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. Hematopoietic stem cell transplantation is a known treatment method 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 a starting population of cells, EPCR + The procedure involves selecting and culturing a population of cells. EPCR is a hematopoietic stem cell marker, also known as CD201.

[0004] Furthermore, methods for culturing hematopoietic stem cells are being researched. Generally, albumin-containing culture media have been used to induce cell division in hematopoietic stem cell culture. However, albumin has problems with stable inhibition of undifferentiation and biological contamination. Therefore, research is being conducted on albumin-free culture methods. For example, Patent Document 2 describes the cultivation of human hematopoietic stem cells by adding PVA or Soluplus® instead of albumin. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2017 / 205977 [Patent Document 2] WO2021 / 149799 [Overview of the project] [Problems that the invention aims to solve]

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

Means for Solving the Problems

[0007] In repeated studies, the inventors examined a method for culturing hematopoietic stem cells. As a result, by culturing human CB CD34 + cells in an albumin-free medium with a specific composition different from the conventional one, hematopoietic stem cells could be proliferated well. Furthermore, the obtained culture fraction was a culture fraction enriched with hematopoietic stem cells of CD201 + or CD90 + (both are hematopoietic stem cell markers). Based on this finding, the inventors completed the present invention.

[0008] According to one aspect of the present invention, there is provided a composition comprising (a) a PI3K activator 、(b) a TPO receptor agonist, and ( c) ranilcipromine (hereinafter also referred to as 2-PCPA) or a salt thereof. By using this composition, a culture fraction containing many cells of CD201 + or CD90 + can be efficiently produced.

[0009] According to one aspect of the present invention, there is provided a method for producing cells, which includes a culturing step of culturing cells using the above composition. By using this method, a culture fraction containing many cells of CD201 + or CD90 + can be efficiently produced.

Brief Description of the Drawings

[0010] [Figure 1] It is a diagram showing the measurement results of the cell yield of CD201+CD45RA-CD34+CD38-Lin- cells after culturing for 10 days or 17 days in Medium A (UM729). [Figure 2]This figure 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). [Figure 3] This figure shows the results of measuring the cell proliferation rate (total cells) after 10 days of culture in medium B (UM729), medium C (UM171), or medium D (UM171 (SFEM)). [Figure 4] This figure shows the results of measuring the cell proliferation rate (CD34+ cells) after 10 days of culture in medium B (UM729), medium C (UM171), or medium D (UM171 (SFEM)). [Figure 5] This figure shows the expression analysis results for CD34 and CD201 in cells cultured in medium A (UM729) for 10 days. [Figure 6] This figure shows the expression analysis results for CD41, CD45RA, CD201, and CD90 in cells cultured in medium A (UM729) for 10 days. The cells analyzed were CD34+Lin- cells. [Figure 7] This figure shows the results of the CD34 and Lin expression analysis of cells cultured in medium B (UM729) for 10 days. [Figure 8] This figure shows the expression analysis results for CD41, CD45RA, CD201, and CD90 in cells cultured in medium B (UM729) for 10 days. The cells analyzed were CD34+Lin- cells. [Figure 9] This figure shows the results of the CD34 and Lin expression analysis of cells cultured in medium C (UM171) for 10 days. [Figure 10] This figure shows the expression analysis results for CD41, CD45RA, CD201, and CD90 in cells cultured in medium C (UM171) for 10 days. The cells analyzed were CD34+Lin- cells. [Figure 11] This figure shows the results of the CD34 and Lin expression analysis of cells cultured in medium D (UM171 (SFEM)) for 10 days. [Figure 12] This figure shows the expression analysis results for CD41, CD45RA, CD201, and CD90 in cells cultured for 10 days in medium D (UM171 (SFEM)). The cells analyzed were CD34+Lin- cells. [Figure 13] This figure shows the expression analysis results for CD34 and CD201 in control cells before culture. [Figure 14] This figure shows the expression analysis results for CD34 and CD201 in cells cultured in medium B (UM729) for 10 days. [Figure 15] This figure shows the expression analysis results for CD34 and CD201 in cells cultured for 10 days in medium D (UM171 (SFEM)). [Figure 16] This figure shows the expression analysis results for CD34 and CD201 in cells cultured in medium E (2-PCPA) for 10 days. [Modes for carrying out the invention]

[0011] The embodiments of the present invention will be described in detail below. To avoid repetition and unnecessary complexity, similar content will be omitted from explanation as appropriate.

