Stem cell chromosome stabilizer

Abstract

To provide a stem cell chromosome stabilizer.SOLUTION: The present invention provides a stem cell chromosome stabilizer, characterized by containing as an active ingredient β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof; and a method for culturing stem cells, characterized by culturing pluripotent stem cells in a culture medium containing β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a material capable of preventing the occurrence of chromosomal abnormalities during the process of culturing and passaging stem cells, and a chromosomal stabilizing agent for stem cells that uses said material. [Background technology]

[0002] Stem cells, such as pluripotent stem cells, are undifferentiated cells with the ability to self-replicate and differentiate into various types of cells. In recent years, there has been active research into regenerative medicine, in which stem cells or cells induced to differentiate from stem cells are transplanted into damaged tissues of patients to restore their functions. Since regenerative medicine requires the preparation of large quantities of stem cells and their differentiated cells, there has been active development of methods for efficiently proliferating and differentiating stem cells.

[0003] However, when stem cells are cultured for a long period of time or passaged many times, chromosomal abnormalities may occur. Chromosomal abnormalities include aneuploidy, such as monosomy, in which a pair of two chromosomes becomes one, or trisomy, in which a pair of two chromosomes becomes three, as well as structural abnormalities, such as translocation, inversion, partial duplication, and partial deletion. When such chromosomal abnormalities occur in stem cells, not only do they lose their proliferation and differentiation abilities, but there is also a risk that cells or tissues differentiated from the stem cells may mutate into cancer cells or develop into tumors when used in regenerative medicine or the like. Chromosomal abnormalities may occur in cancer cells or tumors, and in recent years, methods for detecting chromosomal abnormalities more accurately have been reported (see, for example, Patent Documents 1 and 2).

[0004] On the other hand, nicotinamide mononucleotide (NMN) is a coenzyme called NAD +NMN is an intermediate metabolic product in the biosynthesis of insulin. In recent years, it has been reported that NMN has the effect of improving insulin secretion in aged mice, the effect of dramatically improving insulin sensitivity and secretion in mouse models of type 2 diabetes caused by a high-fat diet and aging (see, for example, Patent Document 3), and the effect of significantly enhancing mitochondrial function in aged muscles. Furthermore, it has been reported that culturing pluripotent stem cells in the presence of NMN can improve their proliferation and differentiation abilities (see, for example, Patent Documents 4 and 5). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2019-213537 A [Patent Document 2] JP 2019-144097 A [Patent Document 3] U.S. Patent No. 7,737,158 [Patent Document 4] International Publication No. 2018 / 143258 [Patent Document 5] International Publication No. 2019 / 182044 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a material capable of preventing the occurrence of chromosomal abnormalities during the process of culturing and passaging stem cells, as well as a chromosomal stabilizer for stem cells that uses said material, a method for culturing stem cells, and a method for stabilizing the chromosomes of stem cells. [Means for solving the problem]

[0007] As a result of intensive research by the inventors to solve the above problems, they discovered that in the presence of β-NMN, chromosome stability in stem cells is improved and chromosomal abnormalities are suppressed and prevented, thereby completing the present invention.

[0008] Specifically, the present invention provides the following chromosome stabilizing agent for stem cells, method for culturing stem cells, and method for stabilizing chromosomes for stem cells. [1] A chromosome stabilization agent for stem cells, characterized in that it contains as an active ingredient β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof. [2] The chromosome stabilizer according to [1] above, which is added to a culture medium for stem cells in an amount of 0.01 to 5 mM calculated as β-nicotinamide mononucleotide. [3] The chromosome stabilizing agent according to [1] or [2] above, which is used for chromosome stabilization of one or more types of stem cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and hematopoietic stem cells. [4] A method for culturing stem cells, comprising culturing pluripotent stem cells in a culture medium containing β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof. [5] The culture method according to [4] above, wherein the culture medium has a β-nicotinamide mononucleotide concentration of 0.01 to 5 mM. [6] The culture method according to [4] or [5] above, wherein the stem cells are one or more types selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and hematopoietic stem cells. [7] A method for chromosomal stabilization of stem cells, comprising culturing the stem cells in a culture medium containing β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof. Effect of the Invention

