WO2009096049A1 - Cellules différenciées ayant pour origine des cellules souches pluripotentes artificielles - Google Patents

Cellules différenciées ayant pour origine des cellules souches pluripotentes artificielles Download PDF

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WO2009096049A1
WO2009096049A1 PCT/JP2008/059590 JP2008059590W WO2009096049A1 WO 2009096049 A1 WO2009096049 A1 WO 2009096049A1 JP 2008059590 W JP2008059590 W JP 2008059590W WO 2009096049 A1 WO2009096049 A1 WO 2009096049A1
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cells
ips
cell
gene
pluripotent stem
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Shinya Yamanaka
Takashi Aoi
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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
    • 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
    • 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
    • C12N2501/604—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to cells and tissues produced by inducing differentiation from induced pluripotent stem cells obtained by reprogramming differentiated somatic cells.
  • Embryonic stem cells are stem cells established from early embryos of humans and mice, and can be cultured over a long period of time while maintaining the pluripotency that can differentiate into all cells present in the living body. Have. Using this property, human ES cells are expected as a resource for cell transplantation for many diseases such as Parkinson's disease, juvenile diabetes and leukemia. However, transplantation of ES cells has the problem of causing rejection similar to organ transplantation. There are also many disagreements from the ethical point of view regarding the use of ES cells established by destroying human embryos.
  • ES-like cells induced pluripotent stem cells
  • iPS cell induced pluripotent stem cells
  • ES-like cells an ideal without rejection or ethical problems It is expected that it can be used as a typical pluripotent cell.
  • An object of the present invention is to provide means for reducing or eliminating the risk of tumor development in tissues and individuals obtained by inducing differentiation of induced pluripotent stem cells produced by reprogramming somatic cells.
  • the present inventors have found that tumors in tissues and individuals produced by inducing differentiation of induced pluripotent stem cells obtained using hepatocytes or gastric epithelial cells. We found that the incidence was significantly lower.
  • the present invention has been completed based on the above findings.
  • the present invention provides a cell, tissue, organ, or individual obtained by inducing differentiation of an induced pluripotent stem cell obtained by nuclear reprogramming of a hepatocyte or gastric epithelial cell.
  • the nuclear reprogramming factor is a single substance or a combination of a plurality of substances that is positive by the nuclear reprogramming factor screening method described in International Publication WO ⁇ 2005/80598.
  • Cell, tissue, organ, or individual gene product of a single gene or a gene product of multiple genes whose nuclear reprogramming factor is positive by the screening method for nuclear reprogramming factor described in International Publication WO ⁇ 2005/80598.
  • the cell, tissue, organ, or individual that is a combination; the cell, tissue, organ, or individual that performs nuclear reprogramming by nuclear reprogramming factor by introducing the gene into the somatic cell; the gene into the somatic cell Cells, tissues, organs or individuals as described above, which are introduced by a recombinant vector, preferably a viral vector, more preferably a retroviral vector;
  • a recombinant vector preferably a viral vector, more preferably a retroviral vector
  • the gene encoding the reprogramming factor is at least one selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene.
  • a combination of two genes selected from genes excluding the Myc family preferably a combination of three genes, more preferably a combination of four genes, and particularly preferably a combination of four or more genes.
  • An organ or an individual is provided.
  • a more preferred combination is (a) a combination of two genes consisting of an Oct family gene and a Sox family gene; (b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene; ) A combination of four genes consisting of Oct family gene, Sox family gene, Lin family gene, and Nanog gene. Furthermore, it is also preferable to combine the TERT gene and / or the SV40 Large T antigen gene. In some cases, it may be preferable to remove the Klf family gene. In some cases, these combinations may include a Myc family gene, but in the present invention, a combination not including a Myc family gene can be preferably used.
  • particularly preferred combinations are combinations of two genes consisting of Oct3 / 4 and Sox2; combinations of three genes consisting of Oct3 / 4, Klf4, and Sox2; and Oct3 / 4, Sox2, Lin28, and It is a combination of four kinds of genes consisting of Nanog, and it is also preferable to combine these with the TERT gene and / or SV40 Large T antigen gene. In some cases, it may be preferable to remove Klf4. In some cases, c-Myc can be combined with these combinations, but in the present invention, a combination not containing c-Myc can be preferably used.
  • the somatic cell is a hepatocyte or gastric epithelial cell of a mammal including a human, preferably a human or mouse hepatocyte or gastric epithelial cell, particularly preferably a human hepatocyte or gastric epithelial cell.
