WO2010131747A1 - Virus-producing cell - Google Patents

Abstract

Disclosed is a virus-producing cell which can produce homogeneous RNA viruses carrying a gene encoding a reprogramming factor and of the same lot in a large quantity. The virus-producing cell carries, on the chromosome thereof, a gene encoding a reprogramming factor and a gene encoding a virus-constituting protein containing vesicular stomatitis virus-G (VSV-G) protein so that RNA viruses carrying the gene encoding the reprogramming factor and containing vesicular stomatitis virus-G (VSV-G) protein as an envelope protein can be produced.

Description

Virus-producing cells

The present invention relates to a virus-producing cell capable of producing an RNA virus containing a gene encoding an reprogramming factor. The present invention further relates to a method for producing the virus-producing cell, an RNA virus containing a gene encoding an reprogramming factor, a method for producing the RNA virus, and a method for producing induced pluripotent stem cells using the RNA virus.

Induced pluripotent stem cells (iPS cells) are pluripotent cells obtained by reprogramming somatic cells. Induced pluripotent stem cells were created by a group of Prof. Shinya Yamanaka at Kyoto University, a group of Rudolf Jaenisch et al. At Massachusetts Institute of Technology, a group of James Thomson et al. At University of Wisconsin, Harvard University Several groups have been successful, including the group of Konrad Hochedlinger et al. Induced pluripotent stem cells have great expectations as ideal pluripotent cells without rejection and ethical problems. For example, Patent Document 1 discloses somatic cell nuclear reprogramming factors including Oct family gene, Klf family gene, and Myc family gene gene products, as well as Oct family gene, Klf family gene, Sox family gene and Myc family gene. A method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, comprising a step of contacting a nuclear reprogramming factor of a somatic cell containing a gene product with the nuclear reprogramming factor. Are listed.

Since the establishment of induced pluripotent stem cells (iPS) sputum cells was reported, research on their establishment methods and application methods has been conducted all over the world. As a basic method for establishing iPS cells, a method of introducing a gene encoding an reprogramming factor into a somatic cell using a retroviral vector is used. Currently, in Japan, the method of producing ecotropic virus by transfecting two types of plasmids into the retrovirus packaging cell plat-E 用 い is mainly used. To promote iPS cell research, it is necessary to reliably establish more human iPS cells from somatic cells of healthy individuals or disease patients, but the establishment of human iPS cells using ecotropic retroviruses involves the following: There are technical problems as described in (1) to (3) below. (1) It is necessary to introduce a receptor for ecotropic virus into a target cell using a lentiviral vector prior to infection with a retrovirus, and each time there is a possibility that the establishment will not succeed unless both infection efficiencies are sufficiently high. is there. (2) It is an absolute condition for successful establishment that the virus to be used is fresh, and the virus cannot be stored frozen. Therefore, since preparation at the time of use is required in accordance with the culture of the target cells, the procedure becomes complicated, and the establishment efficiency may vary depending on trials. (3) In the method using transient virus production by transfection, it is difficult to obtain a large amount of the same lot, and it is necessary to conduct tests such as the occurrence of wild-type virus each time, considering clinical application. It is disadvantageous for ensuring security at times. As described above, in order to establish various human iPS cells easily and reliably in many facilities, it is necessary to develop a new iPS cell establishment method.

On the other hand, Non-Patent Document 1 describes a stable human-derived packaging cell capable of producing high-titer vesicular stomatitis virus G (VSV-G) pseudotyped retrovirus. The packaging cell described in Non-Patent Document 1 has a gag gene, a pol gene, and a VSV-G gene in a chromosome, and expresses a VSV-G gene depending on the presence or absence of a drug, thereby producing a VSV-G pseudotyped retrovirus. Is a cell capable of producing

International Publication No. WO2007 / 069666

An object of the present invention is to provide a virus-producing cell that is an RNA virus containing a gene encoding an reprogramming factor and can produce a large amount of a uniform and the same lot of virus.

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found an early stage on the chromosome of a packaging cell having a gene encoding a viral constitutive protein including vesicular stomatitis virus G (VSV-G) protein on the chromosome Production of an RNA virus containing a gene encoding a reprogramming factor by introducing a gene encoding a reprogramming factor, wherein the RNA virus contains a vesicular stomatitis virus G (VSV-G) protein as an envelope protein Succeeded in establishing cells. Furthermore, reprogramming somatic cells (hematopoietic cells), which were difficult to establish iPS cells by other existing methods, using RNA viruses containing genes encoding reprogramming factors produced by established virus-producing cells Also succeeded. The present invention has been completed based on these findings.

That is, according to the present invention, an RNA virus containing a gene encoding an reprogramming factor, which can be produced so that an RNA virus containing vesicular stomatitis virus G (VSV-G) protein as an envelope protein can be produced. Provided is a virus-producing cell having a gene encoding a factor and a gene encoding a viral constitutive protein including vesicular stomatitis virus G (VSV-G) protein on the chromosome.

Preferably, the gene encoding the reprogramming factor is at least one of the Oct gene, Sox gene, Klf gene, and Myc gene.
Preferably, the virus-producing cell of the present invention cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a predetermined drug, and vesicular stomatitis virus G (VSV-G) in the absence of the drug. ) Packaging cells capable of expressing the protein, or vesicular stomatitis virus G (VSV-G) protein in the absence of a given drug and vesicular stomatitis virus G (VSV-G) in the presence of the drug It is obtained by introducing a gene encoding a reprogramming factor into a packaging cell capable of expressing a protein.

Preferably, the gene encoding the reprogramming factor is introduced as a virus.
Preferably, the predetermined drug is tetracycline.
Preferably, a packaging cell that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a predetermined drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug Or packaging cells that are unable to express vesicular stomatitis virus G (VSV-G) protein in the absence of a given drug and are capable of expressing vesicular stomatitis virus G (VSV-G) protein in the presence of the drug, A cell obtained by introducing a gene encoding vesicular stomatitis virus G (VSV-G) protein into a cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus. The gene cannot be expressed in the presence of a given drug and in the absence of the drug. Or has been introduced to allow Oite expression, or the gene can not be expressed in the absence of a given agent is a cell that has been introduced to allow expression in the presence of the agent.

Preferably, a cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus is HEK293 cells.
Preferably, a packaging cell that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a predetermined drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug Are HEK293GPG cells.
Preferably, the virus-producing cell of the present invention is a cloned cell derived from a single cell.

Furthermore, according to the present invention, vesicular stomatitis virus G (VSV-G) protein cannot be expressed in the presence of a predetermined drug, and vesicular stomatitis virus G (VSV-G) protein is expressed in the absence of the drug. Can not express vesicular stomatitis virus G (VSV-G) protein in the absence of a given drug, or can express vesicular stomatitis virus G (VSV-G) protein in the presence of the drug There is provided a method for producing the virus-producing cell of the present invention described above, which comprises introducing a gene encoding an reprogramming factor into a packaging cell.
Preferably, a gene encoding a reprogramming factor is introduced as a virus.

According to the present invention, there is further provided an RNA virus comprising a gene encoding a reprogramming factor, wherein the RNA virus comprises vesicular stomatitis virus G (VSV-G) protein as an envelope protein.
Preferably, the gene encoding the reprogramming factor is any one or more of the Oct gene, Sox gene, Klf gene, and Myc gene.

Preferably, the RNA virus of the present invention is produced by the virus-producing cell of the present invention described above.
Preferably, the RNA virus of the present invention is used for the production of induced pluripotent stem cells.
Preferably, the RNA virus of the present invention is stored frozen after production.