[0009] The chromosome stabilizing agent for stem cells according to the present invention acts on stem cells to improve the stability of the chromosomes of the stem cells and suppress or prevent the occurrence of chromosomal abnormalities. Therefore, by including the chromosome stabilizing agent in a culture medium, the stability of the chromosomes of stem cells can be improved, the occurrence of chromosomal abnormalities can be suppressed or prevented, and stem cells can be cultured more stably. In addition, the use of the chromosome stabilizing agent can reduce the risk of canceration or tumorigenesis of cells or tissues differentiated from stem cells. [Brief description of the drawings]

[0010] [Figure 1] (a) is a fluorescent photograph of cells stained with PI in Example 2, (b) is a fluorescent photograph of cells immunostained with anti-γH2A.X antibody in Example 2, and (c) is a merged photograph of (a) and (b). [Diagram 2] This is a graph showing the ratio of γH2A.X-positive cell nuclei among all cells stained with PI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In the present invention and the present specification, stem cells refer to undifferentiated cells that have the ability to self-replicate and have the ability to differentiate (the ability to differentiate into various cell types), and examples of such stem cells include pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells), as well as somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and skin stem cells. Preferred are stem cells that can differentiate into any of cells derived from the ectoderm, mesoderm, and endoderm, and stem cells that can differentiate into cells derived from the mesoderm.

[0012] The chromosome stabilizing agent for stem cells according to the present invention (hereinafter sometimes referred to as the "chromosome stabilizing agent according to the present invention") is NMN (chemical formula: C 11 H 15 N2O8P) is an active ingredient and is added to the culture medium when culturing and subculturing stem cells. By culturing and subculturing stem cells in the presence of NMN, the stability of the chromosomes of the stem cells can be improved and the occurrence of chromosomal abnormalities can be suppressed or prevented.

[0013] NMN has two optical isomers, α and β, but the NMN that is the active ingredient of the chromosome stabilizing agent of the present invention is β-NMN (CAS number: 1094-61-7). The structure of β-NMN is shown below.

[0014] [ka]

[0015] The β-NMN used as an active ingredient may be prepared by any method. For example, β-NMN artificially synthesized by chemical synthesis, enzymatic method, fermentation method, etc., and purified β-NMN can be used as an active ingredient. In addition, since β-NMN is a component that is widely present in living organisms, β-NMN obtained by extraction and purification from natural raw materials such as animals, plants, and microorganisms can also be used as an active ingredient. Commercially available purified β-NMN may also be used.

[0016] As a chemical synthesis method for synthesizing β-NMN, for example, NAM can be reacted with L-ribose tetraacetate and the resulting nicotinamide mononucleoside can be phosphorylated to produce β-NMN. As an enzymatic method, for example, β-NMN can be produced from NAM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) using nicotinamide phosphoribosyltransferase (NAMPT). As a fermentation method, for example, β-NMN can be produced from NAM using the metabolic system of a microorganism expressing NAMPT.

[0017] The active ingredient of the chromosome stabilizer according to the present invention may be a pharmacologically acceptable salt of β-NMN. The pharmacologically acceptable salt of β-NMN may be an inorganic acid salt or an organic acid salt having a basic site such as an amine. Examples of acids constituting such acid salts include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. The pharmacologically acceptable salt of β-NMN may be an alkali salt or an organic salt having an acidic site such as a carboxylic acid. Examples of bases that constitute such acid salts include alkali metal salts or alkaline earth metal salts, such as those derived from bases such as sodium hydride, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, and tetramethylammonium hydroxide.

[0018] The active ingredient of the chromosome stabilizing agent of the present invention may be a solvate of free β-NMN or a pharmacologically acceptable salt of β-NMN. Solvents that form the solvate include water, ethanol, etc.

