Cell culture

Abstract

To provide a method for efficiently obtain a hematopoietic stem cell.SOLUTION: A composition contains (a) PI3K activator or TPO receptor agonist, and (b) methyl 4-(3-piperidine-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate or a salt thereof or Tranylcypromine or a salt thereof. A method for producing cells including a culturing process of culturing cells by using the composition may be used. The composition may be an albumin-free culture medium.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The technical field of the present invention relates to cell culture. [Background technology]

[0002] Hematopoietic stem cells are stem cells that can differentiate into blood cells. Transplantation of hematopoietic stem cells is known to be a treatment for diseases such as cancer and immunodeficiency.

[0003] Human umbilical cord blood (CB) is known as a source of hematopoietic stem cells. For example, Patent Document 1 describes a method for obtaining hematopoietic stem cells from human CB. Specifically, human CB CD34 + From the starting population of cells, EPCR + Selection and culturing of populations of cells is described. EPCR is a hematopoietic stem cell marker, also known as CD201.

[0004] In addition, methods for culturing hematopoietic stem cells have been studied. In general, in culturing hematopoietic stem cells, albumin-containing media have been used to induce cell division. However, albumin has problems such as the inhibition of stable undifferentiation and biological contamination. Therefore, research on albumin-free culture methods has been conducted. For example, Patent Document 2 describes the culturing of human hematopoietic stem cells by adding PVA or Soluplus (registered trademark) instead of albumin. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2017 / 205977 [Patent Document 2] WO2021 / 149799 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventional methods were not sufficient to efficiently obtain human hematopoietic stem cells. In addition, there were few reports on albumin-free culture methods for human hematopoietic stem cells, and available media were limited. [Means for solving the problem]

[0007] In the course of our research, we have investigated a method for culturing hematopoietic stem cells. As a result, we have found that human CB CD34 cells are cultured in an albumin-free medium with a specific composition different from conventional methods. + By culturing the cells, hematopoietic stem cells were successfully proliferated. Furthermore, the obtained culture fraction expressed CD201 + or CD90 + (all of which are hematopoietic stem cell markers) were enriched in the culture fraction. The present inventors completed the present invention based on this finding.

[0008] According to one aspect of the present invention, there is provided a composition comprising (a) a PI3K activator or a TPO receptor agonist, and (b) methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate (hereinafter, also referred to as UM729) or a salt thereof, or tranylcypromine (hereinafter, also referred to as 2-PCPA) or a salt thereof. The composition can be used to inhibit CD201 + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0009] According to one aspect of the present invention, there is provided a method for producing cells, comprising a culture step of culturing cells using the above composition. + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of measuring the cell yield of CD201+CD45RA-CD34+CD38-Lin- cells after 10 or 17 days of culture in medium A (UM729). [Diagram 2] FIG. 1 shows the results of measuring the cell yield of CD201+CD45RA-CD34+CD38-Lin- cells after 10 or 17 days of culture in medium B (UM729). [Diagram 3] FIG. 1 shows the results of measuring the proliferation rate (total cells) of cells after 10 days of culture in medium B (UM729), medium C (UM171), or medium D (UM171 (SFEM)). [Figure 4] FIG. 13 shows the results of measuring the proliferation rate (CD34+ cells) of cells after 10 days of culture in medium B (UM729), medium C (UM171), or medium D (UM171 (SFEM)). [Diagram 5] FIG. 1 shows the results of expression analysis of CD34 and CD201 in cells cultured in medium A (UM729) for 10 days. [Figure 6] 1 shows the results of expression analysis of CD41, CD45RA, CD201, and CD90 in cells cultured in medium A (UM729) for 10 days. CD34+Lin- cells were analyzed. [Figure 7] FIG. 1 shows the results of CD34 and Lin expression analysis of cells cultured in medium B (UM729) for 10 days. [Figure 8] 1 shows the results of expression analysis of CD41, CD45RA, CD201, and CD90 in cells cultured for 10 days in medium B (UM729). CD34+Lin- cells were analyzed. [Figure 9] FIG. 1 shows the results of CD34 and Lin expression analysis of cells cultured in medium C (UM171) for 10 days. [Figure 10] 1 shows the results of expression analysis of CD41, CD45RA, CD201, and CD90 in cells cultured in medium C (UM171) for 10 days. CD34+Lin- cells were analyzed. [Figure 11] FIG. 1 shows the results of CD34 and Lin expression analysis of cells cultured in medium D (UM171 (SFEM)) for 10 days. [Figure 12]1 shows the results of expression analysis of CD41, CD45RA, CD201, and CD90 in cells cultured in medium D (UM171 (SFEM)) for 10 days. CD34+Lin- cells were analyzed. [Figure 13] FIG. 1 shows the results of expression analysis of CD34 and CD201 in control cells before culture. [Figure 14] FIG. 1 shows the results of expression analysis of CD34 and CD201 in cells cultured in medium B (UM729) for 10 days. [Figure 15] FIG. 1 shows the results of expression analysis of CD34 and CD201 in cells cultured in medium D (UM171 (SFEM)) for 10 days. [Figure 16] FIG. 1 shows the results of expression analysis of CD34 and CD201 in cells cultured in medium E (2-PCPA) for 10 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiments of the present invention will be described in detail. Note that, in order to avoid repetition and complexity, the description of similar contents will be omitted as appropriate.

