Culture medium for human cells, and method for culturing human hematopoietic stem cells

Abstract

The present invention provides: a culture medium for culturing hematopoietic stem cells in vitro, the culture medium making it possible to selectively culture hematopoietic stem cells when a cell population containing the hematopoietic stem cells and cancer cells is cultured; and a method for culturing hematopoietic stem cells using the culture medium. The present invention relates to: a culture medium for human cells, the culture medium being intended to be used for culturing a cell population containing human cells, and the culture medium comprising a polyalkylene glycol modified by a copolymer of a polyvinylcaprolactam block and a polyvinyl acetate block, a phosphatidylinositol-3 kinase (PI3K) activator, a TPO receptor agonist, and one or more components selected from the group consisting of a proteasome inhibitor, a TNF-related apoptosis-inducing ligand and a cereblon modulator; and a method for culturing human hematopoietic stem cells, the method comprising culturing a cell population containing human hematopoietic stem cells in the aforementioned culture medium for human cells.

Description

Human cell culture medium and method for culturing human hematopoietic stem cells

[0001] The present invention relates to a culture medium suitable for culturing human hematopoietic stem cells and a method for culturing human hematopoietic stem cells. This application claims priority to Japanese Patent Application No. 2023-125823, filed on August 1, 2023, the contents of which are incorporated herein by reference.

[0002] Hematopoietic stem cells are stem cells that can differentiate into blood cells. Transplantation of hematopoietic stem cells is known as a treatment for diseases such as cancer and immunodeficiency. The main sources of hematopoietic stem cells are known to be human umbilical cord blood (CB), bone marrow cells, and peripheral blood hematopoietic stem cells (PBSC). In particular, autologous hematopoietic stem cell transplantation is the primary treatment for multiple myeloma (MM), a hematological tumor.

[0003] To achieve a sufficient therapeutic effect in hematopoietic stem cell transplantation, a sufficient number of hematopoietic stem cells must be transplanted. Therefore, methods have been attempted in which hematopoietic stem cells for transplantation are cultured and expanded ex vivo before transplantation. For example, Patent Document 1 discloses a method for culturing hematopoietic stem cells ex vivo in a culture medium supplemented with an agent that reduces the level of ikaros family member transcription factors and an agent that inhibits TGFβ receptors, in order to increase the proportion of hematopoietic stem cells in a cultured cell population. Patent Document 2 discloses a method for culturing hematopoietic stem cells in a culture medium supplemented with a histone demethylase inhibitor, a TGFβ receptor inhibitor, and a histone deacetylase inhibitor.

[0004] In general, albumin-containing media have been used to induce cell division in hematopoietic stem cell culture. However, albumin has problems such as the inhibition of stable undifferentiated state and biological contamination. Therefore, research has been conducted on culture methods using albumin-free chemically defined media. For example, Patent Document 3 reports that human hematopoietic stem cells can be proliferated in an albumin-free serum-free medium in the presence of polyvinyl alcohol (PVA), tissue factor (SCF), thrombopoietin (TPO) or a TPO receptor agonist, and by further adding a phosphatidylinositol 3-kinase (PI3K) activator. It also reports that long-term culture of human hematopoietic stem cells in this medium facilitates differentiation into megakaryocytic lineage cells, but that differentiation into megakaryocytic lineage cells can be suppressed by further adding UM171 to the medium. Furthermore, Patent Document 4 reports that human hematopoietic stem cells exhibit remarkable proliferation while maintaining stemness in the presence of polyethylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, even in a serum-free medium that does not contain albumin, and that proliferation of hematopoietic stem cells can be maintained even under cytokine-free conditions, but that various blood cell lineages other than hematopoietic stem cells can also proliferate under the same conditions.

[0005] On the other hand, the biggest challenge in autologous hematopoietic stem cell transplantation is recurrence, and one of the causes is said to be contamination with the patient's own cancer cells during transplantation. Human peripheral blood mononuclear cells (PBMCs) are very useful as a transplant source in autologous hematopoietic stem cell transplantation. However, unlike umbilical cord blood from healthy donors, there is a concern that cancer cells contaminating the collected patient's peripheral blood may contribute to post-transplant recurrence. To avoid this risk, for example, Patent Document 5 reports that cancer cells can be efficiently removed (purged) by culturing PBMCs derived from cancer patients in a culture medium supplemented with a soluble fragment of FasL in an amount sufficient to kill cancer cells without substantially affecting cell viability, thereby obtaining a culture consisting of a cell population with a reduced proportion of cancer cells.

[0006] Japanese Patent No. 7080179 Japanese Patent No. 6980534 International Publication No. 2021 / 049617 International Publication No. 2021 / 149799 International Publication No. 2006 / 037762

[0007] In culturing cell populations for transplantation, it is most important to efficiently proliferate functional hematopoietic stem cells while maintaining their function in vitro. However, all of the methods reported for the in vitro culture of autologous graft-derived cells only aim to remove or inhibit the proliferation of cells other than hematopoietic stem cells in order to increase the proportion of hematopoietic stem cells in the cell culture.

[0008] The present invention aims to provide a culture medium for culturing hematopoietic stem cells in vitro, which is capable of selectively culturing hematopoietic stem cells when culturing a cell population containing hematopoietic stem cells and cancer cells, and a method for culturing hematopoietic stem cells using the culture medium.

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that when a cell population containing human PBMC-derived hematopoietic stem cells (PBSCs) is cultured in an albumin-free culture medium containing polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and the medium is supplemented with a PI3K activator, a TPO receptor agonist, and one or more selected from the group consisting of a proteasome inhibitor, a TNF-related apoptosis-inducing ligand, and a cereblon modulator, the human hematopoietic stem cells can be selectively proliferated, resulting in a culture comprising a cell population with an increased proportion of human hematopoietic stem cells, even when the cell population also contains cancer cells, thereby completing the present invention.

[0010] The present invention is as follows: [1] A culture medium for culturing a cell population containing human cells, comprising: a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block; a phosphatidylinositol 3-kinase (PI3K) activator; a TPO receptor agonist; and one or more selected from the group consisting of a proteasome inhibitor, a TNF-related apoptosis-inducing ligand, and a cereblon modulator. [2] The human cell culture medium of [1], wherein the proteasome inhibitor is one or more selected from the group consisting of bortezomib and carfilzomib. [3] The human cell culture medium of [1], wherein the cereblon modulator is one or more selected from the group consisting of lenalidomide, pomalidomide, avadomide, iverdomide, medignomide, and thalidomide. [4] The human cell culture medium according to any one of [1] to [3] above, further comprising a pyrimidoindole compound. [5] The human cell culture medium according to [4] above, wherein the pyrimidoindole compound is one or more selected from the group consisting of UM171 and UM729. [6] The human cell culture medium according to any one of [1] to [5] above, further comprising a FLT3 ligand. [7] The human cell culture medium according to any one of [1] to [6] above, wherein the culture medium is a serum-free medium. [8] The human cell culture medium according to any one of [1] to [7] above, wherein the culture medium is substantially free of albumin. [9] A method for culturing human hematopoietic stem cells, comprising culturing a cell population containing human hematopoietic stem cells in the human cell culture medium according to any one of [1] to [8] above.

[10] The method for culturing human hematopoietic stem cells according to [9] above, wherein the cell population is cultured in the human cell culture medium for 7 days or more.

[11] The method for culturing human hematopoietic stem cells according to [9] or

[10] above, wherein the cell population is PBMC.

[12] The method for culturing human hematopoietic stem cells according to any of [9] to

[111] above, wherein the cell population is a cell population derived from a cancer patient.

[13] The method for culturing human hematopoietic stem cells according to any of [9] to

[12] above, wherein the proportion of cancer cells in the cell population is lower at the end of the culture than at the start of the culture.

[14] The method for culturing human hematopoietic stem cells according to any of [9] to

[13] above, wherein the proportion of human hematopoietic stem cells in the cell population is higher at the end of the culture than at the start of the culture.

[15] A human hematopoietic stem cell population obtained by culturing a cell population collected from a human in a human cell culture medium according to any of [1] to [8] above.

[16] A human hematopoietic stem cell population containing human hematopoietic stem cells and a human cell culture medium according to any of [1] to [8] above.

[0011] The culture medium of the present invention and the culture method using the same enable efficient removal of cancer cells and enrichment of human hematopoietic stem cells, even when a cell population derived from a human cancer patient is cultured. That is, the cell population obtained by the present invention has an increased proportion of hematopoietic stem cells and a reduced amount of cancer cells, and therefore can reduce the risk of cancer recurrence when transplanted, and is therefore useful as transplant cells for autologous hematopoietic stem cell transplantation.

