WO2021200545A1 - 幹細胞の染色体安定化剤 - Google Patents

幹細胞の染色体安定化剤 Download PDF

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WO2021200545A1
WO2021200545A1 PCT/JP2021/012557 JP2021012557W WO2021200545A1 WO 2021200545 A1 WO2021200545 A1 WO 2021200545A1 JP 2021012557 W JP2021012557 W JP 2021012557W WO 2021200545 A1 WO2021200545 A1 WO 2021200545A1
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stem cells
nmn
cells
chromosomal
medium
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French (fr)
Japanese (ja)
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あゆみ 何
啓太 黄瀬
賀也 富盛
保田　尚孝
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Oriental Yeast Co Ltd
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Oriental Yeast Co Ltd
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Priority to CN202180022602.5A priority Critical patent/CN115315507A/zh
Priority to JP2022512069A priority patent/JP7677949B2/ja
Priority to AU2021247767A priority patent/AU2021247767A1/en
Priority to KR1020227036502A priority patent/KR20220160611A/ko
Priority to EP21780670.2A priority patent/EP4130250A4/en
Priority to US17/907,491 priority patent/US20230117880A1/en
Priority to IL296817A priority patent/IL296817A/en
Publication of WO2021200545A1 publication Critical patent/WO2021200545A1/ja
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Priority to JP2025008641A priority patent/JP2025061552A/ja
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Definitions

  • the present invention relates to a material capable of preventing the occurrence of chromosomal abnormalities in the process of culturing and subculturing stem cells, and a chromosomal stabilizer for stem cells using the material.
  • Stem cells represented by pluripotent stem cells are undifferentiated cells having self-renewal ability and capable of differentiating into various cells.
  • regenerative medicine has been actively studied in which stem cells and cells induced to differentiate from stem cells are transplanted into damaged tissues of patients to regenerate their functions.
  • it is necessary to prepare a large amount of stem cells and their differentiated cells, and therefore, a method for efficiently proliferating stem cells and a method for efficiently differentiating stem cells are being actively developed.
  • stem cells may have chromosomal abnormalities if the culture period is long or the number of passages is large.
  • Chromosomal abnormalities include aneuploidy abnormalities such as monosomy in which two pairs of chromosomes become one and trisomy in which three chromosomes become one, as well as structures such as translocation, inversion, partial duplication, and partial deletion. There is an abnormality.
  • a chromosomal abnormality occurs in a stem cell, not only the proliferative ability and differentiation ability held by the stem cell are lost, but also when the cell or tissue differentiated from the stem cell is used for regenerative medicine or the like, it is mutated into a cancer cell. Or there is a risk of developing a tumor.
  • Chromosomal abnormalities may occur in cancer cells and tumors, and in recent years, methods for more accurately detecting chromosomal abnormalities have been reported (see, for example, Patent Documents 1 and 2).
  • NMN nicotinamide mononucleotide
  • NAD + nicotinamide mononucleotide
  • the present invention relates to a material capable of preventing the occurrence of chromosomal abnormalities in the process of culturing and subculturing stem cells, a stem cell chromosomal stabilizer using the material, a stem cell culturing method, and a stem cell chromosomal stabilizing method.
  • the purpose is to provide.
  • the present invention provides the following stem cell chromosome stabilizer, stem cell culture method, and stem cell chromosome stabilization method.
  • a chromosomal stabilizer for stem cells which comprises ⁇ -nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient.
  • the chromosomal stabilizer according to the above [1] which is added to a stem cell culture medium at 0.01 to 5 mM in terms of ⁇ -nicotinamide mononucleotide.
  • Culture of stem cells which comprises culturing pluripotent stem cells in a culture medium containing ⁇ -nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, or a solvate thereof.
  • Method. [5] The culture method according to [4] above, wherein the ⁇ -nicotinamide mononucleotide concentration in the culture medium is 0.01 to 5 mM.
  • 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.
  • a method for stabilizing stem cells which comprises culturing stem cells in a culture medium containing ⁇ -nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, or a solvate thereof. ..
  • the stem cell chromosomal stabilizer according to the present invention can improve the chromosomal stability of the stem cell and suppress or prevent the occurrence of chromosomal abnormalities by acting on the stem cell. Therefore, by containing the chromosomal stabilizer in the culture medium, the chromosomal stability of the stem cells can be improved, the occurrence of chromosomal abnormalities can be suppressed or prevented, and the stem cells can be cultured more stably. In addition, by using the chromosome stabilizer, the risk of canceration / tumorigenesis of cells or tissues differentiated from stem cells can be reduced.