[0012] (1) Composition According to one embodiment of the present invention, there is provided a composition comprising (a) a PI3K activator or a TPO receptor agonist, and (b) methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate (hereinafter, also referred to as UM729) or a salt thereof, or tranylcypromine (hereinafter, also referred to as 2-PCPA) or a salt thereof. Here, the composition may be a culture medium. The composition can be used to inhibit CD201 + or CD90 + For example, in the Examples described below, this composition can be used to efficiently produce a culture fraction rich in human CB CD34 cells. + As a result of cell culture, CD201 + Cells or CD90 +It is described that a culture fraction enriched in cells was obtained. In addition, according to one embodiment of the present invention, a method for producing cells, a method for culturing cells, a method for producing a medium, and a method for culturing CD201 using the composition are disclosed. + or CD90 + A method for concentrating cells of the present invention and a container containing the composition are provided.

[0013] (2) Culture medium According to one embodiment of the present invention, there is provided a medium comprising (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0014] (3) Method According to one embodiment of the present invention, there is provided a method comprising contacting a cell with a composition comprising (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. The method can be, for example, a method for producing a cell, a method for culturing a cell, or a method for detecting CD201 + or CD90 + The present invention also includes a method for concentrating cells.

[0015] (4) Production method According to one embodiment of the present invention, there is provided a method for producing cells, comprising a culture step of culturing cells using a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof. + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0016] (5) Production method According to one embodiment of the present invention, a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof is used to induce CB CD34 +A method for producing cells is provided, which includes a step of culturing the cells. + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0017] (6) Production method According to one embodiment of the present invention, CD201 is induced using a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. + or CD90 + The present invention provides a method for producing cells, the method comprising the step of culturing cells of the present invention. + or CD90 + The cells can be grown well.

[0018] (7) Production method According to one embodiment of the present invention, there is provided a method for producing cells, comprising a culture step of culturing human hematopoietic stem cells in a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof. Using this method, human hematopoietic stem cells can be successfully proliferated.

[0019] (8) Production method According to one embodiment of the present invention, there is provided a method for producing a medium, comprising the step of mixing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof. The medium obtained by this method can be used to detect CD201 + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0020] (9) Production method According to one embodiment of the present invention, a composition containing a population of cells obtained by any one of the methods described in (3) to (8) above is used to detect hematopoietic stem cell markers. + and a hematopoietic stem cell marker after the cell separation. +A step of culturing the cells of the present invention or a hematopoietic stem cell marker after the culturing + The present invention provides a method for producing a cell population, comprising the step of recovering the cells of the present invention. By using this method, it is possible to further enrich for hematopoietic stem cells or recover a larger amount of hematopoietic stem cells.

[0021] (10) Culture method According to one embodiment of the present invention, there is provided a method for culturing cells, comprising a culture step of culturing cells in a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof. + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0022] (11) Concentration method According to one embodiment of the present invention, a method for detecting CD201, comprising culturing cells in a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof, is provided. + or CD90 + The present invention provides a method for enriching cells containing CD201 + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0023] (12) Concentration method According to one embodiment of the present invention, there is provided a method for enriching hematopoietic stem cells, comprising a culture step of culturing cells in a medium containing (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof. By using this method, a culture fraction rich in hematopoietic stem cells can be efficiently produced.

[0024] (13) Container According to one embodiment of the present invention, there is provided a container containing a composition comprising (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. Using this container, CD201 + or CD90 + This makes it possible to efficiently produce a culture fraction containing a large number of cells of the abovementioned type.

[0025] (14) Container According to one embodiment of the present invention, there is provided a container containing cells or a cell population thereof obtained by the method according to any one of (3) to (12) above. This container can be used for hematopoietic stem cell transplantation. Prior to hematopoietic stem cell transplantation, desired cells (e.g., hematopoietic stem cell markers) can be isolated from the cell population. + The method may also include a step of separating the cells (the cells), a step of culturing the separated cells, a step of recovering the cultured cells, or a step of retaining the recovered cells in the container.

[0026] (15) Cell According to one embodiment of the present invention, there is provided a cell or a cell population thereof obtained by the method according to any one of (3) to (12) above. The cell or the cell population thereof can be used for hematopoietic stem cell transplantation. Prior to hematopoietic stem cell transplantation, a desired cell (e.g., a hematopoietic stem cell marker) can be isolated from the cell population. + The method may also include a step of separating the cells (the cells), a step of culturing the separated cells, a step of recovering the cultured cells, or a step of retaining the recovered cells in the container.

[0027] (16) Cell According to one embodiment of the present invention, CD201 + or CD90 + The present invention provides a cell population comprising 5% or more of the above-mentioned cells. This cell population can be used for the production of a pharmaceutical composition for hematopoietic stem cell transplantation.

[0028] (17) Culture medium According to one embodiment of the present invention, CD201 on total cells in culture medium + or CD90 +The present invention provides a cell-containing medium having a cell ratio of 5% or more. The medium can be used for producing a pharmaceutical composition for hematopoietic stem cell transplantation.

[0029] (18) Composition According to one embodiment of the present invention, there is provided a composition for use in any of the methods described in any of (3) to (12) above, comprising (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof, or 2-PCPA or a salt thereof.

[0030] (19) Composition According to one embodiment of the present invention, there is provided a composition comprising the cell or cell population thereof according to (15) or (16) above, (a) a PI3K activator or a TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. + or CD90 + Since it contains a large amount of these cells, it is useful for producing a pharmaceutical composition for use in hematopoietic stem cell transplantation.

