JP2019201599A - Culture medium for maintaining and culturing hematopoietic stem cell, and culture method using the same - Google Patents

Abstract

To provide culture medium for culturing a hematopoietic stem cell in a resting period while maintaining an undifferentiated state, and a culture method using the medium .SOLUTION: A resting period state and an undifferentiated state of a hematopoietic stem cell can be maintained by culturing the cell in culture medium containing 1 to 10ng/mL of stem cell factor, 0.02 to 6ng/mL of thrombopoietin concentration, and 10 to 100 mg/mL of fatty acid carrying albumin.SELECTED DRAWING: None

Description

  The present invention relates to a medium for maintaining and culturing hematopoietic stem cells, a culture method using the medium, and cells obtained by culture using the medium.

  Hematopoietic stem cells are cells that are present in the bone marrow in mammals and maintain blood cell production throughout life. In vivo, it is also known that the cell cycle is in a quiescent state and maintains an undifferentiated state (a state having both self-renewal ability and ability to produce differentiated cells (multipotency)). In addition, transplantation of hematopoietic stem cells is a radical treatment method for diseases including hematopoietic malignant tumors, and is a cell that has established clinical application.

  However, the number of donors that provide hematopoietic stem cells is limited. Although hematopoietic stem cells can be obtained from umbilical cord blood, the number of cells is small, and engraftment failure occurs at a certain rate. Therefore, attempts have been made to amplify hematopoietic stem cells outside the body in order to solve such problems. As a result, hematopoietic stem cell amplification was observed in the expression of the surface marker (hematopoietic stem cell-specific marker), and in that respect, some success was achieved (Non-Patent Documents 1 to 3). However, the amplified cells are inferior in functions such as engraftment and hematopoietic ability, and the clinical course has not been improved by using these cells.

  This is probably because functional hematopoietic stem cells cannot be amplified by such a method, and a method for maintaining undifferentiation in a culture system is not sufficiently clarified. In fact, as in vivo, the conditions for maintaining hematopoietic stem cells while maintaining quiescence (the property of keeping the cell cycle in quiescence) and undifferentiated have been rarely studied.

Science. 2010 Sep 10; 329 (5997): 1345-8 pages. Science. September 19, 2014; 345 (6203): 1509-12 pages. Cell Stem Cell. January 7, 2016; 18 (1): 144-55 Cell Stem Cell. January 3, 2013; 12 (1): 49-61

  The present invention has been made in view of the above-described problems of the prior art, and culturing hematopoietic stem cells in a stationary phase while maintaining undifferentiation (pluripotency and self-renewal ability). An object of the present invention is to provide a medium for maintaining and culturing hematopoietic stem cells reflecting the conditions, and a culture method using the same.

  The present inventors have previously found that activation of the glycolytic system in a hypoxic environment is involved in maintaining the quiescent and undifferentiated nature of hematopoietic stem cells in the body (in the bone marrow) ( Non-patent document 4). Therefore, further studies were made focusing on this hypoxic environment and two cytokines (stem cell factor (SCF) and thrombopoietin (TPO)) which are considered as essential factors for maintaining hematopoietic stem cells.

  As a result, in the culture of mouse-derived hematopoietic stem cells, the oxygen concentration is lower than the atmospheric concentration, the SCF concentration in the medium is 1 to 10 ng / mL, and the TPO concentration is 0.02 to 0.02. It was revealed that when 6 ng / mL, the stationary phase and undifferentiation of the cells are easily maintained.

  It has also been found that albumin loaded with fatty acids is essential for maintaining these properties of hematopoietic stem cells, and that these properties are more easily maintained in the presence of insulin and / or cholesterol. Became.

  Furthermore, even when hematopoietic stem cells cultured for a long time (about 1 month) under such conditions are transplanted, the cells are differentiated into lymphocytes (T cells, B cells) and myeloid in the recipient mouse. It has also been clarified that the hematopoietic stem cells obtained after the culture are functionally maintained.

  In addition, the present inventors have found that the conditions found in the mouse can be applied to the maintenance of quiescence and undifferentiation of human-derived hematopoietic stem cells, and have completed the present invention.

Therefore, the present invention relates to a medium for maintaining and culturing hematopoietic stem cells, a culture method using the medium, and cells obtained by culture using the medium, and more specifically, the following.
<1> The concentration of stem cell factor (SCF) is 1 to 10 ng / mL, the concentration of thrombopoietin (TPO) is 0.02 to 6 ng / mL, and the concentration of fatty acid-carrying albumin is 10 to 100 mg / mL. A medium for maintaining and culturing hematopoietic stem cells.
<2> The medium according to <1>, further containing at least one substance selected from the group consisting of insulin and cholesterol.
<3> The medium according to <1> or <2>, wherein the fatty acid-carrying albumin is albumin carrying at least a saturated fatty acid.
<4> A culture method for maintaining hematopoietic stem cells, comprising a step of culturing hematopoietic stem cells in the medium according to any one of <1> to <3>.
<5> The culture method according to <4>, wherein hematopoietic stem cells are cultured under an oxygen concentration of 5% by volume or less.
<6> A cell obtained by culturing hematopoietic stem cells in the medium according to any one of <1> to <3>.
<7> The cell according to <6>, wherein the culture is performed under an oxygen concentration of 5% by volume or less.
<8> A kit for producing the medium according to any one of <1> to <3>, comprising at least SCF, TPO, the fatty acid-carrying albumin, and a basal medium. kit.

  According to the present invention, hematopoietic stem cells can be cultured in a stationary phase for a long period (for example, for 1 month) while maintaining undifferentiation (multipotency and self-replication ability).

