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  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

• the present invention relates to a method of inducing the differentiation of primordial germ cell (PGC)-like cells from a pluripotent stem cell, a method of maintaining and expanding the PGC-like cells, and a culture medium used for the methods.
• PGC primordial germ cell
• Anticancer agents and radiations used for anticancer therapy are known to cause serious adverse reactions on normal highly active cells and tissues. Anticancer agents mostly exhibit their therapeutic effects by selective toxicity based on the higher
• anticancer agents even attack normal cells in vigorous proliferation, such as gastrointestinal mucosa, hair root cells, myelocytes, and spermatogonia.
• adriamycin is known to cause orchiopathies characterized by necrosis of spermatogonia.
• G-CSF granulocyte colony stimulating factor
• the infertile male mouse was mated with a female mouse, and offspring derived from the donor was obtained. Furthermore, Shinohara et al. established a method of culturing spermatogonial stem cells in vitro for a long time using a culture medium containing glial cell-derived neurotrophic factor (GDNF) , leukemia inhibitory factor (LIF) , epithelial cell growth factor (EGF) , and basic fibroblast growth factor (bFGF) , and named the cell line obtained
• GDNF glial cell-derived neurotrophic factor
• LIF leukemia inhibitory factor
• EGF epithelial cell growth factor
• bFGF basic fibroblast growth factor
• transplantation of the GS cells to the testis after treatment makes it possible to avoid infertility due to adverse
• testicular tissue specimens poses the risk of causing
• iPS cells mouse and human induced pluripotent stem cells
• Human iPS cells can be differentiated into cells of various tissues after being generated using cells derived from a patient to be treated, and are therefore expected to serve as transplantation materials free of graft rejections in the field of regenerative medicine.
• iPS cells can easily be established from, for example, dermal
• fibroblasts and the like it would be possible to avoid the risky procedure of collecting a source of cells, such as by multiple biopsies of the testis, provided that sperms or progenitor cells thereof are efficiently differentiation- induced from human iPS cells and expanded. This is expected to lead to more safe spermiogenesis in male infertility patients.
• pluripotent stem cells such as iPS cells
• a method of maintaining and expanding the germline stem cells and culture media used therefor.
• the present inventors inserted reporter genes (GFP and RFP) downstream of the respective expression control regions of the undifferentiated cell-specific Oct3/4 (Oct4) gene and the mouse homolog of the germ cell lineage-specific Vasa gene (Mouse vasa homolog; hereinafter referred to as Mvh) to create a transgenic (Tg) mouse wherein undifferentiated cells and germ cells are visualized.
• GFP and RFP reporter genes downstream of the respective expression control regions of the undifferentiated cell-specific Oct3/4 (Oct4) gene and the mouse homolog of the germ cell lineage-specific Vasa gene (Mouse vasa homolog; hereinafter referred to as Mvh) to create a transgenic (Tg) mouse wherein undifferentiated cells and germ cells are visualized.
• the inventors conducted extensive investigations using iPS cells induced from the mouse and ES cells as an experimental system, in an attempt to establish culturing conditions that allow Oct4-GFP-positive Mvh-RFP- positive (Oct4 + /Mvh + ) cells to be induced efficiently, and culturing conditions that allow the cells to be maintained and expanded in the Oct4 + /Mvh + state.
• Oct4 + /Mvh + cells can be induced efficiently by culturing pluripotent stem cells in the presence of BMP4, GDNF, EGF, and stem cell factor (SCF) , and that Oct4 + /Mvh + cells can be expanded, while maintaining their double-positive state, by culturing the cells in the presence of GDNF, EGF, bFGF, and SCF, preferably in the further presence of one or more factors selected from the group consisting of hepatocyte growth factor (HGF) ,
• HGF hepatocyte growth factor
• GR cells revealed the identity of the cells as a cell line reflecting the characteristics of primordial germ cells (PGC) in the later stage of migration. Furthermore, the present inventors found that these GR cells, when transplanted into the seminal duct of an infertile mouse, survived for a long time and did not show tumor formation, confirming their identity as unipotent stem cells destined to become germline cells, and have developed the present invention.
• PSC primordial germ cells
• the present invention relates to the following:
• a method of producing an Oct4-positive Vasa-positive germline stem cell comprising culturing a pluripotent stem cell in the presence of (a) bone morphogenetic protein 4 (BMP4) and (b) one or more growth factors selected from among glial cell-derived neurotrophic factor (GDNF) , epithelial cell growth factor (EGF), and stem cell factor (SCF).
• BMP4 bone morphogenetic protein 4
• GDNF glial cell-derived neurotrophic factor
• EGF epithelial cell growth factor
• SCF stem cell factor
• the pluripotent stem cell is an iPS cell or ES cell.
• GDNF selected from among GDNF, EGF, and SCF, in combination.
• germline stem cell supplemented with the inducer according to (8) or (9) above.
• kits for inducing differentiation from a pluripotent stem cell to an Oct4-positive Vasa-positive germline stem cell comprising a medium containing a basal medium and one or more components selected from Table 1 and the inducer according to any one of (8) to (10) above.
