WO2017082294A1 - Milieu de culture de cellules souches, promoteur de prolifération et procédé de culture, composition cellulaire comprenant des cellules souches et son procédé de production - Google Patents

Milieu de culture de cellules souches, promoteur de prolifération et procédé de culture, composition cellulaire comprenant des cellules souches et son procédé de production Download PDF

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WO2017082294A1
WO2017082294A1 PCT/JP2016/083232 JP2016083232W WO2017082294A1 WO 2017082294 A1 WO2017082294 A1 WO 2017082294A1 JP 2016083232 W JP2016083232 W JP 2016083232W WO 2017082294 A1 WO2017082294 A1 WO 2017082294A1
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peptide
amino acid
bfgf
seq
stem cells
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PCT/JP2016/083232
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Japanese (ja)
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義基 中島
健史 大政
正義 塚原
誠 柿谷
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協和発酵キリン株式会社
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

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  • the present invention relates to a medium for culturing stem cells, a growth promoter and a culture method, a cell composition containing stem cells, and a method for producing the same. More specifically, the present invention relates to a medium for culturing stem cells and the like containing a substance that exhibits excellent growth promoting activity on stem cells.
  • bFGF basic fibroblast growth factor
  • fibroblast growth factor 2 basic fibroblast growth factor 2
  • Patent Documents 1 to 4 As described above, since bFGF is very expensive, an alternative substance has been developed (for example, Patent Documents 1 to 4), but a substance having a sufficient proliferation promoting function for stem cells has been obtained. Absent.
  • the main object of the present invention is to provide a substance that exhibits excellent growth promoting activity against stem cells.
  • [BFGF heparin-binding region-containing peptide] In order to solve the above problems, the present invention provides the following [1] to [40].
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10.
  • the growth promoter according to [8], wherein the bFGF heparin-binding region-containing peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10.
  • B including any amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10, and proliferates stem cells Peptides with promoting activity.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a method for culturing stem cells comprising a step of culturing stem cells in a medium containing a peptide containing a bFGF heparin binding region.
  • the culture method according to [14], wherein the bFGF heparin-binding region-containing peptide is the following peptide (A), (B), or (C).
  • B including any amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10, and proliferates stem cells Peptides with promoting activity.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • a method for producing a cell composition containing stem cells comprising a step of culturing stem cells in a medium containing a peptide containing a bFGF heparin binding region.
  • a method for producing a cell composition containing stem cells comprising a step of culturing stem cells in a medium containing a peptide containing a bFGF heparin binding region.
  • the bFGF heparin-binding region-containing peptide is the following peptide (A), (B) or (C).
  • a method for producing a cell composition containing differentiated cells comprising a step of culturing stem cells in a medium containing a bFGF heparin-binding region-containing peptide and a step of inducing stem cell differentiation.
  • the production method of [28], wherein the bFGF heparin-binding region-containing peptide is the following peptide (A), (B), or (C).
  • a peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10.
  • the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • the stem cell is derived from a human.
  • the medium is a serum-free medium.
  • a cell composition comprising a bFGF heparin-binding region-containing peptide and stem cells and / or differentiated cells derived from stem cells.
  • B including any amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6 to 10, and proliferates stem cells Peptides with promoting activity.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • [BFGF fragment peptide] The present invention also provides the following [41] to [68].
  • [41] A medium for culturing stem cells, comprising a bFGF fragment peptide.
  • A) A peptide comprising any one amino acid sequence shown in SEQ ID NOs: 2, 5, and 16.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a stem cell proliferation promoter comprising a bFGF fragment peptide.
  • the growth promoter according to [46], wherein the bFGF fragment peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising any one amino acid sequence shown in SEQ ID NOs: 2, 5, and 16.
  • B an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 2, 5 and 16, and has an activity of promoting the proliferation of stem cells Having peptide.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a method for culturing stem cells comprising a step of culturing stem cells in a medium containing a bFGF fragment peptide.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a method for producing a cell composition containing a stem cell comprising a step of culturing the stem cell in a medium containing a bFGF fragment peptide.
  • A A peptide comprising any one amino acid sequence shown in SEQ ID NOs: 2, 5, and 16.
  • B an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 2, 5 and 16, and has an activity of promoting the proliferation of stem cells Having peptide.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • [57] The production method of [55] or [56], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • the stem cells are derived from a human.
  • the medium is a serum-free medium.
  • a cell composition comprising a bFGF fragment peptide and stem cells and / or differentiated cells derived from stem cells.
  • A A peptide comprising any one amino acid sequence shown in SEQ ID NOs: 2, 5, and 16.
  • B an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 2, 5 and 16, and has an activity of promoting the proliferation of stem cells Having peptide.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a stem cell proliferation promoter comprising an FGF receptor-binding peptide.
  • the growth promoter according to [74], wherein the FGF receptor-binding peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14.
  • B a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14 and having an activity of promoting the proliferation of stem cells .
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • [76] The proliferation promoter according to [74] or [75], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • a method for culturing a stem cell comprising a step of culturing the stem cell in a medium containing an FGF receptor binding peptide.
  • the culture method according to [78], wherein the FGF receptor-binding peptide is the following peptide (A), (B) or (C).
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a method for producing a cell composition containing stem cells comprising a step of culturing stem cells in a medium containing an FGF receptor binding peptide.
  • [84] The production method of [83], wherein the FGF receptor-binding peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14.
  • B a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14 and having an activity of promoting the proliferation of stem cells .
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the production method of [83] or [84], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • the production method of any of [83] to [85], wherein the stem cell is derived from a human.
  • the production method of any of [83] to [86], wherein the medium is a serum-free medium.
  • a method for producing a cell composition containing differentiated cells comprising a step of culturing stem cells in a medium containing a bFGF receptor-binding peptide and a step of inducing differentiation of stem cells.
  • [89] The production method of [88], wherein the FGF receptor-binding peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14.
  • B a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14 and having an activity of promoting the proliferation of stem cells .
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • A A peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14.
  • B a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 11 to 14 and having an activity of promoting the proliferation of stem cells .
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • [95] The cell composition according to [93] or [94], wherein the stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • [BFGF binding protein fragment peptide] The present invention also provides the following [97] to [124].