It was produced by culturing 300 mouse hematopoietic stem cells (CD150 + CD48-CD41-CD34-Flt3-Lin-Sca1 + cKit + cells (LSK)) for 7 days under various concentrations of cytokines (stem cell factor (SCF) and thrombopoietin (TPO)). It is a plot figure which shows the total cell number (log10). For the culture, a medium in which 4% by volume of bovine serum albumin (BSA) and 2-mercaptoethanol (2-ME) are added to SF-O3 medium in addition to SCF and TPO added at various concentrations is used. It was. In the figure, the left side shows the result of culturing in a 1% by volume O ₂ environment and the right side shows the result of culturing in a 20% by volume O ₂ environment. The broken line in the figure indicates the condition where the number of cells is about 300 (n = 3, from three independent experiments). It is a plot figure which shows the cell number (log10) of the megakaryocyte produced on the culture conditions similar to FIG. In the figure, the left side shows the result of culturing in a 1% by volume O ₂ environment and the right side shows the result of culturing in a 20% by volume O ₂ environment. The broken line in the figure indicates the condition that the number of megakaryocytes is about 10. It is a plot figure which shows the ratio (%) of the cell number of the hematopoietic stem cell fraction (CD150 + CD48-CD41-CD34-Flt3-LSK) with respect to the total cell number after culture | cultivating on the conditions similar to FIG. In the figure, the left side shows the result of culturing in a 1% by volume O ₂ environment and the right side shows the result of culturing in a 20% by volume O ₂ environment. It is a plot figure which shows the cell number of a hematopoietic stem / progenitor cell fraction after culture | cultivating on the conditions similar to FIG. In the figure, the upper part shows the result of culturing in a 1% by volume O ₂ environment, and the lower part shows the result in a 20% by volume O ₂ environment. The broken lines in the figure indicate the conditions under which the number of hematopoietic stem cells is about 100. “CD150 + CD48-LSK” indicates hematopoietic stem cells, and “CD150-CD48-LSK”, “CD150 + CD48 + LSK” and “CD150-CD48 + LSK” indicate pluripotent progenitor cells (MPP). FIG. 5 is a plot diagram in which “CD150 + CD48−LSK” in FIGS. 1, 2 and 4 is superimposed to show the conditions under which the total number of cells and the number of megakaryocytes are not increased and the number of hematopoietic stem cells is maintained. Each broken line in the figure shows them in FIGS. Moreover, those intersections are shown by x character. It is a microscope picture (magnification x4) which shows the result of having culture | cultivated 300 hematopoietic stem cells (CD150 + CD48-CD41-CD34-Flt3-LSK) on each condition shown in the figure for 7 days. The scale bar indicates 200 μm. In addition to the SCF and TPO shown in the figure, 4 wt% BSA and 2-ME were added to the SF-O3 medium and used for the culture. Hematopoietic stem cell fraction (CD150 + CD48-CD41-CD34 -Flt3-LSK) 300 pieces of, after culturing for 7 days under 1 vol% _{O 2} environment, shows the results of analysis in flow cytometry. For the culture, a medium prepared by adding 3 ng / mL SCF, 0.1 ng / mL TPO, 4 wt% BSA and 2-ME to SF-O3 medium was used. It is a figure which shows the result of having analyzed 300 hematopoietic stem cells (CD150 + CD48-CD41-CD34-Flt3-LSK) for 16 hours on each condition shown in the figure, and analyzing the cell cycle by EdU incorporation assay ( Mean ± standard deviation, n = 4). In the figure, the left side is a histogram showing the results of flow cytometry analysis of cells cultured for 16 hours under each condition, and the right side is a graph summarizing those results. In addition, the culture medium which added 4 weight volume% BSA and 2-ME other than SCF and TPO added at each density | concentration to SF-O3 culture medium was used for culture | cultivation. It is a scheme figure which shows the outline | summary of a hematopoietic stem cell transplantation experiment. It is a graph which shows a donor cell origin peripheral blood chimerism in a recipient mouse for 4 months after hematopoietic stem cell transplantation (mean +/- standard deviation, n = 6). For transplantation, hematopoietic stem cells cultured for 14 days under various cytokine concentration conditions were used. As a control, fresh hematopoietic stem cells were also transplanted. It is a graph which shows a donor cell origin peripheral blood chimerism in a recipient mouse for 4 months after hematopoietic stem cell transplantation (mean +/- standard deviation, n = 6). For transplantation, hematopoietic stem cells cultured for 27 days under various cytokine concentration conditions were used. As a control, fresh hematopoietic stem cells were also transplanted. Hematopoietic stem cells (CD150 + CD48-LSK) 300 pieces of, BSA, and with medium supplemented with formulation consisting water-soluble fatty acids and cholesterol, is a graph showing the number of cells after culturing for 7 days under 1 vol% _{O 2} environment (N = 3). In the figure, the numerical values (× 0 to × 1/125) on the right side of each graph indicate the dilution ratio of the preparation in each medium. In addition, the culture medium which added SCF 3ng / mL, TPO 0.1ng / mL, and 2-ME in addition to BSA and each said formulation which were added at each density | concentration to SF-O3 culture medium was used for culture | cultivation. It is a scheme figure which shows the outline | summary of a cDNA microarray experiment. It is a dot figure which shows the result of having analyzed and compared the gene expression in a fresh hematopoietic stem cell and the hematopoietic stem cell after culture | cultivation by a cDNA microarray. In the figure, gene expression data of fresh hematopoietic stem cells is shown on the x-axis, and gene expression data of hematopoietic stem cells after culture is shown on the y-axis. In addition, fatty acid biosynthesis-related genes are shown in red. Cultivation was performed for 7 days in a 1% by volume O ₂ environment in a medium in which 4% by volume BSA, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO were added to SF-O3 medium. It was. It is a dot figure which shows the result of having analyzed and compared the gene expression in a fresh hematopoietic stem cell and the hematopoietic stem cell after culture | cultivation by a cDNA microarray. In the figure, gene expression data of fresh hematopoietic stem cells is shown on the x-axis, and gene expression data of hematopoietic stem cells after culture is shown on the y-axis. In addition, fatty acid biosynthesis-related genes are indicated by red dots. For the culture, SF-O3 medium was supplemented with 4 wt% fatty acid-free BSA, sodium palmitate 200 μg / mL, sodium oleate 200 μg / mL, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO. For 7 days in a 1% by volume O ₂ environment. It is a dot figure which shows the result of having analyzed and compared the gene expression in the hematopoietic stem cell after culture | cultivation by a cDNA microarray. In the figure, gene expression data of hematopoietic stem cells after culturing in the presence of BSA is shown on the x-axis, and gene expression data of hematopoietic stem cells after culturing in the presence of fatty acid-free BSA and fatty acid is shown on the y-axis. In addition, fatty acid biosynthesis-related genes are indicated by red dots. The culture in the presence of BSA was carried out in a 1% by volume O ₂ environment in a medium in which 4% by volume BSA, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO were added to SF-O3 medium. 7 days under. In addition, the culture in the presence of fatty acid-free BSA and fatty acid was carried out in SF-O3 medium with 4 wt% fatty acid-free BSA, sodium palmitate 200 μg / mL, sodium oleate 200 μg / mL, 2-ME, 3 ng / mL SCF. And a medium supplemented with 0.1 ng / mL TPO in a 1% by volume O ₂ environment for 7 days. It is a dot figure which shows the result of having analyzed two times (# 1, # 2) independently on the same conditions, and analyzing and comparing the gene expression in the hematopoietic stem cell after those cultures by a cDNA microarray. In the figure, fatty acid biosynthesis-related genes are indicated by red dots. For the culture, SF-O3 medium was supplemented with 4 wt% fatty acid-free BSA, sodium palmitate 200 μg / mL, sodium oleate 200 μg / mL, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO. For 7 days in a 1% by volume O ₂ environment. It is a plot figure which shows the result analyzed by the gene set concentration analysis (GSEA) method about a fresh hematopoietic stem cell and a hematopoietic stem cell on the 7th day of culture. In the figure, the left side shows a GSEA plot of a hematopoietic stem cell-related gene set, and the right side shows a GSEA plot of a gene set whose expression decreases with cell proliferation. Regarding hematopoietic stem cells on day 7 of culture, hematopoietic stem cells cultured in the presence of BSA, hematopoietic stem cells cultured in the presence of fatty acid-free BSA and fatty acid # 1, and fatty acid-free BSA and The results of analysis together with hematopoietic stem cells cultured in # 2 in the presence of fatty acids are shown. Hematopoietic stem cell fractionation in the presence of a fatty acid-free BSA loaded (reconstituted) with fatty acids (sodium palmitate, sodium oleate, sodium linoleate) at various mixing ratios in a 1% by volume O ₂ environment for 7 days Is a graph showing the total number of cells and the number of hematopoietic stem cells after culturing (mean ± standard deviation, n = 4). In addition, the table | surface which shows the mixing ratio of a fatty-acid sodium salt is collectively shown on the left side in the figure. For the culture, 4 wt% fatty acid-free BSA, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO are added to SF-O3 medium, and the fatty acid salts are added at a ratio of 400 μg / mL in the ratio shown in the above table. It carried out in the culture medium further added so that it might become mL. As a control, the result of culturing without adding a fatty acid is shown as “FA-free” in the figure. Furthermore, instead of fatty acid-free BSA supporting fatty acid, the result of adding 4% by volume BSA and culturing is shown as “FA +” in the figure. Hematopoietic stem cell fraction in a 1% by volume O ₂ environment for 7 days in the presence of a fatty acid-free BSA loaded with fatty acids (sodium palmitate and sodium oleate mixed in equal mass) at various concentrations It is a graph which shows the total cell number after culture | cultivation, and the number of hematopoietic stem cells (mean +/- standard deviation, n = 4). P value is based on one-way ANOVA. In the culture, SF-O3 medium was supplemented with 4 wt% fatty acid-free BSA, 2-ME, 3 ng / mL SCF and 0.1 ng / mL TPO, and further added with fatty acids at the concentrations shown in the figure. It was done in. As a control, the result of culturing without adding a fatty acid is shown as “4% FA-free BSA” in the figure. Furthermore, the result of adding 4% by volume BSA instead of fatty acid-free BSA carrying fatty acid and culturing is shown as “4% BSA” in the figure. The number of cells (total number of cells and the number of hematopoietic stem cells) after culturing 300 hematopoietic stem cells (CD150 + CD48-Flt3-LSK) for 7 days in the presence or absence of insulin in a 1% by volume O ₂ environment, It is a graph (n = 4). Culturing was carried out in a medium with 4 wt% BSA, 3 ng / mL SCF and 0.1 ng / mL TPO added to αMEM, and with or without 4000 ng / mL insulin. Shows a hematopoietic stem cell (CD150 + CD48-Flt3-LSK ) 300 pieces of 7 days the number of cells after culturing under 1 vol% _{O 2} environment (total cell number and the number of hematopoietic stem cells) is a graph (n = 4). The P value is based on 2-way ANOVA divided by two factors, the insulin concentration and the presence or absence of cholesterol, except for the nBSA group. Regarding “nBSA” in the figure, the culture was carried out in a medium in which αMEM was added with 4 wt% BSA, 3 ng / mL SCF, 0.1 ng / mL TPO, 4000 ng / mL and insulin. For “nBSA + FA”, the culture was added to αMEM with 4 wt% BSA, 3 ng / mL SCF, 0.1 ng / mL TPO and fatty acid, and insulin was added at the indicated concentrations in the figure, and 20 μg / mL. Was performed in a medium with or without the addition of cholesterol. The fatty acids used for the culture of “nBSA + FA” are sodium palmitate 320 μg / mL, sodium oleate 240 μg / mL, sodium linoleate 160 μg / mL, and sodium stearate 80 μg / mL. Human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA- ) 600 pieces of each concentration under the SCF and TPO, under 1 vol% _{O 2} environment shows after culturing for 7 days, total cell number and the number of hematopoietic stem cells, plot (N = 3). In addition, the culture medium which added 4 weight volume% BSA and 2-ME other than SCF and TPO added at each density | concentration to SF-O3 culture medium was used for culture. Human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA- cells) each concentration under the conditions of 600 SCF and TPO, under 1 vol% _{O 2} environment, and cultured for 4 weeks, total cell count and hematopoietic undifferentiated cells per week (CD34 + CD38 -A graph showing the number of cells (mean ± standard deviation, n = 4). In the figure, “S3T3”, “S10T10” and “S100T100” indicate that the concentrations of SCF and TPO added to the medium in each culture were 3 ng / mL and 3 ng / mL, 10 ng / mL and 10 ng / mL, and 100 ng / mL, respectively. mL and 100 ng / mL. After culturing 600 human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA− cells) in the presence or absence of BSA in a 1% by volume O ₂ environment for 7 days, the total cell, hematopoietic undifferentiated cell fraction (CD34 + CD38−) ) And the number of cells in the hematopoietic stem cell fraction (mean ± standard deviation, n = 4). In addition, in addition to SCF and TPO added at the concentrations shown in the figure, 2-ME is added to the SF-O3 medium, and further 4 wt% BSA is added to the culture ("+ BSA" in the figure). ) Or not added (indicated by “-BSA” in the figure). In the figure, “S3T3” and “S10T10” indicate that the addition concentrations of SCF and TPO to the medium in each culture are 3 ng / mL and 3 ng / mL, and 10 ng / mL and 10 ng / mL, respectively. . After culturing 600 human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA− cells) in the presence or absence of cholesterol in a 1% by volume O ₂ environment for 7 days, the total cell, hematopoietic undifferentiated cell fraction (CD34 + CD38−) ) And the number of cells in the hematopoietic stem cell fraction (mean ± standard deviation, n = 4). For the culture, in addition to fatty acid (FA) added to the SF-O3 medium at the concentration shown in the figure, 4 wt% BSA, 3 ng / mL SCF, 3 ng / mL TPO and 2-ME were added. A medium with or without added cholesterol was used. The fatty acid used was a mixture of sodium palmitate, sodium oleate, sodium linoleate and sodium stearate in a weight ratio of 4: 3: 2: 1. Further, in the figure, “FA0”, “FA250”, “FA750”, and “FA1000” indicate that the concentration of fatty acid added to the medium is 0, 250.750, and 1000 μg / mL. P value is based on 2-way ANOVA divided by two factors: fatty acid concentration and the presence or absence of cholesterol. Total cells, hematopoietic undifferentiated cell fraction (CD34 + CD38-) and hematopoietic stem cell fraction cells after culturing 600 human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA- cells) in a 1% by volume O ₂ environment for 7 days It is a graph which shows a number (mean +/- standard deviation, n = 4). In addition, for cultivation, in addition to fatty acid (FA) and cholesterol (Chol) added to SF-O3 medium at the concentrations shown in the figure, 2-ME, 3 ng / mL SCF, and 3 ng / mL TPO And a medium supplemented with 4 wt% BSA or 4 wt% FA-free BSA was used. The fatty acid used was a mixture of sodium palmitate, sodium oleate, sodium linoleate and sodium stearate in a weight ratio of 4: 3: 2: 1. In the figure, “FA0”, “FA100”, “FA200”, “FA300” and “FA400” indicate that the concentration of fatty acid added to the medium is 0, 100.200, 300 and 400 μg / mL. Show. “Chol0”, “Chol20” and “Chol40” indicate that the concentrations of cholesterol added to the medium are 0, 20 and 40 μg / mL. P value is based on 2-way ANOVA divided by two factors of fatty acid concentration and the presence or absence of cholesterol (mean ± standard deviation, n = 4). Total cells, hematopoietic undifferentiated cell fraction (CD34 + CD38-) and hematopoietic stem cell fraction cells after culturing 600 human hematopoietic stem cells (CD34 + CD38-CD90 + CD45RA- cells) in a 1% by volume O ₂ environment for 7 days It is a graph which shows a number (mean +/- standard deviation, n = 4). In addition, for culture, a medium in which 2-ME, 4% by volume BSA, fatty acid, and 20 μg / mL cholesterol were added to αMEM medium in addition to SCF, TPO and insulin added at the concentrations shown in the figure. Was used. As the fatty acid, a mixture of sodium palmitate 80 μg / mL, sodium oleate 60 μg / mL, sodium linoleate 40 μg / mL and sodium stearate 20 μg / mL was used. In the figure, “S3T3”, “S3T6”, “S6T3” and “S6T6” indicate that the addition concentrations of SCF and TPO to the medium in each culture were 3 ng / mL and 3 ng / mL, 3 ng / mL and 6 ng / mL, 6 ng / mL and 3 ng / mL, and 6 ng / mL and 6 ng / mL. The addition concentration of insulin was 0, 0.4, 40, or 4000 ng / mL. P value is based on 2-way ANOVA divided by two factors: cytokine conditions and insulin concentration.