According to the present invention, there is further provided an RNA virus comprising a gene encoding a reprogramming factor, comprising culturing the above-described virus-producing cell of the present invention, wherein vesicular stomatitis virus G (VSV-G) is used as an envelope protein. A method for producing an RNA virus comprising a protein is provided.

The present invention further provides a method for producing induced pluripotent stem cells, which comprises introducing the above-described RNA virus of the present invention into somatic cells.
Preferably, the somatic cell is a hematopoietic cell.
Preferably, the hematopoietic cells are derived from bone marrow.
Preferably, the hematopoietic cells are derived from cord blood.
Preferably, the somatic cell is a peripheral blood cell.
Preferably, the somatic cell is a peripheral blood CD34 positive cell.

The virus-producing cells of the present invention can be used stably and semipermanently for a long time, and can always produce a virus for expressing a uniform and high titer reprogramming factor by a simple culture method. In addition, the virus-producing cells of the present invention can be cryopreserved, making it possible to prepare a large amount of the same uniform lot semipermanently.

Furthermore, the virus-producing cells of the present invention produce vesicular stomatitis virus G (VSV-G) protein-coated virus, not ecotropic virus, and this VSV-G protein-coated virus can be stored frozen. Further, since the VSV-G protein-coated virus is excellent in physical strength, a high-titer virus lot can be obtained by centrifugation.

Furthermore, since the virus production method using the virus-producing cells of the present invention is easy to operate, technical differences among engineers are difficult to occur, the difference between virus lots produced is small, and the efficiency is the same in any facility. This makes it possible to establish iPS sputum cells.

Furthermore, the virus-producing cells of the present invention can be stored as a master cell bank of cell lines or clones that have undergone quality test, and the virus supernatant produced and prepared using them can also be safe. It has the advantage of being easily guaranteed. This ensures the safety of established iPS cells, thereby enabling the production of clinically applicable virus lots.

FIG. 1 shows the state of cell growth under conditions in which virus production of HEK293GPG clone introduced with mouse Klf4 gene was induced. FIG. 2 shows the state of cell growth of the HEK293GPG clone into which the mouse Klf4 gene was introduced under conditions that did not induce virus production. FIG. 3 shows the results of PCR analysis of 20 clones derived from 293GPG cells into which the mouse Klf4 gene was introduced, using a primer pair that can specifically detect a vector provirus containing the Klf4 gene. FIG. 4 shows the results of evaluating the infectivity of a retrovirus having a Klf4 gene produced by a 293GPG clone in which the presence of a vector provirus containing the Klf4 gene was confirmed. FIG. 5 shows the induction of iPS cells from hematopoietic cells in a single hematopoietic stem cell transplantation model. (A) A schematic diagram of the experimental procedure is shown. Single hematopoietic stem cells obtained from B6 Ly5.1 mice (CD150 ⁺ CD34 ^{neg / low} KSL cells) together with bone marrow cells from B6 Ly5.1 / 5.2 F1 mice were irradiated with a lethal dose of B6 Ly5.2 Mice were transplanted. Bone marrow hematopoietic stem / progenitor cells are obtained from recipient mice that have demonstrated long-term (10 months) stable Ly5.1 chimeras (~ 80%), enriched with Ly5.1 ⁺ cells, and used to generate iPS cells It was. (B) A schematic diagram of iPS cell preparation from bone marrow hematopoietic stem / progenitor cells is shown. (I) Plot of lineage marker (Lin) vs. c-Kit is shown for cells before purification (whole bone marrow) and after purification (Lin ⁻ c-Kit ⁺ ). 98% of the purified hematopoietic stem / progenitor cells were positive for the panblood cell marker CD45. (Ii) Schematic diagram of the process of creating iPS cells from bone marrow hematopoietic stem / progenitor cells. (C) Typical ES cell-like appearance of sHSC-iPS cell colonies (left) with high ALP activity (right). The bar is 100 μm. (D) Determination of cell origin of sHSC-iPS cell clone. The upper figure shows an overview of the PCR method performed using the single nucleotide polymorphism in CD45 exon (Ex) 25. The black triangle indicates the position of the primer. Ly5.1 and Ly5.2 strains have a single base substitution in the 301 bp amplicon as shown in the 12 bp sequence presented in the figure. Treatment with the restriction enzyme KpnI leaves the Ly5.1 ⁺ cell-derived amplicon undigested, but produces two small fragments (113 bp +188 bp) from Ly5.2 ⁺ cells. The gel image (below) shows analysis images of four sHSC-iPS cell clones. One is derived from Ly5.1 / 5.2 F1 cells and three (# 9-5, -6 and -8) are derived from a single Ly5.1 ⁺ hematopoietic stem cell-derived Ly5.1 ⁺ cell. Is. (E) A chimeric mouse obtained by transplanting sHSC-iPS cell clone # 9-5 into an ICR host blastocyst. FIG. 6 shows characteristics of iPS cells derived from primary bone marrow hematopoietic cells prepared using four kinds of reprogramming factors. (A) RT-PCR analysis showing ES marker gene expression in primary bone marrow hematopoietic stem / progenitor cell-derived iPS cell clones (pHPC-iPS cells). H ₂ O: control without template; ES: ES cells as positive control; RT (−): control without reverse transcriptase. (B) PCR analysis of Ig reconstitution of DJ fragment (DJ1-DJ3) in pHPC-iPS cell clone. GL shows amplification of a fragment showing the unrearranged germline arrangement of the Ig heavy chain gene. EL4 is a T lymphocyte cell as a non-reconstituted control. (C) Teratoma tissue section derived from pHPC-iPS cell clone. (D) Image of pHPC-iPS cell colonies derived from EGFP transgenic mice. The bar is 100 μm (CD). (E) A chimeric fetus produced using one representative EGFP ⁺ iPS cell clone. FIG. 7 shows the production of iPS cells from bone marrow hematopoietic cells. (A) Appearance of iPS cell induction from bone marrow hematopoietic stem / progenitor cells. The bar is 100 μm. (B) SSEA-1 expression in bone marrow hematopoietic cell-derived iPS cells (BM derived iPSCs). Representative flow cytometry plots are shown for established BM derived iPSCs (right), whole bone marrow (left), and ES cells (middle). The expression of SSEA-1 in BM derived iPS cells is at the same level as that in ES cells, and no expression is seen in whole bone marrow cells. FIG. 8 shows evaluation of reprogramming factor genes in sHSC-iPS cells. (A) PCR analysis showing the presence of reprogramming factor in each sHSC-iPS clone. H ₂ O: control without template, Control: positive control using each vector DNA as template. (B) RT-PCR analysis using a specific primer set for examining the expression of four reprogramming factors. In each sHSC-iPS clone, a transcript derived from an endogenous gene can be detected (Endo), but a transcript derived from a retrovirus is not detected (Tg). RT (-) is a control without reverse transcriptase. FIG. 9 shows an evaluation of the expression of pluripotency markers in sHSC-iPS cells. (A) RT-PCR analysis showing expression of ES marker gene in sHSC-iPS clone. H ₂ O: control without template, ES: ES cell as positive control, RT (-): control without reverse transcriptase. (B) Nanog expression in sHSC-iPS clones by immunostaining. NC: Negative control using isotype control antibody. Nuclei were stained with DAPI. The bar is 100 μm. FIG. 10 shows the developmental potential of sHSC-iPS cells. The teratoma tissue section derived from the sHSC-iPS clone is shown. The bar is 100 μm. FIG. 11 shows evaluation of reprogramming factor genes in pHPC-iPS cells. (A) PCR analysis showing the presence of reprogramming factor in each pHPC-iPS clone. H ₂ O: control without template, Control: positive control using each vector DNA as template. (B) RT-PCR analysis using a specific primer set for examining the expression of four reprogramming factors. In each pHPC-iPSC clone, a transcript derived from an endogenous gene can be detected (Endo), but a transcript derived from a retrovirus is not detected (Tg). RT (-) is a control without reverse transcriptase. FIG. 12 shows the establishment of human iPS cell-derived 4 factor retrovirus producing 293GPG cell line (KLF4). FIG. 13 shows a process for establishing iPS cells derived from human skin cells. FIG. 14 shows the process of establishing umbilical cord blood-derived sputum iPS sputum cells. FIG. 15 shows human skin cell-derived iPS cells (immunostaining, G-banding). A: Human iPS cell immunostaining: Expression of SSEA-4, an undifferentiated marker, was observed in the same manner as the control human ES cell line (KhES-3). B: Chromosome maintained normal karyotype 46XY. FIG. 16 shows the expression of ES related genes in human skin cell-derived iPS cells. Expression of ES gene-related genes (semi-quantitative RT-PCR) The expression of ES-related genes was observed to the same extent as that of human control ES cells (KhES-3). On the other hand, the expression of foreign genes used for the production of iPS cells was strongly suppressed. Tg: Transgene FIG. 17 shows the process of establishing iPS cells derived from peripheral blood CD34-positive cells (non-mobilized). FIG. 18 shows normal donor peripheral blood CD34-positive cell-derived iPS cells (TkPBV1-1).