• a method of expanding an Oct4-positive Vasa-positive germline stem cell comprising culturing the germline stem cell in the presence of GDNF, EGF, SCF, and basic fibroblast growth factor (bFGF) . . -
• HGF hepatocyte growth factor
• IL-2 interleukin 2
• FGF9 fibroblast growth factor 9
• a culture medium for expanding an 0ct4-positive Vasa- positive germline stem cell consisting of a medium containing a basal medium and one or more optionally chosen components selected from Table 1, and one or more components selected from Table 2 added thereto.
• a method of allowing an infertile animal to form sperms comprising transplanting an Oct4-positive Vasa-positive germline stem cell obtained by the method according to any one of (1) to (6) above, the Oct4-positive Vasa-positive germline stem cell according to (7) above, or an Oct4-positive Vasa- positive germline stem cell expanded by the method according to any one of (12) to (15) above, to the testis of the
• a therapeutic agent for male infertility comprising an Oct4-positive Vasa-positive germline stem cell obtained by the method according to any one of (1) to (6) above, the 0ct4- positive Vasa-positive germline stem cell according to (7) above, or an Oct4-positive Vasa-positive germline stem cell expanded by the method according to any one of (12) to (15) above.
• germline stem cells capable of spermiogenesis from iPS cells that can easily be generated from somatic cells such as skin cells, and to maintain and expand the same induced.
• Fig. 1 is a photographic representation of iPS cells established from EFs derived from an Oct4-GFP/Mvh-RFP Tg fetal mouse.
• the upper panel shows images of the iPS clone 522A3, established by transfection with the three genes Oct3/4, Sox2 and Klf4.
• the lower panel shows images of the iPS clone 522B2, established by transfection with the four genes Oct3/4, Sox2, Klf4 and Nanog.
• Fig. 2 is a photographic representation of
• Fig. 3 is a photographic representation of
• electrophoresis showing the results of an examination of the expression of undifferentiation markers by RT-PCR analysis in the established iPS clones 522A1 to A4 and 522B1 to 522B4.
• Fig. 4 is a photographic representation showing the results of a histological analysis of teratomas formed by subcutaneously injecting an established iPS clone into
• Fig. 5 is a photographic representation of embryoid bodies (EB) formed from ES cells and iPS cells (clone 522B2) by a conventional method of differentiation induction.
• EB embryoid bodies
• Fig. 6 shows a photograph showing the morphology of M15- 4GF cells obtained by transferring the GDNF, mSCF and EGF genes to M15-BMP4 cells (left panel) , and an electrophoregram showing the results of RT-PCR analysis of M15-4GF (right panel) .
• Fig. 7 is a photographic representation of cell masses obtained by suspension-culturing established iPS cells and M15-4GF cells in the medium of Fig. 27 (without Supplement) (upper panel) , and Oct4-GFP-positive / Mvh-RFP-positive colonies obtained by dissociating the cell masses with trypsin and collagenase, and culturing them in the medium of Fig. 27 (with Supplement) on feeder cells (lower panel) .
• Fig. 8 is a photographic representation of Oct4-GFP- positive / Mvh-RFP-positive colonies that are stably
• Fig. 9 is a photographic representation showing that an
• Oct4-GFP-positive / Mvh-RFP-positive colony exhibited an alkaline phosphatase activity, which serves as a cell surface marker both in undifferentiated cells and in germ cells.
• Fig. 10 is a photographic representation of cells obtained by culturing an Oct4-GFP-positive / Mvh-RFP-positive colony under iPS cell culturing conditions (upper panel) , and cells obtained by culturing an Oct4-GFP-positive / Mvh-RFP- positive colony under germ stem (GS) cell culturing conditions (lower panel) .
• Fig. 11 is a photographic representation of cells obtained by culturing undifferentiated iPS cells under iPS cell culturing conditions (upper panel) , and cells obtained by culturing undifferentiated iPS cells directly under the maintenance culture conditions of Fig. 27 without the process of differentiation induction (lower panel) .
• Fig. 12 is a photographic representation of Oct4-GFP- positive / Mvh-RFP-positive germ cell-like cells (GR cells) (upper panel), and 0ct4-GFP-positive / Mvh-RFP-negative apparently undifferentiated cells (Gsp cells) (lower panel) .
• Fig. 13 is a photographic representation of electrophoresis showing the results of genomic PCR analysis of
• GR cells and Gsp cells were also analyzed.
• Fig. 14 is a photographic representation of
• Fig. 15 is a graphic representation of the results of total cell counting of iPS cells and GR cells in culture as counted every two days.
• Fig. 16 is a photographic representation of the results of cultivation of GR cells on feeder cells (MEF) or in the absence of feeders on gelatin-, laminin- or fibronectin-coated plates.
• Fig. 17 is a photographic representation of the results of an examination of immunodeficient nu/nu mice receiving subcutaneously transplanted GR cells or Gsp cells to determine whether tumors would be formed at transplantation sites. For control, iPS cells and EK cells were also transplanted in the same way.
• Fig. 18 is a photographic representation of
• iPS cells endogenous genes in GR cells and Gsp cells.
• EK cells endogenous genes in GR cells and Gsp cells.
• Fbx-iPS cells [Cell, 126, 663-676 (2006)] were also analyzed.
