Serum-free medium without albumin and culture method without albumin suitable for culturing human hematopoietic stem cells

Abstract

Disclosed are: a composition of a serum-free medium not containing albumin and suited for culturing human hematopoietic stem cells; and an albumin-free culturing method. The present invention provides a method for culturing human hematopoietic stem cells, the method comprising bringing human hematopoietic stem cells into contact with PVA and a PI3K activator.

Description

Technical Field

[0001] The present invention discloses a composition of a serum-free medium without albumin suitable for culturing human hematopoietic stem cells and a culture method without albumin. According to the present invention, a method for culturing human hematopoietic stem cells is provided, which method includes contacting human hematopoietic stem cells with PVA and a PI3K activator.

Background Art

[0002] In the culture of hematopoietic stem cells, research has been carried out on proliferation using a chemically defined medium, and it has been clarified that the culture of mouse hematopoietic stem cells using a chemically defined medium is possible (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

[0004] The present invention provides a composition of a serum-free medium without albumin suitable for culturing human hematopoietic stem cells and a culture method without albumin.

[0005] The inventors have found and reported that mouse hematopoietic stem cells (sometimes called KSL cells due to their isolation method) can proliferate massively over a long period by adding polyvinyl alcohol (PVA) in a serum-free medium without albumin (Wilkinson et al., Nature, 571:117-121, 2019). However, regarding human hematopoietic stem cells, a method for culturing them in a serum-free medium without albumin has not been established. Now, the inventors have found that human hematopoietic stem cells have weak activation of various signaling pathways including the PI3K pathway and Akt pathway in a serum-free medium without albumin in the presence of PVA, stem cell factor (SCF), and thrombopoietin (TPO). The inventors have also found that human hematopoietic stem cells proliferate by adding a PI3K activator in a serum-free medium without albumin in the presence of PVA, SCF, and TPO. The inventors have further found that in a serum-free medium without albumin in the presence of PVA, a PI3K activator can completely replace SCF. The inventors have also found that in a serum-free medium without albumin in the presence of PVA and a PI3K activator, TPO can be replaced with a TPO receptor agonist. The inventors have also found that in a serum-free medium without albumin in the presence of PVA, a PI3K activator, and TPO or a TPO receptor agonist, human hematopoietic stem cells are more likely to differentiate into cells of the megakaryocyte lineage when cultured for a long time. The inventors have also found that the differentiation into cells of the megakaryocyte lineage is inhibited by adding UM171 to the medium, or the number of cells of the megakaryocyte lineage decreases, and that UM171 is beneficial for the maintenance and proliferation of human hematopoietic stem cells which are CD34+ cells.

[0006] According to the present invention, the following inventions are provided. [1] A method for culturing human hematopoietic stem cells, comprising: culturing human hematopoietic stem cells in a culture medium, the culture medium contains polyvinyl alcohol, does not contain albumin, and (1) Comprising a phosphatidylinositol 3-kinase (PI3K) activator and one or more 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) Comprising a PI3K activator and a TPO receptor agonist, wherein the number of human hematopoietic stem cells is increased by the above culture, Method. [2] The method according to [1] above, comprising obtaining the increased human hematopoietic stem cells. [3] The method according to [1] or [2] above, wherein the culture medium contains a PI3K activator and 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 an agonist of TPO. [5] The method according to [4] above, wherein the culture medium does not contain both SCF and TPO. [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, comprising human hematopoietic stem 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 a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist, Composition. [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 obtained by the method according to any one of [1] to [7] above.

[12] A culture medium for human hematopoietic stem cells, which contains polyvinyl alcohol, does not contain albumin, and (1) contains one or more selected from the group consisting of a PI3K activator and one or more selected from the group consisting of thrombopoietin (TPO) and a TPO receptor agonist; (2) contains one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) contains a PI3K activator and a TPO receptor agonist. A culture medium for human hematopoietic stem cells.

[0007] [1] A method for culturing or producing human hematopoietic stem cells, comprising culturing human hematopoietic stem cells in a culture medium, wherein the culture medium contains polyvinyl alcohol and (1) contains a phosphatidylinositol 3-kinase (PI3K) activator and one or more selected from the group consisting of thrombopoietin (TPO) and a TPO receptor agonist; (2) contains one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (3) contains a PI3K activator and a TPO receptor agonist, and the number of human hematopoietic stem cells increases by the above culturing. A 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 substantially does not contain albumin. The method according to any one of [1] to [4] above, comprising obtaining increased human hematopoietic stem cells. The method according to any one of [1] to [5] above, wherein the culture medium contains a PI3K activator and does not contain stem cell factor (SCF). The method according to any one of [1] to [6] above, wherein the culture medium contains a PI3K activator and an agonist of TPO. The method according to [7] above, wherein the culture medium does not contain both SCF and TPO. 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). The method according to [9] above, wherein the culture period is 7 days or more. A composition comprising human hematopoietic stem cells, polyvinyl alcohol as an alternative to albumin, (1) one or more selected from the group consisting of 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 a PI3K activator, and a TPO receptor agonist; or (3) a PI3K activator and a TPO receptor agonist, and comprising. The composition according to

[11] above, comprising a PI3K activator and a TPO receptor agonist. 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). Human hematopoietic stem cells obtained by the method according to any one of [1] to

[10] above. As an alternative to albumin, comprising polyvinyl alcohol, and (1) comprising 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)A PI3K activator and a TPO receptor agonist, A culture medium for human hematopoietic stem cells.