[0012] (1) Composition According to one embodiment of the present invention, a composition is provided comprising (a) a PI3K activator or a TPO receptor agonist, and (b) methyl 4-(3-piperidine-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate (hereinafter sometimes referred to as UM729) or a salt thereof, or tranylcypromine (hereinafter sometimes referred to as 2-PCPA) or a salt thereof. The composition may also be a culture medium. Using this composition, CD201 + Or CD90 + This allows for the efficient production of a culture fraction containing a large amount of human CB CD34 cells. For example, in the examples described later, this composition is used to produce human CB CD34 + As a result of culturing the cells, CD201 + Cells or CD90 + It is stated that a culture fraction with concentrated cells was obtained. Furthermore, according to one embodiment of the present invention, a method for producing cells using this composition, a method for culturing cells, a method for producing a culture medium, and CD201 + Or CD90 +A method for concentrating cells and a container containing this composition are provided.

[0013] (2) Culture medium According to one embodiment of the present invention, a culture medium is provided 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 medium, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

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

[0015] (4) Production method According to one embodiment of the present invention, a method for producing cells is provided, 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. Using this method, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0016] (5) Production method According to one embodiment of the present invention, (a) a PI3K activator or TPO receptor agonist, and (b) a culture medium containing UM729 or a salt thereof or 2-PCPA or a salt thereof, are used to obtain CB CD34 + A method for producing cells is provided, which includes a cell culture step. Using this method, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0017] (6) Production method According to one embodiment of the present invention, CD201 is prepared using a medium containing (a) a PI3K activator or TPO receptor agonist and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. + Or CD90 + A method for producing cells is provided, which includes a culture step for culturing the cells of CD201. + Or CD90 + It can promote the healthy proliferation of these cells.

[0018] (7) Production method One embodiment of the present invention provides a method for producing cells, comprising a culture step of culturing human hematopoietic stem 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. This method allows for the efficient proliferation of human hematopoietic stem cells.

[0019] (8) Production method According to one embodiment of the present invention, a method for producing a culture medium is provided, comprising the steps of (a) mixing a PI3K activator or a TPO receptor agonist and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. Using the culture medium obtained by this method, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0020] (9) Production method According to one embodiment of the present invention, a hematopoietic stem cell marker is obtained from a composition containing a population of cells obtained by any of the methods described in (3) to (8) above. + The process of separating cells, and hematopoietic stem cell markers after separation. + The process of culturing cells, or hematopoietic stem cell markers after culture + A method for producing a cell population is provided, which includes a step of recovering the cells. This method allows for greater concentration of hematopoietic stem cells or recovery of a larger number of hematopoietic stem cells.

[0021] (10) Culture method According to one embodiment of the present invention, a cell culture method is provided, 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. Using this method, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0022] (11) Concentration method According to one embodiment of the present invention, CD201 includes 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 + A method for enriching the cells of CD201 is provided. Using this method, + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0023] (12) Concentration method One embodiment of the present invention provides 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. This method allows for the efficient production of a culture fraction rich in hematopoietic stem cells.

[0024] (13) Container According to one embodiment of the present invention, a container is provided comprising (a) a PI3K activator or TPO receptor agonist, and (b) a composition comprising UM729 or a salt thereof, or 2-PCPA or a salt thereof. Using this container, CD201 + Or CD90 + This allows for the efficient production of culture fractions containing a large number of these cells.

[0025] (14) Container According to one embodiment of the present invention, a container is provided containing cells or a population of cells obtained by any of the methods described in (3) to (12) above. This container can be used for hematopoietic stem cell transplantation. Before hematopoietic stem cell transplantation, desired cells (e.g., hematopoietic stem cell markers) are selected from the cell population. + The process may involve separating cells, culturing the separated cells, recovering the cultured cells, or holding the recovered cells in a container.