  • a human preferably a human or mouse hepatocyte or gastric epithelial cell, particularly preferably a human hepatocyte or gastric epithelial cell.
  • the above-mentioned cells, tissues, organs or individuals as described above, wherein the somatic cells are hepatocytes or gastric epithelial cells derived from adult humans; the hepatocytes or stomach collected from patients as the somatic cells;
  • Provided is the above cell, tissue, organ or individual that is an epithelial cell; the above cell, tissue, organ or individual in which the occurrence of a tumor is substantially reduced or eliminated.
  • Another aspect of the present invention is a method for reducing or eliminating tumor development in a cell, tissue, organ, or individual that has been induced to differentiate from an induced pluripotent stem cell, comprising reprogramming nuclear cells of hepatocytes or gastric epithelial cells.
  • a method comprising the step of inducing differentiation of the induced pluripotent stem cell obtained by the above is provided by the present invention.
  • stem cell therapy is performed, and cells, tissues, or organs obtained by inducing differentiation of induced pluripotent stem cells obtained from hepatocytes or gastric epithelial cell vesicles isolated and collected from a patient are transplanted into the patient.
  • a therapy comprising the steps of:
  • a method for evaluating the physiological action and toxicity of compounds, drugs, toxicants, etc. using cells, tissues, or organs obtained by inducing differentiation of induced pluripotent stem cells obtained from hepatocytes or gastric epithelial cell vesicles are also provided by the present invention.
  • clones labeled A EGF and HGF were added to the serum-free medium.
  • clones labeled B used 10% serum medium and did not use EGF or HGF.
  • a primer set that amplifies only the endogenous transcript (endo) was used.
  • NAT1 was used (Genes Dev., 11, 321, 1997).
  • PCR was also performed on a template without reverse transcription (RT-) for Sox2. It is the figure which showed the transfection efficiency by a retrovirus, and iPS cell induction
  • EGFP Enhanced Green Fluorecent Protein
  • pMXs-EGFP Enhanced Green Fluorecent Protein
  • control virus mock
  • EGFP Enhanced Green Fluorecent Protein
  • pMXs-EGFP Enhanced Green Fluorecent Protein
  • mock control virus
  • b) shows transfection efficiency and iPS induction by retroviruses in MEF. The top row is seeded with 100,000 MEF cells per well of a gelatin-coated 6-well plate, infected the next day with a 10-fold serial dilution of EGFP-expressing retrovirus, and transfection efficiency was determined by flow cytometry 48 hours later. Results (average and standard deviation of 3 independent experiments) are shown.
  • the bottom row covers 100,000 Fbx15-reporter MEF cells per well of a 6-well plate with STO feeder cells and contains 10-fold serial dilutions of retrovirus (Oct3 / 4, Sox2, c-Myc, and Klf4) 2), and 2 days after infection, cells were selected with 0.3 mg / ml G418 for 12 days (average value and standard deviation of the number of colonies of iPS cells in three independent experiments). It is the figure which showed the characteristic of the adult mouse
  • iPS-Hep cells (clone 92A-3, -5, and -6) and ES cells.
  • b) shows RT-PCR analysis results of ES marker gene expression in iPS-Stm cells and ES cells. For Oct3 / 4 and Sox2, primer sets that only amplify endogenous transcription were used (endo). The morphology of clone iPS-Stm-99-4 was different from that of ES cells.
  • c) Results of bisulfite genome sequencing of the promoter regions of Oct3 / 4, Nanog, and Fbx15 in iPS cells (iPS-Hep-98A-2 and iPS-Stm-99-1), ES cells, and hepatocytes Show.
  • iPS-Hep-98A2 cells and iPS-Stm-99-1 cells were microinjected into C57BL / 6 blastocysts and transplanted into pseudopregnant female mice.
  • the iPS cell portion can be identified by dark gray hair.
  • b) shows iPS-Stm cell germline transmission. Male chimeric mice derived from iPS-Stm-99-1 cells were mated with C57BL / 6 female mice. The fluorescence photograph of F1 mouse
  • the position of the RIS on the chromosome in two iPS-Stm clones (99-1 and -3) and two iPS-Hep clones (98A-1 and -2) are shown. It is the figure which showed the presumed function of the gene which received retrovirus insertion.
  • the dark gray arrow indicates the insertion site of 4 factors.
  • the light gray arrow indicates the translation start site.
  • Black arrows indicate the transcription start site and gene direction.