[16] The culture medium according to

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

[17] The culture medium according to

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

[18] The culture medium according to any one of

[15] to

[17] above, which does not contain albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] [Figure 1] FIG. 1 shows the results of culturing mouse hematopoietic stem cells (mouse KSL cells) and human hematopoietic stem cells (CD34+CD38− cells) in a medium containing polyvinyl alcohol (PVA) in an albumin-free serum-free medium in the presence of 10 ng / mL of stem cell factor (SCF) and 100 ng / mL of thrombopoietin (TPO) for mouse and human, respectively. [Diagram 2] FIG. 2 is a diagram showing the degree of phosphorylation of signal factors downstream of SCF and TPO in hematopoietic stem cells cultured in the presence of 10 ng / mL of stem cell factor (SCF) and 100 ng / mL of thrombopoietin (TPO) for hematopoietic stem cells of mouse and human, respectively, in the absence of albumin and in the presence of PVA. The symbol "m" indicates mouse hematopoietic stem cells, and "h" indicates human hematopoietic stem cells. [Diagram 3] FIG. 3 shows the results of culturing human hematopoietic stem cells in the absence of albumin and in the presence of PVA in the presence of 10 ng / mL of human stem cell factor (SCF) and 100 ng / mL of human thrombopoietin (TPO), and in the presence of AKT Activator II (AKTa) or PI3K activator (PI3Ka). [Figure 4]Figure 4 shows the growth rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells are cultured in the absence of albumin, in the presence of PVA, and in the presence of 100 ng / mL of human thrombopoietin (TPO). In Figure 4, conditions with SCF and without SCF are compared, indicating that there is no statistically significant difference between the conditions in terms of the total number of cells and the number of CD34+ cells on day 7 of culture. [Diagram 5] Figure 5 shows the change in the number of cells when human hematopoietic stem cells are cultured by replacing TPO with various TPO receptor agonists in the presence of albumin or PVA. The symbol "Buty" represents butyzamide, "Elt" represents eltrombopag, and "Ava" represents avatrombopag. [Figure 6] Figure 6 shows the growth rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells are cultured by replacing TPO with various TPO receptor agonists in the presence of either albumin or PVA. [Figure 7] Figure 7 shows the total number of cells, the number of CD34+ cells, and the number of GEmM colonies on day 7 when human hematopoietic stem cells are cultured in a medium containing a PI3K activator and TPO or butyzamide (Buty) in the absence of albumin and in the presence of PVA. The types of colonies were determined under a microscope after collecting the colonies and preparing cytospin specimens, followed by Giemsa staining. G means "granulocyte", E means "erythroblast", m means "macrophage", and M means "megakaryocyte". [Figure 8] Figure 8 shows the growth rates of total cells and CD34+ cells on day 7 when human hematopoietic stem cells are cultured in a culture medium that does not contain SCF and TPO but contains a PI3K activator or a TPO receptor agonist or a combination thereof in the absence of albumin and in the presence of PVA. [Figure 9] Figure 9 shows the growth rates of each cell population in the culture obtained on day 7 when human hematopoietic stem cells are cultured in a culture medium that does not contain SCF and TPO but contains a PI3K activator and a TPO receptor agonist in the absence of albumin and in the presence of PVA. [Figure 10]Figure 10 shows the changes in the total cell count and the number of CD34+ cells on the 7th and 14th days when human hematopoietic stem cells are cultured in a culture medium that does not contain albumin and contains PVA, does not contain SCF and TPO, but contains a PI3K activator and a TPO receptor agonist. [Figure 11] Figure 11 shows the results of gating the cells obtained on the 14th day when human hematopoietic stem cells are cultured in a culture medium that does not contain albumin and contains PVA, does not contain SCF and TPO, but contains a PI3K activator and a TPO receptor agonist. 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 photograph in Figure 11 is an optical micrograph of the obtained culture. [Figure 12] Figure 12 shows the results of a colony assay of the cells obtained on the 14th day when human hematopoietic stem cells are cultured in a culture medium that does not contain albumin and contains PVA, does not contain SCF and TPO, but contains a PI3K activator and a TPO receptor agonist. The types of colonies were determined under a microscope after collecting the colonies under a microscope, preparing cytospin specimens, performing Giemsa staining, and observing under a microscope. G means colonies containing "granulocytes", E means "erythroblasts", m means "macrophages", and M means colonies containing "megakaryocytes". [Figure 13] Figure 13 shows the proliferation rates of the total cells and CD34+ cells on the 14th day when human hematopoietic stem cells are cultured in a culture medium that does not contain albumin and contains PVA, does not contain SCF and TPO, but contains a PI3K activator and a TPO receptor agonist, and either or both of SR-1 and UM171. [Figure 14] Figure 14 shows the proliferation rates of the total cells and CD34+ cells, and the number of CD41+ cells on the 14th day when cultured under each of Conditions 1 to 3. [Figure 15] Figure 15 shows the results of flow cytometry of the cells in the culture on the 14th day when cultured under each of Conditions 1 to 3. The upper part shows the results using CD34 (horizontal axis) and CD38 (vertical axis), and the lower part shows the results using CD41a (horizontal axis) and CD42b (vertical axis). [Figure 16] FIG. 16 is a diagram showing 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 the 7th day into mice. [Figure 17] FIG. 17 shows the total cell number, the percentage (%) of CD34+ cells, the percentage (%) of viable cells, and the number of CD34+ cells of a culture obtained by culturing for 14 days in a medium containing neither albumin, SCF nor TPO but containing PVA, a PI3K activator and a TPO receptor agonist, in the presence or absence of UM171, after mononuclear cells were isolated from donated cord blood. DETAILED DESCRIPTION OF THE INVENTION

[0009] As used herein, a "hematopoietic stem cell" is a stem cell capable of differentiating into hematopoietic 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, it is known that hematopoietic stem cells are abundantly contained in the CD34-positive CD38-negative cell fraction. Therefore, human hematopoietic stem cells can be CD34-positive CD38-negative. Hematopoietic stem cells may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells ex vivo.

[0010] As used herein, "ex vivo" means outside the living body. As used herein, ex vivo is used in contrast to in vivo (inside the living body) and means a state in which cells present in the living body are taken out from the living body to the outside of the living body. Culturing can be performed ex vivo.

[0011] As used herein, "positive" means that a cell is recognized as expressing a molecule specified by the term immediately preceding it. As used herein, "positive" may be simply denoted as "+".

[0012] In this specification, "polyvinyl alcohol" means a polymer of vinyl alcohol. Polyvinyl alcohol can be obtained by saponifying polyvinyl acetate obtained by polymerizing vinyl acetate monomer. The weight-average molecular weight (M W ) of polyvinyl alcohol 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 polyvinyl acetate is saponified by the above method to obtain PVA, the saponification rate can be 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more.

