WO2011135969A1

WO2011135969A1 - Method for producing induced pluripotent stem cells

Info

Publication number: WO2011135969A1
Application number: PCT/JP2011/058027
Authority: WO
Inventors: 健一磯部
Original assignee: 国立大学法人名古屋大学
Priority date: 2010-04-28
Filing date: 2011-03-30
Publication date: 2011-11-03
Also published as: JPWO2011135969A1

Abstract

Disclosed is a method for efficiently and conveniently producing iPS cells. Pluripotent stem cells are obtained by culturing bone marrow in the presence of granulocyte-monocyte colony-stimulating factor, and then genetically transforming the propagated cells.

Description

Method for producing induced pluripotent stem cells

The present invention relates to a method for producing induced pluripotent stem cells and uses thereof. This application claims priority based on Japanese Patent Application No. 2010-103211 filed on Apr. 28, 2010, the entire contents of which are incorporated by reference.

The establishment of induced pluripotent stem cells (iPS cells) from mice or humans is a major breakthrough in regenerative medicine (Non-Patent Documents 1 to 5). This technology is expected to enable treatment using patients' own cells (regenerative medicine). Elderly people create iPS cells from their own cells and modify them as necessary to use them. The era of repairing organs and tissues whose functions have declined and treating diseases associated with aging is about to come.

One of the issues to be overcome in clinical application of iPS cells is their low production efficiency. In order to use iPS cells for treatment of diseases and repair of organs and tissues that have deteriorated due to aging, it is necessary to develop a technique for efficiently producing iPS cells. In particular, in the present age of aging, it is required to provide a technique for efficiently producing iPS cells from somatic cells of elderly people (aging individuals). Then, this invention makes it a subject to provide the method of producing an iPS cell efficiently and simply.

The present inventors have intensively studied to solve the above problems, adopt bone marrow that can be collected relatively easily as a cell source, and perform transformation operations (transformation into iPS cells) by introducing transcription factors and the like. Previously, we devised a strategy of culturing cells in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF; Macrophage-colony-stimulating Factor). In order to verify the effectiveness of this strategy, experiments were performed using bone marrow from aged mice. Specifically, the bone marrow of an aged mouse was cultured in a medium supplemented with GM-CSF for a short period, and then four transcription factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced. As a result, the appearance of iPS cell-like colonies was observed after about 1 month. When clones were established from the formed colonies and their characteristics were examined, they showed the ability to differentiate into three germ layers as well as iPS cells prepared from fibroblasts and highly expressed pluripotent stem cell markers. Thus, iPS cells were successfully established from the bone marrow of an aging individual, and the above strategy was confirmed to be effective. Here, the age of the old mouse used in the experiments by the present inventors (21 months of age) corresponds to 60s to 80s in terms of human age. The successful establishment of iPS cells from such aged cells is a major breakthrough for the practical application of iPS cells.

On the other hand, it was shown that iPS cell colonies appeared earlier when bone marrow of young individuals was used than when bone marrow of old individuals was used (see Examples described later). This result confirms that the bone marrow activity of young individuals is high, while the above strategy is also effective when the bone marrow of young individuals is used as the cell source.

By the way, many iPS cells in past reports have been prepared from fetal fibroblasts in mice and young individual fibroblasts in humans. Even when producing from fibroblasts, the production efficiency of iPS cells is not good. Furthermore, it is more difficult to produce iPS cells from other cells. For example, in order to establish iPS cells from T cells, it is necessary to delete the p53 gene (Non-patent Document 6). On the other hand, although it has been reported that iPS cells have been successfully established from bone marrow (Non-patent Documents 7 and 8), the bone marrow of an aged individual is not used. Although the method employed by the present inventors is relatively simple, it increases iPS cell production efficiency and enables the establishment of iPS cells from the bone marrow of aged individuals. In other words, it is different from past reports, and provides extremely important and useful technology for the practical application and application of iPS cells in the field of regenerative medicine, and its significance is great. On the other hand, according to the method of the present invention, iPS cells are prepared from myeloma cells induced by stimulation with GM-CSF. Therefore, iPS cells suitable for application to regenerative medicine and free from gene rearrangement can be obtained. This is also an advantage unique to the production method of the present invention, and is worthy of special mention.
The present invention listed below is based on the above findings and results.
[1] A method for producing induced pluripotent stem cells, comprising the following steps (1) and (2):
(1) culturing bone marrow in the presence of granulocyte monocyte colony-stimulating factor;
(2) Transforming the proliferated cells into pluripotent stem cells.
[2] The method for producing induced pluripotent stem cells according to [1], wherein the bone marrow is human bone marrow.
[3] The method for producing induced pluripotent stem cells according to [1], wherein the bone marrow is human bone marrow after the middle age.
[4] The method for producing induced pluripotent stem cells according to [1], wherein the bone marrow is mouse bone marrow.
[5] The transformation according to any one of [1] to [4], wherein the transformation in step (2) is performed by introducing four factors Oct3 / 4, Sox2, Klf4 and c-Myc into the cell. Of Induced Pluripotent Stem Cells
[6] An induced pluripotent stem cell produced by the production method according to any one of [1] to [5].
[7] The induced pluripotent stem cell according to [6], which expresses Nanog gene, Oct4 gene, FgF4 gene, Esg-1 gene and Cript gene, which are pluripotent stem cell markers.
[8] The induced pluripotent stem cell according to [6] or [7], wherein gene rearrangement is not performed.
[9] A method for producing a cell differentiated into a specific cell lineage, which comprises subjecting the induced pluripotent stem cell according to any one of [6] to [8] to a differentiation induction treatment.
[10] A method for preparing cells for producing induced pluripotent stem cells, comprising culturing bone marrow in the presence of granulocyte monocyte colony-stimulating factor.

