WO2011158967A1 - Procédé d'établissement efficace de cellules souches pluripotentes induites - Google Patents

Procédé d'établissement efficace de cellules souches pluripotentes induites Download PDF

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WO2011158967A1
WO2011158967A1 PCT/JP2011/064330 JP2011064330W WO2011158967A1 WO 2011158967 A1 WO2011158967 A1 WO 2011158967A1 JP 2011064330 W JP2011064330 W JP 2011064330W WO 2011158967 A1 WO2011158967 A1 WO 2011158967A1
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mutant
cells
lin28a
hmga2
ips cells
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Shinya Yamanaka
Koji Tanabe
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Kyoto University
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
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    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/608—Lin28
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a method for improving efficiency in the establishment of induced pluripotent stem cells (hereinafter, referred to as iPS cells) and an agent used for the method. More specifically, the present invention relates to a method for improving efficiency in the establishment of iPS cells, comprising a step of introducing HMGA2 and/or Lin28A mutant proteins or nucleic acids encoding the proteins into somatic cells and an agent for improving efficiency in the establishment of iPS cells, comprising HMGA2 and/or Lin28A mutant proteins or nucleic acids encoding the proteins, for example.
  • iPS cells induced pluripotent stem cells
  • iPS cells In recent years, mouse and human iPS cells have been established in succession. Yamanaka et al have induced iPS cells by introducing Oct3/4, Sox2, Klf4 and c-Myc genes into mouse-derived fibroblasts to cause forced expression of the genes (WO 2007/069666 Al and Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)). Thereafter, it was also revealed that iPS cells can also be established using three of the above factors excluding the c-Myc gene (Nakagawa, M. et al., Nat.
  • Lin28 is not essential for reprogramming, although is a factor that improves reprogramming efficiency (WO 2007/069666 Al).
  • HMGA2 HMGI-C
  • HMGA2 is involved in aging and cancer progression (Tzatsos A. and Bardeesy N., Cell Stem Cell. 3: 469-70 (2008)).
  • iPS cell induction has been neither disclosed nor suggested.
  • An object of the present invention is to provide a novel method for improving efficiency in the establishment of iPS cells and an agent for improving establishment efficiency.
  • HMGA2 has the effect of improving (increasing the number and/or sizes of colonies) efficiency in the establishment of iPS cells.
  • HMGA2 is subjected to transcriptional regulation by upstream LIN28A via the zinc-finger domain in a step for establishment of iPS cells, so that HMGA2 regulates in an inhibitory manner the expression of pl 6IN 4a and pl9Arf which are further downstream factors.
  • the present inventors have completed the present invention.
  • the present invention is as follows.
  • a method for improving efficiency in the establishment of iPS cells comprising a step of introducing the following proteins or nucleic acids encoding the proteins and a nuclear reprogramming substance into somatic cells:
  • An agent for improving efficiency in the establishment of iPS cells comprising the following proteins or nucleic acids encoding the proteins in somatic cells:
  • a method for producing iPS cells comprising a step of introducing the following proteins or nucleic acids encoding the proteins and a nuclear reprogramming substance into somatic cells:
  • nuclear reprogramming substance is selected from the group consisting of an Oct family member, an Sox family member, a Klf4 family member, an Myc family member, Nanog, and an Lin family member, and nucleic acids encoding them.
  • An agent for inducing iPS cells from somatic cells comprising the following proteins or nucleic acids encoding the proteins and a nuclear reprogramming substance in somatic cells: 1 ) HMGA2; and/or
  • nuclear reprogramming substance is selected from the group consisting of an Oct family member, an Sox family member, a Klf4 family member, an Myc family member, Nanog, and an Lin family member, and nucleic acids encoding them.
  • a method for producing somatic cells comprising carrying out differentiation induction for the iPS cells according to [26] or [27] so as to cause the differentiation of the iPS cells into somatic cells.
  • a method for producing somatic cells comprising the following steps of: (1) producing iPS cells by the method according to any one of [11] to [18]; and
  • step (2) carrying out differentiation induction for the iPS cells obtained in step (1) above, so as to cause the differentiation of the iPS cells into somatic cells.
  • HMGA2 and/or a foreign nucleic acid encoding HMGA2 or an Lin28A mutant and/or a nucleic acid encoding the Lin28A mutant for improving efficiency in the establishment of iPS cells.
  • HMGA2 and/or a nucleic acid encoding HMGA2 or an Lin28A mutant and/or a nucleic acid encoding the Lin28A mutant for production of iPS cells, wherein the production comprises introducing the nucleic acid and a nuclear reprogramming substance together into somatic cells.
  • nuclear reprogramming substance is selected from the group consisting of an Oct family member, an Sox family member, a Klf4 family member, an Myc family member, Nanog, and an Lin family member, and nucleic acids encoding them.
  • nuclear reprogramming substances include Oct3/4, Sox2, and Klf4, or nucleic acids encoding them.
  • iPS cells can be highly efficiently established with the use of HMGA2.
  • Fig. 1 shows a graph showing the number of human iPS cell colonies established by introduction of each factor shown below the graph together with four factors into adult human skin-derived fibroblasts (HDF: human dermal fibroblasts 1503 cell line) using retrovirus vectors.
  • the longitudinal axis indicates the numbers of iPS cell colonies.
  • “O” indicates human Oct3/4,
  • “S” indicates human Sox2,
  • K indicates human Klf4,
  • "M” indicates human c-Myc.
  • the longitudinal axis indicates the numbers of iPS cell colonies confirmed on 10-cm petri dishes.
  • FIG. 2 shows graphs showing the sizes of the colonies of human iPS cells established by introduction of each factor shown below each graph together with four factors into adult human skin-derived fibroblasts (HDF) using retrovirus vectors.
  • the left graph shows the results for the 1606 cell line and the right graph shows the results for the 1503 cell line.
  • the longitudinal axis indicates the size ratio found when the average colony size of iPS cells confirmed on 10-cm petri dishes upon addition of DsRed was determined to be 1. "O, S, K, and M" have the same meanings as they do in Fig. 1.
  • Fig. 3 shows the results for Lin28A (wild-type and mutant) and Lin28B (wild- type).
  • Lin28A (A) indicates wild-type Lin28A
  • ACSD indicates cold- shock domain-deficient mutant Lin28A
  • AZinc indicates zinc finger domain-deficient mutant Lin28A
  • Lin28B (B) indicates wild-type Lin28B.
