JP2014523733A - Mitochondrial disease-specific induced pluripotent stem cells, production method thereof and use - Google Patents

Abstract

The present invention relates to an artificial pluripotent stem (iPS) cell produced from a mitochondrial disease patient having a mutation in mitochondrial DNA and exhibiting the degree of bimodal mutant heteroplasmy, that is, a mutation having an increased frequency of the mutation Provided are rich mitochondrial disease-specific iPS (Mt-iPS) cells and mutant-free Mt-iPS cells in which the frequency of the mutation has been reduced to an undetectable level. The present invention also provides a method for producing mutant-rich Mt-iPS cells and mutant-free Mt-iPS cells, and the use of the former as a model cell source for mitochondrial disease, and the latter as a cell source for autologous therapy. The use of is also provided.
[Selection figure] None

Description

  The present invention is an induced pluripotent stem (iPS) cell produced from a mitochondrial disease patient having a mutation in mitochondrial DNA, and exhibits a bimodal mutation heteroplasmy degree, that is, a certain clone has the mutation And other clones relate to iPS cells that are substantially free of the mutation. The invention also relates to methods for producing mutation-rich and mutation-free mitochondrial disease-specific iPS (Mt-iPS) cells and uses thereof.

  Mitochondrial DNA (mtDNA) is present in mitochondria and encodes components essential for cellular energy production. mtDNA mutations can cause human degenerative diseases. The tRNA (Leu) A3243G mutation is one of the most frequently observed mtDNA mutations and has a wide range of clinical phenotypes including diabetes, hearing loss, cardiomyopathy and mitochondrial encephalopathy, lactic acidosis, and stroke-like seizure syndrome (MELAS) Accompanied by [1].

  The inherited form of mitochondrial disease is maternal, but the disease penetration rate is variable [2]. Because mtDNA segregation tends to follow a genetic floating pattern, it is impossible to predict a child's phenotype based on maternal genotype and phenotype [2]. This is true for both somatic and germ cells: it is impossible to predict to which cell type the mutated mtDNA will preferentially migrate during development. To date, there is no specific treatment or care for mitochondrial disease, only supportive care exists. Attempts to understand the genetics and pathophysiology underlying mitochondrial disease have been hampered by the lack of disease models.

  In recent years, iPS cells have been prepared from fibroblasts obtained from patients with various diseases [3-14], but none from patients with mitochondrial diseases.

Finsterer, Int. J. Cardiol., 137: 60-62 (2009) Shoubridge and Wai, Curr. Top. Dev. Biol., 77: 87-111 (2007) Dimos et al., Science, 321: 1218-1221 (2008) Ebert et al., Nature, 457: 277-280 (2009) Park et al., Cell, 134: 877-886 (2008) Soldner et al., Cell, 136: 964-977 (2009) Rowntree and Mcneish, Regen. Med., 5: 557-568 (2010) Ghodsizadeh et al., Stem Cell Rev., 6: 622-632 (2010) Meng et al., Proc. Natl. Acad. Sci. USA, 107: 7886-7891 (2010) Liu et al., Stem Cell Rev., 2010 Dec 22. [Epub ahead of print] Kang, Korean J. Pediatr., 53: 786-789 (2010) Carvajal-Vergara et al., Nature, 465: 808-812 (2010) Raya et al., Nature, 460: 53-59 (2009) Maehr et al., Proc. Natl. Acad. Sci. USA, 106: 15768-15773 (2009)

  The object of the present invention is to obtain pluripotent stem cells from patients with mitochondrial diseases and to provide a cell source for in vitro human mitochondrial disease models useful for the discovery of new drugs for human mitochondrial diseases.

  The present inventors established Mt-iPS cells by transfecting fibroblasts obtained from patients with the mtDNA A3243G mutation with four reprogramming genes (Oct3 / 4, Sox2, Klf4 and c-Myc). did. Surprisingly, Mt-iPS cells showed a bimodal mutant heteroplasmy degree. The mutation frequency decreased to a level that was not detectable in 6 out of 14 clones (<2%), while the other clones compared to the original fibroblast mutation frequency (18-24%). The mutation frequency increased several times (51-87%). Stem cell properties were indistinguishable between mutation-free Mt-iPS cells and mutation-rich Mt-iPS cells. No mutation recurrence is observed in mutation-free Mt-iPS clones during continuous subculture of cells or after spontaneous differentiation, and there is no significant change in heteroplasmy levels in mutation-rich Mt-iPS clones. I couldn't see it.

  As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing iPS cells from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the frequency of the mutation is increased or decreased to an undetectable level compared to the somatic cell. There,
(1) contacting a somatic cell obtained from the patient with an reprogramming substance;
(2) selecting an iPS cell colony from the cell culture; and (3) determining the frequency of the mutation of the iPS cell.
[2] The method according to [1] above, wherein the mutation is a substitution of adenine to guanine at position 3243 (A3243G).
[3] The method according to [1] or [2] above, wherein the mitochondrial disease is associated with diabetes.
[4] The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The method according to any one of [3].
[5] The method according to [4] above, wherein the nuclear reprogramming substance is Oct3 / 4, Sox2 and Klf4, or a nucleic acid encoding them.
[6] The method according to [4] above, wherein the nuclear reprogramming substance is Oct3 / 4, Sox2, Klf4 and c-Myc or L-Myc, or a nucleic acid encoding them.
[7] An iPS cell established from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the frequency of the mutation is increased compared to the somatic cell.
[8] The iPS cell according to [7] above, wherein the mutation is a substitution of adenine to guanine at position 3243 (A3243G).
[9] The iPS cell according to [7] or [8] above, wherein the mitochondrial disease is associated with diabetes.
[10] A method for producing a mitochondrial disease model cell, comprising differentiating the iPS cell according to any one of [7] to [9] above into a somatic cell.
[11] The method according to [10] above, wherein the somatic cells are pancreatic β cells, nerve cells, cardiomyocytes or skeletal muscle cells.
[12] A mitochondrial disease model cell obtained from the method described in [10] or [11] above.
[13] A method for screening for a therapeutic and / or prophylactic agent for mitochondrial diseases,
(1) A step of bringing the mitochondrial disease model cell according to [12] into contact with a test substance;
(2) determining one or more phenotypes of mitochondrial disease in the cells; and (3) treating at least one test substance with improved phenotype as compared with the absence of the test substance for treating mitochondrial disease And / or selecting as a prophylactic agent candidate.
[14] An iPS cell established from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the mutation frequency is reduced to an undetectable level.
[15] A method for producing a therapeutic and / or prophylactic agent for mitochondrial diseases, comprising differentiating the iPS cells according to the above [14] into somatic cells.
[16] The method according to [15] above, wherein the somatic cells are pancreatic β cells, nerve cells, cardiomyocytes or skeletal muscle cells.
[17] A somatic cell obtained by the method according to [15] or [16] above.
[18] A therapeutic and / or prophylactic agent for mitochondrial diseases, comprising the somatic cell according to [17] above.
[19] A method for treating or preventing mitochondrial disease, comprising transplanting the somatic cell according to [17] above to a patient having a mutation in mitochondrial DNA.
[20] Use of the iPS cell according to [14] above for the manufacture of a therapeutic and / or prophylactic agent for mitochondrial diseases.
[21] The iPS cell according to [14] above as a source of a therapeutic and / or prophylactic agent for mitochondrial diseases.