The figure which shows typically the preparation procedure of an iPS cell. Bone marrow cells were removed from 21-month-old EGFP (enhanced green fluorescent protein) positive C57BL / 6 mice, cultured in the presence of GM-CSF for 4 days, and then 4 factors necessary for iPS production were introduced. Several IPS-like colonies appeared. These were picked up using trypsin and cultured in 24-well plates. Among them, two clones grew while maintaining iPS-like morphology. One clone was used for later analysis while stored under liquid nitrogen. The figure which shows the morphological change of a cell. Three morphological changes are shown. Left: Morphology after culturing bone marrow cells of 21-month-old C57BL / 6 mice in the presence of GM-CSF (culture day 4). Center: Appearing iPS cell colony. Right: Established bone marrow cell-derived iPS cells (BM-M-iPS). Comparison of iPS cell production efficiency. C57BL / 6 mouse fibroblasts (MEF), 2-month-old mouse bone marrow cells cultured in the presence of GM-CSF (4 days), or 23-month-old mouse bone marrow cells cultured in GM-CSF (4 days ) Were cultured on feeder cells in a 10 cm culture dish, 4 factors for iPS production were introduced, and the number of colonies that appeared was counted over time. The figure which shows the tumor formed by the subcutaneous injection of BM-M-iPS cell. Left: A tumor formed on the back of the mouse. Right: Removed mass. The figure which shows the differentiation ability in the body of the iPS cell established from the aging bone marrow myeloma cell. ^Seven BM-M-iPS cells 1 × 10 ⁷ were injected subcutaneously into the back of C57BL / 6 mice. A. The tumor was confirmed 21 days after injection (FIG. 3) and fixed with OCT for preparation of tissue sections. B. H & E stained image is shown. Ectoderm; stratum corneum of skin; mesoderm; smooth muscle cells; endoderm; gastrointestinal epithelial cells. The figure which shows the gene expression of the iPS cell established from the aging bone marrow myeloma cell. Bone marrow cells cultured for 4 days in the presence of GM-CS (BMD4) are cultured for 7 days (BMD7; cells obtained under normal conditions to induce bone marrow dendritic cells). α, IL-1b), chemokine (ccl7), transcription factors (C / EBPa, Pu-1) are expressed, but iPS cells (BM-M-iPS) established from senescent bone marrow myeloma cells are produced from MEF Similar to iPS cells, the expression of these cytokines is reduced. Instead, the expression of pluripotent stem cell markers Nanog, Oct4, FgF4, Esg-1 and Cript increased. Gene expression was also examined for BM-M-iPS (BM-i-DC) and MEF-iPS (MEF-i-DC) after differentiation induction. In vitro differentiation of iPS cells (BM-M-iPS) derived from senescent (21 months old) bone marrow myelocytes. (A) Scheme of embryoid body (EB) formation and in vitro differentiation induction (× 40). BM-M-iPS cells were cultured for 8 days by the hanging drop method and then transferred to a 24-well plate. The culture by the hanging drop method (left) and the formed EB (right) are shown (using an Olympus phase contrast microscope). (B) Immunostaining of differentiated BM-M-iPS (× 100). The upper row shows mesoderm tissue expressing α-actin. The middle row shows endoderm tissue expressing α-fetoprotein. The lower row shows ectoderm tissue expressing neurofilament H. (C) iPS cells were cultured on OP9 seeded in a 6-well plate coated with 0.1% gelatin. After culturing for 5 days, a phase contrast microscope image was taken (× 100). Upper row: MEF-iPS on day 5 of co-culture with OP9. Bottom: BM-M-iPS on day 5 of co-culture with OP9. Phase contrast microscope image and Giemsa stained image after differentiation induction. Upper row: Differentiation of MEF-iPS. Bottom: BM-M-iPS differentiation. Analysis results of cell surface markers before and after differentiation induction. BM-M-iPS cells and MEF-iPS cells were induced to differentiate into myeloma using GM-CSF, and then the myeloma cell surface marker was detected. Chimeric mice obtained using BM-M-iPS cells.

