JP2011160661A - Reprogramming method of blood cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a pluripotent stem cell from a somatic cell. <P>SOLUTION: The method for producing the pluripotent stem cell from the somatic cell comprises the following steps (a) and (b): (a) culturing the somatic cell in a cell culture medium containing an IL-6 signaling factor-stimulating factor and at least one kind of cytokines; and (b) dedifferentiating the somatic cell. In the method, the step (b) may be carried out subsequent to the step (a), or the steps (a) and (b) may be carried out simultaneously. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to methods and kits for producing pluripotent stem cells by reprogramming or dedifferentiating differentiated somatic cells, particularly blood cells.

BACKGROUND ART Embryonic stem cells (ES cells) are cell lines that can maintain unlimited cell growth over a long period of time while maintaining the pluripotency that can differentiate into almost all types of cells that make up tissues and organs in the body. is there. Due to this property, ES cells are used as a cell resource for transplantation therapy for many diseases such as Parkinson's disease, type I diabetes, spinal cord injury, and heart failure. It is expected to be used as a supply source of human cells used for basic research and drug discovery research. However, since ES cells are cell lines established by destroying early embryos of humans and mice, there are many objections to the use of ES cells from an ethical point of view. In addition, cell transplantation therapy involves the risk of inducing a rejection reaction in the same manner as organ transplantation because it involves transplanting another person's cells. As a method for avoiding these problems, it is expected to use induced pluripotent stem cells (iPS cells) having a pluripotency and proliferative ability similar to those of ES cells, in which the differentiated somatic cells of patients are initialized.

  As an initialization method for differentiated somatic cells, for example, iPS cells are induced by co-infection of mouse embryonic fibroblasts (MEF cells) with four genes of Oct4, Sox2, Klf4, and c-myc with a retroviral vector. Non-patent document 1: K. Takahashi, S. Yamanaka, Cell, 126, pp.663-676, 2006; Non-patent document 2: K. Okita, T. Ichisaka, S. Yamanaka, Nature , 448, pp. 313-317, 2007; Non-Patent Document 3: M. Wernig et al., Nature, 448, pp. 318-324, 2007). A technique for inducing iPS cells from mouse hepatocytes and gastric epithelial cells by a similar method has also been reported (Non-patent Document 4: T. Aoi et al., Science Express on 14 Feb. 2008). The screening method for reprogramming factors in these reports has been proposed in WO 2005/080598 pamphlet (Patent Document 1), and the reprogramming factors include other ES cell-specific genes (reprogramming). This is proposed in the pamphlet of International Publication No. 2007/069666 (Patent Document 2) together with candidate genes for factors). In these reports and proposals, human cells are partially described, but at this stage, the establishment of human iPS cells having the same properties as human ES cells is incomplete. In addition, induction from hepatocytes and gastric epithelial cells is highly invasive when applied to humans and is expected to increase the burden on the patient to be collected.

  As a method for establishing human iPS cells, a method has been reported in which four factors of Oct4, Sox2, Klf4, and c-myc are introduced into adult skin-derived fibroblasts (HDF cells) using a retrovirus (non-native). Patent Document 5: K. Takahashi, S. Yamanaka, Cell, 131, pp.861-872, 2007). In this method, in order to infect a retrovirus with high efficiency, a cell in which a mouse ecotropic virus receptor, sodium carrier family 7 (Slc7a1), is expressed by a lentivirus is used. As a result, human ES cell-like colonies were recognized around 25 days after retrovirus infection, and colonies were picked up after 30 days. In this method, since a mouse ecotropic virus receptor is newly introduced by lentivirus, the risk of canceration of host cells increases due to the expression of genes derived from heterologous animals and increased opportunities for inserting DNA fragments into chromosomes. I have a problem. Furthermore, since the induction period is one month, there is a problem with the culture cost. In this report, neonatal foreskin-derived fibroblasts (BJ1 cells) are also used to induce iPS cells. In this case, the appearance frequency of human ES cell-like colonies is higher than when HDF is used. The number of days has not been shortened.

  In another group, a report was also made of induction of iPS cells by retrovirus-introducing the same four factors using human neonatal foreskin-derived fibroblasts that expressed the mouse ecotropic virus receptor mCAT1 gene by adenovirus. (Non-patent document 6: H. Masaki et al., Stem Cell Research, 1, pp. 105-115, 2007). In this report, the appearance of iPS-like colonies is observed on the 17th day after retrovirus infection. In this method, the expression of a viral receptor in adenovirus reduces the risk of inserting a heterologous gene into chromosomal DNA, but the risk associated with introduction of the heterologous gene into the cell remains as before. . There is also a report that iPS cells were induced by introducing the same four factors into human fetal lung fibroblasts (MRC5 cells) by retrovirus (Non-patent Document 7: IH Park et al., Nature, 451, pp.141-). 146, 2008). However, in this report, iPS cell induction by four factors from BJ1 cells, adult mesenchymal stem cells (MSC cells), and HDF cells has not been successful. In addition, ES cell-like colonies were obtained from fetal fibroblast cells (IMR90 cells) and newborn foreskin fibroblasts by expressing them in cells with lentivirus using Oct4, Sox2, NANOG, and Lin28 as reprogramming factors. (Non-patent document 8: J. Yu et al., Science, 318, pp.1917-1920, 2007). When fetal and neonatal cells are used, iPS cells can be induced in a shorter period of time than when adult epidermal fibroblasts are used, but ethical issues as a cell source for treatment and research Remains. This report further reports that iPS cells can be induced using blood cells differentiated from human ES cells. However, cells induced to differentiate from ES cells have not undergone a normal differentiation process and cannot be said to be the same cells as adult-derived blood cells. At present, there is no report on the induction of iPS cells from blood cells collected from adults.

  In addition to the four factors Oct4, Sox2, Klf4, and c-myc, there is also a report that the induction of iPS cells is made more efficient by co-expressing Nanog (Non-patent Document 9: WE Lowry et al., Proc. Natl. Acad. Sci. USA, 105, pp.2883-2888, 2008). It has been conventionally reported that the reprogramming (initialization) efficiency is increased in the presence of Nanog (Non-Patent Document 10: J. Silva et al., Nature, 441, pp.997-1001, 2006). Gene transfer is performed on HDF cells using retrovirus, and ES cell-like colonies appear on the 21st day after the end of virus infection. The efficiency of iPS cell induction has been increased by using Nanog, but increasing the type of factor also increases the type of gene inserted into the chromosome, improving the efficiency with a method that does not increase the type of reprogramming factor Is desired.

  There is also a report that iPS cells are induced from terminally differentiated mature B cells in mice (Non-patent Document 11: J. Hanna et al., Cell, 133, pp. 250-264, 2008). In this report, iPS cells are first induced by introducing a unit expressing four nuclear reprogramming factors, Oct4, Sox2, Klf4, and c-myc in a doxycycline-dependent manner using a lentiviral vector. Furthermore, transgenic mice are prepared using induced iPS cells. For induction of iPS cells from mature B cells, B cells prepared from the transgenic mice thus obtained are used, and it is impossible to apply the same method to humans. In order to induce iPS cells from human terminally differentiated blood cells, this method cannot be applied, and an initialization method applicable to human primary cells is desired.

  On the other hand, in the medical field where therapeutic effects are expected by using pluripotent stem cells such as ES cells, somatic stem cells that exist in vivo in parallel with the study of treatment methods using cells induced to differentiate from ES cells The use of has been considered. Among such efforts, there is a report of a technique for amplifying somatic stem cells in vitro for the purpose of cell transplantation therapy. Among them, for hematopoietic stem cells, stem cell factor (SCF), Flt ligand (FL), thrombopoietin (TPO), interleukin-6 (IL-6) and solubilized IL-6 receptor α chain (IL-6R) There has been a report of amplification of cord blood-derived hematopoietic stem cells and bone marrow-derived hematopoietic stem cells using cytokines such as fusion proteins (Non-patent Document 12: T. Ueda et al., J. of Clinical Investigation, 105, pp) -1013-1021, 2000; Non-Patent Document 13: L. Gammaitoni et al., Experimental Hematology, 31, pp. 261-270, 2003). Furthermore, there is a technique for improving the amplification factor using stromal cells (Patent Document 3: International Publication No. 2003/014336 pamphlet). Both are technologies for amplifying blood cell stem cells or progenitor cells, and cannot be differentiated into other cell lineages at the time of amplification, and do not have somatic cell initialization or dedifferentiation. Moreover, there is a limit to the amplification of hematopoietic stem cells, and it cannot be expanded indefinitely while maintaining pluripotency like ES cells.

International Publication No. 2005/080598 Pamphlet International Publication No. 2007/069666 Pamphlet International Publication No. 2003/014336 Pamphlet K. Takahashi, S. Yamanaka, Cell, 126, pp.663-676, 2006 K. Okita, T. Ichisaka, S. Yamanaka, Nature, 448, pp. 313-317, 2007 M. Wernig et al., Nature, 448, pp.318-324, 2007 T. Aoi et al., Science Express on 14 Feb. 2008 K. Takahashi, S. Yamanaka, Cell, 131, pp.861-872, 2007 H. Masaki et al., Stem Cell Research, 1, pp.105-115, 2007 I. H. Park et al., Nature, 451, pp.141-146, 2008 J. Yu et al., Science, 318, pp.1917-1920, 2007 W. E. Lowry et al., Proc. Natl. Acad. Sci. USA, 105, pp. 2883-2888, 2008 J. Silva et al., Nature, 441, pp.997-1001, 2006 J. Hanna et al., Cell, 133, pp.250-264, 2008 T. Ueda et al., J. of Clinical Investigation, 105, pp-1013-1021, 2000 L. Gammaitoni et al., Experimental Hematology, 31, pp. 261-270, 2003

Summary of the Invention

  The present inventors efficiently pluripotent stem cells from somatic cells by combining culture in the presence of IL-6 signaling factor (IL6ST) stimulating factor and cytokine cocktail and cell dedifferentiation. I found out that it can be established. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide a method for efficiently producing pluripotent stem cells from somatic cells.

  The method for producing pluripotent stem cells according to the present invention is a method for producing pluripotent stem cells from somatic cells, wherein (a) the somatic cells are treated with IL-6 signaling factor stimulating factor and at least one cytokine. And (b) a step of dedifferentiating somatic cells, step (b) is performed after step (a), or steps (a) and ( b) is performed simultaneously.

  According to the present invention, pluripotent stem cells can be established from a somatic cell that can be collected with a lighter burden than the human body, such as peripheral blood cells, in a shorter period of time and with higher efficiency than in the conventional method. The period required for establishment of pluripotent stem cells can be shortened to, for example, about ½ of the conventional method. Thus, the produced pluripotent stem cells can be used for treatment of urgent diseases such as spinal cord injury, and the cost for producing pluripotent stem cells can be reduced.

Detailed description of the invention

  The “pluripotent stem cell” produced in the present invention means a cell that can differentiate into various differentiation lineages, and is not necessarily limited to a cell that can differentiate into all cell lineages equivalent to ES cells, For example, endoderm stem cells, mesoderm stem cells, or ectoderm stem cells may be used. For example, in order to produce pluripotent stem cells used for the treatment of heart failure, it is only necessary to produce cells that can differentiate into cardiomyocytes. Alternatively, in order to produce pluripotent stem cells used for the treatment of type I diabetes, it is only necessary to produce cells that can differentiate into β cells. By using a reporter gene according to the purpose of use or an antibody against a specific surface antigen, it is possible to efficiently separate the target cells.