[0026] (15) Cell According to one embodiment of the present invention, cells or a population of cells obtained by any of the methods described in (3) to (12) above are provided. These cells or a population of cells can be used for hematopoietic stem cell transplantation. Before hematopoietic stem cell transplantation, desired cells (e.g., hematopoietic stem cell markers) are selected from the cell population. + The process may involve separating cells, culturing the separated cells, recovering the cultured cells, or holding the recovered cells in a container.

[0027] (16) Cell According to one embodiment of the present invention, CD201 + Or CD90 + A cell population containing 5% or more of the specified cells is provided. This cell population can be used to produce pharmaceutical compositions for hematopoietic stem cell transplantation.

[0028] (17) Culture medium According to one embodiment of the present invention, CD201 in total cells in the culture medium + Or CD90 + A cell-containing culture medium is provided, containing a cell percentage of 5% or more. This medium can be used for the production of pharmaceutical compositions for hematopoietic stem cell transplantation.

[0029] (18) Composition According to one embodiment of the present invention, a composition is provided for use in any of the methods described in (3) to (12) above, comprising (a) a PI3K activator or 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, a composition is provided comprising the cells or cell population described in (15) or (16) above, (a) a PI3K activator or TPO receptor agonist, and (b) UM729 or a salt thereof or 2-PCPA or a salt thereof. This composition is CD201 + Or CD90 + Because it contains a large number of these cells, it is useful for producing pharmaceutical compositions for use in hematopoietic stem cell transplantation.

[0031] (20) Composition According to one embodiment of the present invention, a pharmaceutical composition for use in hematopoietic stem cell transplantation or a method for producing the same is provided, comprising cells or a population of the same obtained by any of the methods described in (3) to (12) above. 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 purifying hematopoietic stem cell markers from the composition described in (19) above. + The process of separating cells, and hematopoietic stem cell markers after separation. + The process of culturing cells, or hematopoietic stem cell markers after culture + The step may include a step of recovering the cells. In another aspect, according to one embodiment of the present invention, the cells or cell population described in (15) or (16) above, or a pharmaceutical composition containing such cells or cell population, is provided for use in hematopoietic stem cell transplantation. In another aspect, according to one embodiment of the present invention, the use of the cells or cell population described in (15) or (16) above in the production of a pharmaceutical composition for hematopoietic stem cell transplantation is provided.

[0032] (21) Transplant method According to one embodiment of the present invention, a transplantation method is provided. This transplantation method may include, for example, the step of transplanting the cells or cell population described in (15) or (16) above, or a pharmaceutical composition containing such cells or cell population, into a target. This transplantation method allows for the effective engraftment of hematopoietic stem cells, thereby enabling the treatment of diseases.

[0033] In embodiments of the present invention (including, for example, (3) to (12) or (21) above), the method may include one or more of the following steps (i) to (v): (i) placing or holding in a container a composition comprising (a) PI3K activator, (b) TPO receptor agonist, and (c) UM729 or a salt thereof or 2-PCPA or a salt thereof before culturing; (ii) bringing the cells into contact with the composition in the container; (iii) culturing the cells in the container; (iv) CD201 + Or CD90 + A step of generating a cell population in which the cells are concentrated, or (v) CD201 in total cells in culture medium + Or CD90 + A step of producing a cell-containing medium in which the proportion of cells is 5% or more. The methods of embodiments of the present invention (for example, including (3) to (12) or (21) above) may include one or more of the following steps (vi) to (xx): (vi) a step of preparing a cell population including hematopoietic stem cells, (vii) CB CD34 + (viii) Steps to prepare a cell population containing cells, (viii) CD34 + (ix) The process of separating the cells, CD34 after separation + (x) A step of culturing cells, (x) a step of mixing (a) PI3K activator, (b) TPO receptor agonist, and (c) UM729 or a salt thereof or 2-PCPA or a salt thereof before culturing to produce a composition containing the mixed components, (xi) a step of incubating the cells in a culture medium, (xii) a step of seeding the cells in a culture medium in a container and incubating them, (xiii) a step of taking CD201 from the cell population + Or CD90 + (xiv) A step of separating or recovering cells, (xv) A step of separating or recovering hematopoietic stem cells from the cell population, (xv) CD201 after separation or recovery + Or CD90 + (xvi) A step of culturing the cells, (xvii) A step of culturing the hematopoietic stem cells after sorting or recovery, (xvii) CD201 after sorting or recovery and culture + Or CD90 +(xviii) Steps to collect the cells, (xix) Steps to collect the hematopoietic stem cells after sorting or collection and culture, (xix) Steps to collect the CD201 + Or CD90 + The process of preserving the cells (e.g., cryopreservation), or (xx) CD201 after recovery + Or CD90 + The process of transplanting cells. Employing one or more of these processes is considered a CD201 procedure. + Or CD90 + This is useful for efficiently producing a culture fraction containing a large amount of cells. When two or more steps from the embodiments of the present invention (for example, including the methods of (3) to (12), (21) above, or the steps of (i) to (xx)) are adopted, the order is arbitrary and can be determined according to the desired operation. Each method or step of the embodiments of the present invention (for example, including the methods of (3) to (12), (21) above, or the steps of (i) to (xx)) may be carried out in vitro or ex vivo.