[0012] In Example 1, MM.1S cells (FIG. 1(A)) and PB-derived CD34 cells were cultured using modified 3a medium supplemented with bortezomib or carfilzomib. ＋1(B)) for one week. This figure shows the results of measuring the cell viability (%) when cells (PBSCs) ( FIG. 1(B)) were cultured for one week. This figure shows the results of measuring the relative cell number of the human HSC fraction when PBSCs were cultured for one week using modified 3a medium supplemented with bortezomib in Example 1. This figure shows the results of measuring the cell viability (%) when MM.1S cells were cultured for one week using culture media supplemented with 2.0 μM bortezomib or 0.2 μM carfilzomib in SSUM, SSSR, modified 2a medium, and modified 3a medium, respectively, or culture media supplemented with neither bortezomib nor carfilzomib in Example 1. In Comparative Example 1, MM.1S cells (FIG. 4(A)) and PBSCs (FIG. 4(B)) were cultured for one week using modified 2a medium or modified 3a medium supplemented with panobinostat, and the results of measuring the relative cell number of the human HSC fraction (FIG. 4(C)) are shown. In Example 3, the CD34 expression level was increased when PBSCs were cultured for one week using modified 3a medium supplemented with each cereblon modulator. ＋ EPCR ＋ CD90 ＋ CD45RA － ITGA3 ＋ 6(A) shows the results of measuring the relative viable cell count of MM.1S cells among all viable cells when MM.1S cells and PBSCs were co-cultured for 14 days using modified 3a medium, BL-containing modified 3a medium, SSUM, or SSSR in Example 4. ＋ EPCR ＋ 6(B) shows the time course of the percentage (%) of viable cells (FIG. 6(B)). In Example 4, the CD34 expression level was measured when PBSCs were cultured for 14 days using modified 3a medium supplemented with DMSO alone, modified 3a medium containing 2 μM bortezomib alone (B-containing modified 3a medium), modified 3a medium containing 2 μM bortezomib and 1 μM lenalidomide (BL(1)-containing modified 3a medium), or modified 3a medium containing 2 μM bortezomib and 1 μM lenalidomide (BL(10)-containing modified 3a medium). ＋ EPCR ＋ CD90 ＋ CD45RA － ITGA3＋ 8(A) and 8(B) show the results of measuring the relative viable cell count of cells. In Example 5, the figure shows the results of measuring the cell viability (%) when MM.1S cells were cultured for one week in modified 3a medium containing bortezomib and TRAIL ( FIG. 8(A) ) or SSUM containing bortezomib and TRAIL ( FIG. 8(B) ).

[0013] In the present invention and this specification, "X to Y (X and Y are real numbers satisfying X<Y)" means a numerical range of "not less than X and not more than Y."

[0014] 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 be cells obtained by ex vivo differentiation of pluripotent stem cells such as ES cells and iPS cells. Furthermore, as used herein, "functional hematopoietic stem cells" refer to hematopoietic stem cells that retain the functions inherent to hematopoietic stem cells, particularly undifferentiated state and pluripotency.

[0015] As used herein, "ex vivo" means outside of a living body. As used herein, ex vivo is used in contrast to in vivo (inside a living body) and refers to a state in which cells present in a living body are removed from the living body to outside the living body. Culturing can be performed ex vivo.

[0016] As used herein, "positive" means that a cell is recognized as expressing the molecule identified by the term immediately preceding it. As used herein, "positive" may be simply represented as "+".

[0017] In this specification, "polyvinyl alcohol" refers to 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 ) 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 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.

[0018] In this specification, "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 and culture of hematopoietic stem cells. Human serum albumin (hereinafter sometimes referred to as "HSA") is human serum albumin, and may 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 thereto.

[0019] As used herein, "comprising B as a substitute for A" means including B instead of A. Including B in place of A means partially or completely replacing A with B. "Comprising B as a substitute for A" can mean substantially free of A and including B. "Substantially free" allows for unavoidable carryover into the system and carryover below the limit of detection. "Substantially free" can also allow for no preventable carryover.

[0020] As used herein, the term "agonist" refers to a substance that activates proteins such as receptors and enzymes.

[0021] As used herein, "culturing" means incubating cells under conditions suitable for their growth or maintenance, preferably at 37°C and 5% CO for human cells. ２ The "culturing" may be carried out under an atmosphere. When "culturing" involves proliferation, it is understood that the "culturing" is the production of proliferated cells. As used herein, the "culturing" may be carried out in a serum-free medium. As used herein, the "culturing" may be carried out in a chemically defined culture medium (or in a synthetic medium). A chemically defined culture medium is a serum-free medium.

[0022] As used herein, the term "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 and streptomycin), essential amino acids such as glutamine, and culture additives such as insulin, transferrin, selenium (e.g., sodium selenite), and ethanolamine. The culture medium can be a liquid medium.

[0023] As used herein, "cytotoxicity" refers to the property of reducing cell numbers or killing cells during culture. As used herein, "non-cytotoxicity" refers to the property of not reducing cell numbers during culture. Some substances can become cytotoxic by increasing their concentration. In this case, if the substance's expected beneficial effect occurs even when its concentration is reduced to a level that does not cause cytotoxicity, it can be defined as non-cytotoxic.

[0024] 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.

[0025] 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 formal maintenance of hematopoietic stem cells. Human thrombopoietin may be, for example, a protein having the amino acid sequence registered under GenBank Accession No. AAB33390.1 or a thrombopoietin having an amino acid sequence corresponding thereto. TPO may include functional homologs of TPO and proteins having an amino acid sequence with 90% or more, 95% or more, or 100% identity to the amino acid sequence registered under GenBank Accession No. AAB33390.1 and having the function of TPO. As used herein, "TPO receptor agonist" refers to a substance other than TPO that activates the TPO receptor. TPO receptor agonists include TPO variants other than TPO, peptides, and compounds that activate the TPO receptor. In this specification, the term "compound" is a concept that encompasses organic compounds.

[0026] As used herein, stem cell factor (SCF) refers to a hematopoietic cell growth factor that acts in the early stages of hematopoiesis. Human stem cell factor may be, for example, a protein having the amino acid sequence registered under GenBank Accession No. AAA85450.1 or an SCF having a corresponding amino acid sequence. SCF may include functional homologs of SCF and proteins having an amino acid sequence with 90% or more, 95% or more, or 100% identity to the amino acid sequence registered under GenBank Accession No. AAB33390.1 and having the function of SCF.

[0027] Proteasomes are enzymes that degrade ubiquitinated proteins. As used herein, the term "proteasome inhibitor" refers to a substance that inhibits the degradation of proteins by the proteasome. When proteasomes are inhibited, unnecessary proteins accumulate in cells, resulting in the induction of apoptosis. For this reason, proteasome inhibitors are also used as anticancer drugs.

[0028] TNF-related apoptosis-inducing ligand (TRAIL) is a death receptor activator that transmits apoptosis-inducing signals through activation of caspase 8 and caspase 3 by binding to TRAIL receptors present on the cell membrane surface. There are two main types of TRAIL receptors expressed in human cells: DR4 (death receptor 4) (also known as TRAIL-R1, CD261) and DR5 (death receptor 5) (also known as TRAIL-R2). Many cancer cells, including myeloma cells, are sensitive to TRAIL, but DR4 and DR5 are rarely expressed in normal cells, and therefore TRAIL is also used as an anticancer drug.

[0029] Cereblon is one of the components of the ubiquitin ligase E3 complex and is involved in the removal of unnecessary proteins from cells. As used herein, "cereblon modulator" refers to a substance that binds to cereblon and affects its proteolysis-inducing activity. In multiple myeloma, when a cereblon modulator binds to cereblon, transcription factors (e.g., Ikaros, Aiolos) necessary for the survival of multiple myeloma are recognized as neosubstrates, and the ubiquitin ligase E3 complex promotes proteasomal degradation, inducing apoptosis. For this reason, some cereblon modulators are used as anticancer drugs.

[0030] FLT3 is a cell surface receptor (also known as CD135) known as FMS-related tyrosine kinase 3. The FLT3 receptor is also expressed on the surface of hematopoietic stem cells. As used herein, "FLT3 ligand (FLT3L)" refers to a ligand of the FLT3 receptor, which is involved in, for example, the normal development of hematopoietic stem cells and hematopoietic progenitor cells.

[0031] <Human Cell Culture Medium> The human cell culture medium of this embodiment is a culture medium for culturing a cell population containing human cells, and contains a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block (hereinafter, sometimes referred to as "PCL-PVAc-polyalkylene glycol"), a PI3K activator, a TPO receptor agonist, and one or more selected from the group consisting of a proteasome inhibitor, TRAIL, and a cereblon modulator. By containing PCL-PVAc-polyalkylene glycol, a PI3K activator, and a TPO receptor agonist, the human cell culture medium of this embodiment is capable of expanding human functional hematopoietic stem cells ex vivo. Furthermore, the human cell culture medium of this embodiment further contains one or more compounds selected from the group consisting of a proteasome inhibitor, TRAIL, and a cereblon modulator, thereby enabling the proliferation of human functional hematopoietic stem cells while suppressing the proliferation of cancer cells, thereby enabling the selective removal of cancer cells.