Hereinafter, the present invention will be described in more detail.
The virus-producing cell of the present invention is an RNA virus containing a gene encoding an reprogramming factor, and is capable of producing an RNA virus containing vesicular stomatitis virus G (VSV-G) protein as an envelope protein. A cell having a gene encoding a factor and a gene encoding a viral constitutive protein including vesicular stomatitis virus G (VSV-G) protein on a chromosome.

The gene encoding the reprogramming factor used in the present invention is a gene encoding a reprogramming factor having the action of reprogramming somatic cells to become induced pluripotent stem cells. In the present invention, a gene encoding at least one reprogramming factor can be used. The gene encoding the reprogramming factor used in the present invention can be selected from, for example, Oct gene, Klf gene, Sox gene, Myc gene, Nanog gene, Lin28 gene, htert gene, SV40hlarge T gene and the like. Among the above, preferably, the Oct gene, Klf gene, Sox gene and Myc gene can be used.

The Oct gene, Klf gene, Sox gene and Myc gene each contain multiple family genes. As specific examples of each family gene, those described in pages 11 to 13 of the specification of International Publication No. WO2007 / 069666 can be used. Specifically, it is as follows.

Specific examples of genes belonging to the Oct gene include Oct3 / 4 (NM_002701), Oct1A (NM_002697), and Oct6 (NM_002699) (in parentheses indicate the NCBI accession number of the human gene). Oct3 / 4 is preferable. Oct3 / 4 is a transcription factor belonging to the POU family, is known as an undifferentiated marker, and has been reported to be involved in maintaining pluripotency.

Specific examples of genes belonging to the Klf gene include Klf1 (NM — 006563), Klf2 (NM — 016270), Klf4 (NM — 004235), Klf5 (NM — 001730), etc. (in parentheses indicate NCBI accession numbers of human genes) ). Klf4 is preferred. Klf4 (Kruppel like factor-4) has been reported as a tumor suppressor.

Specific examples of genes belonging to the Sox gene include, for example, Sox1 (NM_005986), Sox2 (NM_003106), Sox3 (NM_005634), Sox7 (NM_031439), Sox15 (NM_006942), Sox17 (NM_0022454), and Sox18 (NM_018419). (In parentheses indicate the NCBI accession number of the human gene). Preferably it is Sox2. Sox2 is a gene that is expressed during early development and encodes a transcription factor.

Specific examples of genes belonging to the Myc gene include c-Myc (NM_002467), N-Myc (NM_005378), and L-Myc (NM_005376) (in parentheses the NCBI accession number of the human gene). Show). Preferably, c-MycMy. c-Myc is a transcriptional regulator involved in cell differentiation and proliferation, and has been reported to be involved in maintaining pluripotency.

The genes described above are genes that exist in common in mammals including humans, and genes derived from any mammal (eg, derived from mammals such as humans, mice, rats, monkeys) can be used in the present invention. . In addition, several (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3) of wild type genes. It is also possible to use a mutated gene having a base substitution, insertion and / or deletion and having a function similar to that of a wild-type gene.

The virus-producing cell of the present invention cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a predetermined drug, and does not express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug. Packaging cells that can be expressed, or cannot express vesicular stomatitis virus G (VSV-G) protein in the absence of a given drug, but express vesicular stomatitis virus G (VSV-G) protein in the presence of the drug It can be produced by introducing a gene encoding a reprogramming factor into a packaging cell that can be produced. As packaging cells, for example, HEK293 cells derived from human fetal kidney (HEK293 cells are cell lines established by transforming human fetal kidney cells with adenovirus E1 gene), a package based on mouse fibroblast NIH3T3 Cells, preferably packaging cells based on HEK293 cells can be used.

In the present invention, the gene encoding the reprogramming factor is preferably introduced as a virus from the viewpoint of integration into the chromosome of the packaging cell. The method for introducing a virus into a packaging cell is not particularly limited, and can be performed using a known gene introduction method such as a calcium phosphate method, a lipofection method, or an electroporation method.

Packaging cells that are unable to express vesicular stomatitis virus G (VSV-G) protein in the presence of a given drug and that are capable of expressing vesicular stomatitis virus G (VSV-G) protein in the absence of the drug or Examples of packaging cells that cannot express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug and can express vesicular stomatitis virus G (VSV-G) protein in the presence of the drug include, for example, A cell obtained by introducing a gene encoding vesicular stomatitis virus G (VSV-G) protein into a cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus. The gene cannot be expressed in the presence of the given drug and the absence of the drug Or it has been introduced to allow expression in, or the gene can not be expressed in the absence of a given agent can be used a cell that has been introduced to allow expression in the presence of the agent. As the predetermined drug mentioned here, it is preferable to use an antibiotic such as tetracycline (or doxycycline).

In order to allow a target protein to be expressed in the absence of tetracycline (or doxycycline) and not in the presence of tetracycline (or doxycycline) in a host cell, the Tet-Off Gene Expression System (Clontech ) Can be used. The Tet-Off® System is based on two regulatory factors, the Tet repressor protein (TetR) and the Tet operator DNA sequence (tetO) obtained from the Escherichia coli tetracycline resistance operon. A bi-stable cell line incorporating the factor into the host genome is obtained. Established cell lines can express target genes in a dose-dependent response to tetracycline (or doxycycline) (Proc Natl Acad Sci USA. 1992 Jun 15; 89 (12): 5547-51. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M, Bujard H). In addition, in order to allow the target protein to be expressed in the presence of tetracycline (or doxycycline) and not to be expressed in the absence of tetracycline (or doxycycline) in the host cell, Tet-On に お い て Gene Expression System (Clontech) can be used. Tet-on system is a system that induces gene expression by binding tetracycline-dependent transcriptional regulatory factor (rtTA) to TRE (tetracycline responsive element) in the promoter region when doxycycline, a tetracycline derivative, is present. is there.