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

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

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

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

[0038] In embodiments of the present invention (including, for example, (1) to (21) above), methyl 4-(3-piperidine-1-ylpropylamino)-9H-pyrimide[4,5-b]indole-7-carboxylate includes the compound with CAS registry number 1448723-60-1 or PubChem CID 71714933. Methyl 4-(3-piperidine-1-ylpropylamino)-9H-pyrimide[4,5-b]indole-7-carboxylate is sometimes referred to as UM729. In this specification, it may be written as UM729 for abbreviation, but these are interchangeable.

[0039] In embodiments of the present invention (including, for example, (1) to (21) above), tranylcypromine comprises the compound with CAS registry number 155-09-9 or PubChem CID 96025309. Trannylcypromine is sometimes referred to as 2-PCPA. Trannylcypromine comprises (1R,2S)-2-phenylcyclopropane-1-amine. In this specification, it may be written as 2-PCPA for abbreviation, but these are interchangeable.

[0040] In embodiments of the present invention (including, for example, (1) to (21) above), culture includes incubating cells under conditions suitable for growth or maintenance. Culture may include the steps of contacting a culture medium and cells to produce a cell-containing composition, and incubating the cell-containing composition. The incubation step may be carried out with the cell-containing composition standing or agitated. Incubation may be carried out at about 37°C and in an atmosphere of about 5% CO2. Contact includes, for example, seeding cells into a culture medium or mixing cells with a culture medium. Culture may be carried out 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 does not contain the target component at all, or a state in which the medium does not contain the component at a concentration above the detection limit. Albumin-free includes being substantially albumin-free.

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

[0042] In embodiments of the present invention (including, for example, (1) to (21) above), culture may include growing hematopoietic stem cells under conditions sufficient for the maintenance or proliferation of hematopoietic stem cells. Proliferation may include, for example, growing hematopoietic stem cells tenfold or more from the start of culture. This multiplier may be 10, 50, 100, 200, 300, 400, or 500 times or more, and may be within the range of any two of these values.

[0043] In embodiments of the present invention (including, for example, (1) to (21) above), the cells used for culture may be umbilical cord blood (CB) cells, peripheral blood cells, bone marrow cells, or a population of these cells. The cells include cells collected from CB, cells collected from peripheral blood, and cells collected from bone marrow. These cells or cell populations are CD34 + It may be a cell. For example, a cell is CB CD34 + Cells (CD34 derived from CB) + Cells), peripheral blood CD34 + Cells (CD34 derived from peripheral blood) + (Cells) or bone marrow CD34 + Cells (CD34 derived from bone marrow) + These may be cells. These cells may be purchased from a manufacturer or distributor (e.g., StemExpress or Lonza) and obtained from CB, peripheral blood, or bone marrow. + They may also be obtained by separating cells. In another embodiment of the present invention, the cells used in culture or obtained in culture are stem cells, hematopoietic stem cells, or CD34 + CD201 + , or CD90 +It contains cells. The cells or cell population used for culturing can also be referred to as starting cells or starting cell population. In embodiments of the present invention (including, for example, the above (1) to (21)), the cells used for culturing or the cells obtained by culturing may be mammalian cells. Mammals include, for example, humans, monkeys, rodents (such as mice, hamsters, etc.), rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, etc. In embodiments of the present invention (including, for example, the above (1) to (21)), the cells may exist in the state of a cell population.