  • Fig. 4 shows the results of examining the effects of introduction of various Lin28 mutants on HMGA2 expression upon induction of iPS cells.
  • a photograph on the left shows the results of HMGA2 protein expression upon induction of iPS cells.
  • Various mutants were introduced together with four factors into adult human skin- derived fibroblasts (HDF), the 1503 cell line, using retrovirus vectors. Seven (7) days after infection, the proteins were extracted using an RIPA buffer and then detected by Western blot.
  • HDF adult human skin- derived fibroblasts
  • "O, S, K, and M" have the same meanings as they do in Fig. 1.
  • the name of each factor introduced together with the four factors is described above each lane.
  • Fig. 1 The name of each factor introduced together with the four factors is described above each lane.
  • ES indicates the expression in the human ES cell line (H9 cell line).
  • the right graph shows the numbers of colonies of human iPS cells established via introduction of each factor shown below the graph together with four specific factors into human skin-derived fibroblasts (TIG-119 cell line) using retrovirus vectors.
  • the longitudinal axis shows the colony number ratio when the average number of iPS cell colonies confirmed on 10-cm petri dishes upon addition of DsRed was determined to be 1.
  • the present invention provides a method for improving efficiency in the establishment of iPS cells, comprising a step of introducing HMGA2 and/or Lin28A mutant proteins or nucleic acids encoding the proteins into somatic cells.
  • nuclear reprogramming for somatic cells is carried out by introducing a nuclear reprogramming substance into somatic cells.
  • the present invention also provides a method for producing iPS cells, comprising introducing HMGA2 and/or Lin28A mutant proteins or nucleic acids encoding the proteins and a nuclear reprogramming substance into somatic cells.
  • iPS cells cannot be established via introduction of a nuclear reprogramming substance alone, but iPS cells are established via introduction of HMGA2 and/or Lin28A mutant proteins or nucleic acids encoding the proteins together with a nuclear reprogramming substance into somatic cells, is treated as being relevant to "improvement of efficiency in establishment.”
  • Somatic cells that can be used as starter materials for production of iPS cells in the present invention may be any cells other than germ cells from mammals (e.g., humans, mice, monkeys, cattle, pigs, rats, and dogs).
  • somatic cells include keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the surface layer of tongue), exocrine epithelial cells (e.g., mammary glandular cells), hormone-secreting cells (e.g., adrenal medullary cells), cells for metabolism and/or storage (e.g., hepatocytes), boundary-forming luminal epithelial cells (e.g., type I alveolar cells), luminal epithelial cells of inner chain tube (e.g., vascular endothelial cells), ciliated cells having transport capacity (e.g., airway epithelial cells), cells for secretion to extracellular matrix (e
  • undifferentiated precursor cells also including somatic stem cells
  • terminally- differentiated mature cells can be similarly used as origins for somatic cells in the present invention.
  • undifferentiated precursor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • somatic cells are particularly preferably collected directly from a patient or another subject whose HLA type is identical to or substantially identical to that of the patient, in order not to cause rejection.
  • the phrase "(the HLA type is) substantially identical to” means that when cells obtained by induced differentiation of the somatic-cell-derived iPS cells with the use of an immunosuppressive agent or the like are transplanted into a patient, the HLA types agree to such an extent that the transplanted cells can survive.
  • HLA-A major HLA types
  • HLA-B major HLA types
  • HLA-DR major HLA types
  • iPS cells when no administration (transplantation) is carried out for a human, for example when iPS cells are used as a source of cells for screening for evaluation of the presence or the absence of a patient's drug sensitivity or adverse reaction, it is similarly desirable to collect somatic cells directly from a patient or another subject whose gene polymorphism correlating with drug sensitivity or adverse reaction is identical to that of the patient.
  • Somatic cells separated from a mammal can be subjected to preculture (prior to the nuclear reprogramming step) in a medium known per se appropriate for the culture depending on cell types.
  • a medium include, but are not limited to, a minimum essential medium (MEM) containing about 5%-20% fetal calf serum, Dulbecco's modified Eagle's medium (DMEM), an RPMI1640 medium, a 199 medium, and an F12 medium.
  • a medium may be preferably exchanged in advance with a serum free medium, so as to avoid a decrease in introduction efficiency
  • HMGA2 in the present invention is a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or 4.
  • the HMGA2 protein may be isolated and purified from cells or tissues [e.g., cells or tissue of thymus gland, bone marrow, spleen, brain, spinal cord, heart, skeletal muscle, kidney, lung, liver, pancreas, or prostate gland, precursor cells of these cells, and stem cells or cancer cells] of humans or other mammals (e.g., mice, rats, monkeys, pigs, and dogs) by protein separation and purification techniques known per se.
  • the HMGA2 protein may also be a recombinant protein produced by gene recombination techniques known per se using a nucleic acid encoding HMGA2 or a protein produced by cell-free protein synthesis.
  • protein comprising an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 2 or 4" is a protein consisting of an amino acid sequence having about 80% or more, more preferably about 90% or more, and furthermore preferably about 95% or more identity with the amino acid sequence of human HMGA2 shown in SEQ ID NO: 2 or 4 and having activity of the same quality as that of the amino acid sequence shown in SEQ ID NO: 2 or 4.
  • activity refers to the effect of improving efficiency in the establishment of iPS cells.
  • substantially the same quality refers to that the effects are exerted equivalently to or more effectively than the effects to be exerted by HMGA2 on arbitrary somatic cells.
  • the effect of improving efficiency in the establishment of iPS cells can be verified by comparing the number of iPS colonies that have appeared in a case in which only predetermined reprogramming factors (e.g., three factors; Oct3/4, Sox2, and Klf4) were introduced into somatic cells and such number in a case in which HMGA2 was introduced in addition to the reprogramming factors.