<Hematopoietic stem cell culture medium>
The present invention relates to a medium for maintaining and culturing hematopoietic stem cells, and more particularly, containing 1 to 10 ng / mL stem cell factor (SCF), 0.02 to 6 ng / mL thrombopoietin, and 10 to 10 It is a culture medium characterized by containing 100 mg / mL fatty acid-carrying albumin.

  And by using such a medium, hematopoietic stem cells are maintained in an undifferentiated state (pluripotency and self-renewal ability) in a stationary phase, even for a long period (for example, for one month) It can be cultured.

  In the present invention, the “hematopoietic stem cell” is a cell having multipotency capable of differentiating into all blood cell differentiation lineages of blood cells and capable of self-replication while maintaining the multipotency.

  The animal species from which hematopoietic stem cells are derived is not particularly limited, but vertebrates are preferred, and mammals are more preferred. Examples of mammals include primates such as humans, monkeys and marmosets, rodents such as mice and rats, rabbit eyes such as rabbits, ungulates such as pigs, sheep, cows, goats and horses, and dogs And cats such as cats, but are not limited thereto. The hematopoietic stem cells according to the present invention are preferably hematopoietic stem cells derived from rodents such as mice and primates such as humans, more preferably hematopoietic stem cells derived from humans.

  The tissue derived from hematopoietic stem cells is not particularly limited as long as the cells are present, but is preferably a hematopoietic tissue, and examples include bone marrow, umbilical cord blood, peripheral blood, and liver.

  Hematopoietic stem cells are usually isolated from the tissue using the expression of a surface marker (hematopoietic stem cell-specific marker) specific to the cell as an index. Regarding the expression of such markers, hematopoietic cells derived from humans are usually CD34 positive (+) CD38 negative (−) CD90 positive (+) CD45RA negative (−) cells, preferably CD34 + CD38−CD90 + CD45RA−CD49f + cells. . Further, the mouse-derived hematopoietic cells are usually CD150 + CD48-Flt3-LSK, preferably CD150 + CD48-CD41-CD34-Flt3-LSK. “LSK” refers to Lin-Sca1 + cKit + cells.

  In the present invention, “stem cell factor (SCF)” added to the medium to maintain hematopoietic stem cells is a kind of hematopoietic factor (cytokine) and is a ligand of a tyrosine kinase receptor encoded by the KIT locus. . It is also referred to as KIT ligand or KITLG. Typically, it is a protein consisting of the amino acid sequence described in NCBI reference sequence: NP_000890 or NP_003985 if derived from human, and NCBI reference sequence: NP_001334085 or derived from mouse. A protein comprising the amino acid sequence described in NP_038626.

  The concentration of SCF in the culture medium of the present invention is 1 to 10 ng / mL, but the upper limit is preferably 7.5 ng / mL from the viewpoint that it is easier to maintain the resting phase and undifferentiation of hematopoietic stem cells. More preferably, it is 5 ng / mL, More preferably, it is 3.5 ng / mL. From the same viewpoint, the lower limit of the SCF concentration is preferably 1.5 ng / mL, more preferably 2 ng / mL, and further preferably 2.5 ng / mL. The SCF concentration is particularly preferably about 3 ng / mL, and most preferably 3 ng / mL.

  In the present invention, “about” is used to mean ± 10%. Further, the “concentration” according to the present invention means the final concentration in the medium of the present invention, except for the oxygen and carbon dioxide concentrations described below, unless otherwise specified.

  In the present invention, “thrombopoietin (TPO)” added to a medium to maintain hematopoietic stem cells is a kind of hematopoietic factor (cytokine), and is a glycoprotein involved in the proliferation and differentiation of platelet progenitor cells (megakaryocytes) It is. Also referred to as THPO, thrombopoiesis stimulating factor (TSF), megakaryocyte colony stimulating factor (MK-CSF), megakaryocyte stimulating factor or megakaryocyte (megakaryocyte) amplification factor, NCBI reference sequence: NP_000451, NP_001171068, NP_001171069, NP_001276926 or NP_001276927 is a protein consisting of the amino acid sequence described in NCBI reference sequence: NP_001166976, NP_001276823, NP_001276825 or NP_001276805 is there.

  The concentration of TPO in the medium of the present invention is 0.02 to 6 ng / mL, but from the viewpoint that it is easier to maintain the stationary phase and undifferentiation of hematopoietic stem cells, the upper limit is preferably 5 ng / mL. Yes, more preferably 4.5 ng / mL, still more preferably 4 ng / mL, more preferably 3.5 ng / mL. From the same viewpoint, the lower limit of the TPO concentration is preferably 0.04 ng / mL, more preferably 0.05 ng / mL, still more preferably 0.06 ng / mL, more preferably 0.08 ng. / ML. When maintaining mouse-derived hematopoietic stem cells, the concentration of TPO is preferably 0.08 to 0.12 ng / mL, particularly preferably about 0.1 ng / mL, and most preferably 0.1 ng. / ML. When maintaining human-derived hematopoietic stem cells, the concentration of TPO is preferably 2 to 4 ng / mL, particularly preferably about 3 ng / mL, and most preferably 3 ng / mL.

  In the present invention, “albumin” used to maintain hematopoietic stem cells is a protein having a high ability to bind to various substances (fatty acids, enzymes, hormones, inorganic ions). Any albumin may be used as long as it has an ability to bind to a fatty acid. Examples include albumin derived from serum, albumin derived from egg white, albumin derived from milk, and albumin derived from serum is preferable. It should be noted that serum-derived albumin is bound to various substances contained in serum, and in the case of fatty acids, it has the ability to bind to two normal fatty acid molecules per albumin molecule.

  Moreover, as shown in the below-mentioned Example, in order to maintain a hematopoietic stem cell, it is necessary to supply a fatty acid via albumin. Therefore, albumin needs to carry a fatty acid in the medium of the present invention. The fatty acid carried by albumin is not particularly limited, and may be a saturated fatty acid (for example, a saturated fatty acid having 8 to 20 carbon atoms such as palmitic acid or stearic acid), or an unsaturated fatty acid (for example, oleic acid, It may be an unsaturated fatty acid having 16 to 20 carbon atoms such as linoleic acid. From the viewpoint of easier maintenance of hematopoietic stem cells, saturated fatty acids are preferable, saturated fatty acids having 12 to 20 carbon atoms are more preferable, saturated fatty acids having 14 to 18 carbon atoms are more preferable, and palmitic acid is particularly preferable.

  Fatty acids to be supported on albumin are in the form of salts (for example, alkali metal salts such as fatty acid sodium and fatty acid potassium, alkaline earth metal salts such as fatty acid magnesium, fatty acid calcium and fatty acid barium, and ammonia salt (fatty acid ammonium)). It may also be in the form of an ester.

  In addition, the fatty acid supported on albumin may be a single type (for example, palmitic acid only) or a plurality of types (for example, a mixture of palmitic acid and oleic acid). Preferable examples of the fatty acid supported on albumin include a fatty acid mixture containing at least palmitic acid, a mixture of palmitic acid and oleic acid (1: 1 by weight), palmitic acid, oleic acid, linoleic acid and And a mixture of stearic acid (4: 3: 2: 1 by weight). The former ratio is based on the fact that bovine serum albumin carries saturated fatty acid and unsaturated fatty acid at a ratio of about 1: 1 as a result of measurement by the present inventors. The latter is based on the fatty acid composition ratio in human bone marrow (see J Cell Biochem. October 2016; 117 (10): 2370-2376).

  In addition, if the fatty acid-carrying albumin is derived from nature such as serum, since the fatty acid is usually carried by albumin, it can be used as it is, but as described above, when a predetermined fatty acid is used in combination. Can be prepared by loading a desired fatty acid on albumin (such as fatty acid-free (free) albumin). There is no restriction | limiting in particular about such preparation, If it is an expert, it can carry out by selecting a well-known method suitably. For example, as shown in the examples described later, a desired fatty acid is solid-phased in advance in an incubator, a medium containing albumin (fatty acid-free albumin, etc.) is added to the incubator, and treatment such as ultrasonic disruption is performed. By dissolving the fatty acid, the fatty acid can be supported on the albumin.

  The concentration of the fatty acid-carrying albumin in the medium of the present invention is 10 to 100 mg / mL (1 to 10% by weight), but from the viewpoint that hematopoietic stem cells are more easily maintained, the upper limit is preferably 80 mg / mL. Yes, more preferably 70 mg / mL, still more preferably 60 mg / mL, more preferably 50 mg / mL. From the same viewpoint, the lower limit of the TPO concentration is preferably 15 mg / mL, more preferably 20 mg / mL, still more preferably 25 mg / mL, and more preferably 30 mg / mL. The concentration of fatty acid-carrying albumin is particularly preferably 35 to 45 mg / mL, more preferably about 40 mg / mL, and most preferably 40 mg / mL.

  The upper limit of the fatty acid concentration in the medium of the present invention is preferably 1 mg / mL, more preferably 500 μg / mL, and even more preferably 200 μg / mL, from the viewpoint that hematopoietic stem cells can be more easily maintained. It is. From the same viewpoint, the lower limit of the fatty acid concentration is preferably 10 μg / mL, more preferably 50 μg / mL, still more preferably 100 μg / mL, and more preferably 250 μg / mL.

  In addition, regarding the above-mentioned SCF, TPO and albumin, which are essential components of the medium of the present invention, there is no particular limitation on the origin of these animal species, and vertebrates are preferred, and mammals are more preferred, as is the origin of the above hematopoietic stem cells. . In addition, when the cells obtained in the present invention are used for treatment in humans described later, it is desirable that they are derived from humans from the viewpoint of suppressing rejection. In addition, natural proteins isolated from the animals may be used as these proteins, but genetic recombinants (so-called recombinant proteins) prepared by genetic engineering may be used. In addition, as long as hematopoietic stem cells can be maintained in quiescent and undifferentiated state, it is not necessary to include the full length of these proteins, and part of their amino acid sequences may be deleted or replaced by other amino acids, Or a derivative in which one or more amino acids are bonded to the N-terminal and / or C-terminal, or a sugar chain is deleted or substituted.