  • (A) is a fluorescence photograph of cells stained with PI in Example 2, and (b) is an anti- ⁇ H2A. It is a fluorescent photograph immunostained with an X antibody, and (c) is a photograph in which (a) and (b) are merged. ⁇ H2A. In all PI-stained cells. It is a graph which shows the ratio of the cell nucleus of X positive.
  • stem cells are undifferentiated cells having self-renewal ability and differentiation ability (ability to differentiate into various cell types), for example, ES cells (embryonic stem cells).
  • pluripotent stem cells such as iPS cells (induced pluripotent stem cells)
  • somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, nerve stem cells, and skin stem cells can be mentioned.
  • the stem cell chromosomal stabilizer according to the present invention (hereinafter, may be referred to as “chromosome stabilizer according to the present invention”) contains NMN (chemical formula: C 11 H 15 N 2 O 8 P) as an active ingredient. It is added to the culture medium when stem cells are cultured and subcultured. By culturing and subculturing stem cells in the presence of NMN, the stability of the stem cells' chromosomes can be improved, and the occurrence of chromosomal abnormalities can be suppressed or prevented.
  • NMN chemical formula: C 11 H 15 N 2 O 8 P
  • NMN optical isomers
  • ⁇ and ⁇ the NMN that is the active ingredient of the chromosomal stabilizer according to the present invention
  • ⁇ -NMN CAS number: 1094-61-7
  • the structure of ⁇ -NMN is shown below.
  • the ⁇ -NMN as the active ingredient may be prepared by any method.
  • ⁇ -NMN that has been artificially synthesized by a chemical synthesis method, an enzyme method, a fermentation method, or the like can be used as an active ingredient.
  • ⁇ -NMN is a component widely present in a living body
  • ⁇ -NMN obtained by extracting and purifying from natural raw materials such as animals, plants and microorganisms can also be used as an active ingredient.
  • commercially available purified ⁇ -NMN may be used.
  • ⁇ -NMN can be produced by reacting NAM with L-ribose tetraacetate and phosphorylating the obtained nicotinamide mononucleoside.
  • ⁇ -NMN can be produced from NAM and 5'-phosphoribosyl-1'-pyrrolic acid (PRPP) by nicotinamide phosphoribosyl transferase (NAMPT).
  • PRPP 5'-phosphoribosyl-1'-pyrrolic acid
  • NAMPT nicotinamide phosphoribosyl transferase
  • ⁇ -NMN can be produced from NAM by utilizing the metabolic system of a microorganism expressing NAMPT.
  • the active ingredient of the chromosomal stabilizer according to the present invention may be a pharmacologically acceptable salt of ⁇ -NMN.
  • the pharmacologically acceptable salt of ⁇ -NMN may be an inorganic acid salt or an organic acid salt having a basic moiety such as amine.
  • the 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 and ISEthione.
  • the pharmacologically acceptable salt of ⁇ -NMN may be an alkaline salt or an organic salt having an acidic moiety such as a carboxylic acid.
  • Examples of the base constituting such an acid salt are alkali metal salts or alkaline earth metal salts, such as sodium hydride, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, and magnesium hydroxide.
  • Zinc hydroxide ammonia, trimethylamonia, triethylammonia, ethylenediamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocine, procaine, diethanolamine, N-benzylphenethylamine, diethylamine, piperazin, tris ( Those derived from bases such as hydroxymethyl) -aminomethane and tetramethylammonium hydroxide can be mentioned.
  • the active ingredient of the chromosomal stabilizer according to the present invention may be a solvate of a free ⁇ -NMN or a pharmacologically acceptable salt of ⁇ -NMN.
  • the solvent for forming the solvate include water, ethanol and the like.
  • the chromosomal stabilizer according to the present invention may contain other active ingredients in addition to ⁇ -NMN.
  • the other active ingredient used in combination with ⁇ -NMN may be only one kind or a combination of two or more kinds.
  • Other active ingredients are known to enhance the survival efficiency and proliferation efficiency of stem cells such as albumin, ascorbic acid, ⁇ -tocopherol, insulin, transferase, sodium selenate, ethanolamine, and Rock inhibitor.
  • Ingredients such as valproic acid, dimethylsulfoxide, dexamethasone, butyric acid, tricostatin A, GSK3 inhibitor, BMP inhibitor, Wnt inhibitor, activin, nogin and other components known to enhance the differentiation efficiency of stem cells. It can be appropriately selected from the above and used.
  • the culture medium contains the chromosomal stabilizer according to the present invention to improve the chromosomal stability of the stem cells and to proliferate the stem cells while suppressing or preventing the occurrence of chromosomal abnormalities. Can be done.