[0013] In this specification, "albumin" is a protein known as a plasma component. Albumin is said to account for 60% of plasma proteins and is present in large amounts in blood, serum, and plasma. Albumin is considered to play a role in maintaining the osmotic pressure of blood in vivo and in 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 under GenBank accession number: AAN17825.1 or human serum albumin having an amino acid sequence corresponding thereto.

[0014] In this specification, "including B as an alternative to A" means including B instead of A. Including B instead of A means partially or completely replacing A with B. "Including B as an alternative to A" can mean not substantially including A and including B. "Not substantially including" allows an amount of introduction that cannot be prevented in the system and an amount of introduction below the detection limit. "Not substantially including" may not allow an amount of introduction that can be prevented.

[0015] In this specification, an "agonist" refers to a substance that activates proteins such as receptors and enzymes.

[0016] As used herein, "PI3K" means phosphatidylinositol 3-kinase. PI3K is an enzyme that phosphorylates inositol phospholipids, which are components of cells. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) generated by phosphorylation phosphorylates Akt (also known as protein kinase B) and transmits its signal downstream. As used herein, "PI3K activator" means an agonist of receptor tyrosine kinase and a substance that activates PI3K.

[0017] As used herein, "TPO" means thrombopoietin. TPO is a protein responsible for the differentiation of hematopoietic stem cells into megakaryocytes. TPO is known to be involved in the formal maintenance of hematopoietic stem cells. Human thrombopoietin can be, for example, a protein having the amino acid sequence registered under GenBank accession number: AAB33390.1 or thrombopoietin having an amino acid sequence corresponding thereto. TPO may include functional homologs of TPO and proteins having an amino acid sequence having 90% or more, 95% or more, or 100% identity with the amino acid sequence registered under GenBank accession number: AAB33390.1 and having the function of TPO. As used herein, "TPO receptor agonist" means a substance other than TPO that activates the TPO receptor. Examples of TPO receptor agonists include variants of TPO other than TPO, peptides, and compounds that activate the TPO receptor. As used herein, "compound" is a concept that includes organic compounds.

[0018] As used herein, stem cell factor (SCF) is a hematopoietic cell growth factor that acts at the initial stage of hematopoietic function. Human stem cell factor can be, for example, a protein having the amino acid sequence registered under GenBank accession number: AAA85450.1 or SCF having a corresponding amino acid sequence. SCF may include functional homologs of SCF and proteins having an amino acid sequence having 90% or more, 95% or more, or 100% identity with the amino acid sequence registered under GenBank accession number: AAB33390.1 and having the function of SCF.

[0019] As used herein, "culturing" means incubating cells under conditions suitable for their growth or maintenance. Incubation can preferably be carried out under an atmosphere of 37 °C and 5% CO2 in the case of human cells. When "culturing" involves growth, "culturing" is understood to be the production of proliferated cells. As used herein, "culturing" can be carried out in a serum-free medium. As used herein, "culturing" can be carried out in a chemically defined culture medium (or in a completely synthetic medium). A chemically defined culture medium is a serum-free medium.

[0020] As used herein, "culture medium" means a medium used for culturing cells. A culture medium can be prepared by adding components necessary for a basal medium. The necessary components can be a pH adjuster, a sugar source such as glucose, antibiotics (e.g., penicillin and streptomycin, etc.), essential amino acids such as glutamine, and culture additives such as insulin, transferrin, selenium (e.g., sodium selenite) and ethanolamine. A culture medium can be a liquid medium.

[0021] As used herein, "cytotoxicity" means the property of having an effect of reducing the number of cells or killing cells during culture. As used herein, "non-cytotoxicity" refers to the property of not reducing the number of cells by culture. Depending on the substance, cytotoxicity may occur by increasing the concentration. In this case, if the advantageous effects expected of the substance occur even when the concentration is decreased to such an extent that cytotoxicity does not occur, it can be defined as non-cytotoxic.

[0022] The inventors have found and reported that mouse hematopoietic stem cells (sometimes called KSL cells due to their isolation method) can proliferate in large quantities over a long period by adding polyvinyl alcohol (PVA) in a serum-free medium without albumin (Wilkinson et al., Nature, 571:117-121, 2019). However, regarding human hematopoietic stem cells, a method for proliferation in a serum-free medium without albumin has not been established. Now, the inventors have found that human hematopoietic stem cells have weak activation of various signaling pathways including the PI3K pathway and Akt pathway in a serum-free medium without albumin in the presence of PVA, SCF, and TPO. The inventors have also found that human hematopoietic stem cells proliferate by adding a PI3K activator in a serum-free medium without albumin in the presence of PVA, SCF, and TPO. The inventors have further found that in a serum-free medium without albumin in the presence of PVA, a PI3K activator can completely replace SCF. The inventors have also found that in a serum-free medium without albumin in the presence of PVA and a PI3K activator, TPO can be replaced with a TPO receptor agonist. The inventors have further found that in a serum-free medium without albumin in the presence of PVA, a PI3K activator, and TPO or a TPO receptor agonist, human hematopoietic stem cells are more likely to differentiate into cells of the megakaryocyte lineage when cultured for a long time. The inventors have also found that differentiation into cells of the megakaryocyte lineage is inhibited by adding UM171 to the medium, or the number of cells of the megakaryocyte lineage decreases, and UM171 is beneficial for the maintenance and proliferation of human hematopoietic stem cells which are CD34+ cells.

[0023] Therefore, according to the present invention, there is provided a method for culturing human hematopoietic stem cells, comprising: culturing human hematopoietic stem cells in a culture medium, wherein the culture medium contains polyvinyl alcohol, does not contain albumin, and (1) comprising one or more selected from the group consisting of phosphatidylinositol 3-kinase (PI3K) activator, thrombopoietin (TPO), and TPO receptor agonist; (2) comprising one or more selected from the group consisting of stem cell factor (SCF) and PI3K activator, and a TPO receptor agonist; or (3) comprising a PI3K activator and a TPO receptor agonist, A method is provided. In this embodiment, the number of human hematopoietic stem cells can be increased or maintained by the above culture.