Examples of packaging cells that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of tetracycline drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of tetracycline , HEK293GPG cells (Ory DS, Neugeboren BA, Mulligan RC. A stable human-derived packaging cell line for production of high titer retrovirus / vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci 1996 Can be mentioned.

In the present invention, a retroviral vector (for example, pMXs vector) having a gene encoding a reprogramming factor (for example, an Oct gene, Sox gene, Klf gene, or Myc gene) and a vector having a VSV-G cDNA are 293GP cells with viral gag and pol genes on the chromosome (Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vector: concentration gene and nonmammalian cells. Proc Natl Acad Sci U S A. 1993; 90: 8033-8037) to transfect genes that reprogram the factor (eg, Oct gene, Sox gene, Klf gene, or Myc gene) Can be prepared. Next, by introducing the obtained retrovirus having a gene encoding a reprogramming factor into the above HEK293GPG cell, an RNA virus containing the gene encoding the reprogramming factor, and vesicular stomatitis virus as an envelope protein The virus-producing cell of the present invention capable of stably producing an RNA virus containing G (VSV-G) protein can be produced.

According to the present invention, an RNA virus comprising a gene encoding a reprogramming factor by culturing the above-described virus-producing cells of the present invention, wherein vesicular stomatitis virus G (VSV-G) protein is used as an envelope protein. RNA viruses containing can be produced. An RNA virus containing the gene encoding the reprogramming factor produced as described above and containing the vesicular stomatitis virus G (VSV-G) protein as an envelope protein is also included in the present invention. The RNA virus of the present invention is a virus containing a gene encoding a reprogramming factor, and can be used to produce induced pluripotent stem cells from somatic cells. In addition, the RNA virus of the present invention contains vesicular stomatitis virus G (VSV-G) protein as an envelope protein and is excellent in physical strength. Therefore, it can be stored frozen after production. The RNA virus of the present invention may be provided in the form of a virus solution containing the virus, or may be used by preparing a virus solution by dissolving a cryopreserved virus at the time of use.

The present invention further provides a method for producing induced pluripotent stem cells, which comprises introducing the above-described RNA virus of the present invention into somatic cells.

The type of somatic cell used for initialization in the present invention is not particularly limited, and any somatic cell can be used. That is, the somatic cells referred to in the present invention include all cells other than germ cells among the cells constituting the living body, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the somatic cell may be any of mammals, birds, fishes, reptiles, amphibians, and is not particularly limited, but is preferably a mammal (for example, a rodent such as a mouse or a primate such as a human). Preferably it is a human. Moreover, when using human somatic cells, any fetal, neonatal or adult somatic cells may be used. As an example, hematopoietic cells, particularly hematopoietic stem cells may be used.

As another example of human somatic cells used for reprogramming, peripheral blood cells, preferably peripheral blood CD34 positive cells, can be used. In particular, if induced pluripotent stem cells can be established from a small amount of blood collected from human peripheral blood, banking of induced pluripotent stem cells for each human leukocyte antigen (HLA) becomes very easy. There is no report yet on the generation of induced pluripotent stem cells from peripheral blood under conditions that do not give stress such as G-CSF administration. G-CSF administration may induce leukemia at a low frequency, and requires hospitalization for about one week, which places a heavy burden on the donor. In the present invention, induced pluripotent stem cells can be produced by introducing RNA viruses produced by the virus-producing cells of the present invention into peripheral blood cells (preferably, peripheral blood CD34-positive cells). .・

In addition, when the induced pluripotent stem cell produced by the method of the present invention is used for treatment of diseases such as regenerative medicine, it is preferable to use somatic cells isolated from the patient suffering from the disease.

The induced pluripotent stem cell referred to in the present invention has a self-replicating ability over a long period of time under a predetermined culture condition (for example, under the condition of culturing ES cells), and also has an ectoderm, A stem cell having multipotency into germ layers and endoderm. The induced pluripotent stem cell in the present invention may be a stem cell capable of forming a teratoma when transplanted to a test animal such as a mouse.

The method for introducing a gene encoding an reprogramming factor into a somatic cell is not particularly limited as long as the introduced reprogramming gene is expressed to achieve somatic cell reprogramming. For example, the virus of the present invention containing at least one reprogramming gene can be introduced into a somatic cell according to a conventional method known to those skilled in the art. When a gene encoding two or more types of reprogramming factors is introduced into a somatic cell, a gene encoding two or more types of reprogramming factors may be incorporated into one virus and the virus introduced into the somatic cell. Alternatively, two or more types of viruses incorporating one type of reprogramming gene may be prepared and introduced into somatic cells.

A medium capable of maintaining the undifferentiation and pluripotency of ES cells is known in the art, and induced pluripotent stem cells can be separated and cultured by using a suitable medium in combination. The medium for culturing induced pluripotent stem cells includes various growth factors, cytokines, hormones (eg, FGF-2, TGFb-1, activin A, Noggin, BDNF, NGF, NT-1, Ingredients involved in the growth and maintenance of human ES cells such as NT-2 and NT-3 may be added. In addition, the differentiation ability and proliferation ability of the isolated induced pluripotent stem cells can be confirmed by using confirmation means known for ES cells.

The use of the induced pluripotent stem cells produced by the method of the present invention is not particularly limited, and can be used for various tests / researches and disease treatments. For example, by treating the induced pluripotent stem cell obtained by the method of the present invention with a growth factor such as retinoic acid, EGF, or glucocorticoid, a desired differentiated cell (for example, neuronal cell, cardiomyocyte, hepatocyte, Pancreatic cells, blood cells, etc.) can be induced, and stem cell therapy by autologous cell transplantation can be achieved by returning the differentiated cells thus obtained to the patient.

The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Establishment of VSV-G pseudotyped retrovirus-producing cells and production of VSV-G pseudotyped retroviral particles pMXs vectors encoding Onc3 / 4, Sox2, Klf4, and c-Myc (Onishi M, Kinoshita S, Construction of Morikawa Y, et al. Applications of retrovirus-mediated expression cloning. Exp Hematol. 1996; 24: 324-329) has been reported (Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126: 663-676). Retroviral vectors used in this example pMXs-human OCT3 / 4, pMXs-human SOX2, pMXs-human KLF4, pMXs-human cMYC, pMXs-murine Oct3 / 4, pMXs-murine Sox2, pMXs-murine Klf4, pMXs- murine cMyc was provided by Dr. Yamanaka, Kyoto University.

Highly enriched VSV-G pseudotyped retrovirus (RV) supernatant was prepared as previously described (Hamanaka S, Nabekura T, Otsu M, et al. Stable transgene expression in mice generated from retrovirally transduced embryonic stem cells. Mol Ther. 2007; 15: 560-565 ； Nabekura T, Otsu M, Nagasawa T, Nakauchi H, Onodera M. Potent vaccine therapy with dendritic cells genetically modified by the gene-silencing-resistant retroviral vector GCDNsap. Mol 13: 301-309; and Sanuki S, Hamanaka S, Kaneko S, et al. A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells. J Gene Med. 2008; 10: 965-971). VSV-G pseudotyped retroviral particles were provided by 293GP cells (Richard Dr. Mulligan (Harvard University); Burns JC, Friedmann T, Driver W, Burrascano M, Yee JK. high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A. 1993; 90: 8033-8037), pMXs vector containing genes encoding each reprogramming factor and VSV-G cDNA It was prepared by transfection with a vector. The resulting retroviral particles were centrifuged at 6,000 × g for 16 hours, resuspended in 1/100 volume α- MEM and concentrated. Next, 293GPG cells (provided by Dr. Richard Mulligan (University of Harvard)); Ory DS, Neugeboren BA, Mulligan RC, A stable human-derived packaging cell line for production of high titer retrovirus / vesicular stomatitis virus G pseudocci S A. 1996; 93: 11400-11406), the above-described concentrated virus supernatant was introduced to establish a stable 293GPG cell line capable of producing VSV-G pseudotyped retroviral particles upon induction. Specifically, the following procedure was used.