  • [97] A medium for stem cell culture, comprising a bFGF-binding protein fragment peptide.
  • A) A peptide comprising the amino acid sequence shown in SEQ ID NO: 15.
  • B A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 15 and having an activity of promoting proliferation of stem cells.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the medium according to [97] or [98], wherein the stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • the medium according to any one of [97] to [99], wherein the stem cells are derived from human.
  • the medium according to any one of [97] to [100], wherein the medium is a serum-free medium.
  • a stem cell proliferation promoter comprising a bFGF binding protein fragment peptide.
  • the growth promoter according to [102], wherein the bFGF binding protein fragment peptide is the following peptide (A), (B) or (C).
  • A) A peptide comprising the amino acid sequence shown in SEQ ID NO: 15.
  • (B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 15 and having an activity of promoting proliferation of stem cells.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B).
  • [104] The growth promoter according to [102] or [103], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell.
  • a method for culturing stem cells comprising a step of culturing stem cells in a medium containing a bFGF binding protein fragment peptide.
  • the culture method of [106], wherein the bFGF binding protein fragment peptide is the following peptide (A), (B) or (C).
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the production method of [111], wherein the bFGF-binding protein fragment peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising the amino acid sequence shown in SEQ ID NO: 15.
  • B A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 15 and having an activity of promoting proliferation of stem cells.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B). [113] The production method of [111] or [112], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell. [114] The production method of any one of [111] to [113], wherein the stem cells are derived from a human.
  • (B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 15 and having an activity of promoting proliferation of stem cells.
  • (C) A peptide obtained by dimerizing the peptide according to (A) or (B). [118] The production method of [116] or [117], wherein the stem cell is an inducible pluripotent stem cell or a mesenchymal stem cell. [119] The production method of any of [116] to [118], wherein the stem cells are derived from human. [120] The production method of any one of [116] to [119], wherein the medium is a serum-free medium.
  • a cell composition comprising a bFGF-binding protein fragment peptide and stem cells and / or differentiated cells derived from stem cells.
  • the cell composition of [121], wherein the bFGF binding protein fragment peptide is the following peptide (A), (B) or (C).
  • A A peptide comprising the amino acid sequence shown in SEQ ID NO: 15.
  • B A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 15 and having an activity of promoting proliferation of stem cells.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • stem cell is derived from a human.
  • [Low molecular compounds] provides the following [125] to [152].
  • [125] A medium for culturing stem cells, comprising one or more low-molecular compounds selected from the group consisting of a cholinesterase inhibitor, a phenothiazine derivative, a steroid, an acetylcholine receptor agonist, and a prostaglandin receptor agonist.
  • the medium according to [125] wherein the low molecular compound is epiboxidine hydrochloride.
  • the medium according to [125] or [126], wherein the stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • a stem cell proliferation promoter comprising one or more low molecular weight compounds selected from the group consisting of a cholinesterase inhibitor, a phenothiazine derivative, a steroid, an acetylcholine receptor agonist, and a prostaglandin receptor agonist.
  • the growth promoter according to [130], wherein the low molecular compound is epiboxidine hydrochloride.
  • a method for culturing stem cells comprising a step of culturing stem cells in a medium containing one or more low molecular weight compounds selected from the group consisting of cholinesterase inhibitors, phenothiazine derivatives, steroids, acetylcholine receptor agonists, and prostaglandin receptor agonists. .
  • a cell containing a stem cell comprising a step of culturing the stem cell in a medium containing one or more low molecular weight compounds selected from the group consisting of a cholinesterase inhibitor, a phenothiazine derivative, a steroid, an acetylcholine receptor agonist, and a prostaglandin receptor agonist.
  • a method for producing the composition [140] The production method of [139], wherein the low-molecular compound is epiboxidine hydrochloride.
  • the production method of [139] or [140] wherein the stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • the medium is a serum-free medium.
  • a step of culturing stem cells in a medium containing one or more low-molecular compounds selected from the group consisting of cholinesterase inhibitors, phenothiazine derivatives, steroids, acetylcholine receptor agonists, and prostaglandin receptor agonists, and induction of stem cell differentiation A method for producing a cell composition containing differentiated cells.
  • the production method of [144], wherein the low-molecular compound is epiboxidine hydrochloride.
  • stem cells are inducible pluripotent stem cells or mesenchymal stem cells.
  • stem cells are derived from a human.
  • medium is a serum-free medium.
  • one or more low molecular weight compounds selected from the group consisting of cholinesterase inhibitors, phenothiazine derivatives, steroids, acetylcholine receptor agonists, and prostaglandin receptor agonists, and stem cells and / or differentiated cells derived from stem cells, A cell composition comprising.
  • the cell composition according to [149], wherein the low-molecular compound is epiboxidine hydrochloride.
  • bFGF heparin-binding region-containing peptide refers to bFGF such as human full-length bFGF peptide (GenBank accession number: NP_001997) or mouse bFGF full-length peptide (GenBank accession number: NP_032032) containing the heparin-binding region of bFGF.
  • BFGF fragment peptide means a partial peptide of bFGF that does not include the heparin-binding region of bFGF.
  • the “FGF receptor-binding peptide” means a peptide that binds to the FGF receptor and has an amino acid sequence not derived from bFGF.
  • the “bFGF binding protein fragment peptide” means a partial peptide of a protein (bFGF binding protein) having a function of binding to bFGF to form a bFGF complex protein and increasing the binding affinity of bFGF to the bFGF receptor.
  • the “activity for promoting the proliferation of stem cells” refers to the survival and proliferation of stem cells cultured in the presence of the substance for a substance, and the survival and proliferation of stem cells cultured in the absence of the substance. It means that it is promoted compared to proliferation.
  • the number of stem cells after culturing for a certain period in the presence of the substance is 5% or more, 10% or more, or 20% as compared to the number of stem cells after culturing for the same period in the absence of the substance. Or more, preferably 30% or more, 40% or more, 50% or more, more preferably 60% or more, 70% or more or 80% or more, still more preferably 90% or more, 100% or more or more. .
  • a substance that exhibits excellent growth promoting activity against stem cells is provided.
