WO2013062140A1 - Procédé pour l'induction efficace de la différenciation de cellules souches pluripotentes en des cellules du lignage hépatique - Google Patents

Procédé pour l'induction efficace de la différenciation de cellules souches pluripotentes en des cellules du lignage hépatique Download PDF

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WO2013062140A1
WO2013062140A1 PCT/JP2012/078407 JP2012078407W WO2013062140A1 WO 2013062140 A1 WO2013062140 A1 WO 2013062140A1 JP 2012078407 W JP2012078407 W JP 2012078407W WO 2013062140 A1 WO2013062140 A1 WO 2013062140A1
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cell
cells
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differentiation
hepatic
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Shinya Yamanaka
Masatoshi Kajiwara
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Kyoto University
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    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for efficiently inducing differentiation of a pluripotent stem cell into a hepatocyte. More particularly, the present invention relates to a method for efficiently inducing differentiation of an induced pluripotent stem cell (hereinafter to be referred to as iPS cell) into a hepatocyte by using an iPS cell derived from a blood cell, and a method for efficiently inducing differentiation of a pluripotent stem cell into a hepatocyte.
  • iPS cell an induced pluripotent stem cell
  • Yamanaka et al induced iPS cells by introducing the Oct3/4, Sox2, Klf4 and c-Myc genes into fibroblasts derived from a mouse and human, and forcing the cells to express the genes [WO 2007/069666 Al; Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)].
  • a group of Thomson et al. produced human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (4, 5) .
  • the iPS cells obtained as mentioned above can be any suitable material.
  • the iPS cells obtained as mentioned above can be any suitable material.
  • differentiated into the cells of respective tissues can be a tool for in vitro drug discovery screening and a cell to be transplanted for
  • Hepatocyte produced from a human iPS cell also becomes a useful resource for the development of pharmaceutical products and regenerative medicine.
  • a method for inducing differentiation of an iPS cell into a mature hepatocyte in vitro has not been established yet.
  • various attempts have been made to improve the differentiation method (6-16) whether or not there exists a human iPS cell appropriate for hepatocyte differentiation, namely, the relationship between hepatocyte differentiation and derived cell, has not been studied yet.
  • An object of the present invention is to . search for an iPS cell suitable for hepatocyte differentiation, with which to provide a method for inducing differentiation of a human iPS cell into a hepatocyte efficiently and with good reproducibility.
  • the present inventors first somewhat modified the existing methods of ES cell (Hay, D.C. et al., Stem Cells, 26(4): 894-902 (2008); Hay, D.C. et al., Proc. Natl. Acad. Sci . USA, 105(34): 12301-12306 (2008)) to induce differentiation of various human iPS cell lines into hepatocytes.
  • ES cell Host Cell Cells, 26(4): 894-902 (2008)
  • Hay, D.C. et al. Proc. Natl. Acad. Sci . USA, 105(34): 12301-12306 (2008)
  • the present inventors developed a new method by improving an existing method (Hay, D.C. et al., Stem Cells (2008), supra; Hay, D.C. et al., Proc. Natl. Acad. Sci. USA (2008), supra) to perform induction of differentiation of human iPS cell into hepatocyte more efficiently and with good reproducibility.
  • an undifferentiated human iPS cell colony is directly used to induce
  • the present inventors developed a new method capable of inducing superior differentiation of a human iPS cell in a single-cell state into a hepatocyte by adjusting the number of days of the first step (Step 1) of differentiation induction and
  • the present inventors studied the differentiation tendency (differentiation propensity) toward hepatocyte by using this new differentiation induction method and 22 kinds of human iPS cell lines established from various somatic cell origins by the retrovirus method or episomal method.
  • a blood cell- derived iPS cell shows a significantly high differentiation ability (albumin secretion potential) toward hepatocyte as compared to iPS cells derived from fibroblast and dental pulp stem cell, and human ES cells.
  • PBMC peripheral blood mononuclear cell
  • iPS cell maintains the memory (epigenetic memory) of the somatic cell origin from which it was derived and, in fact, a report has documented that a blood cell-derived iPS cell is easily differentiated into a blood cell ⁇ Nature, 467, 285-290 (2010) ) . Also from such aspect, the present finding is epoch-making and overturns conventional theories.
  • the present invention provides the following.
  • a method for producing a cell belonging to an endodermal lineage comprising inducing differentiation of a blood cell- derived induced pluripotent stem (iPS) cell.
  • iPS blood cell- derived induced pluripotent stem
  • a method for producing a cell belonging to an endodermal lineage comprising the following steps (1) and (2):
  • step (1) into a cell belonging to an endodermal lineage.
  • pluripotent stem cell into a CXCR4 positive endoderm cell characterized by the following (1) and (2) :
  • differentiation of iPS cell into cells belonging to an endodermal lineage such as hepatocyte can be induced efficiently by using a blood cell- derived iPS cell as a cell source.
  • a blood cell- derived iPS cell as a cell source.
  • an iPS cell derived from a peripheral blood mononuclear cell shows high differentiation propensity toward hepatocyte
  • an iPS cell having hepatic differentiation propensity equal to or. more than that of a liver-derived iPS cell can be obtained by ordinary blood sampling, without using a method including inducing an iPS cell from a hepatocyte collected from a hepatic tissue, which is highly burdensome for the donor.
  • physiological functions of hepatocyte in a living organism than established hepatocyte can be prepared without a
  • the cell can be utilized as a high-throughput in vitro screening system for the evaluation of efficacy and toxicity in drug discovery.
  • Fig. 1 shows directed hepatic differentiation of hiPSCs and hESCs.
  • A Schematic presentation of directed hepatic differentiation protocol in this study.
  • B Time course expression of undifferentiated and hepatocyte differentiation marker genes in the hepatic differentiated hiPSCs (201B6) .
  • Embryoid body (EB) was -obtained with a floating culture for 8 days followed by a monolayer culture on gelatin-coated plate for another 8 days, and used as positive control of early lineage differentiated markers. HepG2 and adult liver were used as positive control for hepatic markers.
  • Fig. 2 shows marked hepatic differentiation diversity among hESC and sibling hiPSC lines.
  • A Percentage of albumin positive cells by flow cytometry analysis after 17 days of hepatic differentiation.
  • C Albumin secretion potential of hepatic differentiated hiPSCs and hESCs at day 17 and other control cells analyzed by ELISA.
  • Fig. 3 shows characterization of hepatic differentiated hiPSCs (201B6) and hESCs (KhES3) .