[0032] The human cell culture medium in this embodiment may be a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, and a proteasome inhibitor, a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, and TRAIL, or a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, and a cereblon modulator. Because selective removal of cancer cells can be performed more efficiently, a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, a proteasome inhibitor, and TRAIL, a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, a proteasome inhibitor, and a cereblon modulator, or a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, TRAIL, and a cereblon modulator is more preferred, and a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, a proteasome inhibitor, TRAIL, and a cereblon modulator is particularly preferred.

[0033] The PCL-PVAc-polyalkylene glycol contained in the human cell culture medium of this embodiment is preferably a polymer in which a polymer of alkylene glycol having 1 to 10 carbon atoms has been modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, more preferably a polymer in which a polymer of alkylene glycol having 1 to 5 carbon atoms has been modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, even more preferably a polymer in which a polymer of alkylene glycol having 2 to 4 carbon atoms has been modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, even more preferably a polymer in which a polymer of ethylene glycol or propylene glycol has been modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and particularly preferably a polymer in which a polymer of ethylene glycol has been modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0034] The PCL-PVAc-polyalkylene glycol contained in the human cell culture medium of this embodiment is preferably, for example, a polymer (PCL-PVAc-PEG) represented by the following general formula (1): In general formula (1), 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.

[0035]

[0036] In general formula (1), the natural number n is preferably 5 or more, more preferably 10 or more, even more preferably 20 or more, and even more preferably 30 or more. In general formula (1), the natural number n is preferably 45 or less, more preferably 40 or less, and even more preferably 30 or less.

[0037] In general formula (1), the natural number m is preferably 15 or more, more preferably 20 or more, and even more preferably 30 or more. In general formula (1), the natural number m is preferably 80 or less, more preferably 60 or less, even more preferably 50 or less, and even more preferably 40 or less.

[0038] In general formula (1), the natural number 1 is preferably 15 or more, more preferably 20 or more, even more preferably 30 or more, and even more preferably 50 or more. In general formula (1), the natural number 1 is preferably 180 or less, more preferably 150 or less, even more preferably 120 or less, even more preferably 100 or less, and particularly preferably 60 or less.

[0039] The PCL-PVAc-polyalkylene glycol contained in the human cell culture medium of this embodiment may be, for example, a compound in which the ethylene glycol unit in the above general formula (1) is replaced with an alkylene glycol unit. The number of carbon atoms in the alkylene glycol unit is preferably 1 to 10, more preferably 1 to 5, even more preferably 2 to 4, and particularly preferably 2 (i.e., ethylene glycol unit) or 3.

[0040] The content of PCL-PVAc-polyalkylene glycol in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that allows the proliferation of human hematopoietic stem cells. In the human cell culture medium of this embodiment, the content of PCL-PVAc-polyalkylene glycol relative to the total volume of the culture medium is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.008% by mass or more, and even more preferably 0.01% by mass or more. In the human cell culture medium of this embodiment, the content of PCL-PVAc-polyalkylene glycol relative to the total volume of the culture medium is preferably 1.0% by mass or less, more preferably 0.8% by mass or less, even more preferably 0.5% by mass or less, even more preferably 0.2% by mass or less, and particularly preferably 0.1% by mass or less. The content of PCL-PVAc-polyalkylene glycol in the cell culture medium can be calculated from the amounts of the raw materials used.

[0041] The PI3K activator contained in the human cell culture medium of this embodiment may be any substance that activates PI3K, and is not particularly limited thereto. However, it is preferably a non-cytotoxic PI3K activator that is not cytotoxic to human hematopoietic stem cells in the presence of PCL-PVAc-polyalkylene glycol. The PI3K activator contained in the human cell culture medium of this embodiment may be one type, or two or more types may be combined. The PI3K activator contained in the human cell culture medium of this embodiment is particularly preferably the phosphorylated peptide 740Y-P (CAS RN: 1236188-16-1).

[0042] The content of the PI3K activator in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that allows the proliferation of human hematopoietic stem cells. For the human cell culture medium of this embodiment, the PI3K activator content relative to the total volume of the culture medium is preferably 0.1 μM or more, more preferably 0.5 μM or more, even more preferably 0.8 μM or more, and even more preferably 1.0 μM or more. For the human cell culture medium of this embodiment, the PI3K activator content relative to the total volume of the culture medium is preferably 50 μM or less, more preferably 25.0 μM or less, even more preferably 20.0 μM or less, even more preferably 10.0 μM or less, and particularly preferably 5.0 μM or less. The PI3K activator content in the cell culture medium can be calculated from the amounts of the raw materials used.

[0043] The TPO receptor agonist contained in the human cell culture medium of this embodiment may be any substance that activates the TPO receptor, and is not particularly limited thereto. However, it is preferably a non-cytotoxic TPO receptor agonist that is not cytotoxic to human hematopoietic stem cells in the presence of PCL-PVAc-polyalkylene glycol. The TPO receptor agonist contained in the human cell culture medium of this embodiment may be one type, or two or more types may be combined. The TPO receptor agonist contained in the human cell culture medium of this embodiment is particularly preferably 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") (PubChem CID: 44602781) or a salt thereof.

[0044] The content of the TPO receptor agonist in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that allows the proliferation of human hematopoietic stem cells. The content of the TPO receptor agonist in the human cell culture medium of this embodiment relative to the total volume of the culture medium is preferably 0.01 μM or more, more preferably 0.05 μM or more, even more preferably 0.08 μM or more, and even more preferably 0.10 μM or more. The content of the TPO receptor agonist in the human cell culture medium of this embodiment relative to the total volume of the culture medium is preferably 2.0 μM or less, more preferably 1.5 μM or less, even more preferably 1.0 μM or less, even more preferably 0.5 μM or less, and particularly preferably 0.2 μM or less. The content of the TPO receptor agonist in the cell culture medium can be calculated from the amounts of the raw materials used.

[0045] Those skilled in the art can appropriately confirm whether a PI3K activator and a TPO receptor agonist are non-cytotoxic by a human hematopoietic stem cell culture test. Here, a human hematopoietic stem cell culture test to examine the non-cytotoxicity of a PI3K activator can be performed using an IMDM medium containing 1% ITS-X (insulin-transferrin-selenium-ethanolamine), 1% penicillin-streptomycin-glutamine (PSG), 0.10 μM TPO receptor agonist, and 0.1% PCL-PVAc-polyalkylene glycol. A human hematopoietic stem cell culture test to examine the non-cytotoxicity of a TPO receptor agonist can be performed using an IMDM medium containing 1% ITS-X, 1% PSG, 1.0 μM PI3K activator, and 0.1% PCL-PVAc-polyalkylene glycol.

[0046] The proteasome inhibitor contained in the human cell culture medium of this embodiment is not particularly limited as long as it is a substance that inhibits the degradation of ubiquitinated proteins by the proteasome. For example, proteasome inhibitors used in anticancer treatments for various cancers can be used as the proteasome inhibitor contained in the human cell culture medium of this embodiment. Examples of the proteasome inhibitor include bortezomib ({(1R)-3-methyl-1-[(2S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido]butyl}boronic acid, CAS RN: 179324-69-7), carfilzomib ((2R)-2-methyl-2-[N-[(2S)-2-[(morpholinoacetyl)amino]-4-phenylbutyryl]-L-Leu-L-Phe-L-Leu-]oxirane, CAS RN: 868540-17-4), ixazomib (B-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]boronic acid, CAS RN: RN: 1072833-77-2), MG-312 (benzyloxycarbonylleucylleucine aldehyde, CAS RN: 133407-82-6), etc. The proteasome inhibitor contained in the human cell culture medium of this embodiment may be one type, or two or more types may be combined. The proteasome inhibitor contained in the human cell culture medium of this embodiment is preferably one or more types selected from the group consisting of bortezomib and carfilzomib.

[0047] The content of the proteasome inhibitor in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that can inhibit cancer cell proliferation and enable the proliferation of human hematopoietic stem cells. The preferred content of the proteasome inhibitor, i.e., the "content that can inhibit cancer cell proliferation and enable the proliferation of human hematopoietic stem cells," can be determined experimentally for each type of proteasome inhibitor. For example, a multiple myeloma cell line (e.g., MM.1S cells) and hematopoietic stem cells (PBSCs) recovered from PBMCs recovered from healthy individuals are cultured in culture media containing various concentrations of the proteasome inhibitor, and the viability is examined. The concentration at which the viability of PBSCs is high but the viability of multiple myeloma cell lines is low can be selected as the concentration of the proteasome inhibitor in the human cell culture medium of this embodiment.