• Fig. 19 is a graphic representation of the results of quantitation by realtime PCR of the expression of exogenous transgenes and corresponding endogenous genes in GR cells and Gsp cells. For control, iPS cells and EK cells were also analyzed.
• Fig. 20 is a photographic representation of
• iPS cells iPS cells, EK cells and the testis were also analyzed.
• Fig. 21, like Fig. 20, is a photographic representation of electrophoresis showing the results of RT-PCR of the expression of germ cell marker genes.
• Fig. 22 shows the results of a comparison of the
• Fig. 23 is a photographic representation of
• electrophoresis showing the results of Western blot analysis of the expression of the proteins of germ cell marker genes in GR cells and Gsp cells.
• iPS cells and EK cells were also analyzed.
• Fig. 24 shows the results of an analysis of DNA
• iPS cells and EK cells were also analyzed.
• Fig. 25 is a photographic representation of the results of a histological analysis of the testis from a W/Wv mouse receiving a GR cell graft transplanted into the testis.
• Fig. 26 is a photographic representation of 0ct4-GFP- negative / Mvh-RFP-positive EK cells cultured under the culturing conditions of Fig. 27 (upper panel) and ES cell- derived 0ct4-GFP-positive / Mvh-RFP-positive GR cells cultured under the culturing conditions of Fig. 27 (lower panel) .
• Fig. 27 shows a table of components of the medium used to induce the differentiation of Oct4-GFP-positive / Mvh-RFP- positive cells from iPS cells or ES cells (without Supplement) and to maintain-culture the same (with Supplement) .
• the present invention provides a method of inducing germline stem cells from a pluripotent stem cell, and a method of maintaining and expanding the germline stem cells .
• the germline stem cells differentiation-induced from a pluripotent stem cell by the present invention are characterized by the co-expression of both the Oct3/4(Oct4) gene, which is a marker of undifferentiated cells, and the Vasa gene, which is
• the Vasa gene is a gene that encodes ATP-dependent RNA helicase, which has a DEAD box, identified by an analysis of the germ cell aplasia mutation in the drosophila. Its homologs have been cloned in various mammals, including mice (Proc. Natl. Acad. Sci. USA, 91:
• the germline stem cells (also referred to as GR cells) of the present invention are PGC-like cells that reflect the characteristics of primordial germ cells (PGC) in the later stage of migration (just before or just after penetration into the fetal genital primordium) .
• the germline stem cells are stem cells that can be maintained and expanded under the maintain-culturing conditions of the present invention (possessing the capability of self- regeneration) , and are destined to become germline cells because they are capable of survive for a long time without forming tumors and without being eliminated when transplanted to the testis.
• the pluripotent stem cells that serve as a starting material in the present invention are not particularly limited, as far as they are undifferentiated cells having "a potential for self-regeneration" to proliferate while maintaining the undifferentiated state, and "pluripotency", the capability of differentiating into all the three primary germ layers of the embryo; examples include iPS cells and ES cells, as well as embryonic germ (EG) cells derived from primordial germ cells, multipotent germline stem (mGS) cells isolated in the process of establishment and cultivation of GS cells from testicular tissue, multipotent adult progenitor cells (MAPC) isolated from the bone marrow and the like.
• the ES cells may be ones resulting from nuclear reprogramming of somatic cells.
• iPS cells or ES cells are preferred, mGS cells and MAPC are also preferred because they can be acquired from live-born individuals.
• the methods of the present invention are applicable to optionally chosen mammals for which any pluripotent stem cells have been established or are
• mice examples include humans, mice, monkeys, pigs, rats, dogs and the like, with preference given to humans or mice .
• pluripotent stem cells in the present invention is described below, but is not to be construed as limiting the scope of the present invention.
• A Source of somatic cells Any cells other than germ cells of mammalian origin (e.g., humans, mice, monkeys, pigs, rats and the like) can be used as starting material for producing iPS cells in the present invention. Examples include keratinizing epithelial cells
• keratinized epidermal cells e.g., keratinized epidermal cells
• mucosal epithelial cells e.g., keratinized epidermal cells
• intimal cells e.g., epithelial cells of the superficial layer of tongue
• exocrine gland epithelial cells e.g., mammary gland cells
• hormone-secreting cells e.g., adrenomedullary cells
• cells for metabolism or storage e.g., liver cells
• epithelial cells constituting interfaces e.g., type I
• intimal epithelial cells of the obturator canal e.g., vascular endothelial cells
• cells having cilia with transporting capability e.g., airway epithelial cells
• cells for extracellular matrix secretion e.g., fibroblasts
• constrictive cells e.g., smooth muscle cells
• cells of the blood and the immune system e.g., T lymphocytes
• sense- related cells e.g., rod cells
• autonomic nervous system neurons e.g., cholinergic neurons
• sustentacular cells of sensory organs and peripheral neurons e.g., satellite cells
• nerve cells and glia cells of the central nervous system e.g., astroglia cells
• pigment cells e.g., retinal pigment
• progenitor cells tissue progenitor cells thereof and the like.