  Mutant-rich Mt-iPS cells may provide a suitable cell source for in vitro human mitochondrial disease models. Furthermore, the mutant-free iPS cells may provide an unlimited disease-free cell supply for autograft therapy.

FIG. 1 shows the mtDNA mutation frequency in Mt-iPS cells. Patient-derived blood cells (Mt1-blood and Mt2-blood), fibroblasts (Mt1-fibro and Mt2-fibro), and Mt-iPS clones (Mt1-1 to Mt1-4 and Mt2-1 to Mt2-10) MtDNA A3243G mutation frequency. The passage numbers when Mt-iPS cells were collected were as follows: Mt1-1: passage (p) 12, Mt1-2: p17, Mt1-3: p17, Mt1-4: p14, Mt2- 1: p9, Mt2-2: p8, Mt2-3: p10, Mt2-4: p9, Mt2-5: p10, Mt2-6: p10, Mt2-7: p7, Mt2-8: p7, Mt2-9: p9, Mt2-10: p11. FIG. 2 shows the characterization of the prepared Mt-iPS cells. (A) Mt-iPS cell (Mt2-3 and Mt2-6) colonies grown on mouse fibroblast STO cell line (SNL) feeder cells showed human ES cell-like morphology. Figure 8 shows detection of alkaline phosphatase activity and immunofluorescence analysis for the presence of pluripotency markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, NANOG and SSEA-1. Scale bar = 200 μm. (B) Karyotype analysis of Mt-iPS cells (Mt2-3 and Mt2-6) at passage 22. FIG. 3 shows the pluripotency of Mt-iPS cells by in vitro and in vivo differentiation. (A) The in vitro differentiation of Mt-iPS cells (Mt2-3 and Mt2-6) reveals the possibility of generating cell derivatives of all three primary germ layers. Immunofluorescence analysis shows the expression of neuroectodermal marker (β3-tubulin), mesoderm marker (a-SMA) and endoderm marker (FOXA2). Shows overlay with nuclear staining (DAPI). Scale bar = 100 μm. (B) Teratoma formation occurred after Mt-iPS cells (Mt1-2 and Mt1-3) were injected into the testes of SCID mice. Hematoxylin and eosin staining of teratoma sections show ectoderm derivatives (pigment epithelial cells), mesoderm derivatives (cartilage) and endoderm derivatives (intestinal tract-like glandular structure). Scale bar = 100 μm. FIG. 4 shows the influence of culture passage number and differentiation on mtDNA mutation frequency in Mt-iPS cells. (A) mtDNA A3243G mutation frequency in undifferentiated Mt-iPS clones at various culture passage numbers. (B) mtDNA A3243G mutation frequency in undifferentiated (UD) and differentiated EB (EB) Mt-iPS clones. Culture passage numbers are shown in parentheses. FIG. 5 shows the mtDNA content in Mt-iPS cells. MtDNA content (copy / cell) in blood cells (dotted bar graph), fibroblasts (hatched bar graph), mutation-free iPS clones (white bar graph) and mutation-rich iPS clones (black bar graph). Also shown are mtDNA from two human ES cell lines (H9 and KhES1) and two human iPS cell lines (B7 and G1) (white bar graph). Approximately p30 Mt-iPS cells were used for embryoid body formation and analyzed for mtDNA content (Mt2-3 EB and Mt2-6 EB). FIG. 6 shows the induction of embryoid bodies from mutation-rich Mt-iPS clones (Mt2-5) and mutation-free Mt-iPS clones (Mt2-4) established from the same patient with the A3243G mutation in mtDNA, The production amount of the protein involved in the respiratory chain complex encoded by mtDNA in the embryoid body is shown. FIG. 7 shows the differentiation of mutation-rich Mt-iPS clone (Mt2-5) and mutation-free Mt-iPS clone (Mt2-4) into intestinal and pancreatic endocrine cells.

Detailed Description of the Invention
The present invention relates to a mitochondrial disease-specific iPS (Mt-iPS) in which mtDNA mutation frequency is increased compared to the original somatic cell, including reprogramming a somatic cell derived from a patient having a mutation in mitochondrial DNA (mtDNA). ) Cells, or Mt-iPS cells that are substantially free of the mutation (ie, the frequency of the mutation has been reduced to an undetectable level).

1． Somatic cell donors and somatic cell sources The methods of the invention can be applied in any mammal in which iPS cells are established or are capable of being established, eg, humans, mice, monkeys, pigs, rats. , Dogs and the like, and preferably a human.

  Mitochondrial disease may be caused by abnormalities in mtDNA or abnormalities in nuclear genomic DNA, but the patient serving as a somatic donor in the present invention has a mutation in mtDNA that causes mitochondrial diseases It is a mammal. For example, mitochondrial encephalomyopathy, lactic acidosis, stroke-like seizure syndrome (MELAS), Kearns-Sayre syndrome (KSS), and Leigh encephalopathy account for 70% of all mitochondrial diseases It is considered as the three major disease types in clinical disease classification. 80% of MELAS patients have the A3243G mutation and 10% have the T3274 mutation. Regarding KSS, the A3243G mutation is reported to be the most common in Europe and the United States, but it is said that there are many large-scale deletion mutations in Japan. Although there are various abnormalities in the gene causing Leigh's encephalopathy, the T8993G mutation in the ATPase subunit 6 gene of mtDNA is known as a severe form. Although it is not a mitochondrial disease patient in a narrow sense, many patients (about 2% of all diabetic patients; about 130,000 in Japan) who have clinical features such as diabetes and hearing loss have the A3243G mutation. The A3243G mutation is a mutation in the leucine transfer RNA (tRNA) gene. This mutation causes an abnormality in the three-dimensional structure of tRNA and decreases the leucine addition reaction during translation. As a result, the amount of all proteins encoded by genes on mtDNA is decreased, resulting in abnormal mitochondrial function. Thus, in a preferred embodiment of the present invention, A3243G mutation can be mentioned, but the method of the present invention is applicable to any mutation on mtDNA.

  There are no particular limitations on the clinical pathology of the patient as the donor, and examples include MELAS, KSS, Leigh's encephalopathy, and diabetes with mtDNA mutation. As long as it has an mtDNA mutation, a mammal that has not yet developed symptoms may be a donor of somatic cells.