  • bFGF substitute A medium for culturing stem cells according to the present invention contains a substance (hereinafter referred to as “bFGF substitute”) in place of bFGF conventionally used for the purpose of promoting the proliferation of stem cells.
  • bFGF substitute means that it can be used alternatively for bFGF, and does not exclude the use of bFGF substitute together with bFGF. That is, the bFGF substitute can be used together with bFGF.
  • bFGF heparin-binding region-containing peptide means a partial peptide of bFGF containing the heparin-binding region of bFGF.
  • bFGF is known to have a domain (heparin binding region) that strongly binds to heparan sulfate, and includes amino acid sites 166 to 210 and 235 to 262 from the N-terminus of mouse bFGF (GenBank accession number: NP_032032). The site of the second amino acid corresponds to the domain (Non-patent Document 2).
  • bFGF secreted from the stem cells themselves or bFGF added to the medium is adsorbed to heparin sulfate, the growth promoting function of bFGF on the stem cells may be inhibited.
  • the peptide containing bFGF heparin-binding region may promote the survival and proliferation of stem cells by competitively inhibiting the adsorption of bFGF secreted from stem cells themselves or bFGF added to the medium to heparin sulfate. Sex is conceivable.
  • the derived species of the bFGF heparin-binding region-containing peptide is not particularly limited as long as it has a stem cell proliferation promoting activity, and besides humans, rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cows, It may be ungulates such as goats or sheep, cats such as dogs or cats, primates such as monkeys, rhesus monkeys, marmosets, orangutans or chimpanzees.
  • the length of the bFGF heparin binding region-containing peptide is preferably 9 to 19 in terms of the number of amino acid residues, but may be 9 or less or 19 or more as long as the stem cell proliferation promoting activity is maintained. It may be about ⁇ 9 or 19 ⁇ 50.
  • the bFGF heparin-binding domain-containing peptide has a conventionally known modifying group or protecting group such as an amide at the side chain and / or amino terminus and / or carboxy terminus of the amino acid residue as long as the stem cell proliferation promoting activity is maintained. It may be.
  • bFGF heparin-binding region-containing peptide examples include, for example, the following peptides (A), (B) or (C).
  • A a peptide comprising any one amino acid sequence represented by SEQ ID NO: 1, 3, 4, 6 to 10, preferably from any one amino acid sequence represented by SEQ ID NO: 1, 3, 4, 6 to 10 Peptide.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • a particularly preferred bFGF heparin-binding region-containing peptide is a peptide consisting of the amino acid sequence “YRSRKYSSSWY” (SEQ ID NO: 1) at positions 106 to 115 from the N-terminal of the mouse bFGF shape body.
  • SEQ ID NO: 1 A peptide comprising an amino acid sequence represented by SEQ ID NO: 1 has been reported as an FGF antagonist (Patent Document 6), and a method for reducing skin wrinkles using this peptide (Patent Document 5) has been proposed. It has not been known so far that the peptide promotes the proliferation of stem cells.
  • the number of amino acids to be deleted, substituted, inserted or added is 10 or less, preferably 7 or less, more preferably 5 or less, and most preferably 1 or 2.
  • amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added can be deleted, substituted, inserted or added by known mutant polypeptide production methods such as site-directed mutagenesis. Means an amino acid sequence in which a certain number of amino acids are deleted, substituted, inserted or added (preferably 10 or less, more preferably 7 or less, even more preferably 5 or less, most preferably 1 or 2) .
  • amino acids in an amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein.
  • polymorphisms polymorphism
  • proteins that do not significantly change the structure or function of the protein. Therefore, in addition to the modified sequence obtained by artificially introducing mutations by a known mutant polypeptide production method, the “amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added” Naturally occurring polymorphic sequences are also included.
  • bFGF fragment peptide means a partial peptide of bFGF that does not contain the heparin-binding region of bFGF.
  • the species from which the bFGF fragment peptide is derived is not particularly limited as long as it has stem cell proliferation promoting activity.
  • rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cows, goats or sheep May be ungulates such as dogs, cats such as dogs or cats, primates such as monkeys, rhesus monkeys, marmosets, orangutans or chimpanzees.
  • the length of the bFGF fragment peptide is preferably 15 to 25 in terms of the number of amino acid residues, but may be 15 or less or 25 or more as long as the stem cell proliferation promoting activity is maintained, for example, 9 to 15 or It may be about 25-50.
  • the bFGF fragment peptide may have a conventionally known modifying group or protecting group such as an amide at the side chain and / or amino terminus and / or carboxy terminus of the amino acid residue as long as the stem cell proliferation promoting activity is maintained. Good.
  • bFGF fragment peptide examples include the following peptides (A), (B) or (C).
  • a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in any one of the amino acid sequences shown in SEQ ID NOs: 2, 5, 16 and promotes proliferation of stem cells A peptide having activity.
  • the number of amino acids to be deleted, substituted, inserted or added is 10 or less, preferably 5 or less, more preferably 3 or less, and most preferably 1 or 2.
  • amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added can be deleted, substituted, inserted or added by known mutant polypeptide production methods such as site-directed mutagenesis. Means an amino acid sequence in which a certain number of amino acids are deleted, substituted, inserted or added (preferably 10 or less, more preferably 7 or less, even more preferably 5 or less, most preferably 1 or 2) .
  • amino acids in an amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein.
  • polymorphisms polymorphism
  • proteins that do not significantly change the structure or function of the protein. Therefore, in addition to the modified sequence obtained by artificially introducing mutations by a known mutant polypeptide production method, the “amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added” Naturally occurring polymorphic sequences are also included.
  • FGF receptor binding peptide means a peptide that binds to FGF receptor and has an amino acid sequence not derived from bFGF.
  • the FGF receptor-binding peptide is not particularly limited as long as it has a stem cell proliferation promoting activity.
  • rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cattle It may bind to FGF receptors such as ungulates such as goats or sheep, cats such as dogs or cats, primates such as monkeys, rhesus monkeys, marmosets, orangutans or chimpanzees.