[0048] The TRAIL contained in the human cell culture medium of this embodiment is not particularly limited, as long as it binds to DR4 or DR5 to transmit so-called death signaling in human cells and induce apoptosis. For example, it may be a full-length human TRAIL protein, a full-length TRAIL protein derived from a non-human animal species, or a partial peptide of human TRAIL or a non-human animal-derived TRAIL that has the ability to bind to a TRAIL receptor. TRAIL or a partial peptide thereof may also be a mutant in which one or more amino acids have been substituted, inserted, or deleted while retaining the ability to bind to a TRAIL receptor. Furthermore, these may be substances in which various tag peptides, fluorescent substances, etc. are bound to these. In addition, TRAIL used in anti-cancer treatments for various cancers can also be used as the TRAIL contained in the human cell culture medium of this embodiment. The TRAIL contained in the human cell culture medium of this embodiment may be one type of TRAIL or a combination of two or more types of TRAIL.

[0049] The TRAIL content in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that can suppress cancer cell proliferation and enable the proliferation of human hematopoietic stem cells. The preferred TRAIL content, i.e., the "content that can suppress cancer cell proliferation and enable the proliferation of human hematopoietic stem cells," can be determined experimentally for each type of TRAIL. For example, multiple myeloma cell lines (e.g., MM.1S cells) and PBSCs collected from healthy individuals are cultured in culture media containing various concentrations of TRAIL, and the viability is examined. The concentration at which the viability of PBSCs is high but the viability of multiple myeloma cell lines is low can be set as the TRAIL concentration in the human cell culture medium of this embodiment.

[0050] The cereblon modulator contained in the human cell culture medium of this embodiment is not particularly limited, as long as it is a substance that binds to cereblon intracellularly and ultimately induces apoptosis. Examples of cereblon modulators include lenalidomide, pomalidomide, avadomide, iverdomide, mezignomide, and thalidomide. The human cell culture medium of this embodiment may contain one type of cereblon modulator or a combination of two or more types. The cereblon modulator contained in the human cell culture medium of this embodiment is preferably one or more selected from the group consisting of lenalidomide, pomalidomide, avadomide, iberdomide, mezignomide, and thalidomide, more preferably one or more selected from the group consisting of lenalidomide, pomalidomide, avadomide, iberdomide, and mezignomide, and even more preferably one or more selected from the group consisting of lenalidomide and pomalidomide.

[0051] When the human cell culture medium of this embodiment contains a cereblon modulator, the amount of cereblon modulator contained in the human cell culture medium is not particularly limited, as long as it is an amount that can inhibit cancer cell proliferation and enable the proliferation of human hematopoietic stem cells. The preferred amount of cereblon modulator, i.e., the amount that can inhibit cancer cell proliferation and enable the proliferation of human hematopoietic stem cells, can be determined experimentally for each type of cereblon modulator. For example, a multiple myeloma cell line (e.g., MM.1S cells) and a cell population containing hematopoietic stem cells (PBSC fraction) prepared from PBMCs collected from healthy individuals are cultured in culture medium containing various concentrations of cereblon modulator, and their viability is examined. The concentration at which the cell viability of the PBSC fraction is high but the viability of the multiple myeloma cell line is low can be selected as the concentration of cereblon modulator in the human cell culture medium of this embodiment.

[0052] The human cell culture medium of this embodiment preferably further contains a pyrimidoindole compound in addition to at least one of a proteasome inhibitor, TRAIL, and a cereblon modulator. By containing a pyrimidoindole compound, the human cell culture medium of this embodiment suppresses differentiation of human hematopoietic stem cells into megakaryocytic lineage cells (e.g., megakaryocyte progenitor cells and megakaryocytes) during culture, thereby tending to maintain the undifferentiated state of human hematopoietic stem cells. Examples of pyrimidoindole compounds contained in the human cell culture medium of this embodiment include UM171 (4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine, CAS RN: 1448724-09-1) and UM729 (CAS RN: 1448723-60-1). The human cell culture medium of this embodiment may contain one type of pyrimidoindole compound, or two or more types may be combined.

[0053] When the human cell culture medium of this embodiment contains a pyrimidoindole compound, the content of the pyrimidoindole compound in the human cell culture medium is not particularly limited, as long as it is an amount capable of inhibiting the differentiation of human hematopoietic stem cells. The preferred content of the pyrimidoindole compound, i.e., the "content capable of inhibiting the differentiation of human hematopoietic stem cells," can be experimentally determined for each type of pyrimidoindole compound. For example, PBSC fractions collected from healthy individuals are cultured in culture media containing various concentrations of the pyrimidoindole compound, and the proportion of undifferentiated human hematopoietic stem cells is examined. The concentration at which the proportion of undifferentiated human hematopoietic stem cells in the cell population obtained by culture ([number of undifferentiated human hematopoietic stem cells in the cell population] / [total number of cells in the cell population] × 100%) is equal to or higher than that before culture can be determined as the concentration of the pyrimidoindole compound in the human cell culture medium of this embodiment.

[0054] The human cell culture medium of this embodiment preferably further contains a FLT3 ligand in addition to PCL-PVAc-polyalkylene glycol, a PI3K activator, and a TPO receptor agonist. By containing a combination of PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, and a FLT3 ligand, the human cell culture medium of this embodiment can proliferate human functional hematopoietic stem cells ex vivo while maintaining their undifferentiated state and differentiation potential. It is particularly preferable that the human cell culture medium of this embodiment contains PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, a pyrimidoindole compound, and a FLT3 ligand.

[0055] The FLT3 ligand contained in the human cell culture medium of this embodiment is not particularly limited, as long as it is a substance that can bind to the FLT3 receptor and thereby proliferate hematopoietic stem cells while maintaining their undifferentiated state. For example, it may be a full-length protein of human FLT3 ligand, a full-length protein of FLT3 ligand derived from an animal species other than human, or a partial peptide of human FLT3 ligand or a FLT3 ligand derived from an animal species other than human that has the ability to bind to the FLT3 receptor. Furthermore, the FLT3 ligand or its partial peptide may be a mutant in which one or more amino acids are substituted, inserted, or deleted while retaining the ability to bind to the FLT3 receptor. Furthermore, it may be a substance in which various tag peptides, fluorescent substances, etc. are bound to these. In addition, FLT3 ligands used in anti-cancer treatments for various cancers can also be used as the FLT3 ligand contained in the human cell culture medium of this embodiment. The FLT3 ligand contained in the human cell culture medium of this embodiment may be one type, or two or more types may be combined.

[0056] The content of FLT3 ligand in the human cell culture medium of this embodiment is not particularly limited, as long as it is an amount that allows human hematopoietic stem cells to proliferate while maintaining their undifferentiated state and differentiation potential. The preferred content of FLT3 ligand, i.e., the "content that allows human hematopoietic stem cells to proliferate while maintaining their undifferentiated state and differentiation potential," can be experimentally determined for each type of FLT3 ligand. For example, PBMCs collected from healthy individuals or PBSC fractions prepared therefrom are cultured in culture media containing various concentrations of FLT3 ligand, and the proportion of undifferentiated human hematopoietic stem cells is examined. The concentration at which the proportion of undifferentiated human hematopoietic stem cells in the cell population obtained by culture ([number of undifferentiated human hematopoietic stem cells in the cell population] / [total number of cells in the cell population] × 100%) is equal to or higher than that before culture can be determined as the concentration of FLT3 ligand in the human cell culture medium of this embodiment.

[0057] The human cell culture medium of this embodiment is preferably a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, at least one of a proteasome inhibitor, TRAIL, and a cereblon modulator, and a pyrimidoindole compound, and more preferably a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, at least one of a proteasome inhibitor, TRAIL, and a cereblon modulator, a pyrimidoindole compound, and a FLT3 ligand. The medium preferably contains CL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, at least two selected from the group consisting of a proteasome inhibitor, TRAIL, and a cereblon modulator, and a pyrimidoindole compound, and more preferably contains PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, at least two selected from the group consisting of a proteasome inhibitor, TRAIL, and a cereblon modulator, a pyrimidoindole compound, and a FLT3 ligand. In particular, the human cell culture medium of this embodiment is more preferably a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, at least one of a proteasome inhibitor and TRAIL, a cereblon modulator, a pyrimidoindole compound, and a FLT3 ligand, and even more preferably a medium containing PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, a proteasome inhibitor, TRAIL, a cereblon modulator, a pyrimidoindole compound, and a FLT3 ligand.