[0044] In embodiments 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. In embodiments of the present invention, the ratio of CD201 + or CD90 + cells in the total cells in the culture medium after culturing may be, for example, 5% or more. This ratio may be, for example, 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more, and may be within the range of any two of these values. This ratio may be, for example, 5 to 50%, 15 to 40%, or 25 to 40%. The ratio of CD201 + 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 ratio of CD90 + cells is preferably 13% or more, more preferably 15% or more in order to efficiently obtain hematopoietic stem cells. In embodiments of the present invention, the ratio of CD201 + or CD90 + cells in the cell population after culturing may be 5 times or more compared to before culturing. This multiple may be, for example, 10, 20, 50, 100, or 200 times or more, and may be within the range of any two of these values. The ratio of cells may also be the ratio at the time of culturing the cells for the culture time described later.

[0045] In embodiments of the present invention (including, for example, the above (1) to (21)), hematopoietic stem cells include stem cells that can differentiate into hematopoietic cells. Hematopoietic stem cells can be collected from umbilical cord, bone marrow, placenta, and peripheral blood. Human hematopoietic stem cells include cells of CD34 + and may be CD34 + CD38-. Human hematopoietic stem cells may be CD34 + CD201 + CD90 + or CD34 + CD201 + Human hematopoietic stem cell markers include CD34 + , CD201 + or CD90 + Hematopoietic stem cells include long-term hematopoietic stem cells.

[0046] In embodiments of the present invention (including, for example, the above (1) to (21)), the culture time of 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, and may also be within the 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 days, 5 to 14 days. Culturing includes the time for incubating a medium containing cells.

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

[0048] It seems there is a tag "? " in your original text which is not clear. I've translated the rest as accurately as possible. If you can clarify that tag, I can provide a more perfect translation.In embodiments of the present invention (including, for example, (1) to (21) above), the composition or culture medium includes a culture medium used for cell culture. The base components of the culture medium may be any common base components, and its composition is not limited. The base components of the culture medium may include, for example, amino acids, peptides, proteins, inorganic salts, vitamins, minerals, or carbon sources (e.g., glucose). The base components of the culture 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 culture medium may include, for example, a basic culture medium for cell culture. Basic culture media include, for example, 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 proliferation medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), and MyeloCult medium (StemCell This includes HPG serum-free medium (Lonza Technologies), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen, etc.), MEM medium (Gibco Invitrogen, etc.), MEMα (Gibco Invitrogen, etc.), DMEM medium (Gibco Invitrogen, etc.), IMDM medium (Wako Pure Chemical, etc.), PRMI1640 medium (Gibco Invitrogen, etc.), Ham F-12 medium (Gibco, etc.), RD medium, etc.The culture medium is preferably albumin-free (e.g., GenBank registry number AAN17825.1) or serum-free. In this case, stable inhibition of undifferentiation and problems of biological contamination can be suppressed. The medium may contain Soluplus® (CAS registry number 402932-23-4). The medium may contain antibiotics (e.g., penicillin or streptomycin). The medium contains hematopoietic stem cells and CD201. + cells, CD90 + The medium may include cells, including CB cells. The culture medium may be a medium for culturing these cells. The culture medium may be a liquid medium or a solid medium. Culturing cells using a culture medium includes culturing cells in a culture medium.

[0049] In embodiments of the present invention (including, for example, (1) to (21) above), the composition or culture medium is CB CD34 + The composition or culture medium may further contain cells. By incubation, the composition or medium will produce CD201 + Or CD90 + The composition or culture medium contains cells that are concentrated.

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

[0051] In embodiments of the present invention (including, for example, (1) to (21) above), 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, and may be within the 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. 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, in order to efficiently obtain hematopoietic stem cells.

[0052] In embodiments of the present invention (including, for example, (1) to (21) above), the concentration of UM729 or its salt in the composition or culture 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, and may be within the 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. 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, in order to efficiently obtain hematopoietic stem cells.

