor cells and megakaryocytes (see Figure 37).

[0128] Example 5: Transplantation experiment of CD34+ cells after culture Human CD34+ cells cultured for 7 days under each of conditions 1 to 3 were transplanted into irradiated NOG mice to confirm cell engraftment. Specifically, 1 × 10 human CD34+ cells were transplanted into NOG mice irradiated with 1.5 Gy of gamma rays. 4Cells were transplanted. Twelve weeks after transplantation, peripheral blood was collected from the NOG mice, and the cellular components in the peripheral blood were analyzed using a flow cytometer. The results are shown in Figure 38. As shown in Figure 38, under conditions 1 and 2, the hematopoietic stem cell engraftment rates were 14.9% and 11.1%, respectively, when pre-culture CD34+ cells were transplanted, but increased to 66.6% and 54.9%, respectively, when cultured CD34+ cells were transplanted. Furthermore, under condition 3, the hematopoietic stem cell engraftment rate was 17% when pre-culture CD34+ cells were transplanted, but increased to 67% when cultured CD34+ cells were transplanted.

[0129] These results demonstrate that in serum-free medium without albumin and in the presence of PVA, PI3K activators and TPO receptor agonists effectively proliferate human hematopoietic stem cells, and that the expanded human CD34+ cells maintain their hematopoietic stem cell properties and improve engraftment rates after transplantation into other individuals. Furthermore, when human hematopoietic stem cells are cultured for long periods under these conditions, some differentiate into megakaryocytes, thereby producing megakaryocytes, while others tend to proliferate as CD34+ cells. The addition of UM171 to these cells also improved the proliferation rate of CD34+ cells and suppressed their differentiation into megakaryocytes.

[0130] Next, human mononuclear cells were isolated from fresh human umbilical cord blood. The cells obtained under conditions 1 and 3 (5 × 10 5 The total cell count, CD34+ cell count, percentage of CD34+ cells among total cells, and cell viability were determined 7 and 14 days after the start of culture. The results are shown in Figure 39. As shown in Figure 39, under all conditions, the total cell count of mononuclear cells obtained from human umbilical cord blood decreased with culture. On the other hand, the percentage of CD34+ cells among total cells under Condition 3 was significantly higher than under Condition 1. Cell viability was also significantly higher under Condition 3 than under Condition 1. Furthermore, the number of CD34+ cells showed a significant increase under Condition 3, whereas under Condition 1, the cell count was maintained, but no increase in cell count was observed.

[0131] Example 6: Culture experiment of human hematopoietic stem cells in the presence of polymer A The above examples revealed that human hematopoietic stem cells, unlike mouse hematopoietic stem cells, require activation of the PI3K pathway and that TPO can be substituted with butizamide, a TPO receptor agonist. Furthermore, it was also revealed that human hematopoietic stem cells can be cultured in the absence of UM171 when not cultured for a long period of time, but that when cultured for a long period of time, differentiation into the megakaryocytic lineage can be suppressed by culturing them in the presence of UM171.

[0132] Therefore, in this example, an experiment was conducted in which human hematopoietic stem cells were cultured in the presence of polymer A, a PI3K activator, a TPO receptor agonist, and UM171, replacing PVA with polymer A. As a control, human hematopoietic stem cells were cultured in the presence of PVA, a PI3K activator, a TPO receptor agonist, and UM171. PI3Ka was used as the PI3K activator, and butizamide was used as the TPO receptor agonist, at the concentrations described in the above examples.

[0133] The results are shown in Figure 22. Human hematopoietic stem cells cultured in the presence of polymer A instead of PVA proliferated well, and as shown in Figure 22, the percentage of CD34-positive cells was higher. Furthermore, the percentage of CD34-positive cells was higher in the presence of polymer A than in the presence of PVA. This suggests that human hematopoietic stem cells proliferate well in the presence of polymer A under conditions in which the PI3K pathway is activated (and in the absence of SCF). It also suggests that human hematopoietic stem cells can proliferate well under these conditions even when TPO is replaced with a TPO receptor agonist. It was also revealed that human hematopoietic stem cells can proliferate well in the presence of UM171.

[0134] Furthermore, changes in cell number were monitored over time. In this experiment, human hematopoietic stem cells were cultured for three weeks in a culture medium supplemented with a T cell expansion kit (Milteny Biotec) and human IL-2 (Peprotech). The results are shown in Figure 23. As shown in Figure 23, human hematopoietic stem cells proliferated equally well in the presence of PVA and polymer A.