  • predetermined reprogramming factors e.g., three factors; Oct3/4, Sox2, and Klf4
  • an example of HMGA2 in the present invention is: a protein comprising: (i) an amino acid sequence having a deletion of 1 or 2 or more (e.g., about 1-10, preferably 1 to several (5, 4, 3, or 2)) amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2 or 4; (ii) an amino acid sequence having an addition of 1 or 2 or more (e.g., about 1-10, preferably 1 to several (5, 4, 3, or 2)) amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2 or 4; (iii) an amino acid sequence having an insertion of 1 or 2 or more (e.g., about 1-10, preferably 1 to several (5, 4, 3, or 2)) amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2 or 4; (iv) an amino acid sequence having a substitution of 1 or 2 or more (e.g., about 1-10, preferably 1 to several (5, 4, 3, or 2)) amino acids with other amino acids, with respect to the amino acid sequence shown
  • HMGA2 protein examples include human HMGA2 (including isoforms) (RefSeq Accession NO. NP_003474.1 , NP_003475.1) comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or an ortholog thereof in another mammal (e.g., a mouse ortholog registered with GenBank under RefSeq Accession No. NP 034571.1 and a rat ortholog registered with GenBank under RefSeq Accession No. NP_1 14459.1), and furthermore, natural allelic mutants thereof or polymorphism. It is desirable to use HMGA2 of the same species depending on the animal species of somatic cells to be subjected to introduction.
  • the Lin28A mutant is a protein comprising an amino acid sequence that has an insertion, a deletion, or a substitution with respect to and having activity of substantially the same quality as that of a wild-type Lin28A (NCBI Accession No. NP 078950 (SEQ ID NO: 8).
  • a preferable Lin28A mutant retains a zinc finger domain and has an insertion, a deletion, or a substitution other than this domain.
  • An example of such an Lin28A mutant is, but is not particularly limited to, cold-shock domain-deficient mutant Lin28A (SEQ ID NO: 10).
  • the zinc finger domain is a domain containing the sequence shown in SEQ ID NO: 6 and the sequence corresponds to an amino acid sequence from Asp at position 137 to Leu at position 176 of the amino acid sequence of the wild-type Lin28A (SEQ ID NO: 8).
  • the nucleotide sequence encoding the zinc finger domain is shown in SEQ ID NO: 5.
  • the HMGA2 protein and/or Lin28 mutant can be introduced into somatic cells using a method known per se for introducing a protein into cells.
  • a method known per se for introducing a protein into cells examples include a method using a reagent for protein introduction, a method using a protein transduction domain (PTD) or a cell penetrating peptide (CPP) fusion protein, and microinjection.
  • PTD protein transduction domain
  • CPP cell penetrating peptide
  • a cationic lipid-based BioPOTER Protein Delivery Reagent Gene Therapy Systems
  • a Pro-JectTM Protein Transfection Reagent PIERCE
  • ProVectin IGENEX
  • a lipid-based Prefect- 1 Targeting Systems
  • a membrane-permeable peptide-based Penetrain Peptide Q biogene
  • Chariot Kit Active Motif
  • GenomONE Ishihara Sangyo Kaisha Ltd.
  • HVJ envelope inactivated Sendai virus
  • HMGA2 is diluted in an appropriate solvent (e.g., buffer such as PBS or HEPES), a reagent for introduction is added, incubation is carried out at room temperature for about 5-15 minutes to cause the formation of a complex.
  • an appropriate solvent e.g., buffer such as PBS or HEPES
  • a reagent for introduction is added, incubation is carried out at room temperature for about 5-15 minutes to cause the formation of a complex.
  • the resultant is added to cells after medium exchange with a serum free medium and then incubation is carried out at 37°C for 1 or several hours. Thereafter the medium is removed and exchanged with a serum-containing medium.
  • PTD PTD prepared using a cell penetrating domain of a protein has been developed, such as Drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988); Green, M. & Loewenstein, P.M. Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J.
  • PTD-derived CPP examples include polyarginines such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)).
  • a fusion protein expression vector in which HMGA2 cDNA, a PTD sequence, and a CPP sequence have been incorporated is constructed and then recombination and expression are carried out.
  • the fusion protein is collected and then used for introduction.
  • Introduction can be carried out in a manner similar to the above except that no reagent for protein introduction is added.
  • Microinjection is a method that involves filling a glass needle having a tip diameter of about 1 ⁇ with a protein solution and then injecting the solution into cells via a puncture, which can ensure protein introduction into cells.
  • protein introduction methods such as electroporation, a semi-intact cell method (Kano, F. et al. Methods in Molecular Biology, Vol. 322, 357-365 (2006)), and a method for introduction using a Wr-t peptide (Kondo, E. et al., Mol. Cancer Then 3(12), 1623-1630(2004)) can also be employed herein.
  • the protein introduction operation can be carried out once or more optionally chosen times (e.g., once or more to 10 times or less, or once or more to 5 times or less, and the like); preferably, the introduction operation can be performed twice or more (e.g., 3 times or 4 times) repeatedly.
  • the time interval for repeated introduction is, for example, 6 to 48 hours, and preferably 12 to 24 hours.
  • a nucleic acid encoding HMGA2 in the present invention is not particularly limited, as long as it encodes any one of the above HMGA2 proteins in the present invention.
  • a nucleic acid may be DNA or RNA, or a DNA/RNA chimera.
  • a nucleic acid may be synthetic RNA.
  • a preferable example is DNA.
  • the nucleic acid may be double-stranded or single-stranded. In the case of a double- stranded nucleic acid, such a nucleic acid may be double-stranded DNA, double- stranded RNA, or a DNA : RNA hybrid.
  • the definition of the term "nucleic acid" for HMGA2 described above is also applied to a nucleic acid encoding Lin28A mutant.
  • DNA encoding HMGA2 may be cloned from cDNA from, for example, the cells or tissues [e.g., cells or tissues of thymus gland, bone marrow, spleen, brain, spinal cord, heart, skeletal muscle, kidney, lung, liver, pancreas, or prostate gland, precursor cells of these cells, stem cells, or cancer cells of these cells] of humans or other mammals (e.g., mice, rats, monkeys, pigs, and dogs) according to a conventional method.
  • the cells or tissues e.g., cells or tissues of thymus gland, bone marrow, spleen, brain, spinal cord, heart, skeletal muscle, kidney, lung, liver, pancreas, or prostate gland, precursor cells of these cells, stem cells, or cancer cells of these cells
  • mammals e.g., mice, rats, monkeys, pigs, and dogs
  • Examples of a nucleic acid encoding HMGA2 include a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3, and a nucleic acid encoding a protein that comprises a nucleotide sequence capable of hybridizing under stringent conditions to a complementary strand sequence of the nucleotide sequence shown in SEQ ID NO: 1 or 3 and has activity of substantially the same quality as that of HMGA2 above.