  • the amount of the chromosomal stabilizer according to the present invention contained in the stem cell culture medium is lower than that in the case of culturing in the culture medium not containing the chromosomal stabilizer.
  • the amount is not particularly limited as long as the concentration is sufficient to suppress the suppression, and can be appropriately adjusted in consideration of the type of stem cells, the balance with other components of the culture medium, and the like.
  • the chromosomal stabilizing effect may be weak, and if an excessive amount of ⁇ -NMN is contained, cell proliferation may be suppressed.
  • the content of the chromosomal stabilizer according to the present invention in the culture medium is preferably such that the ⁇ -NMN concentration is 0.01 to 5 mM, and more preferably 0.05 to 2 mM. , 0.1 to 1 mM, more preferably.
  • the ⁇ -NMN concentration is within the above range, the chromosome of the stem cell can be sufficiently stabilized.
  • Stem cell culture in the presence of the chromosomal stabilizer according to the present invention can be carried out by a conventional method except that the culture medium contains the chromosomal stabilizer according to the present invention.
  • the culture medium a medium used for maintaining or proliferating stem cells or a medium used for culturing animal cells can be generally used.
  • commercially available culture media for various stem cells can also be used.
  • examples of the medium containing the chromosomal stabilizer according to the present invention and used for culturing stem cells include Eagle's minimum essential medium (MEM), Dalbeco's modified Eagle's medium (DMEM), and ⁇ Eagle's minimum essential medium.
  • MEM Eagle's minimum essential medium
  • DMEM Dalbeco's modified Eagle's medium
  • ⁇ Eagle's minimum essential medium ⁇ Eagle's minimum essential medium.
  • ⁇ MEM Iscover modified Dalveco medium
  • IMDM Iscover modified Dalveco medium
  • F-12 medium F-10 medium
  • DMEM / F12 medium RPMI-1640 medium
  • MSCBM Membrane cell basal medium
  • E8 (Essential 8) medium E8 (Essential 8) medium
  • TeSR -E8 medium mTeSR1 medium
  • MCDB medium MCDB medium and the like
  • these culture media may have components known to enhance the survival efficiency and proliferation efficiency of stem cells, and have an action of maintaining an undifferentiated state of stem cells.
  • Known components and the like may be appropriately contained. As these components, the above-mentioned ones can be used.
  • the culture conditions can generally be the culture conditions for culturing animal cells, and may be appropriately modified as necessary.
  • the culture can be performed at a culture temperature of 30 to 40 ° C., a CO 2 concentration of 1 to 10% by volume, and an O 2 concentration of 0.1 to 25% by volume.
  • stem cells whose chromosomes are stabilized by the chromosome stabilizer according to the present invention animal-derived stem cells are preferable, mammalian-derived stem cells are more preferable, and human-derived stem cells are even more preferable.
  • the stem cells whose chromosomes are stabilized by the chromosomal stabilizer according to the present invention are preferably animal-derived ES cells, iPS cells, or mesenchymal stem cells, and are mammalian-derived ES cells and iPS cells. , Or mesenchymal stem cells, more preferably human-derived ES cells, iPS cells, or mesenchymal stem cells.
  • Example 1 The effect of ⁇ -NMN on chromosomes during culture of mesenchymal stem cells was investigated.
  • mesenchymal stem cells two donors of human adipose tissue-derived mesenchymal stem cells (ADSC) (product number: PT-5006, manufactured by Lonza) were used.
  • ADSC human adipose tissue-derived mesenchymal stem cells
  • a medium obtained by mixing DMEM and MCDB medium at a ratio of 1: 1 is used as a basal medium (see Patent No. 5804385), and FGF, PDGF, TGF- ⁇ , HGF, etc. are used in the basal medium.
  • the medium to which EGF, phospholipids, fatty acids and the like were added was used as the basal medium.
  • the ⁇ -NMN-added medium was prepared by adding ⁇ -NMN to the basal medium so as to have a concentration of 0.25 mM.
  • ⁇ Subculture> Mesenchymal stem cells were seeded at 5 ⁇ 10 3 cells / cm 2 in a culture dish previously coated with 2.5 ⁇ g / cm 2 fibronectin, and cultured using a basal medium. One day after sowing, the medium was replaced with basal medium or ⁇ -NMN-added medium. After that, the medium was changed once every 2 or 3 days. Subculture was performed when the cell density in the culture dish reached a confluency of about 80 to 90%.