  • A RT-PCR analysis of various CYP450 expression, ABC transporters and enzyme of gluclonidation in hepatic differentiated hiPSC and hESCs at day 17.
  • B Periodic acid-Schiff (PAS) staining was performed to detect intracellular glycogen, (bar 100 ⁇ )
  • PAS Periodic acid-Schiff
  • Fig. 4 shows close comparison of hepatic differentiation propensity between sibling hiPSC lines, 201B6 and 201B7.
  • A Shematic presentation of modified protocol for hepatic differentiation. In this protocol, hiPSCs/hESCs were
  • Fig. 5 shows time course of endodermal and hepatic differentiation from single hiPSCs.
  • Phase contrast images of endodermal differentiation from single hiPSCs (201B6, 201B7) and hepatic differentiation with further cultivation.
  • 0.5 mM NaB was added for 3 days (day 1 - day 3) .
  • bar 100 ⁇ At day 21 of hepatic differentiation, albumin staining was performed.
  • Fig. 6 shows characterization of undifferentiated cells, CXCR4 positive cells at day 7 and hepatic differentiated cells at day 21 derived from 201B6 and 201B7 hiPS clones by
  • Microarray-based gene expression analysis showed that the global gene expression pattern including 10 liver-related transcription factors (orange dots and letters) is similar between 201B6 and 201B7 hiPSC lines at both
  • Fig. 7 shows comparison of albumin secretion potential of hepatic differentiated cells at day 21 among various
  • Fig. 8 shows comparison of endoderm and hepatic
  • the present invention provides a method for producing a cell belonging to an endodermal lineage comprising inducing differentiation of a blood cell-derived iPS cell.
  • An iPS cell can be prepared by transferring a nuclear reprogramming substance to a somatic cell.
  • the somatic cell to be used as a starting material for the production of an iPS cell in the present invention may be derived from any mammal such as human, mouse, monkey, bovine, swine, rat, dog and the like, as long as it is derived from blood.
  • specific examples of the blood cell include peripheral blood mononuclear cell (PBMC, also referred to as peripheral mononuclear cell (PM C) ) and cord blood cell . (CB cell).
  • PBMC peripheral blood mononuclear cell
  • PM C peripheral mononuclear cell
  • CB cell cord blood cell .
  • T cells, B cells, NK cells, NKT cells and the like obtained by separation from peripheral blood mononuclear cells can also be used as a somatic cell source in the present invention.
  • PBMC can be prepared by diluting the peripheral blood collected from a mammal with PBS etc.
  • T cells, B cells, NK cells, NKT cells and the like can be isolated by flow cytometry and the like using an antibody against a cell surface marker specific to each of them.
  • the choice of mammal individual as a source of somatic cells is not particularly limited; however, when the endoderm cells as a product are to be used for the treatment of diseases such as liver disfunction in humans, it is preferabl from the viewpoint of prevention of graft rejection and/or GvHD, that blood cells are patient's own cells or collected from another person having the same or substantially the same HLA type as that of the patient.
  • substantially the same HLA type as used herein means that the HLA type of donor matches with that of patient to the extent that the transplanted cell which have been obtained by inducing differentiation of iPS cells derived from the donor's blood cells, can be engrafted when they are transplanted to the patient with use of
  • immunosuppressor and the like.
  • it includes an HLA type wherein major HLAs (the three major loci of HLA-A, HLA-B and HLA-DR or four loci further including HLA-Cw) are
  • endoderm cells are not to be administered (transplanted) to a human, but used as, for example, a source of cells for screening for evaluating a patient's drug
  • Blood cells separated from a mammal can be pre-cultured using a medium known per se suitable for the cultivation thereof, depending on the kind of the cells.
  • a medium known per se suitable for the cultivation thereof include, but are not limited to, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum,
  • MEM minimal essential medium
  • DMEM Dulbecco's modified Eagle medium
  • RPMI1640 medium 199 medium
  • F12 medium F12 medium
  • transfection reagent such as a cationic liposome in contacting the cell with nuclear reprogramming substance (s) and iPS cell establishment efficiency improver (s)
  • the medium be previously replaced with a serum-free medium to prevent a reduction in the transfer efficiency.
  • a nuclear reprogramming substance refers to any substance (s) capable of inducing an iPS cell from a somatic cell, which may be composed of any substance such as a proteinous factor or a nucleic acid that encodes the same (including forms incorporated in a vector) , or a low-molecular compound.
  • the nuclear reprogramming substance is a proteinous factor or a nucleic acid that encodes the same, the following combinations, for example, are preferable (hereinafter, only the names for proteinous factors are shown) .
  • 2007/069666 for information on replacement of Sox2 with Soxl8 and replacement of Klf4 with Klfl or Klf5 in the combination (2) above, see Nature Biotechnology, 26, 101-106 (2008)); for the combination "Oct3/4, Klf4, c-Myc, Sox2", see also Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007) and the like; for the combination "Oct3/4, Klf2 (or Klf5) , c-Myc, Sox2", see also Nat. Cell Biol., 11, 197-203 (2009); for the combination "Oct3/4, Klf4, c-Myc, Sox2, hTERT, SV40 LT”, see also Nature, 451, 141-146 (2008).]
  • Oct3/4 may be replaced with another member of the Oct family, for example, OctlA, Oct6 or the like.
  • Sox2 (or Soxl, Sox3, Soxl5, Soxl7, Soxl8) may be replaced with another member of the Sox family, for example, Sox7 or the like.
  • Klfl, Klf2 and Klf5 substitutes, for example, members of the Esrr family such as Esrrb, Esrrg and the like, members of the IRX family such as IRX1, IRX2, IRX3, IRX4, IRX5, IRX6 and the like, members of the GLIS family such as GLISl, GLIS2, GLIS3 and the like, members of the PTX family such as PITX1, PITX2, PITX3 and the like, and DMRTB1 can also be used.
  • members of the Esrr family such as Esrrb, Esrrg and the like
  • members of the IRX family such as IRX1, IRX2, IRX3, IRX4, IRX5, IRX6 and the like
  • members of the GLIS family such as GLISl, GLIS2, GLIS3 and the like
  • members of the PTX family such as PITX1, PITX2, PITX3 and the like
  • DMRTB1 DMR
  • Lin28 is included as a nuclear reprogramming factor, L-Myc or Lin28B can be used in place of c-Myc or Lin28, respectively.
  • Members of the GLIS family such as GLISl and GLIS3 may also be used in place of c-Myc.