[0058] The human cell culture medium of this embodiment can be produced by adding PCL-PVAc-polyalkylene glycol, a PI3K activator, a TPO receptor agonist, and at least one selected from the group consisting of a proteasome inhibitor, TRAIL, and a cereblon modulator to a basal medium containing the minimum nutrients necessary for the survival of hematopoietic stem cells, and, if necessary, further adding one or more selected from the group consisting of a pyrimidoindole compound and an FLT3 ligand. Examples of basal media used in preparing the human cell culture medium of this embodiment include IMDM (Iscove's Modified Dulbecco's Medium) medium, DMEM (Dulbecco's Modified Eagle Medium) medium, EMEM (Eagle's Minimal Essential Medium) medium, MEM (Minimum Essential Medium) medium, MEMα medium, Opti-MEM medium, RPMI-16 medium, and modified media thereof.

[0059] The human cell culture medium of this embodiment preferably contains various components typically contained in culture media for mammalian cells, to the extent that the effects of the present invention are not impaired. To enable more efficient proliferation of hematopoietic stem cells, the human cell culture medium of this embodiment preferably contains one or more components selected from the group consisting of insulin, transferrin (apo), sodium selenite, and ethanolamine, and more preferably contains all of these. Furthermore, the human cell culture medium of this embodiment may contain HEPES, sodium pyruvate, vitamins, amino acids, heparin, heparan sulfate, chondroitin sulfate, etc., and is also preferably a medium containing antibiotics such as penicillin and streptomycin.

[0060] The human cell culture medium of this embodiment is preferably an albumin-free medium (a medium that does not substantially contain albumin), such as a serum-free medium. Because the human cell culture medium of this embodiment contains PCL-PVAc-polyalkylene glycol, hematopoietic stem cells can grow efficiently even in an albumin-free medium.

[0061] The human cell culture medium of this embodiment is preferably a medium that is substantially free of SCF and TPO. Because it does not contain SCF or TPO, the proliferation of cancer cells and cells other than hematopoietic stem cells is suppressed in the human cell culture medium. Furthermore, since the human cell culture medium of this embodiment contains a PI3K activator, hematopoietic stem cells can be efficiently proliferated without SCF. Furthermore, since the human cell culture medium contains a TPO receptor agonist, hematopoietic stem cells can be efficiently proliferated without TPO. As a result, even if a cell population to be cultured contains cancer cells as well as hematopoietic stem cells, after culture, cells other than hematopoietic stem cells, including cancer cells, are selectively removed, resulting in a cell population with a high hematopoietic stem cell content ([number of hematopoietic stem cells in the cell population] / [total number of cells in the cell population] × 100%).

[0062] Granulocyte monocyte colony-stimulating factor (GM-CSF) is a cytokine that promotes differentiation of hematopoietic stem cells, and acts cooperatively with hematopoietic growth factors interleukin-3 (IL-3) and IL-5 to differentiate hematopoietic stem cells into myeloid progenitor cells. Therefore, the human cell culture medium of this embodiment preferably does not contain GM-CSF, and more preferably does not contain GM-CSF, IL-3, or IL-5. Because the human cell culture medium of this embodiment does not contain these cytokines, the undifferentiated state of hematopoietic stem cells can be maintained, and a cell population with a high percentage of functional hematopoietic stem cells can be obtained.

[0063] The human cell culture medium of this embodiment preferably contains substantially no cytokines other than a PI3K activator, a TPO receptor agonist, a proteasome inhibitor, TRAIL, a cereblon modulator, a pyrimidoindole compound, and a FLT3 ligand. Because the human cell culture medium is substantially free of cytokines, cells other than hematopoietic stem cells are selectively removed during culture and differentiation of hematopoietic stem cells is also suppressed, resulting in a cell population with a high percentage of functional hematopoietic stem cells.

[0064] The human cell culture medium of this embodiment is preferably a chemically defined medium, more preferably a chemically synthesized medium, which can prevent cytokines that promote proliferation of cells other than hematopoietic stem cells or differentiation of hematopoietic stem cells from unintentionally being mixed into the culture medium.

[0065] <Method for Culturing Human Hematopoietic Stem Cells> The method for culturing human hematopoietic stem cells of this embodiment is a method for culturing a cell population containing human hematopoietic stem cells in the human cell culture medium of this embodiment. The human cell culture medium of this embodiment is a culture medium optimized for the proliferation of human functional hematopoietic stem cells and is not suitable for the proliferation of other cells. Therefore, by culturing a cell population containing human hematopoietic stem cells using the human cell culture medium of this embodiment, human hematopoietic stem cells can be selectively proliferated while maintaining their functionality (undifferentiated state and differentiation potential). Therefore, the method for culturing human hematopoietic stem cells of this embodiment enriches human hematopoietic stem cells in the cell population, making the proportion of human hematopoietic stem cells in the cell population higher at the end of the culture than at the start of the culture. Furthermore, if the cell population contains cancer cells, the method for culturing human hematopoietic stem cells of this embodiment selectively removes the cancer cells from the cell population, making the proportion of cancer cells in the cell population lower at the end of the culture than at the start of the culture.

[0066] In the method for culturing human hematopoietic stem cells of this embodiment, the cells can be cultured under general conditions for culturing human hematopoietic stem cells or cell populations containing the same, except that the culture medium for human cells described above is used as the culture medium. For example, in the method for culturing human hematopoietic stem cells of this embodiment, the cells can be cultured under conditions generally used for culturing human hematopoietic stem cells or cell populations containing the same, such as 5% CO ２The cells can be cultured in an incubator controlled at 37°C under an ambient temperature. The culture time is not particularly limited, and can be, for example, 1 day or more, preferably 4 days or more, and more preferably 7 days or more. In the method for culturing human hematopoietic stem cells of this embodiment, by appropriately adjusting the culture time, the hematopoietic stem cells can be expanded 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. The upper limit of the culture time is not particularly limited, and can be, for example, 30 days or less, 25 days or less, 20 days or less, or 15 days or less. The method for culturing human hematopoietic stem cells of this embodiment allows for the production of a human hematopoietic stem cell population containing human hematopoietic stem cells and the human cell culture medium of this embodiment.

[0067] In the method for culturing human hematopoietic stem cells of this embodiment, the culture can be performed in the presence of fibronectin, i.e., under conditions that allow contact between the hematopoietic stem cells and fibronectin. For example, the culture can be performed using a culture vessel whose inside (e.g., bottom) is coated with fibronectin.

[0068] Human hematopoietic stem cells in the cell population, which is the culture obtained by the method for culturing human hematopoietic stem cells of this embodiment, can be detected and purified using cell surface markers of human hematopoietic stem cells, such as CD34, CD38, CD133, CD45RA, and CD49. ｆ Examples of such markers include CD34-positive, CD38-negative, CD133-positive, CD45RA-negative, CD49-negative, and CD133-positive human hematopoietic stem cells. These markers include CD34-positive, CD38-negative, CD133-positive, CD45RA-negative, and CD49-negative human hematopoietic stem cells. These markers are useful as cell surface markers for human hematopoietic stem cells cultured in vitro, since they are rarely expressed on the cell surface of human hematopoietic stem cells in vivo but are confirmed to be expressed on the cell surface of human hematopoietic stem cells cultured in vitro. Therefore, human hematopoietic stem cells cultured by the human hematopoietic stem cell culture method of this embodiment are CD34-positive, CD38-negative, CD133-positive, CD45RA-negative, and CD49-negative human hematopoietic stem cells. ｆThese cells are moderately positive and EPCR positive. Detection and purification using cell surface markers can be performed by immunological techniques using antibodies against cell surface markers, such as flow cytometry and immunostaining. For example, CD34 ＋ Fraction, CD34 ＋ CD38 － Fraction, CD34 ＋ CD38 － CD133 ＋ Fractionation and CD34 ＋ CD38 － CD45RA － CD49 ｆ ＋ By collecting the fraction, human hematopoietic stem cells can be purified.

[0069] By culturing PBMCs or a PBSC fraction prepared therefrom using the human hematopoietic stem cell culture method of this embodiment, human hematopoietic stem cells, which are contained in relatively small amounts in PBMCs, can be selectively expanded while maintaining their functionality. Therefore, the human hematopoietic stem cell culture method of this embodiment is particularly suitable for culturing cells that will serve as a transplant source for autologous hematopoietic stem cell transplantation. For example, a cell population obtained by culturing PBMCs collected from a donor cancer patient using the human hematopoietic stem cell culture method of this embodiment has a reduced number of cancer cells and an increased proportion of functional hematopoietic stem cells, which are essential for transplant engraftment. Therefore, transplanting the cell population obtained by this culture method can reduce the risk of cancer recurrence, improve transplant safety, and increase the transplant engraftment rate.