  • the scale bar indicates the size (bp) of the gene, and the gene abbreviation is an abbreviation in UCSC and the ensemble database.
  • Three cases (Oct3 / 4 in Stm99-1, c-Myc in Stm99-3, and c-Myc in Hep98A-2) detected more RIS than the number of bands detected in FIG. 7 (these In some cases, the band may be too large or too small to be detectable by Southern blotting, or there may be band overlap).
  • FIG. 1 It is a schematic diagram of a retrovirus insertion site. It is a schematic diagram of a retrovirus insertion site. It is the figure which showed the influence at the time of remove
  • the present invention is a cell, tissue, organ, or individual obtained by inducing differentiation of an induced pluripotent stem cell, and the induced pluripotent stem cell is obtained by nuclear reprogramming of hepatocytes or gastric epithelial cells.
  • the induced pluripotent stem cell is obtained by nuclear reprogramming of hepatocytes or gastric epithelial cells.
  • somatic cell that can be obtained by inducing differentiation of an induced pluripotent stem cell according to the present invention is not particularly limited, and any somatic cell can be prepared, and any tissue or organ can be prepared.
  • Artificial pluripotent stem cells have pluripotency, and for example, means for inducing differentiation into specific somatic cells, tissues, or organs known for embryonic stem cells can be appropriately employed.
  • nerve cells For example, nerve cells, cardiomyocytes, bone marrow cells, insulin-producing cells, blood cells, etc., nerve tissue, cornea, retina, lens, muscle, skin, bone, blood vessel, lymphatic vessel, lymph node, or lymph node tissue, or heart, Arbitrary somatic cells, tissues, or organs can be produced, including organs such as kidney, pancreas, liver, stomach, large intestine, small intestine, esophagus, gallbladder, or lung.
  • a complete individual can be generated from the induced pluripotent stem cell, and this embodiment is also encompassed in the “induction of differentiation of induced pluripotent stem cell” in the present invention (except for a human individual). .
  • a screening method for a nuclear reprogramming factor described in International Publication WO 2005/80598 can be used.
  • the entire disclosures of the above publications are incorporated herein by reference.
  • Those skilled in the art can screen for nuclear reprogramming factors by referring to the above-mentioned publications and use them in the present invention.
  • the nuclear reprogramming factor can be confirmed using a method in which appropriate modification or alteration is added to the above screening method.
  • a gene encoding a reprogramming factor that can be used in the present invention, one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene
  • one or more genes selected from the group consisting of the Oct family gene, Klf family gene, Sox family gene, Lin family gene, and Nanog gene excluding the Myc family gene more preferably two types
  • a combination of genes more preferably a combination of three genes, particularly preferably a combination of four genes can be mentioned.
  • an initialization factor encoded by one or more genes selected from the group consisting of the Oct family gene, the Klf family gene, the Sox family gene, the Myc family gene, the Lin family gene, and the Nanog gene is replaced with, for example, a cytokine. Or may be replaced with one or more other low molecular weight compounds.
  • a low molecular weight compound for example, an action of promoting the expression of one or more genes selected from the group consisting of Oct family gene, Klf family gene, Sox family gene, Myc family gene, Lin family gene, and Nanog gene
  • a low molecular weight compound having the above can be used, but such a low molecular weight compound can be easily screened by those skilled in the art.
  • genes are genes that exist in common in mammals including humans, and in order to use the genes in the present invention, they are derived from any mammal (for example, human, mouse, rat, cow, sheep, Genes derived from mammals such as horses and monkeys) can be used.
  • any mammal for example, human, mouse, rat, cow, sheep, Genes derived from mammals such as horses and monkeys
  • the wild-type gene product several (eg, 1 to 10, preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2)
  • a gene product having a function similar to that of a wild-type gene product which is a mutant gene product in which the amino acid is substituted, inserted, and / or deleted.
  • the c-Myc gene product may be a wild type or a stable type (T58A). The same applies to other gene products.
  • genes encoding factors that induce cell immortalization may be further combined.
  • a combination of four genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, and a TERT gene (e) a combination of four genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, and a TERT gene; (f) A combination of four genes consisting of Oct family gene, Klf family gene, Sox family gene, and SV40 Large T antigen gene; (g) A combination of five genes consisting of Oct family gene, Klf family gene, Sox family gene, TERT gene, and SV40 Large T antigen gene can be mentioned. If necessary, the Myc family gene can be combined with these, and the Klf family gene can be excluded from the above combination.