  • nucleic acid capable of hybridizing under stringent conditions to a complementary strand sequence of the nucleotide sequence shown in SEQ ID NO: 1 or 3
  • a nucleic acid comprising a nucleotide sequence that has about 80% or more, preferably about 90% or more, and further preferably about 95% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 or 3 is used.
  • stringent conditions include conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1 - 6.3.6, 1999, such as hybridization under 6 x SSC (sodium chloride/sodium citrate)/45°C, followed by one or more instances of washing under 0.2 x SSC/0.1% SDS/50°C-65°C, for example. Persons skilled in the art can appropriately select hybridization conditions for providing equivalent stringency.
  • Examples of a nucleic acid encoding HMGA2 preferably include a nucleic acid comprising the nucleotide sequence encoding human HMGA2 shown in SEQ ID NO: 1 or 3 (RefSeq Accession No. NM_003483.4, NM_003484.1 ), or orthologs thereof in other mammals (e.g., a mouse ortholog registered with GenBank under RefSeq Accession No. 1.NM 010441.2 and a rat ortholog registered with GenBank under RefSeq Accession No. NM 032070.1), and furthermore, natural allelic mutants thereof, and polymorphism. It is desirable to use a nucleic acid encoding HMGA2 of the same animal species as that of somatic cells to be subjected to introduction.
  • a nucleic acid encoding HMGA2 can be introduced into somatic cells using a method known per se for introducing a gene into cells.
  • a nucleic acid encoding HMGA2 is inserted into an appropriate expression vector containing a promoter that can function in somatic cells as host cells.
  • expression vectors viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus vectors, and plasmids for expression in animal cells (e.g., pAl-11 , pXTl , pRc/CMV, pRc/RSV, and pcDNAI/Neo) can be used, for example.
  • adenovirus vector adenovirus vector, a plasmid vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector, and the like can be used.
  • Examples of a promoter to be used in an expression vector include an EFl oc promoter, a CAG promoter, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, and an HSV-TK (herpes simplex virus thymidine kinase) promoter.
  • an EFloc promoter, a CAG promoter, a MoMuLV LTR, a CMV promoter, an SRa promoter, and the like are preferable.
  • An expression vector may contain, in addition to a promoter, if desired, an enhancer, polyA addition signal, a selection marker gene, an SV40 replication origin, and the like.
  • a selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.
  • a nucleic acid encoding HMGA2 may be independently integrated into an expression vector or may also be integrated in combination with one or more reprogramming genes into one expression vector.
  • the former case is preferably selected, but when a plasmid, an adenovirus, an episomal vector, or the like is used, the latter case may be preferably selected, without particular limitation.
  • the plurality of genes can be integrated into an expression vector via preferably a sequence that enables polycistronic expression.
  • a sequence that enables polycistronic expression a plurality of genes integrated into one type of expression vector can be expressed efficiently.
  • a sequence that enables polycistronic expression for example, a 2A sequence of foot and mouth disease virus (PLoS ONE3, e2532, 2008, Stem Cells 25, 1707, 2007), an IRES sequence (U.S. Patent No. 4,937,190), or preferably the 2A sequence can be used.
  • An expression vector containing a nucleic acid encoding HMGA2 can be introduced into cells by a technique known per se depending on the types of vector.
  • a viral vector a plasmid containing the nucleic acid is introduced into appropriate packaging cells (e.g., Plat-E cells) or a complementary cell line (e.g., 293 cells), the viral vector produced in a culture supernatant is collected, and then cells are infected with the vector by an appropriate method according to each viral vector.
  • appropriate packaging cells e.g., Plat-E cells
  • a complementary cell line e.g., 293 cells
  • cells are infected with the vector by an appropriate method according to each viral vector.
  • a specific means of using a retrovirus vector is disclosed in WO2007/69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007).
  • HMGA2 expression re-activation
  • a gene existing in the vicinity of a site where the HMGA2 gene has been integrated may increase carcinogenic risk in the tissue regenerated from iPS cell- derived differentiated cells.
  • the nucleic acid encoding HMGA2 is not integrated into a chromosome of cells and is preferably expressed transiently.
  • the use of an adenovirus vector that is rarely used for integration into a chromosome is preferable.
  • a specific means of using an adenovirus vector is described in Science, 322, 945-949 (2008).
  • an adeno-associated virus is infrequently integrated into a chromosome and has lower effects of causing cytotoxicity or inflammation compared with an adenovirus vector.
  • a Sendai virus vector can exist stably outside the chromosome and can be degraded and removed by siRNA as necessary. Hence, such a Sendai virus vector can also be similarly used preferably.
  • a Sendai virus vector described in J. Biol. Chem., 282, 27383- 27391 (2007) or JP Patent No. 3602058 can be used.
  • a method that can be preferably employed herein involves using a Cre/loxP system and excising the HMGA2-coding nucleic acid at the time when HMGA2 is no longer required.
  • loxP sequences are located in advance at both ends of the nucleic acid. After induction of iPS cells, Cre recombinase is caused to act on cells using a plasmid vector or an adenovirus vector, and then the region flanked by the loxP sequences can be excised.
  • an enhancer-promoter sequence in an LTR U3 region can upregulate the adjacent host gene by insertion mutation.
  • 3 '-self-inactivating (SIN) LTR in which the relevant sequence has been deleted or substituted with a polyadenylation sequence such as SV40, so as to avoid the expression control for endogenous genes by LTR located outside the unexcised loxP sequences remaining in the genome.
  • the vector in the case of a plasmid vector that is a non-viral vector, the vector can be introduced into cells by lipofection, a liposome method, electroporation, calcium phosphate coprecipitation, a DEAE dextran method, microinjection, a gene gun method, or the like.
  • a specific means of using a plasmid as a vector is described in Science, 322, 949-953 (2008), or the like.
  • gene introduction can be carried out one or more arbitrary instances (e.g., one or more instances to ten or less instances, or one or more instances to 5 or less instances).
  • arbitrary instances e.g., one or more instances to ten or less instances, or one or more instances to 5 or less instances.
  • Introduction operation can be carried out one or more arbitrary instances (e.g., one or more instances to ten or less instances or one or more instances to 5 or less instances) also in this case.
  • introduction operation can be carried out twice or more (e.g., three or four times) repeatedly.
  • a transgene may be integrated into a chromosome.
  • a transgene may be integrated into a chromosome.