[0031] (20) Composition According to one embodiment of the present invention, there is provided a pharmaceutical composition for use in hematopoietic stem cell transplantation, comprising cells or a cell population thereof obtained by the method described in any one of (3) to (12) above, or a method for producing the same. This production method may include, for example, a step of purifying hematopoietic stem cells from the cell population described in (15) or (16) above. This production method may include, for example, a step of isolating hematopoietic stem cell markers from the composition described in (19) above. + and a hematopoietic stem cell marker after the cell separation. + A step of culturing the cells of the present invention or a hematopoietic stem cell marker after the culturing +From another aspect, according to one embodiment of the present invention, there is provided a cell or cell population according to (15) or (16) above, or a pharmaceutical composition comprising said cell or cell population, for use in hematopoietic stem cell transplantation. From another aspect, according to one embodiment of the present invention, there is provided use of the cell or cell population according to (15) or (16) above in the production of a pharmaceutical composition for hematopoietic stem cell transplantation.

[0032] (21) Transplant method According to one embodiment of the present invention, there is provided a transplantation method. This transplantation method may include, for example, a step of transplanting the cells or cell population described in (15) or (16) above, or a pharmaceutical composition containing the cells or cell population, into a subject. According to this transplantation method, a disease can be treated by effectively engrafting hematopoietic stem cells.

[0033] In the embodiments of the present invention (including, for example, the above (3) to (12) or (21)), the method may include any one or more of the following steps (i) to (v): (i) prior to culturing, placing in a container or holding in a container a composition comprising (a) a PI3K activator, (b) a TPO receptor agonist, and (c) UM729 or a salt thereof, or 2-PCPA or a salt thereof, (ii) contacting the cells with the composition in the container, (iii) culturing the cells in the container, (iv) detecting CD201 + or CD90 + or (v) generating a cell population enriched for cells of CD201 relative to the total cells in the culture medium. + or CD90 + The method according to the embodiment of the present invention (including, for example, the above (3) to (12) or (21)) may include any one or more of the following steps (vi) to (xx): (vi) preparing a cell population containing hematopoietic stem cells; (vii) preparing a CB CD34 cell population containing CB CD34 + (viii) obtaining a cell population comprising CD34 cells; and + (ix) isolating the CD34 cells after sorting; +(x) prior to the culturing, mixing (a) a PI3K activator, (b) a TPO receptor agonist, and (c) UM729 or a salt thereof or 2-PCPA or a salt thereof to produce a composition containing the mixed components; (xi) incubating the cells in a culture medium; (xii) seeding the cells in a medium in a container and incubating them; (xiii) isolating CD201 from the cell population; + or CD90 + (xiv) isolating or recovering hematopoietic stem cells from the cell population; (xv) isolating or recovering CD201 + or CD90 + (xvi) culturing the hematopoietic stem cells after sorting or recovery; (xvii) culturing the CD201 cells after sorting or recovery and culture; + or CD90 + (xviii) recovering the hematopoietic stem cells after sorting or recovery and culture; (xix) recovering the CD201 cells after recovery. + or CD90 + (xx) storing (e.g., cryopreserving) the cells; or (xx) storing the CD201 cells after collection. + or CD90 + Transplanting cells of the invention into a subject. + or CD90 + This is useful for efficiently producing a culture fraction rich in cells of the present invention. When two or more steps from the embodiments of the present invention (e.g., the above methods (3) to (12) and (21), or steps (i) to (xx)) are employed, the order may be arbitrary and the order may be determined according to the desired operation. Each method or step of the embodiments of the present invention (e.g., the above methods (3) to (12) and (21), or steps (i) to (xx)) may be carried out in vitro or ex vivo.

[0034] In the embodiments of the present invention (including, for example, the above (1) to (21)), CD201 is a protein known as a hematopoietic stem cell marker (for example, WO2017 / 205977 describes human CB CD34 as a marker for CD201). +(It is described that hematopoietic stem cells were obtained from the cells.) CD201 includes a protein having an amino acid sequence of a protein represented by NCBI RefSeq Accession number NP_006395.2 or UniProt ID Q9UNN8. CD201 is also referred to as cluster of differentiation 201 or EPCR.

[0035] In an embodiment of the present invention (including, for example, the above (1) to (21)), CD90 is a protein known as a hematopoietic stem cell marker (for example, Rix et al., Front Physiol. 2022 Sep 30; 13: 1009160 describes CD90 as a hematopoietic stem cell marker). CD90 includes a protein having an amino acid sequence of a protein represented by NCBI RefSeq Accession number NP_001298089.1 or UniProt ID P04216. CD90 is also referred to as Cluster of Differentiation 90.

[0036] In the embodiments of the present invention (including, for example, the above (1) to (21)), the PI3K activator includes an activator of phosphatidylinositol 3-kinase. The PI3K activator includes an agonist of PI3K, for example, 740Y-P or a salt thereof. 740Y-P includes a compound having CAS Registry Number 1236188-16-1 or PubChem CID 90488730.

[0037] In the embodiment of the present invention (including, for example, the above (1) to (21)), the TPO receptor agonist includes an agonist of the thrombopoietin receptor. The TPO receptor agonist includes, for example, butizamide or a salt thereof. Butizamide can be represented by (E)-3-[2,6-dihydro-4-[[4-[3-(2,2-dimethyl-1-propoxypropyl)-2-methoxyphenyl]-1,3-thiazol-2-yl]carbamoyl]phenyl]-2-methylprop-2-enoic acid. Butizamide includes a compound having a PubChem CID of 44602781.