[0070] The PBMCs used in the method for culturing human hematopoietic stem cells of this embodiment are preferably collected from patients with hematologic malignancies, particularly patients with multiple myeloma. Among these, PBMCs collected from patients with hematologic malignancies who have been treated with chemotherapy, radiation therapy, or the like to reduce the number of tumor cells, and then whose hematopoietic stem cells have been mobilized with G-SCF Mozobil or the like are preferred.

[0071] Next, the present invention will be described in more detail by showing examples, etc., but the present invention is not limited to the following examples, etc., as long as it does not depart from the gist of the present invention. In other words, it will be understood by those skilled in the art that these examples are merely illustrative, that various modifications are possible, and that such modifications are also within the scope of the present invention.

[0072] <Culture Composition> Three types of culture media (3a medium, modified 3a medium, and modified 2a medium) were prepared by adding various additives to IMDM medium. These media consisted of IMDM (albumin-free medium), ITS-X (Gibco), PSG, the polymer "Solplus (registered trademark)" (BASF) represented by the general formula (1) (PCL-PVAc-PEG) in which n is 13, m is 30, and l is 57, the TPO receptor agonist butizamide (Shionogi & Co., Ltd.), the PI3K activator 740Y-P (Scrum), UM729 (Stem Cell Technologies, 72332), and recombinant human FLT3 ligand (Peprotech, AF-300-19). The compositions of both media are shown below.

[0073] (3a medium) IMDM 1% ITS-X 1% PSG 0.1% PCL-PVAc-PEG 0.1 μM butizamide 1 μM 740Y-P 1 μM UM729

[0074] (Modified 3a medium) IMDM 1% ITS-X 1% PSG 0.01% PCL-PVAc-PEG 0.2 μM butizamide 5 μM 740Y-P 1 μM UM729 10 ng / mL FLT3 ligand

[0075] (Modified 2a medium) IMDM 1% ITS-X 1% PSG 0.01% PCL-PVAc-PEG 0.2 μM butizamide 5 μM 740Y-P 10 ng / mL FLT3 ligand

[0076] Furthermore, two types of media (SSUM and SSSR) were prepared by adding various additives to StemSpanSFEM (ST-09600, manufactured by Stem Cell Technologies) containing a UM729 / SR-1-based medium. SSUM and SSSR consisted of StemSpan SFEM (albumin-containing medium), PSG, recombinant human SCF (Peprotech, AF-300-07), recombinant human TPO (Peprotech, AF-300-18), recombinant human FLT3 ligand (Peprotech, AF-300-19), low-density lipoprotein (Stem Cell Technologies, 02698), UM729 (Stem Cell Technologies, 72332), recombinant human IL-6 (Peprotech, AF-200-06), and SR-1 (Chemsheen, CS-1643). The compositions of both media are shown below.

[0077] (SSUM) Stemspan SFEM 1% PSG 100 ng / mL SCF 50 ng / mL TPO 100 ng / mL FLT3 ligand 10 μg / mL Low-density lipoprotein 0.5 μM UM729

[0078] (SSSR) StemspanSFEM 1% PSG 50ng / mL SCF 50ng / mL TPO 50ng / mL FLT3 ligand 50ng / mL IL-6 0.75μM SR-1

[0079] <Mouse> Immunodeficiency NOD / Shi-scid IL-2Rγ ｎｕｌｌ (NOG) mice were developed at the Central Institute for Experimental Animals. All mice were housed under specific pathogen-free conditions with free access to food and water. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Care and Use Committee of the Institute of Medical Science, University of Tokyo, the Laboratory Animal Resource Center, University of Tsukuba, and the Institutional Animal Care and Use Committee of the Central Institute for Experimental Animals.

[0080] <Cells> (MM.1S cells) MM.1S (CRL-2974) cells were purchased from ATCC (American Type Culture Collection) and cultured in RPMI-16 medium (30-2001, ATCC) containing 10% fetal bovine serum under 5% CO according to the manufacturer's instructions or other specified culture conditions. ２ The cells were maintained at 37°C in an environment containing MM.1S cells. For phenotypic analysis of MM.1S cells, the cells were stained with PECy7-labeled anti-human CD38 antibody (BioLegend, 303516) and PE-labeled anti-human CD138 antibody (BioLegend, 356504).

[0081] (Human peripheral blood stem cells (PBSCs)) Fresh human peripheral blood stem cells (PBSCs) were obtained from healthy adult donors for allogeneic transplantation at Tsukuba University Hospital (Ibaraki Prefecture). All donors agreed to the experimental use of PBSCs after informed consent, and our study was approved by the University of Tsukuba Ethics Committee. Mononuclear cells (MCs) were isolated from the total cells after leukocyte removal using Lymphocyte Separation Medium 1077 (PromoCell, C-44010). After isolation, CD34 ＋ The cells were enriched using a human CD34 Microbead Kit UltraPure (Milteny Biotech, 130-100-453) and a MACS LS column (Milteny Biotech, 130-042-401). ＋ Cells were cultured in prewarmed modified 3a medium or StemSpan SFEM bead medium. For phenotypic analysis of cultured PBSCs, cells were stained with FITC-conjugated anti-human CD34 antibody (BioLegend, 343504) and APC-conjugated anti-human CD201 (EPCR) antibody (BD Biosciences, 563266). For more detailed analysis, cells were stained with PE-Cy7-conjugated anti-human CD90 antibody (BioLegend, 329124), APC-Cy7-conjugated anti-human CD45RA antibody (BioLegend, 304128), and PE-conjugated anti-human CD49 antibody (BD Biosciences, 563266). ｆ The cells were also stained with (ITGA3) antibody (BD Biosciences, 556025).

[0082] (Human umbilical cord blood stem cells (CDSCs)) Human umbilical cord blood stem cells (CDSCs) were obtained from StemExpress, Inc. and were derived from human umbilical cord blood CD34 ＋ Cells (CBP3400.5C) were used.

[0083] <Cell Culture> Unless otherwise specified, cell culture was performed as follows: 1 × 10 ４ The cells were seeded onto a Costar 48-well plate (Corning, 3548) and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator.

[0084] <Measurement of Cell Viability> The cell viability was measured using a commercially available measurement kit ("Annexin V Conjugates for Apoptosis Detection," A13201, manufactured by Invitrogen) that uses annexin V and propidium iodide (PI). Specifically, the cell suspension after culture was first centrifuged, and the collected cell precipitate was washed with PBS(-). The cell precipitate was then added to annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl diluted with distilled water). ２ ) was added. The cells were then stained with AlexaFluor488-labeled annexin V (Invitrogen, A13201) and propidium iodide (BioLegend, 421301) and incubated at room temperature for 15 minutes. Finally, the cell samples were resuspended in annexin binding buffer and analyzed using an Attune flow cytometer (Invitrogen). Cells that were both annexin V and PI negative were defined as viable cells.

[0085] Example 1 MM.1S cells and PBSCs were cultured for one week in culture medium supplemented with 0-10 μM bortezomib (S1013, Selleck) or 0-10 μM carfilzomib (S2853, Selleck), both of which are FDA-approved therapeutic agents for multiple myeloma (MM), and the viability was examined. Bortezomib and carfilzomib were added to the culture medium as solutions dissolved in DMSO. Modified 3a medium was used as the culture medium.

[0086] The results of measuring the cell viability (%) of MM.1S cells are shown in Figure 1(A), and the results of measuring the cell viability (%) of PBSCs are shown in Figure 1(B). In the figure, "Bor" and "Car" indicate the results for cells cultured in medium supplemented with bortezomib and carfilzomib, respectively (numbers in parentheses indicate concentrations (μM)). In the figure, "DMSO" indicates the results for cells cultured in medium supplemented with DMSO only. As a result, as shown in Figure 1(A), a concentration-dependent decrease in the viability of MM.1S cells was observed with both bortezomib and carfilzomib. At concentrations of 1.0 μM or higher for bortezomib and 0.1 μM or higher for carfilzomib, the viability was significantly reduced compared to the control (cells in DMSO-supplemented medium). On the other hand, as shown in Figure 1(B), when human PBSCs (PBMC-derived CD34-positive cells) were cultured under the same culture conditions, a concentration-dependent decrease in PBSC viability was observed with both bortezomib and carfilzomib. Furthermore, at concentrations of 5.0 μM or higher for bortezomib and 0.5 μM or higher for carfilzomib, viability was significantly reduced compared to the control. The difference in viability between MM.1S cells and PBSCs indicated that culture conditions with 2.0 μM bortezomib or 0.2 μM carfilzomib were sufficient to selectively remove only MM.1S cells.