  • nuclear reprogramming substances can also be included in the scope of "nuclear reprogramming substances" in the present invention. Provided that the somatic cell to undergo nuclear reprogramming is endogenously expressing one or more of the constituents of any one of (1) to (24) above at a level sufficient to cause
  • nuclear reprogramming a combination of only the remaining constituents excluding the one or more constituents can also be included in the scope of "nuclear reprogramming substances" in the present invention.
  • GLIS3 can be mentioned as examples of preferable nuclear reprogramming substance.
  • iPS cells obtained are to be used for therapeutic purposes
  • a combination of the three factors Oct3/4, Sox2 and Klf [combination (9) above] are preferably used.
  • the iPS cells obtained are not to be used for therapeutic purposes (e.g., used as an investigational tool for drug discovery screening and the like)
  • three factors of Oct3/4, Sox2 and Klf4 as well as four factors additionally further containing c-Myc/L-Myc, five factors additionally further containing Lin28/Lin28B, six factors additionally further containing GLIS1/GLIS3, seven factors additionally further containing Nanog, and the like can be recited as
  • ECAT4 mouse and human cDNA sequence information on L-Myc, Lin28, Lin28B, GLIS1, GLIS3, Esrrb and Esrrg can be acquired by referring to the following NCBI accession numbers,
  • a proteinous factor for use as a nuclear reprogramming substance can be prepared by inserting the cDNA obtained into an appropriate expression vector, introducing the vector into a host cell, and recovering the recombinant proteinous factor from the cultured cell or its conditioned medium.
  • the nuclear reprogramming substance used is a nucleic acid that encodes a proteinous factor
  • the cDNA obtained is inserted into a viral vector, plasmid vector, episomal vector etc. to construct an expression vector, and the vector is subjected to the step of nuclear reprogramming.
  • Transfer of a nuclear reprogramming substance to a somatic cell can be achieved using a method known per se for protein transfer into a cell, provided that the substance is a proteinous factor.
  • the starting material iPS cell be also prepared without gene manipulation.
  • Such methods include, for example, the method using a protein transfer reagent, the method using a protein transfer domain (PTD) - or cell penetrating peptide (CPP)- fusion protein, the microinjection method and the like.
  • Protein transfer reagents are commercially available, including those based on a cationic lipid, such as BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-JectTM Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX) ; those based on a lipid, such as Profect-1 (Targeting Systems) ; those based on a membrane-permeable peptide, such as Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), GenomONE (ISHIHARA SANGYO KAISHA, LTD.) utilizing HVJ envelope (inactivated hemagglutinating virus of Japan) and the like.
  • the transfer 5 can be achieved per the protocols attached to these
  • reprogramming substance (s) is (are) diluted in an appropriate solvent (e.g., a buffer solution such as PBS or HEPES) , a transfer reagent is added, the mixture is incubated at room 10 temperature for about 5 to 15 minutes to form a complex, this complex is added to cells after exchanging the medium with a serum-free medium, and the cells are incubated at 37°C for one to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.
  • an appropriate solvent e.g., a buffer solution such as PBS or HEPES
  • Developed PTDs include those using transcellular domains of proteins such as drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988) or Green, M. & Loewenstein, P. M. Cell 55, 1179-88 (1988)), Penetratin
  • CPPs derived from the PTDs include polyarginines such as 11R ⁇ Cell Stem Cell, 4,381-384 (2009)) and 9R (Cell Stem Cell, 4, 472-476
  • a fused protein expression vector incorporating cDNA of a nuclear reprogramming substance and PTD or CPP sequence is prepared, and recombination expression is performed using the vector.
  • the fused protein is recovered and used for transfer. Transfer can be performed in the same manner as above except that a protein transfer reagent is not added.
  • Microinjection a method of placing a protein solution in a glass needle having a tip diameter of about 1 um, and injecting the solution into a cell, ensures the transfer of the protein into the cell.
  • the protein transferring operation can be performed one 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) .
  • the transferring operation can be performed twice or more (e.g., 3 times or 4 times) repeatedly.
  • the time interval for repeated transferring operation is, for example, 6 hours to 7 days, preferably 12 to 48 hours or 7 days.
  • nuclear reprogramming substance may also be used preferably in the form of a nucleic acid that encodes a proteinous factor, rather than the factor as it is.
  • the nucleic acid may be a DNA or an RNA, or a DNA/RNA chimera, and may be double-stranded or single-stranded.
  • the nucleic acid is a double-stranded DNA, particularly a cDNA.
  • a cDNA of a nuclear reprogramming substance is inserted into an appropriate expression vector comprising a promoter capable of functioning in a host somatic cell.
  • expression vectors include, for example, viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus and Sendai virus, plasmids for the expression in animal cells (e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like.
  • viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus and Sendai virus
  • plasmids for the expression in animal cells e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo
  • a vector for this purpose can be chosen as appropriate according to the intended use of the iPS cell to be obtained.
  • Useful vectors include adenovirus vector, plasmid vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, episomal vector and the like/ " -'
  • promoters used in expression vectors include the EFla promoter, the CAG promoter, the SRa promoter, the SV40 promoter, the LTR promoter, the C V (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney mouse leukemia virus) LTR, the HSV-TK (herpes simplex virus thymidine kinase) promoter and the like, with preference given to the EFla promoter, the CAG promoter, the MoMuLV LTR, the CMV promoter, the SRa promoter and the like.
  • the expression vector may contain as desired, in addition to a promoter, an enhancer, a polyadenylation signal, a
  • selectable marker gene a SV40 replication origin and the like.
  • selectable marker genes include the dihydrofolate reductase gene, the neomycin resistant gene, the puromycin resistant gene and the like.
  • nucleic acids as nuclear reprogramming substances may be separately integrated into
  • an expression vector incorporating two kinds or more of genes and another expression vector incorporating one gene alone can be used in combination.
  • genes when a plurality of genes are incorporated in one expression vector, these genes can be incorporated in one expression vector.
  • an intervening sequence enabling polycistronic expression preferably be inserted into the expression vector via an intervening sequence enabling polycistronic expression.
  • Useful sequences enabling polycistronic expression include, for example, the 2A sequence of foot-and-mouth disease virus ⁇ PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), IRES sequence (U.S. Patent No. 4,937,190) and the like, with preference given to the 2A sequence.
  • An expression vector harboring a nucleic acid as a nuclear reprogramming substance can be introduced into a cell by a technique known per se according to the choice of the vector.