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

[0054] In embodiments of the present invention (including, for example, (1) to (21) above), the composition or culture medium may contain, as other protein components, a protein component at a concentration of 1 ng / mL or higher. This concentration may be, for example, 1, 5, 8, 10, 15, 20, 30, or 50 ng / mL or higher, and may be within the range of 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 embodiments of the present invention (including, for example, (1) to (21) above), the composition or culture medium may contain modified polyalkylene glycol as another additive 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, and 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). Modified polyalkylene glycol may be, for example, a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block. Polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block may be, for example, polyethylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block. Modified polyalkylene glycol may be, for example, a polyvinylcaprolactam-polyvinylacetate-polyethylene glycol graft copolymer. Modified polyalkylene glycol may be, for example, a graft copolymer obtained from (i) N-vinylcaprolactam, (ii) vinyl acetate, and (iii) polyether. The polyether may be polyethylene glycol. Modified polyalkylene glycol may be, for example, a compound obtained by free radical polymerization of a mixture of (i) to (iii) above. 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%, and may be within the range of any two of these values. (ii) may be, for example, 15, 20, 25, 30, or 35 wt%, and may be within the range of any two of these values.(iii) may be, for example, 10, 13, 15, 20, 25, or 30 wt%, and may be within the range of any two of these values. The above (i) to (iii) may total 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, 40-60 wt%, 15-35 wt%, and 10-30 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 of these values. The wt% of m may be, for example, 15, 20, 25, 30, or 35, and may be within the range of any two of these values. The wt% of n may be, for example, 10, 13, 15, 20, 25, or 30, and may be within the range of any two of these values. The wt% of l, m, and n above may total 100 wt%. 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, 60-160, 470-1110, and 480-1130, respectively. The degree of polymerization of l may be, for example, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160, and may be within the 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, and may be within the 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, and may be within the range of any two of these values. The degree of polymerization may also be the average degree of polymerization. The mass-average molecular weight (Mw) of the modified polyalkylene glycol may be, for example, 70,000 g / mol or more. This value may be, for example, 70,000, 80,000, 90,000, 100,000, 120,000, 140,000, 150,000, 160,000, or 170,000 g / mol, and may be within the range of any two of these values. This value may be, for example, 70,000 to 170,000 g / mol, 80,000 to 160,000 g / mol, 90,000 to 140,000 g / mol, 100,000 to 130,000 g / mol, or 110,000 to 125,000 g / mol. This value may also be approximately 118,000 g / mol.The modified polyalkylene glycol may be, for example, Soluplus. Soluplus is a compound having the structure of formula (1) above. l, m, and n of Soluplus may have the wt% or degree of polymerization described above. 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) above may be components described in these documents.

[0056] In embodiments of the present invention (including, for example, (1) to (21) above), the container includes a culture vessel or a medical container. A culture vessel may be used when used for culture, and a medical container may be used when used for hematopoietic stem cell transplantation. The culture vessel 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, (1) to (21) above), the recovery step may include, for example, transferring desired cells or cell populations from a container containing cells or cell populations to another container. Recovery may include a separation or purification step. The recovery step may be carried out in a sterile environment.

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

[0059] In embodiments of the present invention (including, for example, (1) to (21) above), the pharmaceutical composition may be prepared by mixing cells (e.g., hematopoietic stem cells) with one or more pharmaceutically acceptable carriers by any method known in the art of pharmaceuticals. The pharmaceutical composition may contain stabilizers, buffers, or pH adjusters. The dosage, administration interval, method of administration, and route of administration are not particularly limited and can be appropriately selected depending on the patient's age, weight, symptoms, target organ, etc. The pharmaceutical composition preferably contains a therapeutically effective amount or an effective amount of hematopoietic stem cells that exert the desired effect. In one embodiment of the present invention, a therapeutically effective amount includes the amount necessary for clinically observed improvement or suppression of symptoms in the patient. In one embodiment of the present invention, pharmaceutically acceptable includes a state suitable for use in proportion to a reasonable benefit-risk ratio, within the range of reasonable medical judgment.