  • one or more genes selected from the group consisting of Fbx15, ERas, ECAT15-2, Tcl1, and ⁇ -catenin may be combined, and / or ECAT1, Esg1, Dnmt3L, ECAT8
  • One or more genes selected from the group consisting of Gdf3, Sox15, ECAT15-1, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, and Grb2 can also be combined. These combinations are specifically described in International Publication WO2007 / 69666.
  • Particularly preferred gene combinations are: (1) a combination of two genes consisting of Oct3 / 4 and Sox2; (2) A combination of three genes consisting of Oct3 / 4, Klf4, and Sox2; (3) a combination of four genes consisting of Oct3 / 4, Sox2, Lin28, and Nanog; (4) A combination of four genes consisting of Oct3 / 4, Sox2, TERT, and SV40 Large T antigen; (5) A combination of five genes consisting of Oct3 / 4, Klf4, Sox2, TERT, and SV40 Large T antigen, but is not limited thereto. You can combine them with c-Myc as needed.
  • the factor containing the above gene product is combined with one or more gene products selected from the group consisting of the following groups: Fbx15, Nanog, ERas, ECAT15-2, Tcl1, and ⁇ -catenin May be. Further, for example, one or more genes selected from the group consisting of the following groups: ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, and Grb2. It can also be combined with gene products. These gene products are disclosed in International Publication WO2007 / 69666.
  • the gene products that can be included in the nuclear reprogramming factor of the present invention are not limited to the gene products of the genes specifically described above.
  • the nuclear reprogramming factor of the present invention includes, in addition to other gene products that can function as a nuclear reprogramming factor, 1 or 2 factors related to differentiation, development, or proliferation, or other factors having physiological activity. Needless to say, such embodiments can be included, and such embodiments are also included in the scope of the present invention.
  • the gene product of one or more of these genes can be excluded from the factors to be introduced.
  • one or more genes other than the already expressed gene can be introduced into a somatic cell by an appropriate gene introduction method, for example, a method using a recombinant vector.
  • an appropriate gene introduction method for example, a method using a recombinant vector.
  • the remaining one or more genes are appropriately selected.
  • the gene can be introduced by, for example, a method using a recombinant vector.
  • the gene product that is a nuclear reprogramming factor may be, for example, in the form of a fusion gene product of the protein and other proteins or peptides in addition to the protein itself produced from the above gene.
  • a fusion gene product with a peptide such as a fusion protein with green fluorescent protein (GFP) or a histidine tag can also be used.
  • GFP green fluorescent protein
  • a histidine tag can also be used.
  • GFP green fluorescent protein
  • TAT peptide derived from HIV virus it is possible to promote intracellular uptake of nuclear reprogramming factor from the cell membrane, avoiding complicated operations such as gene transfer. It is possible to induce reprogramming simply by adding the fusion protein to the medium. Since methods for preparing such fusion gene products are well known to those skilled in the art, those skilled in the art can easily design and prepare appropriate fusion gene products according to the purpose.
  • an “artificial pluripotent stem cell” is a cell having properties close to those of an ES cell, and more specifically, an undifferentiated cell that has been initialized from a somatic cell. Cell having ability and proliferative ability, but the term should not be interpreted in a limited way in any way, but in the broadest sense.
  • a method for preparing induced pluripotent stem cells using a nuclear reprogramming factor is described in International Publication WO2005 / 80598 (in the above publication, the term ES-like cells is used), and induced pluripotent stem cells The separation means is also specifically described.
  • International publication WO2007 / 69666 discloses specific examples of reprogramming factors and specific examples of somatic cell reprogramming methods using the reprogramming factors. Therefore, it is desirable for those skilled in the art to refer to these publications when practicing the present invention.
  • the method for preparing an induced pluripotent stem cell from a somatic cell by the method of the present invention is not particularly limited, and a method capable of nuclear reprogramming of a somatic cell with a nuclear reprogramming factor in an environment where the somatic cell and the induced pluripotent stem cell can proliferate Any method may be adopted as long as it is.
  • a method capable of nuclear reprogramming of a somatic cell with a nuclear reprogramming factor in an environment where the somatic cell and the induced pluripotent stem cell can proliferate Any method may be adopted as long as it is.
  • means such as introducing a gene into a somatic cell using a vector containing a gene capable of expressing a nuclear reprogramming factor may be employed.
  • two or more genes may be incorporated into the vector and the respective gene products may be expressed simultaneously in somatic cells.