[0024] According to the present invention, a method for culturing human hematopoietic stem cells in a culture medium, contacting human hematopoietic stem cells with polyvinyl alcohol in the culture medium, in the culture medium, human hematopoietic stem cells and (1) contacting with one or more selected from the group consisting of phosphatidylinositol 3-kinase (PI3K) activator, thrombopoietin (TPO), and TPO receptor agonist; (2) contacting with one or more selected from the group consisting of stem cell factor (SCF) and PI3K activator, and a TPO receptor agonist; or (3) contacting a PI3K activator with a TPO receptor agonist, A method comprising this is provided. In this embodiment, the number of human hematopoietic stem cells can be increased or maintained by the above culture.

[0025] The inventors have revealed that in the absence of albumin and in the presence of PVA, some compounds containing certain TPO receptor agonists can exhibit cytotoxicity to human hematopoietic stem cells. Therefore, according to the present invention, in the present invention, as the PI3K activator, those having cytotoxicity to human hematopoietic stem cells in the absence of albumin and in the presence of PVA are not used. That is, the PI3K activator used in the present invention is non-cytotoxic. In addition, the TPO receptor agonist used in the present invention is non-cytotoxic to human hematopoietic stem cells in the absence of albumin and in the presence of PVA. Whether the PI3K activator is non-cytotoxic or not can be appropriately confirmed by those skilled in the art through a culture test of human hematopoietic stem cells. Here, the culture test of human hematopoietic stem cells can be confirmed using an IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 100 ng / mL TPO, and 0.1% PVA. Also, whether the TPO receptor agonist is non-cytotoxic or not can be appropriately confirmed by those skilled in the art through a culture test of human hematopoietic stem cells. Here, the culture test of human hematopoietic stem cells can be confirmed using an IMDM medium containing 1% insulin-transferrin-selenium, 1% penicillin-streptomycin-glutamine, 10 ng / mL SCF, and 0.1% PVA.

[0026] In one aspect of the present invention, the PI3K activator may be a peptide represented by the amino acid sequence RQIKIWFQNRRMKWKKSDGGYMDMS, which is a peptide in which Y is phosphorylated (also referred to as "740Y-P").

[0027] In one aspect of the present invention, the TPO receptor agonist may be 3-[4-[[[4-[2-methoxy-3-(1-tert-butyl-2-oxapentan-1-yl)phenyl]thiazol-2-yl]amino]carbonyl]-2,6-dichlorophenyl]-2-methylprop-enoic acid (hereinafter also referred to as "butizamide").

[0028] In one aspect of the present invention, the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide.

[0029] In certain embodiments of the present invention, the culture medium used in the culture method of the present invention is a serum-free medium. In certain embodiments of the present invention, the culture medium used in the culture method of the present invention is a chemically defined medium. In certain embodiments 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 containing a PI3K activator and a TPO receptor agonist, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide).

[0030] In certain embodiments of the present invention, the culture medium used in the culture method of the present invention is comprising polyvinyl alcohol, not containing albumin, and (1) comprising one or more selected from the group consisting of 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 a PI3K activator and a TPO receptor agonist; or (3) comprising a PI3K activator and a TPO receptor agonist, and can be a culture medium for human hematopoietic stem cells.

[0031] A culture medium suitable for culturing hematopoietic stem cells can be appropriately used. As the basal medium, 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 (Invitorogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Daiichi Pure Chemicals), HPGM medium (Daiichi Pure Chemicals), AIM V medium (Invitorogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM / F-12 medium (Invtrogen), Stemline hematopoietic stem cell proliferation medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen etc.), MEM medium (Gibco Invitrogen etc.), MEMα (Gibco Invitrogen etc.), DMEM medium (Gibco Invitrogen etc.), IMDM medium (Gibco Invitrogen etc.), PRMI1640 medium (Gibco Invitrogen etc.), Ham F-12 medium (Gibco etc.), RD medium, etc. can be used. The culture medium contains a basal medium. The culture medium may contain, for example, one or more, or all, selected from insulin, transferrin (apo), sodium selenite, and ethanolamine. The culture medium may contain HEPES, sodium pyruvate, vitamins, amino acids, heparin, heparan sulfate, chondroitin sulfate, etc. The culture medium may 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.

[0032] The culture medium of the present invention may contain an effective amount of a non-cytotoxic PI3K activator and an effective amount of PVA in order to grow human hematopoietic stem cells in an environment without albumin and in the presence of PVA. 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 an effective amount of TPO and a non-cytotoxic TPO receptor agonist, and UM171.

[0033] According to the present invention, the method for culturing human hematopoietic stem cells can be a method for producing cells of the megakaryocyte lineage from human hematopoietic stem cells. In this embodiment, the culture medium used in the culturing method may contain a PI3K activator and a TPO receptor agonist, the PI3K activator may be 740Y-P, and the TPO receptor agonist may be butyzamide. In this specific embodiment, the culture medium can be free of UM171.

[0034] In one aspect of the present invention, the culturing method of the present invention may further 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 aspect of the present invention, the culture medium used in the culturing method of the present invention may further contain UM171. Thereby, human hematopoietic stem cells may be more likely to maintain their properties as human hematopoietic stem cells, or differentiation into cells of the megakaryocyte lineage (e.g., megakaryocyte progenitor cells and megakaryocytes) may be suppressed.

[0035] In certain embodiments of the present invention, the culturing 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 certain embodiments of the present invention, the culture medium used in the culturing method of the present invention further does not contain UM171. Thereby, human hematopoietic stem cells are more likely to differentiate into cells of the megakaryocyte lineage (e.g., megakaryocyte progenitor cells and megakaryocytes).

[0036] In the present invention, human hematopoietic stem cells can be cultured under conditions suitable for the proliferation of human hematopoietic stem cells. The present invention may further include isolating human hematopoietic stem 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 hematopoietic stem cell markers. Human hematopoietic stem cells may be obtained as human hematopoietic stem cells, or may be used after further differentiating into other cells. When differentiating from 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.