  • a viral vector for example, a plasmid containing the nucleic acid is introduced into an appropriate packaging cell (e.g., Plat-E cells) or a complementary cell line (e.g., 293-cells) , the viral vector produced in the culture supernatant is recovered, and the vector is infected to the cell by a method suitable for the viral vector.
  • an appropriate packaging cell e.g., Plat-E cells
  • a complementary cell line e.g., 293-cells
  • a nucleic acid encoding a nuclear reprogramming substance is preferably expressed transiently, without being integrated into the chromosome of the cells. From this viewpoint, use of an adenoviral vector, whose integration into chromosome is rare, is preferred.
  • an adenoviral vector is disclosed in Science, 322, 945-949 (2008) . Because an adeno-associated viral vector is also low in the frequency of integration into chromosome, and is lower than adenoviral vectors in terms of cytotoxicity and inflammation-inducibility, it can be
  • Sendai viral vector is capable of being stably present outside the
  • a method can be used preferably wherein a nucleic acid encoding a nuclear reprogramming substance is cut out using the Cre-loxP system, when becoming unnecessary. That is, with loxP sequences
  • the Cre recombinase is allowed to act on the cells using a plasmid vector or adenoviral vector, and the region sandwiched by the loxP sequences can be cut out. Because the enhancer-promoter sequence of the LTR U3
  • a plasmid vector can be any vector that can be used as a non-viral vector.
  • liposome method liposome method, electroporation method, calcium phosphate co- precipitation method, DEAE dextran method, microinjection method, gene gun method and the like.
  • Specific means using a plasmid as a vector are described in, for example, Science,
  • the transfection can be performed once or more optionally chosen times (e.g., once to 10 times, once to 5 times or the like) .
  • the transfection can be performed once or more optionally chosen times (e.g., once to 10 times, once to 5 times or the like) , preferably the transfection can be repeatedly performed twice or more (e.g., 3 times or 4 times).
  • transgene can get integrated into chromosome; therefore, it is eventually necessary to confirm the absence of insertion of the gene into chromosome by Southern blotting or PCR. For this reason, like the aforementioned Cre-loxP system, it can be advantageous to use a means wherein the transgene is
  • a method can be used wherein the transgene is integrated into chromosome using a transposon, thereafter a transposase is allowed to act on the cell using a plasmid vector or adenoviral vector so as to completely eliminate the transgene from the chromosome.
  • a transposase is allowed to act on the cell using a plasmid vector or adenoviral vector so as to completely eliminate the transgene from the chromosome.
  • preferable transposons piggyBac, a transposon derived from a lepidopterous insect, and the like can be mentioned. Specific means using the piggyBac transposon is disclosed in Kaji, K. et al . , Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766-770 (2009).
  • Another preferred non-recombination type vector is an episomal vector autonomously replicable outside the chromosome.
  • a specific procedure for using an episomal vector is disclosed by Yu et al. in Science, 324, 797-801 (2009).
  • an expression vector may be constructed by inserting a
  • sequences placed in the same orientation at both the 5' and 3' sides of the vector element essential for the replication of the episomal vector may be transferred into a somatic cell.
  • episomal vector examples include vectors
  • the vector element required for its autonomous replication is a replication origin or a gene that encodes a protein that binds to the replication origin to regulate its replication; examples include the replication origin oriP and EBNA-1 gene for EBV, and the replication origin ori and SV40 large T antigen gene for SV40.
  • the episomal expression vector contains a promoter that controls the transcription of the reprogramming gene.
  • the promoter used can be the same promoter as the above.
  • the episomal expression vector may further comprise an enhancer, poly-A addition signal, selection marker gene and the like as desired, as described above. Examples of selection marker gene include the dihydrofolate reductase gene, neomycin resistance gene and the like.
  • An episomal vector can be introduced into a cell using, for example, lipofection method, liposome method,
  • electroporation method calcium phosphate co-precipitation method, DEAE dextran method, microinjection method, gene gun method and the like. Specifically, the method described in Science, 324: 797-801 (2009), for example, can be used.
  • replication of reprogramming gene has been removed from the iPS cell can be determined by performing Southern blot
  • An episome fraction can be prepared using a method well known in the art, for example, the method described in Science, 324: 797-801 (2009) .
  • the nuclear reprogramming substance is a low- molecular compound
  • introduction thereof into a somatic cell can be achieved by dissolving the substance at an appropriate concentration in an aqueous or non-aqueous solvent, adding the solution to a medium suitable for cultivation of somatic cells isolated from human or mouse [e.g., minimal essential medium (MEM) comprising about 5 to 20% fetal bovine serum, Dulbecco' s modified Eagle medium (DMEM) , RPMI1640 medium, 199 medium, F12 medium, and the like] so that the nuclear reprogramming substance concentration will fall in a range that is
  • MEM minimal essential medium
  • DMEM Dulbecco' s modified Eagle medium
  • reprogramming substance used is chosen as appropriate over the range of about 0.1 nM to about 100 nM. Duration of contact is not particularly limited, as far as it is
  • the nuclear reprogramming substance may be allowed to be co-present in the medium until a positive colony emerges.
  • iPS cell establishment efficiency improvers include, but are not limited to, histone deacetylase (HDAC) inhibitors [e.g., valproic acid (VPA) ⁇ Nat. Biotechnol. ,
  • HDAC histone deacetylase
  • VPA valproic acid
  • low-molecular inhibitors such as trichostatin A, sodium butyrate, MC 1293, and M344, nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool ® (Millipore) , HuSH 29mer shRNA Constructs against HDAC1 (OriGene) and the like) , and the like], DNA methyltransferase inhibitors (e.g., 5'- azacytidine) [Nat.
  • siRNAs and shRNAs against HDAC e.g., HDAC1 siRNA Smartpool ® (Millipore) , HuSH 29mer shRNA Constructs against HDAC1 (OriGene) and the like
  • DNA methyltransferase inhibitors e.g., 5'- azacytidine
  • G9a histone methyltransferase inhibitors e.g., low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), nucleic acid-based expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology) and the like) and the like] , L-channel calcium agonists (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 activator (e.g., soluble Wnt3a) [Cell Stem Cell, 3, 132-135 (2008)], 2i/LIF [2i is an enzyme that cata protein sequence.
  • nucleic acid-based expression inhibitors may be in the form of expression vectors harboring a DNA that encodes an siRNA or shRNA.
  • SV40 large T and the like can also be included in the scope of iPS cell
  • auxiliary factors for somatic cell nuclear reprogramming.
  • the auxiliary factors which are not essential for nuclear reprogramming, may be
  • nuclear reprogramming substances conveniently considered as nuclear reprogramming substances or iPS cell establishment efficiency improvers.