  • a method that can be employed herein involves integrating a transgene into a chromosome using transposon, causing transferase to act on cells using a plasmid vector or an adenovirus vector, and thus completely removing the transgene from the chromosome.
  • An example of preferable transposon is lepidopteran insect-derived transposon, piggyBac.
  • a specific means of using piggyBack transposon is disclosed in Kaji, K. et al., Nature, 458: 771 -775 (2009), Woltjen et al., Nature, 458: 766-770 (2009).
  • a preferable non-integrating vector is an episomal vector that is autonomously replicable outside the chromosome.
  • a specific means of using an episomal vector is disclosed in Yu et al., Science, 324, 797-801 (2009).
  • an expression vector is constructed by inserting a nucleic acid encoding HMGA2 into the episomal vector in which loxP sequences are located in the same direction on the 5' and the 3' sides of a vector element required for replication of the episomal vector.
  • the expression vector can also be introduced into somatic cells.
  • An example of the episomal vector is a vector containing sequences required for autonomous replication, which are from EBV, SV40, or the like, as vector elements.
  • a vector element required for autonomous replication include a replication origin and a gene encoding a protein that controls replication via its binding to the replication origin.
  • examples of such vector elements include replication origin oriP and an EBNA-1 gene.
  • examples of such vector elements include replication origin ori and an SV40 large T antigen gene.
  • an episomal expression vector contains a promoter that controls the transcription of a nucleic acid encoding HMGA2.
  • a promoter similar to the above can be used.
  • an episomal expression vector may further contain similarly to the above, an enhancer, poly A addition signal, a selection marker gene, and the like, if desired. Examples of a selection marker gene include a dihydrofolate reductase gene and a neomycin resistance gene.
  • loxP sequences to be used in the present invention include, in addition to the bacteriophage PI -derived wild-type loxP sequence and arbitrary mutant loxP sequences that can cause recombination so as to delete a sequence between the loxP sequences when located in the same direction at positions flanking a vector element required for replication of a transgene.
  • mutant loxP sequences include lox71 having a mutation in the 5' side repeat sequence, lox66 having a mutation in the 3' side repeat sequence, and lox2272 or lox511 having a mutation in the spacer part.
  • the two loxP sequences located on the 5' and the 3' sides of the vector element may be identical to each other or may differ from each other.
  • mutant loxP sequences having a mutation in the spacer part (e.g., a pair of lox2272 and a pair of lox511).
  • a combination of a mutant loxP sequence (e.g., lox71) having a mutation in the 5' side repeat sequence and a mutant loxP sequence (e.g., lox66) having a mutation in the 3' side repeat sequence is used.
  • the loxP sequence remaining on the chromosome as a result of recombination has double mutations in the repeat sequences on the 5' and 3' sides, and is therefore unlikely to be recognized by Cre recombinase.
  • any mutant loxP sequence may be located on the 5' and 3' sides of the aforementioned vector element. However, it is necessary that the mutant loxP sequences be inserted in the direction such that the mutated site is located at the outer ends of the loxP sequences.
  • the two loxP sequences are located in the same direction on the 5' and 3' sides of a vector element essential for replication of a transgene (i.e., a replication origin or a gene sequence encoding a protein that binds to the replication origin to control its replication).
  • the vector element flanked by the loxP sequences may be either a replication origin or a gene sequence encoding a protein that binds to the replication origin to control its replication, or both.
  • An episomal vector can be introduced into cells by lipofection, a liposome method, electroporation, calcium phosphate co-precipitation, a DEAE dextran method, microinjection, a gene gun method, or the like. Specifically, the method described in Science, 324: 797-801 (2009), for example, can be used.
  • Whether or not the vector element required for replication of a transgene has been removed from iPS cells can be confirmed by performing Southern blot analysis or PCR analysis using a nucleic acid comprising a nucleotide sequence inside the vector element and/or in the vicinity of loxP sequences as a probe or a primer, and an episome fraction isolated from the iPS cells as a template, so as to examine the presence or absence of a band or the length of the band detected.
  • An episome fraction can be prepared using a method well known in the art, such as the method described in Science, 324: 797-801 (2009).
  • the "nuclear reprogramming substance" in the present invention may be composed of any substance, such as a protein factor or a nucleic acid encoding a protein factor (including a form included in a vector), or a low molecular weight compound, as long as it is a substance (group) capable of inducing iPS cells from somatic cells through introduction of the substance into the somatic cells or introduction of the same with HMGA2 or a nucleic acid encoding HMGA2 into the somatic cells.
  • a nuclear reprogramming substance is a protein factor or a nucleic acid encoding a protein factor, preferably the following combinations are exemplified (only the names of protein factors are described below).
  • Sox2 can be substituted with Soxl , Sox3, Soxl 5, Sox 17, or Soxl 8.
  • Klf4 can be substituted with Klfl, Klf2, or Klf5.
  • c- Myc can be substituted with T58A (active mutant), N-Myc, or L-Myc.
  • Oct family members such as OctlA, Oct6, and the like can also be used instead of Oct3/4.
  • Sox family members such as Sox7 can also be used instead of Sox2 (or Soxl , Sox3, Soxl 5, Soxl 7, or Soxl 8).
  • combinations that are not relevant to (l)-(24) above, but contain all components in any of (l )-(24) and other arbitrary substances may also fall under the category of the "nuclear reprogramming substance" in the present invention.
  • preferable nuclear reprogramming substances among these combinations include at least one, preferably two or more, and more preferably three or more factors selected from among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and SV40LT.
  • iPS cells for treatment are taken into consideration, a combination (specifically, (9) above) of three factors, Oct3/4, Sox2, and Klf4 is preferable.
  • the use of iPS cells for treatment is not taken into consideration (e.g., the use of iPS cells as research tools for screening in drug discovery)
  • four factors including c-Myc in addition to the three factors, Oct3/4, Sox2, and Klf4 five factors including Nanog in addition to the four factors, and six factors including SV40 Large T in addition to the five factors can be exemplified.
  • a combination in which the above c-Myc is replaced with L-Myc is also a preferable example of a nuclear reprogramming substance.
  • mice and human cDNA sequence information for each of the above protein factors can be obtained by referring to NCBI accession numbers described in WO 2007/069666 (Nanog is described in the publication under the name of "ECAT4.”