[0037] According to the present invention, there are provided human hematopoietic stem cells obtained by the culturing method of the present invention. The human hematopoietic stem cells obtained by the culturing method of the present invention can be purified using CD34 and preferably CD38 as markers. The human hematopoietic stem cells obtained by the culturing method of the present invention have a better engraftment rate after transplantation into a recipient than human hematopoietic stem cells obtained by conventional methods. Therefore, according to the present invention, there are provided human hematopoietic stem cells obtained by the culturing method of the present invention and having an improved engraftment rate after transplantation into a recipient as compared with before culturing.

[0038] According to the present invention, megakaryocyte lineage cells (e.g., megakaryocyte progenitor cells and megakaryocytes) obtained by the culturing method of the present invention are provided. The megakaryocyte lineage cells can be differentiated into multinucleated megakaryocytes by a conventional method. Platelets can be obtained from the multinucleated megakaryocytes. In the present invention, it may further include isolating megakaryocyte lineage cells from the obtained culture. Isolation of megakaryocyte lineage cells can be performed by methods known to those skilled in the art, for example, by flow cytometry using markers of megakaryocyte lineage cells (e.g., CD41a and CD42b).

[0039] In the culturing method of the present invention, the culturing can be carried out in the presence of fibronectin. In the culturing method of the present invention, the culturing can be carried out under conditions where hematopoietic stem cells can contact fibronectin. In the culturing method of the present invention, preferably, for example, the inner side (e.g., the bottom surface) of the culture vessel is coated with fibronectin.

[0040] The albumin-free medium used in the culturing 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)% of serum albumin, or does not contain it at all.

[0041] The medium used in the culturing method of the present invention may contain recombinant TPO. The recombinant TPO can be, for example, recombinant TPO of a mammal and can be recombinant human TPO. In certain embodiments of the present invention, the TPO concentration is 20 - 200 ng / mL, more preferably 30 - 150 ng / mL, still more preferably 40 - 150 ng / mL, and can be, for example, 100 ng / mL.

[0042] The culture medium used in the culture method of the present invention may further contain recombinant SCF. The recombinant SCF can be, for example, a recombinant SCF of a mammal and can be a recombinant human SCF. In certain embodiments of the present invention, the SCF concentration is from 1 to 200 ng / mL, more preferably from 1 to 150 ng / mL, still more preferably from 1 to 100 ng / mL, for example, from 1 to 50 ng / mL, even more preferably from 1 to 30 ng / mL, even more preferably from 1 to 20 ng / mL, for example, it can be from 5 to 15 ng / mL.

[0043] The culture medium used in the culture method of the present invention may contain recombinant TPO and recombinant SCF. In this embodiment, the TPO concentration is from 20 to 200 ng / mL, more preferably from 30 to 150 ng / mL, still more preferably from 40 to 150 ng / mL, for example, it can be 100 ng / mL, and the SCF concentration is from 1 to 200 ng / mL, more preferably from 1 to 150 ng / mL, still more preferably from 1 to 100 ng / mL, for example, from 1 to 50 ng / mL, even more preferably from 1 to 30 ng / mL, even more preferably from 1 to 20 ng / mL, for example, it can be from 5 to 15 ng / mL. In a preferred embodiment, the culture 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, for example, it can be any concentration within the above concentration range, and can be 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more higher.

[0044] The method for culturing human hematopoietic stem cells of the present invention may further include growing the hematopoietic stem cells under conditions sufficient for the maintenance and / or proliferation of the hematopoietic stem cells. In this embodiment, for example, it may include growing the hematopoietic stem cells 10 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, or 500 times or more from the start of the culture.

[0045] Sufficient conditions for the maintenance and / or proliferation of human hematopoietic stem cells may be, for example, conditions for culturing in the above medium. Sufficient conditions for the maintenance and / or proliferation of human hematopoietic stem cells may preferably be, for example, conditions in the presence of fibronectin, and conditions under which human hematopoietic stem cells can come into contact with fibronectin.

[0046] The culturing method of the present invention (C) recovering the proliferated human hematopoietic stem cells from the medium may further include. The recovered human hematopoietic stem cells may be further concentrated or isolated. Concentration or isolation of human hematopoietic stem cells can be performed using cell surface markers. Examples of cell surface markers that can be used for concentration or isolation of human hematopoietic stem cells include CD34 and CD38. Concentration or isolation of hematopoietic stem cells can be performed using a cell sorter.

[0047] According to the present invention, there is provided a method for producing human hematopoietic stem cells ex vivo, a method including the culturing method of the present invention is provided. In the production method of the present invention, functional human hematopoietic stem cells can be obtained. Here, "functional" means that hematopoiesis can be restored in the transplanted human individual (recipient) by human hematopoietic stem cell transplantation.

Example

[0048] Example 1: Hematopoietic stem cell proliferation test using albumin-free culture medium In this example, a test was conducted to proliferate mouse hematopoietic stem cells (KSL) and human hematopoietic stem cells (CD34+CD38-) using a culture medium without albumin. The culture medium contained polyvinyl alcohol (PVA) described in Wilkinson et al., Nature, 571:117-121, 2019 and was a serum-free medium without albumin.

[0049] Specifically, in 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 included in the above medium at a final concentration of 0.1%. In 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 included in the above medium at a final concentration of 0.1%. In all media used in the following examples, unless otherwise specified, 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") was used. Therefore, hereinafter, the medium will be described focusing on the components added to the common medium. For example, since the above medium contains TPO and SCF in addition to the common medium, for convenience, it will be referred to as TPO+SCF medium. When expressing the concentration of each factor, it may be described as SCF10+TPO100, or abbreviated as S10+T100.

[0050] As mouse hematopoietic stem cells, KSL cells from mouse bone marrow were used. Specifically, mouse bone marrow cells were isolated from the tibia, femur, and pelvis and stained with an APC-c-KIT antibody. c-KIT+ cells were enriched using anti-APC magnetic beads and an LS column (Miltenyi Biotec). Subsequently, the c-KIT-enriched cells were 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. Thereafter, the cell population was directly sorted and purified into wells containing medium using FACS AriaII (BD) with propidium iodide used as a dead cell stain.