  As the “somatic cell” used in the present invention, any cell other than germ cells obtained from a patient having a mtDNA mutation can be used. For example, keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface), exocrine glandular epithelial cells (eg, mammary cells), hormone secreting cells (eg, adrenal medullary cells) Cells for metabolism / storage (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells in the inner chain (eg, vascular endothelial cells), transport Ciliated cells (eg, airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T) Lymphocytes), sensory cells (eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory and peripheral neuron support cells (eg, associated cells), central nervous system neurons and glia Cells (eg, astrocytes), pigment cells (eg, retinal pigment epithelial cells) ), And their progenitor cells (tissue progenitor cells) and the like. There is no particular limitation on the degree of cell differentiation and the age of the animal from which the cells are collected. This can be applied to both undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells. It can be used as the source of somatic cells in the invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

  Somatic cells isolated from a patient can be pre-cultured in a medium known per se suitable for culturing according to the type of cells prior to being subjected to the nuclear reprogramming step. Examples of such a medium include a minimum essential medium (MEM) containing about 5 to 20% fetal calf serum (FCS), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, and F12 medium. However, it is not limited to them. When using a nuclear reprogramming substance (and if necessary, the following iPS cell establishment efficiency improving substance) and a somatic cell, for example, when using an introduction reagent such as a cationic liposome, prevent a reduction in the introduction efficiency. Therefore, it may be preferable to replace the serum-free medium.

2． iPS cell production
iPS cells can be created by introducing a specific nuclear reprogramming substance into somatic cells in the form of nucleic acids or proteins, and have characteristics similar to those of ES cells, such as proliferation through pluripotency and self-renewal. (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007) Cell, 131: 861-872; J Yu et al. (2007) Science, 318: 1917-1920; M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106; WO 2007/069666). The nuclear reprogramming substance may be a gene that is specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof. For example, Oct family members (eg, Oct3 / 4), Klf family members (eg, Klf4, Klf1, Klf2, Klf5), Sox family members (eg, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18), Myc family members (C-Myc, L-Myc, N-Myc), Lin family members (eg, Lin28, Lin28b), GLIS family members (eg, GLIS1, GLIS2, GLIS3), TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7 , Bmil, Nanog, Esrrb or Esrrg. These reprogramming substances may be used in combination when iPS cells are established. For example, a combination including at least one, two, or three of these reprogramming substances is preferable, and a combination including four is preferable.

Nucleotide sequences of mouse and human cDNAs of each of the above nuclear reprogramming substances and amino acid sequence information encoded by the cDNAs can be obtained by referring to NCBI accession numbers described in WO 2007/069666. The mouse cDNA sequence and amino acid sequence information of L-Myc, GLIS1, GLIS2, GLIS3, Lin28, Lin28b, Esrrb and Esrrg can be obtained by referring to the following NCBI accession numbers, respectively. A person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.
Gene name mouse human
L-Myc NM_008506 NM_001033081
GLIS1 NM_147221 NM_147193
GLIS2 NM_031184 NM_032575
GLIS3 NM_175459 NM_001042413
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438

  These nuclear reprogramming substances may be introduced into somatic cells in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, and the like. Or you may introduce | transduce in a somatic cell in the form of DNA, for example by techniques, such as a vector, such as a virus, a plasmid, an artificial chromosome, lipofection, a liposome, and microinjection. As viral vectors, retrovirus vectors, lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 85, 348-62, 2009) Etc. are exemplified. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid vector, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008 and WO 2009/032456). The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that a nuclear reprogramming substance can be expressed. Furthermore, if necessary, selectable marker sequences such as drug resistance genes (eg, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene), thymidine kinase gene, diphtheria toxin gene, green fluorescent protein (GFP), β-glucuronidase (GUS), reporter gene sequences such as FLAG, and the like. In addition, in the above expression vector, after introduction into a somatic cell, in order to excise together a gene or promoter encoding a nuclear reprogramming substance and a gene encoding a nuclear reprogramming substance that binds to it, loxP sequences before and after them You may have. Furthermore, the expression vector may contain the EBNA-1 gene and the oriP sequence or the Large T antigen gene and the SV40ori sequence so that they are present episomally (ie, replicated without being incorporated into the chromosome). it can.

In order to increase the induction efficiency of iPS cells at the time of nuclear reprogramming, in addition to the above factors, for example, a histone deacetylase (HDAC) inhibitor [for example, valproic acid (VPA) (Nat. Biotechnol., 26 (7 ): 795-797 (2008)), small molecule inhibitors such as trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg, HDAC1 siRNA Smartpool ^O (Millipore), HuSH 29mer shRNA constructs against HDAC1 Nucleic acid expression inhibitors such as (OriGene) etc.], DNA methyltransferase inhibitors (eg 5'-azacytidine) (Nat. Biotechnol., 26 (7): 795-797 (2008)), G9a histone methyltransferase Inhibitors [eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.), etc. Nucleic acid expression inhibitors, etc.], L-channel calcium agonist (eg Ba yk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors [eg, p53 dominant negatives such as siRNA and shRNA against p53 (Cell Stem Cell, 3, 475-479 (2008), p275S, p53DD) , Small molecule inhibitors such as pifthrin (PFT) -α, -β, -μ (WO 2009/157593)], Wnt Signaling stimulator (eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008) ), Cytokines such as LIF or bFGF, ALK5 inhibitors (eg SB431542) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitors, glycogen synthase kinase-3 inhibitors (PloS Biology) , 6 (10), 2237-2247 (2008)), miR-291-3p, miR-294, miR-295 and other miRNAs (RL Judson et al., Nat. Biotech., 27: 459-461 (2009) ) Etc. can be used.

  Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10 to 15% FBS (these media are LIF, penicillin / streptomycin, puromycin, L-glutamine). , (2) ES cell culture medium containing bFGF or SCF, for example mouse ES cell culture medium (for example, TX-WES medium, thrombos. X) or primate ES cell culture medium (for example, primate (human & monkey) ES cell culture medium, Reprocell, Kyoto, Japan). At this time, in order to increase the induction efficiency of iPS cells, a low protein medium or a cell cycle arrester-containing medium may be used (WO 2010/004989).

In one culture method, for example, a somatic cell and a nuclear reprogramming substance (nucleic acid or protein) are contacted on a DMEM or DMEM / F12 medium containing 10% FBS, and the culture is performed at 37 ° C., 5% CO ₂ for about 4 to about 7 days. Cultivate in the presence, then re-spread on feeder cells (eg, mitomycin C-treated STO cells, SNL cells and other cells), about 10 days after contact of somatic cells with nuclear reprogramming substance To bFGF-containing primate ES cell culture medium, whereby iPS-like colonies can be generated about 30 to about 45 days or more after the contact. In order to increase the induction efficiency of iPS cells, somatic cells may be cultured under conditions of an oxygen concentration as low as 5 to 10% (WO 2010/013845).

  Alternatively, DMEM medium containing 10% FBS on feeder cells (eg, mitomycin C-treated, STO cells, SNL cells, and other cells) (this may also include LIF, penicillin / streptomycin, puromycin, L-glutamine, non- The cells may be cultured with essential amino acids, such as β-mercaptoethanol, etc.), which can generate ES-like colonies after about 25 to about 30 days or more.