  The medium to which the above-mentioned SCF, TPO and fatty acid-carrying albumin are added (so-called basal medium) is not particularly limited, such as a medium used for normal cell culture or a medium prepared for culturing hematopoietic cells. Can be used. For example, examples of a medium used for normal cell culture include αMEM, DMEM, Ham F12 medium, DMEM / F12 medium, RPMI1640 medium, IMEM, McCoy's 5A medium, and EMEM. As a medium used for culturing hematopoietic cells, S-Clone medium (for example, SF-O3 medium, manufactured by AEDIA), StemPro34 (manufactured by Invitrogen), Stemline (manufactured by Sigma-Aldrich), Stemline II (manufactured by Sigma-Aldrich) ), X-VIVO 10 (Lonza), X-VIVO 15 (Lonza), X-VIVO 20 (Lonza), HPGM (Lonza), StemSpan H3000 (Stem Cell Technology), StemSpanSFEM ( Stem Cell Technology Co., Ltd.) and QBSF-60 (Quality Biological Co., Ltd.). In the present invention, these media may be appropriately mixed and used.

  The basal medium according to the present invention is not limited to the above examples, but is preferably SF-O3 medium, αMEM, DMEM / F12 medium from the viewpoint that it has been used in the culture of hematopoietic stem cells. More preferred is SF-O3 medium. Also, DMEM / F12 medium containing insulin, selenite and transferrin instead of SF-O3 medium; insulin, iron nitrate (III), iron sulfate (II), copper sulfate, zinc sulfate, putrescine, lipoic acid RPMI1640 medium containing, alanine, pyruvic acid, selenite and transferrin; αMEM containing insulin and nucleoside is also preferably used as the basal medium according to the present invention.

  The medium of the present invention may further contain a well-known and commonly used medium additive in addition to SCF, TPO, fatty acid-carrying albumin and the like. Examples of such medium additives include functional proteins (insulin, transferrin, lactoferrin, etc.), lipids other than fatty acids (cholesterol, etc.), reducing agents (2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.). Amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides (nucleoside, cytidine, adenosine 5′-monophosphate, hypoxanthine, thymidine, etc.), metal salts ( Iron nitrate (III), iron sulfate (II), copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), Vitamins and inorganic compounds Selenious acid), organic compounds (paraaminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvate, lactic acid, triiodothyronine, etc.), antibiotics (penicillin, streptomycin, etc.), buffer compounds ( HEPES, sodium bicarbonate, etc.) and pH indicators (phenol red, etc.), but are not limited thereto.

  In addition, regarding these medium additives, it is desirable that the medium of the present invention contains insulin and / or cholesterol from the viewpoint that hematopoietic stem cells can be more easily maintained.

  From the above viewpoint, the concentration of insulin in the medium of the present invention is preferably 40 to 4000 ng / mL, more preferably 200 to 800 ng / mL, and further preferably about 400 ng / mL. Moreover, it is preferable that it is 10-100 microgram / mL from the same viewpoint, and, as for the density | concentration of the cholesterol in the culture medium of this invention, it is more preferable that it is 20-40 microgram / mL.

  The medium of the present invention preferably contains 2-mercaptoethanol. In such a case, the concentration in the medium is usually 10 to 100 μM, preferably 30 to 80 μM, more preferably 50 to 60 μM. (For example, 55 μM).

<Culture of hematopoietic stem cells>
As shown in Examples described later, by using the above-mentioned medium, hematopoietic stem cells can be cultured for a long period of time in an quiescent state while maintaining undifferentiation (multipotency and self-renewal ability). . Therefore, this invention provides the culture method for maintaining a hematopoietic stem cell including the process of culturing a hematopoietic stem cell in the above-mentioned culture medium.

  In the culture method of the present invention, from the viewpoint of easier maintenance of hematopoietic stem cells, the oxygen concentration is lower than the normal atmospheric oxygen concentration (20% by volume) (also referred to herein as “hypoxic conditions”). ). As low oxygen conditions, the oxygen concentration in the gas in contact with the culture medium during culture is preferably 1 to 15% by volume, more preferably 1 to 10% by volume, and even more preferably 1 to 5% by volume. %. Moreover, the density | concentration of the carbon dioxide in the said gas is 1-10 volume% normally, Preferably it is 2-5 volume%. The culture under such a low oxygen concentration can be performed, for example, by culturing in a commercially available low oxygen incubator. The culture temperature is not particularly limited, but is usually 30 to 40 ° C, preferably about 37 ° C.

  As the incubator used for culturing hematopoietic stem cells, a known one used for culturing hematopoietic cells and the like can be used as appropriate, and a non-cell-adhesive incubator may be used. An incubator (for example, an incubator coated with a cell support substrate such as ECM, Matrigel, gelatin, collagen) may be used. The culture of hematopoietic stem cells may be non-adherent culture or adhesive culture. Examples of non-adherent culture include dispersion culture, agglutination suspension culture, and suspension culture on a carrier. Furthermore, from the viewpoint of reproducing the environment in the bone marrow and making it easier to maintain hematopoietic stem cells, co-culture with bone marrow stromal cells may be used.

  Moreover, hematopoietic stem cells used for the culture of the present invention are as described above, and can be prepared from mammalian hematopoietic tissues and the like. In this preparation, when the hematopoietic tissue or the like is a cell mass, first, individual cells are dissociated by treatment with protease, collagenase, or the like. On the other hand, if cells are separated like blood (peripheral blood or the like), the cells are subjected to the following hematopoietic stem cell isolation without cell separation treatment.

  Isolation of hematopoietic stem cells is not particularly limited, and can be performed by appropriately using known techniques. For example, as shown in Examples described later, cells having hematopoietic stem cell-specific marker characteristics can be isolated using a cell sorter, magnetic beads, or the like using an antibody against the above-mentioned hematopoietic stem cell-specific marker.

The concentration of the hematopoietic stem cells prepared in this manner is not particularly limited, but is usually 5 × 10 ^{2 to} 5 × 10 ³ cells / mL, preferably 1 × 10 ³ to 3 × 10 ³ pieces / mL.

  More preferable conditions for the culture of the present invention include the following (note that the SF-03 medium used as the basal medium under the following conditions originally contains insulin).

  For example, the conditions for maintaining and culturing mouse-derived hematopoietic stem cells include 2 to 4 ng / mL (eg, 3 ng / mL) SCF, 0.08 to 0.12 ng / mL (eg, 0.1 ng / mL) TPO. In SF-03 medium containing 35-45 mg / mL (eg, 40 mg / mL) serum albumin and 50-60 μM (eg, 55 μM) 2-mercaptoethanol to about 37 ° C. and 1% by volume oxygen concentration And culturing.

  The conditions for maintaining and culturing human-derived hematopoietic stem cells include 2 to 4 ng / mL (eg, 3 ng / mL) SCF, 2 to 4 ng / mL (eg, 3 ng / mL) TPO, and 35 to 45 mg / mL. SF containing (eg 40 mg / mL) serum albumin, 100-250 μg / mL fatty acid containing at least palmitic acid, 20-40 μg / mL cholesterol and 50-60 μM (eg 55 μM) 2-mercaptoethanol Incubating in a -03 medium at about 37 ° C. and 1% by volume oxygen concentration. In this culture condition, αMEM containing about 400 ng / mL insulin can be used as the basal medium instead of the SF-03 medium. Moreover, it is desirable that αMEM used as the basal medium contains 0.2 to 20 μg / mL (more preferably about 2 μg / mL) of thymidine.

<Cultivated hematopoietic stem cells>
As shown in Examples described later, in the cell group obtained by culturing hematopoietic stem cells in the above-mentioned medium, the stationary phase and undifferentiation of the cells are maintained high. Such cells are useful in the treatment and screening of diseases related to the hematopoietic function described below. Therefore, the present invention also provides a cell obtained by culturing hematopoietic stem cells in the above-mentioned medium.

  The method for culturing hematopoietic stem cells is as described above, and the culture period is usually 1 to 31 days, preferably 5 to 14 days.

  In the cells obtained through the above-described culture, the ratio of hematopoietic stem cells is usually 30% or more, preferably 50% or more, more preferably 70% or more. The ratio of hematopoietic stem cells can be identified by a cell sorter or the like using an antibody against hematopoietic stem cell-specific markers, as shown in Examples described later.

  Whether the cultured cells function as hematopoietic stem cells can be confirmed by transplanting the cells into immunodeficient mice (eg, NOG mice, NOD / SCID mice). That is, as shown in the examples described later, by transplanting the cells of interest into the mouse, and detecting the migration / engraftability of the transplanted cells into the bone marrow, pluripotency, bone marrow remodeling ability, etc. It can be confirmed that the function as a hematopoietic stem cell is maintained. Furthermore, the bone marrow of an immunodeficient mouse once transplanted is collected and re-transplanted (secondary transplantation) into another immunodeficient mouse mouse to measure the self-replication ability and long-term bone marrow remodeling ability of the transplanted cells. Is possible.

<Pharmaceutical composition, treatment of diseases related to hematopoietic function>
As shown in the examples below, when cells obtained by culturing using the medium of the present invention are transplanted into a recipient, they can be engrafted and reconstituted with bone marrow function (hematopoietic ability). Can be used as a graft for hematopoietic stem cell therapy in place of conventional bone marrow transplantation or umbilical cord blood transplantation.

  Therefore, the present invention. Provided is a pharmaceutical composition containing, as an active ingredient, cells obtained by culturing hematopoietic stem cells in the medium described above.

  The pharmaceutical composition of the present invention can be produced as parenteral preparations such as injections, suspensions, drops, etc. by mixing pharmaceutically acceptable carriers according to conventional means. Examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride) containing physiological saline, glucose and other adjuvants. An aqueous liquid for injection can be mentioned. The preparation includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride), a stabilizer (eg, serum albumin, polyethylene glycol), You may mix | blend with a preservative, antioxidant, etc.

  Hematopoietic stem cells are effective in the treatment of various diseases in addition to the treatment of leukemia and the like that require bone marrow transplantation. Therefore, the hematopoietic stem cells maintained by the method of the present invention can be used for diseases associated with hematopoietic cell decrease and / or decreased hematopoietic function, diseases associated with hematopoietic dysfunction, immune cell decreased, immune cell increased, diseases associated with autoimmunity, immune dysfunction Effective for ischemic diseases.

  Specific examples of diseases relating to such hematopoietic function include blood cancer such as acute or chronic leukemia, chronic granulomatosis, severe combined immunodeficiency syndrome, adenosine deaminase (ADA) deficiency, agammaglobulinemia, Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, acquired immune deficiency syndrome (AIDS) and other immunodeficiency syndromes, C3 deficiency, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, Gaucher disease, Lysosomal storage diseases such as mucopolysaccharidosis, adrenoleukodystrophy, Fanconi syndrome, aplastic anemia, granulocytopenia, lymphopenia, thrombocytopenia, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura Disease, Kasabach-Merritt syndrome, malignant lymphoma, Hodgkin's disease, multiple myeloma, Sexual hepatopathy, autoimmune hemolytic anemia, myelodysplastic syndromes, polycythemia vera, red rose multi sclerosis, essential thrombocythemia, myeloproliferative disease, spinal cord injury due to trauma or the like, and the like.

  In addition, when bone marrow suppression occurs as a side effect due to chemotherapy, radiation therapy, etc. for solid cancer patients, bone marrow is collected before the operation, and hematopoietic stem cells maintained by the method of the present invention are returned to the patient after the operation, Early recovery from hematopoietic disorders. This method makes it possible to perform more powerful chemotherapy and the like and improve the treatment effect.