[0051] As human hematopoietic stem cells, CD34+CD38− cells from human bone marrow were used. Specifically, commercially available human bone marrow CD34-positive cells (Lonza 2C-101) were purchased.

[0052] The results were as shown in Fig. 1. In Wilkinson et al., 2019, mouse hematopoietic stem cells could not maintain long-term proliferation even in the presence of SCF and TPO in serum-free medium without albumin. However, in Wilkinson et al., 2019, it was shown that by adding PVA to the medium, mouse hematopoietic stem cells could proliferate long-term in serum-free medium without albumin. As shown in Fig. 1, mouse hematopoietic stem cells proliferated well in serum-free medium containing PVA and without albumin, but human hematopoietic stem cells did not show good proliferation in this medium.

[0053] Example 2: Differences in signal transduction between mouse and human hematopoietic stem cells The signaling pathways of mouse and human hematopoietic stem cells in the presence of SCF and TPO were analyzed.

[0054] In the absence of albumin and in the presence of PVA, the degree of phosphorylation of signaling factors downstream of SCF and TPO in hematopoietic stem cells of mice and humans cultured in the presence of 10 ng / mL of stem cell factor (SCF) and 100 ng / mL of thrombopoietin (TPO) was analyzed by phosphorylation immunostaining. First, the phosphorylation states of each factor downstream of the SCF and TPO signals were detected using antibodies specific for the phosphorylated forms of the respective factors. Specifically, after reacting cells stimulated with cytokines with phosphorylation antibodies in representative signaling molecules such as Akt, PI3K, and Stat5, the fluorescence intensity of the antibodies reacting with the cells was quantitatively analyzed using a fluorescence microscope.

[0055] Then, as shown in Figure 2, among the seven factors examined, some factors with different phosphorylation states were found between mouse hematopoietic stem cells and human hematopoietic stem cells. Among them, for PI3K and Akt, more phosphorylated forms were required in mouse hematopoietic stem cells 24 hours after the addition of SCF and TPO, while almost no phosphorylated forms were observed in human hematopoietic stem cells, or the amount was less than that of the phosphorylated forms in mouse hematopoietic stem cells.

[0056] Therefore, human hematopoietic stem cells were cultured in a culture medium supplemented with 0.3 μM AKTa (manufacturer: Sigma-Aldrich, product number 123871) or 20 μM PI3Ka. After 3 days, 5 days, and 7 days of culture, the cells were counted, and the ratio of the number of cells at each time point was determined with the number of cells on the first day of culture set as 1. In the following examples, 740Y-P (manufacturer: Tocris, product number 1983) was used as PI3Ka. Then, as shown in Figure 3, in the presence of PI3Ka, human hematopoietic stem cells showed clear proliferation. On the other hand, for AKTa, no proliferative effect on human hematopoietic stem cells was observed under the conditions of this experiment.

[0057] Next, regarding the culture of human hematopoietic stem cells, it was examined whether PI3Ka could completely replace SCF. When 20 μM of PI3Ka was added to the above culture medium for human hematopoietic stem cells (S10 + PI3Ka20 + T100), and when 20 μM of PI3Ka was added to the above culture medium and SCF was removed (PI3Ka20 + T100), after culturing human hematopoietic stem cells for 7 days, the total number of cells and the number of isolated CD34+ cells by cell sorting using an anti-CD34 antibody were determined. The results were as shown in FIG. 4. As shown in FIG. 4, when PI3Ka was added, no significant difference was observed in the total number of cells and the number of CD34+ cells depending on the presence or absence of SCF.

[0058] Next, regarding the culture of human hematopoietic stem cells, it was examined whether TPO could be replaced by a TPO receptor agonist. As TPO receptor agonists, butizamide, eltrombopag, and avatrombopag are known. Human hematopoietic stem cells were cultured in a medium obtained by removing TPO from the above culture medium and adding 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 in the presence of PVA. In this experiment, Mpl32D cells were used as human hematopoietic stem cells. The results were as shown in FIG. 5. As shown in FIG. 5, in the presence of albumin, butizamide, eltrombopag, and avatrombopag were able to support the growth 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 growth effect on human hematopoietic stem cells in the absence of TPO, while the effect of avatrombopag was small and almost no effect was observed with eltrombopag.

[0059] Furthermore, purchased human bone marrow CD34+ cells (vendor name Lonza, product number 2C-101) or transferred fresh cord blood from which CD34+ cells were separated using microbeads were used in the culture experiment. In a 24-well plate, 0.2 - 1.0×10 per well 5Cells were dispensed, thrombopoietin (TPO) was removed from the culture medium for human hematopoietic stem cells, and replaced with any of the above TPO receptor agonists (hereinafter sometimes referred to as "TPOago") for experiments. The ratio of the number of cells on day 7 of culture to that on the first day of culture was determined. The results were as shown in Fig. 6. As shown in Fig. 6, human hematopoietic stem cells showed significant cell growth 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 avatrombopag or eltrombopag. However, these TPO receptor agonists are highly safe compounds used for the treatment of patients with surgical treatment of liver cirrhosis and patients with aplastic anemia in the in vivo environment. From the results of this example, it is shown that in the absence of albumin and in the presence of PVA, some TPO receptor agonists can be cytotoxic to human hematopoietic stem cells. Also, from this, it is suggested that there are TPO receptor agonists that are cytotoxic to human hematopoietic stem cells, and that for culturing human hematopoietic stem cells, those that are not cytotoxic to human hematopoietic stem cells may be used.