(1) Establishment of virus-producing 293GPG cells 293GP cells were transfected with 30 μg of retrovirus vector and 10 μg of vesicular stomatitis virus G protein (VSV-G) expression vector, and after 48 hours, the virus supernatant was collected and filtered through a 0.45 μm filter. And a primary virus preparation was obtained. Transfection was performed using a calcium phosphate method with a kit manufactured by Promega. Next, 293GPG cells were infected with the primary virus solution to establish a virus-producing cell bulk strain.

(2) Cloning of virus-producing 293GPG cells After confirming the virus-producing ability of the bulk strain, a cell line derived from a single cell was cloned. EGFP-expressing 293 cells were seeded in a 96-well flat-bottom plate with DMEM 10% (D-10) fetal calf serum + tetracycline 1 μg / ml, 200 μl per well, and after establishing a feeder layer, 15 Gy X-ray irradiation was performed. Bulk cells were sorted on this feeder at 1 cell / well using Moflo ^™ . After 7 to 10 days, the medium was changed to teracycline (−) D-10, and the culture was further continued, and the monoclonal clones showing proliferation were sequentially expanded.

As an example of the 293GPG clone selected above, FIG. 1 shows the state of cell proliferation of a clone in which the mouse Klf4 gene was introduced to induce virus production, and the mouse Klf4 gene was introduced under conditions that did not induce virus production. The state of cell growth of the clone is shown in FIG.

(3) Selection of 293GPG clone (3-1) Retention of vector sequence Genomic DNA was prepared from the obtained clone, and PCR analysis was performed using a primer pair that can be detected specifically for the vector provirus. As an example, FIG. 3 shows the results of selecting 293GPG clone (9 clones) introduced with the mouse Klf4 gene by PCR analysis using a primer pair capable of specifically detecting a vector provirus containing the mouse Klf4 gene. . MXs-Klf4 gene sequence was positive in 9 clones (45%) of 20 clones. From the above results, it can be seen that by the method of the present invention, a virus-producing cell carrying a gene encoding an reprogramming factor can be produced with a certain probability. As a positive control, β-globin genomic gene was amplified, and clones in which the control and proviral sequences were clearly amplified were tested for virus production ability.

(3-2) Virus production ability Each candidate clone was seeded in the same number in a 6-well plate, and virus production was induced by changing to a medium without tetracycline after 2 days. After changing to a fresh medium, the supernatant after 1 day was collected and infected with Jurkat cells. Two days after the infection, genomic DNA was prepared from Jurkat cells, and the number of provirus copies in the genome was compared semi-quantitatively using the same primer pair as in the PCR method. As an example, FIG. 4 shows the results of evaluating the infectivity of a retrovirus having a Klf4 gene produced by a 293GPG clone into which a mouse Klf4 gene has been introduced. No. Viruses produced by 2, 10, 14, and 19 clones were shown to have high infectivity for Jurkat cells.

Human gene-transferred 293GPG cells were prepared using the same method as mouse gene-transferred 293GPG cells. As an example, FIG. 12A shows a result of selecting 293GPG clone (13 clones) introduced with the human Klf4 gene by PCR analysis using a primer pair capable of specifically detecting a vector provirus containing the human Klf4 gene. . MXs-Klf4 gene sequence was positive in 8 clones (62%) out of 13 clones. As an endogenous control, the GAPDH genomic gene was amplified, and clones in which the control and proviral sequences were clearly amplified were tested for virus production ability. FIG. 12B shows the results of evaluating the infectivity of a retrovirus having a Klf4 gene produced by a 293GPG clone into which a human Klf4 gene has been introduced. No. investigated Among the 7, 8, and 9 clones, no. The virus produced by 9 clones was shown to have a high infectivity to Jurkat cells. The 293GPG clone into which the human OCT3 / 4 gene, human SOX2 gene, and human cMYC gene were introduced was also established in the same manner as described above.

Example 2: Preparation of iPS cells from mouse bone marrow HSPC (A) Material and method (1) Mouse C57BL / 6 (B6) -Ly5.2 and B6 EGFP transgenic mice were manufactured by Nippon SLC Co., Ltd. (Shizuoka, Japan) B6-Ly5.1 mice were purchased from Sankyo Lab Service Co., Ltd. (Tsukuba, Japan). NOD / CB17-Prkdc ^scid / J (NOD / Scid) mice were purchased from Nippon Charles River Co., Ltd. (Kanagawa, Japan).

(2) Purification of hematopoietic stem / progenitor cells (HSPCs) Bone marrow cells were isolated from the femur, tibia and pelvic bones of adult mice (20-30 weeks of age), and CD4, CD8, Incubated with a mixture of biotinylated monoclonal antibodies specific for B220, IL-7R, Gr-1, Mac-1, and Ter-119 (e-Bioscience, San Diego, CA). Lineage marker negative (Lin ⁻ ) cells were enriched by negative selection using Dynabeads ^™ MyOne ^™ Streptavidin C ₁ (Dynal Biotech, Lake Success, NY). Cells expressing c-Kit were obtained using a MACS ^™ LS column after incubation with anti-c-Kit MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany). When concentration of Ly5.1 ⁺ cells was required, biotin anti-CD45.2 monoclonal antibody was added to the above mixture to obtain hematopoietic stem / progenitor cells.

(3) Preparation and maintenance of iPS cell clones Established iPS cells and ES cells are 15% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), 1,000 U / ml leukemia inhibitory factor (LIF; Chemicon, Temecula, CA), 20 mM HEPES buffer (pH 7.3), 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids (GIBCO, Grand Island, NY), 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin (Sigma -Maintained on the cell layer of mouse embryo fibroblasts (MEF) treated with mitomycin C in complete ES medium consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with -Aldrich, St. Louis, MO).

Hematopoietic stem / progenitor cells were seeded at 5 × 10 ⁵ cells / well in a 24-well plate coated with fibronectin fragment CH296 (Takara Bio), and 20 ng / ml mouse stem cell factor (SCF), 100 ng / ml human thrombopoietin ( Pre-stimulated in α-MEM supplemented with TPO), 1% FBS, 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin. On the first day, a mixture of Oct3 / 4 gene, Sox2 gene, cMyc gene and Klf4 gene retrovirus (prepared in Example 1) was added to the hematopoietic stem / progenitor cell culture. Four hours after virus contact, the cells were washed and replaced with fresh media. On the second day, the cells were subjected to the same gene transfer treatment as described above, and the cells were collected after 4 hours. The transfected cells were plated again on the MEF layer in a 6-well plate containing complete ES medium. 10 ng / ml SCF was included in the culture so that the cells survived until days 5-7. The medium was changed daily from day 14 to day 16. Cells were collected, stained with phycoerythrin (PE) -conjugated anti-SSEA-1 monoclonal antibody (R & D Systems, Minneapolis, Minn.), And fractionated for SSEA-1-positive fractions with a MoFlo cell sorter (Dako, Glostrup, Denmark). The sorted cells were grown again on the MEF feeder layer in complete ES medium, and iPS cell-like colonies were individually picked up to establish stable iPS cell clones.