  Furthermore, the cells contained in the pharmaceutical composition of the present invention may be cells that have been genetically modified in order to perform gene therapy. The gene to be modified may be selected according to the target disease, and is not particularly limited. Examples thereof include genes such as hormones, cytokines, receptors, enzymes, and polypeptides. In addition, genetic modification can be appropriately performed by those skilled in the art using known methods. For example, gene introduction using a viral vector, genome editing, knockout method, siRNA method, shRNA method, ribozyme method, Sense methods can be used as appropriate. Furthermore, such treatment by genetic modification for hematopoietic stem cells can be applied to the treatment of congenital genetic diseases. Examples of such treatment include introduction of ADA gene for adenosine deaminase (ADA) deficiency, X-linked severe combined immunodeficiency Introduction of the γc chain gene in the disease (X-SCID), introduction of the gp91-phox gene in the chronic granulomatosis, and introduction of the glucocerebrosidase gene in the Gaucher disease.

  The present invention also provides a method for treating a disease related to hematopoietic function, wherein a cell obtained in the present invention or a pharmaceutical composition containing the cell as an active ingredient is administered to a patient.

  The cell, the pharmaceutical composition and the disease relating to the hematopoietic function are as described above. In addition, the administration method of the cells and the like varies depending on the age, weight, sex, condition, etc. of the administration subject, but can be administered through any administration route of oral administration or parenteral administration (eg, intravenous administration, arterial administration, local administration). Can be administered. The dose of the cells also varies depending on the age, weight, sex, condition, etc. of the subject, but can be adjusted as appropriate by those skilled in the art.

<Screening method>
As shown in Examples described later, the culture method of the present invention can maintain its quiescent and undifferentiated properties as in the case of hematopoietic stem cells in vivo. Therefore, the present invention can be suitably used for screening a compound having an activity of amplifying hematopoietic stem cells while maintaining the function.

Therefore, the present invention
(I) a step of culturing hematopoietic stem cells in the presence of a test compound in the medium of the present invention described above,
(Ii) measuring the number of hematopoietic stem cells after the culture;
(Iii) If the number of cells detected in the step is greater than the number of hematopoietic stem cells after culturing in the absence of the test compound, the test compound is a compound having an activity of amplifying hematopoietic stem cells. A screening method for a compound having an activity of amplifying hematopoietic stem cells, comprising a step of determining is provided.

  The culture medium and the culture method using the same in such screening are as described above. The “test compound” is not particularly limited, but is a synthetic low molecular weight compound library, gene library expression product, peptide library, antibody, bacterial release substance, cell (microorganism, plant cell, animal cell) extract. The compound can be screened by using culture supernatant, purified or partially purified polypeptide, marine organism, plant or animal extract, soil, random phage peptide display library. Moreover, there is no restriction | limiting in particular about a culture period, Usually, it is 1-31 days, Preferably it is 5-14 days. Furthermore, the number of hematopoietic stem cells after culture can be measured by a cell sorter using an antibody against a hematopoietic stem cell-specific marker, as described above.

  In addition, when a hematopoietic stem cell to be maintained and cultured in the present invention is derived from a disease related to a hematopoietic function, a compound having therapeutic activity against the disease can be screened.

Therefore, the present invention
(I) a step of culturing hematopoietic stem cells derived from a patient suffering from a disease related to hematopoietic function in the medium of the present invention described above in the presence of a test compound;
(Ii) detecting the state of the hematopoietic stem cells after the culture;
(Iii) If the state of the hematopoietic stem cell detected in the step is better than the state of the hematopoietic stem cell after culturing in the absence of the test compound, the test compound is a compound having an activity of treating the disease And a method for screening a compound having a therapeutic activity against a disease relating to hematopoietic function, comprising the step of determining.

  The culture medium, the culture method using the same, the test compound, and the culture period in such screening are as described above. In addition, the “disease related to hematopoietic function” is not particularly limited, and examples thereof include the diseases described above in <Pharmaceutical composition, treatment of diseases related to hematopoietic function, etc.> Furthermore, the “state of hematopoietic stem cells” to be detected may be appropriately selected depending on the target disease, and is not particularly limited. For example, the number of hematopoietic stem cells, the size of colonies formed, the cell surface marker Expression patterns, hematopoietic stem cell undifferentiation, stationary phase and function (engraftability, hematopoietic ability) can be mentioned.

<Kit etc. for preparing medium for hematopoietic stem cells>
Physiologically active substances such as SCF, TPO and fatty acid-carrying albumin gradually lose their activity in the medium. Therefore, it is desirable to prepare the medium of the present invention at the time of culturing hematopoietic stem cells, as shown in the Examples described later. Therefore, the present invention provides a kit for producing a culture medium for maintenance culture of hematopoietic stem cells, comprising SCF, TPO, fatty acid-carrying albumin, and basal medium.

  The components of the kit (SCF, TPO, fatty acid-carrying albumin, basal medium, etc.) are as described above, but fatty acid-carrying albumin is contained in the kit in a form in which fatty acid and albumin are separated. Also good. Moreover, the culture medium preparation kit of the present invention may further contain the above-mentioned culture medium additive, and can further include instructions indicating the concentration of each substance in the culture medium.

  The kit may contain the predetermined amount of SCF, TPO, and fatty acid-carrying albumin so that the medium of the present invention can be prepared simply by mixing. That is, the culture medium preparation kit of the present invention includes “SCF, TPO, fatty acid-carrying albumin, and basal medium, and the amount of the SCF, TPO, and fatty acid-carrying albumin is 1 mL of the basal medium. A mode of “a kit for preparing a culture medium for maintenance culture of hematopoietic stem cells, which is 1 to 10 ng, 0.02 to 6 ng, and 10 to 100 mg” can also be taken. For more preferable amounts of SCF, TPO and fatty acid-carrying albumin, see the description of <Hematopoietic stem cell medium> above.

  The present invention also provides a kit for maintaining and culturing hematopoietic stem cells, including the medium preparation kit. In the kit, in addition to the above-mentioned components, the above incubator, bone marrow stromal cell, hypoxic incubator, etc. can be included, and for confirming that hematopoietic stem cells are maintained in the cultured cells. Reagents can also be included. Examples of the reagent include an antibody that recognizes a hematopoietic stem cell-specific marker, a labeled secondary antibody that recognizes the antibody, and magnetic beads. Further, the kit can include an instruction manual describing conditions (oxygen concentration) for maintaining and culturing hematopoietic stem cells.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example. First, materials and methods used in this example will be described below.

(mouse)
C57BL / 6J mice (C57BL / 6-Ly5.2 mice, 8-14 weeks old, purchased from Japan SLC or Japan Clare) were used in all experiments. In addition, C57BL / 6-Ly5.1 congenic mice purchased from CLEA Japan were used as recipient mice in the hematopoietic stem cell transplantation experiment (competitive reconstitution assay) described below.

  All mice were housed under specific pathogen-free (SPF) conditions in an animal facility at the National Center for Global Health and Medicine in a freely drinkable environment. For experiments, both male and female mice were used. During the experiment, mice were euthanized by cervical dislocation. The animal experiment was conducted after approval by the National International Medical Research Center Animal Experiment Committee.

(Preparation of mouse c-Kit positive cells)
Mouse bone marrow cells were isolated from two femurs and tibia. Specifically, first, using a 10 mL syringe (manufactured by Terumo) equipped with a 21 gauge needle (manufactured by Terumo), 2% by volume fetal bovine serum (FBS) -containing phosphate buffered saline (PBS) was added to the femur. Bone marrow plugs were collected by flushing into the tibia and. Next, the bone marrow plug was finely crushed by putting it in and out of a 21 gauge needle, and further subjected to centrifugation at 4 ° C. and 680 g for 5 minutes by a centrifuge (KUBOTA 5930, manufactured by KUBOTA). Then, the supernatant was removed, and the obtained cells were suspended in a buffer solution (0.17 M NH ₄ Cl, 1 mM EDTA, 10 mM NaHCO ₃ ) at room temperature for 5 minutes. Further, twice the volume of 2% FBS-containing PBS was added, and the cells were washed by centrifugation at 680 g for 5 minutes at 4 ° C. The cells were then resuspended in PBS containing 2% FBS and filtered through a 40 μm nylon mesh (BD Biosciences). The obtained filtrate was again subjected to 680 g centrifugation at 4 ° C. for 5 minutes to recover bone marrow cells.

  An anti-CD16 / 32 antibody that blocks the binding between non-specific IgG and Fc receptor (hereinafter also referred to as “Fc block”; BD biosciences, 2 μL used per mouse) is added to the recovered bone marrow cells. Incubated at 4 ° C for 5 minutes. Subsequently, anti-c-Kit magnetic beads (manufactured by Miltenyi) were added to 20% by volume of the total volume of the reaction solution, and incubated at 4 ° C. for 15 minutes. Thereafter, the antibody was removed by washing twice with PBS containing 2% by volume FBS, and then c-Kit + cells were isolated using a cell sorter (manufactured by Miltenyi, product name: autoMACS Pro, Possel-s mode). . The isolated cells were subjected to centrifugation at 340 g for 5 minutes and then stained with the following antibody for flow cytometry (antibody panel).

(Preparation of mouse hematopoietic stem cells, etc.)
After selecting c-Kit + cells using the magnetic beads, the isolated cells were subjected to centrifugation at 340 g for 5 minutes, and the cell fractions of mouse hematopoietic stem cells and hematopoietic progenitor cells were labeled using the following antibody panel.

  Antibody panel: differentiation antigen markers (CD4, CD8a, Gr-1, Mac-1, Ter-119, B220) -PerCP-Cy5.5, c-Kit-APC-Cy7, Sca-Cy7, CD150-PE, CD41- FITC, CD48-FITC, Flt3-APC, CD34-BV421. All antibodies were used in an amount of 0.5 μL per mouse.

  The labeled cells were resuspended in 0.5 to 2 mL of PBS containing 2% by volume FBS and 0.1% by volume PI, and subjected to a cell sorter (product name: FACS Aria (registered trademark) II, manufactured by BD Biosciences). Sorted cells were collected in SF-O3, αMEM or DMEM / F12 described later, containing 4% by volume BSA. In addition, when the mouse-derived cells were subjected to the culture in the absence of fatty acid, the sorted cells were collected using a medium containing fatty acid-free BSA instead of the medium containing 4% by weight BSA.

  In this sorting, mouse hematopoietic stem cells were defined as CD150 + CD41-CD48-CD34-Flt3-LSK cells, CD150 + CD41-CD48-LSK cells, or CD150 + CD48-LSK cells. In addition, MPP1 (CD150-CD41-CD48-Flt3-LSK), MPP2 (CD150 + CD41 / CD48 + Flt3-LSK), MPP3 (CD150-CD41 / CD48 + Flt3-LSK) and MPP4 (Flt3 + LSK) are divided into pluripotent progenitor cells (MPP). Defined as a stroke.

