WO2009096049A1

WO2009096049A1 - Differentiated cells originating in artificial pluripotent stem cells

Info

Publication number: WO2009096049A1
Application number: PCT/JP2008/059590
Authority: WO
Inventors: Shinya Yamanaka; Takashi Aoi
Original assignee: Kyoto University
Priority date: 2008-02-01
Filing date: 2008-05-23
Publication date: 2009-08-06

Abstract

Cells, a tissue, an organ or an individual having a high safety that are obtained via the induction of the differentiation of artificial pluripotent stem cells, which have been obtained by the nucleus initialization of hepatic cells or gastric epithelial cells, and have a reduced or no risk of tumorigenesis.

Description

Artificial pluripotent stem cell-derived differentiated cells

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 (ES 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.

Dedifferentiation is induced using the patient's own differentiated somatic cells, and the cells have pluripotency and proliferative ability similar to those of ES cells (this cell is referred to as “artificial pluripotent stem cell” or “iPS cell” However, if these cells can be called “induced pluripotent stem cells”, “embryonic stem cell-like cells”, or “ES-like cells”), an ideal without rejection or ethical problems It is expected that it can be used as a typical pluripotent cell. Recently, it has been reported that this iPS cell can be produced from mouse and human differentiated cells, and has been called a huge response (International Publication WO2007 / 69666; Cell, 126, pp.1-14, 2006; Cell, 131, pp .1-12, 2007; Science, 318, pp.1917-1920, 2007; Nature, 451, pp.141-146, 2008).

In these methods, multiple specific factors (Cell, 126, pp.1-14, 2006 uses four factors, Oct3 / 4, Sox2, Klf4, and c-Myc) are introduced into somatic cells. A virus vector such as a retrovirus or a lentivirus is used for the introduction of factors. However, since retroviruses are introduced into somatic cells, there is a concern about the risk of tumor development in cells, tissues, or individuals produced by inducing differentiation of the obtained induced pluripotent stem cells.

On the other hand, it is known that artificial pluripotent stem cells can be produced not only from skin cells but also from stomach cells or hepatocytes (International Publication WO2007 / 69666, Example 10 in Examples). However, the above publication does not disclose a tissue or an individual obtained by inducing differentiation from an induced pluripotent stem cell produced from a stomach cell or a hepatic cell, and the tumor or the individual in the differentiated tissue or individual is not disclosed. There is no suggestion or teaching about the incidence.
International Publication WO2007 / 69666 Cell, 126, pp.1-14, 2006 Cell, 131, pp.1-12, 2007 Science, 318, pp.1917-1920, 2007 Nature, 451, pp.141-146, 2008

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.

As a result of intensive studies to solve the above problems, 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.

That is, 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.

According to a preferred embodiment of the present invention, 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 公開 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 公開 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; There is provided the cell, tissue, organ or individual described above, wherein the staging is carried out by introduction of a gene product of the gene into a somatic cell.

According to a further preferred aspect of the invention, 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.

Of these, 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.

According to another preferred embodiment, 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. 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.

Furthermore, according to 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:

Also, 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.