  • the expression vector When a gene is introduced into a somatic cell using a vector capable of expressing the above gene, the expression vector may be introduced into the somatic cell cultured on the feeder cell, but the expression vector only in the somatic cell. You may introduce. The latter method may be suitable for increasing the efficiency of introducing the expression vector.
  • feeder cells feeder cells used for culturing embryonic stem cells can be used as appropriate. For example, mouse 14-15 day embryonic fibroblast primary cultured cells, fibroblast-derived cell lines such as STO cells, etc. Cells treated with a drug such as mitomycin C or cells treated with radiation can be used.
  • nuclear reprogramming progresses autonomously, and artificial pluripotent stem cells can be produced from hepatocytes or gastric epithelial cells.
  • the step of obtaining an induced pluripotent stem cell by introducing it into a somatic cell using a vector expressing a gene encoding a nuclear reprogramming factor can be performed according to a method using a retrovirus, for example, Cell, 126 , Pp.1-14, 2006; Cell, 131, pp.1-12, 2007; Science, 318, pp.1917-1920, 2007 and the like.
  • the culture density of the cells after introduction of the expression vector lower than in the case of normal animal cell culture. For example, it is preferable to continue the culture at a cell density of about 100,000 to 100,000, preferably about 50,000 per cell culture dish.
  • the medium used for the culture is not particularly limited and can be appropriately selected by those skilled in the art.
  • the above publications can also be referred to for the selection of culture media and culture conditions.
  • the generated induced pluripotent stem cells can be confirmed with various markers peculiar to undifferentiated cells, and the means thereof are described specifically and in detail in the above-mentioned publications.
  • Various media that can maintain the undifferentiation and pluripotency of ES cells or media that cannot maintain the properties thereof are known in the art.
  • artificial pluripotent stem cells can be separated efficiently.
  • the differentiation ability and proliferation ability of the separated induced pluripotent stem cells can be easily confirmed by those skilled in the art by using confirmation means widely used for ES cells.
  • a colony of induced pluripotent stem cells can be obtained, and the presence of the induced pluripotent stem cell can be specified from the shape of the colony.
  • mouse induced pluripotent stem cells form raised colonies
  • human induced pluripotent stem cells are known to form flat colonies
  • these colony shapes are mouse ES cells and Since it is very similar to a colony of human ES cells, those skilled in the art can identify induced pluripotent stem cells generated from the colony shape.
  • hepatocytes or gastric epithelial cells to be initialized is not particularly limited.
  • cells derived from any mammal eg, mammals such as human, mouse, rat, cow, sheep, horse, monkey
  • hepatocytes or gastric epithelial cells in mature individuals may be used.
  • hepatocytes or gastric epithelial cells isolated and collected from adult human individuals can be used.
  • Hepatocytes and gastric epithelial cells can be separated and collected by biopsy or surgery, and gastric epithelial cells can also be collected by, for example, endoscopy.
  • hepatocytes or gastric epithelial cells isolated from patients When artificial pluripotent stem cells are used for treatment of diseases, it is desirable to use hepatocytes or gastric epithelial cells isolated from patients. For example, when stem cell transplantation is performed for diseases such as heart failure, insulin-dependent diabetes, Parkinson's disease, or spinal cord injury, it is preferable to use hepatocytes or gastric epithelial cells isolated and collected from patients.
  • Example 1 induced pluripotent stem cells are abbreviated as iPS cells.
  • iPS cells induced pluripotent stem cells.
  • Example 1 ⁇ Method> 1. Primary culture of adult mouse hepatocytes and gastric epithelial cells Separated mouse hepatocytes were isolated by a two-stage collagenase perfusion method, and hepatocytes were isolated by literature methods (Hepatology, 31, 65, 2000). The stomach of the mouse was excised and incised, washed with PBS and divided into small pieces. The glandular gastric mucosa was minced and digested for 60 minutes with shaking at 37 ° C.
  • bovine serum albumin 2.0 g / L, glucose 2.0 g / L, galactose 2.0 g / l, ornithine 0.1 g / L, proline 0.030 g / L, nicotinamide 0.610 g / L, ZnCl 2 0.025 mg / L, ZnSO 4 ⁇ 7H 2 O 0.750 mg / l, CuSO 4 ⁇ 5H 2 O 0.20 mg / l, MnSO 4 0.025 mg / l, Glutamine 5.0 mM, Insulin 5.0 mg / l, Human transferrin 5.0 mg / l , Selenium 5.0 ⁇ g / l, dexamethasone 10 ⁇ 7 M, penicillin 100 mg / l
  • Retroviruses derived from pMXs were prepared for hepatocytes and gastric epithelial cells with some modifications to the methods described in the literature (Cell, 126, 663, 2006).