During the culture, the medium is replaced with a fresh medium once a day from the second day after the start of the culture. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

  Selection of iPS cell candidate colonies includes a method using drug resistance and reporter activity as indicators and a method based on visual morphological observation. When a drug resistance gene is used as a marker gene, a marker gene-expressing cell can be selected by culturing in a medium (selective medium) containing the corresponding drug. When the marker gene is a fluorescent protein gene, the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, and by adding a chromogenic substrate in the case of a chromogenic enzyme gene. Can be detected. On the other hand, examples of a method for selecting candidate colonies by visual morphological observation include the method described in Takahashi et al., Cell, 131, 861-872 (2007). Although a method using a reporter cell is simple and efficient, when iPS cells are produced for the purpose of human therapeutic use, visual colony selection is desirable from the viewpoint of safety.

  Confirmation that the cells of the selected colony are iPS cells can also be performed by the above-mentioned Nanog (or Oct3 / 4) reporter positive (puromycin resistance, GFP positive, etc.) and visual formation of ES cell-like colonies; However, for the sake of more accuracy, tests such as alkaline phosphatase staining, expression of various ES cell-specific genes, and transplantation of selected cells to mice to confirm teratoma formation may also be performed. it can.

3． Identification of mtDNA mutation frequency in Mt-iPS cells The present invention shows that iPS cell clones established from somatic cells derived from patients with mutations in mtDNA exhibit a bimodal mutant heteroplasmy degree, ie The clone has an increased frequency of the mutation compared to the original somatic cell (mutation-rich Mt-iPS cells), and the other clones have substantially no mutation (mutation-free Mt- Based on discovery with iPS cells). Therefore, according to the present invention, by a method similar to the conventional establishment of disease-specific iPS cells, the source of disease model cells in which the gene abnormality causing the disease is further accumulated, and having a normal mitochondrial function, And a source of autologous cells for somatic cell donors.

  Therefore, the method of the present invention further comprises the step of measuring the mutation frequency of mtDNA in the cells in order to determine mutation-rich Mt-iPS cells and mutation-free Mt-iPS cells.

  Methods for measuring the mutation frequency of mtDNA are known. For example, the invader assay described in Nat Biotechnol. 1999; 17: 292-296, Clin Biochem. 2004; 37: 268-276, Jpn J Ophthalmol. 2006; 50: 128-134, etc. is preferably used. Any other method known in the art may be used. For confirming that the test Mt-iPS cells are mutation-free, for example, PCR-RFLP method or fluorescence correlation spectroscopy (FCS) (Biophys J. 2000; 79: 2858-2866, Diabetologia. 1994; 37: 504 -510, N Engl J Med. 1994; 330: 962-968).

Four. Other methods for producing mitochondrial disease-specific pluripotent stem cells The mutation frequency in the Mt-iPS cells of the present invention shows a bimodality when the mutation frequency of mtDNA passes through generations, from heteroplasmy to homoplasmy. This is thought to be because the rapid separation phenomenon, which changes rapidly, is reproduced in vitro. Several models have been proposed to explain the sudden separation. It is known that 10 ³ to 10 ⁴ copies of mtDNA exist in somatic cells, mature egg cells, and early embryos, but temporarily decrease to 10 to 100 copies in primordial germ cells (mtDNA bottleneck effect). In the preparation of ES cells, it has been reported that the copy number of mtDNA rapidly decreases when changing from an inner cell mass (ICM) to an ES cell. From these findings, ES cells (Mt-ES cells) established from early embryos derived from patients with mitochondrial diseases are also considered to exhibit bimodal mtDNA mutation frequency.

  In in vitro fertilization in infertility treatment, a preimplantation diagnosis (PGD) is performed on a family with a pathogenic mutation, and if the fertilized egg has a pathogenic mutation, it may be processed as a surplus embryo. Overseas, human ES cells established from surplus embryos having such pathogenic mutations are used for pharmaceutical research as disease-specific ES cells. Therefore, Mt-ES cells can be obtained from early embryos that have been found to have surplus embryos after mtDNA mutations have been discovered.

  As a method for producing ES cells, for example, a method of culturing an inner cell mass in a mammalian blastocyst stage (see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)) ) And the like, but is not limited thereto.

  For each ES cell clone obtained, the above 3. In the same manner as described above, the mutation frequency of mtDNA in Mt-ES cells can be measured, and mutation-rich Mt-ES cells and mutation-free Mt-ES cells can be determined.

Five. Method for Inducing Differentiation of Somatic Cells from Pluripotent Stem Cells The mitochondrial disease-specific pluripotent stem cells obtained as described above can be differentiated into desired somatic cells / tissues using known differentiation induction methods. Can do. The affected organs of mitochondrial diseases range from pancreatic β cells, nerves, myocardium, skeletal muscles in diabetic patients. Mutation-rich Mt-iPS / ES cells with accumulated mtDNA mutations are differentiated into cells and tissues of organs that can develop these pathological conditions, making them useful models for drug discovery screening as model cells for mitochondrial diseases Can do. On the other hand, normal-mitochondrial function is achieved by differentiating mutation-free Mt-iPS / ES cells from which the mtDNA mutation has disappeared into cells / tissues of affected organs of patients who are donors of somatic cells from which the cells are derived. , You can create your own cells and tissues. Therefore, it becomes possible to treat mitochondrial diseases by autotransplanting these cells and tissues.

  Examples of the method for inducing differentiation of Mt-iPS / ES cells into pancreatic β cells include, for example, JP 2004-121165, Cell Res. 2009 Apr; 19 (4): 429-38, Sci China C Life Sci, 2009 Jul; 52 (7): 615-21 and the like. Examples of methods for inducing differentiation into nerve cells include, for example, JP 2002-291469, Cell Mol Life Sci. 2010 Nov; 67 (22): 3837-47, Proc Natl Acad Sci US A. 2010 Mar 2; 107 (9). : 4335-40, Stem Cells. 2009 Oct; 27 (10): 2427-34, Stem Cells. 2009 Apr; 27 (4): 806-11, Proc Natl Acad Sci US A. 2008 Apr 15; 105 (15) : The method described in 5856-61 etc. is mentioned. Methods for inducing differentiation into cardiomyocytes include, for example, Nature. 2008 May 22; 453 (7194): 524-8. Epub 2008 Apr 23., Exp Cell Res. 2010 Oct 1; 316 (16): 2555-9, 2009 Oct 13; 120 (15): 1513-23, Cell Biol Int. 2009 Nov; 33 (11): 1184-93, Cell Physiol Biochem. 2009; 24 (1-2): 73-86, Circ Res 2009 Feb 27; 104 (4): e30-41, Circulation. 2008 Jul 29; 118 (5): 498-506, Circulation. 2008 Jul 29; 118 (5): 507-17, etc. It is done. Examples of the method for inducing differentiation into skeletal muscle cells include the method described in FASEB J. 2010 Jul; 24 (7): 2245-53 and the like.