  According to a preferred embodiment of the present invention, a “pluripotent stem cell” is a cell that can differentiate into all cell lineages equivalent to ES cells. Such cells are cells that express marker molecules such as SSEA-3, SSEA-4, TRA1-60, TRA1-81 in humans. In mice, it is a cell that expresses a marker molecule such as SSEA-1. It is known that human and mouse cells form teratomas when transplanted subcutaneously into immunodeficient mice such as SCID mice and nude mice. Teratomas formed from pluripotent stem cells include differentiated cells of the endoderm system, mesoderm system, and ectoderm system, and their respective lineages. Thus, pluripotent stem cells that can differentiate into all cell lineages equivalent to ES cells are characterized by the formation of such teratomas.

  The method for producing pluripotent stem cells according to the present invention comprises a step of culturing in a cell culture medium containing an IL-6 signaling factor (IL6ST) stimulating factor and at least one cytokine (hereinafter referred to as “step (a)”). Comprising.

  In the present invention, “IL6 signaling factor (IL6ST)” means interleukin 6 signal transducer and is also called gp130. IL6ST means a protein having a total length of 918 amino acid residues composed of a signal region, an extracellular region, a transmembrane region, and an intracellular region. Regarding IL6ST, it is known that signal transduction via IL6ST is maintained even when the signal peptide from the N-terminal to the 22nd glycine residue is deleted. IL-6R bound to IL-6 binds to two molecules of IL6ST, and IL6ST forms a homodimer. It is known that IL6ST that forms a homodimer is phosphorylated on a tyrosine residue in the intracellular region and activated signal transduction. IL6ST is known to bind to IL-11R and transmit a signal. IL6ST also binds to leukocyte migration inhibitory factor receptor (LIFR), oncostatin M receptor (OSMR), ciliary neurotrophic factor receptor (CNTFR), cardiotropin receptor (CT-1R), etc., and heterodimer It is known that it is activated by forming.

  In the present invention, “IL6ST stimulating factor” means a molecule that transmits a signal via human IL6ST, and more specifically, refers to a molecule that causes IL6ST activation by associating with IL6ST. Examples of IL6ST stimulating factors include IL-6R or a mutant thereof and an IL-6 receptor (hereinafter referred to as “IL-6R”) or a fusion protein thereof, IL-6R (preferably soluble IL-6R (sIL -6R)) or a variant thereof and a combination of IL-6 or a variant thereof (Japanese Patent Publication No. 10-509040), a complex of IL-6 and the α chain of IL-6 receptor, IL6ST Antibodies to IL6ST acting as agonists (ZJ Gu et al., Leukemia, 14, pp.188-197, 2000), interleukin-11 (IL-11), leukocyte migration inhibitory factor (LIF), oncostatin M, and Examples include cardiotropin.

  In the present invention, “IL-6” means a protein of 212 amino acid residues in total length composed of four helices, commonly known as IL-6. In the present invention, “IL-6 mutant” refers to an IL-6 mutant having one or more modifications selected from substitution, deletion, insertion, and addition and having IL6ST stimulating activity. means. Regarding IL-6, it is known that IL6ST stimulating activity is maintained even if the signal peptide is deleted from the N-terminal to the 28th alanine residue (WO00 / 01731). In addition, even if the C-terminal side of the 37th lysine residue of IL-6 is digested with protease and the 10 amino acid residues from the 28th alanine residue to the 37th lysine residue are deleted. It is known that there is no hindrance to IL-6 activity (WO00 / 01731). Therefore, in the present invention, modified IL-6 lacking the N-terminus and having IL6ST stimulating activity may be used as the IL-6 mutant. Examples of such IL-6 variants include IL-6 from which the N-terminal sequence up to the 28th is deleted, and IL-6 from which the N-terminal sequence up to the 37th is deleted.

  In the present invention, “IL-6R” is a protein having a total length of 468 amino acid residues, which is generally known as an IL-6 receptor, and is composed of a signal region, an extracellular region, a transmembrane region, and an intracellular region. Means. In the present invention, “IL-6R mutant” refers to an IL-6R mutant having one or more modifications selected from substitution, deletion, insertion, and addition and having IL6ST stimulating activity. means. For IL-6R, the cytokine receptor region (region from the 112th valine residue to the 323rd alanine residue) existing in the extracellular region is necessary and sufficient for binding to IL-6. Are known. Therefore, in the present invention, modified IL-6R comprising a region from the vicinity of the 112th valine residue to the vicinity of the 333rd alanine residue of IL-6R may be used as an IL-6R mutant.

  According to a preferred embodiment of the present invention, the IL6ST stimulating factor is a fusion protein in which IL-6 or a variant thereof and IL-6R or a variant thereof are linked directly or via a linker. Such fusion proteins can be obtained by methods known in the art, for example, WO00 / 01731, WO99 / 02552, JP 2000-506014, or Fischer et al., Nature Biotech., 15, pp. 142-145, It can be produced according to the method described in 1997.

  According to a particularly preferred embodiment of the present invention, the fusion protein comprises a C-terminal fragment of the IL-6R fragment from the 112th valine residue to the 333rd alanine residue, and the 38th aspartic acid of IL-6. A protein (hereinafter referred to as “FP6”) in which the N-terminal of the fragment from the residue to the 212th methionine residue is directly linked. In this case, a fusion protein is prepared by introducing a vector in which a DNA sequence encoding the fusion protein is linked so that it can be expressed in the host, culturing the transformed host, and collecting FP6 from the culture. FP6 can be produced (see WO00 / 01731).

  The amount of IL6ST stimulating factor added to the cell culture medium is not particularly limited, but is preferably 1 ng / ml to 100 μg / ml, more preferably 10 ng / ml to 20 ng / ml.

  In the method for producing pluripotent stem cells according to the present invention, the use of IL-6ST stimulating factor is one main feature. Without being bound by any particular theory, IL6ST activation can be achieved, for example, by activation of JAK kinase, phosphorylation of STAT3, and the genes such as JunB and JAB by STAT3 that formed a homodimer by phosphorylation. It is considered that the transcription is promoted sequentially, resulting in cell proliferation, cell differentiation regulation and the like. Therefore, in the present invention, instead of IL6ST stimulating factor, STAT3C (JFBromberg et. Al., Cell, 98, pp) in which a mutation that forms activated JAK kinase, phosphorylated STAT3, or homodimer is introduced. -295-303, 1999), STAT3ER (T.Matsuda et al., EMBO Journal, 18, pp-4261-4269, 1999), an inhibitor of JAK kinase, which induces homodimers inducibly by estrogen analogs It is also conceivable to introduce an SOCS molecule inhibitor or the like into a cell in the form of a gene or protein.

  “Cytokines” used in step (a) include thrombopoietin (TPO) and variants thereof and derivatives thereof, c-mpl ligand (excluding TPO) and derivatives thereof, stem cell factor (SCF), Flt-3 Ligand (FL), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage Growth factors such as colony stimulating factor (M-CSF) and interleukin-7 (IL-7); antibodies having growth stimulating activity such as anti-human CD40 antibody, anti-human CD3 antibody and anti-human CD28 antibody; Growth stimulating factors such as polysaccharides (LPS); and combinations thereof.

  In the present invention, “thrombopoietin” (TPO) refers to a protein consisting of an amino acid sequence having a total length of 332 amino acid residues and having an activity of specifically stimulating or increasing platelet production (WO95 / 21919). In the present invention, the “TPO mutant” refers to a TPO mutant that has one or more modifications selected from substitution, deletion, insertion, and addition, and that specifically stimulates or increases platelet production. Means. Examples of TPO mutants include those described in WO95 / 18858, JP-A-8-277296, JP-A-8-228781, and WO95 / 21919, and preferably amino acid residues. Examples include TPO variants (fragments) consisting of the first to 163rd amino acid sequences.

  In the present invention, “derivatives of TPO and its mutants” mean TPO and its mutants formed by linking water-soluble polymers. The water soluble polymer includes polyethylene glycol, preferably polyethylene glycol having an average molecular weight of about 5 kDa to about 50 kDa. Polyethylene glycolation (PEGylation) of TPO and its variants can be carried out according to well-known methods (see, for example, Focus on Growth Factors, 3, pp. 4-10, 1992). The molar ratio of water-soluble polymer to “TPO and its variants” protein can be 1: 1 to 100: 1, 1: 1 to 20: 1 in the case of polyPEGylation, in the case of monoPEGylation Can be 1: 1 to 5: 1.

  In the present invention, “c-mpl ligand” means a peptidic ligand, a proteinaceous ligand, and a non-peptidic ligand that can bind to the mpl receptor. Proteinaceous c-mpl ligands (excluding TPO) include agonist antibodies (WO99 / 03495 and WO99 / 10494) as stimulators of megakaryocytes and other proteins such as hematopoietic receptor agonists (WO96 / 23888, WO97 / 12978, WO97 / 12985, WO98 / 17810, WO96 / 34016 and WO00 / 24770). Peptide c-mpl ligands include those described in WO96 / 40189, WO96 / 40750, WO98 / 25965, JP-A-10-72492, WO99 / 42127, and WO00 / 24770. Non-peptide c-mpl ligands include benzodiazepine derivatives (JP-A-11-1477 and JP-A-11-152276) and other small molecule ligands (WO99 / 11262, WO99 / 22733, WO99 / 22734, WO00 / 35446 and WO00 / 35446). WO00 / 28987).

  In the present invention, the “derivative of c-mpl ligand” means a c-mpl ligand formed by linking a water-soluble polymer and a variant thereof. The water soluble polymer includes polyethylene glycol, preferably polyethylene glycol having an average molecular weight of about 5 kDa to about 50 kDa. Polyethylene glycolation (PEGylation) of c-mpl ligand can be carried out according to a well-known method (see, for example, Focus on Growth Factors, 3, pp. 4-10, 1992). The molar ratio of water soluble polymer to c-mpl ligand protein can be 1: 1 to 100: 1, 1: 1 to 20: 1 for polyPEGylation, 1: 1 to 5: 1.

  In the present invention, “stem cell factor” (SCF) refers to a protein consisting of an amino acid sequence having a total length of 273 amino acid residues that plays an important role in hematopoiesis and reproductive development. Further, in the present invention, the “SCF variant” means one or more modifications selected from substitution, deletion, insertion and addition, and specifically stimulates a c-kit molecule which is a receptor for SCF. Means a mutant of SCF to be activated. It is known that SCF retains activity even when the signal peptide from the N-terminal to the 25th threonine residue is deleted. SCF is digested by proteolytic enzymes between the 189th alanine residue and the 190th alanine residue from the N-terminal, or between the 190th alanine residue and the 191st serine residue. Is known to exist as a solubilized SCF. In addition, it is known that there are a plurality of alternative forms, and it is known that there is a membrane-bound SCF from which the 175th serine residue to the 202th isoleucine residue are deleted from the N-terminus. Yes. In the present invention, any of these mutants can be used as the “SCF mutant”.