  • mouse and human cDNA sequence information for Lin28, Esrrb, Esrrg, and L-Myc can each be obtained by referring to the following NCBI accession numbers.). Persons skilled in the art can easily isolate these cDNAs.
  • the protein factor can be prepared by inserting the thus obtained cDNA into an appropriate expression vector, introducing the vector into host cells, and then collecting the recombinant protein factor from the culture product obtained by culturing the cells.
  • the thus obtained cDNA is inserted into a viral vector, an episomal vector, or a plasmid vector in a manner similar to the case for the above nucleic acid encoding HMGA2, so as to construct an expression vector and then the resultant is subjected to the nuclear reprogramming step.
  • the above Cre- loxP system or piggyBack transposon system can also be used.
  • the nucleic acids when nucleic acids encoding two or more protein factors as nuclear reprogramming substances are introduced into cells, the nucleic acids may be separately carried by different vectors. Alternatively, a plurality of nucleic acids may be connected in tandem to form a polycistronic vector. In the latter case, to enable efficient polycistronic expression, it is desirable to ligate a 2A self-cleaving peptide of foot and mouth disease virus between nucleic acids (see Science, 322, 949-953, 2008, for example).
  • a nuclear reprogramming substance can be brought into contact with somatic cells in a manner similar to that for the above HMGA2 protein when (a) the substance is a protein factor or in a manner similar to that for the above nucleic acid encoding HMGA2 when (b) the substance is a nucleic acid encoding the protein factor in (a).
  • a nuclear reprogramming substance when (c) a nuclear reprogramming substance is a low molecular weight compound, a nuclear reprogramming substance can be brought into contact with somatic cells by dissolving the substance into an aqueous or a non-aqueous solvent at an appropriate concentration, adding the substance solution to a medium appropriate for culturing somatic cells isolated from a human or another mammal (e.g., about 5%- 20% fetal calf serum-containing minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, 199 medium, and F12 medium) to a concentration of the nuclear reprogramming substance, so that it.
  • a medium appropriate for culturing somatic cells isolated from a human or another mammal e.g., about 5%- 20% fetal calf serum-containing minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, 199 medium,
  • the concentration of a nuclear reprogramming substance differs depending on the type of a nuclear reprogramming substance to be used herein, which is appropriately selected from the range of about 0.1 nM-about 100 nM.
  • the contact period is not particularly limited, as long as it is sufficient for achievement of cell nuclear reprogramming. In general, such a nuclear reprogramming substance may be caused to coexist with cells in a medium until positive colonies appear.
  • HDAC histone deacetylase
  • VPA valproic acid
  • trichostatin A e.g., HDAC l siRNA Smartpool ® (Millipore) and HuSH 29mer shRNA Constructs against HDAC 1 (OriGene)
  • DNA methyltransferase inhibitors e.g., 5'-azacytidine
  • G9a histone methyltransferase inhibitors e.g., low-molecular- weight inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)) and nucleic acid expression inhibitors such as siRNA and shRNA against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology))], an L-channel calcium agonist (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (e.g., siRNA and shRNA against p53 (Cell Stem Cell, 3, 475-479 (2008)), UTF1 (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (e.g., soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i/LIF (2i is an inhibitor for mitogen-activated protein kinase signaling and glycogen syntha
  • nucleic acid expression inhibitors may be in the form of expression vectors containing DNA encoding siRNA or shRNA.
  • nuclear reprogramming substances SV40 large T or the like can also fall under the category of the substance for improving efficiency in the establishment of iPS cells, since it is not essential for somatic cell nuclear reprogramming, but is a cofactor.
  • cofactors other than essential factors for nuclear reprogramming may be conveniently defined as nuclear reprogramming substances or substances for improving efficiency in the establishment of iPS cells.
  • the somatic cell nuclear reprogramming process can be understood as an overall phenomenon caused by the contact of somatic cells with a nuclear reprogramming substance and a substance for improving efficiency in the establishment of iPS cells. Persons skilled in the art are not always required to clearly distinguish the nuclear reprogramming substance from the substance for improving efficiency in the establishment of iPS.
  • a substance for improving efficiency in the establishment of iPS cells can be brought into contact with somatic cells by a method similar to each of the above methods described for nuclear reprogramming substances depending on the following cases: (a) the substance is a protein factor, (b) the substance is a nucleic acid encoding the protein factor, and (c) the substance is a low molecular weight compound.
  • a substance for improving efficiency in the establishment of iPS cells containing HMGA2 and/or an Lin28A mutant or nucleic acids encoding them may be brought into contact with somatic cells simultaneously with a nuclear reprogramming substance, or prior to or after the contact of a nuclear reprogramming substance with somatic cells, as long as the efficiency in the establishment of iPS cells from somatic cells is significantly improved compared with a case under the absence of the substance.
  • a nuclear reprogramming substance is a nucleic acid encoding a protein factor and a substance for improving efficiency in the establishment of iPS cells is a chemical inhibitory substance
  • the former substance results in a time lag (a given period) between gene introduction treatment and mass-expression of the protein factor
  • the latter substance can immediately act on cells. Accordingly, cells are cultured for a given period after gene introduction treatment and then a substance for improving efficiency in the establishment of iPS cells can be added to the medium.
  • both a nuclear reprogramming substance and a substance for improving efficiency in the establishment of iPS cells are used in the form of viral vectors or plasmid vectors, both substances may be simultaneously introduced into cells,
  • Efficiency in the establishment of iPS cells can be further improved by culturing cells under low-oxygen conditions in the nuclear reprogramming step for somatic cells.
  • low-oxygen conditions refers to that the oxygen concentration in an atmosphere when cells are cultured is significantly lower than that in ambient air.
  • an example of such oxygen conditions is a condition of an oxygen concentration lower than that in a 5-10% CO 2 /95-90% ambient air atmosphere that is generally employed for general cell culture.
  • conditions where an oxygen concentration in an atmosphere is 18% or less are applicable.
  • an oxygen concentration in an atmosphere is 15% or less (e.g., 14% or less, 13% or less, 12% or less, and 11% or less), 10% or less (e.g., 9% or less, 8% or less, 7% or less, and 6% or less), or 5% or less (e.g., 4% or less, 3% or less, and 2% or less).
  • an oxygen concentration in an atmosphere is preferably 0.1% or more (e.g., 0.2% or more, 0.3% or more, and 0.4% or more), 0.5% or more (e.g., 0.6% or more, 0.7% or more, 0.8%» or more, and 0.95 or more), or 1% or more (e.g., 1.1 % or more, 1.2% or more, 1.3% or more, and 1.4% or more).