  A tissue can also be constructed from somatic cells obtained as described above. For example, when a myocardial tissue is constructed from cardiomyocytes, the myocardial tissue can be constructed by purifying the myocardial cells from an ascorbic acid-treated embryoid body with a Percoll gradient and culturing in the presence of type I collagen and Matrigel. (Circulation 2006; 113: 2237). For other tissues, a cell having an appropriate three-dimensional structure can be produced by seeding and engrafting cells on an appropriate biocompatible (biodegradable) scaffold. As the scaffold, a porous sintered body, sponge, mesh, gel or the like is used.

6． Screening for Mitochondrial Disease Prevention / Treatment Agents Using Mutant-Rich Mt-iPS / ES Cells The present invention is a somatic cell derived from a mutant-rich Mt-iPS / ES cell obtained as described above, preferably pancreatic β Cell, nerve cell, cardiomyocyte, skeletal muscle cell or tissue constructed by them and contact with test substance to improve pathological condition of mitochondrial disease expressed by the cell or tissue, that is, normal cell or tissue trait A candidate for a prophylactic and / or therapeutic drug for mitochondrial disease, comprising selecting the test substance that changes the cell or tissue so as to approach (phenotype) or prevents or delays the development of the pathological condition A method for screening a substance is provided.

  The test substance in the present invention may be any known compound or novel compound, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry technology And random peptide libraries prepared by solid phase synthesis and phage display methods, and natural components derived from microorganisms, animals and plants, marine organisms, and the like.

  In the screening, for example, ATP synthesis, oxygen consumption, lactic acid / pyruvate ratio, and mitochondrial matrix redox potential in somatic cells are measured in the presence and absence of the test substance, and in the presence of the test substance. Compare the measured value with the value measured in the absence of the test substance, and select the test substance that has improved the measured value (closer to the normal value) as a candidate drug for the prevention and / or treatment of mitochondrial disease Can be implemented.

  In particular, when the somatic cell is a pancreatic β-cell, improvement of pancreatic β-cell function such as improvement of insulin secretion and insulin sensitivity can be used as an index. Further, when the somatic cell is a nerve cell, improvement in the degree of demyelination, glial degeneration, and necrosis can be used as an index. When the somatic tissue is a myocardial tissue, candidate substances for prophylactic and / or therapeutic agents for mitochondrial diseases are myocardial tissue when not in contact with the test substance and myocardial tissue when in contact with the test substance. Comparing) muscle contraction force, 2) cardiomyocyte loss, and 3) fibrosis, the decrease in muscle contraction force in the presence of the test substance, cardiomyocyte loss, and fibrosis are higher than those in the absence Can be obtained by selecting the test substance as a candidate substance. When somatic cells / tissues are skeletal muscle cells / tissues, candidate drugs for the prevention and / or treatment of mitochondrial diseases include red boro fiber (RRF) staining, succinate dehydrogenase (SDH) activity staining, cytochrome C oxidase ( COX) staining can be selected using as an index the decrease in RRF, the decrease in SDH activity, and the increase in COX activity.

7． Agent for preventing and treating mitochondrial disease A substance selected by the above screening method can be used as an agent for preventing and / or treating mitochondrial disease.

  Selected substances that are active ingredients can be formulated for oral or parenteral administration as is or as a pharmaceutical composition in an appropriate dosage form. The pharmaceutical composition used for administration may comprise a selected substance and a pharmacologically acceptable carrier, diluent or excipient.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like. Examples of the composition for parenteral administration include injections and suppositories. Injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions and the like. These formulations include excipients (eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid metal salts such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; wax like beeswax and gay wax Borax; adipic acid; sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate; anhydrous silicic acid, silicic acid hydrate Such as silicic acids; and starch derivatives as described above), binders (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the excipients), disintegrants (eg, low degree of substitution) Cellulose derivatives such as hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, internally crosslinked sodium carboxymethylcellulose; carboxymethyl starch, carboxymethyl starch sodium, crosslinked polyvinylpyrrolidone Such as chemically modified starch and cellulose), emulsifiers (eg, colloidal clays such as bentonite and bee gum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; sodium lauryl sulfate, calcium stearate Anionic surfactants; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters ), Stabilizers (paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol) Thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (for example, commonly used sweeteners, acidulants, fragrances, etc.) and additives such as diluents. obtain.

  The dose of the active ingredient of the mitochondrial disease preventive / therapeutic agent of the present invention varies depending on various conditions such as the patient's symptoms, age, weight, etc. For example, in the case of oral administration, the active ingredient is usually per dose. 0.1 mg or more (preferably 0.5 mg or more) and 1000 mg or less (preferably 500 mg or less) are administered to adults 1 to 6 times per day. In the case of parenteral administration, 0.01 mg or more (preferably 0.05 mg or more) and 100 mg or less (preferably 50 mg or less) can be administered per administration. If symptoms are particularly severe, the dosage may be increased depending on the symptoms.

  Furthermore, the preventive / therapeutic agent for mitochondrial disease of the present invention includes other drugs such as a therapeutic drug for diabetes (eg, insulin, etc.), L-arginine, carnitine, uridine, vitamin C, vitamin E, vitamin K, coenzyme Q, succin You may use together with an acid, cytochrome C, pyruvic acid, dichloroacetic acid, etc. The agent for preventing and treating mitochondrial disease of the present invention and these other agents can be administered simultaneously, sequentially or separately.

8． Mutation-free autotransplantation therapy using Mt-iPS / ES cells Mutation-free somatic cells differentiated from Mt-iPS / ES cells are mixed with a pharmaceutically acceptable carrier for injection, suspension It can be manufactured as a parenteral preparation such as an agent or an instillation. Pharmaceutically acceptable carriers that can be included in the parenteral preparation include, for example, isotonic solutions containing physiological saline, glucose and other isotonic agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc. ) And the like. The parenteral preparation of the present invention includes, for example, a buffer (eg, phosphate buffer, acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives, antioxidants and the like. When the agent of the present invention is formulated as an aqueous suspension, somatic cells are suspended in one of the aqueous solutions so that the cell density is about 1 × 10 ⁶ to about 1 × 10 ⁸ cells / mL. The agent of the present invention can be cryopreserved under the conditions usually used for stem cell cryopreservation and thawed at the time of use. In that case, the agent of the present invention may further contain serum or an alternative thereof, an organic solvent (eg, DMSO) and the like. The concentration of serum or its substitute is not particularly limited, but can be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

Since the thus obtained preparation is stable and has low toxicity, it can be safely administered to mammals such as humans. The administration method is not particularly limited, but is preferably injection or infusion, and can be administered into the target organ. Alternatively, the preparation can be embedded in a biocompatible gel and applied to the affected area. For example, it is usually convenient to administer an agent having an amount of about 1.0 × 10 ⁵ to about 1.0 × 10 ⁷ cells as a somatic cell dose once or 2 to 10 times at intervals of about 1 to about 2 weeks.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

[Example 1. Preparation of iPS cells derived from diabetic patients with mtDNA A3243G mutation]
Skin biopsies were obtained from two Japanese diabetic patients carrying the mtDNA A3243G mutation. Patient 1 (Mt1) was a 38 year old male and patient 2 (Mt2) was a 46 year old female. Patient clinical data are shown in Table 1.