The present invention provides a novel method for producing induced pluripotent stem cells (hereinafter also referred to as the production method of the present invention). “Induced pluripotent stem cells” are cells having pluripotency (multipotency) and proliferative ability, which are produced by reprogramming somatic cells by introduction of reprogramming factors. Induced pluripotent stem cells exhibit properties similar to embryonic stem cells (ES cells). For convenience of explanation, in the present specification, induced pluripotent stem cells may be abbreviated as iPS cells.

In the production method of the present invention, the following two steps, namely, step (1) “step of culturing bone marrow in the presence of granulocyte monocyte colony-stimulating factor” and step (2) “proliferated cells as pluripotent stem cells” Step of transforming into Details of each step will be described below.

(1) Cultivation in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF) In the present invention, prior to the step of transforming into iPS cells, bone marrow cells are cultured in the presence of GM-CSF. To do. Performing this culture is the greatest feature of the present invention. By incorporating this culture step, iPS cell production efficiency is improved. Without being bound by theory, application of stimulation with GM-CSF leads to induction in the myeloma cell lineage, resulting in highly proliferative cells. Moreover, gene rearrangement can be prevented. This feature is clearly different from the technology for producing iPS cells derived from B cells and T cells that have undergone gene rearrangement (reference documents 6 and 13), and can provide iPS cells suitable for use in regenerative medicine. Important and beneficial in terms.

In the present invention, first, bone marrow is prepared. Bone marrow can be collected in the usual way (for example, the bone marrow transplant promotion foundation donor safety committee edited by the bone marrow transplant promotion foundation third edition is helpful). For example, it can be collected from the iliac bone using a bone marrow puncture needle. The collected bone marrow may be subjected to pretreatment. Examples of the pretreatment here include removal of impurities by filter treatment, separation of cell components by centrifugation, washing with PBS, and the like.

The animal species of bone marrow is not particularly limited. Bone marrow collected from humans is preferably used, but animals other than humans (including pet animals, domestic animals, laboratory animals. Specifically, examples include mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep. Bone marrow collected from dogs, cats, chickens, etc.).

In general, the activity of a cell depends on the age of the individual from which it is derived. That is, the cells of old individuals are usually less active than the cells of young individuals. For this reason, when producing iPS cells from cells of old individuals, the success rate (production efficiency) is low. According to the present invention, iPS cell production efficiency can be improved, and iPS cells can be obtained relatively stably from cells of old individuals. In one embodiment of the present invention, this feature is utilized to produce iPS cells using human bone marrow after the middle age. In another embodiment, human bone marrow after middle age is used, and in another embodiment, human bone marrow after senior age is used. Human life is childhood (0-4 years), boyhood (5-14 years), adolescence (15-24 years), middle age (25-44 years), middle age (45-64 years), Can be divided into older age (65 years old and over). In general, it is said that it becomes easy to suffer from lifestyle-related diseases after the middle age, and the prevalence of various diseases caused by aging (for example, muscle atrophy, osteoporosis, arthritis) increases. The present invention that improves the production efficiency of iPS cells greatly contributes to the development of medical treatment for patients after the middle age when the risk of disease increases. As can be seen from the above description, the present invention is particularly effective when the cells of old individuals, in other words, cells with low activity, are used as the source, but the scope of application is not particularly limited. You may use a cell of an age individual as a source.

Bone marrow prepared as described above is subjected to culture in the presence of GM-CSF. Specifically, bone marrow is cultured in a medium supplemented with GM-CSF. Prior to culturing under such conditions, culturing may be performed in a medium not containing GM-CSF. For example, GM-CSF-free medium is used for primary culture (or primary culture and several subsequent passages), and GM-CSF-containing medium is used for subsequent subcultures. The animal species of GM-CSF to be used may not be the same as the animal species of bone marrow, but the animal species are preferably combined so that the action of GM-CSF can be exhibited well. For example, when human bone marrow is used, it is preferable to use human GM-CSF. Matching animal species in this way is also preferable in terms of preventing the introduction of components derived from different animals and improving safety.

GM-CSF is a cytokine (hematopoietic growth factor) that promotes differentiation into hematopoietic stem cells, and promotes proliferation and differentiation of progenitor cells of granulocytes and monocytes / macrophages. GM-CSF is mainly secreted from activated T cells. GM-CSF also has colony-forming activity of erythroblasts, eosinophils, and megakaryocytes, and has been reported to be widely involved in the hematopoietic mechanism of living organisms. GM-CSF separated and purified from a living body may be used, or recombinantly produced GM-CSF (recombinant GM-CSF) may be used. Several GM-CSFs are commercially available (for example, provided by Cosmo Bio Co., Ltd., Biochemical Bio Business Co., Ltd., Miltenyi Biotech Co., Ltd., etc.), and such commercially available products can also be used.