[0060] In embodiments of the present invention (including, for example, (1) to (21) above), the subject (including the patient) includes one or more non-human mammals (for example, one or more of the following: mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cattle, horse, cat, dog, marmoset, monkey, or chimpanzee). 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 embodiments of the present invention (including, for example, (1) to (21) above), "concentrated" means an increase in the proportion of a particular type of cell in the culture medium or cell population. CD201 + Or CD90 + The culture fraction in which the cells are concentrated contains CD201 relative to the total cells in the culture medium. + Or CD90 + The percentage of 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, and may be within the range of any two of these values. This percentage may be, for example, 8-50%, 15-40%, or 25-40%. CD201 +The cell percentage 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. CD90 + The cell percentage is preferably 13% or more, and more preferably 15% or more, in order to efficiently obtain hematopoietic stem cells. The culture fraction includes the cell-containing composition obtained by culturing the cells. The culture fraction includes the fraction before sorting or purification after culturing the cells.

[0062] In embodiments of the present invention (including, for example, (1) to (21) above), the salt is not particularly limited and includes, for example, inorganic salts or organic salts (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 pharmaceutically acceptable salts. In one embodiment of the present invention, "pharmaceutically acceptable" includes a form having reasonable benefits for pharmaceutical use.

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

[0064] All publications cited herein are referred to in their entirety. In this specification, “or” is used when “at least one” of the items listed in the text can be adopted. The same applies to “or.” For example, CD201 + Or CD90 + CD201 + CD90 + CD201 + and CD90 + This includes any of the following. Where it is specified herein that “within the range of two values” includes the two values ​​themselves. Where it is specified herein that “A to B” includes A and B. Where it is specified herein that “(1) to (21) above” includes reference to 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 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 other configurations can be adopted. Furthermore, the present invention can be adopted by combining or independently the configurations or features described in the above embodiments. [Examples]

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

[0067] <Example 1> 1. Proliferation of hematopoietic stem cells 1.1 Method 1.1.1 Culture 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. Human umbilical cord blood CD34, thawed according to the manufacturer's instructions, was added to the resulting medium (Medium A). + The cells were suspended and plated in 96-well plates at a density of 10,000 cells / well. The culture medium was changed every 3-4 days. On days 10 and 17, all CD34 cells in the wells were removed. + Cell progeny were collected for flow cytometry analysis. 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 analyzed using CD34 + Other markers were used to determine the proliferation rate of cells. CD34 was grown ex vivo. + It was used to confirm the characteristics of the cells.

[0068] 1.1.2 Medium B In section 1.1.1 above, the culture medium was changed to medium B, which is medium A to which an additional 0.01% (w / v) of Soluplus® at a final concentration was added, and the same procedure was carried out.

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

[0070] [Table 2]

[0071] [Table 3]

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

[0073] <Example 2> 2. Analysis of hematopoietic stem cell proliferation and hematopoietic stem cell markers 2.1 Method 2.1.1 Culture 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. Human umbilical cord blood CD34, thawed according to the manufacturer's instructions, was added to the resulting medium (Medium A). + The cells were suspended and plated in 96-well plates at a density of 10,000 cells / well. The culture medium was changed every 3-4 days. On days 10 and 17, all CD34 cells in the wells were removed. + Cell progeny were collected for flow cytometry analysis. 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 analyzed using CD34 + Other markers were used to determine the proliferation rate of cells. CD34 was grown ex vivo. + It was used to confirm the characteristics of the cells.

[0074] 2.1.2 Medium B In section 2.1.1 above, the culture medium was changed to medium B, which is medium A to which an additional 0.01% (w / v) of Soluplus® at a final concentration was added, and the same procedure was carried out.

[0075] 2.1.3 Medium C In section 2.1.1 above, the same procedure was performed using medium (Culture Medium C) in which 1 μM UM729 in the culture medium was replaced with 35 nM UM171 (Stemcell Technologies).

[0076] 2.1.4 Medium D In section 2.1.1 above, the culture medium was supplemented with 35 nM UM171, 10 μg / mL human low-density lipoprotein, 100 ng / mL Flt3-ligand, 100 ng / mL stem cell factor, and 50 ng / mL thrombopoietin in a StemSpan. TM The same procedure was performed using Serum-Free Expansion Medium (SFEM) (ST-09600, STEMCELL Technologies) (Culture Medium D) instead.