  Mt1 showed thirst, heavy drinking, polyuria, fatigue and weight loss at age 31 years. The blood glucose concentration was 692 mg / dl (3.84 mmol / l), and the hemoglobin A1c (HbA1c) level was 14.3%. He started insulin therapy to improve diabetes control. Mt2 was diagnosed with gestational diabetes at age 24. At the same time, he presented with progressive hearing impairment. He has been suffering from epilepsy since age 27 and has been treated with valproic acid. At age 31, she developed diabetic ketoacidosis and started insulin therapy. Both patients had a positive mother's family history of diabetes. A3243G mitochondrial DNA mutations were identified by sequencing polymerase chain reaction (PCR) products amplified from peripheral blood genomic DNA from both patients (data not shown).

Skin biopsy was performed after informed consent under a protocol approved by the Kyoto University Ethics Committee. A 4 mm skin sample was further cut into small pieces with a scalpel, the tissue fragment was placed in a tissue culture dish, and a sterile coverslip was placed on top. Medium (DMEM supplemented with 10% FBS and penicillin / streptomycin; Invitrogen, Carlsbad, Calif.) Was added to completely soak the coverslips and the dishes were incubated at 37 ° C. in a humidified incubator (5% CO ₂ ). When fibroblasts grew out of the tissue fragments and proliferated and became sufficiently large, the cells were trypsinized and expanded. Two fibroblast cell lines (Mt1-fibro and Mt2-fibro) were obtained from skin biopsies of Mt1 and Mt2, respectively.

  iPS cells were prepared according to the protocol of Ohnuki et al. [Curr Protoc Stem Cell Biol. 2009; Chapter 4: Unit 4A 2]. Briefly, the mouse ecotropic retroviral receptor Slc7a1 gene (Addgene, www.addgene.org) was introduced into patient-derived fibroblasts by infection with lentivirus for 24 hours. Retrovirus production was performed in Plat-E packaging cells for 24 hours via transfection with pMXs-hOCT3 / 4, pMXs-hSOX2, pMXs-hKLF4, pMXs-hc-MYC (Addgene) [Gene Ther 2000; 7: 1063-1066]. The fibroblasts expressing the mouse Slc7a1 gene were then infected with the retroviral cocktail. The next day, the medium was replaced with DMEM supplemented with 10% FBS. Six days after transduction, infected fibroblasts were re-sown on SNL feeder cells [Int J Dev Biol. 1990; 48: 1149-1154]. On the next day, the medium was replaced with human embryonic stem (ES) medium (ReproCELL; ReproCELL, Tokyo, Japan) supplemented with 4 ng / ml bFGF (Wako, Osaka, Japan), and the medium was changed every two days. From 4 weeks post infection, colonies were picked based on morphological similarity to human ES cell colonies and transferred onto SNL feeder cells in 6-well plates: this stage was defined as one passage. From Mt1-fibro and Mt2-fibro, 4 (Mt1-1 to 1-4) and 10 (Mt2-1 to 2-10) putative iPS (Mt-iPS) clones could be isolated, respectively. Cultures were maintained on SNL feeders and passaged enzymatically every 5-7 days using 0.25% trypsin with 0.1 mg / ml type IV collagenase.

[Example 2. MtDNA mutation frequency in Mt-iPS cells]
The presence and level of heteroplasmy in blood cells from patients, fibroblasts (Mt1-fibro and Mt2-fibro) and putative iPS clones (Mt1-1-1-4 and Mt2-1-2-10) were evaluated . To quantify the degree of heteroplasmy of the mtDNA A3242G mutation in Mt-iPS cells, an invader assay was applied [Nat Biotechnol. 1999; 17: 292-296, Clin Biochem. 2004; 37: 268-276, Jpn. J Ophthalmol. 2006; 50: 128-134]. The passage numbers when Mt-iPS cells were collected for invader assay were as follows: Mt1-1: passage (p) 12; Mt1-2: p17; Mt1-3: p17; Mt1-4 : P14; Mt2-1: p9; Mt2-2: p8; Mt2-3: p10; Mt2-4: p9; Mt2-5: p10; Mt2-6: p10; Mt2-7: p7; Mt2-8: p7 Mt2-9: p9; Mt2-10: p11.

The primary probes and invader oligos used to detect A3243G heteroplasmy are as follows:
3243A primary probe:
5'- CGCGCCGAGG AGCCCGGTAATCGC <amino> -3 ',
3243G primary probe:
5'- ACGGACGCGGAG GGCCCGGTAATCG <amino> -3 ',
Common invader oligos:
5'-CCCACCCAAGAACAGGGTTTGTTAAGATGGCAGT-3 '.

  The underlined sequence of the primary probe indicates the 5 'flap of the invader reaction. Cleavage enzyme, fluorescence resonance energy transfer (FRET) probe, signal probe and invader oligo were added to the microplate containing the diluted plasmid containing the primary probe / invader oligo binding region and then the invader assay was performed as previously reported [Int J Hematol. 2009; 89: 482-488]. Plates were incubated at 63 ° C. in a fluorescent microplate reader (FluoDia-T70; Otsuka Electronics, Osaka, Japan). FAM (carboxyfluorescein) fluorescence value for 3243A (wavelength / bandwidth: excitation, 485/20 nm; emission, 530/25 nm) and RED (REDmond RED) fluorescence value for 3243G (excitation, 560/20 nm; emission) 620/40 nm) was measured every 2 minutes for 4 hours. For detection of A3243G heteroplasmy, the copy numbers of 3243A and 3243G were calculated using a standard curve with a quantitative invader assay as described [Int J Hematol. 2009; 89: 482-488; Proc Natl Acad Sci USA. 2000; 97: 8272-8277]. The A3243G ratio was based on the ratio of all copies (3243A and 3243G) to 3243A copies. In this assay, the minimum detection limit for mutation frequency is 2%. The mtDNA A3243G mutation was also analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) or fluorescence correlation spectroscopy (FCS) [Biophys J. 2000; 79: 2858-2866; Diabetologia. 1994; 37: 504- 510; N Engl J Med. 1994; 330: 962-968]. The results are shown in Figure 1.

  The mutation frequency in peripheral blood cells from both patients was 24% (Mt1-blood: 24%, Mt2-blood: 24%). Skin derived fibroblasts from both patients showed similar mutation frequency levels compared to blood cells from the same patient (Mt1-fibro: 18%, Mt2-fibro: 24%). However, 2 out of 4 Mt1-iPS clones (Mt1-1 and Mt1-2) and 6 out of 10 Mt2-iPS clones (Mt2-1, Mt2-2, Mt2-3, Mt2-4, Mt2 -7, Mt2-8) showed an undetectable level (<2%) of A3243G mutation. The loss of these mutations was confirmed by gene analysis by PCR-RFLP and FCS. Furthermore, in other iPS strains, a marked increase in mutation frequency was observed compared to the original fibroblast cell line (Mt1-3: 51%, Mt1-4: 87%, Mt2-5: 83%) Mt2-6: 69%, Mt2-9: 79%, Mt2-10: 74%). There was no significant relationship between the number of passages of Mt-iPS cells and the mutation frequency.