(4) PCR and RT-PCR analysis In order to recover iPS cells, MEF cells were removed by adhesion selection on a gelatin-coated plate (adhesion selection). Genomic DNA samples from iPS cells were prepared using the QIAmp DNA Mini Kit (QIAGEN, Valencia, CA). Total RNA samples were purified with Trizol reagent and reverse transcribed using a ThermoScript ^™ RT-PCR System (Invitrogen, Carlsbad, Calif.). Quantitative PCR analysis was performed with the cDNA product by Rodent GAPDH Control Reagents (Applied Biosystems, Foster City, Calif.). The amount of template used was made equal using the calculated copy number of GAPDH cDNA in each sample. PCR reaction was performed using ExTaq HS and LA Taq HS (Takara Bio) under the optimum conditions for each primer set. The primer sequences used are shown below. The primer sequences described in Table 1 are also described in SEQ ID NOs: 1 to 36 in the Sequence Listing.

(5) PCR analysis of immunoglobulin gene DJ rearrangement and single nucleotide polymorphism of Cd45 The immunoglobulin heavy chain gene rearrangement was analyzed according to a previously reported method (Kawamoto H, Ikawa T, Ohmura K, Fujimoto S, Katsura Y. T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver. Immunity. 2000; 12: 441-450; and Schlissel MS, Corcoran LM, Baltimore D. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J Exp Med. 1991; 173: 711-720). Single nucleotide polymorphisms in exon (EX) 25 of Cd45 were analyzed as previously reported (Ramos CA, Zheng Y, Colombowala I, Goodell MA. Tracing the origin of non-hematopoietic cells using CD45 PCR restriction fragment length polymorphisms. Biotechniques 2003; 34: 160-162). A PCR amplified genomic DNA sample was obtained from an iPS cell clone (polymorphic sequence sandwiched between a pair of primers). The amplicon was purified and a portion was digested with 10 U Kpn I (Takara Bio) at 37 ° C. for 1 hour. The digested sample and the undigested sample were electrophoresed on a 1% agarose gel.

(6) Flow cytometry analysis For flow cytometry analysis and cell sorting, cells were treated with FITC-conjugated anti-CD45 monoclonal antibody and FITC-streptavidin (BD PharMingen, San Diego, CA), PE-conjugated anti-SSEA-1 monoclonal antibody. , And PE-cyanin7 (Cy7) anti-CD117 monoclonal antibody (e-Bioscience). Dead cells were removed by propidium iodide staining. Stained cells were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

(7) Embryoid body (EB) formation iPS cells are treated with trypsin to complete embryoid body differentiation (EBD) medium (Kennedy M, Firpo M, Choi K, et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature. 1997; 386: 488-493). The cells were transferred to a 100 mm petri dish at 2 x 10 ⁵ cells per 10 ml EBD. The medium was replaced with fresh EBD on the 4th day, and then changed every 2 days until stable embryoid body formation was observed.

(8) Teratoma formation and histological analysis iPS cells were prepared at 1 × 10 ⁷ cells / ml in PBS. Suspended cells (1-3 × 10 ⁶ ) were injected subcutaneously into the flank of anesthetized NOD / Scid mice. Six weeks after injection, mice were killed and tumors were dissected. Tumor samples were fixed with 10% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.

(9) Preparation of chimeric mice Approximately 10 to 15 iPS cells were injected into blastocysts obtained from crosses of B6 and B6 x DBA2 F1 mice. Blastocysts were transplanted into pseudopregnant ICR female mice. Fetuses were removed by cesarean section on day 10.5 and analyzed.

(B) Results FIG. 1A shows the outline of the experiment. Preparation of iPS cells was attempted from bone marrow hematopoietic stem / progenitor cells collected after reconstitution of the hematopoietic system from a single hematopoietic stem cell of C57BL6 (B6) Ly1,1- origin. In this example, concentrated VSV-G pseudotyped retrovirus (made in Example 1) was used. Lin ^- Kit ⁺ cells, which are hematopoietic stem / progenitor cells containing CD45-expressing cells (˜98%), were purified from the bone marrow of the reconstituted mouse (B6Ly5,2) (FIG. 1B). These cells are transfected with a mixture of retroviral vectors containing each of Oct4, Sox2, Klf4 and c-Myc, transferred onto mouse embryonic fibroblast (MEF) cells, and leukemia inhibitory factor (LIF) until cell sorting (FIG. 1B). On days 9-11, visible iPS-like colonies appeared in most hematopoietic cells that were not reprogrammed. These colonies grew stably (FIG. 7A). To enrich iPS cells, cells expressing SSEA-1 were sorted on days 14-16 and grown for an additional 7-12 days (FIG. 1B). IPS cell-like colonies showing typical embryonic stem (ES) cell-like appearance were picked on days 21-28. These cells showed a very stable phenotype, had high alkaline phosphatase (ALP) activity (FIG. 1C), and expressed SSEA-1 at a level similar to that of ES cells (FIG. 7B). . In the absence of LIF, embryoid bodies were readily formed. Among the established iPS clones using the single nucleotide polymorphism of CD45, it was found that 3 clones were derived from Ly5.1 + cells and 1 clone was derived from Ly5.1 / 5.2 cells (FIG. 1D). These results demonstrated that direct reprogramming of bone marrow hematopoietic cells is possible. When iPS cells are established from bone marrow hematopoietic stem progenitors reconstituted from a single hematopoietic stem cell, these iPS cells are named sHSC-iPS cells.

Each sHSC-iPS cell clone had a proviral sequence of four reprogramming factors (FIG. 8A), but due to gene silencing, expression of the transgene could not be detected (FIG. 8B). All sHSC-iPS cells expressed each reprogramming factor gene endogenously (FIG. 8B). All sHSC-iPS cells expressed the ES cell marker genes Nanog, Eras, Rex1, and Gdf3 (FIG. 9A). Nanog expression was also confirmed by immunostaining (FIG. 9B). Despite low expression levels of other ES cell marker genes, Ecat1 and Zfp296, these sHSC-iPS cells showed teratoma formation (FIG. 10) and contribution to chimeric mice (FIG. 1E).

Next, the reproducibility of direct reprogramming of primary bone marrow hematopoietic stem / progenitor cells was confirmed. Initialization with retrovirus was performed using Lin ⁻ Kit ⁺ bone marrow cells obtained from adult B6 mice (FIG. 1B). From 5 × 10 ⁵ hematopoietic stem / progenitor cells, there were always 10-30 colonies showing ALP activity and having a typical ES cell-like appearance. An iPS cell clone (pHPC-iPS) established from primary bone marrow hematopoietic stem / progenitor cells expressed an ES cell marker gene more strongly than sHSC-iPS cells (FIGS. 2A and 9A). The expression level of the endogenous reprogramming factor gene was also stronger in the pHPC-iPS cells than in the sHSC-iPS cells (FIG. 9B) (FIG. 11). This result confirms the possibility that the enormous stress imparted to a single hematopoietic stem cell by reconstitution of the hematopoietic system limits the effective reprogramming of target cells that are thought to be aging. is there. In addition, confirmation of the germline non-recombinant structure in the immunoglobulin gene revealed the non-B cell origin of pHPC-iPS cells (FIG. 2B). The pHPC-iPS cells had the ability to differentiate into multiple lineages including various tissues representing all three germ layers, as demonstrated by teratoma formation (FIG. 2C). Moreover, it succeeded in producing the pHPC-iPS cell which constitutively expresses green fluorescent protein (GFP) from an EGFP transgenic mouse (FIG. 2D). These iPS cells showed a high contribution to fetal development when microinjected into blastocysts (FIG. 2E).