  • the length of the FGF receptor-binding peptide is preferably 14 to 29 in terms of the number of amino acid residues, but may be 14 or less or 29 or more as long as the stem cell proliferation promoting activity is maintained, for example, 9 to 14 Or it may be about 29-105.
  • the bFGF fragment peptide may have a conventionally known modifying group or protecting group such as an amide at the side chain and / or amino terminus and / or carboxy terminus of the amino acid residue as long as the stem cell proliferation promoting activity is maintained. Good.
  • FGF receptor binding peptide examples include, for example, the following peptides (A), (B) or (C).
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the number of amino acids to be deleted, substituted, inserted or added is 10 or less, preferably 5 or less, more preferably 3 or less, and most preferably 1 or 2.
  • amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added can be deleted, substituted, inserted or added by known mutant polypeptide production methods such as site-directed mutagenesis. Means an amino acid sequence in which a certain number of amino acids are deleted, substituted, inserted or added (preferably 10 or less, more preferably 7 or less, even more preferably 5 or less, most preferably 1 or 2) .
  • amino acids in an amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein.
  • polymorphisms polymorphism
  • proteins that do not significantly change the structure or function of the protein. Therefore, in addition to the modified sequence obtained by artificially introducing mutations by a known mutant polypeptide production method, the “amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added” Naturally occurring polymorphic sequences are also included.
  • bFGF binding protein fragment peptide is a protein having a function of binding to bFGF to form a bFGF complex protein and increasing the binding affinity of bFGF to the bFGF receptor (bFGF binding protein). Of the partial peptide.
  • the species from which the bFGF binding protein fragment peptide is derived is not particularly limited as long as it has stem cell proliferation promoting activity.
  • rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cows, goats
  • rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cows, goats
  • rodents such as rats, mice, hamsters or guinea pigs, rabbits such as rabbits, pigs, cows, goats
  • they may be ungulates such as sheep, cats such as dogs or cats, primates such as monkeys, rhesus monkeys, marmosets, orangutans or chimpanzees.
  • the length of the bFGF binding protein fragment peptide is preferably 41 to 43 in terms of the number of amino acid residues, but may be 41 or less or 43 or more as long as the stem cell proliferation promoting activity is maintained. It may be 41 or about 43-50. As long as the stem cell proliferation promoting activity is maintained, the bFGF binding protein fragment peptide has a conventionally known modifying group or protecting group such as an amide at the side chain of amino acid residue and / or amino terminal and / or carboxy terminal. May be.
  • bFGF binding protein examples include, for example, FGF-BP1 protein.
  • bFGF binding protein fragment peptide include, for example, the following (A) and (B) derived from a partial peptide of human FGF-BP1 protein: Or the peptide of (C) is mentioned.
  • C A peptide obtained by dimerizing the peptide according to (A) or (B).
  • the number of amino acids to be deleted, substituted, inserted or added is 10 or less, preferably 5 or less, more preferably 3 or less, and most preferably 1 or 2.
  • amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added can be deleted, substituted, inserted or added by known mutant polypeptide production methods such as site-directed mutagenesis. Means an amino acid sequence in which a certain number of amino acids are deleted, substituted, inserted or added (preferably 10 or less, more preferably 7 or less, even more preferably 5 or less, most preferably 1 or 2) .
  • amino acids in an amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein.
  • polymorphisms polymorphism
  • proteins that do not significantly change the structure or function of the protein. Therefore, in addition to the modified sequence obtained by artificially introducing mutations by a known mutant polypeptide production method, the “amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added” Naturally occurring polymorphic sequences are also included.
  • Each peptide described above can be prepared by a conventionally known chemical technique or genetic engineering technique.
  • the chemical synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a dichloromethane method, an active ester method, a carboimidazole method, and a redox method.
  • the synthesis method any of a solid phase synthesis method and a liquid phase synthesis method can be applied, and an automatic peptide synthesizer may be used.
  • the binding of the modifying group or protecting group to the peptide may be performed by a conventionally known method, such as a method of chemically modifying the peptide after synthesis, a method of synthesizing the peptide using chemically modified amino acids, or the final deprotection of peptide synthesis A method of appropriately selecting reaction conditions can be applied.
  • a recombinant vector is prepared by inserting DNA encoding a peptide downstream of a promoter of an appropriate expression vector by a conventionally known technique, and the recombinant vector is introduced into an appropriate host cell. It can be produced by expression.
  • Peptide purification can also be performed by a conventionally known method, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, or the like can be performed in combination as necessary.
  • the low molecular compound includes one or more compounds selected from the group consisting of cholinesterase inhibitors, phenothiazine derivatives, steroids, acetylcholine receptor agonists, and prostaglandin receptor agonists.
  • cholinesterase inhibitor examples include ethopapropine hydrochloride.
  • phenothiazine derivatives examples include aceprozine maleate, benzoyl leuco methylene blue, chloropromide hydride, mesoside benzine hydrate, and phenoxide.
  • steroids examples include dexamethasone.
  • acetylcholine receptor agonists examples include epiboxidine hydrochloride.
  • prostaglandin receptor agonists examples include fluprostenol.
  • acetylcholine receptor agonists are preferable, and epiboxidine hydrochloride is particularly preferable.
  • FGFR1 is known to be activated by the binding of bFGF, and the cells are used in an assay system for evaluating the activity of a bFGF recombinant protein (Cancer Res., 1988, Aug 1, 48 ( 15), p.4266-71).
  • the same assay system can also be used in screening evaluation of bFGF substitutes.
  • BALB / 3T3 clone A31 is known as a mouse fetal fibroblast.
  • the composition of the medium for culturing stem cells according to the present invention may be the same as the medium conventionally used for culturing stem cells except that it contains the above-mentioned bFGF substitute. That is, the medium according to the present invention can be prepared by adding a bFGF substitute to a basal medium conventionally used for culturing cells derived from animal tissues. Specific examples of the basal medium include the following.
  • Hybridoma Serum free medium Chemically Defined Hybrids' medium Medium F-12, Ham's Medium F-10, Ham's Medium F12K, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7 MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymout 's MB752 / 1, CMRL-1066, William's medium E, Brinster's BMOC-3 Medium, Essential8 Medium (above Thermo Fisher Scientific), mTeSR1 (Stem Cell Technologies), TeSR-E8 Medium ), StemSure (Wako Pure Chemical Industries, Ltd.