It is the figure which showed the characteristic of the iPS sputum cell produced from the liver cell and stomach epithelial cell of the adult mouse | mouth. a) The morphology of primary hepatocytes and gastric epithelial cells on a gelatin-treated plate is shown. Scale bar = 50 μm. b) The morphology of iPS-HepH cells and iPS-Stm cells on STOS feeder cells. Scale bar = 500 μm. c) Shows the expression analysis of ES cell marker gene in iPS-Hep cells, iPS-TTF cells, and ES cells by RT-PCR. Two kinds of culture media were used for gene introduction by retrovirus. For clones labeled A, EGF and HGF were added to the serum-free medium. On the other hand, clones labeled B used 10% serum medium and did not use EGF or HGF. For expression of Oct3 / 4 and Sox2, a primer set that amplifies only the endogenous transcript (endo) was used. As a control of the migration amount, NAT1 was used (Genes Dev., 11, 321, 1997). As a negative control, 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 | guidance | derivation efficiency. a) shows the efficiency of retroviral transfection in hepatocytes. EGFP (Enhanced Green Fluorecent Protein) -expressing retrovirus (pMXs-EGFP) or control virus (mock) is transfected into hepatocytes, and the cells are cultured on a gelatin-coated plate in a medium supplemented with HGF and EGF. After 48 hours, cells were analyzed by flow cytometry. 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 | mouth hepatocyte and the iPS cell derived from a stomach epithelial cell. a) Proliferation of iPS-Hep cells (clone 92A-3, -5, and -6) and ES cells. 200,000 cells per well of a 6-well plate were passaged every 3 days (in parentheses indicate division time). 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. White circles indicate unmethylated CpG dinucleotides, and black circles indicate methylated CpGs. It is the figure which showed the teratoma derived from iPS-Hep / Stm cell. iPS-Hep-98A-2 and iPS-Stm-99-1 cells were transplanted subcutaneously into nude mice, the tumor was excised 4 weeks later, and histological analysis by hematoxylin-eosin staining was performed. It is the figure which showed the chimeric mouse derived from iPS-Hep / Stm cell. a) An iPS-Hep / Stm cell-derived adult chimeric mouse is shown. 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 | mouth and the result of a genomic PCR analysis are shown. It is a figure which shows the pluripotency of iPS-Hep / Stm cell. a) Tumor incidence and mortality of iPS sputum cell-derived mice. The cumulative mortality (left side) and total mortality (right side) due to tumors of chimeric mice (upper) and F1 mice (lower) prepared from iPS-MEF cells or iPS-Hep / Stm cells are shown. “Death” includes slaughter cases due to weakness. All deaths were dissected to investigate the cause of death. The numbers below each figure indicate the number of mice analyzed at each time point. b) High perinatal mortality in chimeric mice derived from iPS-HepH cells and iPS-Stm cells, showing the number of surviving and dead individuals at birth. It is a figure which shows the retrovirus insertion site in iPS-HepH cell and iPS-Stm cell. Southern blot analysis of Oct3 / 4, Sox, Klf4, and c-Myc genes inserted using retrovirus was shown for clones of iPS-Hep cells and iPS-Stm cells. Genomic DNAs isolated from iPS-Stm cells, iPS-Hep cells, iPS-MEF cells, and wild-type ES cells were analyzed. The number of detected bands is shown in the lower part. The arrow indicates the band corresponding to the endogenous locus. The white arrow indicates the band corresponding to the Oct3 / 4 pseudogene. It is the figure which showed the retrovirus insertion site (RIS) in iPS-Hep / Stm cell. 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. a) Histogram showing the molecular function of a gene with a retrovirus insertion. The functions were classified according to Gene Ontology (GO) in Mouse Genome Informatics (MGI) of the Jackson Laboratory (http://www.jax.org/) website. It showed molecular function detected at least twice. b) GO classification of the cellular compartment of a gene that has undergone retroviral insertion. It is a schematic diagram of a retrovirus insertion site. 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). It is a schematic diagram of a retrovirus insertion site. It is a schematic diagram of a retrovirus insertion site. 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 | excluding c-Myc when producing an iPS cell from a Nanog-reporter hepatocyte. a) The production of iPS cells from Nanog-GFP reporter hepatocytes by 4 or 3 factors is shown (the number of GFP-positive clones is shown). b) Comparison of the number of GFP-positive clone colonies by 4 factors or 3 factors except c-Myc (mean value and standard deviation in 3 independent experiments), depending on 4 factors in each experiment The number of colonies was 1. The result of protein expression in hepatocytes and MEF is shown. Four types of factors or DsRed (control) were introduced into hepatocytes and MEF by retrovirus, and after 4 days, cell supernatants were collected and analyzed by SDS-PAGE and Western blot. It is the figure which showed the iPS-Hep cell derived from an albumin expression cell. a) Demonstrate how to track cell fate. b) Pictures of phase contrast microscope, fluorescence microscope, and X-gal staining of iPS sputum cell colonies derived from triple transgenic mouse hepatocytes are shown. Arrows indicate β-gal negative colonies. Scale bar = 2mm.

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.

For example, International Publication WO2007 / 69666, Cell, 126, pp.1-14, 2006, Cell, 131, pp.1-12, 2007, Science, 318, pp.1917-1920, 2007, Nature, 451, pp. Induced nuclear reprogramming of hepatocytes or gastric epithelial cells according to the methods described in 141-146, 2008, etc., or by appropriate modifications or alterations to those methods, to prepare induced pluripotent stem cells Inducing any tissue, organ, or individual mammal by inducing differentiation of the obtained induced pluripotent stem cells into somatic cells and adding appropriate culture, tissue or organ formation processes as necessary Can do.