[0019] The chromosome stabilizer according to the present invention may contain other active ingredients in addition to β-NMN. The other active ingredients used in combination with β-NMN may be one type only, or may be a combination of two or more types. The other active ingredients may be appropriately selected from, for example, ingredients known to increase the survival efficiency and proliferation efficiency of stem cells, such as albumin, ascorbic acid, α-tocopherol, insulin, transferrin, sodium selenite, ethanolamine, and Rock inhibitors; and ingredients known to increase the differentiation efficiency of stem cells, such as valproic acid, dimethyl sulfoxide, dexamethasone, butyric acid, trichostatin A, GSK3 inhibitors, BMP inhibitors, Wnt inhibitors, activin, and noggin.

[0020] When culturing stem cells, the chromosome stabilizing agent according to the present invention is added to the culture medium, thereby improving the stability of the chromosomes of the stem cells and suppressing or preventing the occurrence of chromosomal abnormalities while allowing the stem cells to proliferate. The amount of the chromosome stabilizing agent according to the present invention added to the culture medium for stem cells is not particularly limited as long as it is an amount sufficient to suppress the number of stem cells with chromosomal abnormalities to a low level compared to the case of culturing in a culture medium not containing the chromosome stabilizing agent, and can be appropriately adjusted in consideration of the type of stem cells and the balance with other components of the culture medium. If the β-NMN concentration in the culture medium is too low, the chromosome stabilizing effect may be weak, and if an excessive amount of β-NMN is added, cell proliferation may be suppressed. The content of the chromosome stabilizing agent according to the present invention in the culture medium is preferably an amount that results in a β-NMN concentration of 0.01 to 5 mM, more preferably an amount that results in a β-NMN concentration of 0.05 to 2 mM, and even more preferably an amount that results in a β-NMN concentration of 0.1 to 1 mM. By having the β-NMN concentration within the above range, the chromosomes of the stem cells can be sufficiently stabilized.

[0021] Culturing stem cells in the presence of the chromosome stabilizing agent according to the present invention can be carried out by a conventional method, except that the chromosome stabilizing agent according to the present invention is contained in the culture medium. For example, as the culture medium, a medium generally used for the maintenance or proliferation of stem cells or a medium used for the culture of animal cells can be used. In addition, various commercially available culture media for stem cells can also be used. In the present invention, examples of media used for culturing stem cells containing the chromosome stabilizing agent according to the present invention include Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), α Eagle's minimum essential medium (αMEM), Iscove's modified Dulbecco's medium (IMDM), F-12 medium, F-10 medium, DMEM / F12 medium, RPMI-1640 medium, mesenchymal cell basal medium (MSCBM), E8 (Essential 8) medium, TeSR-E8 medium, mTeSR1 medium, MCDB medium, etc. Amino acids, inorganic salts, vitamins, antibiotics, etc. may be added to these media as necessary.

[0022] In addition to the chromosome stabilizing agent according to the present invention, these culture media may appropriately contain components known to increase the survival efficiency or proliferation efficiency of stem cells, components known to have the effect of maintaining the undifferentiated state of stem cells, etc. As these components, the above-mentioned ones can be used.

[0023] The culture conditions can be the same as those generally used for culturing animal cells, and may be appropriately modified as necessary. For example, the culture temperature is 30 to 40°C, the CO2 concentration is 1 to 10% by volume, and the O2 concentration is 0.1 to 25% by volume.

[0024] The stem cells whose chromosomes are stabilized by the chromosome stabilizer of the present invention are preferably animal-derived stem cells, more preferably mammal-derived stem cells, and even more preferably human-derived stem cells. The stem cells whose chromosomes are stabilized by the chromosome stabilizer of the present invention are preferably animal-derived ES cells, iPS cells, or mesenchymal stem cells, more preferably mammal-derived ES cells, iPS cells, or mesenchymal stem cells, and even more preferably human-derived ES cells, iPS cells, or mesenchymal stem cells. EXAMPLES

[0025] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0026] [Example 1] The effect of β-NMN on chromosomes during the culture of mesenchymal stem cells was investigated. Two donors of human adipose tissue-derived mesenchymal stem cells (ADSC) (product number: PT-5006, manufactured by Lonza) were used as mesenchymal stem cells.