• degree of cell differentiation the age of an animal from which cells are collected and the like; even undifferentiated progenitor cells (including somatic stem cells) and finally
• differentiated mature cells can be used alike as sources of somatic cells in the present invention. Examples of
• tissue stem cells such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
• the choice of mammalian individual as a source of somatic cells is not particularly limited; however, when the desired germline stem cells (GR cells) are to be used for the treatment of infertility, it is preferable to collect somatic cells from the patient. Meanwhile, when the GR cells are to be used for gene therapy for germ cells and the like, it is preferable, from the viewpoint of preventing graft rejections, to collect somatic cells from the patient or another person with the same or substantially the same HLA type as that of the patient.
• GR cells germline stem cells
• the HLA type of the donor matches with that of the patient to the extent that the transplanted cells, which have been obtained by inducing the differentiation of iPS cells derived from the donor's somatic cells, can engraft when transplanted to the patient with the use of an immunosuppressant and the like.
• it includes an HLA type wherein major HLAs (e.g., the three loci of HLA-A, HLA-B and HLA-DR, four loci further including HLA-Cw) are identical and the like (the same applies below) .
• the GR cells are not to be administered (transplanted) to a human, but used as, for example, a source of cells for screening for evaluating drug susceptibility or adverse reactions in a patient's testis, it is likewise desirable to collect the somatic cells from the patient or another person with the same genetic polymorphism correlating with the drug susceptibility or adverse reactions.
• Somatic cells isolated from a mammal can be pre-cultured using a medium known per se suitable for their cultivation according to the choice of cells before being subjected to the step of nuclear reprogramming.
• a medium known per se suitable for their cultivation according to the choice of cells before being subjected to the step of nuclear reprogramming.
• such media include, but are not limited to, minimal essential medium (MEM)
• FCS fetal calf serum
• DMEM Dulbecco's modified Eagle medium
• RPMI1640 medium 199 medium, F12 medium, and the like.
• a nuclear reprogramming substance can be a proteinous factor (s) capable of inducing iPS cells from a somatic cell or a nucleic acid that encodes the same (including forms incorporated in a vector) .
• a nuclear reprogramming substance used in the present invention may be a gene described in WO 2007/069666.
• examples include Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb and Esrrg.
• These reprogramming substances may be used in combination when establishing iPS cells; the combination comprises at least one, two, or three of the aforementioned reprogramming substances, with preference given to a
• combination comprising four.
• examples include the following combinations (hereinafter, only the names for proteinous factors are shown) .
• L-Myc can be used in place of c-Myc
• Lin28b can be used in place of Lin28.
• the somatic cell to undergo nuclear reprogramming is endogenously expressing one or more of the constituents of any one of (1) to (22) above at a level sufficient to cause
• nuclear reprogramming a combination of only the remaining constituents excluding the one or more constituents can also be included in the scope of "nuclear reprogramming substances" in the present invention.
• the four factors Oct3/4, Sox2, Klf4 and c-Myc (or L-Myc) and the three factors Oct3/4, Sox2, and Klf4 exemplify preferable nuclear reprogramming substances.
• the five or four factors consisting of these combinations plus Lin28 (or Lin28b) , and the six or five factors consisting of these combinations plus SV40 Large T antigen are also included.
• a proteinous factor When used as it is as a nuclear reprogramming substance, it can be prepared by inserting the cDNA obtained into an appropriate expression vector,
• the cDNA obtained is inserted into a viral vector, plasmid vector, episomal vector or the like to construct an expression vector, which is subjected to the step of nuclear
• nuclear reprogramming substance to a somatic cell can be achieved using a method known per se for protein transfer into a cell.
• Such methods include, for example, the method using a protein transfer reagent, the method using a protein transfer domain (PTD) or cell penetrating peptide (CPP) fusion protein, the microinjection method and the like.
• PTD protein transfer domain
• CPP cell penetrating peptide
• Protein transfer reagents are commercially available, including those based on a cationic lipid, such as BioPOTER Protein Delivery Reagent (Gene Therapy Systems) , Pro-JectTM Protein Transfection Reagent (PIERCE) and ProVectin (I GENEX) ; those based on a lipid, such as Profect-1 (Targeting Systems) ; those based on a membrane- permeable peptide, such as Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), GenomONE (ISHIHARA SANGYO KAISHA, LTD.) utilizing HVJ envelope (inactivated hemagglutinating virus of Japan) and the like.
• a cationic lipid such as BioPOTER Protein Delivery Reagent (Gene Therapy Systems) , Pro-JectTM Protein Transfection Reagent (PIERCE) and ProVectin (I GENEX)
• those based on a lipid such as Profect-1 (
• a nuclear reprogramming substance is diluted in an appropriate solvent (e.g., a buffer solution such as PBS or HEPES) , a transfer reagent is added, the mixture is incubated at room temperature for about 5 to 15 minutes to form a complex, this complex is added to cells after exchanging the medium with a serum-free medium, and the cells are incubated at 37°C for one to several hours.
• an appropriate solvent e.g., a buffer solution such as PBS or HEPES
• the medium is removed and replaced with a serum- containing medium.