The type of 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. 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. Further, in the method of the present invention, 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). .

As a means for confirming a nuclear reprogramming factor, for example, 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. In addition, the nuclear reprogramming factor can be confirmed using a method in which appropriate modification or alteration is added to the above screening method.

An example of a combination of genes encoding reprogramming factors is disclosed in International Publication WO2007 / 69666. The entire disclosures of the above publications are incorporated herein by reference. Those skilled in the art can appropriately select genes that can be suitably used in the present invention by referring to the above-mentioned publications. Another example of a combination of genes encoding reprogramming factors is described in Science, 318, pp.1917-1920, 2007. Accordingly, those skilled in the art can understand the diversity of combinations of genes encoding nuclear reprogramming factors, and by using the nuclear reprogramming factor screening method described in International Publication WO 2005/80598, Appropriate gene combinations other than those described in WO2007 / 69666 and Science, 318, pp.1917-1920, 2007 can be used in the present invention.

As 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 Preferably, 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.

For the Oct family gene, the Klf family gene, the Sox family gene and the Myc family gene, specific examples of family genes are shown in International Publication WO2007 / 69666. Those skilled in the art can similarly extract a family gene for the Lin family gene.

In addition, 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. As such 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.

As a more preferable combination of genes,
(a) Combination of two genes consisting of Oct family gene and Sox family gene
(b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene;
(c) a combination of four genes consisting of an Oct family gene, a Sox family gene, a Lin family gene, and a Nanog gene;
However, it is not limited to these. These can be combined with Myc family genes.

These 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. In addition to 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) It is also possible to use 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. For example, the c-Myc gene product may be a wild type or a stable type (T58A). The same applies to other gene products.

Moreover, in addition to the above genes, genes encoding factors that induce cell immortalization may be further combined. As disclosed in International Publication WO2007 / 69666, for example, a TERT gene and one or more genes selected from the group consisting of the following genes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmil alone Or they can be used in appropriate combinations.
As a preferred combination, for example,
(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.

Furthermore, in addition to the above genes, one or more genes selected from the group consisting of Fbx15, ERas, ECAT15-2, Tcl1, and β-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 β-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. However, 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.

If the gene product of one or more of these genes has already been expressed in the somatic cell to be initialized, such gene product can be excluded from the factors to be introduced. For example, 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. Alternatively, when one or more of the gene products of these genes are introduced into the nucleus by a technique such as fusion protein or microinjection into the nucleus, the remaining one or more genes are appropriately selected. The gene can be introduced by, for example, a method using a recombinant vector.

In addition, 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. For example, 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. In addition, by preparing and using a fusion protein with a 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.

As used herein, an “artificial pluripotent stem cell” (iPS 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. For example, 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. When such a vector is used, two or more genes may be incorporated into the vector and the respective gene products may be expressed simultaneously in somatic cells.

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. As 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. In some cases, it is preferable to perform initialization in the presence of specific microRNAs to increase the efficiency of initialization (Koyanagi et al., 30th Annual Meeting of the Molecular Biology Society of Japan, 1T7-7, Abstracts published November 2007) 25th).

By culturing somatic cells into which a nuclear reprogramming factor has been introduced under appropriate conditions, 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. When producing human induced pluripotent stem cells, it is desirable to set 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. For example, when producing human induced pluripotent stem cells, it may be preferable to use a medium suitable for human ES cell culture. 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. By using a combination of appropriate media, 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. Further, when the generated induced pluripotent stem cells are grown under appropriate conditions, 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. For example, it is known that mouse induced pluripotent stem cells form raised colonies, whereas human induced pluripotent stem cells are known to form flat colonies, and 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.

The type of hepatocytes or gastric epithelial cells to be initialized is not particularly limited. For example, cells derived from any mammal (eg, mammals such as human, mouse, rat, cow, sheep, horse, monkey) are used. In addition to fetal hepatocytes or gastric epithelial cells, hepatocytes or gastric epithelial cells in mature individuals may be used. Preferably, 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. 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.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example. In the following examples, induced pluripotent stem cells are abbreviated as iPS 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. using a digestion solution containing 0.05% collagenase type I, 150 U / ml Dispase (Gibco), and 0.2% bovine serum albumin. Primary hepatocytes and gastric epithelial cells are 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 μg / l, dexamethasone 10 ⁻⁷ M, penicillin 100 mg / l, and streptomycin 100 mg / l, and cultured in a basic chemically defined medium consisting of DMEM (manufactured by Nacalai Tesque).