  In the present invention, “IL-3” is a protein that acts on hematopoietic stem / progenitor cells and has an activity to support colony formation. IL-3 has an important function as a growth factor for differentiated cells such as mast cells, eosinophils, basophils, neutrophils and monocytes. IL-3 consists of an amino acid sequence having a total length of 152 amino acid residues. In the present invention, “IL-3 mutant” has one or more alterations selected from substitution, deletion, insertion and addition, and specifically stimulates the IL-3 receptor to activate. It means a mutant of IL-3 to be activated. It is known that IL-3 retains activity even when the signal peptide from the N-terminus to the 19th glutamine residue is deleted. In the present invention, such a mutant can be used as “IL-3 mutant”.

  In the present invention, “Flt-3 ligand” (FL) is a protein having an important function as a growth factor of hematopoietic stem / progenitor cells. FL is a membrane-bound protein consisting of an amino acid sequence having a total length of 235 amino acid residues. In the present invention, the “FL mutant” has one or more modifications selected from substitution, deletion, insertion and addition, and is specifically activated by stimulating the Flt-3 tyrosine kinase receptor. A FL mutant (fragment) consisting of an amino acid sequence from the first amino acid residue to the 185th proline residue. It is known that FL retains its activity even when the signal peptide from the N-terminal to the 26th glycine residue is deleted. In the present invention, any of these mutants can be used as the “FL mutant”.

  According to a preferred embodiment of the invention, the cytokine is stem cell factor (SCF), thrombopoietin (TPO) and variants thereof and derivatives thereof, Flt-3 ligand (FL), interleukin-3 (IL-3) and It is selected from its variants, and combinations of two or more thereof, more preferably selected from SCF, TPO, IL-3 and Flt-3 ligands, and combinations of two or more thereof. According to a particularly preferred embodiment of the invention, the cytokine is a combination of SCF, TPO and IL-3 or a combination of SCF, TPO, IL-3 and Flt-3 ligand.

  The cell culture medium used in step (a) is, for example, the following group: granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-7 (IL-7) A growth factor such as anti-human CD40 antibody, anti-human CD3 antibody and anti-human CD28 antibody; one selected from the group consisting of growth stimulating factors such as lipopolysaccharide (LPS) The above growth stimulating factor may be further included. In some cases, the induction of pluripotent stem cells may be more efficient due to enhanced expression of IL6ST by these factors.

  The amount of these cytokines added to the cell culture medium is not particularly limited, but is preferably 1 ng / ml to 100 μg / ml, more preferably 10 ng / ml to 100 ng / ml.

The basal medium used for the culture is not particularly limited as long as the proliferation and survival of somatic cells such as blood cells and pluripotent cells are not impaired, and examples thereof include D-MEM medium, D-MEM / F12 medium, and MEM-α medium. Opti-MEM medium, IMDM medium, RPMI 1640 medium, StemPro medium (Invitrogen), mTeSR 1 medium (StemCell Technologies), HEScGRO medium (Millipore), and N2B27 medium are preferably used. The culture temperature is usually 25 to 39 ° C, preferably 33 to 39 ° C. Examples of substances added to the medium include fetal bovine serum, human serum, knockout serum replacement (KnockOutSerumReplacement (KSR): Invitrogen), insulin, transferrin, lactoferrin, ethanolamine, sodium selenite, monothioglycerol, 2- Examples include mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, and various amino acids. CO ₂ is usually 4-15%, preferably 5-10%. O ₂ is usually 5% to 25%. In addition, thrombopoietin (TPO) and its mutants and their derivatives, c-mpl ligand (excluding TPO) and its derivatives, stem cell factor (SCF), Flt-3 ligand (FL), interleukin-3 ( IL-3), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Growth factors such as interleukin-7 (IL-7); antibodies having growth stimulating activity such as anti-human CD40 antibody, anti-human CD3 antibody and anti-human CD28 antibody; growth stimulating factors such as lipopolysaccharide (LPS) In addition to cytokines such as transforming growth factor-β (TGF-β), bone morphogenetic factor (BMP) factor Millie, hedgehog factor (Hh) family, growth factors represented by bFGF, CC chemokines such as MIP-1α and SDF-1, or hematopoietic hormones such as CXC chemokine, erythropoietin (EPO), Wnt family gene products, Notch Compounds such as a ligand family gene product, lithium chloride, pipecolic acid, and γ-aminobutyric acid (GABA) can be added in the range of 10 ng / ml to 100 μg / ml.

  In the culture in the present invention, culture using a petri dish, flask, or culture bag for culture is possible, but the culture is controlled by a bioreactor capable of culturing at high density by mechanically controlling the medium composition, pH, and the like. Can also be improved.

  As feeder cells used for the culture, mouse embryonic fibroblasts (MEF), SNL cells (A.P.McMahon and A.Bradley, Cell, 62, pp-1073-1085, 1990), STO cells and the like can be used. As a method for suppressing the growth of feeder cells, methods such as mitomycin C treatment and radiation irradiation can be used. Further, instead of feeder cells, a method of coating a culture substrate with Matrigel such as BD Matrigel (BD Bioscience), extracellular matrix proteins such as collagen, laminin, fibronectin, gelatin, or the like can also be used.

  The method for producing pluripotent stem cells according to the present invention further comprises a step of dedifferentiating somatic cells (hereinafter referred to as “step (b)”). This step (b) may be performed after the step (a) or may be performed simultaneously with the step (a).

  In an embodiment in which step (a) and step (b) are performed simultaneously, somatic cell dedifferentiation is performed in the cell culture medium containing the IL6ST stimulating factor and cytokine described above. That is, step (a) and step (b) are started simultaneously. In this embodiment, the medium can be changed to another medium, for example, a medium for ES cell culture, in the process of dedifferentiation. That is, in this embodiment, only step (a) may be terminated first and only step (b) may be continued.

  In an embodiment in which step (b) is performed after step (a), somatic cells are cultured in the cell culture medium containing the above-described IL6ST stimulating factor and cytokine, and then the somatic cells are dedifferentiated. Is called. Here, the dedifferentiation of somatic cells can be performed using the same medium as that used in step (a). That is, in this embodiment, step (a) is started before step (b), but step (a) does not necessarily have to be completed when step (b) is started. Furthermore, in this embodiment, the medium can be changed to another medium, for example, a medium for ES cell culture, in the process of dedifferentiation. That is, in this embodiment, only step (a) may be terminated first and only step (b) may be continued.

  In the present invention, “dedifferentiation” means that cells of differentiated tissues and organs return to a more undifferentiated state, in other words, a process opposite to differentiation. The somatic cell dedifferentiation in the step (b) can be performed by a method known in the art.

  According to a preferred embodiment of the present invention, the somatic cell dedifferentiation in the step (b) is performed by nuclear reprogramming treatment of the somatic cell. Here, “nuclear initialization” means that the somatic cell nucleus returns to the fertilized egg state. Such a nuclear initialization process can be performed according to a method known in the art, for example, a method described in WO2007 / 069666.

  The nuclear reprogramming factor used for the nuclear reprogramming process includes an Oct family gene (for example, Oct3 / 4 gene), a Klf family gene (for example, Klf4 gene), a Sox family gene (for example, Sox2 gene), and a Myc family gene (for example, c- Each gene product of (Myc gene) is known (WO2007 / 069666). Thus, nuclear reprogramming can be performed by contacting somatic cells with these gene products. Alternatively, the nuclear reprogramming treatment can be performed by activating or expressing these genes in somatic cells. According to a preferred embodiment of the present invention, nuclear reprogramming treatment of somatic cells comprises Oct family gene (eg Oct3 / 4 gene), Klf family gene (eg Klf4 gene), Sox family gene (eg Sox2 gene) and Myc family. Introducing a gene (eg, c-Myc gene) into a somatic cell in an expressible form.

  Examples of the medium used for nuclear reprogramming treatment include the following groups: granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and interleukin-7 (IL-7). One or more selected from the group consisting of growth stimulating factors such as lipopolysaccharide (LPS); growth factors such as anti-human CD40 antibody, anti-human CD3 antibody and anti-human CD28 antibody. A growth stimulating factor may be included. Increased expression of the nuclear reprogramming gene family by these factors may lead to more efficient induction of pluripotent stem cells.

  As a nuclear reprogramming factor used for the nuclear reprogramming treatment, a ribonucleic acid such as cytokines, chemokines, growth factors, low molecular weight compounds, microRNA, or the like may be used instead of the above genes. Examples of such nuclear reprogramming factors include transforming growth factor-β (TGF-β), bone morphogenetic factor (BMP) family, hedgehog factor (Hh) family, growth factors represented by bFGF, MIP, and the like. CC chemokines such as -1α and SDF-1 or CXC chemokines, hematopoietic hormones such as erythropoietin (EPO), Wnt family gene products, Notch ligand family gene products, lithium chloride, pipecolic acid, γ-aminobutyric acid (GABA), etc. Examples thereof include compounds, low-molecular compounds that enhance the expression of nuclear reprogramming factors, and microRNA molecules such as siRNA of factors involved in the transcriptional regulation of nuclear reprogramming factors.

  The method for introducing the nuclear reprogramming factor used in the nuclear reprogramming process is not particularly limited. For example, retrovirus vector, lentivirus vector, adeno-associated virus vector, adenovirus vector, herpes simplex virus vector, feline endogenous virus Examples thereof include a method using a vector, a vector for animal cells, etc., preferably a method using a retrovirus vector or a lentivirus vector, more preferably a method using a retrovirus vector. Alternatively, a method of injecting a nuclear reprogramming factor into cells may be used, or a method of adding each factor to cells by modifying membrane permeability may be used.

  The method for introducing a gene used for nuclear reprogramming is not particularly limited, and a general method used for gene introduction into animal cells, for example, retrovirus vectors such as Moloney murine leukemia virus, adenovirus vectors, Methods using animal cell vectors derived from viruses such as adeno-associated virus (AAV) vector, herpes simplex virus vector, lentivirus vector, calcium phosphate coprecipitation method, DEAE-dextran method, electroporation method, liposome method, lipofection method, A microinjection method, an HVJ liposome method, or the like can be used. According to a preferred embodiment of the present invention, the method of introducing a gene used for nuclear reprogramming is a method using a virus-derived animal cell vector, more preferably a retrovirus vector.

  In the method for producing pluripotent stem cells according to the present invention, somatic cells used as a starting material are not particularly limited, and any somatic cells may be used. In addition, as the somatic cell, a cell that has been once frozen and then stored may be used.

  According to a preferred embodiment of the present invention, the somatic cell is a blood cell. In the present invention, “blood cell” means a group of cells equivalent to blood cells that may be present in the bloodstream in a living body, and is not necessarily limited to peripheral blood cells. For example, in bone marrow or umbilical cord blood. It may exist. More specifically, “blood cell” refers to a cell that expresses a blood cell marker molecule such as CD45 or CD11a. In addition, it has been found herein that pluripotent stem cells can be established more efficiently than other tissue cells by using blood cells that have been confirmed to be present in the bone marrow. Therefore, according to a preferred embodiment of the present invention, the blood cell is a bone marrow-derived mononuclear cell. Furthermore, according to another preferred embodiment of the present invention, the blood cell is a differentiated blood cell. Such blood cells may be cells that have been collected and then cryopreserved.