• Developed PTDs include those using transcellular domains of proteins such as drosophila-derived AntP, HIV-derived TAT [Frankel, A. et al., Cell 55, 1189-93 (1988); Green, M. and Loewenstein, P.M., Cell 55, 1179-88 (1988)], Penetratin
• CPPs derived from the PTDs include polyarginines such as 11R [Cell Stem Cell, 4:381-384 (2009)] and 9R [Cell Stem Cell, 4:472-476
• a fusion protein expression vector incorporating a cDNA of the nuclear reprogramming substance and the PTD or CPP sequence is prepared to allow recombinant expression, and the fusion protein is recovered and used for transfection.
• the transfection can be performed in the same manner as the above except that no protein transfer reagent is added.
• Microinjection a method of placing a protein solution in a glass needle having a tip diameter of about 1 ⁇ , and
• injecting the solution into a cell ensures the transfer of the protein into the cell.
• the protein transferring operation can be performed one or more optionally chosen times (e.g., once or more to 10 times or less, or once or more to 5 times or less and the like) .
• the transferring operation can be performed twice or more (e.g., 3 times or 4 times) repeatedly.
• the time interval for repeated transferring operation is, for example, 6 to 48 hours, preferably 12 to 24 hours.
• the nuclear reprogramming substance be used not as a proteinous factor, but in the form of a nucleic acid that encodes the same.
• the nucleic acid may be a DNA or RNA, and may be a DNA/RNA chimera.
• the nucleic acid may be double- stranded or single-stranded.
• the nucleic acid is a double-stranded DNA, particularly a cDNA.
• a cDNA of the nuclear reprogramming substance is inserted i-n-fee—a-n—appr-opr-i-a-t-e-expression vector comprising a promoter capable of functioning in the host somatic cell.
• Useful expression vectors include, for example, viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesvirus and Sendai virus, plasmids for the expression in animal cells (e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like.
• the type of a vector to be used can be chosen as
• useful vectors include adenoviral vectors, plasmid vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, episomal vectors and the like.
• promoters used in expression vectors include the EFla promoter, the CAG promoter, the SRa promoter, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV
• HSV-TK herpes simplex virus thymidine kinase
• the expression vector may contain as desired, in addition to a promoter, an enhancer, a polyA addition signal, a
• selection marker gene a SV40 replication origin and the like.
• selection marker genes include the dihydrofolate reductase gene, the neomycin resistance gene, the puromycin resistance gene and the like.
• Nucleic acids that are nuclear reprogramming substances may be separately integrated onto respective expression vectors, and two or more, preferably two or—h-ree—di-f-£erent—gen ⁇ ⁇ ⁇ " expression vector. It is preferable to choose the former mode when using a retrovirus or lentiviral vector, which offers high gene transfer efficiency, and to choose the latter mode when using a plasmid, adenovirus or episomal vector or the like. Furthermore, an expression vector incorporating two or more different genes and an expression vector incorporating one gene alone may be used in combination.
• a plurality of reprogramming genes e.g., two or more, preferably two or three, genes selected from among Oct3/4, Sox2, Klf4, and c- Myc
• these genes can preferably be integrated into the expression vector via a sequence enabling polycistronic expression.
• a sequence enabling polycistronic expression makes it possible to more efficiently express a plurality of genes integrated in one expression vector.
• Useful sequences enabling polycistronic expression include, for example, the 2A sequence of foot-and- mouth disease virus (PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), the IRES sequence (U.S. Patent No. 4,937,190) and the like, with preference given to the 2A sequence.
• An expression vector comprising a reprogramming gene can be introduced into a cell by a technique known per se
• a viral ' vector for example, a plasmid containing the nucleic acid is introduced into an appropriate packaging cell (e.g., Plat-E cell) or a complementary cell line (e.g., 293-cells) , the viral vector produced in the culture supernatant is recovered, and the vector is infected to the cell by a method suitable for the viral vector.
• an appropriate packaging cell e.g., Plat-E cell
• a complementary cell line e.g., 293-cells
• the viral vector produced in the culture supernatant is recovered, and the vector is infected to the cell by a method suitable for the viral vector.
• specific means using a retroviral vector are disclosed in WO2007/69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007).
• Specific means using a lentiviral vector is disclosed in Science, 318, 1917-1920 (2007).
• iPS cells from iPS cells are utilized for medical purposes such as infertility treatment and germ cell gene therapy, the
• the reprogramming gene is preferably
• an adenoviral vector whose integration into chromosome is rare, is preferred. Specific means using an adenoviral vector is described in Science, 322, 945-949 (2008). Because an adeno- associated viral vector is also low in the frequency of
• a Sendai viral vector is capable of being stably present outside the chromosome, and can be degraded and removed using an siRNA as required, it is preferably utilized as well.
• a Sendai viral vector one described in J. Biol. Chem. , 282, 27383-27391 (2007) or Japanese Patent 3602058 can be used.
• a method can be used preferably wherein the nucleic acid that encodes the nuclear reprogramming substance is cut out using the Cre-loxP system, when the vector becomes no longer necessary. That is, with loxP sequences arranged on both ends of the nucleic acid in advance, iPS cells are induced, thereafter Cre recombinase is allowed to act on the cells using a plasmid vector or adenoviral vector, and the region sandwiched by the loxP sequences can be cut out. Because the enhancer-promoter
• sequence of the LTR U3 region possibly upregulates the host gene in the vicinity thereof by insertion mutation, it is more preferable to avoid the expression control of the endogenous genes by the LTR outside of the loxP sequence remaining in the genome without being cut out, using a 3' -self-inactivated
• a plasmid vector can be transferred into a cell using the lipofection method, liposome method, electroporation method, calcium phosphate co- precipitation method, DEAE dextran method, microinjection method, gene gun method and the like.