  • the bFGF substitute may be added alone to the medium, or a plurality of bFGF substitutes may be added to the medium. Moreover, a bFGF substitute may be added in addition to bFGF.
  • the addition concentration of the bFGF substitute is not particularly limited as long as it exhibits stem cell proliferation promoting activity, but is, for example, 1 to 500 ⁇ M, preferably 5 to 250 ⁇ M, more preferably 10 to 200 ⁇ M.
  • the species derived from the peptide can be appropriately selected according to the species derived from the stem cells to be cultured, preferably The same species as the stem cells to be cultured. For example, when culturing human-derived cells, it is preferable to use human-derived bFGF heparin-binding region-containing peptides. Moreover, what is necessary is just to select suitably the peptide couple
  • Physiologically active substances and / or nutrient factors necessary for cell survival or proliferation can be added to the medium. These additives may be added in advance to the medium, or may be added during cell culture.
  • the method to be added during the culture may be in any form such as one solution or a mixed solution of two or more, and may be continuous or intermittent addition.
  • Nutritional factors include sugars, amino acids, vitamins, hydrolysates or lipids.
  • examples of the sugar include neutral sugars such as glucose, mannose, and fructose, acidic sugars such as sialic acid, amino sugars, and sugar alcohols, and one kind or a combination of two or more kinds is used.
  • vitamins examples include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin or DL- ⁇ -tocopherol, and one or a combination of two or more Used.
  • hydrolyzate examples include hydrolyzed soybeans, wheat, rice, peas, corn, cotton seeds or yeast extracts.
  • lipids include cholesterol, linoleic acid, and linolenic acid.
  • an antibiotic such as kanamycin, streptomycin, penicillin or hygromycin may be added to the medium as necessary.
  • an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to pH 5 to 9, preferably 6 to 8, which is a neutral range suitable for cell growth.
  • the medium according to the present invention may be a serum-containing medium or a serum-free medium. From the viewpoint of preventing contamination with heterogeneous animal-derived components, it is preferable not to contain serum or to use serum derived from the same species as the cultured stem cells.
  • the serum-free medium means a medium containing no unadjusted or unpurified serum.
  • the serum-free medium may contain purified blood-derived components, animal tissue-derived components such as growth factors, proteins produced by gene recombination techniques, or synthesized peptides.
  • the medium according to the present invention may or may not contain a serum substitute, like serum.
  • Serum substitutes include, for example, albumin substitutes such as albumin, lipid-rich albumin and recombinant albumin, plant starch, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace amounts Examples thereof include element, 2-mercaptoethanol, 3′-thioglycerol and equivalents thereof.
  • serum substitutes include, for example, those prepared by the method described in International Publication No. 98/30679, commercially available knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Life Technologies max) and Gluten. (Life Technologies).
  • KSR knockout Serum Replacement
  • Chemically-defined Lipid concentrated Life Technologies max
  • Gluten (Life Technologies).
  • the “stem cell” targeted by the present invention refers to an immature cell having self-replication ability and differentiation / proliferation ability, and depending on the differentiation ability, a pluripotent stem cell, a multipotent stem cell (multipotent stem cell). cell) or a unipotent stem cell.
  • a “stem cell” is generally defined as an undifferentiated cell having “self-renewal ability” capable of proliferating while maintaining an undifferentiated state and “differentiation pluripotency” capable of differentiating into all three germ layers.
  • a pluripotent stem cell means a cell having an ability to differentiate into all tissues and cells constituting a living body.
  • a multipotent stem cell means a cell having the ability to differentiate into multiple types of tissues and cells, although not all types.
  • a unipotent stem cell means a cell having the ability to differentiate into a specific tissue or cell.
  • the stem cell origin species is not particularly limited, for example, rodents such as rats, mice, hamsters or guinea pigs, rabbit eyes such as rabbits, ungulates such as pigs, cows, goats or sheep, cats such as dogs or cats, etc.
  • rodents such as rats, mice, hamsters or guinea pigs
  • rabbit eyes such as rabbits
  • ungulates such as pigs, cows, goats or sheep
  • cats such as dogs or cats, etc.
  • Cells such as eyes, humans, monkeys, rhesus monkeys, marmosets, orangutans or chimpanzees can be used.
  • stem cells include myoblasts, vascular endothelial cells, osteoblasts, adipocytes, myocytes, cardiomyocytes, mesenchymal stem cells that differentiate into chondrocytes, neural stem cells that differentiate into neurons and glial cells, leukocytes Hematopoietic stem cells or bone marrow stem cells that differentiate into red blood cells, platelets, mast cells, dendritic cells, etc., and differentiation / induction to various tissues through formation of pseudo embryos called embryoid bodies (EB bodies) from the spheroid state Embryonic stem cells (embryonic stem cells: ES cells), inducible pluripotent stem cells (iPS cells), embryonic germ cells (EG) cells derived from primordial germ cells, Multipotent germline stem (isolated in the process of culturing GS cells from testis tissue) GS) cells, multipotent adult progenitor cell (MAPC) pluripotent stem cells, such as isolated from the bone marrow and the like.
  • pluripotent stem cells examples include the aforementioned ES cells or iPS cells.
  • Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 1997, 385, p. 810, Science, 1998, 280, p. 1256, Nature Biotechnology, 1999, 17, p.456, Nature, 1998, 394, p.369, Nature Genetics, 1999, 22, p.127, Proc. Natl. Acad. Sci. USA, 1999, 96, p. 14984, Nature Genetics, 2000, 24, p. 109).
  • Human ES cell lines are obtained from, for example, WA01 (H1) and WA09 (H9) from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan). Is possible.
  • iPS cells include cells that have acquired multipotency similar to ES cells obtained by introducing a plurality of genes (reprogramming factors) into somatic cells such as skin cells.
  • iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene, C-Myc gene and Sox2 gene iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nature Biotechnology, 2008) 26, p.101-106).
  • genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, and ECAT15.
  • -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. are exemplified, and these initialization factors may be used alone or in combination.
  • Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 0775119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 045 626, WO 2010/056831, WO 2010/068955, WO 2010/098419, WO 2010/102267, WO 2010
  • iPS cells can be obtained from predetermined institutions (RIKEN BioResource Center, Kyoto University, etc.).
  • multipotent stem cells examples include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and reproductive stem cells.
  • the multipotent stem cell is preferably a mesenchymal stem cell.
  • the mesenchymal stem cell broadly means a group of stem cells or progenitor cells that can differentiate into all or some mesenchymal cells such as osteoblasts, chondroblasts, and lipoblasts.
  • mesenchymal stem cells include human bone marrow-derived mesenchymal stem cells (hMSC-BM, Takara), human umbilical cord matrix-derived mesenchymal stem cells (hMSC-UC, Takara), human adipose tissue-derived mesenchymal stem cells. (HMSC-AT, Takara) can be mentioned.
  • the medium according to the present invention can be suitably used for the proliferation of any stem cell, but preferably for culturing mesenchymal stem cells, ES cells or iPS cells, more preferably for culturing iPS cells.
  • it can be used for culturing human iPS cells. More specifically, as human iPS cells, 253G1 strain (RIKEN Cell Bank No. HPS0002), 201B7 strain (RIKEN Cell Bank No. HPS0063), 409B2 strain (RIKEN Cell Bank No. HPS0076), 454E2 strain (RIKEN Cell Bank No. HPS0077), HiPS -RIKEN-1A strain (RIKEN Cell Bank No.
  • HPS0003 HiPS-RIKEN-2A strain (RIKEN Cell Bank No. HPS0009), HiPS-RIKEN-12A strain (RIKEN Cell Bank No. HPS0029) or Nips-B2 strain (RIKEN Cell Bank No. HPS0223).
  • the stem cell culturing method according to the present invention includes a procedure for culturing stem cells in a medium containing a bFGF substitute. Specifically, in the culture method according to the present invention, in the presence of any one or more of the above-mentioned bFGF fragment peptide, FGF receptor binding peptide, bFGF heparin binding region-containing peptide, bFGF binding protein fragment peptide and low molecular weight compound, The stem cells are cultured by batch culture (Bach Culture), fed-batch culture, continuous culture, perfusion culture, or the like.
  • Batch culture is a method of culturing without supplementing a medium containing a bFGF substitute to a culture tank during culture.
  • Fed-batch culture is a method of culturing while supplementing a medium containing a bFGF substitute to a culture tank during culture.
  • Continuous culture is a method in which a part of the medium containing the bFGF substitute is withdrawn from the culture tank being cultured, and the medium is cultured while supplementing the medium corresponding to the amount of withdrawal.
  • the perfusion culture is a method in which only the liquid is extracted from the culture tank being cultured, and the culture is performed while supplementing a medium containing a bFGF substitute equivalent to the amount of withdrawal.
  • the survival and proliferation of stem cells can be promoted by the action of the bFGF substitute. That is, in the present invention, bFGF fragment peptide, FGF receptor binding peptide, bFGF heparin binding region-containing peptide, bFGF binding protein fragment peptide, cholinesterase inhibitor, phenothiazine derivative, steroid, acetylcholine receptor agonist and prostaglandin receptor agonist are selected.
  • the low molecular weight compound can function as a proliferation promoter for stem cells.
  • the incubator used in the culture method according to the present invention is not particularly limited, and examples thereof include flasks, dishes, petri dishes, microwell plates, microslides, chamber slides, tubes, trays, culture vessels such as culture bags or tanks, and the like.
  • the substrate of these incubators is not particularly limited, and examples thereof include glass, various plastics such as polypropylene and polystyrene, metals such as stainless steel, or combinations thereof.
  • collagen, gelatin, poly-L-lysine, poly-D-lysine, and the like are used on the substrate surface in order to induce adhesion of anchorage-dependent cells to the substrate surface of the incubator.
  • Adhesive cultures coating scaffolds such as laminin, partial structures of laminin, fibronectin and mixtures thereof (eg, Matrigel) and lysed cell membrane preparations (Lancet, 2005, 365, p1636-1641), and polymer polymers such as methylcellulose ( Stem Cell Reports, 2014, 2, 5, 734-745), MFG- Examples thereof include suspension culture using E8 (Milk fat globule-EGF factor 8) or a fragment of the protein without the above scaffold.
  • scaffolds such as laminin, partial structures of laminin, fibronectin and mixtures thereof (eg, Matrigel) and lysed cell membrane preparations (Lancet, 2005, 365, p1636-1641), and polymer polymers such as methylcellulose ( Stem Cell Reports, 2014, 2, 5, 734-745), MFG- Examples thereof include suspension culture using E8 (Milk fat globule-EGF factor 8) or a fragment of the protein without the above scaffold.
  • the stem cells to be cultured can be dispersed cells or non-dispersed cells.
  • Dispersed cells refer to cells that have been treated to promote cell dispersion. Examples of the dispersed cells include cells forming a small cell mass composed of 2 to 50 cells, 2 to 20 cells, or 2 to 10 cells.
  • the dispersed cells can be floating (suspension) cells or adherent cells.
  • the culture density of the stem cells is not particularly limited as long as the density can achieve the effect of promoting cell survival and proliferation.
  • the culture density is, for example, 1.0 ⁇ 10 1 to 1.0 ⁇ 10 7 cells / ml, preferably 1.0 ⁇ 10 2 to 1.0 ⁇ 10 7 cells / ml, more preferably 1.0 ⁇ 10 3. ⁇ 1.0 ⁇ 10 7 cells / ml, more preferably 3.0 ⁇ 10 4 to 1.0 ⁇ 10 7 cells / ml.
  • Temperature, dissolved CO 2 concentration, culture conditions, such as dissolved oxygen concentration and pH can be appropriately set on the basis of the techniques conventionally used for the cultivation of cells derived from animal tissue.
  • the culture temperature is not particularly limited, but may be 33 to 39 ° C., preferably 36 to 37 ° C.
  • the dissolved CO 2 concentration can be 1-10%, preferably 2-5%.
  • the oxygen partial pressure can be 10-22%.
  • the cells When performing adherent culture of stem cells, the cells may be cultured in the presence of feeder cells.