[0038] In the embodiments of the present invention (including, for example, the above (1) to (21)), methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate includes a compound having a CAS registration number of 1448723-60-1 or a PubChem CID of 71714933. Methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate is sometimes referred to as UM729. In this specification, it may be written as UM729 for shorthand, but they can be used interchangeably.

[0039] In the embodiments of the present invention (including, for example, the above (1) to (21)), tranylcypromine includes a compound having a CAS Registry Number of 155-09-9 or a PubChem CID of 96025309. Tranylcypromine is also referred to as 2-PCPA. Tranylcypromine includes (1R,2S)-2-phenylcyclopropan-1-amine. In this specification, it may be written as 2-PCPA for shorthand, but they can be used interchangeably.

[0040] In the embodiments of the present invention (including, for example, the above (1) to (21)), the culturing includes incubating the cells under conditions suitable for growth or maintenance. The culturing may include a step of contacting the medium and the cells to produce a cell-containing composition, and a step of incubating the cell-containing composition. The incubating step may be performed while the cell-containing composition is left stationary or stirred. The incubation may be performed at about 37°C and in an atmosphere of about 5% CO2. The contacting step includes, for example, seeding the cells in the medium or mixing the cells with the medium. The culturing may be performed in an albumin-free or serum-free medium. Free includes a state in which the target component is not added to the medium, a state in which the medium is completely free of the target component, or a state in which the medium does not contain the component at a concentration above the detection limit. Albumin-free includes being substantially free of albumin.

[0041] In the embodiments of the present invention (including, for example, the above (1) to (21)), the culture may be performed in the presence of a cell adhesion factor (e.g., fibronectin). The culture may be performed under conditions that allow contact between the hematopoietic stem cells and the cell adhesion factor. The culture may be performed, for example, with the inside (e.g., the bottom surface) of the culture vessel coated with the cell adhesion factor.

[0042] In embodiments of the present invention (including, for example, (1) to (21) above), the culturing may include expanding the hematopoietic stem cells under conditions sufficient for the maintenance or proliferation of the hematopoietic stem cells. The expansion may include, for example, expanding the hematopoietic stem cells 10-fold or more from the start of the culture. This expansion may be 10, 50, 100, 200, 300, 400, or 500-fold or more, or may be within a range of any two of these values.

[0043] In the embodiments of the present invention (including, for example, the above (1) to (21)), the cells used for culture may be umbilical cord blood (CB) cells, peripheral blood cells, bone marrow cells, or cell populations thereof. The cells include cells harvested from CB, cells harvested from peripheral blood, and cells harvested from bone marrow. These cells or cell populations include those that express CD34 + The cell may be, for example, a CB CD34 + Cells (CB-derived CD34 + cells), peripheral blood CD34 + Cells (CD34 from peripheral blood + cells) or bone marrow CD34 + Cells (bone marrow-derived CD34 + These cells may be purchased from a manufacturer or distributor (e.g., StemExpress or Lonza) or may be isolated from CB, peripheral blood, or bone marrow using CD34 + In another embodiment of the present invention, the cells used for the culture or the cells obtained by the culture are stem cells, hematopoietic stem cells, or CD34 + , CD201 + , or CD90 +The cells used for culture or the cell population thereof may also be referred to as starting cells or a starting cell population. In the embodiments of the present invention (including, for example, the above (1) to (21)), the cells used for culture or the cells obtained by culture may be mammalian cells. Mammals include, for example, humans, monkeys, rodents (mouse, hamsters, etc.), rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, etc. In the embodiments of the present invention (including, for example, the above (1) to (21)), the cells may exist in the form of a cell population.

[0044] In an embodiment of the present invention (including, for example, the above (1) to (21)), the cell population includes, for example, a plurality of cells generated by cell division. + or CD90 + The percentage of CD201 cells may be, for example, 5% or more. This percentage may be, for example, 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more, or may be within a range of any two of these values. This percentage may be, for example, 5 to 50%, 15 to 40%, or 25 to 40%. + In order to efficiently obtain hematopoietic stem cells, the percentage of cells is preferably 10% or more, more preferably 15% or more, and even more preferably 25% or more. + In order to efficiently obtain hematopoietic stem cells, the percentage of CD201 cells is preferably 13% or more, more preferably 15% or more. + or CD90 + The ratio of cells may be 5 times or more compared to before the culture. This ratio may be, for example, 10, 20, 50, 100, or 200 times or more, or may be within a range of any two of these values. The ratio of cells may be the ratio when the cells are cultured for the culture time described below.

[0045] In the embodiments of the present invention (including, for example, the above (1) to (21)), hematopoietic stem cells include stem cells that can differentiate into blood cells. Hematopoietic stem cells can be collected from the umbilical cord, bone marrow, placenta, and peripheral blood. Human hematopoietic stem cells express CD34 + Human hematopoietic stem cells contain CD34 + Human hematopoietic stem cells may be CD34 + CD201 + CD90 + or CD34 + CD201 + The human hematopoietic stem cell marker may be CD34. + , CD201 + or CD90 + Hematopoietic stem cells include long-term hematopoietic stem cells.