(Preparation of human hematopoietic stem cells, etc.)
Human CD34 + bone marrow cells were purchased from Lonza and stored in liquid nitrogen until used. In the experiment, first, frozen cells in the vial were thawed in a 37 ° C. water bath and transferred to a 15 mL tube. Subsequently, a medium (SF-O3 (manufactured by Aedia)), αMEM (Thermo) containing 10% by volume fetal bovine serum (FBS: purchased from Biowest or Thermo Fisher Scientific) and 2 U / mL DNase I (manufactured by Sigma Aldrich). Fisher Scientific) or DMEM / Ham's F12 (Nacalai Tesque)) was slowly added to the thawed cells and the tubes were filled with gentle mixing. The cell suspension obtained by thawing was subjected to centrifugation at 200 g for 15 minutes with a centrifuge (product name: Ex-125, manufactured by TOMY) at room temperature. Next, the supernatant was aspirated and the washing step by centrifugation using the medium was repeated once more.

Next, an antibody cocktail (50 μL of phosphate buffered saline (PBS: manufactured by Nacalai Tesque), 2% by volume FBS and each of the following antibodies) is added to the cells from which the supernatant has been removed after washing, and maintained on ice for 30 minutes. did. Cells were washed once with PBS containing 2% by volume FBS and resuspended in PBS containing 0.1% by volume PI and 2% by volume FBS.
Anti-CD34-FITC (BD Biosciences) 10 μL
Anti-CD38-PerCP-Cy5.5 (BD Biosciences) 2 μL
Anti-CD90-PE-Cy7 (BD Biosciences) 5 μL
10 μL of anti-CD45RA-PE (BD Biosciences).

  The cells labeled with the antibody are subjected to a cell sorter (FACS Aria), and the sorted cells contain 4 wt% BSA (manufactured by Sigma Aldrich) and 55 μM 2-ME (manufactured by Life Technologies). , Recovered in SF-O3 or αMEM. In addition, when subjected to culture in the absence of fatty acid, unlike cells derived from the above-mentioned mice, human-derived cells have a lower survival rate when cultured using fatty acid-free BSA. Was recovered in a medium containing the 4 wt% BSA.

  In this sorting, human hematopoietic stem cells were defined as CD34 + CD38-CD90 + CD45RA- cells.

(Culture medium)
For cell culture, SF-O3 medium, αMEM or DMEM / F12 medium was used. In the preparation of SF-O3 medium, first, the supplement provided by the same manufacturer and bovine serum albumin (with a final concentration of 0.1 wt. BSA: manufactured by Eadia or Sigma Aldrich) was added. Furthermore, after carbon dioxide was blown in and sufficiently acidified, what was preserve | saved at 4 degreeC through the 0.22 micrometer filter was used as SF-O3 culture medium.

  The SF-O3 basal medium was prepared by mixing RPMI 1640 supplemented with cytidine, glutathione, paraaminobenzoic acid and cholesterol, Dulbecco's MEM (DMEM), and Ham's F-12 medium at a ratio of 2: 1: 1. Medium.

  In addition, recombinant human insulin, recombinant human transferrin, sodium selenite, ethanolamine, HEPES and 2.2 g / L sodium bicarbonate were added to the SF-O3 basal medium as the supplement.

  BSA crystals (manufactured by Sigma Aldrich) or fatty acid-free BSA (manufactured by Sigma Aldrich) were directly dissolved in the medium to the indicated concentration in each experiment and filtered using a 0.22 μm filter. Before filtration, 2-mercaptoethanol (2-ME: manufactured by Life Technologies) was also added so as to have a final concentration of 55 μM. In addition, after filtration, a preparation composed of SCF (Peprotech), TPO (Peprotech), water-soluble fatty acid and cholesterol (product name: 250 × cholesterol lipid concentrate, manufactured by Thermo Fisher Scientific Co., Ltd.) is used so as to obtain the indicated concentration in each experiment. Added to the medium.

  For media other than SF-O3 (αMEM or DMEM / F12 media), human recombinant insulin (manufactured by Nacalai Tesque) was added to the indicated concentration in each experiment before the filtration, and the final concentration 2-ME was added so that it might become 55 micromol.

(Cell culture)
Mouse or human-derived hematopoietic stem cells prepared as described above were subjected to centrifugation at 340 g for 5 minutes, and the supernatant was discarded. Then, the medium prepared above is added to a concentration of (number of cells per well) / 10 μL, and 10 μL of the obtained cell suspension is added to each well with an 8-well pipette and 200 μL of the medium to each well. Dispense into pre-added 96 well plates.

Culture conditions are 1% by volume O ₂ and 5% by volume CO ₂ at 37 ° C. in a humidified incubator (low oxygen incubator APM-30DR: adjusted using Astech) or 20% by volume O ₂ and 5% by volume CO 2. ₂ (SCI-165D: adjusted using ASTEC).

  In the case of 7-day culture, the medium was not changed during the culture period. However, for longer periods of culture (ie, 11-31 days), half the medium was changed twice a week (every 3-5 days).

(Transplantation of hematopoietic stem cells)
C57BL / 6-Ly5.2 mouse-derived hematopoietic stem cells (CD150 + CD41-CD48-CD34-Flt3-LSK) prepared as described above or cells cultured under predetermined conditions (cultured hematopoietic stem cell-derived cells), A competitor (4 × 10 ⁵ C57BL / 6-Ly5.1 mouse bone marrow cells) was transplanted into the orbital venous plexus of C57BL / 6-Ly5.1 mice irradiated with a lethal dose (9.5 Gy) of radiation.

  Irradiation was performed using MBR-1520R-3 (Hitachi Power Solutions) (using 125 kV 10 mA, 0.5 mm Al, 0.2 mm Cu filter). Transplantation into the orbital venous plexus was performed using a 1 mL syringe with a 27 gauge needle (manufactured by Terumo).

  1, 2, 3 and 4 months after transplantation of hematopoietic stem cells, peripheral blood was collected, and the proportion of donor-derived cells and their differentiation state were determined by MACS Quant (Miltenyi).

Specifically, 40-80 μL of peripheral blood was collected from the orbital venous plexus using a glass capillary and suspended in 1 mL of heparin-containing PBS. After centrifuging the obtained blood suspension at 340 g for 3 minutes, the supernatant was discarded and resuspended in 1 mL of PBS containing 1.2 wt% dextran (200 kDa, manufactured by Nacalai Tesque), and then at room temperature. Incubate for 45 minutes to allow most of the red blood cells to settle to the bottom of the tube. The supernatant is subjected to 340 g centrifugation for 3 minutes, and the resulting pellet is resuspended in 0.17 M NH ₄ Cl solution and the remaining red blood cells are allowed to flow for 5-10 minutes until the cell suspension becomes clear. Dissolved. Centrifugation was performed again, and the obtained cells were resuspended in 50 μL of PBS containing 0.3 μL of Fc block and 2% by volume FBS.

Next, after surface antigen staining using the following antibody, it was subjected to analysis by MACS Quant.
Antibodies used for surface antigen staining: Gr-1-PE-Cy7, Mac-1-PE-Cy7, B220-APC, CD4-PerCP-Cy5.5, CD8a-PerCP-Cy5.5, CD45.1-PE, CD45.2-FITC. All antibodies used 0.3 μL per sample.

  The donor-derived cells are Ly5.2 (CD45.2) + Ly5.1 (CD45.1) − cells, and the cells derived from competitors or recipients are Ly5.2 (CD45.2) −Ly5.2. 1 (CD45.1) + cells. In addition, myeloid, B cell, and T cell were identified as Gr-1 + or Mac-1 + cell, B220 + cell, CD4 + or CD8a + cell, respectively.

Based on the data obtained, the frequency (%) of donor-derived cells was calculated as follows:
Frequency of donor-derived cells (%) = donor-derived cells (%) / [donor-derived cells (%) + competer and recipient-derived cells (%)] × 100.

  In addition, as the total cell chimerism, the frequency of donor-derived Ly5.2 + Ly5.1-cells relative to the frequency of Ly5.2-Ly5.1 + cells in mononuclear cells was calculated.

  Further, secondary transplantation was performed 4 months after the transplantation of the hematopoietic stem cells (primary transplantation). Specifically, bone marrow cells were collected from recipient mice 4 months after primary transplantation by the method described above (Preparation of mouse c-Kit positive cells). After counting the number of bone marrow cells using an automatic cell counter TC10 (manufactured by BioRad), 20 to 30% of the total cell suspension prepared from each recipient was pooled.

In addition, 2 × 10 ⁶ bone marrow cells were suspended in 200 μL of SF-O3 medium (containing 0.1% weight volume BSA) for each recipient. The cell suspension (200 μL / recipient) thus prepared was injected into the orbital venous plexus of irradiated C57BL / 6-Ly5.1 mice in the same manner as the primary transplantation, and transplanted.

(Treatment treatment of fatty acid, etc. on BSA)
Fatty acid sodium salt (palmitate, oleate, linoleate, stearate, all manufactured by Tokyo Chemical Industry Co., Ltd.), cholesterol (manufactured by Tokyo Chemical Industry Co., Ltd.), or a combination of these, glass It melt | dissolved in methanol (made by Wako) so that it might become a density | concentration of 4-20 mg / mL or 4 mg / mL within a tube.

  The fatty acid salt and / or cholesterol solution thus prepared was blown with air using an electric pipette, dried in the glass tube, and then methanol was completely evaporated on a 50 ° C. water bath ( Heated (5-10 minutes). Next, a medium containing 4 wt% fatty acid-free BSA or BSA crystals (both from Sigma Aldrich) was added directly to the glass tube and sonicated until the fatty acid salt and cholesterol were dissolved. Then, it was subjected to filtration using a 0.22 μm filter (Millipore). In addition, as described in the above (medium), when using SF-O3 culture medium, 2-ME was added just before filtration. Moreover, when using other culture media, insulin was added just before filtration as needed.

(EdU incorporation assay)
Hematopoietic stem cells were cultured for 16 hours under the conditions described below, 10 mM EdU was added to the medium to a final concentration of 10 μM, and incubated at 37 ° C. for 2 hours. The cells were then analyzed using the Click-iT EdU Alexa Fluor 647 flow cytometry assay kit (Thermo Fisher Scientific). Moreover, in order to detect intracellular EdU, it fixed and membrane permeabilized and analyzed using MACS Quant (made by Miltenyi Biotec).

(Analysis of cultured cells by MACS Quant)
From each well of a 96-well plate seeded with hematopoietic stem cells as described above (cell culture), the medium was aspirated to 170 μL, followed by addition of the following antibody cocktail and incubation at 4 ° C. for 30 minutes.

  In experiments targeting mice-derived cells, differentiation antigen markers (CD4, CD8a, Gr-1, Mac-1, B220, Ter-119) -PerCP-Cy5.5, anti-c-Kit-APC-Cy7, anti- An antibody cocktail consisting of Sca-1-PE-Cy7, anti-CD150-PE, anti-CD48-FITC and anti-CD41-APC was used. All antibodies were added to the plate so as to be 0.1 μL / well.