The concentration of GM-CSF in the medium is not particularly limited, and can be set as appropriate by preliminary experiments or the like. An example of the GM-CSF concentration is 1 ng / ml to 100 ng / ml, and a preferred concentration is 5 ng / ml to 20 ng / ml. The GM-CSF concentration need not be constant throughout the entire culture period. For example, it is possible to employ a culture condition in which the added concentration becomes higher in the later stage of culture. The culture period using the GM-CSF-containing medium is, for example, 1 to 20 days, preferably 2 to 14 days, more preferably 3 to 7 days, and even more preferably 3 to 5 days.

Except for the addition of GM-CSF, basically, normal culture conditions for mammalian cells may be followed. The medium used in the present invention can be constituted by adding necessary components to the basic medium. Iscov modified Dulbecco medium (IMDM) (GIBCO, etc.), ham F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco's modified Eagle medium (D-MEM) (Nacalai Tesque, Sigma, Gibco, etc.), Glasgow basic medium (Gibco, etc.), RPMI1640 medium, etc. can be used. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (Gibco)) can be mentioned. Examples of components that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum replacement (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, 2-mercapto Examples include ethanol, leukemia inhibitory factor (LIF), PVA, L-glutamine, insulin, transferrin, and selenium. Other culture conditions such as culture temperature may be in accordance with normal culture conditions for mammalian cells. That is, for example, it may be cultured in an environment of 37 ° C. and 5% CO ₂ .

(2) Transformation into iPS cells (initialization, reprogramming)
As described above, without being bound by theory, bone marrow derived myeloid cells grow when bone marrow is cultured in the presence of GM-CSF. In the present invention, the myeloma cells are transformed into iPS cells. Various iPS cell production methods reported so far can be used for transformation into iPS cells. In addition, it is naturally assumed that an iPS cell production method developed in the future will be applied.

The most basic method of iPS cell production is to introduce four factors, transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). Three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), Oct3 / 4 and Klf4, two factors (Kim J B, et al: Nature 454 (7204 ), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136 (3), 411-419, 2009) has been reported to establish iPS cells. In addition, a method of introducing a gene expression product into a cell (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al : Cell Stem Cell 4, 472-476, 2009) has also been reported. On the other hand, by using the inhibitor BIX-01294 for histone methyltransferase G9a, the histone deacetylase inhibitor valproic acid (VPA) or BayK8644, production efficiency can be improved and factors to be introduced can be reduced. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J Et al: PLoS. Biol. 6 (10), e 253, 2008). Studies on gene transfer methods are also underway. In addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766- 770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or Techniques using episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed.

Cells that have undergone transformation (reprogramming) into iPS cells are expressed pluripotent stem cell markers (undifferentiation markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript Etc. can be selected as an index. The selected cells are collected as iPS cells.