Example 3: Production of iPS cells from human skin cells and hematopoietic progenitor cells (A) Materials and methods
(1) Human skin cells Human adult and neonatal skin cells were purchased from Cell Applications, Inc. (San Diego, CA) and used.
(2) Human umbilical cord blood Human umbilical cord blood was purchased from the Tokyo Umbilical Cord Blood Bank with a sample obtained from the consent form for research use.
(3) Human bone marrow CD34-positive cells Human bone marrow CD34-positive cells were purchased from Lonza and used.

(4) Purification of hematopoietic stem / progenitor cells (HSPCs) Human umbilical cord blood was separated into mononuclear cells using Lymphosepar solution (Immunobiology Laboratories), and then anti-CD34 antibody microbeads. CD34 expressing hematopoietic stem / progenitor cells were purified by (Miltenyi Biotech, Bergisch Gladbach, Germany).

(5) Creation and maintenance of iPS cell clones Established iPS cells and ES cells are 20% knockout serum replacement (KSR; Invitrogen, Carlsbad, CA), 5 ng / ml basic fibroblast growth factor (bFCF; Upstate Biotech., Waltham, MA), 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid (GIBCO, Grand Island, NY), 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin (Sigma-Aldrich, St. Cell layer of mouse feeder mouse embryo fibroblasts (SNL MEF cell line) irradiated with 50 Gy in human undifferentiated maintenance medium consisting of F-12 mixed Dulbecco's modified Eagle medium (DMEM / F12) supplemented with Louis, MO) Maintained above.

(6) Establishment of iPS cells from human skin cells A frozen vial of human skin cells was thawed at 37 ^° C., and culture was started in DMEM medium containing 10% FBS. Cells in the logarithmic growth phase were collected by trypsin treatment and seeded at 8 × 10 ⁵ cells / dish in a gelatin-coated 10 cm dish. On the first day, a mixture of 3 types of OCT3 / 4 gene, SOX2 gene, hKLF4 gene, or 4 types of retroviruses added with cMYC gene (produced in Example 1) was added to the culture of skin cells. After removal of the medium on the second day, the medium was replaced with a new medium, the same gene introduction treatment as described above was performed, and the medium was replaced on the third day. The medium was changed again on the 5th day, and the cells transfected with the gene on the 7th day were collected by trypsin treatment. On the 10 cm dish of the irradiated SNLMEF feeder cell layer prepared on the previous day (6th day), 5 -10 x 10 ⁴ seeds / dish. On the 8th day, the medium was replaced with human undifferentiated maintenance medium, and thereafter, medium replacement with human undifferentiated maintenance medium was continued every two days. Around 20-30 days, colonies that stably maintain undifferentiated ES cell-like morphology were picked up as needed and seeded on 6 cm dishes containing 50 Gy irradiated mouse embryo fibroblasts (MEF). Until growth was stabilized, 10 (M Rock inhibitor (Y27632, Chemicom) was added to human undifferentiated maintenance medium and used. Subsequently, a strain that stably maintained the ES cell-like morphology was selected and transferred. Instead, human iPS cell clones were established.

(7) Establishment of iPS cells from human hematopoietic stem / progenitor cells Human umbilical cord blood or bone marrow blood CD34 positive cells were seeded at 5 × 10 ⁵ cells / well in a 24-well plate coated with fibronectin fragment CH296 (Takara Bio). 50 ng / ml mouse human stem cell factor (SCF), 50 ng / ml human thrombopoietin (TPO), 50 ng / ml human Flt3-L, 115% FBS, 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin Pre-stimulation in supplemented ISCOVE'S MODIFIED DULBECCO'S MEDIUM (IMDM) medium. On the first day, stain with anti-human CD34APC antibody (BD) and anti-human CD45Alexa405 antibody, and use Moflo to detect CD34 positive (CD45 + / CD34 +) and CD34 negative (CD45 + / CD34-) cells in the CD45 positive cell fraction. They were collected and seeded again in 1 well of a 6-well plate coated with retronectin. A mixture of 3 types of OCT3 / 4 gene, SOX2 gene, hKLF4 gene and 4 types of retroviruses (prepared in Example 1) with the above medium added to the culture medium of CD34 positive cells was added. . By the 2nd day, repeat the same gene transfer treatment as described above 1-3 times, and then add 5 ng / ml basic fibroblast growth factor (bFCF) to the medium and pick up valproic acid for colonies. The culture was continued until the addition. From day 7 onwards, 20% knockout serum replacement (KSR), 5 ng / ml basic fibroblast growth factor (bFCF), 50 ng / ml mouse stem cell factor (SCF), 0.1 mM 2-mercaptoethanol, F-12 mixed Dulbecco's modified Eagle medium supplemented with 0.1 mM non-essential amino acids (GIBCO, Grand Island, NY), 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin (Sigma-Aldrich, St. Louis, MO) It was maintained on a cell layer of mouse feeder cells (SNL MEF cell line) irradiated with 50 Gy in a human undifferentiated maintenance medium consisting of (DMEM / F12). From day 15 onward, colonies that stably maintain undifferentiated ES cell-like morphology were picked and seeded on 6 cm dishes containing 50 Gy irradiated mouse embryonic fibroblasts (MEF). Until growth was stabilized, 10 (M Rock inhibitor (Y27632) was added to human undifferentiated maintenance medium. After that, a strain that stably maintained the ES cell-like morphology was selected and passaged. A human iPS cell clone was established.

(8) PCR and RT-PCR analysis In order to recover iPS cells, MEF cells were removed by diminishing adherent cells on gelatin-coated plates. Genomic DNA samples from iPS cells were prepared using the QIAmp DNA Mini Kit (QIAGEN, Valencia, CA). Total RNA samples were purified with Trizol reagent and reverse transcribed using a ThermoScript ^™ RT-PCR System (Invitrogen, Carlsbad, Calif.). Semi-quantitative PCR analysis was performed using the cDNA product by GAPDH Control Reagents (Applied Biosystems, Foster City, Calif.) Using the GAPDH gene as an endogenous control. The amount of template used was made equal using the calculated copy number of GAPDH cDNA in each sample. The amount of cDNA in each sample was adjusted. PCR reaction was performed using ExTaq HS and LA Taq HSrTaq (Takara Bio) under the optimum conditions for each primer set. The primer sequences used are shown below. The primer sequences described in Table 2 are also described in SEQ ID NOs: 37 to 58 in the Sequence Listing.

(9) Teratoma formation and histological analysis iPS cells were prepared at 1 × 10 ⁷ cells / ml in PBS. Suspended cells (1-3 × 10 ⁶ ) were injected into the testes of anesthetized NOD / Scid mice. Six to eight weeks after injection, mice were sacrificed and tumors were dissected. Tumor samples were fixed with 10% paraformaldehyde 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.

(B) Results FIG. 13 and FIG. 14 show the outline of the experiment. By operating according to the method, human ES cells were obtained from dermal fibroblasts from around day 20 (FIG. 13) and from cord blood CD34-positive cells and bone marrow blood CD34-positive cells (FIG. 14) from around day 12, respectively. The appearance of a colony was confirmed. Like ES cells, an undifferentiated marker (SSEA-4 as an example, FIG. 15A) was expressed, and no chromosomal abnormality was observed by the G-band method (FIG. 15B). The number of ES cell-like colonies formed in the experiments so far is shown in the table (Table 3).