  In experiments involving human-derived cells, anti-CD34-FITC (0.1 μL / well), anti-CD38-PerCP-Cy5.5 (0.1 μL / well), anti-CD90-PE-Cy7 (0.25 μL / well) Well) and anti-CD45RA-PE (0.5 μL / well) antibody cocktail was used.

  After incubation (staining with the antibody), 100 μL of 2% FBS-containing PBS was added to each well and subjected to centrifugation at Accelerated Low and Decelerated Medium, 400 g, 4 ° C. for 5 minutes. Thereafter, 100 μL of the supernatant was aspirated, 200 μL of PBS containing 2% by volume FBS, 0.1% by volume PI and 0.25% by volume Flow-Check Fluorespheres (Beckman Coulter) was added to each well, and the cells were suspended. Made cloudy.

  In the Fast mode of MACS Quant, the cell suspension was collected, and 100 μL (large colony) or 150-170 μL (small colony) was aspirated and analyzed. Data was exported as FCS file and analyzed using FlowJo software. The number of cells was corrected by the number of beads of Flow-Check (about 1000 / μL). Megakaryocytes were defined as cells with high forward and side scatter and high CD150 and CD41 expression.

(CDNA microarray analysis)
First, according to the method described in (Preparation of mouse c-Kit positive cells) and (Preparation of mouse hematopoietic stem cells and the like), hematopoietic stem cells (CD150 + CD41-CD48-CD34-Flt3-LSK cells) from the bone marrow of 10 mice. Were isolated and pooled in SF-O3 medium.

  The pooled hematopoietic stem cells were divided into two equal parts, and half were dissolved in 75 μL of RLT buffer (containing 0.75 μL 2-ME) and subjected to the following microarray analysis and the like as fresh hematopoietic stem cell samples. The remaining half was cultured for 7 days under the conditions shown in FIG. 13, and then centrifuged at 340 g for 5 minutes at 4 ° C. to recover the cultured cells. 75 μL RLT buffer (containing 0.75 μL 2-ME) It melt | dissolved in 75 microliters and used for the following microarray analysis etc. as a hematopoietic stem cell sample after culture | cultivation.

  Then, RNA extraction, cDNA synthesis, microarray analysis and data normalization (data reading and standardization) from each hematopoietic stem cell sample prepared in this way were commissioned to the DNA chip laboratory.

  Specifically, RNA was extracted from the hematopoietic stem cell sample using RNeasy micro (QIAGEN). Using a WT-Ovation Pico RNA amplification system (manufactured by NuGen), cDNA was synthesized from 2 ng of the extracted RNA and amplified. The amplified cDNA was labeled with Genomic DNA Enzymatic Labeling Kit (manufactured by Agilent), followed by Gene Expression Hybridization Kit (manufactured by Agilent) and Gene Express Wash Pack (manufactured by Agilent). Was hybridized with Gene chip Mouse 8 × 60k (manufactured by Agilent).

  The hybridized gene chip was scanned using DNA MicroArray Scanner (manufactured by Agilent). The scanned image was recorded on Feature Extraction Ver. Analysis was performed with 9.5.3 (manufactured by Agilent).

  Furthermore, normalization was performed using GeneSpring software (manufactured by Agilent). Specifically, the raw data was first converted to the log2 scale and the expression level less than 1.0 was set to 1.0. And the gene expression level between samples was standardized by 75th percentile shift algorithm.

  In addition, the standardized expression data was visualized as a scatter plot by gene name annotation. The visualization was performed using the R software ggplot2 package. As a result, genes with a log doubling difference of gene expression level> 1.5 were highlighted by coloring in dark blue, and the genes of interest (ie fatty acid related genes) were colored in red. Genes with the “not detected” flag were removed from the scatter plot.

(Gene set enrichment analysis: GSEA)
Expression data normalized as described above was evaluated using GSEA v2.0.13 software (Broad Institute). The gene set was obtained from Molecular Signatures Database v4.0 distributed on the GSEA website (http://www.roadinstate.org/gsea/msgigb/index.jsp). The number of permutations was set to 1000. Gene sets with a nominal p value of less than 0.05 and a false discovery rate q value <0.25 were considered statistically significant.

(Creation of 2D and 3D plots of cytokine responses)
After the number of cells after culturing at various cytokine concentrations was exported to an FCS file, the data was smoothed using R software. Local regression fits a polynomial surface to data points using the loess function. The parameters were set to degree = 2 and span = 0.25. The X-axis and Y-axis were expanded from a 7 × 7 matrix to a 61 × 61 matrix, and data prediction was performed using a predict function. When the data was on a logarithmic scale (ie, total cell number and megakaryocyte count), 1 was added to the measurement data so that the data after logarithmic conversion was positive. From all data, 2D contour plots were generated using the ggplot2 package and 3D plots were generated using the rgl package.

(Statistical analysis)
Data are presented as mean ± standard deviation unless otherwise noted. When performing multiple comparison test, statistical significance is obtained by Tukey's multiple comparison test using the Tukey HSD function of R software, one-way analysis of variance (1-way ANOVA) or two-way analysis of variance (2-way ANOVA) using an anova function. It was determined. An unpaired two-tailed Student's t-test was used in the comparison of the two groups. The false discovery rate (FDR) was calculated using the qvalue function of the qvalue package of Bioconductor (http://www.bioconductor.org).

  Next, the results of experiments conducted using the above materials and methods are shown below.

<Example 1> Examination of culture conditions (oxygen, SCF and TPO concentrations) for maintaining mouse-derived hematopoietic stem cells Hematopoietic stem cells are in a hypoxic environment in a bone marrow environment and are maintained in a stationary phase It remains undifferentiated. Furthermore, it is also known that stem cell factor (SCF) and thrombopoietin (TPO) are essential factors for maintaining hematopoietic stem cells.

  Thus, in order to reproduce the conditions that allow the hematopoietic stem cells to remain stationary and undifferentiated even in culture, the oxygen concentration and the concentrations of SCF and TPO were first examined.

  Specifically, in order to simplify the conditions, only SCF and TPO were added to the medium as cytokines. Then, in order to determine the optimum conditions for SCF and TPO, hematopoiesis under low oxygen conditions (1 vol% oxygen) or high oxygen conditions (20 vol% oxygen, atmospheric concentration) in SF-O3 medium containing 4 wt% BSA. Stem cells are cultured for 7 days, in total cell count (Fig. 1), megakaryocyte count (Fig. 2), ratio of hematopoietic stem cell fraction to total cell count (Fig. 3) and undifferentiated cell fraction (LSK fraction) The number of cells at each differentiation stage (FIG. 4) was measured by MACS Quant.

  As a result, as shown in FIG. 1, the total cell number increased in proportion to both the SCF and TPO addition concentrations. Blood. 1993 Oct 1; 82 (7): 2031-2037, there were fewer cells under hypoxic conditions. In addition, as shown in FIG. 2, for megakaryocytes, the number of cells increased depending on the concentration of SCF and TPO under high oxygen conditions while strongly dependent on the concentration of TPO under low oxygen conditions. On the other hand, as shown in FIG. 3, the ratio of hematopoietic stem cells was higher when the SCF and TPO concentrations were lower, and the ratio of hematopoietic stem cells was higher under hypoxic conditions. Furthermore, looking at the number of cells in the LSK fraction, as shown in FIG. 4, the number of differentiated cells (CD150 + CD48 + LSK cells and CD150-CD48 + cells) is significantly increased under high oxygen conditions as compared with hypoxic conditions, Blood. As described in 1993, it was confirmed that hematopoietic stem cells are maintained more undifferentiated under hypoxic conditions.

In addition, as shown in FIG. 3, hematopoietic stem cells had a TPO concentration that decreased when the SCF concentration was 3 ng / mL or less under the culture conditions where the TPO concentration was 0 to 0.5 ng / mL. However, it was found that the SCF concentration is desirably 3 ng / mL or more under the low conditions. On the other hand, the hematopoietic stem cells were alive under the culture conditions where the TPO concentration was 2.5 ng / mL or more, even when the SCF concentration was 3 ng / mL or less. In particular, an increase in the number of hematopoietic stem cells was observed under the conditions where the concentration of SCF was 1 ng / mL and the concentration of TPO was 62.5 ng / mL (“1% O ₂ CD150 + CD48−LSK” in FIG. 4). reference).

  On the other hand, for pluripotent progenitor cells (MPP) that are derived from hematopoietic stem cells and have lost their self-renewal ability but have multipotency, when the SCF concentration is 3 ng / mL or more, the CD150-CD48-LSK fraction (MPP1) increased. Furthermore, the CD150 + CD48 + LSK fraction (MPP2) increased under conditions where the TPO concentration was 2.5 ng / mL or more. Moreover, few cells differentiated into CD150-CD48 + LSK (MPP3) under high oxygen conditions (see FIG. 4).

  Combined the above results, conditions that maintain quiescence (total number of cells is low) and maintain undifferentiated cells (low number of megakaryocytes / differentiated cells and high number of hematopoietic stem cells) As a result, as shown in FIG. 5, the oxygen concentration is lower than the atmospheric concentration, the SCF concentration is 1 to 10 ng / mL, and the TPO concentration is 0.02 to 6 ng / mL. It is desirable that the concentration be 1 mL by volume, and that the oxygen concentration is 1% by volume, SCF is added to the medium at a concentration of 3 ng / mL, and TPO is added to the medium at a concentration of 0.1 ng / mL. Has been found to be optimal in maintaining its stationary phase and undifferentiation.

  In fact, as shown in FIG. 6, when the oxygen concentration is 1% by volume and the cells are cultured in a medium supplemented with 3 ng / mL SCF and 0.1 ng / mL TPO (hereinafter, this culture condition is referred to as “S3T0.1”). Compared with the case of culturing under existing conditions (high oxygen, high cytokine, for example, Cell Stem Cell. January 3, 2013; 12 (1): 49-61). It became clear that the cell colonies were kept small.

  Furthermore, as a result of the flow cytometry analysis, as shown in FIG. 7, as a result of culturing under the S3T0.1 condition, it is clear that many cells maintain the surface marker CD150 + CD48-LSK before the start of culture. became.

Subsequently, the cell cycle 16 hours after the start of the culture was analyzed by an EdU incorporation assay, and S3T0.1 conditions were compared with high cytokine (SCF 100 ng / mL, TPO 100 ng / mL) conditions. Furthermore, for these conditions, it was also investigated for high oxygen (20 vol% _{O 2)} conditions.

  As a result, as shown in FIG. 8, consistent with the analysis results for the number of cells shown in FIGS. 1 to 5, EdU positive cells were kept low at low cytokine concentrations, and the cell cycle of hematopoietic stem cells was low in rotation. It became clear that there was. However, the oxygen concentration did not significantly affect the cell cycle at this time point.