2. Preparation of retroviruses 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. The next day, 9 μg of retroviral vector was introduced into PLAT-E cells using 27 μl of FuGENE6 transfection reagent (Roche). After 24 hours, the medium was replaced with 10 ml of the above basal chemically defined medium. The next day, the virus-containing supernatant from the PLAT-E cell culture was collected and filtered through a 0.45 μm cellulose acetate filter (Fatman).

3.Transfection of retrovirus into primary cultures of hepatocytes and gastric epithelial cells One day after the start of primary culture, the medium was mixed with hepatocyte growth factor (HGF, peprotech) 40 ng / ml and epidermal growth factor (EGF, (Peprotech) The basal chemically defined medium was replaced with 20 ng / ml. On day 2 for the culture of gastric epithelial cells and on day 3 for the culture of hepatocytes, the medium was replaced with medium containing retrovirus obtained from PLAT-E cell culture (polybrene (final concentration 8 μg / ml), HGF (40 ng / ml) and EGF (20 ng / ml) supplemented).

4. 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. 24 hours after gene transfer, replace cells with basal chemically defined medium (supplemented) supplemented with 40 ng / ml HGF and 20 ng / ml EGF, then replace medium every 2 days and select Fbx15 On day 3 after gene introduction, and on day 7 for gene selection, the medium was replaced with ES medium supplemented with LIF (Proc. Natl. Acad. Sci. USA, 93, 14041, 1996). For selection of iPS cells by Fbx15, G418 was added at a final concentration of 0.3 mg / ml, and for selection of iPS cells by Nanog, puromycin 1.5 μg / ml was added. In one experiment, G418 selection was started 10 days after gene transfer and in another experiment no drug selection was performed (Table 1). Teratoma formation, RT-PCR analysis, and bisulfite genome sequencing were performed according to literature methods (Cell, 126, 663, 2006).

5. Generation of triple transgenic mice for follow-up analysis of genetic lines First, homozygous GAG-CAT-Z mice (Biochem. Biophys. Res. Commun., 237, 318, 1997) were crossed with homozygous Nanog reporter mice. did. Next, the obtained F1 mice were crossed with homozygous Alb-Cre mice (J. Biol. Chem., 274, 305, 1999) to obtain triple transgenic mice. Primary hepatocytes were isolated from the triple transgenic mice for induction of iPS cells.

6. X-gal staining of cultured cells Cells were washed with PBS and fixed with 1% glutaraldehyde for 5 minutes. After treatment with 1% Triton-X100 and washing with PBS, staining solution (0.04% X-gal / DMSO, 1 mM MgCl ₂ , 3 mM K ₄ [Fe (CN) ₆ ], 3 mM K ₃ [ The cells were stained for 12 hours at 37 ° C. with Fe (CN) ₆ ].

7.Decision of retrovirus introduction site 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). Using this ligated template, 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. The alignment of flanking sequences to the mouse genome is the University of California, Santa Cruz (UCSC) BLAT Genome Browser (2006Feb and / or 2007Jul, http://genome.ucsc.edu/cgi-bin/hgblat?command=start), European Bio Informatics Institute (FBI) and Welcome Trust Sanger Institute (WTSI) Ensemble Database (Build m36, http://www.ensembl.org), and National Institute of Health (NIH) National Center for Biotechnology Information (NCBI) at BLASTNni (http://www.ncbi.nlm.nih.gov/).

In order to perform both the insertion site and retrovirus identification, selective inverse PCR (SIPCR) was also performed. Chromosomal DNA was digested with a 6-base cleavage enzyme such as PstI, NsiI, BamHI, or BglIIgl (Toyobo). Amplified by nested PCR using a ligated product of these fragments as a template. PMXsU3F1 (5′-AAATGACCCTGTGCCTTATTTG-3 ′) was used as a common forward primer in the first PCR. As reverse primers in the first PCR, Oct3 / 4R1 (5'-CTGAAGGTTCTCATTGTTGTCG-3 '), Sox2R1 (5'-AGTGGGAGGAAGAGGTAACCAC-3'), c-MycR1 (5'-TTCTTGCTCTTCTTCAGAGTCG-3 '), or Klf4R1 (5' -CGGAATGTATACTGGGTCCAAC-3 ') 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.