The iPS cells obtained by the production method of the present invention are used for drug discovery research tools (eg, development of cell-based assay systems such as screening systems for drug efficacy and safety testing), research tools for elucidating disease states (eg, human diseases) Production of model cells or model animals), or a raw material or material for cell medicine / regenerative medicine. In order to produce disease model cells from iPS cells or to use iPS cells for cell therapy / regenerative medicine, iPS cells are differentiated into a desired cell lineage in principle. Therefore, the present invention also provides a method for producing a cell differentiated into a specific cell lineage, characterized by subjecting the iPS cell produced by the production method of the present invention to differentiation induction treatment. As a means for inducing differentiation of iPS cells obtained by the production method of the present invention into a specific cell lineage, various differentiation induction methods reported in the past for iPS cells can be used, as well as embryonic stem cells (ES cells) The differentiation induction method reported for) can also be applied with modifications as necessary. For example, embryoid body (EB) formation method (Chinzei R, Tanaka Y, et al: Hepatology 36, 22-29, 2002; Yamada T, Yoshikawa M, et al: Stem Cells 20, 146-154, 2002; Asahina K , Fujimori H, et al: Genes Cells 9, 1297-1308, 2004; Choi D, Lee HJ, et al: Stem Cells 23, 817-827, 2005) is the most common method for differentiating ES cells in vitro Is. The EB formation method may be applied when inducing differentiation of the iPS cells obtained by the production method of the present invention. Hanging drop method (Rundnick MA, Mcburney MW, Cell culture methods and induction of differentiation of embryonal carcinoma cell lines, p. 19-49. In Robertson, E. J. (ed.), Teratocarcinomas and embryonic stem cells: a practical approach, IRL press, Washington DC, 1987 etc., Bacterial dish method (Doetschman TC, Eistetter H, Katz M, et al: Expembol , 27-45, 1985, etc.), U-bottom multi-well plate method (Yasuda E, Seki Y, Higuchi T, et al: J Biosci Bioeng 107, 442-446, 2009; Karp JM, Yeh J, Eng G, et al: Lab Chip 7, 786-794, 2007; Moeller HC, Mian MK, Shrivastava S, et al: Biomaterials 29, 752-763, 2008, etc.), SFEBq method (Eiraku M, Watanabe K, Matsuo-Takasaki) M, “etal”: “Cell” Stem “Cell” 3, “519-532,” 2008, etc.) have been developed. It is possible to induce differentiation of various tissue cells from EB (Doetschman.T.C, et al: J. Embryol. Exp. Morphol. 87, 27-45, 1985). ES cell differentiation induction methods include EB formation, differentiation induction by forced expression of specific genes (Levinson-Dushnik M, Benvenisty N, et al: Mol Cell BioI 17, 3817-3822, 1997; Ishizaka S, Shiroi A, et al: FASEB J 16, 1444-1446, 2002; Fujikura J, Yamato E, et al:: Genes Dev 16, 784-789, 2002; Blyszczuk P, Czyz J, et al: Proc Sat cad 100, 998-1003, 2003; Miyazaki S, Yamato E, et al: Diabetes 53, be1030-1037, 2004), co-culture with stromal cells (Shiraki 方法 N, Lai CJ, et al: Genes Cells 10, 503) -516, 2005; Ishii T, Yassuchika K, et al: Exp Cell Res 309, 68-77, 2005; Teratani T, Yamamoto H, et al: Hepatology 41, 836-846, 2005; Soto-Gutierrez A, Kobayashi N , Et al: Nat Biotechnol 24, 1412-1419, 2006), method using humoral factors (SchuldinerM, Yanuka 0, et al: Proc Natl Acad Sci USA 97, 11307-11312, 2000; Lumelsky N, Blondel 0, et al: Science 292, 1389-1394, 2001; Hamazaki T, Iiboshi Y, et al: FEBS Lett 497, 15-19, 2001; Hori Y, Rulifson IC, et al: Proc Natl Acad Sci USA 99, 16105-16110, 2002; Kim D, Gu Y, et al: Pancreas 27, e34-41, 2003). According to these methods, iPS cells obtained by the production method of the present invention may be induced to differentiate. The following are some examples of reports on differentiation induction methods.

To induce differentiation of EBs into cardiomyocytes, the embryoid body is attached to a culture dish and cultured, and the self-pulsating myocytes are separated (Boheler KR, et al: Circ. Res. 91, 189-201) , 2002 etc.) is common. A method for obtaining cardiomyocytes with high purity has also been reported (KlugluM. G, et al: J. Clin. Invest. 98, 216-224 (1996);) Muller M, et al: FASEB. J. 14 , 2540-2548, 2000, etc.). It is also possible to induce differentiation from EBs into pancreatic insulin-producing cells (D'Amour KA, Bang AG, Eliazer S, et al: Nat Biotechnol 24, 1392-1401, 2006, etc.). On the other hand, differentiation induction methods to vascular endothelial cells and vascular smooth muscle cells have also been reported (Vittet D, et al: Proc. Natl. Acad. Sci. USA 94, 6273-6278, 1997; Bloch W, et al: J. Cell Biol. 139, 265-278, 1997; Yamashita J, et al: Nature-408, 92-96, 2000; Feraud O, et al: Lab. Invest. 81, 1669-1681, 2001, etc.). There are also several reports on the induction of differentiation into retinal cells (Ikeda H, Osakada F, Watanabe K, Mizuseki K, Haraguchi T, Miyoshi H, Kamiya D, Honda Y, Sasai N, Yoshimura N, Takahashi M, Sasai Y Proc. Natl. Acad. Sci. USA 102, 11331-11336, 2005; Osakada F, Ikeda H, Mandai M, Wataya T, Watanabe K, Yoshimura, N, Akaike A, Sasai Y, techNolh. 26, 215-224, 2008; Osakada F, Ikeda H, Sasai Y, Takahashi M, Nat. Protoc. 4, 811-824, 2009; Hirami Y, Osakada F, Takahashi K, Okita K, Yked Yoshimura N, Takahashi M, Neurosci. Lett. 458, 126-131, 2009; Osakada F, Jin ZB, Hirami Y, Ikeda H, Danjyo T, Watanabe K, Sasai Y, Takahashi M. 179 , 2009 etc.). In addition, a method of culturing in the presence of bFGF followed by suspension aggregation culture system (StudertuL, et al: Nat. Neurosci. 1, 290-295, 1998, etc.), in the presence of bFGF and glial cell line culture supernatant (Daadi M. M, and Weiss S. J .: Neuroscience 19, 4484-4497, 1999, etc.), a method using FGF8, Shh, bFGF, ascorbic acid, etc. (Lee S. H. et al: Nat. Biotechnol. 18, 675-679, 2000 etc.), co-culture with bone marrow stromal cells (Kawasaki H, et al: Neuron 28, 31-40, 2000 etc.), differentiation induction method into dendritic cells ( Many methods for inducing differentiation into dopaminergic neurons have been reported, such as Senju S, Haruta M, Matsunaga Y, et al: Stem Cells 27, 1021-1031, 2009). The differentiation induction methods described above are merely examples, and the differentiation induction methods applicable to the present invention are not limited to these.

