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WO2010150650A1 - Nouveau procédé pour maintenir des cellules souches dans un état indifférencié - Google Patents

Nouveau procédé pour maintenir des cellules souches dans un état indifférencié Download PDF

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Publication number
WO2010150650A1
WO2010150650A1 PCT/JP2010/059705 JP2010059705W WO2010150650A1 WO 2010150650 A1 WO2010150650 A1 WO 2010150650A1 JP 2010059705 W JP2010059705 W JP 2010059705W WO 2010150650 A1 WO2010150650 A1 WO 2010150650A1
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cells
cell
undifferentiated state
expression vector
maintaining
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Japanese (ja)
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雅規 本田
剛和 三上
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Nihon University
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Nihon University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention uses CD271 (NGFR, nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR, p75 neurotrophin receptor: p75 nerve growth factor receptor, hereinafter referred to as CD271), mesenchymal stem cells, ES cells,
  • CD271 nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR, p75 neurotrophin receptor: p75 nerve growth factor receptor, hereinafter referred to as CD271
  • NGFR nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR
  • p75 neurotrophin receptor p75 nerve growth factor receptor
  • the present invention relates to a method for maintaining an undifferentiated state such as iPS cells.
  • MSCs mesenchymal stem cells
  • ES cells ES cells
  • iPS cells are undifferentiated and are known as pluripotent cells.
  • MSCs mesenchymal stem cells
  • ES cells ES cells
  • iPS cells are undifferentiated and are known as pluripotent cells.
  • methods for maintaining various undifferentiated states have been studied. For example, methods for introducing genes encoding Fibrillalin, AOF2, TIF1 ⁇ , etc. into these cells (Patent Documents 1 to 3), and proteins of the Wnt family
  • Patent Document 4 A method of culturing using a maintenance medium in an undifferentiated state containing lysine
  • Fibrillarin, AOF2, and TIF1 ⁇ are known as genes that are specifically expressed in mouse ES cells in the presence of LIF (leukemia inhibitor factor), which is necessary when mouse ES cells proliferate while maintaining an undifferentiated state. ing.
  • LIF leukemia inhibitor factor
  • genes are introduced, resistance genes for drugs such as geneticin and hygromycin are simultaneously introduced, and cells are selected using these drugs.
  • this method merely confirms the expression of the drug resistance gene, and does not directly confirm the expression of the target gene itself.
  • cells that have become drug resistant must be cultured from a single colony, but this requires long-term culture, There is also the possibility of affecting differentiation. Therefore, methods for introducing genes expressed in the cytoplasm or nucleus, such as Fibrillalin, AOF2, and TIF1 ⁇ , have various problems.
  • CD271 has been conventionally known to be localized and expressed in the cell membrane of cells of the nervous system and to be involved in the development, survival and differentiation of nerve cells. In recent years, it has been shown that it is specifically expressed in the cell membrane of MSCs, and a method for concentrating MSCs from a bone marrow extract using CD271 has been developed as an optimal marker for MSCs (Patent Literature). 5). Since CD271 is localized and expressed in the cell membrane, it differs from the case of introducing a gene expressed in the cytoplasm or nucleus such as Fibrillarin, AOF2, TIF1 ⁇ .
  • CD271 when CD271 is introduced, there is an advantage that cells expressing CD271 and non-expressing cells can be selected alive by using a fluorescently labeled antibody against CD271.
  • CD271 in MSCs has not been clarified, and it has not been predicted that the use of CD271 can maintain undifferentiated states such as mesenchymal stem cells, ES cells, and iPS cells.
  • An object of the present invention is to provide a method for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like using CD271.
  • the present inventors introduce a vector that expresses CD271 into pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells.
  • pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells.
  • bone marrow-derived mesenchymal stem cells or dental pulp-derived mesenchymal stem cells that are forcibly expressed by introducing a vector that expresses CD271 can be used for osteoblasts, adipocytes, and the like. It was confirmed that differentiation and differentiation into osteoblasts / odontoblasts were suppressed.
  • the present invention relates to the following methods (1) to (9) for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like.
  • (1) A method of maintaining an undifferentiated state of a cell by introducing a CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein into an undifferentiated state cell.
  • (2) The method according to (1) above, wherein the CD271 expression vector is an expression vector into which all or part of the gene encoding CD271 described in SEQ ID NO: 1 of the Sequence Listing is incorporated.
  • the undifferentiated cells are any of mesenchymal stem cells, ES cells, or iPS cells.
  • a cell in which an undifferentiated state is maintained by the method according to any one of (1) to (4) above.
  • a pharmaceutical for treating metabolic syndrome by inhibiting cell differentiation into adipocytes comprising the composition according to (8) above.
  • other preferred embodiments according to the present invention are as follows.