8. Immunoblotting Four days after retrovirus infection, the cells were washed with PBS (−), treated with 1 × SDS sample buffer, and boiled. Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). The blot was blocked with TBST buffer containing 4% skim milk and then incubated overnight at 4 ° C. with the primary antibody solution. After washing with TBST buffer, the membrane was incubated with a horseradish peroxidase (HPR) labeled secondary antibody. The signal was detected with an Immobilon Western Chemiluminescent Luminescent HRP substrate (Millipore) and a LAS3000 imaging device (Fuji Film). As an antibody for immunoblotting, anti-Sox2 antiserum (J. Biol. Chem., 280, 24371, 2005), 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-β-catenin antibody (sc-7199, Santa Cruz), anti-β- Actin antibody (A5441, Sigma), anti-mouse IgG-HRP antibody (# 7076, cell signaling), and anti-rabbit IgG-HRP antibody (# 7074, cell signaling) were used.

<Result>
IPS cells were established from epithelial cells. Primary cells from hepatocytes and gastric epithelial cells from mice in which β-geo gene (a fusion gene of β-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).

4 types of transcription factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced into the hepatocytes and gastric epithelial cells using a retroviral vector. The efficiency of retrovirus gene transfer into epithelial cells (30-45%, Figure 2a) was lower than the efficiency of transfer into MEF (> 85%). When the gene was introduced into MEF with diluted retrovirus, iPS cells were not obtained when the efficiency was around 30% (Fig. 2b). Three days after the introduction of the gene into the epithelial cells, the medium was replaced with a medium for ES cells containing serum and G418 (neomycin). Two weeks later, in spite of such low retroviral gene transfer efficiency, both ES cells and gastric epithelial cells were observed with multiple ES cell-like colonies that are resistant to G418 and have large nuclei and poor cytoplasm It was done.

When G418 -resistant colonies were grown, about 60% of the cells showed a form that could not be distinguished from mouse ES cells (Fig. 1b). These cells were named iPS-Hep (iPS-Hepatic) and iPS-Stm (iPS-Stomach), respectively. These cells also showed the same proliferative ability as ES cells (Fig. 3a). When RT-PCR was performed, iPS-Hep cells (Fig. 1c) and iPS-Stm cells (Fig. 3b) were found to express endogenous Oct3 / 4 cells and Sox2 cells, similar to ES cells. In addition, 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. On the other hand, TPS-derived iPS cells (iPS-TTF) selected by the Fbx15 reporter had a low expression level of ES cell marker gene (FIG. 1c).

Next, two types of culture conditions were examined when iPS-Hep cells and iPS-TTF cells were prepared. One with the addition of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) but no serum (Figure 1c, A), the other with 10% serum but no EGF or HGF Conditions (Fig. 1c, B) were used. As a result, under both culture conditions, iPS-Hep cells expressed more ES cell marker genes than iPS-TTF cells. In iPS-Hep cells and iPS-Stm cells, most of the promoter regions of the Oct3 / 4, Nanog, and Fbx15 genes were not complete but were unmethylated (FIG. 3c). This was also in contrast to iPS-TTF cells, and only partial demethylation was observed in iPS-TTF cells (Cell, 126, 663, 2006). Thus, as with iPS-MEF and iPS-TTF, 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).

4 weeks after transplantation, tumors composed of various tissues derived from the three germ layers such as nerve tissue, muscle, cartilage, and intestinal-like epithelial tissue were formed (FIG. 4). From this result, it was demonstrated that iPS-HepPS cells and iPS-Stm cells have pluripotency.

Next, these cells were also transplanted into blastocysts by microinjection (Table 2). Eight kinds of 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. Meissner et al., Nat., Biotechnol, 2007; Cell Stem Cell ,, 2007; M. Nakagawa et al., Nat. Biotechnol, 2007) was also transplanted. Among these, adult chimeric mice (confirmed by hair color) derived from 10 clones of iPS-Hep cells and 2 clones of iPS-Stm cells were obtained (Fig. 5a).

1 clone of 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). In this regard as well, only 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).