The following experiments were conducted to establish iPS cells from aged individuals.
1. Method (1) Mouse C57BL / 6 (B6) mice were purchased from Japan SLC. EGFP-C57BL / 6 mice (C14-Y01-FM131Osb) expressing GFP (Green Fluorescent Protein) throughout the body were sold from RIKEN with permission from Mr. Okabe (Reference 7). These mice were bred at the Experimental Animal Center of Nagoya University School of Medicine based on the animal experiment guidelines of Nagoya University.

(2) Gene Plasmids pMXs-c-Myc, pMXs-Klf4, pMXs-Sox2, and pMXs-OCT3 / 4, which hold transcription factors, were purchased from Addgene.

(3) Cells In order to prepare mouse embryo fibroblasts (MEF), the uterus was removed from a B6 mouse at 13.5 gestation and washed with PBS. The head and abdominal tissues of the separated embryos were removed, the remainder was finely cut with scissors, and the cells were dispersed using 0.25% trypsin / 1 mM EDTA. The cells thus obtained were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FCS.

Bone marrow derived myeloid cells (BM-M) are obtained from 0.3% GM-CSF supernatant (from Toray Dow Corning Silicone Co., Ltd.) based on the method of Inaba et al. RPMI1640 medium (10% FBS, 300 μg / mL glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin and 50 μM 2-mercaptoethanol) supplemented with GM-CSF-producing hamster cell culture supernatant) Obtained). Plat-E packaging cells were purchased from Professor Kitamura (Non-patent Document 9). SNL / 76/7 cells producing LIF were obtained by introducing a LIF expression construct into STO cells (Non-patent Document 10). Two days before gene transfer, Plat-E cells were cultured in a 6-well plate (cell density of 5 × 10 ⁵ per well) and 4 transcription factor genes (Oct3 / 4, Sox2, Klf4 and c) cloned into pMXs vector -Myc) was introduced using Fugene 6 gene introduction reagent (Roche). Specifically, vector and Fugene 6 were mixed and sprinkled drop by drop onto Plat-E cells. After incubation for 24 hours at 37 ° C. and 5% CO ₂ , the medium (MEM) was changed. After 24 hours, a virus-containing supernatant was obtained by passing through a 0.45 μm mesh cellulose acetate filter (Schleicher & Schuell). On the other hand, MEF and BM-M cells were seeded at a cell density of ⁵ × 10 ⁵ cells on mitomycin C-treated SNL / 76/7 cells (1 × 10 ⁶ cells / well). After 24 hours, the virus-containing supernatant was sprinkled. After the treatment, the medium (MEM) was changed (24 hours after MEF and 48 hours after BM-B), and the culture was continued until colonies appeared.

(4) Embryoid body formation (EB) and in vitro differentiation Previous protocol (Ohnuki, M., Takahashi, K., and Yamanaka, S. (2009) Curr. Protoc. Stem Cell Biol., 9, 4A. In vitro differentiation was induced through EB formation by a method with slight modification to 2.1-4A.2.25.). In brief, cells were suspended in a culture solution for iPS cells at a density of 3 × 10 ³ cells / 20 μL, and hanging drop culture was performed for 8 days. The formed EB was transferred to a 0.1% gelatin-coated dish and further induced to differentiate for 10 days. Immunohistochemical staining was performed using α-fetoprotein (R & D MAB1368) as the endoderm marker, α-smooth muscle actin (Sigma A2547) as the mesoderm marker, and neurofilament H (Cell signaling No. 2836) as the ectoderm marker. .

2. Results (1) Establishment of iPS cells from aged bone marrow-derived myeloma cells An experiment was conducted with the aim of establishing iPS cells from aged bone marrow. I tried to make iPS cells several times by removing bone marrow cells from old C57BL / 6 mice that express EGFP systemically and introducing 4 factors (OCT3 / 4, Sox2, Klf4, c-Myc). Both failed. Therefore, bone marrow was cultured in GM-CSF for 4 days, and switched to an experiment in which 4 factors were introduced into cells (BM-M; bone marrow derived myeloid cells) when myeloma cells were differentiated and proliferating. Specifically, 21-month-old EGFPC57BL / 6 mouse bone marrow was cultured in GM-CSF for 4 days, and then 4 factors were introduced (introduction was performed twice with the expectation that efficiency would increase). After one month, several colonies were observed. When these colonies were removed with a pipette and transferred onto mitomycin C-treated SNL / 76/6 cells that had been cultured in a 24-well plate, two clones grew (FIG. 1). The morphology of these clones was similar to iPS cells and ES cells prepared from fetal fibroblasts (MEF) (FIG. 2).