  • feeder cells stromal cells such as fetal fibroblasts can be used (for example, Manipulating the Mouse Embryo A, Laboratory Manual, Secondary IR, Cold Spring Harbor, Pt. Oxford University Press, 1993, Proc. Natl. Acad. Sci. USA, 1981, 78, 12, p.7634-7638, Nature, 1981, 292, 5819, p.154-156, J. Virol., 1969, 4 , 5, p. 49-553, Science, 1996, 272, 5262, p. 722-724, J. Cell. Physiol., 1982, 112, 1, p. 89-95, International Publication No. 01/088100, 2005/080554. Issue).
  • Stem cell suspension culture refers to culturing stem cells in a medium under non-adherent conditions as compared with the case of using an incubator or feeder cells.
  • Stem cell suspension culture includes stem cell dispersion culture and stem cell aggregation suspension culture.
  • Stem cell dispersion culture refers to culturing suspended stem cells, and includes, for example, dispersion culture of small cell clusters composed of 2 to 20 stem cells. When the dispersion culture is continued, the cultured dispersion cells form a larger stem cell mass, and then aggregated suspension culture can be performed.
  • Aggregation suspension culture includes embryoid body culture method (see Curr. Opin. Cell Biol., 1995, 7, 6, p. 862-869), SFEB method (Nature Neuroscience, 2005, 8, 3, p. 288-). 296, International Publication No. 2005/123902), and a sphere culture method (Stem Cell Reports, 2014, 2, 5, p. 734-745) in which cell lines are passaged by mechanical treatment using a mesh
  • the survival and proliferation of the stem cells can be promoted by the procedure of culturing the stem cells in the presence of the bFGF substitute, so that the cells are vulnerable to stress such as stem cells, especially suspension culture after dispersion and dispersion. Can be used for efficiently culturing iPS cells.
  • a cell composition comprising a low molecular weight compound selected from agonists and stem cells can be used for further stem cell culture or for cell sources for regenerative medicine.
  • the culture method according to the present invention can be applied to a method for producing a cell composition containing stem cells or a method for producing a cell composition containing differentiated cells derived from stem cells.
  • the method for producing a cell composition containing stem cells according to the present invention includes a step of culturing stem cells in the presence of the above-described bFGF substitute, and a step of substituting stem cells as necessary.
  • the method for producing a cell composition containing differentiated cells according to the present invention includes a step of culturing stem cells in the presence of a bFGF substitute and a step of inducing differentiation of stem cells, and subcultures cells as necessary. Process.
  • the step of culturing stem cells in the presence of a bFGF substitute can be performed in the same manner as the above-described stem cell culture method.
  • Stem cell passage and differentiation induction may be performed by a conventionally known technique.
  • a BMP inhibitor, a Wnt inhibitor, a Nodal inhibitor, retinoic acid, or the like can be used.
  • mesenchymal stem cells are used in differentiation induction medium (90% ⁇ MEM medium, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 ⁇ M dexamethasone). Can be performed by culturing.
  • cardiomyocytes are cultured for 1 day in a medium in which 0.5 ng / ml BMP-4 is added to STEMdiff APEL Medium (STEMCELL), and then 10 ng / ml BMP in STEMdiff APEL Medium. -4, 10 ng / ml Activin A, 5 ng / ml Replaced with a medium supplemented with bFGF, and continued the culture for another 4 days. Thereafter, the medium was replaced with medium supplemented with 10 ng / ml VEGF and 150 ng / ml Dkk1 in STEMdiff APEL Medium, and cultured for 8 days.
  • STEMdiff APEL Medium STEMdiff APEL Medium
  • the stem cell in the method for producing a cell composition containing a stem cell or a differentiated cell according to the present invention, the stem cell can be promoted to survive and proliferate by the step of culturing the stem cell in the presence of a bFGF substitute.
  • a cell composition containing differentiated cells can be efficiently produced.
  • BFGF fragment peptide, FGF receptor binding peptide, bFGF heparin binding region-containing peptide, bFGF binding protein fragment peptide, or cholinesterase inhibitor, phenothiazine derivative obtained by the method for producing a cell composition containing stem cells or differentiated cells according to the present invention .
  • a steroid, an acetylcholine receptor agonist, or a low molecular weight compound selected from a prostaglandin receptor agonist, and stem cells and / or differentiated cells, for further stem cell culture or for cell sources for regenerative medicine Can be used.
  • the cell composition according to the present invention may be a composition containing dispersed stem cells such as small cell clusters.
  • the cell composition according to the present invention can be used, for example, for preservation, transportation, and passage of stem cells by cryopreservation.
  • the cell composition according to the present invention may further contain serum or an alternative thereof, or an organic solvent such as DMSO.
  • the cell composition according to the present invention may include feeder cells.
  • Mouse fetal fibroblasts (BALB / 3T3 clone A31) were obtained from RIKEN.
  • BALB / 3T3 cells were cultured on plastic culture dishes in accordance with the manual attached to the cells (Resource No. RBRC-RCB00005). Specifically, culture was performed at 37 ° C. under 5% CO 2 using a culture solution in which FBS (Sigma) having a final concentration of 10% was added to DMEM (Sigma D5796). Transplanting was performed every 3 days, cells were dissociated using dissociation solution (0.25% trypsin, Life Technologies) and plated on new plastic culture dishes.
  • Candidate substances include bFGF heparin binding region-containing peptide (SEQ ID NO: 1), bFGF fragment peptide (SEQ ID NO: 2), FGF receptor binding peptide (SEQ ID NO: 11, 13, 14), low molecular weight compound (Fluprostenol (SANTACRUZ), Epiboxidine hydrochloride).
  • SIGMA Dexamethasone (Wako Pure Chemical Industries), Perphenazine (Tokyo Kasei Kogyo), Proximine hydrochloride (SIGMA), Promethazine hydrogenide SIGMA (Hako Pure Chemical Industries, Ltd.) Eucomethyl blue (Tokyo Kasei Kogyo), Aceprozine maleate (Wako Pure Chemical Industries), and Chlorpromine hydrochloride (Wako Pure Chemical Industries) were used.