  According to another preferred embodiment of the present invention, the somatic cells (particularly blood cells) are cultured in the presence of an IL6ST stimulating factor and the above cytokines, and the retrovirus having the vesicular stomatitis virus G protein as a coating protein. The cells are easily infected by viruses. Such cells are advantageous for introducing genes by retroviral vectors.

  According to another preferred embodiment of the present invention, the somatic cells (particularly blood cells) are cells that highly express IL6ST. Specifically, although IL6ST is expressed in any blood cell, it is conceivable to use T cells, monocytes, DC cells, CD34 positive cells, etc. that are highly expressed among them. Also, pluripotent stem cells can be efficiently produced by using somatic cells that highly express IL6ST other than blood cells. Specifically, cells such as liver, kidney, skeletal muscle, lung, pancreas, heart, small intestine, spinal cord and salivary gland can also be used.

  According to another preferred embodiment of the present invention, the somatic cells (particularly blood cells) are cells expressing a receptor for the cytokine used in step (a) or (b). Specifically, it is considered that CD34 positive cells and the like are used as cells expressing c-kit (SCF receptor), c-mpl (TPO receptor), and Flt3. Moreover, it is possible to use a monocyte etc. as a cell which expresses IL-3R (alpha) (IL-3 receptor) etc. Examples of cells other than blood cells include cells that express c-kit.

  According to another preferred embodiment of the present invention, the somatic cells (particularly blood cells) are cells that express at least one nuclear reprogramming factor. For example, examples of cells that express Klf4, which is one of the nuclear reprogramming factors, include monocytes, DC cells, CD34 positive cells and the like among blood cells, and in other tissues, lung, skeletal Examples include muscle and small intestine. In addition to Klf4, somatic cells that express a Klf family gene, Oct3 / 4 family gene, Myc family gene, or Sox family gene can be used. For reprogramming genes such as Oct3 / 4 family genes and Nanog genes, cells expressing those pseudogenes can also be used as somatic cells.

  According to another preferred embodiment of the present invention, the somatic cells (particularly blood cells) are cells in which the expression of a nuclear reprogramming factor is increased in the presence of an IL6ST stimulating factor and the above cytokine. For example, it is conceivable to use cells expressing c-kit (SCF receptor), c-mpl (TPO receptor), Flt3, IL-3Rα (IL-3 receptor), etc., which are receptors for each cytokine. Among blood cells, CD34 positive cells, DC cells and the like can be mentioned. In addition, by using cells in which expression of Oct3 / 4 family gene, Klf family gene, Myc family gene, and / or Sox family gene, which are nuclear reprogramming factors, is increased by stimulation of these receptors with the above cytokines, Without using all these reprogramming factors, pluripotent stem cells can be produced.

  According to a preferred embodiment of the present invention, the somatic cells (particularly blood cells) are human cells. In this embodiment, the IL6ST stimulating factor, cytokine, nuclear reprogramming factor or gene thereof used in the present invention is preferably all derived from human. This makes it possible to efficiently produce human pluripotent stem cells.

  Confirmation of dedifferentiation or reprogramming in cells produced according to the present invention can be performed by methods known in the art. As such a method, for example, a method using an antibody against an undifferentiated marker expressed on the cell surface and analyzing using a fluorescence excitation cell separation device (FACS) can be mentioned. Specifically, antibodies against undifferentiated markers such as SSEA-3, SSEA-4, TRA1-60, TRA1-81 can be used. In addition, reporter genes of undifferentiated markers such as Nanog and Oct3 / 4 gene can be introduced into cells and used.

  The cell culture medium containing IL6ST stimulating factor and at least one cytokine used in step (a) of the present invention can also be used to maintain the properties of the produced pluripotent stem cells. That is, the IL6ST stimulating factor and the above cytokines can be used not only for promoting the nuclear reprogramming process but also for maintaining the reprogrammed state after the nuclear reprogramming.

  Reagents used in the method for producing pluripotent stem cells according to the present invention can be collected into a kit. Thus, according to another aspect of the present invention, there is provided a kit for producing pluripotent stem cells from somatic cells, the kit comprising (a) an IL-6 signaling factor stimulator and at least one cytokine. A cell culture medium for culturing somatic cells, and (b) a reagent for dedifferentiating somatic cells.

  A suitable IL-6 signaling factor stimulating factor and a suitable cytokine are as described above. The reagent for dedifferentiating somatic cells is also as described above. Preferably, the Oct family gene, the klf family gene, the Sox family gene, and the Myc family gene are introduced into the somatic cell in an expressible form. A vector for The kit according to the present invention may further contain reagents, reaction containers, instructions, etc. depending on the specific method used for the method for producing pluripotent stem cells.

  The pluripotent stem cells produced according to the present invention can be induced in various differentiated cells and can be used for cell regenerative medicine. That is, this pluripotent stem cell can be used for both transplantation systems of autologous cell transplantation using the patient's own somatic cells and the same type of cell transplantation using cells obtained from a cell bank or the like. A cell bank of pluripotent stem cells can also be constructed from a large cell bank of blood cells that already exist.

  Moreover, according to the present invention, it is possible to construct a pluripotent stem cell bank corresponding to disease information from blood cell samples from patients with various diseases. Furthermore, by inducing various differentiated cells using pluripotent stem cells in which healthy human-derived blood cells are reprogrammed, or inducing disease target cells using blood cells obtained from patients with intractable diseases Therefore, it is possible to construct various human model cell lines as a drug discovery screening tool.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example 1: Induction of pluripotent cells from human bone marrow-derived mononuclear cells (1)
A. Culture of Human Bone Marrow-derived Mononuclear Cells Human bone marrow-derived mononuclear cells (hBMMNCs) 2.5 × 10 ⁶ cells (Allcells), which have been cryopreserved, are seeded in two 100 mm Petri dishes, 37 ° C., 5% CO ₂ and the culture was started in DMEM medium containing 10% FBS. In one dish, human SCF (100 ng / ml), human TPO (100 ng / ml), and human IL-3 (100 ng / ml) are added, and in the other dish, SCF, TPO and IL-3 are added. FP6 (20 ng / ml) was added. Here, a cytokine cocktail comprising SCF, TPO and IL-3 is referred to as “ST3”, and a cocktail obtained by adding FP6 to ST3 is referred to as “ST3FP6” (the same applies hereinafter). 24 hours after the start of the culture, each cytokine cocktail was added again to each dish at the same concentration. 48 hours after the start of the culture, each cell was collected and suspended in 500 μl of DMEM medium containing 10% FBS. ST3 was added at a concentration of 300 ng / ml to the cell suspension cultured in the presence of ST3, and 60 ng of FP6 was added to the cell suspension cultured in the presence of ST3FP6 in addition to ST3 (each 300 ng / ml). Added at a concentration of / ml. The prepared hBMMNCs were used for the next virus infection experiment.

B. Preparation of recombinant retrovirus vector containing nuclear reprogramming gene 12 × 10 ⁶ G3T-hi cells (Takara Bio Inc.) were seeded on 12 collagen type I-coated 100 mm dishes, respectively, at 37 ° C. and 5% CO _2. The cells were cultured in a DMEM medium containing 10% FBS. After 24 hours of culturing, a viral vector was introduced using three dishes for one type of nuclear reprogramming gene. For each dish, 6 μg of pGP vector (Takara Bio), 6 μg of pVSV-G vector (CLONTECH), and retroviral vector pMXs (T. Kitamura et al., Experimental Hematology, 31, pp. 1007-1014, 2003; Tokyo) 12 μg of a plasmid into which a nuclear reprogramming gene was inserted was co-introduced using a gene introduction reagent FuGene6 (Roche Diagnostics). The four nuclear reprogramming genes used were human Oct3 / 4, human Sox2, human Klf4, and human c-myc. Nuclear reprogramming gene expression retroviral vectors: pMXshOct3 / 4, pMXshSox2, pMXshKlf4, and pMXshc-myc were constructed according to the method of Yamanaka et al. (K. Takahashi, S. Yamanaka, Cell, 131, pp.861-872). , 2007). Approximately 48 hours after gene introduction, a culture solution is collected for each type of nuclear reprogramming gene, the culture solution is filtered through a 0.45 μm filter, centrifuged at 5800 × g for 4 hours at 4 ° C., and the culture supernatant is discarded. A virus precipitate was obtained. All virus precipitates were combined and suspended in 2 ml of DMEM medium containing 10% FBS. Finally, the liquid volume became about 2.4 ml.

C. Treatment of human bone marrow-derived mononuclear cells with virus infection To a 12-well plate previously coated with 50 μg / ml of RetroNectin (Takara Bio Inc.), 1.2 ml of the suspended virus precipitate was added per well, and 1080 × g And centrifuged at 20 ° C. for 2 hours. 500 μl of the cell suspension of hBMMNCs was added to each well on the plate after centrifugation, and culture was started at 37 ° C. and 5% CO ₂ . Approximately 24 hours after the start of the culture, cytokines were added again.

Approximately 48 hours after the start of virus infection, cells in each well were collected with trypsin EDTA solution (GIBCO), and 10 × 10 × 10 × 10 gelatin dish containing 5 × 10 ⁵ / dish mouse embryonic fibroblasts as feeder cells in advance. Cells were reseeded using DMEM medium with% FBS. Cytokines were added again to each dish. Further, after about 24 hours and about 72 hours, the medium was exchanged with DMEM medium containing 10% FBS containing cytokine. Thereafter, after 6 days from the start of virus infection, the medium was replaced with a medium for culturing human ES cells not containing ST3 or ST3FP6. The composition of the medium used was: DMEM-F12 medium (SIGMA) 500 ml, non-essential amino acid solution (SIGMA) 5 ml, 200 mM L-Glutamine (SIGMA) 6.25 ml, KNOCKOUT ^™ Serum Replacement (KSR) (Invitrogen) 125 ml, 2-mercaptoethanol (SIGMA) 5 μl, 5N NaOH 638 μl, Human bFGF (UBI) final concentration 5 ng / ml.

Thereafter, the medium was changed every day. Ten days after the start of virus infection, several colonies were confirmed in both dishes (FIG. 1B). In the case of hBMMNCs cultured in the presence of ST3FP6 at this time, relatively large squamous colonies were included, whereas in the case of hBMMNCs cultured in the presence of ST3, the colonies that appeared were still small and resembling mouse ES cells. The colony. After 15 days, 24 colonies were picked up, and the remaining cells were replated in a gelatin-coated 100 mm dish previously seeded with feeder cells. After 20 days, the colonies were picked up again, half of the remaining cells were seeded, and the expression of the human ES cell marker SSEA-4 was confirmed by FACS for the remaining half of the cells (FIG. 1A). In the analysis by FACS, the collected cells were washed with _{3 to} 5 ml of staining medium (SM) (PBS containing 5% FBS, 0.05% NaN ₃ and 0.5 mM EDTA), and then phycoerythrin-conjugated anti-SSEA4 antibody ( (BD Bioscience) 5 μl was added and allowed to stand in ice for 15 minutes. After washing with 6 ml of SM, the suspension was resuspended with 1 ml of SM solution containing 1 μg / ml of propidium iodide (PI) for detecting dead cells, and FACS Calibur (BD Bioscience) or FACS Aria SORP (BD Bioscience) Measurement was performed using As a result, when hBMMNCs cultured in the presence of ST3FP6 were used, about 20% of cells expressing stem cell marker SSEA-4 reached 20 days after retrovirus infection, and FP6 was not added. It was shown that it reached 2.5 times or more compared to about 15%. Further, it is known that alkaline phosphatase is expressed in human ES cells. Therefore, at the time after 27 days, alkaline phosphatase activity staining was performed according to a conventional method (FIG. 1C). As a result, it was confirmed that most of the colonies at this point were positive for alkaline phosphatase. From the above, it was found that when hBMMNCs cultured in the presence of ST3FP6 were used, the appearance time and the number of cells of pluripotent stem cells were markedly higher than when hBMMNCs cultured in the presence of ST3 were used. .