  • the culture supernatant was removed, washed with 1 ⁇ PBS (-), and then the cells were detached using 1 ⁇ Triple select (product number: 12563011, manufactured by Thermo Fisher) to produce 37 ° C. and 5% CO 2 . It was allowed to stand for 4 minutes under the conditions. After the cells were singled by pipetting, basal medium or ⁇ -NMN-added medium was added, and the cells were centrifuged. After suspending the collected cells in each medium, cell counting was performed, and the cells were seeded in a pre-coated culture dish so as to have a concentration of 5 ⁇ 10 3 cells / cm 2. Medium exchange was performed once every 2 or 3 days using basal medium or ⁇ -NMN-added medium.
  • Frozen cells were seeded in a coated culture dish at 5 ⁇ 10 3 cells / cm 2 in basal medium or ⁇ -NMN-added medium, and the medium was replaced 1 day after seeding. After that, the medium was changed once every two or three days.
  • Subculture was performed once and seeded in a T25 flask at 5 ⁇ 10 3 cells / cm 2. Three days after seeding, the T25 flask was filled with a medium and sent to a chromosomal safety test consignment organization (Japan Genetic Research Institute Co., Ltd.) for chromosome analysis.
  • ⁇ Chromosome stability test> The chromosomal status of the mesenchymal stem cells after culturing was confirmed by the G banding method.
  • Table 1 shows the measurement results of the number of chromosomes
  • Table 2 shows the results of karyotype analysis. The analysis results are described in accordance with the International System of Human Cytogenomic Nomenclature (ISCN) based on the International Code.
  • ISCN International System of Human Cytogenomic Nomenclature
  • Test group 1 shows mesenchymal stem cells (ADSC donor 1, male) cultured in the basal medium
  • test group 2 shows mesenchymal stem cells (ADSC donor 2, female) cultured in the basal medium
  • Test Group 3 contains mesenchymal stem cells (ADSC donor 1, male) cultured in ⁇ -NMN-added medium
  • Test Group 4 contains mesenchymal stem cells (ADSC donor 2, female) cultured in ⁇ -NMN-added medium. show.
  • test groups 1 and 2 In the mesenchymal stem cells cultured in the basal medium, abnormal numbers 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 of 50 cells in each test group, in test group 1, the number of chromosomes increased by 1 from 46 to 47 in 4 cells. Also, in Test Group 2, the number of chromosomes decreased by 1 from 46 to 45 in one cell. On the other hand, in the mesenchymal stem cells cultured in the ⁇ -NMN-added medium, the number of chromosomes was 46 in all the cells of both donors (test groups 3 and 4), which was the normal number of human chromosomes.
  • ⁇ -NMN human pluripotent stem cells
  • iPSCs human pluripotent stem cells
  • 201B7 strain was used, and 5% until the cell density reached about 80% on a 24-well cell culture plate pre-coated with extracellular matrix (Matrigel: Corning). Those cultured at CO 2 and 37 ° C. were subjected to the test.
  • cytokines and salts (insulin, transferase, TGF ⁇ , FGF, sodium selenate, ascorbic acid, hydrogen carbonate) known to be necessary for maintenance subculture of iPSC in the basal medium (DMEM-F12)
  • NMN (-) A ⁇ -NMN-free group (NMN (-)) in which culture is performed in the basal medium using a basal medium containing sodium) and a ⁇ -NMN-added group in which ⁇ -NMN is added to the basal medium so as to have a final concentration of 1 mM. (NMN (+)) was provided.
  • NCX4016 (Sigma-Aldrich, product number SML1669) was added to each of the ⁇ -NMN-free group and the ⁇ -NMN-added group to a final concentration of 5 ⁇ M, and the mixture was incubated for 2 hours to induce chromosomal damage. The cells with damaged nuclear genomic DNA were then subjected to phosphorylated histone H2A. It was detected by labeling with X ( ⁇ H2AX).
  • ⁇ H2A The number of X-stained cell nuclei (indicated by circles) was significantly smaller in the ⁇ -NMN-added group (NMN (+)) than in the ⁇ -NMN-free group (NMN ( ⁇ )).
  • ⁇ H2A In whole cell nuclei stained with propidium iodide (PI). The proportion of X-positive cell nuclei was statistically significantly lower in the ⁇ -NMN-added group (NMN (+)) than in the ⁇ -NMN-free group (NMN (-)) (test of difference in population ratio, p. ⁇ 0.05, n> 8000).
  • PI propidium iodide
  • the bar graph shows ⁇ H2A.
  • the ratio of X-positive cell nuclei is shown, and the error bars indicate the range of the standard error. This result indicates that in stem cells, the addition of ⁇ -NMN alleviates damage to genomic DNA that causes chromosomal abnormalities.

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