[0046] In the embodiments of the present invention (including, for example, the above (1) to (21)), the culture time of the cells may be, for example, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 17, 18, 20, 30, or 35 days or more, or may be within a range of any two of these values. This culture time may be, for example, 1 to 20 days, 3 to 17 days, 4 to 16, or 5 to 14 days. Culturing includes the time for incubating the medium containing the cells.

[0047] In the embodiments of the present invention (including, for example, the above (1) to (21)), the seeding density of cells during culture may be 1000 cells / mL. This value may be 1000, 3000, 5000, 10000, 30000, 50000, 70000, 100000, or 150000 or more, or may be within a range of any two of these values.

[0048] In the embodiments of the present invention (including, for example, the above (1) to (21)), the composition or medium includes a medium used for culturing cells. The base component of the medium may be any general base component, and the composition is not limited. The base component of the medium may include, for example, amino acids, peptides, proteins, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose). The base component of the medium may include, for example, GlutaMAX, L-alanine L-glutamine dipeptide, L-glutamine, ITS-X, insulin, transferrin (apo), sodium selenite, ethanolamine, or Flt3 ligand. The medium may include, for example, a basal medium for cell culture. Examples of basic media include S-clone SF-3 medium, F12 medium, StemSpan (Stem Cell technologies), STEMα (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium (Invitrogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitrogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM / F-12 medium (Invitrogen), Stemline hematopoietic stem cell growth medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen and others), MEM medium (Gibco Invitrogen and others), MEMα (Gibco Invitrogen and others), DMEM medium (Gibco Invitrogen and others), IMDM medium (Wako Pure Chemical and others), PRMI1640 medium (Gibco Invitrogen and others), Ham F-12 medium (Gibco and others), RD medium, etc.The medium is preferably albumin (e.g., GenBank Accession No. AAN17825.1)-free or serum-free. In this case, stable inhibition of undifferentiation and problems of biological contamination can be suppressed. The medium may contain Soluplus® (CAS Registration No. 402932-23-4). The medium may contain an antibiotic (e.g., penicillin or streptomycin). The medium may be a medium for hematopoietic stem cells, CD201. + cells, CD90 + The cell may include a CB cell. The medium may be a medium for culturing these cells. The medium may be a liquid medium or a solid medium. Culturing the cell with the medium includes culturing the cell in the medium.

[0049] In an embodiment of the present invention (including, for example, the above (1) to (21)), the composition or medium is CB CD34 + The composition or medium may further comprise CD201 cells upon incubation. + or CD90 + The present invention includes a composition or medium in which the cells of the present invention are concentrated.

[0050] In the embodiments of the present invention (including, for example, the above (1) to (21)), the concentration of the PI3K activator in the composition or medium may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or 50 μM or more, or may be within a range of any two of these values. This concentration may be, for example, 1 to 25 μM, 2 to 20 μM, or 3 to 10 μM. In order to efficiently obtain hematopoietic stem cells, this concentration is preferably 2 μM or more, more preferably 3 μM or more, and even more preferably 4 μM or more.

[0051] In the embodiments of the present invention (including, for example, the above (1) to (21)), the concentration of the TPO receptor agonist in the composition or culture medium may be, for example, 0.01, 0.03, 0.05, 0.08, 0.1, 0.12, 0.15, 0.2, 0.3, 0.5, 1, or 2 μM or more, or may be within a range of any two of these values. This concentration may be, for example, 0.01 to 0.3 μM, 0.05 to 0.2 μM, or 0.08 to 0.15 μM. In order to efficiently obtain hematopoietic stem cells, this concentration is preferably 0.03 μM or more, more preferably 0.05 μM or more, and even more preferably 0.08 μM or more.

[0052] In the embodiments of the present invention (including, for example, the above (1) to (21)), the concentration of UM729 or a salt thereof in the composition or medium may be, for example, 0.1, 0.3, 0.5, 0.8, 1, 1.3, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 30 μM or more, or may be within a range of any two of these values. This concentration may be, for example, 0.1 to 3 μM, 0.5 to 2 μM, or 0.8 to 1.5 μM. In order to efficiently obtain hematopoietic stem cells, this concentration is preferably 0.3 μM or more, more preferably 0.5 μM or more, and even more preferably 0.8 μM or more.

[0053] In the embodiments of the present invention (including, for example, the above (1) to (21)), the concentration of 2-PCPA or a salt thereof in the composition or medium may be, for example, 1, 3, 5, 8, 10, 13, 15, 18, 20, 30, 40, or 50 μM or more, or may be within a range of any two of these values. This concentration may be, for example, 1 to 30 μM, 5 to 20 μM, or 8 to 15 μM. In order to efficiently obtain hematopoietic stem cells, this concentration is preferably 3 μM or more, more preferably 5 μM or more, and even more preferably 8 μM or more. 2-PCPA or a salt thereof may be, for example, tranylcypromine hydrochloride.

[0054] In the embodiments of the present invention (including, for example, the above (1) to (21)), the composition or medium may contain, as another protein component, a protein component at a concentration of 1 ng / mL or more. This concentration may be, for example, 1, 5, 8, 10, 15, 20, 30, or 50 ng / mL or more, or may be within a range between any two of these values. This concentration may be, for example, 1 to 30 μM, 5 to 20 μM, or 8 to 12 ng / mL. The other protein component may be, for example, an Flt3 ligand.