Example 2: Induction of pluripotent cells from human bone marrow-derived mononuclear cells (2)
A. Culture of Human Bone Marrow-derived Mononuclear Cells Human bone marrow-derived mononuclear cells (hBMMNCs) 2.5 × 10 ⁶ cells (Allcells), which have been cryopreserved, are seeded in a 100 mm Petri dish, 37 ° C., 5% CO ₂ and the culture was started in DMEM medium containing 10% FBS. Human SCF (100 ng / ml), human TPO (100 ng / ml), human IL-3 (100 ng / ml), human Flt3 ligand (100 ng / ml), and FP6 (20 ng / ml) were added to the medium. Here, a cytokine cocktail consisting of SCF, TPO, IL-3, Flt3 ligand, and FP6 is referred to as “ST3FLFP6” (the same applies hereinafter). 24 hours after the start of the culture, ST3FLFP6 was added again at the same concentration. Cells were collected 48 hours after the start of culture, and 5 × 10 ⁵ cells were suspended in 500 μl of DMEM medium containing 10% FBS. In addition to ST3FL (each 300 ng / ml), FP6 was added to the cell suspension at a concentration of 60 ng / ml. The prepared hBMMNCs were used for the next virus infection experiment.

B. Production of recombinant retrovirus vector containing nuclear reprogramming gene Sixteen collagen type I-coated 100 mm dishes were seeded with 2.5 × 10 ⁶ G3T-hi cells (Takara Bio Inc.) at 37 ° C., 5% CO ₂ under were cultured in 10% FBS-containing DMEM medium. After 24 hours of culturing, viral vectors were introduced using 4 dishes of one type of nuclear reprogramming gene. For each dish, 6 μg of pGP vector (Takara Bio), 6 μg of pVSV-G vector (CLONTECH), and 12 μg of a plasmid in which the nuclear reprogramming gene was inserted into the retroviral vector pMXs were used as a gene transfer reagent FuGene6 (Roche Diagnostics) ). The four nuclear reprogramming genes used were human Oct3 / 4, human Sox2, human Klf4, and human c-myc. Approximately 48 hours after gene introduction, the culture solution is collected for each type of nuclear reprogramming gene, the culture solution is filtered through a 0.75 μm filter, centrifuged at 5000 × g for 4 hours at 4 ° C., and the culture supernatant is discarded. A virus precipitate was obtained. All virus precipitates were combined into one, and 500 μl of DMEM medium containing 10% FBS was added and resuspended. As a result, the liquid volume finally reached about 1 ml.

C. Treatment of human bone marrow-derived mononuclear cells with virus infection To one well in a 12-well plate previously coated with 50 μg / ml of RetroNectin (Takara Bio Inc.), the suspended virus precipitate was added to 1080 × g, 20 Centrifugation was performed at 0 ° C. for 2 hours. To the plate after centrifugation, 500 μl of the cell suspension of hBMMNCs was added, and culture was started at 37 ° C. and 5% CO ₂ . About 24 hours after the start of the culture, the medium was replaced with a medium containing ST3FLFP6.

Approximately 48 hours after the start of virus infection, cells were collected with trypsin EDTA solution (GIBCO), and 10% FBS was added to a gelatin-coated 100 mm dish in which mouse embryonic fibroblasts of 5 × 10 ⁵ cells / dish were previously used as feeder cells. Cells were reseeded using DMEM medium supplemented with ST3FLFP6. Further, after about 48 hours, the medium was exchanged with a DMEM medium supplemented with 10% FBS and ST3FLFP6. Thereafter, from 6 days after the start of virus infection, the medium was replaced with a medium for human ES cell culture (not described in Example 1C) not containing ST3FLFP6.

  Thereafter, the medium was changed every day. Eleven days after the start of virus infection, several colonies were confirmed in both dishes. After 18 days, 24 colonies were picked up and the remaining cells were re-seeded in a gelatin-coated 100 mm dish previously seeded with feeder cells. The cells after 22 days were collected, and the expression of human ES cell marker SSEA-4 was analyzed by FACS following the method of Example 1. The result is shown in FIG.

Example 3: Analysis of retrovirally infected human bone marrow-derived mononuclear cells Analysis of Retrovirus Infectivity of Human Bone Marrow-Derived Mononuclear Cells The retroviral infection efficiency was examined using cryopreserved hBMMNCs (Allcells). Following the method of Example 1, a pMXsIRES-EGFP (pMXsIG) vector expressing green fluorescent protein EGFP (T. Kitamura et al., Experimental Hematology, 31, pp.1007-1014) instead of the nuclear reprogramming factor expression pMXs vector. , 2003; provided by Professor Toshio Kitamura of the University of Tokyo).

  The culture conditions of hBMMNCs were carried out in the following three types: (i) Retrovirus infection was performed using the cells after thawing without adding cytokine; (ii) As in Example 1, containing 10% FBS Pre-culture and retroviral infection were performed by adding human SCF (100 ng / ml), human TPO (100 ng / ml), and human IL-3 (100 ng / ml) to DMEM medium (ST3); (iii) Examples Similar to 1, human SCF (100 ng / ml), human TPO (100 ng / ml), human IL-3 (100 ng / ml), and FP6 (20 ng / ml) were added to DMEM medium containing 10% FBS (ST3FP6). ), Preculture and retroviral infection were performed.

  Retrovirus preparation and infection methods were performed as in Example 1. Two days after infection, the cells were collected, washed with SM solution, resuspended in 1 ml of SM solution containing PI (1 μg / ml), and FACS analysis was performed using FACS Calibur (BD Bioscience) ( FIG. 3). As a result, in the case of (i) in which no culture was added with cytokines, no cells infected with retrovirus were detected, whereas by adding ST3 ((ii)) 4% of the cells became infected with retrovirus, and the addition of FP6 followed by addition of ST3FP6 ((iii)) increased the number of cells infected with retrovirus to about 8%. On the other hand, when the retrovirus used in this example was infected to NIH3T3 cells as control cells, infection was possible with an efficiency of 99%, and thus the infection ability of the retrovirus itself was sufficient.

B. Analysis of surface antigen markers of infected cells Next, in the same manner as in Example 1 and Example 2, hBMMNCs cultured in the presence of ST3, ST3FP6, or ST3FLFP6 were positive for EGFP when a retrovirus prepared from the pMXsIG vector was infected. The infected cells were immunostained using antibodies against various surface antigen markers and analyzed using FACS.

  The collected cells were washed with 3-5 ml of SM solution, 5 μl of various antibodies were added, and the mixture was allowed to stand in ice for 15 minutes. After washing with 6 ml of SM, the suspension was resuspended with 1 ml of SM solution containing PI (1 μg / ml), and measurement was performed with FACS Calibur (BD Bioscience). The antibodies used were allophycocyanin-conjugated anti-human CD45 antibody, allophycocyanin-conjugated anti-human CD34 antibody, phycoerythrin-conjugated anti-human CD38 antibody, allophycocyanin-conjugated anti-human CD11b antibody (above, BD Biosciences), phycoerythrin-conjugated. They were anti-human CD3 antibody, phycoerythrin-conjugated anti-human CD117 antibody, and phycoerythrin-conjugated anti-human CD19 antibody (above, IMMUNOTECH). The CD45 antigen is expressed on the surface of all human leukocytes, ie lymphocytes, eosinophils, monocytes, basophils, and neutrophils. CD34 antigen is expressed on the most undifferentiated hematopoietic stem cells and hematopoietic progenitor cells. CD38 antigen is expressed on activated T and B lymphocytes, NK cells, monocytes, plasma cells and thymic medullary cells. CD11b antigen is found in bone marrow monocytic cells and is strongly expressed in NK cells, granulocytes, monocytes / macrophages. CD3 antigen is expressed on mature T cells and thymocytes. The CD117 antigen is expressed on only a few normal bone marrow cells, but is also expressed on mast cells. The CD19 antigen is expressed on all normal B cells, including early B cells, but disappears when maturing into plasma cells.

  As a result of analysis, CD45, which is a leukocyte marker, was positive in almost all retrovirus-infected cells, almost all were CD34 positive cells in ST3, and about 60% were CD34 positive cells in ST3FP6 or ST3FLFP6. On the other hand, markers such as CD11b, CD3, and CD19 were all negative (FIG. 4).

Example 4: Analysis of retrovirally infected human peripheral blood-derived mononuclear cells Using cryo-preserved human peripheral blood-derived mononuclear cells (hPBMNCs) (Allcells) using the pMXsIG vector in the same manner as in Example 3. A retrovirus infection experiment was conducted. The infection efficiency when hPBMNCs were cultured using a cytokine cocktail and EGFP-positive infected cells were immunostained using antibodies against various surface antigen markers, and the infected cells were identified using FACS.

  For the culture of hPBMNCs, human SCF (100 ng / ml), human TPO (100 ng / ml), human IL-3 (100 ng / ml), FP6 (20 ng / ml), Flt3 ligand (100 ng) in DMEM medium containing 10% FBS. / Ml), human GM-CSF (100 ng / ml), GM culture system added with human M-CSF (50 ng / ml), human SCF (100 ng / ml), human TPO (100 ng / ml), Human IL-3 (100 ng / ml), FP6 (20 ng / ml), LPS (1 μg / ml), anti-human CD40 antibody # 341 (Kirin Pharma) (100 ng / ml), anti-human CD3 antibody (OKT3 Janssen Pharma) (1 μg / ml) and anti-human CD28 antibody (eBioscience) (1 μg / ml) were added. Using a culture system of two types of pre-cultured TB culture system, it was carried out retroviral infection. Retrovirus preparation and infection methods were performed as in Example 1. Two days after infection, the cells were collected, and the collected cells were washed with 3 to 5 ml of SM solution, 5 μl of various antibodies were added, and the mixture was allowed to stand in ice for 15 minutes. After washing with 6 ml of SM, the suspension was resuspended with 1 ml of SM solution containing PI (1 μg / ml), and measurement was performed with FACS Calibur (BD Bioscience).

  The antibodies used were allophycocyanin-conjugated anti-human CD45 antibody, allophycocyanin-conjugated anti-human CD34 antibody, phycoerythrin-conjugated anti-human CD38 antibody, allophycocyanin-conjugated anti-human CD11b antibody, phycoerythrin-conjugated anti-human CD13 antibody (hereinafter referred to as BD). Bioscience), phycoerythrin-conjugated anti-human CD3 antibody, phycoerythrin-conjugated anti-human CD117 antibody, and phycoerythrin-conjugated anti-human CD19 antibody (hereinafter, IMMUNOTECH). CD13 antigen is expressed on most myeloid cells such as normal peripheral blood neutrophils, eosinophils, basophils and monocytes.