  • somatic cell nuclear reprogramming process is understood as an overall event resulting from contact of nuclear
  • contact of an iPS cell establishment efficiency improver with a somatic cell can be achieved as described above for each of three cases: (a) the improver is a proteinous factor, (b) the improver is a nucleic acid that encodes the proteinous factor, and (c) the improver is a low-molecular compound.
  • An iPS cell establishment efficiency improver may be brought into contact with a somatic cell simultaneously with a nuclear reprogramming substance, or either one may be
  • the nuclear reprogramming substance is a nucleic acid that encodes a proteinous factor and the iPS cell establishment efficiency improver is a chemical inhibitor
  • the iPS cell establishment efficiency improver can be added to the medium after the cell is cultured for a given length of time after the gene transfer treatment, because the nuclear reprogramming substance
  • establishment efficiency improver are both used in the form of a viral, plasmid or episomal vector, for example, both may be simultaneously introduced into the cell.
  • hypoxic conditions means that the oxygen concentration in the ambient atmosphere during cell culture is significantly lower than that in the air.
  • such conditions include lower oxygen
  • the oxygen concentration in the ambient atmosphere is 15% or less (e.g., 14% or less, 13% or less, 12% or less, 11% or less and the like), 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less and the like) , or 5% or less (e.g., 4% or less, 3% or less, 2% or less and the like).
  • the oxygen concentration in the ambient atmosphere is preferably 0.1% or more (e.g., 0.2% or more, 0.3% or more, 0.4% or more and the like), 0.5% or more (e.g., 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more and the like), or 1% or more (e.g., 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more and the like) .
  • C0 2 incubators are commercially available from a number of manufacturers of equipment (e.g., C0 2 incubators for hypoxic culture
  • the timing of beginning cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells compared with that obtained at a normal oxygen concentration (20%) .
  • the starting time may be before or after contact of nuclear reprogramming substances with a somatic cell, and may be at the same time as the contact.
  • cell culture under hypoxic conditions be begun just after contacting a nuclear reprogramming substance with a somatic cell, or after a given time (e.g., 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8 or 9) days) following the contact.
  • the duration of cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells
  • examples include, but are not limited to, between 3 days or more, 5 days or more, 7 days or more or 10 days or more, and 50 days or less, 40 days or less, 35 days or less or 30 days or less.
  • the preferred duration of cell culture under hypoxic conditions also varies depending on the oxygen
  • iPS cell candidate colonies are selected with drug resistance as an indicator, it is
  • a normal oxygen concentration be restored from hypoxic conditions by the start of drug selection.
  • the preferred starting time and duration of cell culture under hypoxic conditions also vary depending on the choice of nuclear reprogramming substances used, the efficiency of establishment of iPS cells under conditions involving a normal oxygen concentration, and other factors.
  • the cell can be cultured under conditions suitable for the cultivation of, for example, ES cells.
  • LIF Leukemia Inhibitory Factor
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • the cells are cultured in the co-presence of mouse embryo-derived fibroblasts (MEFs) treated with radiation or an antibiotic to terminate the cell division thereof, as feeder cells.
  • MEFs mouse embryo-derived fibroblasts
  • STO cells and the like are commonly used as MEFs, but for inducing iPS cells, SNL cells [McMahon, A. P. & Bradley, A. Cell 62, 1073-1085
  • Co-culture with feeder cells may be started before contact of the nuclear
  • reprogramming substance at the time of the contact, or after the contact (e.g., 1-10 days later).
  • a candidate colony of iPS cells can be selected by a method with drug resistance and reporter activity as
  • a colony positive for drug resistance and/or reporter activity is selected using a recombinant somatic cell wherein a drug resistance gene and/or a reporter gene is targeted to the locus of a gene highly expressed specifically in pluripotent cells (e.g.,
  • Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4) examples of such recombinant somatic cells include blood cells from a mouse having the Pgeo (which encodes a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase) gene knocked- in to the Fbxl5 locus [Takahashi & Yamanaka, Cell, 126, 663- 676 (2006) ] , blood cells from a transgenic mouse having the green fluorescent protein (GFP) gene and the puromycin
  • Pgeo which encodes a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase
  • examples of the method of selecting candidate colonies based on visual examination of morphology include the method described by
  • iPS cells selected by visual examination when iPS cells are prepared for the purpose of human treatment.
  • the three factors Oct3/4, Klf4 and Sox2 are used as nuclear reprogramming substances, the number of clones established decreases but the resulting colonies are mostly of iPS cells of high quality comparable to ES cells, so that iPS cells can efficiently be established even without using reporter cells.
  • the identity of the cells of a selected colony as iPS cells can be confirmed by positive responses to a Nanog (or Oct3/4) reporter (puromycin resistance, GFP positivity and the like) as well as by the formation of a visible ES cell-like colony, as described above.
  • a Nanog or Oct3/4 reporter
  • puromycin resistance or GFP positivity and the like
  • the "cell belonging to the endodermal lineage" is specifically classified into a hepatic lineage cell, a pancreatic lineage cell, and other lineage cell.
  • a hepatic lineage cell include hepatocyte, bile duct epithelial cell and the like.
  • the pancreatic lineage cell include endocrine pancreas cell ( ⁇ cell etc.), exocrine pancreas cell, pancreatic duct epithelial cell and the like.
  • Specific examples of other lineage cell include gastrointestinal tract epithelial cell, alveolar
  • the cell belonging to the endodermal lineage efficiently induced to differentiate from a blood cell-derived iPS cell is a hepatic lineage cell, more preferably a
  • induction include, but are not limited to, serum-free minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM) , RPMI1640 medium, 199 medium, F12 medium and a mixed medium thereof, any of the aforementioned media supplemented with an appropriate concentration of a well-known
  • conventionally-used medium additive e.g., serum albumin, 2- mercaptoethanol, insulin, transferrin, sodium selenite,
  • a serum may or may not be added as
  • Cultivation is performed by seeding iPS cells in a culture container known per se (e.g., 10 cm cell culture dish coated with gelatin, atrigel, collagen etc., or seeded with a suitable feeder cell, and the like) at a cell density of, for example, about 3- about 10 x 10 4 cells/mL, preferably about 4- about 8 x 10 4 cells/mL (about 3- about 10 x 10 5 cells/10 cm dish, preferably about 4- about 8 x 10 5 cells/10 cm dish, and cultivating in an incubator at 5% C0 2 /95% air, about 30- about. 40°C, preferably about 37°C.