[0055] In the embodiments of the present invention (including, for example, the above (1) to (21)), the composition or medium may contain modified polyalkylene glycol as another added component. The concentration of the modified polyalkylene glycol may be, for example, 0.001, 0.005, 0.008, 0.01, 0.012, 0.015, 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 1, 2, or 5% (w / v) or more, or may be within the range of any two of these values. This concentration may be, for example, 0.001 to 0.1% (w / v), 0.005 to 0.05% (w / v), 0.005 to 0.02% (w / v), 0.008 to 0.02% (w / v), or 0.008 to 0.015% (w / v). The modified polyalkylene glycol may be, for example, a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The modified polyalkylene glycol may be, for example, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. The modified polyalkylene glycol may be, for example, a graft copolymer obtained from (i) N-vinyl caprolactam, (ii) vinyl acetate, and (iii) a polyether. The polyether may be a polyethylene glycol. The modified polyalkylene glycol may be, for example, a compound obtained by free radical polymerization of the mixture of the above (i) to (iii). For example, (i) may be 40-60 wt%, (ii) may be 15-35 wt%, and (iii) may be 10-30 wt%. (i) may be, for example, 40, 45, 50, 55, 57, or 60 wt%, or may be within a range of any two of these values. (ii) may be, for example, 15, 20, 25, 30, or 35 wt%, or may be within a range of any two of these values.(iii) may be, for example, 10, 13, 15, 20, 25, or 30 wt%, or may be within the range of any two of these values. The above (i) to (iii) may be a total of 100 wt%. The modified polyalkylene glycol may be, for example, a compound represented by the following structural formula or a salt thereof. [ka] Here, the wt% of l, m, and n may be, for example, 40 to 60 wt%, 15 to 35 wt%, and 10 to 30 wt%, respectively. The wt% of l may be, for example, 40, 45, 50, 55, 57, or 60, and may be within the range of any two values ​​thereof. The wt% of m may be, for example, 15, 20, 25, 30, or 35, and may be within the range of any two values ​​thereof. The wt% of n may be, for example, 10, 13, 15, 20, 25, or 30, and may be within the range of any two values ​​thereof. The wt% of l, m, and n may be 100 wt% in total. The wt% of l, m, and n may be, for example, about 57, about 30, and about 13 wt%, respectively. The degrees of polymerization of l, m, and n may be, for example, 60 to 160, 470 to 1110, and 480 to 1130, respectively. The degree of polymerization of l may be, for example, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160, or may be within a range of any two of these values. The degree of polymerization of m may be, for example, 470, 500, 550, 600, 650, 700, 750, 780, 800, 850, 900, 950, 1000, 1050, or 1110, or may be within a range of any two of these values. The degree of polymerization of n may be, for example, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or 1130, or may be within a range of any two values ​​thereof. The degree of polymerization may be an average degree of polymerization. The mass average molecular weight (Mw) of the modified polyalkylene glycol may be, for example, 70000, 80000, 90000, 100000, 120000, 140000, 150000, 160000 or 170000 g / mol, or may be within a range of any two values ​​thereof. This value may be, for example, 70000 to 170000 g / mol, 80000 to 160000 g / mol, 90000 to 140000 g / mol, 100000 to 130000 g / mol, or 110000 to 125000 g / mol. This value may be about 118000 g / mol.The modified polyalkylene glycol may be, for example, Soluplus. Soluplus is a compound having the structure of the above formula (1). The l, m, and n of Soluplus may have the above wt% or degree of polymerization. The method for producing the modified polyalkylene glycol may be, for example, the method described in US 2008 / 0293828 A1, US 2010 / 0204425 A1, or US 2018 / 0305636 A1. In addition, the components (i) to (iii) may be the components described in these documents.

[0056] In the embodiments of the present invention (including, for example, the above (1) to (21)), the container includes a culture container or a medical container. When used for culture, a culture container may be used, and when used for hematopoietic stem cell transplantation, a medical container may be used. The culture container may be, for example, a plate type, a petri dish type, a flask type, or a bottle type. The medical container may be, for example, a bottle type, a bag type, or a syringe type.

[0057] In embodiments of the present invention (including, for example, the above (1) to (21)), the recovering step may include, for example, transferring the desired cells or cell population from a container containing the cells or cell population to another container. The recovering may include a fractionating or purifying step. The recovering step may be performed in a sterile environment.

[0058] In the embodiments of the present invention (including, for example, the above (1) to (21)), the composition includes, for example, a medium or an additive. The medium is, for example, a medium for hematopoietic stem cell culture or CB CD34 + The additive may be an additive for providing additional nutrients to the basal medium.

[0059] In the embodiments of the present invention (including, for example, the above (1) to (21)), the pharmaceutical composition may be prepared by mixing cells (e.g., hematopoietic stem cells) with one or more pharma- ceutically acceptable carriers and by any method known in the technical field of pharmaceuticals. The pharmaceutical composition may contain a stabilizer, a buffer, or a pH adjuster. The dosage, administration interval, administration method, and administration route are not particularly limited and may be appropriately selected depending on the age and weight, symptoms, target organ, and the like of the patient. The pharmaceutical composition preferably contains a therapeutically effective amount of hematopoietic stem cells, or an effective amount for exerting a desired effect. In one embodiment of the present invention, the therapeutically effective amount includes an amount necessary for clinically observing improvement or suppression of symptoms in a patient. In one embodiment of the present invention, being pharma- ceutically acceptable includes a state suitable for use within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0060] In the embodiments of the present invention (including, for example, the above (1) to (21)), the subject (including a patient) includes a human or a non-human mammal (for example, one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, chimpanzee, etc.). The patient may also be a patient diagnosed with a disease (for example, cancer or immunodeficiency) or a patient in need of treatment for a disease.