  • C0 2 incubators capable of regulating oxygen concentration are marketed by various equipment manufacturers (e.g., C0 2 incubators for low-oxygen culture manufactured by manufacturers such as Thermo scientific, Ikemoto Scientific Technology, Juji Field Inc., and Wakenyaku Co., Ltd. can be used).
  • the time for initiating cell culture under low-oxygen conditions is not particularly limited, as long as it does not inhibit improvement in efficiency in the establishment of iPS cells compared with a case of normal oxygen concentration (20%).
  • the time for initiating cell culture under low-oxygen conditions may be before the contact, simultaneously with the contact, or after the contact of somatic cells with HMGA2 and/or an Lin28A mutant or nucleic acids encoding them and a nuclear reprogramming substance.
  • the relevant cells are preferably cultured under low-oxygen conditions immediately after or after a given period (e.g., between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) days) after the contact of somatic cells with HMGA2 and/or the Lin28A mutant or nucleic acids encoding them and a nuclear reprogramming substance.
  • a given period e.g., between 1 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) days
  • the period for culturing cells under low-oxygen conditions is also not particularly limited, as long as it does not inhibit improvement in efficiency in the establishment of iPS cells compared with a case of normal oxygen concentration (20%).
  • Examples of the period include, but are not limited to, 3 days or more, 5 days or more, 7 days or more, and 10 days or more, and 50 days or less, 40 days or less, 35 days or less, and 30 days or less.
  • a preferable culture period under low-oxygen conditions is also varied depending on an oxygen concentration in an atmosphere. Persons skilled in the art can appropriately adjust the culture period depending on an oxygen condition to be employed. Also, in an embodiment, when candidate iPS cell colonies are selected using drug resistance as an index, it is preferable to recover normal oxygen concentration from the low-oxygen conditions, prior to the initiation of drug selection.
  • the preferable time for initiating cell culture under low-oxygen conditions and the preferable culture period are varied depending on the types of nuclear reprogramming substance to be used, efficiency in the establishment of iPS cells under normal oxygen concentration conditions, for example.
  • HMGA2 and/or the Lin28A mutant or nucleic acids encoding them are brought into contact with a nuclear reprogramming substance and then cells can be cultured under conditions appropriate for culturing ES cells, for example.
  • a general medium is supplemented with a Leukemia Inhibitory Factor (LIF) as a differentiation inhibitory factor and then cells are cultured.
  • LIF Leukemia Inhibitory Factor
  • a medium is desirably supplemented with a basic fibroblast growth factor (bFGF) and/or a stem cell factor (SCF) instead of LIF.
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • MEF mouse embryo-derived fibroblasts
  • STO cells or the like are often used as MEF.
  • SNL cells McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)
  • Coculture with feeder cells may be initiated before, at the time of, or after (e.g., after 1 - 10 days) the contact between HMGA2 or a nucleic acid encoding HMGA2 and a nuclear reprogramming substance.
  • Examples of a method for selecting candidate iPS cell colonies include a method that uses drug resistance and reporter activity as indices and a method that involves morphological observation via visual inspection.
  • An example of the former method is carried out as follows. For example, drug resistance and/or reporter activity- positive colonies are selected using recombinant somatic cells prepared by targeting a drug resistance gene and/or a reporter gene to the locus of a gene (e.g., Fbxl 5, Nanog, and Oct3/4 and preferably Nanog or Oct3/4) that is specifically expressed at a high level in pluripotent cells.
  • a gene e.g., Fbxl 5, Nanog, and Oct3/4 and preferably Nanog or Oct3/4
  • Examples of such recombinant somatic cells include mouse- derived MEF in which a Pgeo (encoding a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase) gene has been knocked-in to the Fbxl 5 locus (Takahashi & Yamanaka, Cell, 126, 663-676 (2006)) and transgenic mouse-derived MEF in which a green fluorescent protein (GFP) gene and a puromycin resistance gene have been integrated into the Nanog locus (Okita et al., Nature, 448, 313-317 (2007)).
  • Pgeo encoding a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase
  • an example of a method for selecting candidate colonies by morphological observation via visual inspection is a method described in Takahashi et al., Cell, 131 , 861 -872 (2007).
  • a method using a reporter cell is convenient and efficient.
  • colonies are desirably selected by visual inspection in view of safety.
  • Cells of the thus selected colonies can be identified as iPS cells based on a positive result for the above Nanog (or Oct3/4) reporter (e.g., having puromycin resistance and being positive for GFP) and ES cell-like colony formation when confirmed by visual inspection.
  • a positive result for the above Nanog (or Oct3/4) reporter e.g., having puromycin resistance and being positive for GFP
  • ES cell-like colony formation when confirmed by visual inspection.
  • tests such as alkaline phosphatase staining, analysis of the expression of various ES cell-specific genes, and confirmation of teratoma formation after transplantation of selected cells into mice can be performed.
  • the thus obtained iPS cells are novel cells differing from conventionally known iPS cells in that they contain the relevant foreign nucleic acid.
  • the foreign nucleic acid is introduced into somatic cells using retrovirus, lentivirus, or the like, such a trait of containing the foreign nucleic acid is stably retained since the foreign nucleic acid is generally integrated into the genome of the thus obtained iPS cells.
  • the thus established iPS cells can be used for various purposes. For example, differentiation of iPS cells into various cells (e.g., cardiac muscle cells, blood cells, nerve cells, vascular endothelial cells, and insulin-secreting cells) can be induced using differentiation induction methods reported for ES cells. Therefore, induction of iPS cells using somatic cells collected directly from a patient or another subject whose HLA type is identical to or substantially identical to the patient enables stem cell therapy involving autologous transplantation such that the resulting iPS cells are differentiated into desired cells (specifically, cells of an affected organ of the patient or cells exerting therapeutic effects against the disease, for example) and then the resultants are transplanted into the patient.
  • desired cells specifically, cells of an affected organ of the patient or cells exerting therapeutic effects against the disease, for example
  • functional cells e.g., hepatocytes
  • iPS cells resulting from differentiation of iPS cells
  • functional cells can also be appropriately used for in vitro screening or the like for drug efficacy or toxicity of a drug candidate compound.