  • PLAT-E cells were spread at a rate of 8 ⁇ 10 6 cells per 100 mm plate in DMEM medium containing 10% FBS.
  • 9 ⁇ g of retroviral vector was introduced into PLAT-E cells using 27 ⁇ l of FuGENE6 transfection reagent (Roche). After 24 hours, the medium was replaced with 10 ml of the above basal chemically defined medium.
  • the virus-containing supernatant from the PLAT-E cell culture was collected and filtered through a 0.45 ⁇ m cellulose acetate filter (Fatman).
  • Induction of iPS cells Induction of iPS cells from primary cultures of hepatocytes and gastric epithelial cells was performed by slightly modifying the methods described in the literature (Cell, 126, 663, 2006). Hepatocytes and gastric epithelial cells were isolated and cultured on feeder cells, and the above-described pMXs-Oct3 / 4, -Sox2, -c-Myc, and -Klf4 were introduced.
  • Retrovirus introduction site in iPS cells was performed by inverse polymerase chain reaction (IPCR) with some modifications to the method described in the literature (Nat. Genet., 23, 348, 1999; Proc. Natl. Acad. Sci. USA, 93, 2414, 1996). That is, genomic DNA from iPS cells was digested using restriction enzymes TaqI and HpyCH4IV (New England Biolabs), respectively, to obtain a fragment containing the junction between the retroviral vector and the flanking sequence. The fragment digested with the restriction enzyme was subjected to a ligation reaction at 16 ° C. for 30 minutes using a half amount of Ligation High (Toyobo).
  • IPCR inverse polymerase chain reaction
  • a vector end containing a flanking sequence was amplified by PCR using EX taq polymerase (Takara). 5'-AGGAACTGCTTACCACA-3 'and 5'-CTGTTCCTTGGGAGGGT-3' were used as primers for the first PCR, and 5'-TCCTGACCTTGATCTGA-3 'and 5'-CTGAGTGATTGACTACC-3' were used for the second PCR. .
  • the PCT product was purified on a gel and directly sequenced with an ABI3130 DNA sequencer (Applied Biosystems). If direct sequencing was not possible, the PCR product was subcloned into pCR2.1 using TOPO TA cloning (Invitrogen) and sequenced again. To confirm which of the four factors are present at each virus insertion site, a primer designed from the corresponding flanking sequence and another one designed from the coding sequence of one of the four factors Chromosomal fragments were amplified by PCR using primers.
  • SIPCR selective inverse PCR
  • Oct3 / 4R1 (5'-CTGAAGGTTCTCATTGTTGTCG-3 '), Sox2R1 (5'-AGTGGGAGGAAGAGGTAACCAC-3'), c-MycR1 (5'-TTCTTGCTCTTCTTCAGAGTCG-3 '), or Klf4R1 was used.
  • PMXsRF1 (5'-TCCAATAAACCCTCTTGCAGTT-3 ') is used as a common forward primer in the second PCR, and Oct3 / 4R2 (5'-AGGTGATCCTCTTCTGCTTCAG-3'), Sox2R2 (5'- CTGCGAGTAGGACATGCTGTAG-3 ′), c-mycR2 (5′-AATCGGACGAGGTACAGGATTT-3 ′), or Klf4R2 (5′-GCAGATTCTCGGCTGTAGAGGA-3 ′) was used. The sequence was determined directly or after subcloning into pCR2.1.
  • the signal was detected with an Immobilon Western Chemiluminescent Luminescent HRP substrate (Millipore) and a LAS3000 imaging device (Fuji Film).
  • Immobilon Western Chemiluminescent Luminescent HRP substrate Millipore
  • LAS3000 imaging device Fuji Film
  • anti-Sox2 antiserum J. Biol.
  • anti-Oct3 / 4 antibody (sc-5279, Santa Cruz), anti-Klf4 antibody (sc-20691) , Santa Cruz), anti-c-Myc antibody (sc-764, Santa Cruz), anti-E-cadherin antibody (610182, BD Bioscience), anti- ⁇ -catenin antibody (sc-7199, Santa Cruz), anti- ⁇ - Actin antibody (A5441, Sigma), anti-mouse IgG-HRP antibody (# 7076, cell signaling), and anti-rabbit IgG-HRP antibody (# 7074, cell signaling) were used.