VPA 0.5mM
By using the 293GPG virus of the present invention, it was possible to establish iPS cells from cord blood CD45 + / CD34 + cells at the same or higher efficiency as skin cells. The results of co-culture with MEF, Matrigel + MEF Conditioned medium, and SNL feeder cells are shown.

Established human iPS cell clones had provirus sequences of 3 or 4 reprogramming factors, but due to gene silencing, transgene expression was detected under RT-PCR trial conditions could not. These human iPS cells endogenously expressed each reprogramming factor gene. In addition, ES cell marker genes NANOG and REX1 were expressed (FIG. 16). These cells also showed teratoma-forming ability.

Example 4: Preparation of iPS cells from peripheral blood CD34 positive cells (method)
100 ml of human peripheral blood was collected, and mononuclear cells were separated using Ficoll (approximately 2-5 × 10e8 cells could be recovered). CD34MACS-beads were used to purify into CD34 positive cells (5-10 × 10e5 cells could be recovered. In addition, 5 × 10e5 cells can be established sufficiently). Blood cell medium (50 ng / ml mouse human stem cell factor [SCF], 50 ng / ml human thrombopoietin [TPO], 50 ng / ml human Flt3-L, 115% FBS, 2 mM L-glutamine, and 100 U / ml After starting culture in ISCOVE'S MODIFIED DULBECCO'S MEDIUM [IMDM] medium supplemented with penicillin / streptomycin, on days 1 and 2, stem cell factor (SCF), thrombopoietin (TPO), and FLT-3 ligand (FL) ( In the presence of 50 ng / ml), a mixture of Oct3 / 4 gene, Sox2 gene, cMyc gene and Klf4 gene retrovirus (produced in Example 1) was added (infected 3 times every 12 hours). . The medium was changed every two days, and cells were seeded on MEF on the seventh day after the start of culture. On day 8 after the start of culture, iPS cell culture medium (20% knockout serum replacement [KSR], 5 ng / ml basic fibroblast growth factor [bFCF], 50 ng / ml mouse stem cell factor [SCF], 0.1 mM F-12 supplemented with 2-mercaptoethanol, 0.1 mM non-essential amino acid [GIBCO, Grand Island, NY], 2 mM L-glutamine, and 100 U / ml penicillin / streptomycin [Sigma-Aldrich, St. Louis, MO] The medium was changed to a human undifferentiated maintenance medium comprising a mixed Dulbecco's modified Eagle medium [DMEM / F12]. When colonies were confirmed, they were recovered using P200 and grown. FIG. 17 shows the process of establishing iPS cells derived from the peripheral blood CD34-positive cells (Non-mobilized).

(result)
FIG. 18 shows normal donor peripheral blood CD34-positive cell-derived iPS cells (TkPBV1-1) established as described above. By introducing the retrovirus derived from the virus-producing cells of the present invention (OCT3 / 4, SOX2, KLF4, c-MYC), human iPS cells (TkPBV) from CD34 positive cells (without mobilization with G-CSF) from normal donors. -1-1) could be induced.

Claims (23)

1. In order to be able to produce an RNA virus comprising a gene encoding a reprogramming factor and comprising the vesicular stomatitis virus G (VSV-G) protein as an envelope protein,
   A virus-producing cell having on its chromosome a gene encoding the reprogramming factor and a gene encoding a virus constitutive protein including vesicular stomatitis virus G (VSV-G) protein.
2. The virus-producing cell according to claim 1, wherein the gene encoding the reprogramming factor is one or more of an Oct gene, a Sox gene, a Klf gene, and a Myc gene.
3. A packaging cell that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a given drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug, or Reprogrammed into packaging cells that are unable to express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug and can express vesicular stomatitis virus G (VSV-G) protein in the presence of the drug The virus-producing cell according to claim 1 or 2, which is obtained by introducing a gene encoding a factor.
4. The virus-producing cell according to claim 3, wherein the gene encoding the reprogramming factor is introduced as a virus.
5. The virus-producing cell according to claim 3 or 4, wherein the predetermined drug is tetracycline.
6. A packaging cell that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a given drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug, or A packaging cell that is unable to express vesicular stomatitis virus G (VSV-G) protein in the absence of any other drug and that is capable of expressing vesicular stomatitis virus G (VSV-G) protein in the presence of the drug is a human fetus. A cell obtained by introducing a gene encoding vesicular stomatitis virus G (VSV-G) protein into a cell line established by transforming renal cells with the E1 gene of adenovirus, A gene cannot be expressed in the presence of a given drug and expressed in the absence of the drug The cell according to any one of claims 3 to 5, wherein the cell is introduced so that the gene can be expressed, or the gene cannot be expressed in the absence of a predetermined drug and is introduced so that the gene can be expressed in the presence of the drug. A virus-producing cell according to claim 1.
7. The virus-producing cell according to claim 6, wherein the cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus is HEK293 cells.
8. Packaging cells that are unable to express vesicular stomatitis virus G (VSV-G) protein in the presence of a given drug and are capable of expressing vesicular stomatitis virus G (VSV-G) protein in the absence of the drug are HEK293GPG The virus-producing cell according to any one of claims 3 to 7, which is a cell.
9. The virus-producing cell according to any one of claims 1 to 8, which is a cloned cell derived from a single cell.
10. A packaging cell that cannot express vesicular stomatitis virus G (VSV-G) protein in the presence of a given drug and can express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug, or Reprogrammed into packaging cells that are unable to express vesicular stomatitis virus G (VSV-G) protein in the absence of the drug and can express vesicular stomatitis virus G (VSV-G) protein in the presence of the drug The method for producing a virus-producing cell according to any one of claims 1 to 9, comprising introducing a gene encoding a factor.
11. The method according to claim 10, wherein the gene encoding the reprogramming factor is introduced as a virus.
12. An RNA virus comprising a gene encoding a reprogramming factor, wherein the RNA virus comprises vesicular stomatitis virus G (VSV-G) protein as an envelope protein.
13. The RNA virus according to claim 12, wherein the gene encoding the reprogramming factor is at least one of Oct gene, Sox gene, Klf gene, and Myc gene.
14. The RNA virus according to claim 12 or 13, which is produced by the virus-producing cell according to any one of claims 1 to 9.
15. The RNA virus according to any one of claims 12 to 14, which is used for producing induced pluripotent stem cells.
16. The RNA virus according to any one of claims 12 to 15, which is cryopreserved after production.
17. 10. An RNA virus comprising a gene encoding a reprogramming factor comprising culturing the virus-producing cell according to any one of claims 1 to 9, wherein vesicular stomatitis virus G (VSV-G) protein is used as an envelope protein A method for producing an RNA virus comprising:
18. A method for producing induced pluripotent stem cells, comprising introducing the RNA virus according to any one of claims 12 to 16 into a somatic cell.
19. The method for producing induced pluripotent stem cells according to claim 18, wherein the somatic cells are hematopoietic cells.
20. The method for producing induced pluripotent stem cells according to claim 19, wherein the hematopoietic cells are derived from bone marrow.
21. The method for producing induced pluripotent stem cells according to claim 19, wherein the hematopoietic cells are derived from cord blood.
22. The method for producing induced pluripotent stem cells according to claim 18, wherein the somatic cells are peripheral blood cells.
23. The method for producing induced pluripotent stem cells according to claim 18, wherein the somatic cells are peripheral blood CD34-positive cells.

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