  As a result of analysis, CD45, which is a leukocyte marker, was positive in almost all retrovirus-infected cells, and about half of them were CD34 positive in the GM culture system, as in the case of using bone marrow cells. In the TB culture system, it was positive for CD3 antigen, which is a T cell marker (FIG. 5). By using this system, there is a possibility that pluripotent cells can be induced from T cells or the like present in peripheral blood.

Example 5: Surface antigen analysis of human induced pluripotent cell clones Results of Example 1 and Example 2 The cloned cell lines were observed for colony morphology and confirmed the expression of human ES cell marker SSEA-4 by FACS. The FACS analysis method was the same as in Example 1.

  Clone # 16 and # 30 obtained using ST3 culture conditions, clone # 22 and # 33 obtained using ST3FP culture conditions, and clones # 1 and # 4 obtained using ST3FLFP culture conditions were analyzed. The results are shown in FIG. All of the clones were confirmed to significantly express stem cell marker SSEA-4.

Example 6: RT-PCR analysis of human-derived pluripotent cell clones Regarding the pluripotent stem cell clones obtained as a result of Examples 1 and 2 including the clones analyzed in Example 5, the expression of mRNA of stem cell-specific genes was determined by RT -Analyzed using PCR method.

  Each clone cell was seed | inoculated on the feeder cell of a mouse embryonic fibroblast, and RNA was collect | recovered from the cell cultured in the culture medium for human ES cells. The composition of the medium used was: DMEM-F12 medium (SIGMA) 500 ml, non-essential amino acid solution (SIGMA) 5 ml, 200 mM L-Glutamine (SIGMA) 6.25 ml, KNOCKOUT ™ Serum Replacement (KSR) (Invitrogen) 125 ml 2-mercaptoethanol (SIGMA) 5 μl, 5N NaOH 638 μl, Human bFGF (UBI) final concentration 5 ng / ml. The collected cells were washed with PBS buffer, and the cell precipitate was frozen in liquid nitrogen. As control cells, BMMNCs after freezing and thawing were cultured on feeder cells for several hours, and feeder cells (MEF cells) were prepared. Total RNA was prepared from the frozen cell sample using RNeasy Plus Mini Kit (QIAGEN) according to the attached protocol. Using the prepared RNA, cDNA was synthesized by SuperScriptIII First Strand Synthesis System (Invitrogen) according to the attached protocol. Using LA Taq DNA polymerase (Takara Bio Inc.) using the synthesized cDNA as a template, 35 cycles of PCR reaction with one cycle consisting of 96 ° C-20 seconds, 55 ° C-30 seconds, and 72 ° C-30 seconds Carried out. The primers used for the PCR reaction are the same as those used by Yamanaka et al. (K. Takahashi, S. Yamanaka, Cell, 131, pp. 861-872, 2007), and the base sequences of the primers are shown in Table 1. Each PCR reaction was performed using a combination of forward (Fw) and reverse (Rv) primers for each gene. As a control for RT reaction and PCR reaction, a housekeeping gene, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene, was used. FIG. 7 shows the results of electrophoresis of the PCR reaction product on an agarose gel containing ethidium bromide and observation with a UV transilluminator. As a result of the analysis, it was confirmed that all the clones expressed the stem cell marker gene.

Example 7: Induction of pluripotent cells from mouse bone marrow derived mononuclear cells (1)
A. Obtaining mouse bone marrow-derived mononuclear cells Oct4-EGFP transgenic mice (10-20 weeks of age) known to express green fluorescent protein EGFP in undifferentiated pluripotent stem cells (K. Ohbo et al. , Dev. Biol., 258, pp.209-225, 2003; provided by Associate Professor Kazuyuki Ohbo, Yokohama City University). The bone marrow in the femur was collected and suspended in PBS. According to a conventional method (Toshishi Takatsu, basic technology of immunological research, Yodosha 1995), the mononuclear cell fraction was concentrated from the bone marrow by specific gravity centrifugation, and this was used as mouse bone marrow mononuclear cell (mBMMNCs).

B. Production of recombinant retroviral vectors containing nuclear reprogramming genes For packaging for 1 × 10 ⁶ each in 10 collagen type I-coated 60 mm dishes and 1 × 10 ⁵ retrovirus in each well of a 6-well plate Cells: PLAT-E cells (S. Morita et al., Gene Therapy, 7, pp.1063-1066, 2000; provided by Professor Toshio Kitamura, University of Tokyo), 37%, 5% CO ₂ , 10% The cells were cultured in DMEM medium containing FBS. After 24 hours of culture, viral vector gene transfer was performed using two 60 mm dishes for one type of mouse nuclear reprogramming gene. For each dish, 3 μg of a plasmid in which a nuclear reprogramming gene was inserted into the retrovirus vector pMXs was introduced using a gene introduction reagent FuGene6 (Roche Diagnostics). The nuclear reprogramming genes used were four types: mouse Oct3 / 4, mouse Sox2, mouse Klf4, and mouse c-myc. Construction of nuclear reprogramming gene expression retroviral vectors, pMXsmOct3 / 4, pMXsmSox2, pMXsmKlf4, and pMXsmc-myc was performed according to the method of Yamanaka et al. (K. Takahashi, S. Yamanaka, Cell, 126, pp. 663-676). , 2006). In addition, 3 μg of each of the above four genes was also introduced into 3 wells of one 60 mm dish and 6 well plate. As a control, 3 μg of pMXsIG vector expressing green fluorescent protein EGFP was introduced into one 60 mm dish. Approximately 48 hours after gene introduction, one 60 mm dish was collected for each nuclear reprogramming gene, filtered through a 0.22 μm filter, centrifuged at 5800 × g for 4 hours at 4 ° C., and the culture supernatant was discarded, followed by 3 ml. A concentrated virus sample (i) resuspended in the mouse ES cell culture medium was obtained. As a medium for mouse ES cells, a DMEM medium containing 20% FBS to which 1/100 volume of 2-mercaptoethanol (SIGMA) and 1/1000 volume of ESGRO (CHEMICON) were added was used. In addition, one 60 mm dish for each nuclear reprogramming gene was collected and filtered through a 0.22 μm filter to obtain 4 virus samples (ii). The culture supernatant for 3 wells of one 60 mm dish and 6 well plate into which 4 types of genes were simultaneously introduced was filtered through a 0.22 μm filter to obtain a 4 gene sample (iii). The culture supernatant into which the control pMXsIG vector was introduced was filtered through a 0.22 μm filter as a control sample (iv).

C. Treatment of mouse bone marrow-derived mononuclear cells with virus infection The above samples (i) to (iv) were added to each two wells of a 12-well plate previously coated with 33 μg / ml of retronectin (Takara Bio Inc.), and 1080 × g, centrifuged at 25 ° C. for 2 hours. The virus sample was removed from the plate after centrifugation, and 1 ml of fresh mouse ES cell culture medium was added. Furthermore, mouse SCF (100 ng / ml), human TPO (100 ng / ml), and mouse IL-3 (100 ng / ml) (ST3) were added to one well of each sample of (i) to (iv) did. Thereafter, mBMMNCs prepared from the above Oct4-EGFP mice were added to each well at 1 × 10 ⁵ cells / well, and culture was started at 37 ° C. and 5% CO ₂ .

In addition, the above-mentioned (i) to (iv) for each well in which Oct4EGFP mouse-derived embryonic fibroblasts (MEF) were seeded at a density of 2 × 10 ⁴ cells / well on the day before virus infection and cultured. ) Was added to each well and centrifuged at 1080 × g and 32 ° C. for 40 minutes, and then the culture was started at 37 ° C. and 5% CO ₂ .

  The percentage of EGFP-expressing cells in the control sample 2 days after virus infection was 34% for MEF cells, 55% for mBMMNCs without cytokines, and 60% for mBMMNCs in the presence of ST3.

  For mBMMNCs infected with 4 virus samples ((ii)), expression of the EGFP gene by the Oct4-EGFP reporter gene was observed 5 days after the start of virus infection, regardless of the addition or non-addition of cytokines (Fig. 8A / B). On the other hand, for MEF, the presence of EGFP gene-expressing cells was not observed at this time (FIG. 8C). Seven days after virus infection, for each sample derived from mBMMNCs, FACS analysis of the expression of Oct4-EGFP reporter gene and the expression of CD45 antigen as a blood cell marker was performed. For this analysis, the collected cells were washed with 3-5 ml of SM solution, 5 μl of allophycocyanin-conjugated anti-mouse CD45 antibody (BD Bioscience) was added, and the mixture was allowed to stand in ice for 15 minutes. After washing with 6 ml of SM, the suspension was resuspended with 1 ml of SM solution containing PI (1 μg / ml), and measurement was performed with FACS Calibur (BD Bioscience). The results are shown in FIG. The state of the cells before FACS analysis is shown in FIG. Almost all mBMMNCs before infection were positive for CD45 antigen, whereas many Oct4-EGFP positive cells at this point were already negative for CD45 antigen. At the same time, EGFP positive cells were sorted by FACS sorting and cloned from these cells to obtain mouse bone marrow-derived induced pluripotent stem cell (mBMiPS) clones # 7 and # 13. FACS analysis was also performed for each acquired clone (FIG. 11).

  In analysis by FACS, the collected cells were washed with 3 to 5 ml of SM solution, 5 μl of various antibodies were added, and the mixture was allowed to stand in ice for 15 minutes. After washing with 6 ml of SM, it was resuspended in 1 ml of SM solution containing PI (1 μg / ml), and measurement was performed using FACS Calibur (BD Bioscience) or FACS Aria SORP (BD Bioscience). As antibodies, allophycocyanin-conjugated anti-mouse CD45 antibody (BD Bioscience) and Alexa Fluor647-conjugated anti-mouse SSEA-1 antibody were used. Alexa Fluor 647-conjugated anti-mouse SSEA-1 antibody was prepared by labeling 100 μg of anti-mouse SSEA-1 antibody (CHEMICON) using Alexa Fluor 647 Protein Labeling Kit (Invitrogen).

Example 8: Induction of pluripotent cells from mouse bone marrow derived mononuclear cells (2)
A. Preparation of recombinant retroviral vector containing nuclear reprogramming gene 7 × 10 ⁵ PLAT-E cells were seeded on 12 collagen type I-coated 60 mm dishes, each containing 37%, 5% CO ₂ and 10% FBS. Incubated in DMEM medium. Various genes were introduced after 24 hours of culture. For four of the twelve, the virus vector was introduced using one 60 mm dish for one type of mouse nuclear reprogramming gene. For each dish, 4 μg of a plasmid containing the nuclear reprogramming gene inserted into the retroviral vector pMYs (T. Kitamura et al., Experimental Hematology, 31, pp.1007-1014, 2003; provided by Professor Toshio Kitamura, The University of Tokyo) The reagent FuGene 6 (Roche Diagnostics) was used for introduction. The four nuclear reprogramming genes used were human Oct3 / 4, human Sox2, human Klf4, and human c-myc. For four 60 mm dishes, 4 μg of each of the above four genes were introduced together. As a control, 16 μg of a pMYsIRES-EGFP (pMYsIG) vector expressing the green fluorescent protein EGFP was introduced into four 60 mm dishes.