  • a mouse ecotropic virus receptor Slc7al gene was expressed in fibroblasts (HDF: human dermal fibroblasts) from the skin of an adult subject (73 -year-old white woman; cell name: 1503) according to the method described in Takahashi, K. et al., Cell, 131 : 861-872 (2007) using a lentivirus (pLenti6/UbC-Slc7al ).
  • the following genes were introduced into the cells (1 x 10 5 cells/well, 6-well plate) according to the method described in Takahashi, K. et al., Cell, 131 : 861-872 (2007) using a retrovirus. These genes were compared with Lin28 for examination of establishment efficiency, which is known to improve establishment efficiency. In addition, controls were not subjected to introduction of the four factors.
  • feeder cells 2.5 x 10 5 cells/100 mm dish.
  • SNL cells McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990) treated with mitomycin C to halt cell division were used.
  • cells were cultured in medium prepared by adding 4 ng/ml recombinant human bFGF (WAKO, Japan) to medium for primate ES cell culture (ReproCELL, Japan).
  • the number of iPS cell colonies was determined, and the results are shown in Fig. 1.
  • HMGA2 to four factors (Oct3/4, Sox2, Klf4, and C-Myc) was found to increase the number of human iPS cell colonies, compared with the results for the control or a group to which DsRed had been added.
  • HMGA2 increased colony size, and the effect thereof was greater than that exerted by the addition of Lin28.
  • HMGA2 was confirmed to have effects of accelerating colony growth and thus to increase establishment efficiency for iPS cells.
  • Lin28A, B wild-type
  • Lin28A mutant Various Lin28 mutants were prepared and examined for domains that had effects on HMGA2 expression so as to be involved in efficiency in the establishment of iPS cells.
  • a mouse ecotropic virus receptor Slc7al gene was expressed in human skin- derived fibroblasts (HDF 1503 cell line) according to the method described in Takahashi, K. et al., Cell, 131 : 861-872 (2007) using a lentivirus (pLenti6/UbC-Slc7al).
  • the following factors were introduced into the cells (l x lO 5 cells/well, 6-well plate) according to the method described in Takahashi, K. et al., Cell, 131 : 861 -872 (2007) using retrovirus vectors. Each cell was treated with IPA buffer on day 7 after infection and then used as a sample for electrophoresis.
  • HMGA2 expression was enhanced through the addition of Lin28A or Lin28B in addition to four factors (Oct3/4, Sox2, Klf4, and C-Myc). Also, whereas the introduction of cold-shock domain-deficient mutant Lin28A resulted in an increased HMGA2 expression level, the effect of enhancing expression could not be obtained with the introduction of zinc- finger domain-deficient mutant Lin28A.
  • a mutant was introduced together with four factors (Oct3/4, Sox2, Klf4, and C-Myc) into human skin-derived fibroblasts (TIG-119 cell line) using retrovirus vectors.
  • the number of iPS cell colonies was determined on day 24 after infection.
  • the addition of Lin28A, Lin28B, or cold-shock domain-deficient mutant Lin28A resulted in increased number of human iPS cell colonies.
  • the effect of increasing the number of human iPS cell colonies could also be obtained with the addition of HMGA2 to the four factors.
  • HMGA2 improves efficiency in the establishment of iPS cells. Also, it was suggested that HMGA2 is transcriptionally regulated at a site downstream of Lin28 via the zinc-finger domain.
  • HMGA2 maintains self-renewal via transcriptional regulation at the downstream side of Lin28, so as to improve efficiency in the establishment of iPS (cells) from somatic cells.
  • the present invention is explained herein with emphasis on preferred embodiments. It is obvious for persons skilled in the art that the preferred embodiments can be changed. It is intended that it be possible to implement the present invention using a method other than those specifically described in the description. Therefore, the present invention includes all changes encompassed within the sprit and the scope of the attached "Claims.”
  • the present invention can be used in the field of production of materials for regenerative medicine.

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Abstract

La présente invention concerne un procédé d'amélioration de l'efficacité de l'établissement de cellules iPS, le procédé comportant une étape d'introduction de protéines mutantes HMGA2 et/ou Lin28A ou d'acides nucléiques codant pour les protéines et une substance de re-programmation nucléaire dans des cellules somatiques. La présente invention concerne également un procédé de production de cellules iPS comportant une étape d'introduction de protéines mutantes HMGA2 et/ou Lin28A ou d'acides nucléiques codant pour les protéines et une substance de re-programmation nucléaire dans des cellules somatiques. De plus, la présente invention concerne un procédé de production de cellules somatiques par l'induction de la différenciation des cellules iPS qui peuvent être obtenues par le procédé de la présente invention et qui contiennent des acides nucléiques codant pour des gènes mutants HMGA2 et/ou Lin28A.
PCT/JP2011/064330 2010-06-16 2011-06-16 Procédé d'établissement efficace de cellules souches pluripotentes induites WO2011158967A1 (fr)

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KR20140121787A (ko) * 2013-04-06 2014-10-16 주식회사 강스템바이오텍 Hmga2를 이용하여 비신경 세포로부터 리프로그래밍된 유도 신경줄기세포를 제조하는 방법
CN105492597A (zh) * 2013-04-06 2016-04-13 首尔大学校产学协力团 利用hmga2制备由非神经元细胞重编程的诱导神经干细胞的方法
US20160215259A1 (en) * 2013-04-06 2016-07-28 Seoul National University R&Db Foundation Method of Preparing Induced Neural Stem Cells Reprogrammed from Non-Neuronal Cells Using HMGA2
EP2982747A4 (fr) * 2013-04-06 2016-09-21 Univ Seoul Nat R & Db Found Procédé pour produire une cellule souche neuronale dérivée reprogrammée à partir d'une cellule non neuronale au moyen de hmga2
KR101870125B1 (ko) * 2013-04-06 2018-06-26 주식회사 강스템바이오텍 Hmga2를 이용하여 비신경 세포로부터 리프로그래밍된 유도 신경줄기세포를 제조하는 방법
CN105492597B (zh) * 2013-04-06 2020-11-10 首尔大学校产学协力团 利用hmga2制备由非神经元细胞重编程的诱导神经干细胞的方法
US11512285B2 (en) 2013-04-06 2022-11-29 Seoul National University R&Db Foundation Method of preparing induced neural stem cells reprogrammed from non-neuronal cells using HMGA2

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