  • IPS cells were established from epithelial cells. Primary cells from hepatocytes and gastric epithelial cells from mice in which ⁇ -geo gene (a fusion gene of ⁇ -galactosidase and neomycin resistance gene) was knocked in at the Fbx15 locus (Mol. Cell Biol., 23, 2699, 2003) 1a) was isolated. The Fbx15 gene is specifically expressed in ES cells and preimplantation embryos. IPS cells derived from fibroblasts selected by the Fbx15 reporter (mouse embryonic fibroblasts [MEF] or caudal fibroblasts [TTF]) are ES cells in gene expression, DNA methylation pattern, and chimera formation. (Cell, 126, 663, 2006).
  • Fbx15 reporter mouse embryonic fibroblasts [MEF] or caudal fibroblasts [TTF]
  • iPS-Hep iPS-Hepatic
  • iPS-Stm iPS-Stomach
  • iPS-Hep cells and iPS-Stm cells expressed ES cell marker genes such as Nanog, Rex1, ECAT1, Cripto, and Gdf3 to the same extent as ES cells.
  • ES cell marker genes such as Nanog, Rex1, ECAT1, Cripto, and Gdf3 to the same extent as ES cells.
  • TPS-derived iPS cells (iPS-TTF) selected by the Fbx15 reporter had a low expression level of ES cell marker gene (FIG. 1c).
  • iPS-Hep cells and iPS-Stm cells are more ES cells than iPS-TTF cells, despite being selected by Fbx15 gene expression. It was very similar. Therefore, iPS-Hep cells and iPS-Stm cells (1 ⁇ 10 6 cells) were transplanted subcutaneously into the posterior abdomen of nude mice (Table 1).
  • iPS-HepPS cells and iPS-Stm cells have pluripotency.
  • iPS-Hep ⁇ clones derived from Fbx15 ⁇ reporter mice and 6 ⁇ iPS-Stm ⁇ clones were used for injection. Furthermore, the injections were similarly performed on clones of 5 types of iPS-Hep cells selected by expression of the Nanog gene. Most of these clones had the GFP transgene expressed by the constitutively activated CAG promoter (Gene, 108, 193, 1991). In addition, one iPS-Hep cell clone selected only by morphology without a selectable marker (A.
  • iPS-Hep cells selected from the expression of Fbx15 gene (derived from 21-week-old mice) and 2 clones of iPS-Stm cells (derived from 12-week-old mice) were introduced into the germline. This was confirmed by the expression of GFP and the presence of the transgene (FIG. 5b).
  • iPS-fibroblasts derived from fibroblasts (MEF) or TTF) selected by expression of Nanog gene become adult or germline chimeras, and in iPS-fibroblast selected by Fbx15 Contrast that they were not obtained (Cell, 126, 663, 2006).
  • mice derived from iPS-Hep cells, iPS-Stm cells, or iPS-MEF cells 46 chimeric adult mice were obtained from iPS-MEF clones of independent 10 clones among the 12 clones injected. Of these chimeric mice, approximately 30% developed tumors by 30 weeks of age (FIG. 6a). On the other hand, in the 65 chimera adult chimeric mice obtained from 12 clones of iPS-Hep sputum cells and iPS-Stm sputum cells, no tumor was observed during this period.
  • chimeric mice derived from iPS-Hep cells and iPS-Stm cells had higher perinatal mortality compared to non-chimeric mice (Fig. 6b).
  • Such high mortality during the perinatal period was not observed in chimeric mice derived from iPS-MEF sputum cells.
  • Necropsy was performed on the dead mice, but the macroscopic appearance was normal and the cause of death was unknown. Although considered common in cloned animals (Nat. N Genet., 39, 295, 2007), some epigenetic anomalies may cause perinatal mortality. On the other hand, there was no increase in mortality in mice that were 1 day old (Fig. 6a).
  • RIS retrovirus insertion sites
  • iPS-Hep cells and iPS-Stm cells differ from iPS-fibroblast in three properties.
  • iPS-Hep cells and iPS-Stm cells contributed to the formation of adult chimeras despite being selected by the expression of the Fbx15 gene.
  • the third difference between iPS-Hep cells and iPS-Stm cells and iPS-MEF and RRF cells is that the RIS of the former two iPS cells is less than that of the latter iPS cells.