  • a culture container known per se e.g., 10 cm cell culture dish coated with gelatin, atrigel, collagen etc., or seeded with a suitable feeder cell, and the like
  • a cell density of, for example, about 3- about 10 x 10 4 cells/mL, preferably about 4- about 8 x 10 4 cells/mL (about 3- about 10 x 10
  • Examples of the known differentiation induction method of iPS cell into hepatocyte include the methods described in Song, Z. et al., Cell Res., 15(11): 1233-1242 (2009); Sullivan, G.J. et al., Hepatology, 52(1): 329-335 (2010); Touboul, T. et al., Hepatology, 52(5): 1754-1765 (2010); Si-Tayeb, K. et al.,
  • a method for inducing differentiation of an ES cell into a cell belonging to an endodermal lineage a method including forming embryoid body (EB) by floating culture is general. For example, using expression of
  • Brachyury which is an early mesodermal marker, as an index, early mesoderm is induced by the EB formation method, and concentrated by flow cytometry, activin A is added under serum-free conditions and attachment culture is performed, whereby differentiation induction into endoderm is performed (Development, 131: 1651-1662 (2004)).
  • Foxa2 and T which are mesendoderm/endoderm markers, as an index, differentiation of anterior primitive streak cells from which endoderm is developed in normal
  • endoderm can also be induced without via EB by cultivating ES cell in an activin A- containing serum-free medium (Nat. Biotechnol., 23: 1542-1550 (2005); Nat. Biotechnol., 23: 1534-1541 (2005)).
  • mesendoderm and endoderm can be induced by normal development, without via EB formation, by using a mesoderm-derived cultured cell (e.g., M15 cell which is a mouse fetal kidney-derived cultured cell etc.) as a feeder cell (Stein Cells, 26: 874-885 (2008)).
  • a mesoderm-derived cultured cell e.g., M15 cell which is a mouse fetal kidney-derived cultured cell etc.
  • bFGF are added to a medium to efficiently induce differentiation into endoderm, since bFGF transfers ES cell from an undifferentiated state to a
  • BMP can be added to the primary medium since it inhibits, like activin A, differentiation of ES cell into ectoderm, but is desirably removed from the medium after expression of an early mesoderm marker since it promotes differentiation of mesendoderm into mesoderm.
  • AFP a-fetoprotein
  • hepatic progenitor cell can be promoted by substituting the serum with Knockout Serum Replacement (KSR) .
  • KSR Knockout Serum Replacement
  • dexamethasone a hepatocyte growth factor (HGF) and oncostatin M (OS ) .
  • HGF hepatocyte growth factor
  • OS oncostatin M
  • albumin positive hepatocyte and DBA positive bile duct cell can be induced, and increase of glycogen storage, expression of drug-metabolizing enzyme (cytochrome P450 group) can also be confirmed (Genes Cells, 13: 731-746 (2008) ) .
  • differentiation induction method of ES cell into hepatic, lineage cell include, but are not limited to, a method including forming EB in a medium containing activin A and BMP4, and performing an attachment culture ⁇ Nat. Biotechnol., 24: 1402-1411 (2006)), a method including
  • liver-derived mesenchymal cell Exp. Cell Res., 309: 68-77 (2005)
  • examples of known differentiation induction method of ES cell into a pancreatic lineage cell include a method including an attachment culture after
  • examples of known differentiation induction method of ES cell into other lineage cell include a method including inducing differentiation of thyroid cell by
  • All the above-mentioned methods for ES cell can be utilized for differentiation induction of iPS cell into hepatocyte in the present invention.
  • a method of inducing differentiation of a cell belonging to an endodermal lineage in which differentiation is not induced by using a colony already grown to some level as in the conventional methods but by using an iPS cell in a single- cell state.
  • Use of iPS cells as a colony namely, in a cell mass state, is defective in that the size and nonuniformity of the colony influence differentiation induction efficiency and reproducibility.
  • problems of easy occurrence of apoptosis and the like are caused.
  • new appropriate culture conditions need to be determined since the sensitivity of the hormonal factors to be added to the medium varies markedly.
  • step 1 a colony grown to subconfluence is cultured in a serum-free medium containing activin A (100 ng/ml), Wnt3a (50 ng/ml) and sodium butyrate (1 mM) for 1 day, sodium butyrate is removed and the colony is further cultivated for 2 days.
  • This step induces differentiation into an endoderm cell characterized by CXCR4 positive (and E-cadherin positive) .
  • CXCR4 positive and E-cadherin positive
  • differentiation of single- celled iPS cell into CXCR4 positive endoderm cell can be induced with high efficiency by prolonging the culture period in an activin A and Wnt3a-containing medium and adjusting concentration and addition period of sodium butyrate.
  • the single-celled iPS cell can be prepared by dissociating an iPS cell colony prepared as mentioned above by pipetting and the like in a suitable cell dissociation solution (e.g., 0.1% collagenase, 0.1-0.2% EDTA, 0.05% trypsin/1 mM EDTA, Ca 2+ - treated trypsin/collagenase, DISPASE, AccutaseTM) .
  • step 1 of the differentiation induction protocol of the present invention single-cell state iPS cell is
  • any of the above-mentioned basic media containing 0- 20%, preferably 0-5%, of serum can be used. More preferred is serum-free.
  • the concentration of activin A to be added to the medium is, without limitation, for example, not less than 50 ng/ml, preferably not less than 70 ng/ml, more preferably not less than 80 ng/ml, and not more than 200 ng/ml, preferably not more than 150 ng/ml, more preferably not more than 120 ng/ml. Particularly preferably, it is about 100 ng/ml.
  • the concentration of nt3a is, without limitation, for example, not less than 10 ng/ml, preferably not less than 20 ng/ml, more preferably not less than 30 ng/ml, and not more than 100 ng/ml, preferably not more than 80 ng/ml, more preferably not more than 70 ng/ml. Particularly preferably, it is about 50 ng/ml.
  • the concentration of NaB to be added to the medium is, without limitation, for example, not less than 0.2 mM, preferably not less than 0.3 mM, more preferably not less than 0.4 mM, and not more than 0.8 mM, preferably not more than 0.7 mM, more preferably not more than 0.6 mM. Particularly preferably, it is about 0.5 mM.
  • period of NaB addition varies depending on the culture period of step 1, it can be appropriately determined between one day and the whole culture period (e.g., 10 days) .
  • the whole culture period e.g. 10 days
  • the addition period of NaB is, for example, not less than 1 day, preferably not less than 2 days, and not more than 7 days, preferably not more than 6 days.