  Next, in order to evaluate whether hematopoietic stem cells cultured under the S3T0.1 conditions found above are functionally maintained as well as the expression of surface markers, as shown in FIG. (Fresh) hematopoietic stem cells, or 500 hematopoietic stem cells cultured for 14 or 29 days, were transplanted into recipient mice (primary transplantation). In addition, bone marrow transplantation experiments (BMT, secondary transplantation) were conducted in which bone marrow cells were collected from recipient mice 4 months after transplantation and transplanted into another mouse.

  As a result, as is apparent from the results shown in FIG. 10, in the hematopoietic stem cells cultured under S3T0.1 conditions for 14 days, peripheral blood chimerism comparable to that of the hematopoietic stem cells immediately after collection in both primary transplantation and secondary transplantation, Any blood cell fraction (myeloid, B cell, T cell) was observed. In addition, regarding hematopoietic stem cells cultured for 29 days under S3T0.1 conditions, the peripheral blood chimerism in the primary transplantation was inferior to that of the Fresh group, but as is clear from the results shown in FIG. The paintings were not inferior in chimerism.

<Example 2> Examination of culture conditions (albumin concentration) for maintaining mouse-derived hematopoietic stem cells Next, cells of hematopoietic stem cells having the bovine serum albumin (BSA) concentration under the S3T0.1 conditions found above The effect on number was examined. As a result, as shown in FIG. 12, it became clear that hematopoietic stem cells can hardly survive under low BSA concentration conditions (BSA less than 1% by volume (10 mg / mL)). On the other hand, no significant difference was observed in the number of hematopoietic stem cells under conditions where the BSA concentration was 1.3 to 4% by volume (13 to 40 mg / mL). However, under the condition of 1.3% by volume of BSA, it was vulnerable to the excessive lipid (cholesterol and fatty acid) loading, and the number of hematopoietic stem cells decreased depending on the volume of the loaded lipid. Under high BSA concentration conditions (BSA 1.3% by volume or more), the number of hematopoietic stem cells peaked at 4-6% by volume (40-60 mg / mL), and 8% by volume (80 mg / mL). Decreased.

  From the above results, it is clear that in order to maintain hematopoietic stem cells, it is desirable to culture at a concentration of 2 to 6% by volume of albumin, and further, a concentration of around 4% by volume is optimal. became.

  Subsequently, in order to examine changes in gene expression in hematopoietic stem cells under the culture conditions found above, as shown in FIG. 13, gene expression of hematopoietic stem cells cultured for 7 days under S3T0.1 conditions was analyzed using a cDNA microarray. Analyzed. The concentration of BSA in the culture was 4% by weight. Furthermore, gene expression of hematopoietic stem cells cultured for 7 days under S3T0.1 conditions in a medium containing fatty acid-free BSA loaded with fatty acids instead of the 4% by volume BSA was also analyzed. For comparison, hematopoietic stem cells immediately after collection were also analyzed.

  As a result, as shown in FIGS. 14 and 15, in the presence of S3T0.1 condition, 4% by volume BSA or fatty acid-free BSA carrying fatty acid, gene expression of fatty acid biosynthetic pathway is suppressed to a level close to that of the Fresh group. It was revealed that fatty acids were supplied from BSA in the culture of hematopoietic stem cells.

  Further, as a result of comparison between the culture in the presence of 4% by weight BSA and the culture in the presence of fatty acid-free BSA loaded with 4% by weight fatty acid, as shown in FIG. Was comparable. 14 and 15, it was suggested that BSA can be replaced with fatty acid-free BSA loaded with fatty acids (palmitic acid and oleic acid) in maintaining hematopoietic stem cells. .

  In addition, it culture | cultivated twice in S3T0.1 conditions and fatty-acid free BSA presence which carried the fatty acid independently, and compared them. As a result, as shown in FIG. 17, gene expression in the fatty acid biosynthetic pathway was similar and confirmed to have reproducibility.

  Furthermore, as shown in FIG. 18, when GSEA was performed using a set of genes related to undifferentiation of hematopoietic stem cells and the cell cycle, both hematopoietic stem cells cultured by the above method were significantly different from the Fresh group. There was no significant change in the gene.

<Example 3> Study on culture conditions (fatty acids) for maintaining mouse-derived hematopoietic stem cells Fatty acids are usually bound to BSA. As described above, it is considered that supplying fatty acids is an important function of BSA in the maintenance culture of hematopoietic stem cells. Therefore, in order to evaluate the effect of fatty acid composition on the number of hematopoietic stem cells, a mixture of fatty acid-free BSA and fatty acids of palmitic acid, oleic acid, and linoleic acid at various ratios is used under the conditions of S3T0.1. Hematopoietic stem cells were cultured for one day.

  As a result, as shown in FIG. 19, it was revealed that the number of viable hematopoietic stem cells was markedly reduced in a medium containing only unsaturated fatty acids (oleic acid and linoleic acid). On the other hand, if palmitic acid was contained, the ratio of other fatty acids did not significantly affect the number of cells. Therefore, palmitic acid is considered important for the maintenance of hematopoietic stem cells.

  Furthermore, in order to evaluate the influence of the concentration of fatty acid supported (reconstituted) on albumin, hematopoietic stem cells were cultured using fatty acid-free BSA in which various amounts of fatty acid were reconstituted. As a result, as shown in FIG. 20, it was revealed that the total number of cells and the number of hematopoietic stem cells after culture tend to be easily maintained depending on the concentration of fatty acid.

<Example 4> Examination about culture conditions (insulin) for maintaining mouse-derived hematopoietic stem cells Insulin is contained in the SF-O3 medium used in the above culture. Therefore, in order to evaluate the influence of insulin on hematopoietic stem cells, 4000 ng / mL of insulin was added to a medium containing no insulin (αMEM medium containing 4 wt% BSA) and cultured under S3T0.1 conditions for 7 days. As a result, as shown in FIG. 21, there was no difference in the number of hematopoietic stem cells after culture depending on the presence or absence of insulin, but it was found that the total number of cells decreased when insulin was not included.

  Furthermore, in order to evaluate the effect of insulin under higher fatty acid conditions, fatty acid-containing BSA (nBSA) further added with fatty acid at a high concentration (800 μg / mL) is added to αMEM, and the number of hematopoietic stem cells is determined as S3T0. .Evaluated under 1 condition. As a result, as shown in FIG. 22, it became clear that both the number of hematopoietic stem cells and the total number of cells tend to be maintained depending on the insulin concentration.

  Next, in order to verify whether the culture conditions for the mouse-derived hematopoietic stem cells found above are applicable to humans, a similar experiment using frozen human CD34 + cells was performed.

<Example 5> Verification of culture conditions (concentrations of SCF and TPO) for maintaining human-derived hematopoietic stem cells First, the dependence of human-derived hematopoietic stem cells on cytokine concentration in the presence of 4 wt% BSA In order to evaluate, SCF and TPO were added at various concentrations and cultured for 7 days, and the total number of cells and the number of hematopoietic stem cells were evaluated.

  As a result, as shown in FIG. 23, as in the case of the mouse, when the concentrations of SCF and TPO are low, it is clear that the total number of cells is suppressed, and the quiescent and undifferentiated nature of hematopoietic stem cells is maintained. Became. It was also revealed that in human-derived hematopoietic stem cells, the concentration of TPO is desirably 0.1 ng / mL or more.

  Subsequently, the number of hematopoietic undifferentiated cells and the total number of cells when human hematopoietic stem cells were cultured for 1 month were measured. As a result, as shown in FIG. 24, it was revealed that the total cell number and the number of undifferentiated cells were easily maintained under the culture conditions in which the SCF concentration was 3 ng / mL and the TPO concentration was 3 ng / mL. .

<Example 6> Verification of culture conditions (albumin) for maintaining human-derived hematopoietic stem cells In order to verify the dependency of human-derived hematopoietic stem cell maintenance on albumin, in the presence or absence of BSA, human Hematopoietic stem cells were cultured. As a result, as shown in FIG. 25, in the absence of BSA, the total number of cells and the number of hematopoietic stem cells both decreased. On the other hand, in the presence of BSA, the number of cells was maintained, and it was revealed that albumin is essential for maintaining human-derived hematopoietic stem cells.

  Next, in order to evaluate the dependence of human-derived hematopoietic stem cells on fatty acids or cholesterol, human hematopoietic stem cells are cultured using BSA in which various amounts of fatty acids are reconstituted in the presence or absence of cholesterol. did.

  As a result, as shown in FIG. 26, it was found that both the total cells after culturing and the hematopoietic stem cells tend to be more easily maintained when cholesterol is present. Furthermore, it became clear that the number of cells was easily maintained by adding fatty acids at a concentration of 250 to 500 μg / mL. On the other hand, it was also found that the number of cells tends to decrease at high concentrations. Moreover, these tendencies were more noticeable in the absence of cholesterol.

  Next, in order to evaluate the influence on human-derived hematopoietic stem cells when the fatty acid to be added is at a lower concentration, 100 to 400 μg / mL of fatty acid in the presence or absence of cholesterol is added with fatty acid-free BSA or ( Human-derived hematopoietic stem cells were cultured using those bound to (containing fatty acid) BSA. As a result, as shown in FIG. 27, it was also clarified that hematopoietic stem cells are most easily maintained when 100 to 200 μg / mL fatty acid is added to BSA in the presence of cholesterol.

<Example 7> Verification about culture conditions (insulin) for maintaining human-derived hematopoietic stem cells In order to evaluate the influence of insulin in maintaining human-derived hematopoietic stem cells, the measurement was performed under several SCF and TPO concentration conditions. Human hematopoietic stem cells were cultured with varying concentrations of insulin. As a result, as shown in FIG. 28, it was clarified that the addition of insulin makes it easy to maintain the total cell number and the number of hematopoietic stem cells depending on the concentration.

  As described above, according to the present invention, hematopoietic stem cells can be cultured for a long period of time while maintaining undifferentiation (pluripotency and self-renewal ability) in the stationary phase. Therefore, the present invention is useful for screening drug discovery targets for diseases relating to hematopoietic functions such as leukemia, and for treating such diseases.

Claims (8)

  Hematopoietic stem cells having a stem cell factor (SCF) concentration of 1 to 10 ng / mL, a thrombopoietin (TPO) concentration of 0.02 to 6 ng / mL, and a fatty acid-carrying albumin concentration of 10 to 100 mg / mL Medium for maintaining culture.   The medium according to claim 1, further comprising at least one substance selected from the group consisting of insulin and cholesterol.   The medium according to claim 1 or 2, wherein the fatty acid-carrying albumin is albumin carrying at least a saturated fatty acid.   The culture method for maintaining a hematopoietic stem cell including the process of culture | cultivating a hematopoietic stem cell in the culture medium as described in any one of Claims 1-3.   The culture method according to claim 4, wherein hematopoietic stem cells are cultured under an oxygen concentration of 5% by volume or less.   The cell obtained by culture | cultivating a hematopoietic stem cell in the culture medium as described in any one of Claims 1-3.   The cell according to claim 6, wherein the culture is performed under an oxygen concentration of 5% by volume or less. A kit for producing the medium according to any one of claims 1 to 3,
A kit comprising SCF, TPO, the fatty acid-carrying albumin, and a basal medium.