  • the expression level of 4 transcription factors by retrovirus was higher in hepatocytes than in fibroblasts (Fig. 16). This fact can explain why, at least in part, there is less RIS in iPS-Hep cells.
  • ES cells have been shown to have epithelial characteristics such as close contact between cells and E- cadherin expression on the cell surface (Mol. Biol. Cell., 18, 2838, 2007).
  • iPS-Hep cells In order to investigate the origin of iPS-Hep cells, we performed a cell lineage tracking analysis using a genetic technique (Fig. 17a).
  • the GFP gene and the puromycin resistance gene are knocked into the Nanog gene for selection of iPS cells.
  • a transgenic mouse that expresses Crel recombinase by this mouse and albumin promoter (J. Biol. Chem. , 274, 305, 1999), and then transgenic mice expressing the loxP-CAT-loxP- ⁇ -gal ( ⁇ - galactosidase) cassette under a constitutively activated promoter (Biochem. Biophys. Res. Commun., 237, 318, 1997).
  • ⁇ -gal activity is induced as the albumin gene is activated, and ⁇ -gal activity persists even if the expression of the albumin gene is stopped.
  • Primary hepatocytes were isolated from these mice, and 4 types of factors were introduced to prepare iPS cells.
  • iPS-Hep cells are derived from hepatocytes or other albumin-expressing cells and derived from undifferentiated cells that do not express albumin I knew it wasn't. Some GFP -positive and ⁇ -gal -negative colonies were also found, but they originated from albumin-negative cells that were mixed in the primary culture of hepatocytes, or simply reflected incomplete recombination with Cre recombinase. It is thought that there is.
  • the present invention provides a means for reducing or eliminating the risk of tumor development in cells, tissues, organs, etc. obtained by inducing differentiation of induced pluripotent stem cells produced by reprogramming somatic cells.
  • pluripotent stem cells By using pluripotent stem cells, it is possible to easily obtain safe cells, tissues, organs or the like in which the risk of tumor development is reduced or eliminated.

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Abstract

La présente invention concerne des cellules, des tissus, un organe ou un individu présentant très peu de risque, obtenus par le biais de l'induction de la différenciation de cellules souches pluripotentes artificielles, qui ont été obtenues par l'initialisation du noyau de cellules hépatiques ou de cellules épithéliales gastriques, et ne présentent pas de risque de tumorigénèse ou un risque réduit.
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WO2011122318A1 (fr) * 2010-03-31 2011-10-06 学校法人東京女子医科大学 Matériau de greffe thérapeutique et son procédé d'utilisation
WO2011121636A1 (fr) * 2010-03-29 2011-10-06 国立大学法人熊本大学 Procédé de production de cellules souches pluripotentes induites
EP2494035A2 (fr) * 2009-10-29 2012-09-05 Janssen Biotech, Inc. Cellules souches pluripotentes
JP2012520660A (ja) * 2009-03-20 2012-09-10 メゾブラスト，インコーポレーテッド 再プログラム化多能性細胞の作製
JP2013078303A (ja) * 2011-10-04 2013-05-02 Masato Sakaki クローン臓器
JP2013523134A (ja) * 2010-03-31 2013-06-17 ザ スクリプス リサーチ インスティチュート 細胞の再プログラム
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US10066203B2 (en) 2008-02-21 2018-09-04 Janssen Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
US10076544B2 (en) 2009-07-20 2018-09-18 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
US10316293B2 (en) 2007-07-01 2019-06-11 Janssen Biotech, Inc. Methods for producing single pluripotent stem cells and differentiation thereof
US10344264B2 (en) 2012-12-31 2019-07-09 Janssen Biotech, Inc. Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US10358628B2 (en) 2011-12-22 2019-07-23 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
JP2019521669A (ja) * 2016-06-03 2019-08-08 中国人民解放軍軍事医学科学院野戦輸血研究所Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｔｒａｎｓｆｕｓｉｏｎ Ｍｅｄｉｃｉｎｅ， Ａｃａｄｅｍｙ Ｏｆ Ｍｉｌｉｔａｒｙ Ｍｅｄｉｃａｌ Ｓｃｉｅｎｃｅｓ， Ｐｅｏｐｌｅ’Ｓ Ｌｉｂｒａｔｉｏｎ Ａｒｍｙ Ｏｆ Ｃｈｉｎａ 消化管由来上皮細胞を内胚葉幹細胞／前駆細胞に初期化するための低分子化合物の組み合わせ、初期化方法および適用
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