  • the timing of addition of NaB to a medium can be optionally selected, and NaB can be preferably added to a medium one day after the start of the culture in step 1.
  • NaB may be intermittently added to a medium, it can be preferably added continuously to a medium for a given period.
  • ROCK inhibitor e.g., Y27632 etc.
  • inhibitor may exert an unpreferable influence on the cell, it is desirably removed from the medium after the period
  • ROCK ROCK
  • inhibitor can be removed one day after the start of the
  • novel differentiation induction protocol of the present invention can also be used for inducing
  • pluripotent stem cells such as ES cell into CXCR4 positive endoderm cell.
  • pluripotent stem cells for which the protocol can be used include, but are not limited to, ES cell, embryonic germ (EG) cell derived from primordial germ cell, multipotent germline stem (mGS) cell isolated from
  • testis tissue during the process of establishment and culture of GS cell multipotent adult progenitor cell (MAPC) isolated from the bone marrow and the like.
  • MPC multipotent adult progenitor cell
  • Pluripotent stem cells can be acquired by methods known per se.
  • available methods of preparing ES cells include, but are not limited to, methods in which a mammalian inner cell mass in the blastocyst stage is cultured [see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)] and methods in which an early embryo prepared by somatic cell nuclear transfer is cultured [Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Iritani et al., Protein, Nucleic Acid and Enzyme, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA
  • ES cells can be obtained from various public and private depositories and are commercially available.
  • human ES cell lines HI and H9 can be obtained from WiCell Institute of University of Wisconsin and KhES-1, -2 and -3 can be obtained from Institute for Frontier Medical
  • somatic cells are the same as those used for producing iPS cells mentioned below.
  • the CXCR4 positive endoderm cell obtained as mentioned above can be further induced to differentiate into a cell belonging to each endodermal lineage by a method known per se.
  • differentiation induction into hepatocyte can be performed according to the method described in Hay, D.C. et al., Stem Cells, 26(4): 894-902 (2008); Hay, D.C. et al., Proc. Natl. Acad. Sci. USA, 205(34): 12301-12306 (2008).
  • CXCR4 positive cell can be differentiated up to a
  • differentiation into cell belonging to endodermal lineage other than hepatocyte can be performed by any of the above- mentioned methods selected as appropriate.
  • the present invention also provides a cell population containing a cell belonging to an endodermal lineage, which is derived from blood cell-derived iPS cell and produced by the aforementioned (2) .
  • the cell population may be a purified cell population (e.g., hepatocyte, endocrine pancreas cell etc.), or one or more kinds of other cell types may be co-present. (4) Use of cell belonging to endodermal lineage derived from blood cell-derived iPS cell
  • the thus-established cell belonging to endodermal lineage and derived from blood cell-derived iPS cell can be used for various purposes.
  • a stem cell therapy by endodermal lineage and derived from blood cell-derived iPS cell can be used for various purposes.
  • a stem cell therapy by endodermal lineage and derived from blood cell-derived iPS cell can be used for various purposes.
  • a stem cell therapy by endodermal lineage and derived from blood cell-derived iPS cell can be used for various purposes.
  • a hepatic lineage or pancreatic lineage cell differentiated from an iPS cell derived from a patient's blood cell is considered to better reflect the actual state of the cell of the patient's liver or pancreas than does the corresponding existing cell line of the liver or pancreas, it can also be suitably used for an in vitro evaluation system for the effectiveness and toxicity of a therapeutic drug for a hepatic disease or a pancreatic disease. Moreover, it can be preferably used as a tool for pathological studies of hepatic diseases and
  • pancreatic diseases with unclarified etiology pancreatic diseases with unclarified etiology.
  • the cell belonging to endodermal lineage of the present invention (including a cell belonging to an endodermal
  • parenteral preparation which is preferably injection, suspension, drip infusion and the like, by mixing with a pharmaceutically acceptable carrier and the like according to a conventional means.
  • pharmaceutically acceptable carrier examples include aqueous liquids for injection such as saline, isotonic solution containing glucose and other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium
  • the preparation of the present invention may also be mixed with, for example,
  • buffering agents e.g., phosphate buffer, sodium acetate buffer
  • soothing agents e.g., benzalkonium chloride
  • procaine hydrochloride and the like stabilizers (e.g., human serum albumin, polyethylene glycol and the like) ,
  • cells belonging to an endodermal lineage only need to be suspended in the above- mentioned aqueous liquid at about 1.0*10 6 - about l.OxlO 7
  • the thus-obtained preparation is stable and of low
  • the administration method is not particularly limited, it is preferably injection or drip administration, and intravenous administration, intraarterial administration, intramuscular administration (topical
  • hiPSCs Human iPS cells
  • hESCs human ES Cells
  • Primate ES medium ReproCELL
  • 4 ng/ml recombinant human basic fibroblast growth factor bFGF, WAKO
  • HepG2 and HuH7 human hepatoma cell lines
  • DMEM Dulbecco' s modified Eagle's medium
  • FBS fetal bovine serum
  • 201B2, 201B6 and 201B7 lines were generated by
  • WiCell Institute for Frontier Medical Sciences, Kyoto University, HI and H9 were obtained from WiCell, and ES03, ES04 and ES06 were obtained from Wicell.
  • ES cell medium supplemented with 4 ng/ml bFGF.
  • the medium was replaced with RPMI1640 (Nacalai Tesque) containing 1> ⁇ B27 supplement (Invitrogen) , 100 ng/ml activin A (PeproTech), 50 ng/ml Wnt3a (R&D systems) and 1 mM sodium butyrate (NaB) (Sigma) , and the cells were cultured for 1 day. On the following 2 days, sodium butyrate was omitted from the medium.
  • hiPSCs/hESCs were incubated with Accutase (Innovative Cell
  • santacruz sc-6556 AFP (1:200, DAKO 0008), ALBUMIN (1:200, Bethyl . A80-229A) , AlAT (1:50, Invitrogen 180002), SOX17 (1:300, R&D systems AF1924) and OCT3/4 (1:100, santacruz sc-5279) .
  • differentiated hiPSCs/hESCs were dissociated in 0.25% Trypsin- EDTA (Invitrogen) and then resuspended in 2% FBS/PBS.
  • Periodic acid-Shiff (PAS) staining was performed.
  • the cultured cells were fixed in 3.3% formalin for 10 minutes, and intracellular glycogen was stained using a PAS staining solution (Muto Pure Chemicals), according to the manufacturer's instructions.