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

[0078] The results of the expression analysis are shown in Figure 5-12. In culture medium A, CD201 + Cells and CD90 + The proportion of cells was significantly higher (Figure 5, 27.6% and 22.9%, respectively). In culture medium B, CD201 + Cells and CD90 + The proportion of cells was significantly higher (Figure 8, 36.5% and 19.7%, respectively). In culture medium C, CD201 + Cells and CD90 + The proportion of cells was very small (Figure 10, 0.81% and 3.27%, respectively). In medium D, CD201 + Cells and CD90+ The proportion of cells was very small (Figure 12, 0.27% and 0.65%, respectively). Therefore, by using 740Y-P or butizamide and UM729, CD201 + Or CD90 + It was shown that a culture fraction concentrated with hematopoietic stem cells could be obtained.

[0079] <Example 3> 3. Analysis of hematopoietic stem cell proliferation and hematopoietic stem cell markers 3.1 Method 3.1.1 Medium B 5 μM 740Y-P PI3K activator, 0.1 μM butizamide TPO receptor agonist, 1 μM UM729 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. Human umbilical cord blood CD34, thawed according to the manufacturer's instructions, was added to the resulting medium (Medium B). + The cells were turbidified and plated in 96-well plates at a density of 10,000 cells / well. The culture medium was changed every 3-4 days. On days 10 and 17, all CD34 cells in the wells were removed. + Cell progeny were collected for flow cytometry analysis. 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 analyzed using CD34 + Other markers were used to determine the proliferation rate of cells. CD34 was grown ex vivo. + It was used to confirm the characteristics of the cells.

[0080] 3.1.2 Medium D In section 3.1.1 above, the culture medium was supplemented with 35 nM UM171, 10 μg / mL human low-density lipoprotein, 100 ng / mL Flt3-ligand, 100 ng / mL stem cell factor, and 50 ng / mL thrombopoietin in a StemSpan.TM The same procedure was performed using Serum-Free Expansion Medium (SFEM) (ST-09600, STEMCELL Technologies) (Culture Medium D) instead.

[0081] 3.1.3 Culture Medium E In section 3.1.1 above, the same procedure was performed using medium (medium E) in which 1 μM UM729 in the medium was replaced with the LSD1 inhibitor 10 μM 2-PCPA (Selleck Chemicals, S4246).

[0082] 3.2 Results The results of the expression analysis are shown in Figure 13-16. In culture medium B, CD201 + The proportion of cells was remarkably high (Figure 14, 33.0%). In culture medium D, CD201 + The proportion of cells was very small (Figure 15, 0.95%). In medium E, CD201 + The proportion of cells was significantly higher (Figure 16, 11.4%). Therefore, using 740Y-P or butizamide and 2-PCPA, CD201 + It was shown that a culture fraction concentrated with hematopoietic stem cells could be obtained.

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

Claims

1. A composition that is a culture medium for cells, comprising (a) 740Y-P or a salt thereof, (b) butizamide or a salt thereof, and (c) tranylcypromine or a salt thereof.

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

3. The composition according to claim 1, which is an albumin-free culture medium.

4. The composition according to claim 1, further comprising hematopoietic stem cells.

5. Umbilical cord blood CD34 + Further inoculation of cells leads to CD201 + The composition according to claim 1, wherein hematopoietic stem cells are concentrated.

6. CD201 + The composition contains hematopoietic stem cells, and CD201 is present in the total cells. + The composition according to claim 1, wherein the proportion of hematopoietic stem cells is 5% or more.

7. A method for producing cells, comprising a cell culture step of culturing cells using the composition described in claim 1.

8. Human cells are cultured using the composition described in claim 1, and CD201 + A method for producing cells, comprising the step of generating a cell population in which hematopoietic stem cells are concentrated.

9. Human cells are cultured using the composition described in claim 1, and CD201 + A method for producing cells for hematopoietic stem cell transplantation, comprising the step of generating a cell population in which hematopoietic stem cells are concentrated.

10. Human cells are cultured using the composition described in claim 1, and CD201 is added to the total cells in the culture medium. + A method for producing cells, comprising the step of generating a cell-containing culture medium in which the proportion of hematopoietic stem cells is 5% or more.

11. A method for culturing cells, comprising a cell culture step of culturing cells using the composition described in claim 1.

12. A method for producing a composition that is a culture medium for hematopoietic stem cells, comprising the step of mixing (a) 740Y-P or a salt thereof, (b) butizamide or a salt thereof, and (c) tranylcypromine or a salt thereof.

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