[0061] In the embodiments of the present invention (including, for example, the above (1) to (21)), enrichment includes increasing the proportion of a particular type of cell in a medium or a cell population. + or CD90 + The enriched culture fraction for CD201 cells was 100% higher than the total cells in the medium. + or CD90 + The percentage of CD201 cells may be, for example, 5% or more. This percentage may be, for example, 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more, or may be within a range of any two of these values. This percentage may be, for example, 8 to 50%, 15 to 40%, or 25 to 40%. +In order to efficiently obtain hematopoietic stem cells, the percentage of cells is preferably 10% or more, more preferably 15% or more, and even more preferably 25% or more. + In order to efficiently obtain hematopoietic stem cells, the percentage of cells is preferably 13% or more, more preferably 15% or more. The culture fraction includes a cell-containing composition obtained by culturing cells. The culture fraction includes a fraction obtained after culturing cells and before carrying out a selection or purification treatment.

[0062] In the embodiments of the present invention (including, for example, the above (1) to (21)), the salt is not particularly limited and includes, for example, an inorganic salt or an organic salt (see, for example, "Bharate et al., Drug Discov Today. 2021 Feb;26(2):384-398." or "Berge et al., J Pharm Sci. 1977 Jan;66(1):1-19."). Salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. Metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. Salts include pharma- ceutically acceptable salts. In one embodiment of the present invention, pharma- ceutically acceptable includes forms that have reasonable benefit for pharmaceutical use.

[0063] In the embodiments of the present invention (including, for example, the above (1) to (21)) + includes that the cell is found to be positive for the molecule specified by the term immediately preceding it.

[0064] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." For example, CD201 + or CD90 + is CD201 + , CD90 + , CD201 + and CD90 + In the present specification, when it is specified that "within a range of two values", the range includes the two values ​​themselves. In the present specification, "A to B" includes A and B. In the present specification, "the above (1) to (21)" includes reference to any one or more of (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), or (21).

[0065] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may be adopted. Furthermore, the present invention may be adopted in combination with each of the configurations or features described in the above embodiments, or independently. EXAMPLES

[0066] The present invention will be further explained below with reference to examples, but is not limited to these.

[0067] <Example 1> 1. Hematopoietic stem cell proliferation 1.1 Method 1.1.1 Medium A 5 μM 740Y-P, a PI3K activator, 0.1 μM butizamide, a TPO receptor agonist, and 1 μM UM729, a synthetic small molecule, were added to IMDM (Iscove's Modified Dulbecco's Media) containing 1x GlutaMAX, 1x ITS-X, and 10 ng / ml Flt3-ligand. The resulting medium (medium A) was incubated with thawed human umbilical cord blood CD34 cells according to the manufacturer's instructions. + The cells were suspended and plated in a 96-well plate at a density of 10,000 cells / well. The medium was changed every 3–4 days. On days 10 and 17, all CD34 + The progeny of the cells were collected for flow cytometry analysis. A FACSVerse (Becton Dickinson, CA, USA) was used for sample analysis. The antibodies shown in Table 3 were used for immunostaining of the samples. CD34 expression was measured using CD34 + Other markers were used to determine the proliferation rate of ex vivo expanded CD34 + Used to confirm the characteristics of the cells.

[0068] 1.1.2 Medium B The same procedure as in 1.1.1 above was carried out, except that the medium was changed to medium A supplemented with Soluplus (registered trademark) at a final concentration of 0.01% (w / v) (medium B).

[0069] The materials used in this experiment are shown below. [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] 1.2 Results The results are shown in Figures 1 and 2. In both media A and B, CD201 + CD45RA - CD34 + CD38 - Lin - The cells proliferated well. CD201 + CD45RA-CD34 + CD38 - Lin - is a marker characteristic of hematopoietic stem cells. This indicates that good proliferation of hematopoietic stem cells can be achieved by using 740Y-P or butizamide, and UM729.

[0073] <Example 2> 2. Hematopoietic stem cell proliferation and analysis of hematopoietic stem cell markers 2.1 Method 2.1.1 Medium A 5 μM 740Y-P, a PI3K activator, 0.1 μM butizamide, a TPO receptor agonist, and 1 μM UM729, a synthetic small molecule, were added to IMDM (Iscove's Modified Dulbecco's Media) containing 1x GlutaMAX, 1x ITS-X, and 10 ng / ml Flt3-ligand. The resulting medium (medium A) was incubated with thawed human umbilical cord blood CD34 cells according to the manufacturer's instructions. + The cells were suspended and plated in a 96-well plate at a density of 10,000 cells / well. The medium was changed every 3–4 days. On days 10 and 17, all CD34 + The progeny of the cells were collected for flow cytometry analysis. A FACSVerse (Becton Dickinson, CA, USA) was used for sample analysis. The antibodies shown in Table 3 were used for immunostaining of the samples. CD34 expression was measured using CD34 + Other markers were used to determine the proliferation rate of ex vivo expanded CD34 + Used to confirm the characteristics of the cells.

[0074] 2.1.2 Medium B The same procedure as in 2.1.1 above was carried out, except that the medium was changed to medium A supplemented with Soluplus® at a final concentration of 0.01% (w / v) (medium B).