[0135] Example 7: Cultivation of human CD3-positive cells Human CD3-positive cells were stimulated with CD28 and CD3 antibodies using a T Cell Activation / Expansion Kit, human (Miltenyi Biotec) under standard conditions and cultured for 6 weeks in albumin-free, cytokine-free basal medium (IMDM medium supplemented with 1% ITSX and 1% penicillin) containing 0.1% PVA or 0.1% SoluPlus™. The initial cell count was 1,000. The results are shown in Figure 40.

[0136] As shown in Figure 40, CD3-positive cells (mainly T cells) proliferated well in both the presence of Soluplus and PVA, demonstrating that the albumin-free culture medium can be used for the maintenance and proliferation of human T cells.

[0137] Example 8: Differentiation of human CD34-positive cells into blood cells Human CD34-positive cells (hematopoietic stem cells) were cultured in an albumin-free basal medium containing PVA or SoluPlus™. The initial cell count was 1,000. Differentiation of the hematopoietic stem cells into blood cells was achieved by adding a cytokine cocktail to the medium. Culture was continued for 10 days. The results are shown in Figure 41.

[0138] As shown in panel A of Figure 41, the cell number in the culture medium in which CD34-positive cells were cultured increased significantly in both the presence of SolPlus and PVA. As shown in panel B of Figure 41, the cells obtained in the culture medium by culturing in the presence of SolPlus included megakaryocytes, erythroblasts, neutrophils, and macrophages. These results demonstrate that cells of almost all blood cell lineages were obtained and each proliferated, demonstrating that human blood cell lineages can be successfully proliferated, maintained, and differentiated in the presence of additives such as PVA or SolPlus, even in an albumin-free environment.

[0139] Example 9: Cultivation of Chronic Myeloid Leukemia (CML) Cells Bone marrow cells from a patient with chronic myeloid leukemia (CML) were cultured for one week under albumin- and cytokine-free conditions. The culture medium used was a basal medium supplemented with 0.1% PVA, 20 μM PI3Ka, and 0.1 μM TPOago. Cultures were performed in the presence and absence of imatinib (IM), an inhibitor of Bcr-Abl, the causative gene for CML. The results are shown in Figure 42.

[0140] As a result, as shown in Figure 42, cell proliferation of 0.5% was confirmed in the absence of IM compared to the total cell number before culture, and in particular, CD34-positive cells, which are a leukemia stem cell fraction, were confirmed to have expanded / maintained by more than 30-fold, and in absolute numbers by more than 6-fold. In contrast, no significant cell expansion was observed in the presence of IM, indicating that the cells expanded in IM+ were CML leukemia stem cells.

Claims

1. 1. A method for culturing human cells, comprising: Culturing human cells in a culture medium; The culture medium is a serum albumin-free medium and contains an additive, the additive being selected from the group consisting of polyvinyl alcohol and modified polyalkylene glycol; (1) A compound comprising a phosphatidylinositol 3-kinase (PI3K) activator and one or more compounds selected from the group consisting of thrombopoietin (TPO) and a TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist; The number of human hematopoietic stem cells or human blood cells is maintained or increased by the above culture. method.

2. The method of claim 1, wherein the human cells are cells selected from the group consisting of human hematopoietic stem cells and human blood cells.

3. The method of claim 1, wherein the human cells are blood cells other than hematopoietic stem cells.

4. The method of claim 3 wherein the additive is polyvinyl alcohol.

5. 3. The method of claim 1 or 2, wherein the additive is a modified polyalkylene glycol.

6. 6. The method of claim 5, wherein the modified polyalkylene glycol is a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

7. 7. The method of claim 6, wherein the modified polyalkylene glycol is a polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

8. 1. A method for culturing human cells, comprising: Culturing human cells in a culture medium; The culture medium comprises a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, method.

9. 9. The method of claim 8, further comprising obtaining the expanded human cells.

10. The method of claim 8 or 9, wherein the human cells are human hematopoietic stem cells.

11. A medium composition for human cell culture, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

12. A composition comprising human cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

13. The composition of claim 11 or 12, wherein the human cells are human hematopoietic stem cells.

14. A human cell obtained by the method according to any one of claims 8 to 10.

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