  • the cells were counted and seeded at a medium density (1 ⁇ 10 4 cells / 0.8 cm 2 , in a medium volume of 1.0 mL) using a 24-well culture plate. The number of cells was counted after culturing for 3 days in a culture medium containing a candidate substance (10 ⁇ M).
  • a culture solution to which human bFGF (Reprocell) was added at 5 ng / ml or 50 ng / ml was used as a negative control (“control” in FIG. 1).
  • the addition of the bFGF heparin-binding region-containing peptide of SEQ ID NO: 1 has a cell growth promoting activity comparable to that of human bFGF (50 ng / ml).
  • the cell growth promoting activity was also confirmed by the addition of the bFGF fragment peptide of SEQ ID NO: 2, the human FGF receptor binding peptide of SEQ ID NO: 11, and Epiboxidine hydrochloride. (FIG. 1).
  • Example 1 Evaluation of iPS cell proliferation promoting activity of peptide containing bFGF heparin binding region
  • the iFGF cell proliferation promoting activity of bFGF heparin-binding domain-containing peptides as bFGF substitutes was evaluated.
  • As the bFGF heparin binding region-containing peptide a peptide having the amino acid sequence shown in SEQ ID NO: 1 derived from mouse bFGF was used by amidating the C-terminus.
  • IPS cells were obtained from RIKEN Cell Bank (No. HPS0063), a human induced pluripotent stem cell (201B7) established by Prof. Shinya Yamanaka, Institute for iPS Cell Research, Kyoto University.
  • IPS cells are Nat. Biotechnol. 2007, 25, p.
  • mouse fetal fibroblasts inactivated by mitomycin treatment, MEF as a feeder layer were cultured in a plastic culture dish.
  • the culture solution is D-MEMF12 (SIGMA, D6421) with a final concentration of 20% KSR (Life Technologies), a final concentration of 1% NON-ESENTIALAMINOACID (SIGMA), 2 mM L-glutamic acid and 80 ⁇ M 2-mercaptoethanol.
  • SIGMA D-MEMF12
  • KSR Life Technologies
  • SIGMA NON-ESENTIALAMINOACID
  • the cells were cultured at 37 ° C. under 5% CO 2 and subcultured every 3 to 4 days.
  • Dissociate the iPS cells from the feeder layer using a dissociation solution phosphate buffer buffered saline with 0.25% trypsin, 1 mg / ml collagenase IV solution, 1 mM CaCl 2 , all Life Technologies
  • a dissociation solution phosphate buffer buffered saline with 0.25% trypsin, 1 mg / ml collagenase IV solution, 1 mM CaCl 2 , all Life Technologies
  • the iPS cells cultured as described above are dissociated as small cell clusters from the feeder cells, and further adsorbed on the bottom of a cell-adhesive culture plate (0.1% gelatin coat) in order to remove the contaminating feeder cells.
  • the culture was incubated at 37 ° C. for 1 hour. Then, iPS cell mass was seed
  • BD Growth Factor Reduced BD Matrigel
  • the cell growth promoting activity of bFGF heparin-binding region-containing peptides against iPS cells was examined as follows.
  • the culture solution was changed to the assay medium (Essential 6, Life Technologies, serum-free) on the day before the assay, and cultured after 24 hours.
  • the iPS cells After dissociating the iPS cells, they were seeded on iMatrix-511 (Nippi) at a density of 5 ⁇ 10 4 cells / 0.32 cm 2 / medium solution volume of 0.2 ml using a 96-well culture plate.
  • the cells were cultured for 4 days in an assay medium supplemented with 10 ⁇ M bFGF heparin-binding domain-containing peptide (SEQ ID NO: 1) or 5 ng / ml recombinant human bFGF (Reprocell).
  • the medium was changed, and further cultured in an assay medium supplemented with 10 ⁇ M bFGF heparin-binding region-containing peptide or 5 ng / ml recombinant human bFGF for 4 days, and the formed colony area of alkaline phosphatase staining positive (ALP +) was determined as the amount of living cells. As measured. For control, cells cultured only in the assay medium were used.
  • SEQ ID NO: 1 Mouse bFGF heparin binding region-containing peptide
  • SEQ ID NO: 2 Human bFGF fragment peptide
  • SEQ ID NO: 3 Human bFGF heparin binding region-containing peptide
  • SEQ ID NO: 4 Human bFGF heparin binding region-containing peptide
  • SEQ ID NO: 5 Human bFGF fragment peptide sequence No.

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Abstract

L'invention concerne, en tant que substances qui présentent un excellent effet favorisant la prolifération sur des cellules souches, des fragments peptidiques de bFGF, des peptides se liant au récepteur de FGF, des peptides comprenant une région de liaison à l'héparine de bFGF, des fragments peptidiques de protéine de liaison à bFGF, ou un composé de faible poids moléculaire sélectionné parmi les inhibiteurs de la cholinestérase, des dérivés de phénothiazine, des stéroïdes, des agonistes des récepteurs de l'acétylcholine, et des agonistes des récepteurs de la prostaglandine ; et un milieu de culture de cellules souches contenant ces éléments.
PCT/JP2016/083232 2015-11-13 2016-11-09 Milieu de culture de cellules souches, promoteur de prolifération et procédé de culture, composition cellulaire comprenant des cellules souches et son procédé de production WO2017082294A1 (fr)

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CN115354023A (zh) * 2022-09-01 2022-11-18 天津永泰生命科技有限公司 一种间充质干细胞的无血清培养基及其制备方法
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WO2009158238A2 (fr) * 2008-06-16 2009-12-30 University Of Rochester Analogues de facteur de croissance de fibroblaste (fgf) et leurs utilisations
JP2012502664A (ja) * 2008-09-19 2012-02-02 ウィスコンシン・アルムニ・リサーチ・ファウンデーション 多能性細胞の長期培養のためのペプチド提示表面

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CN115354023A (zh) * 2022-09-01 2022-11-18 天津永泰生命科技有限公司 一种间充质干细胞的无血清培养基及其制备方法
US20240228949A1 (en) * 2023-01-06 2024-07-11 Joseph CHALIFOUX Method and composition for reprogramming cells

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