Next, tumor incidence was compared between 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. In addition, approximately 20% of the F1 mice derived from 8 iPS-MEF cell clones developed tumors by 30 weeks of age, but tumors were observed in F1 mice derived from iPS-Hep cells and iPS-Stm cells. Not (Figure 6a). Some of the mice derived from iPS-Hep sputum cells and iPS-Stm sputum cells died after moving to a normal housing facility, but no tumor was found even after autopsy of these mice (FIG. 6a).

However, 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).

Next, the number of retrovirus insertion sites (RIS) in iPS-Hep cells and iPS-Stm cells was examined. By Southern blot analysis, 1 to 4 bands were detected for each of 4 types of retroviruses introduced into each clone (FIG. 7). This number is less than the number of bands in MEF-iPS sputum cells, and from 1 to 12 RIS sputum per retrovirus was detected in MEF-iPS sputum cells. When examined using inverse PCR, RIS was randomly distributed on multiple chromosomes in 2 clones of iPS-Hep cells and 2 clones of iPS-Stm cells (Fig. 8).

The gene at the site where the retrovirus was inserted showed no specific bias in functional classification or in the intracellular localization of the molecule (FIG. 9). RIS was mainly present near the transcription start position of these genes (Figs. 10-14). These data indicate that iPS-Hep cells and iPS-Stm cells differ from iPS-fibroblast in three properties. First, iPS-Hep cells and iPS-Stm cells contributed to the formation of adult chimeras despite being selected by the expression of the Fbx15 gene. Second, no increase in tumor incidence was observed in iPS-Hep cell-derived and iPS-Stm -derived chimeric mice during the period up to 30 weeks of age. These two features are similar to iPS-MEF and TTF cells established without using c-Myc retrovirus (M. Nakagawa et al., Nat. Biotechnol., 2007). This suggests that Myc plays a smaller role in the establishment of iPS-Hep cells and iPS-Stm cells than iPS-MEF and RRF cells.

In order to investigate this possibility, 1 type was excluded from 4 types of factors, and the effect on the production of iPS cells from hepatocytes was examined. As a result, when Oct3 / 4, Sox2 or Klf4 was excluded, iPS cell colonies did not appear (FIG. 15a). On the other hand, when c-Myc was excluded, the number of colonies was only reduced by 20 to 40% compared to the case of using all 4 types (Fig. 15a, b). This result is in contrast to iPS-fibroblast cells, whose colonies decreased by more than 90% when c-Myc was excluded (M. Nakagawa et al., Nat. Biotechnol., 2007), and the establishment of iPS cells derived from hepatocytes. Shows the low importance of Myc.

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. In addition, 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). In this analysis, it was confirmed that the expression of E- リン cadherin and β- 発現 catenin in hepatocytes was higher than that of fibroblasts and similar to that of ES cells (Figure 16). This similarity is also considered to contribute to the decrease in the number of RIS in iPS-Hep cells and iPS-Stm cells.

In order to investigate the origin of iPS-Hep cells, we performed a cell lineage tracking analysis using a genetic technique (Fig. 17a). In the Nanog reporter mouse, the GFP gene and the puromycin resistance gene are knocked into the Nanog gene for selection of iPS cells. First, 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-β-gal (β- galactosidase) cassette under a constitutively activated promoter (Biochem. Biophys. Res. Commun., 237, 318, 1997). In this triple transgenic mouse, β-gal activity is induced as the albumin gene is activated, and β-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.

14 days after gene transfer, selection with puromycin was started. By 30 days after gene transfer, 100 or more GFP-positive colonies were obtained (FIG. 17b, left and center). Since most of them were also β-gal -positive (Fig. 17b right), 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 β-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. These results indicate that we succeeded in reprogramming somatic cells that have been differentiated to the stage where the albumin promoter is activated using four types of transcription factors. Furthermore, it was also confirmed that retrovirus insertion into a specific site in the genome is not necessary for the establishment of iPS-Hep cells and iPS-Stm cells.

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. 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.

Claims

A cell, tissue, organ, or individual obtained by inducing differentiation of induced pluripotent stem cells obtained by nuclear reprogramming of hepatocytes or gastric epithelial cells.
A method for reducing or eliminating the occurrence of tumors in cells, tissues, organs, or individuals that have been induced to differentiate from induced pluripotent stem cells, obtained by reprogramming hepatocytes or gastric epithelial cells into a nucleus A method comprising a step of inducing differentiation of a potent stem cell.