[0060] Next, it was examined whether stem cell factor (SCF) and TPO can be replaced with PI3Ka and a TPO receptor agonist in the absence of albumin and in the presence of PVA. In the following examples, butizamide was used as the TPO receptor agonist. The purchased human bone marrow CD34+ cells (vendor name Lonza, product number 2C-101) or the transferred fresh cord blood were separated into CD34+ cells using microbeads as described above and used for the culture experiment. 0.2 - 1.0×10 cells per well were placed in a 24-well plate. 5Cells were dispensed and cultured in media with the composition of replacing SCF with 20 μM PI3Ka (PI3Ka 20 μM + TPO 100), and in media with the composition of replacing SCF with 20 μM PI3Ka and TPO with 0.1 μM butyramide (PI3Ka 20 μM + TPOago 0.1 μM), respectively. After 7 days, the total cell number was determined, and the number of CD34+ cells was determined by flow cytometry. Then, the growth rate compared with the start of culture was determined. The results were as shown in Fig. 7. As shown in Fig. 7, it became clear that butyramide could completely replace TPO. Furthermore, CD34+ cells were sorted with a cell sorter before and after culture, seeded 100 cells each into Methocult H4415, and a colony assay was performed. After 2 weeks, the colonies were picked up, cytospin specimens were prepared, Giemsa staining was performed, and then the types of colonies were determined under a microscope. The number of GEmM colonies per 50 CD34+ cells was counted, and the increase rate of the colony number compared with before culture was determined. Then, it became clear that butyramide could completely replace TPO in terms of the ability to form GEmM colonies.

[0061] Next, a culture experiment of human hematopoietic stem cells was conducted in a culture medium containing neither SCF nor TPO, in a medium supplemented with either or both of 20 μM PI3Ka and 0.1 μM TPOago. On the 7th day of culture, the total cell number and the number of CD34+ cells contained were counted by flow cytometry, and the growth rate relative to the start of culture was determined. Then, as shown in Fig. 8, it became clear that no increase in cells was confirmed with PI3Ka alone or butyramide alone, but the cell number increased significantly when both PI3Ka and TPOago were present.

[0062] Next, we examined which cell population was more likely to proliferate in a medium in which SCF and TPO were replaced with PI3Ka and TPOago. The transferred fresh cord blood was separated into CD34+ cells using microbeads as described above and used for the culture experiment. The cells were fractionated by a cell sorter using anti-CD34-PE-Cy7 antibody (manufacturer BD Biosciences, product number 348791), anti-CD38-V450 antibody (manufacturer BD Biosciences, product number 646851), anti-CD133-PE antibody (manufacturer Miltenyi Biotec, product number 130-080-801), anti-CD45RA-APC antibody (manufacturer BioLegend, product number 304112), and anti-CD49f-PE antibody (manufacturer BioLegend, product number 313611). For each of the CD34+ fraction, CD34+CD38-CD133+ fraction, and CD34+CD38-CD45RA-CD49f+ fraction, which are reported as purification markers for human cord blood-derived hematopoietic stem cells, the total cell number and the number of CD34+ cells contained were counted by flow cytometry on the 7th day after the start of culture, and the growth rate relative to the start of culture was determined. The results were as shown in Fig. 9. As shown in Fig. 9, significant cell proliferation was observed in all cell fractions, but particularly in the CD34+CD38-CD133+ fraction and the CD34+CD38-CD45RA-CD49f+ fraction, especially the CD34+CD38-CD45RA-CD49f+ fraction.

[0063] Example 3: Long-term culture experiment of human hematopoietic stem cells Human hematopoietic stem cells were cultured in the above-described culture medium for human hematopoietic stem cells containing 20 μM of PI3Ka and 0.1 μM of TPOago instead of SCF and TPO. On the 7th and 14th days after the start of culture, the total cell number and the number of CD34+ cells were determined in the same manner as above. The results were as shown in Fig. 10. As shown in Fig. 10, the total cell number increased as the number of culture days elapsed, while the number of CD34+ cells decreased on the 14th day compared to the 7th day.

[0064] When observing the cells cultured using an optical microscope, huge cells were recognized on the 14th day. To confirm the possibility that these huge cells were megakaryocytes or megakaryocyte progenitor cells, the cells were fractionated by a flow cytometer using an anti-CD41a-FITC antibody (manufacturer BD Pharmingen, product number 555466) and an anti-CD42b antibody (manufacturer BD Pharmingen, product number 555473). As a result, as shown in Figure 11, most of the cells on the 14th day after the start of culture were CD41a+CD42b+ cells. However, as shown in Figure 11, even under this condition, CD34+CD38− cells were proliferating.

[0065] Also, 100 CD34+ cells each from the culture obtained on the 14th day after the start of culture were seeded into Methocult H4415, and a colony assay was performed. Two weeks later, the colonies were collected and cytospin specimens were prepared. Thereafter, after performing Giemsa staining on the specimens, the colony types were determined under a microscope. The results were as shown in Figure 12. The types of colonies mean that G is a colony containing "granulocytes", E is a colony containing "erythroblasts", m is a colony containing "macrophages", and M is a colony containing "megakaryocytes". As shown in Figure 12, many of the cell colonies contained megakaryocytes.

[0066] Example 4: Examination of more suitable conditions for long-term culture of human hematopoietic stem cells An attempt was made to perform long-term culture of human hematopoietic stem cells in the presence of a compound capable of proliferating human hematopoietic stem cells.

[0067] A culture medium for the above human hematopoietic stem cells, a culture medium containing 20 μM of PI3Ka and 0.1 μM of TPOago instead of SCF and TPO (PI3Ka 20 μM + TPOago 0.1 μM), and for the medium, a medium prepared by further adding either or both of SR-1 (500 nM) and UM171 (35 nM) (+SR-1, +UM171, and +SR-1+UM171) was prepared. Commercially available human bone marrow CD34+ cells (vendor name Lonza, product number 2C-101) or transferred fresh cord blood were separated into CD34+ cells using microbeads as described above and used for the culture experiment. 0.2 to 1.0×10 5 cells were dispensed per well in a 24-well plate, and CD34+ cells were cultured in the prepared medium. On the 14th day after the start of the culture, the total cell number and CD34+ cells were counted in the same manner as above, and the growth rate from before the start of the culture was determined. The results were as shown in Fig. 13. As shown in Fig. 13, in the culture medium further containing UM171, the total cell number and the number of CD34+ cells increased, and the increase rate of CD34+ cells was significantly increased in the medium containing UM171 compared to the medium not containing UM171. In contrast, SR-1 killed the cells under the experimental conditions.