[0087] Under the same culture conditions, bortezomib reduced the viability of MM.1S cells more than carfilzomib (Fig. 1(A) and (B)). Under conditions that maintained the viability of PBSCs, bortezomib had a greater effect on reducing the viability of MM.1S cells than carfilzomib. Therefore, human PBSCs cultured for one week in modified 3a medium supplemented with bortezomib at a concentration of 0.5-10 μM were analyzed by flow cytometry for the expression of cell surface markers. CD34 ＋ EPCR ＋ CD90 ＋ CD45RA ― ITGA3 ＋The cells were fractionated as human HSCs. The relative cell numbers of the human HSC fraction in the cell populations cultured in each culture medium ([cell number of the human HSC fraction] / [cell number of the human HSC fraction in the control (cell population cultured in DMSO-supplemented medium)] × 1.0) were calculated, and the results are shown in Figure 2. The survival rate of the human HSC fraction cells was extremely poor at bortezomib concentrations of 5.0 μM or higher (Figure 2), suggesting that 2.0 μM is the threshold value used for myeloma cell elimination.

[0088] Next, MM.1S cells were cultured for one week in SSUM, SSSR, modified 2a medium, and modified 3a medium supplemented with 2.0 μM bortezomib or 0.2 μM carfilzomib, respectively, or in medium without either bortezomib or carfilzomib, and viability was examined. The results of measuring the viability (%) of cells cultured in each culture medium are shown in Figure 3. In the case of modified 3a medium ("M3a" in the figure), the addition of bortezomib or carfilzomib reduced the viability of MM.1S cells. In contrast, in the case of SSUM and SSSR, the addition of either bortezomib or carfilzomib did not reduce the viability of MM.1S cells (Figure 3). These results suggest that when SSUM and SSSR were used, the addition of anticancer drugs used to treat MM did not eliminate cancer cells during ex vivo culture, suggesting that the selective elimination of cancer cells by the addition of bortezomib and carfilzomib is an effect achieved in albumin-free media such as modified 3a medium. Furthermore, when MM.1S cells were cultured in modified 2a medium (M2a in the figure), which is modified 3a medium minus UM729, the survival rate was higher than when cultured in modified 3a medium (Figure 3). This suggests that UM729 also contributes to the selective elimination of cancer cells.

[0089] Comparative Example 1: Panobinostat, a histone deacetylase (HDAC) inhibitor approved by the FDA for the treatment of multiple myeloma, has been reported to contribute to the expansion of cultured HSCs at 5 nM as an alternative reagent to UM171 in StemSpan culture medium (Chagraoui et al., Cell Stem Cell, 2021, vol. 28, pp. 48-62). Therefore, MM.1S cells and PBSCs were cultured for 1 week in modified 2a medium or modified 3a medium supplemented with panobinostat (S1030, Selleck) at 0-20 nM, as in Example 1, and their viability was examined.

[0090] The results of measuring the cell viability (%) of MM.1S cells are shown in Figure 4(A), and the results of measuring the cell viability (%) of PBSCs are shown in Figure 4(B). In the figure, "Pano" indicates the results for cells cultured in medium supplemented with panobinostat (numbers in parentheses indicate concentrations (nM)). "DMSO" indicates the results for cells cultured in medium supplemented with DMSO only. As a result, when panobinostat was added to modified 2a medium, a concentration-dependent decrease in the viability of MM.1S cells was observed (Figure 4(A)). The viability of PBSCs was maintained at a constant level or higher in both modified 2a medium and modified 3a medium, regardless of the panobinostat concentration (Figure 4(B)).

[0091] Furthermore, human PBSCs cultured for one week in modified 2a medium and modified 3a medium supplemented with panobinostat at concentrations of 0 to 10 nM were analyzed by flow cytometry for the expression of cell surface markers, and CD34 ＋ EPCR ＋The cells were fractionated as human HSCs. The relative cell number of the human HSC fraction in the cell population cultured in each culture medium ([cell number of human HSC fraction] / [cell number of human HSC fraction in the control (cell population cultured in DMSO-supplemented medium)] × 1.0) was calculated ( Figure 4(C) ). As shown in Figure 4(C) , when cultured in modified 2a medium, very few human HSC fraction cells survived, and the addition of panobinostat did not change this. On the other hand, when cultured in modified 3a medium containing UM729, although some human HSC fraction cells survived, the addition of panobinostat significantly reduced their survival compared to the absence of panobinostat. These results confirmed that the use of panobinostat in ex vivo culture of human hematopoietic stem cells does not result in the selective elimination of cancer cells.

[0092] Example 2: In the treatment of multiple myeloma, cereblon modulators lenalidomide and pomalidomide are administered in combination with bortezomib. We therefore investigated whether cereblon modulators contribute to the efficiency of bortezomib-mediated selective removal of cancer cells.

[0093] Specifically, 1 x 10 ４ MM.1S cells and 1 × 10 ４ PB-derived CD34 ＋ The cells were seeded onto Costar 48-well plates (Corning, 3548) and cultured for one week in modified 3a medium containing bortezomib, lenalidomide (S1029, Selleck), and pomalidomide (S1567, Selleck) at the concentrations listed in Tables 1 to 4. The cells were stained with PE-Cy7-labeled anti-CD38 antibody, PE-labeled anti-CD138 antibody, FITC-labeled anti-CD34 antibody, and APC-labeled anti-CD201 antibody and analyzed using an Attune flow cytometer. The relative viability of each cell type ([cell viability (%)] / [cell viability (%) of the control (cell population cultured in DMSO-supplemented medium)]) was calculated, and the results are shown in Tables 1 to 4.

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[0098] As shown in Tables 1 and 3, even without the addition of bortezomib (0 μM), the viability of MM.1S cells was reduced by adding lenalidomide or pomalidomide to the culture medium, confirming that cereblon modulators also have the effect of eliminating cancer cells. On the other hand, the viability of MM.1S cells was not significantly different when bortezomib was used in combination with lenalidomide or pomalidomide compared to bortezomib alone, indicating that the cancer cell elimination effect was primarily due to the proteasome inhibitor, and the combination of a proteasome inhibitor with a cereblon modulator did not have much of an effect.

[0099] As shown in Tables 2 and 4, PB-derived human hematopoietic stem cells (PB-derived CD34 ＋ The viability of PB-derived human hematopoietic stem cells (PB cells) was significantly increased by adding lenalidomide or pomalidomide to the medium, confirming that the expansion effect of cereblon modulators on human hematopoietic stem cells is also achieved in albumin-free media such as modified 3a medium. Furthermore, the expansion effect of cereblon modulators on human hematopoietic stem cells was also fully achieved in media containing bortezomib, suggesting that cereblon modulators reduce damage to hematopoietic stem cells caused by proteasome inhibitors. The improvement in the viability of PB-derived human hematopoietic stem cells was dependent on the concentration of cereblon modulator, with the combination of 2 μM bortezomib and 1 μM lenalidomide most effectively expanding human hematopoietic stem cells. As shown in Tables 3 and 4, the combination of bortezomib and pomalidomide yielded similar results to the combination of bortezomib and lenalidomide, with the combination of 2 μM bortezomib and 1 μM pomalidomide most effectively expanding human hematopoietic stem cells.

[0100] [Example 3] In Example 2, it was found that the effects of proteasome inhibitors on hematopoietic stem cells can be reduced by cereblon modulators, and therefore the effects of various cereblon modulators were investigated.

[0101] Specifically, 1 x 10 ４ PB-derived CD34 ＋ The cells were seeded on Costar 48-well plates (Corning, 3548) and cultured for 7 days in modified 3a medium containing 0-10 μM avadomide (Selleck, S7892), iverdomide (Selleck, S7892), lenalidomide (Selleck, S1029), and pomalidomide (Selleck, S1567). The cells were incubated with FITC-labeled anti-human CD34 antibody, APC-labeled anti-human CD201 antibody, PE-Cy7-labeled anti-human CD90 antibody, APC-Cy7-labeled anti-human CD45RA antibody, and PE-labeled anti-human CD49 antibody. ｆ The cells were stained with , and analyzed for the expression of cell surface markers using an Attune flow cytometer. ＋ EPCR ＋ CD90 ＋ CD45RA － ITGA3 ＋ The cells were fractionated as human HSCs. The relative cell number of the human HSC fraction in the cell population cultured in each culture medium was calculated ([cell number of the human HSC fraction] / [cell number of the human HSC fraction in the control (cell population cultured in DMSO-supplemented medium)] × 1.0). The results are shown in Figure 5.

[0102] As shown in Figure 5, the expansion efficiency of human HSCs varied depending on the compound, but all cereblon modulators were confirmed to improve human HSC expansion. The relative cell numbers of the human HSC fraction for 100 nM avadomide and 100 nM to 1 μM lenalidomide were 2.23 ± 0.22 and 2.34 ± 0.21, respectively, showing the highest expansion rates.