[0075] 2.1.3 Medium C The same procedure was carried out as in 2.1.1 above, except that the 1 μM UM729 in the medium was replaced with 35 nM UM171 (Stemcell Technologies) (Medium C).

[0076] 2.1.4 Medium D In the above 2.1.1, the medium was changed to StemSpan UM171 supplemented with 35 nM, 10 μg / mL human low-density lipoprotein, 100 ng / mL Flt3-ligand, 100 ng / mL stem cell factor, and 50 ng / mL thrombopoietin. TM The same procedure was carried out, except that the medium was changed to Serum-Free Expansion Medium (SFEM) (ST-09600, STEMCELL Technologies) (Medium D).

[0077] 2.2 Results The results of measuring the proliferation rate are shown in Figures 3 and 4. Total cell count and CD34 + There was no significant difference in cell proliferation rate between media B and C. The proliferation rate in medium D was relatively high.

[0078] The results of expression analysis are shown in Figure 5-12. + Cells and CD90 + The percentage of CD201 cells was significantly higher in medium B (27.6% and 22.9%, respectively). + Cells and CD90 + The percentage of CD201 cells was significantly higher in medium C than in medium D (Fig. 8, 36.5% and 19.7%, respectively). + Cells and CD90 + The percentage of CD201 cells was very low (Figure 10, 0.81% and 3.27%, respectively). + Cells and CD90+ The percentage of CD201 cells was very low (Figure 12, 0.27% and 0.65%, respectively). + or CD90 + It was shown that a culture fraction enriched in hematopoietic stem cells can be obtained.

[0079] <Example 3> 3. Hematopoietic stem cell proliferation and analysis of hematopoietic stem cell markers 3.1 Method 3.1.1 Medium B 5 μM 740Y-P, a PI3K activator, 0.1 μM butizamide, a TPO receptor agonist, 1 μM UM729, a synthetic small molecule, and 0.01% (w / v) Soluplus were added to IMDM (Iscove's Modified Dulbecco's Media) containing 1x GlutaMAX, 1x ITS-X, and 10 ng / ml Flt3-ligand. The resulting medium (medium B) was incubated with thawed human umbilical cord blood CD34 cells according to the manufacturer's instructions. + Cells were suspended and plated in 96-well plates at a density of 10,000 cells / well. The medium was changed every 3–4 days. On days 10 and 17, all CD34 + The progeny of the cells were collected for flow cytometry analysis. A FACSVerse (Becton Dickinson, CA, USA) was used for sample analysis. The antibodies shown in Table 3 were used for immunostaining of the samples. CD34 expression was measured using CD34 + Other markers were used to determine the proliferation rate of ex vivo expanded CD34 cells. + Used to confirm the properties of the cells.

[0080] 3.1.2 Medium D The medium was changed to StemSpan UM171 (35 nM), UM171 (10 μg / mL), human low-density lipoprotein (100 ng / mL), Flt3-ligand (100 ng / mL), stem cell factor (100 ng / mL), and thrombopoietin (50 ng / mL).TM The same procedure was carried out, except that the medium was changed to Serum-Free Expansion Medium (SFEM) (ST-09600, STEMCELL Technologies) (Medium D).

[0081] 3.1.3 Medium E The same procedure was carried out as in 3.1.1 above, except that 1 μM UM729 in the medium was replaced with 10 μM 2-PCPA (Selleck Chemicals, S4246), an LSD1 inhibitor (medium E).

[0082] 3.2 Results The results of expression analysis are shown in Figures 13-16. + The percentage of CD201 cells was significantly higher in medium D (Fig. 14, 33.0%). + The percentage of CD201 cells was very low (Figure 15, 0.95%). + The percentage of CD201 cells was significantly higher (Fig. 16, 11.4%). + It was shown that a culture fraction enriched in hematopoietic stem cells can be obtained.

[0083] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A composition which is a medium for culturing cells, comprising (a) a PI3K activator or a TPO receptor agonist, and (b) tranylcypromine or a salt thereof.

2. The composition according to claim 1, wherein the PI3K activator is 740Y-P or a salt thereof, and the TPO receptor agonist is butizamide or a salt thereof.

3. The composition according to claim 1 or 2, which is a medium for culturing human hematopoietic stem cells.

4. The composition according to claim 1 or 2, which is an albumin-free medium.

5. The composition according to claim 1 or 2, further comprising hematopoietic stem cells.

6. Cord blood CD34 + The composition according to claim 1 or 2, further comprising cells, and by incubating, CD201+ hematopoietic stem cells are concentrated.

7. The composition according to claim 1 or 2, comprising CD201+ hematopoietic stem cells, and the ratio of CD201+ hematopoietic stem cells to the total cells in the composition is 5% or more.

8. A method for producing cells, comprising a culturing step of culturing cells using the composition according to claim 1 or 2.

9. A method for producing cells, comprising a step of culturing human cells using the composition according to claim 1 or 2 to generate a cell population in which CD201+ hematopoietic stem cells are concentrated.

10. A method for producing cells, comprising a step of culturing human cells using the composition according to claim 1 or 2 to generate a cell-containing medium in which the ratio of CD201+ hematopoietic stem cells to the total cells in the medium is 5% or more.

11. A method for culturing cells, comprising a culturing step of culturing cells using the composition according to claim 1 or 2.

12. A method for producing a composition which is a medium for culturing hematopoietic stem cells, the method comprising the step of mixing (a) a PI3K activator or a TPO receptor agonist, and (b) tranylcypromine or a salt thereof.

Images