  Approximately 48 hours after gene transfer, a total of 4 60 mm dishes each individually transfected with each nuclear reprogramming gene were collected to obtain 4 virus samples, and a total of 4 sheets of 4 types introduced simultaneously were collected to form 4 gene samples, and the pMYsIG vector was A total of 4 samples were collected and used as control samples. Each sample was filtered through a 0.22 μm filter, centrifuged at 5800 × g for 4 hours at 4 ° C., the culture supernatant was discarded, and then resuspended in 2.5 ml of mouse ES cell medium. Filtration was performed with a 22 μm filter.

B. 4. Virus infection treatment of cells Four virus samples, four gene samples, and control samples were added to each two wells of a 12-well plate coated with retronectin (Takara Bio Inc.) and centrifuged at 1080 × g at 25 ° C. for 2 hours. . The virus sample is removed from the plate after centrifugation, and Oct4-EGFP mouse BMMNCs or Oct4-EGFP mouse MEF cells suspended in the mouse ES cell medium are 1 × 10 ⁵ cells / well for each sample well. The culture was started at 37 ° C. and 5% CO ₂ .

In addition, for each well in which Oct4-EGFP mouse MEF cells were seeded on a gelatin-coated 12-well plate at 2 × 10 ⁴ cells / well on the day before the virus infection, the above three samples were added to each well. After addition and centrifugation at 1080 × g and 25 ° C. for 40 minutes, culture was started at 37 ° C. and 5% CO ₂ .

  The percentage of EGFP-expressing cells in the control sample 2 days after the start of virus infection was 95% for MEF cells infected with retronectin-coated plates, 65% for MEF cells previously cultured on gelatin-coated plates, and infected with retronectin-coated plates. It was 26% for mBMMNCs (FIG. 12). Thereafter, FACS sorting was performed on the 7th day from the start of virus infection (FIG. 13), and the number of appearance colonies was counted on the 12th day. The results are summarized in Table 3.

Example 9: Pluripotency analysis of mouse bone marrow-derived pluripotent cells In order to evaluate differentiation pluripotency for mBMiPS clones # 7 and # 13, nude mice (Balb / cAJcl-nu) were transplanted subcutaneously. It is known that undifferentiated ES cells form teratomas when transplanted in vivo. Mouse ES cell strain E14 was used as a control cell. 1 × 10 ⁶ cells per nude mouse were suspended in 100 μl serum-free DMEM medium and administered subcutaneously. As a result, a teratocarcinoma having the same size as that obtained when mouse ES cells were transplanted was formed in all cases where mBMiPS cells were transplanted. Tumors 2 weeks and 4 weeks after transplantation were removed and tissue specimens were prepared by formalin fixation. As a result of histological analysis, in all clone-derived teratomas, neural tissue, cartilage tissue, muscle tissue, adipose tissue, esophageal mucosa-like tissue, tracheal epithelium, etc. were observed, and thus were induced from mBMMNCs. Each clone proved to have pluripotency (FIG. 14).

  Furthermore, after injecting mBMiPS clone # 7 cells into a blastocyst prepared from a fertilized egg of a Balb / c mouse, an attempt was made to produce a chimeric individual by transplanting it into a pseudopregnant mouse. As a result, the offspring of the Balb / c mouse should have a white coat color, but a chimeric mouse having a black coat color derived from mBMiPS cells was born (FIG. 15 and Table 4). The hair color reflects a part of the cells of the whole body, and it is considered that cells derived from mBMiPS cells also constitute individuals in other tissues and organs.

Example 10: Expression profile analysis of mouse bone marrow-derived pluripotent cells For mBMiPS clones # 7 and # 13, RNA was prepared from 5 × 10 ⁵ cells using RNAeasy mini plus kit (QIAGEN), and Agilent Expression was used. Expression profile analysis was performed using Array (Agilent). Mouse ES cell strain E14 and mouse BMMNC cells were used as controls. As a result, the expression pattern of each gene as shown in FIG. 16 was shown, and it was clarified that mBMiPS clones # 7 and # 13 were cells very similar to ES cells.