[0103] [Example 4] Modified 3a medium, modified 3a medium supplemented with 2 μM bortezomib and 1 μM lenalidomide, SSUM, and SSSR were used to differentiate MM.1S cells and PB-derived CD34 ＋ The cells were co-cultured and the time course of cell viability was examined.

[0104] Specifically, 1 x 10 ４ MM.1S cells and 1 x 10 ４PB-derived CD34 ＋ Cells were seeded onto Costar 48-well plates (Corning, 3548) and cultured for 14 days in modified 3a medium, modified 3a medium containing 2 μM bortezomib and 1 μM lenalidomide (BL-containing modified 3a medium), SSUM, or SSSR. The cells were stained with PE-Cy7-labeled anti-CD38 antibody, PE-labeled anti-CD138 antibody, FITC-labeled anti-CD34 antibody, and APC-labeled anti-CD201 antibody and analyzed using an Attune flow cytometer on days 1, 4, 7, 10, and 14 of culture.

[0105] The percentage (%) of live MM.1S cells relative to the total live cells cultured in each culture medium was measured over time. The results are shown in Figure 6(A). ＋ EPCR ＋ Figure 6(B) shows the results of measuring the percentage of viable cells over time. After day 7 of culture, the percentage of MM.1S cells cultured in modified 3a medium ("M3a" in the figure) and in BL-containing modified 3a medium ("M3a+BL" in the figure) was lower than that of cells cultured in SSUM or SSSR. Although BL-containing modified 3a medium more efficiently removed MM.1S cells than modified 3a medium, the expansion efficiency of human HSPCs was lower (Tables 1 and 2). Therefore, on days 10 and 14 after the start of culture, the percentage of MM.1S cells was higher in BL-containing modified 3a medium than in modified 3a medium, but there was no significant difference on day 7 after the start of culture (Figure 6(A)). These results suggest that 7 days is the optimal culture period for cancer cell removal in BL-containing modified 3a medium. On the other hand, CD34 ＋ EPCR ＋ The cells appeared after the fourth day of culture, but CD34 ＋ EPCR ＋ The percentage of viable cells remained highest throughout the entire period in culture in BL-containing modified 3a medium (FIG. 6(B)).

[0106] Also, 1 x 10 ４ PB-derived CD34 ＋The cells were seeded on a Costar 48-well plate (Corning, 3548) and cultured for 14 days in modified 3a medium supplemented with DMSO alone, modified 3a medium containing 2 μM bortezomib alone (modified 3a medium containing B), modified 3a medium containing 2 μM bortezomib and 1 μM lenalidomide (modified 3a medium containing BL(1)), or modified 3a medium containing 2 μM bortezomib and 1 μM lenalidomide (modified 3a medium containing BL(10)). The cells were incubated with FITC-labeled anti-human CD34 antibody, APC-labeled anti-human CD201 antibody, PE-Cy7-labeled anti-human CD90 antibody, APC-Cy7-labeled anti-human CD45RA antibody, and PE-labeled anti-human CD49 antibody. ｆ The cells were stained with an antibody against CD34 (ITGA3) and analyzed using an Attune flow cytometer on days 4, 7, 10, and 14 of culture. ＋ EPCR ＋ CD90 ＋ CD45RA － ITGA3 ＋ The cells were fractionated as human HSCs. The relative cell number of the human HSC fraction in the cell population cultured in each culture medium ([cell number of the human HSC fraction] / [cell number of the human HSC fraction in the control (cell population cultured in DMSO-supplemented medium)] × 1.0) was calculated. The results are shown in Figure 7. As a result, throughout the entire period from day 4 to day 14 after the start of culture, both the BL(1)-containing modified 3a medium and the BL(10)-containing modified 3a medium containing bortezomib and lenalidomide showed a higher number of viable human HSC cells than the control or B-containing modified 3a medium.

[0107] [Example 5] The results of Example 4 confirmed that the use of a culture medium containing modified 3a medium in combination with bortezomib and lenalidomide makes it possible to efficiently remove myeloma cells and maintain or expand HSCs. Therefore, to further enhance the efficiency of myeloma cell removal, we focused on TRAIL. It has been reported that myeloma cells express TRAIL receptors, but HSPCs do not. Indeed, expression of CD261 and CD262 was not confirmed in HSPCs cultured for one week in modified 3a medium, but expression of either CD261 or CD262 was observed in more than 70% of MM.1S cells.

[0108] So, 1 x 10４ MM.1S cells were seeded onto Costar 48-well plates (Corning, 3548) and cultured for 7 days in modified 3a medium containing 0 or 2 μM bortezomib and 0, 100, 200, or 500 ng / mL TRAIL to determine viability. MM.1S cells were also cultured in SSUM containing 0 or 2 μM bortezomib and 0, 100, 200, or 500 ng / mL TRAIL to determine viability.

[0109] The results of measuring the cell viability (%) when cultured in modified 3a medium are shown in Figure 8(A), and the results of measuring the cell viability (%) when cultured in SSUM are shown in Figure 8(B). In the figure, "M3a-B0-T0" to "M3a-B0-T500" represent modified 3a medium supplemented with 0 to 500 ng / mL of TRAIL, and "M3a-B2-T0" to "M3a-B2-T500" represent modified 3a medium supplemented with 2 μM bortezomib and 0 to 500 ng / mL of TRAIL. "SSUM-B0-T0" to "SSUM-B0-T500" represent media containing SSUM supplemented with 0 to 500 ng / mL TRAIL, while "SSUM-B2-T0" to "SSUM-B2-T500" represent media containing SSUM supplemented with 2 μM bortezomib and 0 to 500 ng / mL TRAIL. As shown in Figures 8(A) and 8(B), the addition of TRAIL reduced the viability of MM.1S cells in both modified 3a medium and SSUM, regardless of the presence or absence of bortezomib. On the other hand, in SSUM medium, the viability of MM.1S cells did not reach 0%, even with the addition of TRAIL (Figure 8(B)). In contrast, in modified 3a medium, the combined use of 2 μM bortezomib and 100 ng / mL or more of TRAIL resulted in a 0% survival rate of MM.1S cells, and MM.1S cells were completely eradicated (Figure 8(A)).

[0110] These results suggest that the culture medium containing 2 μM bortezomib, 1 μM lenalidomide, and 100 ng / mL TRAIL in modified 3a medium is the optimal culture condition for myeloma cell elimination in PBSC donor grafts, in terms of the efficiency of cancer cell elimination and the proliferation of human hematopoietic stem cells.

Claims

1. A culture medium for culturing a cell population containing human cells, comprising: a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block; a phosphatidylinositol 3-kinase (PI3K) activator; a TPO receptor agonist; and one or more selected from the group consisting of a proteasome inhibitor, a TNF-related apoptosis-inducing ligand, and a cereblon modulator.

2. The culture medium for human cells according to claim 1, wherein the proteasome inhibitor is one or more selected from the group consisting of bortezomib and carfilzomib.

3. The human cell culture medium of claim 1, wherein the cereblon modulator is one or more selected from the group consisting of lenalidomide, pomalidomide, avadomide, iberdomide mezignomide, and thalidomide.

4. The culture medium for human cells according to claim 1, wherein the culture medium further comprises a pyrimidoindole compound.

5. The culture medium for human cells according to claim 4, wherein the pyrimidoindole compound is one or more selected from the group consisting of UM171 and UM729.

6. The culture medium for human cells according to claim 1, wherein the culture medium further contains an FLT3 ligand.

7. The culture medium for human cells according to claim 1, wherein the culture medium is a serum-free medium.

8. The culture medium for human cells of claim 1, wherein the culture medium is substantially free of albumin.

9. A method for culturing human hematopoietic stem cells, comprising culturing a cell population containing human hematopoietic stem cells in a human cell culture medium according to any one of claims 1 to 8.

10. The method for culturing human hematopoietic stem cells according to claim 9, wherein the cell population is cultured in the culture medium for 7 days or more.

11. The method for culturing human hematopoietic stem cells according to claim 9, wherein the cell population is PBMC.

12. The method for culturing human hematopoietic stem cells according to claim 9, wherein the cell population is a cell population derived from a cancer patient.

13. A method for culturing human hematopoietic stem cells as described in claim 9, wherein the proportion of cancer cells in the cell population is lower at the end of the culture than at the start of the culture.

14. The method for culturing human hematopoietic stem cells according to claim 9, wherein the proportion of human hematopoietic stem cells in the cell population is higher at the end of the culture than at the start of the culture.

15. A human hematopoietic stem cell population obtained by culturing a cell population collected from a human in a human cell culture medium according to any one of claims 1 to 8.

16. A human hematopoietic stem cell population comprising human hematopoietic stem cells and a culture medium for human cells according to any one of claims 1 to 8.

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