[Example 3. Characterization of the prepared Mt-iPS cells]
Next, the stem cell characteristics of the Mt-iPS clone were verified by immunochemistry, alkaline phosphatase staining and karyotype analysis.

(1) Immunocytochemistry and alkaline phosphatase staining Immunocytochemistry was performed as previously reported [Zygote. 2006; 14: 299-304]. Anti-human primary antibodies are SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 (all Stemgent, San Diego, CA), NANOG (R & D Systems, Minneapolis, MN), β3 -Tubulin (Millipore, Temecula, CA), α-smooth muscle actin (αSMA) (Sigma-Aldrich, Saint Louis, MO), and FOXA2 (Cell Signaling Technology, Danvers, MA). For immunofluorescence, Alexa Fluor 488 goat anti-mouse IgM, Alexa Fluor 488 goat anti-rat IgM, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 546 rabbit anti-goat IgG, Alexa Fluor 546 goat anti-mouse IgG (all Molecular Probes , OR, USA) was used as the secondary antibody. Alkaline phosphatase activity was detected using Alkaline Phosphatase Staining Kit (Stemgent). Images were captured using an Olympus IX81 microscope (Olympus, Tokyo, Japan).

  All Mt-iPS clones showed typical human ES cell-like morphology (FIG. 2A). Mt-iPS cells are positive for alkaline phosphatase activity, and all 14 clones were confirmed to have pluripotent stem cell markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and NANOG by immunocytochemical analysis. Detected (Figure 2A). Mt-iPS cells did not express SSEA-1 except for a few cells at the end of the colony (FIG. 2A). The morphological and immunocytochemical characteristics of mutant-free Mt-iPS cells and mutant-rich Mt-iPS cells could not be distinguished.

(2) Karyotype analysis
To verify whether Mt-iPS clones are cytogenetically normal, karyotype analysis of Mt-iPS cells (Mt2-3 and Mt2-6) at passage 22 was then performed. Standard G-banding chromosome analysis was performed at the Japan Genetic Research Institute (Sendai, Japan).

  Both the mutant-free Mt-iPS (Mt2-3) clone and the mutant-rich Mt-iPS (Mt2-6) clone maintained normal female karyotype (FIG. 2B).

[Example 4. Pluripotency of Mt-iPS cells by in vitro and in vivo differentiation]
(1) Embryoid body (EB) formation and in vitro differentiation
To determine the in vitro differentiation potential of Mt-iPS cells, embryoid bodies (EBs) were formed using suspension culture [Mol Med. 2000; 6: 88-95]. Mt-iPS cells were dissociated by collagenase / trypsin treatment and transferred to low adhesion multiwell plates in ReproCELL medium. The medium was changed every 2 days. After 8 days in suspension culture, iPS cells formed a ball-like structure. These EBs were transferred to plates coated with 0.1% gelatin and further induced for 8 days. Adherent cells showed various morphological types, including morphologies similar to neurons, paving stone-like cells and epithelial cells (data not shown). Differentiation markers such as β3-tubulin (ectodermal marker), αSMA (mesoderm marker) and FOXA2 (endoderm marker) were analyzed by immunocytochemistry. All Mt-iPS clones were found to be able to differentiate into three germ layers in vitro (FIG. 3A).

(2) Teratoma formation
To determine pluripotency in vivo, Mt-iPS cells were transplanted into testicles of severe combined immunodeficiency (SCID) mice. Approximately 5 × 10 ⁵ iPS cells were collected by collagenase / trypsin treatment and injected into the testes of 7-12 week old immunodeficient SCID mice. Teratomas were collected 9-12 weeks after injection and fixed with 10% neutral buffered formalin. Paraffin-embedded tissue was sectioned and stained with hematoxylin and eosin. Animal experiments were conducted in accordance with the guidelines of our institution and received approval from the Kyoto University Animal Experiment Committee.

  Histological examination showed that the tumor contained various tissues including pigment epithelium (ectoderm), cartilage (mesoderm) and intestinal-like epithelial tissue (endoderm) (FIG. 3B). Therefore, it was observed that the Mt-iPS clone can spontaneously differentiate into all three germ layer derivatives in vivo.

[Example 5. Effects of culture passage number and differentiation on mtDNA mutation frequency in Mt-iPS cells]
Next, it was verified whether the mutation frequency of the Mt-iPS clone was fixed during cell culture and passage. Mutation frequency was measured by the same method as shown in Example 2. Analysis of the same clones at various passage numbers revealed that none of the mutagenesis was observed in the mutation-free Mt-iPS clones (Mt1-2, Mt2-3) (FIG. 4A). Mutation frequency of mutation-rich Mt-iPS clones was relatively constant between passages (Mt1-4, Mt2-6) (FIG. 4A).

  Furthermore, the effect of differentiation on mutation frequency was examined. When the same clone is analyzed in undifferentiated (UD) state and after differentiation (as a result of EB formation) 16 days, mutation-free Mt-iPS clones (Mt1-2, Mt2-1, Mt2-3, Mt2-4) No mutagenesis was shown (Figure 4B). In addition, the mutation frequency of mutation-rich Mt-iPS clones was relatively constant after differentiation (Mt1-3, Mt1-4, Mt2-6) (FIG. 4B).

[Example 6. MtDNA content in Mt-iPS cells]
The mtDNA content (mitochondrial genome copy per cell) in blood cells, fibroblasts (Mt1-fibro and Mt2-fibro) and iPS clones was measured by quantitative genomic PCR. For comparison, two human ES cell lines (H9 and KhES-1) and two human iPS cell lines (B7 and G1) were cultured and collected for mtDNA content analysis [Science. 1998; 282: 1145. Int J Dev Biol. 2004; 48: 1149-1154; Cell. 2007; 131: 861-872; Nat Biotechnol. 2008; 26: 101-106].

  Genomic DNA was extracted from blood, fibroblasts and Mt-iPS cells using the DNeasy Tissue Kit (Qiagen, Valencia, CA) or standard established protocols [Nucleic Acids Res. 1991; 19: 4293]. The extracted DNA sample was stored at 4 ° C. until assayed. Relative mtDNA copy numbers were measured by real-time PCR and corrected with nuclear DNA measurements [Stem Cells. 2010; 28: 721-733]. Primers for the mitochondrial NADH-ubiquinone oxidoreductase chain 5 (ND5) gene were 5'-AGGCGCTATCACCACTCTGTTCG-3 'and 5'-AACCTGTGAGGAAAGGTATTCCTG-3'. Primers for the nuclear cystic fibrosis (CF) gene were 5'-AGCAGAGTACCTGAAACAGGAA-3 'and 5'-AGCTTACCCATAGAGGAAACATAA-3'. PCR was performed with StepOnePlus Real-Time PCR (Applied Biosystems, Carlsbad, CA) using the THUNDERBIRD SYBR qPCR Mix kit (Toyobo, Osaka, Japan). DNA (80 ng) was mixed with 10 μl SYBR qPCR containing 6 pmol forward and reverse primers, and ROX reference dye in a final volume of 20 μl. PCR conditions were 95 ° C for 1 minute, followed by 40 cycles of denaturation (95 ° C, 15 seconds), annealing and primer extension (60 ° C, 60 seconds). A standard curve was generated using serial dilutions of plasmid DNA containing PCR amplicons cloned into pGEM-T Easy (Promega, Madison, Wis.). The threshold cycle number (Ct) values of mitochondrial ND5 gene and CF gene were measured in two DNA duplicate samples. The amplification product was denatured and re-annealed at various temperature points to detect its inherent melting temperature. The sample mtDNA content (mtDNA copy per cell) was calculated using the formula (ND5 gene copy / CF gene copy) × 2 = mtDNA copy per cell.