(2) Comparison of iPS cell production efficiency among fetal fibroblasts (MEF), young bone marrow myelocytes (BM-M), and aged bone marrow myelocytes Fetal fibroblasts (MEF), young (2 months old) bone marrow IPS cells were prepared from myelocytes (BM-M) and senescent (23 months old) bone marrow myelocytes, respectively, and their production efficiencies (iPS cell production frequency) were compared. Colonies appeared in about 15 days from MEF and young (2 months old) BM-M. On the other hand, from aging (23 months old) BM-M, it took 30 days for colonies to appear (FIG. 3). Without GM-CSF, iPS cell colonies could not be obtained for either young bone marrow myelocytes or senescent bone marrow myelocytes.

(3) Formation of three germ layers similar to MEF-iPS by iPS cells (BM-M-iPS) prepared from senescent bone marrow myelocytes. Two-month-old C57BL / 6 mice were treated with BM-M-iPS and MEF-iPS. When 1 × 10 ⁷ were injected subcutaneously, a tumor appeared in about 3 weeks (FIG. 4). After fixation, when stained with H & E, 3 germ layers of ectoderm (corneum of skin, left of Fig. 5), mesoderm (smooth muscle, middle of Fig. 5), endoderm (intestinal epithelium, right of Fig. 5) are observed (FIG. 5).

(4) Gene expression of iPS cells established from senescent bone marrow myelocytes C57BL / 6 mice (21 months old) bone marrow cells were cultured in the presence of GM-CSF for 4 or 7 days. When RNA was prepared from the cells and TNF-α, IL-1β, CCL-7, C / EBPα and PU.1 expression were searched by RT-PCR, strong expression was observed (FIG. 6). On the other hand, in iPS cells (BM-M-iPS) established from senescent bone marrow myelocytes, the expression of these myelic genes was strongly reduced or not observed (FIG. 6). That is, BM-M-iPS showed the same gene expression profile as MEF-iPS prepared from MEF. On the other hand, in BM-M-iPS, the expression of Nanog, Oct / 4, Fgf-4, Esg-1 and Cript, which are known to increase in pluripotent stem cells, is increased in the same way as MEF-iPS. Was.

(5) In vitro differentiation of iPS cells prepared from senescent (21 months old) bone marrow myelocytes Next, an attempt was made to differentiate BM-M-iPS into three germ layers in vitro. BM-M-iPS was cultured for 8 days by the hanging drop method to form embryoid bodies (EB). Subsequently, EB was transferred to a 0.1% gelatin-coated 24-well plate (FIG. 7A). After culturing for 10 days, it was stained with a tissue-specific antibody. Differentiated BM-M-iPS cells expressed GFP (FIG. 7B left) and also expressed α-smooth muscle actin, α-fetoprotein and neurofilament H (middle of FIG. 7B). In the same manner, MEF-iPS was induced to differentiate (data not shown). As a result of counting cells expressing differentiation markers, the proportion of cells expressing α-smooth muscle actin was 50% for BM-M-iPS and 61.2% for MEF-iPS. Regarding α-fetoprotein, expression was observed in 84.1% of BM-M-iPS cells and 51.7% of MEF-iPS cells. Similarly, neurofilament H was expressed in 68% of cells for BM-M-iPS and 73.1% for MEF-iPS. These results indicate that there is no significant difference between BM-M-iPS and MEF-iPS cells with respect to the differentiation ability of the three germ layers.

Furthermore, it was investigated whether BM-M-iPS after differentiation induction could be returned to myeloid cells. Using the culture solution for OP9, BM-M-iPS and MEF-iPS were co-cultured with OP9 cells for 5 days, respectively. Thereafter, the OP9 cells were replaced, and further cultured for 5 days in a culture solution for OP9 supplemented with GM-CSF (FIG. 7C). Subsequently, the cells were transferred to a 6-well plate (no treatment for cell adhesion). At this stage, myeloid-like cells were observed for both BM-M-iPS and MEF-iPS (FIG. 7D). Moreover, in the cells after differentiation induction, the expression of Nanog and Pou5f1 was lost (FIG. 6). On the other hand, increased expression of myelin cytokines (TNF-α, IL-1β), chemokine (CCL-7) and transcription factors (C / EBPα, Pu-1) was observed (FIG. 6). About the cell which induced differentiation, the expression of the myelic cell surface marker was investigated using PE labeled antibody. Expression of CD11b and CD115 was observed for both BM-M-iPS and MEF-iPS induced to differentiate into the myeloma system (FIG. 8). The above results indicate that BM-M-iPS differentiated into myeloma cells.