  • PAS staining solution Moto Pure Chemicals
  • albumin secretion assay the culture media of differentiated cells after 24 h incubation was collected and measured with the Human Albumin ELISA Quantitation Kit (Bethyl Laboratories Inc.) according to the manufacturer's protocol.
  • EZ DNA MethylationTM Kit (Zymo) according to the manufacturer's protocol.
  • the bisulfite treated DNA was amplified by PyroMark PCR kit (QIAGEN) .
  • Pyrosequencing analysis was performed by PyroMarkQ96 ID system (QIAGEN) according to standard procedures. Genomic DNAs of human heart, liver and brain were purchased from BioChain.
  • Step 1 undifferentiated hiPSCs/hESCs grown until reaching nearly 70% confluence were cultivated in serum-free medium supplemented with 100 ng/ml Activin A, 50 ng/ml nt3a and 1 mM sodium butyrate (NaB) toward endoderm lineage for 3 days (NaB was removed from the medium for the last two days) ;
  • Step 2 the cells were cultured in the differentiation medium containing 1% dimethyl sulfoxide (DMSO) and 20% knockout serum replacement (KSR) for 7 days; and
  • DMSO dimethyl sulfoxide
  • KSR knockout serum replacement
  • Step 3 medium was changed to the maturation medium supplemented with 20 ng/ml oncostatin M (OSM) and 20 ng/ml hepatocyte growth factor (HGF) for further maturation for another 7 days.
  • OSM oncostatin M
  • HGF hepatocyte growth factor
  • hiPS clones which had generated from the same fibroblast culture of a single individual donor and two hES clones.
  • Three hiPS clones (201B2, B6 and B7) were generated with retroviral transduction of four reprogramming factors (OCT3/4, SOX2, KLF4 and C-MYC) to adult human dermal fibroblasts (aHDFs) and two clones (253G1 and G4) with three factors devoid of C-MYC to the same aHDFs.
  • Two hESC lines KhESl and 3) established in Japan were used as control.
  • hepatocyte-like cell morphology large cuboidal cell shape with prominent compact nuclei, were observed in 3 clones (201B2, 201B6 and KhES3) . Some binuclear cells indicating mature hepatic phenotype were also found in those clones.
  • cytometry analysis also revealed that the percentage of albumin-positive cells were higher in morphologically good- hepatic-differentiating clones (201B2, 201B6 and KhES3) than poor-hepatic-differentiating clones (201B7, 253G1, 253G4 and KhESl) (Fig. 2A) .
  • RT-PCR Reverse transcription PCR
  • markers including TAT (tyrosine aminotransferase) , TD02
  • Undifferentiated marker OCT3/4 was downregulated in all clones (Fig. 2B) .
  • cytochrome P450 enzymes, which play central roles in drug metabolism in human liver, indicating mature phenotype of hepatocytes.
  • CYP450 cytochrome P450 enzymes, which play central roles in drug metabolism in human liver, indicating mature phenotype of hepatocytes.
  • CYP450 mRNA expression such as CYP1A1, CYP2C9, CYP2C19, CYP2D6 CYP3A4 and CYP7A1.
  • some mature hepatocyte markers ABCC2(MRP2) and ABCB11 (MDR/TAP) ; ATP-binding cassette (ABC) transporter supporting bile acid export in apical surface of the
  • differentiated cells expressed liver-related markers HNF4A at day 10, and AFP, ALBUMIN and A1AT at day 17.
  • hiPSC/hESC colonies are dissociated with Accutase into single cells by pipetting and seeded on the Matrigel-coated culture dish with serum-free medium containing 100 ng/ml Activin A and 50 ng/ml Wnt3a.
  • 10 ⁇ g/ml ROCK inhibitor (Y27632) was added to the culture medium for the first day.
  • endoderm differentiation period was set for 7 days. Because NaB has been reported to be important for endoderm differentiation of hESCs, we added NaB to the culture medium from day 1. Addition of 1 mM NaB showed significant cell death. Accordingly, we set the concentration of NaB at 0.5 mM and varied the
  • Peripheral blood-derived hiPSCs favor hepatic differentiation With this new protocol efficiently generating CXCR4- positive endoderm cells, we compared endoderm and hepatic differentiation propensity among various hiPSC lines with different type of donor cell origin, method of iPS generation or combination of reprogramming factors.
  • aHDF-iPSC aHDF-iPSC
  • DP- iPSC dental pulp cells
  • PB-iPS peripheral blood cells
  • CB-.iPS cord blood cells
  • NaB requirement for generating CXCR4-positive endoderm cells at day 7 was quite different among the original cell types. In nearly half of PB-iPS and CB-iPS clones, NaB
  • NaB was dispensable for endodermal and hepatic differentiation in PB-iPS and CB-iPS clones, whereas 3 or 6 days of NaB
  • HESC lines showed various NaB requirements (Fig. 7A and Fig. 8) .
  • DP-iPSC lines did not respond to this differentiation protocol due to significant cell death or poor cell growth.
  • PB-iPS clones showed significantly high level of albumin secretion compared to aHDF-iPS, DP-iPS and hES clones (Fig. 7B) , suggesting that peripheral blood can be suitable donor cell candidate for generating iPSCs aimed to hepatocyte differentiation.

Abstract

La présente invention concerne un procédé de production d'une cellule appartenant au lignage endodermique, comprenant l'induction de la différenciation d'une cellule iPS issue d'une cellule sanguine, en particulier un procédé de production d'une cellule appartenant à un lignage endodermique, comprenant les étapes suivantes (1) et (2) : (1) une étape de production d'une cellule iPS par la mise en contact d'une substance de reprogrammation nucléaire avec une cellule sanguine, (2) une étape d'induction de la différenciation de la cellule iPS obtenue dans l'étape (1) en une cellule appartenant à un lignage endodermique. De plus, la présente invention concerne un procédé d'induction de la différenciation d'une cellule souche pluripotente humaine en une cellule endodermique positive pour CXCR4, comprenant (1) la mise en culture d'une cellule souche pluripotente humaine dans un état de cellule isolée en présence de l'activine A et de Wnt3a pendant 4-10 jours et (2) la mise en culture en coprésence de 0,1-0,8 mM de butyrate de sodium pendant 0-10 jours au cours de la période de culture.
PCT/JP2012/078407 2011-10-28 2012-10-26 Procédé pour l'induction efficace de la différenciation de cellules souches pluripotentes en des cellules du lignage hépatique WO2013062140A1 (fr)

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