• lipofection method liposome method
• electroporation method calcium phosphate co- precipitation method
• DEAE dextran method DEAE dextran method
• microinjection method gene gun method and the like.
• Specific means using a plasmid as a vector are described in, for example, Science, 322, 949-953 (2008) and the like.
• the transfection can be performed one or more plasmid vector, an adenoviral vector or the like.
• the transfection can be performed one or more optionally chosen times (e.g., once to 10 times, once to 5 times, and the like); preferably, the transfection can be repeatedly performed twice or more (e.g., 3 times or 4 times).
• the transgene can get integrated into chromosome; therefore, it is
• transgene is integrated into chromosome using a transposon, thereafter transposase is allowed to act on the cell using a plasmid vector or adenoviral vector so as to completely eliminate the transgene from the chromosome.
• preferable transposons include piggyBac, a transposon derived from a lepidopterous insect, and the like. Specific means using the piggyBac transposon is disclosed by Kaji, K. et al. in Nature, 458: 771-775 (2009), and by Woltjen et al. in
• Another preferable non-integration type vector is an episomal vector, which is capable of self-replication outside of the chromosome. Specific means using an episomal vector is disclosed by Yu et al. in Science, 324, 797-801 (2009).
• vector components required for autonomous replication are a replication origin and a gene that encodes a protein that binds to the replication origin to control the replication, exemplified by the replication origin oriP and the EBNA-1 gene for EBV, and by the replication origin ori and the SV40 large T antigen gene for SV40.
• An episomal expression vector comprises a promoter that controls the transcription of reprogramming genes.
• the promoter the same promoters as those mentioned above can be used.
• the episomal expression vector may further comprise an enhancer, a polyA addition signal, a selection marker gene and the like if desired, as described above. Examples of selection marker genes include the dihydrofolate reductase gene, the neomycin resistance gene and the like.
• An episomal vector can be transferred into a cell using, for example, the lipofection method, liposome method,
• episome fraction can be prepared by a method obvious in the art; for example, methods described in Science, 324: 797-801 (2009) and the like, can be used.
• a functional inhibitor of p53 may be any substance, as far as it is capable of inhibiting either (a) the function of the p53 protein or (b) the expression of the p53 gene. That is, not only substances that act directly on the p53 protein to inhibit the function thereof and substances that act directly on the p53 gene to inhibit the expression thereof, but also substances that act on a factor involved in p53 signal transduction to result in the inhibition of the function of the p53 protein or the expression of the p53 gene, are also included in the scope of "a functional inhibitor of p53" as mentioned herein.
• the functional inhibitor of p53 is a substance that inhibits the expression of the p53 gene, more preferably an expression vector that encodes an siRNA or shR A against p53.
• substances that inhibit the function of the p53 protein include, but are not limited to, a chemical
• inhibitor of p53 a dominant negative mutant of p53 or a nucleic acid that encodes the same, an anti-p53 antagonist antibody or a nucleic acid that encodes the same, a decoy nucleic acid comprising a consensus sequence of a p53- responsive element, a substance that inhibits the p53 pathway, and the like.
• a chemical inhibitor of p53, a dominant negative mutant of p53 or a nucleic acid that encodes the same, and a p53 pathway inhibitor can be mentioned.
• Examples of chemical inhibitors of p53 include, but are not limited to, p53 inhibitors typified by pifithrin (PFT)-cc and - ⁇ , which are disclosed in WO 00/44364, PFT- ⁇ disclosed by Storm et al. in Nat. Chem. Biol. 2, 474 (2006), analogues thereof and salts thereof (e.g., acid addition salts such as hydrochlorides and hydrobromides, and the like) and the like.
• PFT-a and analogues thereof [2- (2-imino-4, 5, 6, 7- tetrahydrobenzothiazol-3-yl) -1-p-tolylethanone, HBr (trade .
• Pifithrin- ⁇ and 1- (4-nitrophenyl) -2- (4, 5, 6, 7- tetrahydro-2-imino-3 (2H) -benzothiazolyl) ethanone, HBr (trade name: Pifithrin-a, p-Nitro) ] and PFT- ⁇ and analogues thereof
• Pifithrin- ⁇ are commercially available from Merck Company.
• Contact of a chemical inhibitor of p53 with a somatic cell can be performed by dissolving the inhibitor at an
• a medium suitable for cultivation of somatic cells isolated from a human or mouse for example, minimal essential medium (MEM) comprising about 5% to 20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium and the like] so that the inhibitor concentration will fall in a range that fully inhibits the function of p53 and does not cause
• the inhibitor concentration varies depending on the kind of inhibitor used, and is chosen as appropriate over the range of about 0.1 nM to about 100 nM. Duration of contact is not particularly limited, as far as it is sufficient to achieve cell nuclear reprogramming; usually, the inhibitor may be allowed to be co-present in the medium until a positive colony emerges .