(6) Production of chimeric mouse by aggregation method As a further study, production of a chimeric mouse was attempted using BM-M-iPS. First, 5 IU pregnant mare serum gonadotropin (PMSG) was administered to 6-week-old ICR mice on day 1, human placental gonadotropin (hCG) was administered on day 3, and then placed in a cage with male mice . On the morning of 2.5 days after mating, 8-cell embryos were collected and used for the aggregation method on the same day. One day prior to aggregation, a suspension of BM-M-iPS cells in a sub-confluent state (70-80%) was added to the gelatin-coated dish. On the other hand, the 8-cell embryo was removed from the zona pellucida and transferred to an aggregation plate. Immediately before aggregation with the 8-cell stage embryo, a mass of BM-M-iPS cells (8 to 15 cells) was collected by trypsin treatment. Then, the 8-cell embryo from which the transparent body was removed and the mass of BM-M-iPS cells were aggregated. After aggregation, they were transplanted into the uterus of E2.5 pseudopregnant female mice.

As a result of the attempt to produce a chimeric mouse by the above protocol, a pup mouse having a black hair color derived from BM-M-iPS cells was obtained as shown in FIG. This result means that BM-M-iPS shows pluripotency like ES cells and its usefulness is high.

3. Discussion IPS cells were successfully established from aged mouse bone marrow-derived myeloma cells. This achievement is particularly important in the following three points. First, iPS cells were prepared from myeloid cells derived from bone marrow, second, iPS cells were prepared from aged mice, and third, established iPS cells were most commonly used C57BL / Since it is derived from 6 mice and possesses a GFP marker expressed throughout the body, it does not receive immune rejection when used in transfer experiments, and the location (location) of cells after transfer can be easily confirmed.

A simple and safe method is required to establish iPS cells from human autologous cells and use them for treatment of self-diseases and regeneration of tissues damaged by aging (personalized cells and therapies). So far, various cells (sources) have been used for the production of iPS cells. The most commonly used cells are fetal or adult fibroblasts (Refs. 2, 3). When adult neural stem cells are used, iPS cells can be established by using only two factors (Reference 11). IPS cells have also been prepared from liver cells and gastrointestinal cells of adult mice (Reference Document 12). Attempts have also been made to produce iPS cells from immune system cells, and it has been reported that iPS cells have been successfully produced from B cells (reference 13) and T cells (reference 6). However, iPS cells produced by these methods are not suitable for regenerative medicine. This is because the gene has already been recombined (gene rearrangement has occurred). We have succeeded in producing iPS cells from senescent bone marrow cells by adopting a method of culturing for a short time with GM-CSF. Although there are reports (reference documents 14 and 15) that iPS cells were prepared by culturing bone marrow cells, the bone marrows of elderly individuals were not used. The establishment of iPS cells using cells derived from aged individuals is a major breakthrough for the practical application of iPS cells.

According to the method of the present invention, it is possible to produce iPS cells relatively easily from an old individual. Uses of iPS cells obtained by the method of the present invention include treatment of various diseases associated with aging or aging (muscle atrophy, osteoporosis, arthritis, etc.), pulmonary fibrosis, cirrhosis, renal failure, and beauty (keratinocytes). Skin rejuvenation using cells differentiated into (keratinocytes)).

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

References
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Claims

A method for producing induced pluripotent stem cells, comprising the following steps (1) and (2):
(1) culturing bone marrow in the presence of granulocyte monocyte colony-stimulating factor;
(2) Transforming the proliferated cells into pluripotent stem cells.
The method for producing induced pluripotent stem cells according to claim 1, wherein the bone marrow is human bone marrow.
The method for producing induced pluripotent stem cells according to claim 1, wherein the bone marrow is human bone marrow after the middle age.
The method for producing induced pluripotent stem cells according to claim 1, wherein the bone marrow is mouse bone marrow.
The induced pluripotency according to any one of claims 1 to 4, wherein the transformation in step (2) is performed by introducing four factors Oct3 / 4, Sox2, Klf4 and c-Myc into the cell. A method for producing stem cells.
An induced pluripotent stem cell produced by the production method according to any one of claims 1 to 5.
The induced pluripotent stem cell according to claim 6, wherein the pluripotent stem cell marker Nanog gene, Oct4 gene, FgF4 gene, Esg-1 gene and Cript gene are expressed.
The induced pluripotent stem cell according to claim 6 or 7, wherein gene rearrangement is not performed.
A method for producing a cell differentiated into a specific cell lineage, characterized by subjecting the induced pluripotent stem cell according to any one of claims 6 to 8 to differentiation differentiation treatment.
A method for preparing cells for producing induced pluripotent stem cells, comprising culturing bone marrow in the presence of granulocyte monocyte colony-stimulating factor.