[0027] In culturing mesenchymal stem cells, a 1:1 mixture of DMEM and MCDB medium was used as the basal medium (see Patent No. 5804385), and the basal medium was supplemented with FGF, PDGF, TGF-β, HGF, EGF, phospholipids, fatty acids, etc. The β-NMN-supplemented medium was prepared by adding β-NMN to the basal medium to a concentration of 0.25 mM.

[0028] <Subculture> 2.5μg / cm 2 Mesenchymal stem cells were cultured at 5 × 10 3 cells / cm 2 The cells were seeded so that the cells were at a confluency of approximately 80-90%, and cultured in a basal medium. One day after seeding, the medium was replaced with a basal medium or a medium supplemented with β-NMN. Thereafter, the medium was replaced once every 2 or 3 days. When the cell density in the culture dish reached approximately 80-90% confluency, subculture was performed.

[0029] First, the culture supernatant was removed and the cells were washed with 1x PBS(-), after which the cells were detached using 1x Tryple select (product number: 12563011, Thermo Fisher) and allowed to stand at 37°C and 5% CO2 for 4 minutes. After single cell separation by pipetting, basal medium or β-NMN-supplemented medium was added and centrifuged. The collected cells were suspended in each medium, after which a cell count was performed and 5x10 cells were placed in a pre-coated culture dish. 3 cells / cm 2 The medium was replaced once every 2 or 3 days using a basal medium or a β-NMN-supplemented medium.

[0030] After five rounds of subculture, the cells were frozen using a cell freezing medium "STEM-CELLBANKER DMSO Free GMP grade" (product number CB061, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) according to the manufacturer's recommended protocol.

[0031] Frozen cells were plated at 5 × 10 3 cells / cm 2 The cells were seeded in a basal medium or a β-NMN-supplemented medium so that the cells were cultured in the basal medium or the β-NMN-supplemented medium, and the medium was replaced one day after seeding. After that, the medium was replaced once every 2 or 3 days.

[0032] Subculture was performed once, and 5 × 10 3 cells / cm 2 Three days after seeding, the T25 flask was filled with the medium and sent to a contracted testing organization for chromosome safety testing (Nihon Gene Research Institute Co., Ltd.) for chromosome analysis.

[0033] <Chromosomal stability test> The chromosome status of the mesenchymal stem cells after culture was confirmed by G-banding. The measurement results of the number of chromosomes are shown in Table 1, and the karyotype analysis results are shown in Table 2. The analysis results were recorded in accordance with the International System for Human Cytogenomic Nomenclature (ISCN).

[0034] [Table 1]

[0035] [Table 2]

[0036] Test group 1 shows mesenchymal stem cells (ADSC donor 1, male) cultured in basic medium, and test group 2 shows mesenchymal stem cells (ADSC donor 2, female) cultured in basic medium. Meanwhile, test group 3 shows mesenchymal stem cells (ADSC donor 1, male) cultured in β-NMN-supplemented medium, and test group 4 shows mesenchymal stem cells (ADSC donor 2, female) cultured in β-NMN-supplemented medium.

[0037] In mesenchymal stem cells cultured in basic medium, abnormalities in the number of chromosomes were observed in both donors (Test Groups 1 and 2) (Table 1). That is, as a result of measuring the number of chromosomes in 50 cells in each test group, in Test Group 1, the number of chromosomes increased by one in four cells from 46 to 47. In addition, in Test Group 2, the number of chromosomes decreased by one in one cell from 46 to 45. On the other hand, in mesenchymal stem cells cultured in β-NMN-added medium, the number of chromosomes was 46 in all cells from both donors (Test Groups 3 and 4), which is the normal number of chromosomes in humans. As a result of karyotype analysis, abnormalities in chromosomes with different numbers were observed depending on the donor (Table 2). As a result of karyotype analysis of chromosomes in 20 cells in each test group, in Test Group 1, one chromosome 5 was increased in three cells, resulting in trisomy. In Test Group 2, one cell lost one each of chromosomes 7 and 18, and one marker chromosome of unknown origin was increased. In contrast, in mesenchymal stem cells cultured in a medium supplemented with β-NMN, no abnormalities were found in the chromosome karyotypes of either donor (Test Groups 3 and 4) (Table 2). In normal cases, the karyotype is recorded as "46,XY" in the male donor in Test Group 3, and "46,XX" in the female donor in Test Group 4. These results confirmed that β-NMN has the effect of improving the stability of stem cell chromosomes.