  • A) A method of suppressing cell differentiation into adipocytes or maintaining an undifferentiated state of cells by administering the composition according to (8) above.
  • B) A method for preventing or treating metabolic syndrome by suppressing cell differentiation into adipocytes with a pharmaceutical comprising the composition according to (8) above.
  • a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells.
  • Example 2 It is the figure which showed CD271 expression vector (Example 1). It is the figure which confirmed the expression of CD271 in the CD271 positive cell (Test Example 1). It is the figure which showed the correlation of the activity level of the alkaline phosphatase in CD271 positive cell, and the expression level of CD271 (Test Example 2). (Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell. (Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell.
  • the “method for maintaining the undifferentiated state of a cell” of the present invention is achieved by introducing a recombinant gene containing a gene encoding CD271 or a CD271 expression vector into which the recombinant gene has been inserted into a cell such as a stem cell. Any method can be included as long as it can maintain the undifferentiated state.
  • the “method for maintaining the undifferentiated state of cells” of the present invention preferably controls the undifferentiated state of stem cells by forcibly expressing CD271 in the introduced cells such as stem cells by introducing a CD271 expression vector.
  • the “CD271 expression vector” of the present invention may include any recombinant gene containing a gene encoding CD271 or any vector capable of expressing CD271 into which the recombinant gene has been inserted.
  • Commercial products such as FCC117C02 (FIG. 1) can be mentioned.
  • examples include those in which a gene encoding CD271 is incorporated into an expression vector such as pcDNA3.1 (Invitrogen), pCMV5, pEGFP series (Clontech), p3XFLAG-CMV series (SIGMA).
  • the CD271 gene incorporated in the “CD271 expression vector” of the present invention may not be the entire gene encoding CD271, as long as it can maintain the undifferentiated state of cells such as stem cells, and the functional region of CD271, etc. It may be a gene encoding only a necessary sequence.
  • the “cell membrane permeabilizing recombinant CD271 protein” of the present invention refers to a CD271 protein to which a substance having cell membrane permeability such as polyarginine is bound.
  • the CD271 protein introduced into the stem cells or the like is not the entire CD271 protein as long as it can maintain the undifferentiated state of the cells such as the stem cells, but a part thereof is bound to a substance having cell membrane permeability such as polyarginine. It may be a thing.
  • the “undifferentiated state cell” of the present invention refers to a cell that maintains two functions of self-renewal ability and pluripotency, and in mesenchymal stem cells, ES cells, iPS cells, etc. A cell in which these functions are maintained.
  • the “kit for maintaining an undifferentiated state of a cell” of the present invention is a “CD271 expression vector” for expressing CD271 in a cell such as a stem cell or a “kit for introducing CD271 into a cell such as a stem cell”.
  • CD271 expression vector for expressing CD271 in a cell such as a stem cell
  • kit for introducing CD271 into a cell such as a stem cell “Cell membrane permeabilized recombinant CD271 protein” and a kit composed of a combination of one or more of a series of reagents necessary for the introduction thereof.
  • a composition containing CD271 as an active ingredient for inhibiting cell differentiation or maintaining the undifferentiated state of cells means that CD271 is used as an active ingredient to inhibit cell differentiation or maintain the undifferentiated state of cells. Any composition that can be used is included.
  • the CD271 used as an active ingredient may be the “CD271 expression vector” of the present invention for expressing CD271 in cells or the “cell membrane permeabilized recombinant CD271 protein” for introduction into cells.
  • This composition can also contain a pharmaceutically acceptable carrier in addition to CD271 as an active ingredient.
  • composition for suppressing cell differentiation or maintaining the undifferentiated state of cells is a composition capable of suppressing cell differentiation and maintaining the undifferentiated state of cells. Therefore, for example, by inhibiting the differentiation of cells into adipocytes, it can be used for the treatment of metabolic syndrome such as the manufacture of pharmaceuticals such as obesity inhibitors and the prevention and treatment of metabolic syndrome. In addition, by inhibiting the differentiation of cells into osteoblasts, it can also be used for the prevention and treatment of marbleosis and osteosclerosis caused by excessive bone formation, the production of pharmaceuticals therefor, and the like.
  • CD271 as an active ingredient in the present invention can be expected to function as a factor that suppresses hyperplasia caused by the proliferation of differentiated cells.
  • MSCs have pluripotency and differentiate into osteoblasts and adipocytes. Therefore, CD271 specifically expressed in MSCs is considered to have a function related to the multipotency of MSCs, and the difference in differentiation potential between cells expressing CD271 and cells not expressing CD271 was examined. .
  • the dental pulp cells having the ability to differentiate into osteoblasts / odontoblasts / adipocytes / neurons are mesenchymal cells and express CD90. Therefore, CD271 positive cells in dental pulp cells are It may correspond to leaf stem cells (MSCs).
  • MSCs leaf stem cells