• the p53 gene is known as a cancer suppressor gene
• the choice of dominant negative mutant of p53 is not particularly limited, as far as the mutant is capable of competitively acting against the wild-type p53 protein being endogenously present in somatic cells to inhibit the function thereof; examples include p53P275S, resulting from point mutation of the proline at the 275-position (in the case of humans, 278-position) located in the DNA-binding region of mouse p53 to serine [de Vries, A., Proc. Natl. Acad. Sci. USA, 99, 2948-2953 (2002)]; p53DD, resulting from deletion of the amino acids at the 14-301-positions of mouse p53 (in human p53, corresponds to the 11-304-positions) [Bowman, T., Genes
• mutants include, for example, p53S58A, resulting from point mutation of the serine at the 58-position of mouse p53 (in the case of humans, 61-position) to alanine; p53C135Y, resulting from point mutation of the cysteine at the 135-position of human p53 (in the case of mice, 132-position) to tyrosine;
• p53A135V resulting from point mutation of the alanine at the 135-position of mouse p53 (in the case of humans, 138- position) to valine
• p53R172H resulting from point mutation of the arginine at the .172-position (in the case of humans, 175-position) to histidine
• p53R270H resulting from point mutation of the arginine at the 270-position (in the case of humans, 273-position) to histidine
• p53D278N resulting from point mutation of the aspartic acid at the 278-position of ⁇ mouse p53 (in the case of humans, 281-position) to asparagine, and the like; these can be used in the same way.
• a dominant negative mutant of p53 can be obtained by, for example, the technique described below.
• an appropriate oligonucleotide is synthesized as a probe or primer on the basis of mouse or human p53 cDNA sequence information, and a mouse or human p53 cDNA is cloned from a mRNA, cDNA or cDNA library derived from a mouse or human cell or tissue, using the hybridization method or the (RT-)PCR method, and is
• a primer for example, in the case of p53P275S, cct, the codon that encodes Pro at the 275-position
• a codon that encodes another desired amino acid for example, in the. case of p53P275S, tct, the codon that encodes Ser
• a primer may be designed outside the site to be deleted, and inverse PCR may be
• the desired dominant negative mutant can be acquired.
• the use of the mutant protein can be suitable in cases where high safety is required as in the case where the iPS cells obtained are utilized for therapeutic purposes.
• the functional inhibitor of p53 is a nucleic acid that encodes a dominant negative mutant of p53.
• the nucleic acid may be a DNA or RNA or a DNA/RNA chimera, and is
• the nucleic acid may be double-stranded or single-stranded.
• a cDNA that encodes a dominant negative mutant of p53 can be cloned by the technique described above with respect to preparation of the mutant protein.
• the cDNA isolated can be inserted into an appropriate expression vector and transferred to a somatic cell in the same way as the above-described case of nucleic acids that are nuclear reprogramming substances (reprogramming genes) .
• p53 pathway inhibitors include all substances that inhibit any one of the aforementioned signal transduction pathways.
• the p53 pathway inhibitor is a substance that inhibits the expression or function (Myc inhibitory activity) of p21, whose
• nucleic acids that inhibit the expression of p21 can be designed and synthesized and introduced into a somatic cell in the same manner as the method for siRNA, shRNA, antisense nucleic acids, and ribozymes against p53 described below.
• the nucleic acids may be provided in the form of a vector that expresses them; the vector can be constructed and introduced into a somatic cell in the same manner as the method for a vector that expresses an siRNA, shRNA, antisense nucleic acid, or ribozyme against p53 described below.
• the p53 pathway inhibitor is a substance that inhibits the ARF-MDM2-p53 pathway.
• ARF-MDM2-p53 pathway inhibitors include MDM2, which binds directly to p53 to promote the nuclear export or ubiquitination thereof, or a nucleic acid that encodes the same, pl9 ARF , which inhibits the action of MD 2 on p53, a substance that inhibits the expression or function of ATM (ataxia-telangiectasia mutated) (for example, siRNAs and shRNAs against these factors) and the like.
• Examples of other substances that inhibit the function of the p53 protein include an anti-p53 antagonist antibody and a nucleic acid that encodes the same.
• the anti-p53 antagonist antibody may be a polyclonal antibody or a monoclonal antibody.
• the isotype of the antibody is not particularly limited, and is preferably IgG, IgM or IgA, particularly preferably IgG.
• the antibody may be, in addition to a complete antibody
• molecule for example, a fragment such as Fab, Fab', or F(ab' ) 2 r a conjugate molecule prepared by a gene engineering technique, such as scFv, scFv-Fc, minibody, or diabody, or a derivative thereof modified with a molecule having protein-stabilizing action, such as polyethylene glycol (PEG) .
• PEG polyethylene glycol
• antagonist antibody can be produced using p53 or a partial peptide thereof as an antigen, by a method of antibody or anti-serum production known per se.
• Examples of publicly known anti-p53 antagonist antibodies include PAbl801 (Oncogene
• a nucleic acid that encodes an anti-p53 antagonist antibody can be isolated from a hybridoma that produces an anti-p53 monoclonal antibody by a conventional method.