  Skin fibroblast mtDNA content (Mt1-fibro: 553 copies / cell; Mt2-fibro: 4296 copies / cell) is higher than peripheral blood (Mt1-blood: 196 copies / cell; Mt2-blood: 60 copies / cell) it was high. The mtDNA content of the early passage Mt-iPS cells was slightly lower than the original fibroblast culture (Mt1-1 p12: 454 copies / cell; Mt1-4 p14: 151 copies / cell; Mt2- 3 p10: 2100 copies / cell; Mt2-6 p10: 1709 copies / cell; FIG. 5 and data not shown). The mtDNA content of Mt-iPS cells depends on the passage number 20, human ES cells (H9 p87: 79 copies / cell; KhES-1 p78: 78 copies / cell) and iPS cells (B7 p54: 150 copies / cell; G1 p40 : 94 copies / cell) markedly reduced to near levels (Mt1-4 p26: 42 copies / cell; Mt2-3 p20: 49 copies / cell; Mt2-6 p20: 45 copies / cell) and then maintained (Mt2 -3 p30: 68 copies / cell; Mt2-6 p30: 50 copies / cell).

  The mtDNA copy number increased 7 to 10 times after embryoid body differentiation (Mt2-3 p24-derived EB: 384 copies / cell; Mt2-6 p24-derived EB: 484 copies / cell). There was no significant difference in mtDNA content between mutation-free iPS cells and mutation-rich iPS cells (FIG. 5 and data not shown).

[Example 7. Mitochondrial function in mutation-rich and mutation-free Mt-iPS cells]
Mutation-rich Mt-iPS clone (Mt2-5; mtDNA mutation frequency 83%) and mutation-free Mt-iPS clone (Mt2-4) from the same patient with A3243G mutation (Mt2; mtDNA mutation frequency about 20%) MtDNA mutation frequency <1%) was used to evaluate the amount of protein encoded by mtDNA (cytochrome C oxidase subunit 1 (COX1) and cytochrome C oxidase subunit 2 (COX2)). Embryoid bodies (EBs) were derived from Mt2-4 and Mt2-5 clones, and COX1 and COX2 production in each EB was analyzed by Western blotting. The results are shown in FIG.

  Mutation-rich clone Mt2-5 had reduced COX1 and COX2 protein production compared to mutation-free clone Mt2-4. These data suggest that mutant-rich Mt-iPS cells are useful as a source of mitochondrial disease model cells or tissues.

[Example 8. Mutation-rich and mutation-free differentiation from Mt-iPS cells into intestinal and pancreatic endocrine cells]
According to a conventional method, the differentiation-rich Mt-iPS clone (Mt2-5) and the mutation-free Mt-iPS clone (Mt2-4) were induced to differentiate into intestinal and pancreatic endocrine cells. The results are shown in FIG.

  Immunostaining with antibodies directed against intestinal and pancreatic endocrine cell-specific markers reveals that both mutant-rich iPS cells and mutant-free iPS cells can differentiate into intestinal and pancreatic endocrine cells. These data support that mutant-rich iPS cells can be used to create various cell models for mitochondrial disease.

  While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

  The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

  This application is based on US Provisional Patent Application No. 61 / 503,283, filed June 30, 2011, the contents of which are hereby incorporated by reference.

Claims (21)

A method for producing iPS cells from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the frequency of the mutation is increased or decreased to an undetectable level compared to the somatic cell,
(1) contacting a somatic cell obtained from the patient with an reprogramming substance;
(2) selecting an iPS cell colony from the cell culture; and (3) determining the frequency of the mutation of the iPS cell.   2. The method of claim 1, wherein the mutation is an adenine to guanine substitution (A3243G) at position 3243.   The method according to claim 1 or 2, wherein the mitochondrial disease is associated with diabetes.   The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. 2. The method according to item 1.   The method according to claim 4, wherein the nuclear reprogramming substance is Oct3 / 4, Sox2 and Klf4, or a nucleic acid encoding them.   The method according to claim 4, wherein the nuclear reprogramming substance is Oct3 / 4, Sox2, Klf4 and c-Myc or L-Myc, or a nucleic acid encoding them.   An iPS cell established from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the mutation frequency is increased compared to the somatic cell.   The iPS cell according to claim 7, wherein the mutation is a substitution of adenine to guanine at position 3243 (A3243G).   The iPS cell according to claim 7 or 8, wherein the mitochondrial disease is associated with diabetes.   A method for producing a mitochondrial disease model cell, comprising differentiating the iPS cell according to any one of claims 7 to 9 into a somatic cell.   The method according to claim 10, wherein the somatic cells are pancreatic β cells, nerve cells, cardiomyocytes or skeletal muscle cells.   A mitochondrial disease model cell obtained from the method according to claim 10 or 11. A method for screening for a therapeutic and / or prophylactic agent for mitochondrial diseases comprising:
(1) A step of contacting the mitochondrial disease model cell according to claim 12 with a test substance;
(2) determining one or more phenotypes of mitochondrial disease in the cells; and (3) treating at least one test substance with improved phenotype as compared with the absence of the test substance for treating mitochondrial disease And / or selecting as a prophylactic agent candidate.   An iPS cell established from a somatic cell obtained from a patient having a mitochondrial DNA mutation causing mitochondrial disease, wherein the mutation frequency is reduced to an undetectable level.   A method for producing a therapeutic and / or prophylactic agent for mitochondrial diseases, comprising differentiating the iPS cells according to claim 14 into somatic cells.   The method according to claim 15, wherein the somatic cell is a pancreatic β cell, a nerve cell, a cardiomyocyte, or a skeletal muscle cell.   A somatic cell obtained by the method according to claim 15 or 16.   A therapeutic and / or prophylactic agent for mitochondrial diseases, comprising the somatic cell according to claim 17.   A method for treating or preventing mitochondrial disease, comprising transplanting the somatic cell according to claim 17 to a patient having a mutation in mitochondrial DNA.   Use of the iPS cell according to claim 14 for the manufacture of a therapeutic and / or prophylactic agent for mitochondrial diseases.   The iPS cell according to claim 14, as a source of a therapeutic and / or prophylactic agent for mitochondrial diseases.

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