[0038] [Example 2] We investigated the effect of β-NMN on chromosomes during culture of human pluripotent stem cells (iPSCs).

[0039] <Culture medium / culture> The 201B7 strain of human pluripotent stem cells (iPSCs) was used for the test. The cells were cultured in a 24-well cell culture plate pre-coated with extracellular matrix (Matrigel: Corning) at 5% CO2 and 37°C until a cell density of approximately 80% was reached.

[0040] The culture medium used was a basic medium (DMEM-F12) supplemented with cytokines and salts (insulin, transferrin, TGFβ, FGF, sodium selenite, ascorbic acid, sodium bicarbonate) that are known to be necessary for the maintenance and subculture of iPSCs. A β-NMN-free group (NMN(-)) was used in which the cells were cultured in the basic medium, and a β-NMN-added group (NMN(+)) was used in which β-NMN was added to the basic medium to a final concentration of 1 mM.

[0041] NCX4016 (Sigma-Aldrich, product number SML1669) was added to the β-NMN-free group and β-NMN-added group to a final concentration of 5 μM, and chromosome damage was induced by incubating for 2 hours. After that, in order to examine the extent of chromosome damage, cells with damaged nuclear genomic DNA were detected by labeling phosphorylated histone H2A.X (γH2A.X).

[0042] <Immunostaining> The cells incubated with NCX4016 were fixed with 0.99% formaldehyde and permeabilized with 0.10% TritonX-100. Then, they were immunostained with anti-γH2A.X antibody (Thermo Fisher, product number 14-9865-82) and FITC-labeled anti-mouse IgG antibody (Thermo Fisher, product number 11-4015-82) and observed under a fluorescent microscope to detect γH2A.X. In addition, DNA was fluorescently stained with propidium iodide (Nacalai Tesque, product number 19174-31) so that the nuclei of all cells could be observed at the same time.

[0043] <Result> As shown in Figure 1(b), the number of cell nuclei stained with γH2A.X (indicated by circles) was significantly less in the β-NMN-added group (NMN(+)) than in the β-NMN-free group (NMN(-)). Also, as shown in Figure 2, the ratio of γH2A.X-positive cell nuclei among all cell nuclei stained with propidium iodide (PI) was statistically significantly less in the β-NMN-added group (NMN(+)) than in the β-NMN-free group (NMN(-)) (test of difference in population ratios, p<0.05, n>8000). In Figure 2, the bar graph shows the ratio of γH2A.X-positive cell nuclei in each group, and the error bars represent the standard error. This result indicates that the addition of β-NMN alleviates damage to genomic DNA that causes chromosomal abnormalities in stem cells.

Claims

1. A chromosome stabilizing agent for stem cells, comprising as an active ingredient β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof.

2. The chromosome stabilizing agent according to claim 1, which is added to a culture medium for stem cells at a concentration of 0.01 to 5 mM calculated as β-nicotinamide mononucleotide.

3. The chromosome stabilizing agent according to claim 1 or 2, which is used for chromosome stabilization of one or more types of stem cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and hematopoietic stem cells.

4. A method for culturing stem cells, comprising culturing pluripotent stem cells in a culture medium containing β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof.

5. The culture method according to claim 4, wherein the β-nicotinamide mononucleotide concentration in the culture medium is 0.01 to 5 mM.

6. The culture method according to claim 4 or 5, wherein the stem cells are one or more selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and hematopoietic stem cells.

7. A method for chromosome stabilization of stem cells, comprising culturing the stem cells in a culture medium containing β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, or a solvate thereof.

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