As a result of FACS analysis evaluating the expression level of undifferentiated markers of pluripotent stem cells derived from human bone marrow-derived mononuclear cells (hBMMNCs), the morphology of colonies formed by the induced pluripotent stem cells and induced The results of alkaline phosphatase activity staining of pluripotent stem cells are shown. [A] shows the expression of the undifferentiated cell marker SSEA-4 on the 20th day after infection of retrovirus expressing a nuclear reprogramming factor to hBMMNCs. In the figure, the horizontal axis indicates the expression level of SSEA-4, and the cells with low PI staining intensity indicated by the vertical axis indicate live cells. The contour lines in the figure indicate the presence frequency of the analyzed cells. “ST3FP6” indicates that SCF, TPO, IL-3, and FP6 coexist with hBMMNCs as cytokine cocktails in the nuclear reprogramming process for 2 days before retrovirus infection and “ST3” for 2 days before retrovirus infection. It was also shown that SCF, TPO, and IL-3 coexisted with hBMMNCs as cytokine cocktails in the nuclear reprogramming process. [B] showed the morphology of cell colonies formed in the nuclear reprogramming process. “Day 10” and “Day 20” indicate the number of days from the start of retrovirus infection. [C] shows alkaline phosphatase staining on day 27 from the start of retroviral infection of pluripotent stem cells derived from hBMMNCs. The result of the FACS analysis which evaluated the expression level of the undifferentiation marker of the pluripotent stem cell induced | guided | derived from the human bone marrow derived mononuclear cell (hBMMNCs), and the form of the colony which the induced pluripotent stem cell forms were shown. The case where SCF, TPO, IL-3, FP6, and Flt ligand were coexisted with hBMMNCs as cytokine cocktails was shown for 2 days before retrovirus infection and in the nuclear reprogramming process. [A] shows the expression of the undifferentiated cell marker SSEA-4 on day 22 after infection of retrovirus expressing a nuclear reprogramming factor to hBMMNCs. In the figure, the horizontal axis represents the expression level of SSEA-4, and the vertical axis represents the number of cells. [B] showed the morphology of cell colonies formed in the nuclear reprogramming process. The results of FACS analysis for evaluating the infection efficiency of retroviral human bone marrow-derived mononuclear cells (hBMMNCs) prepared using the pMXsIG vector are shown. “PI” indicates the intensity of staining with propidium iodide, and the contour lines in the figure indicate the presence frequency of the analyzed cells. The horizontal axis of each figure showed the expression intensity of green fluorescent protein EGFP expressed from the infected retroviral genome. “No cytokine culture” indicates a case where cytokines were not allowed to coexist before and after retrovirus infection. “ST3-added culture” shows the case where hBMMNCs and cytokines of SCF, TPO, and IL-3 coexisted from 2 days before retrovirus infection to 2 days after retrovirus infection after FACS analysis. “ST3FP6-added culture” shows a case where hBMMNCs and cytokines of SCF, TPO, IL-3, and FP6 coexisted from 2 days before retrovirus infection to 2 days after retrovirus infection after FACS analysis. The results of FACS analysis for identifying and evaluating the cell lineage of human bone marrow-derived mononuclear cells (hBMMNCs) infected with retroviruses prepared using the pMXsIG vector are shown. The case where hBMMNCs were allowed to coexist with SCF, TPO, and IL-3 cytokines from 2 days before retrovirus infection until 2 days after retrovirus infection was shown as ST3, and SCF, TPO, IL-3, When coexisting with FP6 cytokine and ST3FP6, the case coexisting with SCF, TPO, IL-3, FP6, and Flt3 ligand is shown as ST3FLFP6. The horizontal axis of each figure shows the expression intensity of the green fluorescent protein EGFP expressed from the infected retrovirus genome, and the contour lines in the figure show the frequency of the analyzed cells. “CD45APC” showed staining intensity with an allophycocyanin-conjugated anti-human CD45 antibody. “CD34APC” showed staining intensity with an allophycocyanin-conjugated anti-human CD34 antibody. “CD38PE” showed staining intensity with phycoerythrin-conjugated anti-human CD38 antibody. “CD117PE” showed staining intensity with phycoerythrin-conjugated anti-human CD117 antibody. “CD11bAPC” showed staining intensity with an allophycocyanin-conjugated anti-human CD11b antibody. “CD3PE” showed staining intensity with phycoerythrin-conjugated anti-human CD3 antibody. “CD19PE” showed staining intensity with phycoerythrin-conjugated anti-human CD19 antibody. The results of FACS analysis for identifying and evaluating the cell lineage of human peripheral blood-derived mononuclear cells (hPBMNCs) infected with retroviruses prepared using the pMXsIG vector are shown. From 2 days before retrovirus infection until 2 days after retrovirus infection, hPBMNCs were allowed to coexist with human SCF, human TPO, human IL-3, FP6, Flt3 ligand, human GM-CSF, and human M-CSF. In the case of GM culture, human SCF, human TPO, human IL-3, FP6, LPS, anti-human CD40 antibody, anti-human CD3 antibody, and anti-human CD28 antibody coexisting with the factors were shown as TB culture. . The horizontal axis of each figure shows the expression intensity of the green fluorescent protein EGFP expressed from the infected retrovirus genome, and the contour lines in the figure show the frequency of the analyzed cells. “CD45APC” showed staining intensity with an allophycocyanin-conjugated anti-human CD45 antibody. “CD34APC” showed staining intensity with an allophycocyanin-conjugated anti-human CD34 antibody. “CD38PE” showed staining intensity with phycoerythrin-conjugated anti-human CD38 antibody. “CD117PE” showed staining intensity with phycoerythrin-conjugated anti-human CD117 antibody. “CD11bAPC” showed staining intensity with an allophycocyanin-conjugated anti-human CD11b antibody. “CD3PE” showed staining intensity with phycoerythrin-conjugated anti-human CD3 antibody. “CD19PE” showed staining intensity with phycoerythrin-conjugated anti-human CD19 antibody. “CD13PE” showed staining intensity with phycoerythrin-conjugated anti-human CD13 antibody. For the cloned human induced pluripotent cell line, colony morphology and expression analysis results of human ES cell marker SSEA-4 by FACS are shown. In the figure, the horizontal axis represents the expression level of SSEA-4, and the vertical axis represents the number of cells. The RT-PCR analysis results for the cloned human induced pluripotent cell line are shown. Each cell line was arranged in the horizontal direction in the figure. ST3, ST3FP6, and ST3FLFP6 show the induction conditions for each cell, and the numbers below them show each clone number. In the vertical direction of the figure, a gene known as a pluripotent stem cell marker was arranged, and the result of GAPDH as a housekeeping gene was shown as a control gene. The black band in the figure shows the expression for each gene. Morphology and undifferentiation of colonies formed by pluripotent stem cells derived from Oct4-EGFP transgenic mouse bone marrow derived mononuclear cells (mBMMNCs) or Oct4-EGFP transgenic mouse-derived embryonic fibroblasts (MEF) The expression of EGFP protein from Oct4-EGFP reporter gene, which is a marker of [A] shows cell morphology and expression of the undifferentiated marker Oct4-EGFP on the fifth day after infection of mBMMNCs from Oct4-EGFP transgenic mice with retrovirus expressing nuclear reprogramming factor. . The “4 gene sample” shows a case where a retrovirus was prepared by simultaneously introducing four nuclear reprogramming genes into a packaging cell when preparing a retrovirus. The “4 virus sample” refers to the case in which when a retrovirus is prepared, each of the four nuclear reprogramming genes is individually introduced into a packaging cell to produce the retrovirus, and then the retrovirus is mixed to infect the cell. Indicated. [B] shows the morphology of cells on the fifth day after retrovirus infection in the case where cytokines of SCF, TPO, and IL-3 coexist when retrovirus is infected with mBMMNCs derived from Oct4-EGFP transgenic mice. The expression of the undifferentiated marker Oct4-EGFP was shown. [C] shows cell morphology and expression of the undifferentiated marker Oct4-EGFP on day 5 after infection of retrovirus expressing nuclear reprogramming factor to MEF derived from Oct4-EGFP transgenic mice. . For pluripotent stem cells derived from bone marrow-derived mononuclear cells (mBMMNCs) of Oct4-EGFP transgenic mice, expression of EGFP protein from Oct4-EGFP reporter gene, which is an undifferentiated marker, and markers of blood cells The result of analyzing the expression of a certain CD45 antigen by FACS is shown. Analysis of day 7 after infecting mBMMNCs from Oct4-EGFP transgenic mice with retrovirus expressing nuclear reprogramming factor was shown. “4 genes” indicates a case where a retrovirus was prepared by simultaneously introducing four nuclear reprogramming genes into a packaging cell when a retrovirus was prepared. “4 viruses” refers to the case where, when preparing a retrovirus, each of the four nuclear reprogramming genes is individually introduced into a packaging cell to create a retrovirus, and then the retrovirus is mixed to infect the cell. It was. “Concentrated virus” indicates a case where a retrovirus prepared in the same manner as “4 virus” is concentrated and used for infection. The horizontal axis of each figure showed the expression intensity of green fluorescent protein EGFP expressed from the infected retroviral genome. “CD45APC” indicates the staining intensity with the allophycocyanin-conjugated anti-human CD45 antibody, and the contour lines in the figure indicate the frequency of the analyzed cells. Morphology and undifferentiation of colonies formed by pluripotent stem cells derived from Oct4-EGFP transgenic mouse bone marrow derived mononuclear cells (mBMMNCs) or Oct4-EGFP transgenic mouse-derived embryonic fibroblasts (MEF) The expression of EGFP protein from Oct4-EGFP reporter gene, which is a marker of The state on the seventh day after infection of mBMMNCs or MEFs derived from Oct4-EGFP transgenic mice with a retrovirus expressing a nuclear reprogramming factor was shown. “MBMMNCs” indicates cells derived from mBMMNCs derived from Oct4-EGFP transgenic mice. “MEF” indicates cells derived from MEF derived from Oct4-EGFP transgenic mice. Regarding pluripotent stem cell clones derived from bone marrow-derived mononuclear cells (mBMMNCs) of Oct4-EGFP transgenic mice, expression of EGFP protein from Oct4-EGFP reporter gene, which is an undifferentiated marker, and mouse undifferentiated cells Of FEA analysis of expression of SSEA-1 antigen, which is a marker of E. coli, and morphology of colonies formed by pluripotent stem cell clones and expression of EGFP protein from Oct4-EGFP reporter gene, which is an undifferentiated marker in colonies showed that. [A] shows the expression of undifferentiated marker Oct4-EGFP and the expression of SSEA-1 antigen, which is a marker of mouse undifferentiated cells, in FACS for mBMiPS cell clone # 7 and clone # 13 derived from Oct4-EGFP transgenic mice. The analysis results are shown. “IgG APC” showed the staining intensity with allophycocyanin-binding control IgG. “SSEA-1 Alexa647” showed staining intensity with Alexa647-conjugated anti-mouse SSEA-1 antibody. The horizontal axis of each figure shows the expression intensity of the undifferentiated marker Oct4-EGFP, and the contour lines in the figure show the presence frequency of the analyzed cells. [B] shows the results of FACS analysis of the expression of SSEA-1 antigen, which is a marker of mouse undifferentiated cells, for mBMiPS cell clone # 7 and clone # 13 derived from Oct4-EGFP transgenic mice. The horizontal axis represents the expression intensity of SSEA-1 antigen, which is a marker for mouse undifferentiated cells, and the vertical axis represents the number of cells. The solid line in the figure shows the case of staining with Alexa647-conjugated anti-mouse SSEA-1 antibody, and the broken line shows the case of staining with allophycocyanin-binding control IgG. [C] shows the morphology of colonies formed by mBMiPS cell clone # 7 and clone # 13 derived from Oct4-EGFP transgenic mice and the expression of EGFP protein from Oct4-EGFP reporter gene which is an undifferentiated marker. To evaluate the infection efficiency of retroviral Oct4-EGFP transgenic mouse bone marrow-derived mononuclear cells (mBMMNCs) or Oct4-EGFP transgenic mouse-derived embryonic fibroblasts (MEF) prepared using the pMYsIG vector The FACS analysis result of was shown. The horizontal axis of the figure shows the expression intensity of green fluorescent protein EGFP expressed from the infected retroviral genome, and the vertical axis shows the number of cells. The analysis results on the second day after retrovirus infection are shown. “BMMNCs” indicates the case where Oct4-EGFP transgenic mouse-derived mBMMNCs are used, and “MCF pre-cultured on gelatin” indicates the case where Oct4-EGFP mouse MEF cultured on a gelatin-coated plate from the day before virus infection is used. “MEF on retronectin” indicates the case of using MEF infected with a retronectin-coated plate in the same manner as BMMNCs. Bone marrow derived mononuclear cells (mBMMNCs) of Oct4-EGFP transgenic mice or pluripotent stem cells derived from MEF using retrovirus prepared using pMYs nuclear reprogramming gene vector Of FGFP analysis of the expression of EGFP protein from the Oct4-EGFP reporter gene and the expression of SSEA-1 antigen, which is a marker of mouse undifferentiated cells, and the morphology of colonies formed by pluripotent stem cell clones in the colonies The expression of EGFP protein from Oct4-EGFP reporter gene which is an undifferentiated marker was shown. [A] shows the expression of Oct4-EGFP transgenic mouse-derived mBMMNCs and MEF undifferentiated marker Oct4-EGFP and the expression of SSEA-1 antigen, a marker of mouse undifferentiated cells, 7 days after retrovirus infection The results were shown. “SSEA-1 Alexa647” showed staining intensity with Alexa647-conjugated anti-mouse SSEA-1 antibody. The horizontal axis of each figure shows the expression intensity of the undifferentiated marker Oct4-EGFP, and the contour lines in the figure show the presence frequency of the analyzed cells. [B] shows the morphology of colonies formed by mBMMNCs and MEFs derived from Oct4-EGFP transgenic mice 7 days after infection with retrovirus and the expression of EGFP protein from Oct4-EGFP reporter gene, which is an undifferentiated marker. [C] shows the morphology of colonies formed by mBMMNCs and MEFs derived from Oct4-EGFP transgenic mice 12 days after retrovirus infection and the expression of EGFP protein from the Oct4-EGFP reporter gene, an undifferentiated marker. “BMMNCs” indicates the case where Oct4-EGFP transgenic mouse-derived mBMMNCs are used, and “MCF pre-cultured on gelatin” indicates the case where Oct4-EGFP mouse MEF cultured on a gelatin-coated plate from the day before virus infection is used. “MEF on retronectin” indicates the case of using MEF infected with a retronectin-coated plate in the same manner as BMMNCs. Fig. 2 shows teratoma formed from mouse bone marrow-derived iPS # 7 cells transplanted subcutaneously into nude mice. [A] shows a nude mouse in which a teratoma is formed from mouse bone marrow-derived iPS # 7 cells or ES cells transplanted subcutaneously. [B] shows a pathological specimen prepared from the formed teratoma, including undifferentiated nerve tissue, central nerve, cerebral cortex-like tissue, hair root tissue, muscle tissue, cartilage tissue, white adipose tissue, tracheal mucus Epithelial cells and tracheal epithelial cells with cilia, esophageal mucosa-like tissue, pancreatic acinar cells, salivary gland serous cells and mucus cells are observed. A chimeric individual produced by injecting mouse bone marrow-derived iPS # 7 cells into a blastocyst prepared from a fertilized egg of a Balb / c mouse and then transplanting it into a pseudopregnant mouse is shown. Balb / c mice originally show white coat color, and bone marrow-derived iPS cells are derived from C57BL / 6 mice with black coat color. The cells having a black coat color are cells derived from bone marrow-derived iPS cells. It is the figure which showed the comparison of the expression intensity | strength of the gene expressed in each cell about the mouse | mouth bone marrow-derived iPS cell clone, mouse | mouth ES cell, and mouse | mouth bone marrow origin mononuclear cell. “IPS # 7” indicates a mouse iPS cell clone, and “BMMNC” indicates a mouse bone marrow-derived mononuclear cell. The horizontal and vertical axes in each figure indicate the amount of messenger RNA of a gene expressed in each cell, and each point in the figure corresponds to one kind of gene. When the amount of messenger RNA of a gene expressed between cells to be compared is the same, each point in the figure is located on a diagonal line in the figure.

Claims (15)

A method for producing pluripotent stem cells from somatic cells,
(A) culturing a somatic cell in a cell culture medium containing an IL-6 signaling factor stimulating factor and at least one cytokine, and (b) dedifferentiating the somatic cell, Method wherein step (b) is performed after a) or step (a) and step (b) are performed simultaneously.   The method according to claim 1, wherein the IL-6 signaling factor stimulating factor is a fusion protein in which IL-6 or a variant thereof and an IL-6 receptor or a variant thereof are linked.   2. The method of claim 1, wherein the cytokine is selected from the group consisting of SCF, TPO, IL-3 and Flt-3 ligands, and combinations of two or more thereof.   The method according to claim 1, wherein the dedifferentiation of the somatic cell in the step (b) is performed by a nuclear reprogramming treatment of the somatic cell.   The method according to claim 4, wherein the nuclear reprogramming treatment of the somatic cell comprises introducing the Oct family gene, the Klf family gene, the Sox family gene, and the Myc family gene into the somatic cell in an expressible form.   The method according to claim 1, wherein the somatic cell is a bone marrow-derived mononuclear cell.   The method of claim 1, wherein the somatic cell is a differentiated blood cell.   2. The method of claim 1, wherein the somatic cell is a human cell. A kit for producing pluripotent stem cells from somatic cells,
(A) a kit comprising a cell culture medium for culturing somatic cells, comprising IL-6 signaling factor stimulating factor and at least one cytokine, and (b) a reagent for dedifferentiating somatic cells .   The kit according to claim 9, wherein the IL-6 signaling factor stimulating factor is a fusion protein in which IL-6 or a variant thereof and an IL-6 receptor or a variant thereof are fused.   The kit according to claim 9, wherein the cytokine is selected from the group consisting of SCF, TPO, IL-3 and Flt-3 ligands, and combinations of two or more thereof.   The reagent for dedifferentiating somatic cells comprises a vector for introducing an Oct family gene, a klf family gene, a Sox family gene, and a Myc family gene into a somatic cell in an expressible form. The described kit.   The kit according to claim 9, wherein the somatic cell is a bone marrow-derived mononuclear cell.   The kit according to claim 9, wherein the somatic cells are differentiated blood cells.   The kit according to claim 9, wherein the somatic cell is a human cell.