[0068] Furthermore, under three conditions: Condition 1: culturing in a culture medium for human hematopoietic stem cells containing 20 μM of PI3Ka and 0.1 μM of TPOago instead of SCF and TPO for 14 days (PI3Ka 20 μM + TPOago 0.1 μM); Condition 2: culturing under the condition of a medium without butyramide after the 7th day (PI3Ka 20 μM) (Day7-Buty free); and Condition 3: culturing for 14 days in a medium with UM171 further added to the culture medium (PI3Ka 20 μM + TPOago 0.1 μM) (+UM171), CD34+ cells were cultured to determine the increase rates of the total cell number and the CD34+ cell number. Also, the number of CD41+ cells was determined using a flow cytometer. The results were as shown in Fig. 14. As shown in Fig. 14, under Condition 2 where culturing was performed in a medium from which butyramide was removed on the 7th day after the start of culturing, compared with Condition 1, the number of CD34+ cells increased and the number of CD41+ cells decreased. As shown in Fig. 14, under Condition 3 where culturing was performed in a medium with UM171 further added, compared with Conditions 1 and 2, the increase rate of CD34+ cells increased significantly and the number of CD41+ cells decreased significantly. From this, it became clear that in a serum-free medium condition in the presence of PVA, PI3Ka, and TPOago, UM171 can proliferate human hematopoietic stem cells and suppress the differentiation of cells into megakaryocyte progenitor cells and megakaryocytes (see Fig. 15).

[0069] 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, the cultured human CD34+ cells were irradiated with 1.5 Gy of γ-rays and then transplanted into NOG mice at a dose of 1×10 4Cells were transplanted. Twelve weeks after transplantation, the peripheral blood of the NOG mice was collected, and the cell components in the peripheral blood were analyzed using a flow cytometer. The results were as shown in Fig. 16. As shown in Fig. 16, under Conditions 1 and 2, the engraftment rates of hematopoietic stem cells were 14.9% and 11.1% respectively when pre-culture CD34+ cells were transplanted, but increased to 66.6% and 54.9% respectively when post-culture CD34+ cells were transplanted. Also, under Condition 3, the engraftment rate of hematopoietic stem cells was 17% when pre-culture CD34+ cells were transplanted, but increased to 67% when post-culture CD34+ cells were transplanted.

[0070] From this, it became clear that in serum-free medium conditions in the absence of albumin, in the presence of PVA, the PI3K activator and the TPO receptor agonist can proliferate human hematopoietic stem cells well, and the proliferated human CD34+ cells maintain the properties as hematopoietic stem cells and improve the engraftment rate after transplantation into other individuals. Also, when human hematopoietic stem cells are cultured long-term under this condition, a part of them differentiates into megakaryocytes, whereby megakaryocytes can be obtained, while a part has a tendency to proliferate as CD34+ cells. It also became clear that when UM171 is added here, the proliferation rate of CD34+ cells can be improved and the differentiation into megakaryocytes can be suppressed.

[0071] Next, human monocytes were isolated from fresh human umbilical cord blood. Under Conditions 1 and 3, the obtained cells (5×10 5 cells) were cultured, and the total cell number, the number of CD34+ cells, the ratio of CD34+ cells in the total cells, and the cell viability were determined 7 days and 14 days after the start of culture respectively. The results were as shown in Fig. 17. As shown in Fig. 17, under any condition, the monocytes obtained from human umbilical cord blood decreased the total cell number with culture. On the other hand, under Condition 3, the ratio of CD34+ cells in the total cells was significantly improved compared to Condition 1. Also, the cell viability was significantly higher in Condition 3 than in Condition 1. Furthermore, the number of CD34+ cells showed a significant increase under Condition 3, while under Condition 1, although the cell number was maintained well, no increase in the cell number was observed.

Claims

1. A method for culturing or producing human hematopoietic stem cells, comprising: culturing human hematopoietic stem cells in a culture medium, wherein the culture medium contains polyvinyl alcohol and (1) contains one or more selected from the group consisting of stem cell factor (SCF) and a PI3K activator, and a TPO receptor agonist; or (2) contains a PI3K activator and a TPO receptor agonist, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butyramide, and the number of human hematopoietic stem cells increases by the above culturing, method.

2. The method according to claim 1, wherein the culture medium is a serum-free medium.

3. The method according to claim 1, wherein the culture medium is a chemically defined medium.

4. The method according to any one of claims 1 to 3, wherein the culture medium substantially does not contain albumin.

5. The method according to any one of claims 1 to 4, comprising obtaining the increased human hematopoietic stem cells.

6. The method according to any one of claims 1 to 5, wherein the culture medium contains a PI3K activator and does not contain SCF.

7. The method according to any one of claims 1 to 6, wherein the culture medium contains a PI3K activator and a TPO receptor agonist.

8. The method according to claim 7, wherein the culture medium does not contain both SCF and TPO.

9. The method according to any one of claims 1 to 8, 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 claim 9, wherein the culture period is 7 days or more.

11. A composition comprising human hematopoietic stem cells, polyvinyl alcohol as an alternative to albumin, and (1) one or more selected from the group consisting of SCF and a PI3K activator, and a TPO receptor agonist; or (2) a PI3K activator and a TPO receptor agonist, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butyramide.

12. The composition according to claim 11, comprising a PI3K activator and a TPO receptor agonist.

13. The composition according to claim 11 or 12, 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. As an alternative to albumin, it contains polyvinyl alcohol and (1) one or more selected from the group consisting of SCF and PI3K activator, and a TPO receptor agonist; or (2) a PI3K activator and a TPO receptor agonist, The culture medium for human hematopoietic stem cells, wherein the PI3K activator is 740Y-P and the TPO receptor agonist is butizamide.

15. The culture medium according to claim 14, which is a serum-free medium.

16. The culture medium according to claim 14, which is a chemically defined medium.

17. The culture medium according to any one of claims 14 to 16, which does not contain albumin.

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