  • Each of the sorted cells was cultured in ⁇ -MEM medium containing 20% FBS, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin until it became confluent. Next, total RNA was produced from each cell, and based on this, mRNA expression of CD271 was confirmed using a real-time PCR method. As a result, it was confirmed that CD271 positive cells expressed CD271 about 40 times as compared with CD271 negative cells (FIG. 2).
  • Primer Perfect Real Time Primer
  • primer set ID HA036699
  • Takara Bio Reagents SYBR Premix Ex Taq II
  • Product Code RR081A
  • Analytical Equipment Smart Cycler II System C, System C
  • Test Example 2 CD271 positive cells and CD271 negative cells selected and cultured in the same manner as in Test Example 1 were mixed with osteoblast differentiation induction medium (100 ng / ml BMP-2, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 20% FBS, ( ⁇ -MEM medium containing 100 U / ml penicillin, 100 ⁇ g / ml streptomycin) for 6 days, 12 days, and 24 days, and the activity of alkaline phosphatase, which is an index of osteoblast differentiation, was determined to be alkaline phosphatase (ALP) staining solution.
  • osteoblast differentiation induction medium 100 ng / ml BMP-2, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 20% FBS, ( ⁇ -MEM medium containing 100 U / ml penicillin, 100 ⁇ g /
  • ⁇ Method for maintaining the undifferentiated state of mesenchymal stem cells, ES cells, and iPS cells By introducing CD271 into a C3H10T1 / 2 cell, which is an undifferentiated mesenchymal stem cell line derived from a mouse, by constantly expressing CD271, the undifferentiated state of this cell was maintained.
  • CD271 expression vector into C3H10T1 / 2 cells
  • C3H10T1 / 2 cells were made 50% confluent in cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin ( ⁇ -MEM). Pre-cultured in 6 well dishes until complete. Thereafter, the culture solution was replaced with a cell culture solution ( ⁇ -MEM) containing 5% fetal bovine serum, and pre-culture was performed for 2 to 3 hours.
  • a C271 expression vector (TOYOBO cDNA Clones NGFR, Code No. FCC117C02) purchased from TOYOBO was cleaved with DNA restriction enzyme ScaI to generate a linearized vector.
  • the geneticin resistance gene expression region contained in the pcDNA3.1 vector was excised with DNA restriction enzymes KpnI and ApLI.
  • a linearized CD271 expression vector (2 ⁇ g) and a DNA (0.3 ⁇ g) containing a geneticin resistance gene expression region excised from the pcDNA3.1 vector were transfected into cells using lipofectamine LTX of Invitrogen according to the attached manual.
  • the cell culture medium was replaced with a cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, and cultured for 24 hours. Thereafter, the cells were cultured for 10 days in the presence of geneticin. Thereafter, FACS analysis was performed using a fluorescently labeled CD271 antibody, and cells expressing CD271 were selected.
  • Example 1 Induction of differentiation into osteoblasts In the ⁇ -MEM medium containing 10% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, until the C3H10T1 / 2 cells of Example 1 (CD271 forced expression cells) became confluent. Cultured for about 3 weeks.
  • osteoblast differentiation medium 100 ng / ml BMP-2, 10-8M Dexamethasone, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 10% FBS, 100 U / ml penicillin, ⁇ containing 100 ⁇ g / ml streptomycin
  • -MEM medium was further cultured for 12 days, and alkaline phosphatase staining was performed in the same manner as in Test Example 2 above.
  • cells into which only an expression vector not containing CD271 was introduced were used.
  • Example 2 Induction of differentiation into adipocytes
  • the C3H10T1 / 2 cells (CD271 forced expression cells) of Example 1 were cultured until confluent as in 1) above, and then adipocyte differentiation induction medium (100 ng / ml BMP- 2, 10-8 M Dexamethasone, ⁇ -MEM medium containing 100% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin) for 12 days, and formation of fat globules as an indicator of fat cells Observed by staining. Cell nuclei were stained with hematoxylin. For comparison, cells into which only an expression vector not containing CD271 was introduced (control cells) were used.
  • adipocyte differentiation induction medium 100 ng / ml BMP- 2, 10-8 M Dexamethasone, ⁇ -MEM medium containing 100% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin
  • a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells.
  • a composition for suppressing cell differentiation and maintaining an undifferentiated state containing CD271 as an active ingredient to provide a pharmaceutical (eg, a pharmaceutical for treating metabolic syndrome by inhibiting adipocyte differentiation). You can also.

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Abstract

L'invention porte sur un procédé pour maintenir l'état indifférencié de cellules souches mésenchymateuses, de cellules ES et de cellules iPS à l'aide de CD271. Par l'introduction d'un vecteur exprimant CD271 ou par introduction directe de protéine CD271 recombinante à membrane cellulaire perméabilisée dans des cellules non différenciées pluripotentes telles que des cellules souches mésenchymateuses, des cellules ES et des cellules iPS, l'état indifférencié de ces cellules est maintenu.
PCT/JP2010/059705 2009-06-23 2010-06-08 Nouveau procédé pour maintenir des cellules souches dans un état indifférencié Ceased WO2010150650A1 (fr)

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JP2009-148337 2009-06-23
JP2009148337A JP5645197B2 (ja) 2009-06-23 2009-06-23 幹細胞の未分化状態を維持する新規方法

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JP6478418B2 (ja) * 2014-07-11 2019-03-06 国立研究開発法人産業技術総合研究所 細胞分化ポテンシャル判別法

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