• the H-chain and L-chain genes obtained may be joined together to prepare a nucleic acid that encodes a single-chain antibody.
• an anti-p21 antagonist antibody or a nucleic acid that encodes the same can be mentioned.
• An anti-p21 antagonist antibody and a nucleic acid that encodes the same can also be prepared as with the aforementioned anti-p53 antagonist
• Still another substance that inhibits the function of the p53 protein is a decoy nucleic acid comprising a consensus sequence of p53-responsive element [e.g., Pu-Pu-Pu-G-A/T-T/A- C-Py-Py-Py (Pu: purine base, Py: pyrimidine base) ] .
• a nucleic acid can be synthesized on the basis of the
• aforementioned base sequence information using an automated DNA/RNA synthesizer.
• a decoy nucleic acid is commercially available [e.g., p53 transcription factor decoy (GeneDetect.com)].
• An anti-p53 antagonist antibody or an anti-p21 antagonist antibody, as with a dominant negative mutant of p53, and a nucleic acid that encodes the antibody, as with a nucleic acid that encodes the mutant, can be introduced into a cell.
• the aforementioned decoy nucleic acid can be introduced into a cell by the lipofection method and the like.
• examples of substances that inhibit the expression of the p53 gene include siRNAs or shRNAs against p53, vectors that express an siRNA or shRNA against p53, antisense nucleic acids against p53 and ribozymes against p53, and the like, with preference given to siRNAs and shRNAs against p53 and vectors that express an siRNA or shRNA.
• siRNA against p53 can be designed on the basis of mouse or human p53 cDNA sequence information, in accordance with, for example, the rules proposed by Elbashir et al.
• the target sequence for the siRNA is, as a general rule, AA+(N)19, but may be AA+(N)21 or NA+(N)21.
• the 5' end of the sense strand need not to be AA.
• the position of the target sequence is not
• the target sequence be selected between 5'-UTR and about 50 bases from the start codon, as well as from a region other than 3'-UTR.
• the GC content of the target sequence is also not particularly limited, but the content is preferably about 30% to about 50%; a sequence with no irregularity in GC distribution and with only a few repeats is desirable.
• a polIII system promoter is used as the promoter in designing a vector that expresses an siRNA or shRNA of (b2) below, a sequence of 4 or more bases of T or A in succession should not be chosen, so as to prevent polymerase transcription from ceasing.
• the target sequence candidates selected on the basis of the above-described rules are examined for homology to sequences of 16-17 bases in succession in mRNAs other than the target, using a homology search software program such as BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), so as to confirm the specificity of the target sequences selected.
• BLAST http://www.ncbi.nlm.nih.gov/BLAST/
• a double-stranded RNA consisting of a sense strand having a 3'- terminal overhang of TT or UU in 19-21 bases after AA (or NA) , and an antisense strand having a sequence complementary to the 19-21 bases and a 3' -terminal overhang of TT or UU, is designed as an siRNA.
• an shRNA can be designed by optionally choosing as appropriate a linker sequence capable of forming a loop structure (for example, about 8-25 bases) , and ligating the aforementioned sense and
• siRNAs and/or shR As can be searched for using search software programs available at no cost on various websites. Examples of such sites include, but are not limited to, the siRNA Target Finder
• siRNA against p53 can be prepared by synthesizing a sense strand oligonucleotide and antisense strand
• An shRNA against p53 can be prepared by synthesizing oligonucleotides having an shRNA sequence
• nucleotide molecules that constitute the siR A or shRNA may be natural-type RNAs
• the molecules can comprise various chemical modifications in order to increase the stability (chemical and/or to-enzyme) or specific activity (affinity for mRNA) .
• specific activity affinity for mRNA
• each nucleotide that constitutes the antisense nucleic acid can be substituted with, for example, a
• hydroxyl group at the 2 '-position of the sugar (ribose) of each nucleotide may be replaced with -OR [R represents, for example, CH 3 (2 ' -O-Me) , CH 2 CH 2 OCH 3 (2 ' -O-MOE) , CH 2 CH 2 NHC (NH)NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN or the like].
• R represents, for example, CH 3 (2 ' -O-Me) , CH 2 CH 2 OCH 3 (2 ' -O-MOE) , CH 2 CH 2 NHC (NH)NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN or the like].
• a base moiety pyrimidine, purine
• examples of such modifications include introduction of a methyl group or a cationic functional group into the 5- position of the pyrimidine base, substitution of the 2- position carbonyl group with thiocarbonyl and the like.
• RNA derivatives wherein the conformation of the sugar moiety is fixed at the N type by bridging the 2 ' oxygen and 4' carbon so as to confer strong bindability to the target RNA can also be used preferably.
• siRNA against p53 can also be purchased from, for example, Ambion (e.g., Ambion Cat# A 16708, siRNA ID# 69659, 69753, 69843, 187424, 187425, 187426), Santa Cruz (e.g., Santa Cruz Cat# sc-29436, 44219) and the like.
• Ambion e.g., Ambion Cat# A 16708, siRNA ID# 69659, 69753, 69843, 187424, 187425, 187426
• Santa Cruz e.g., Santa Cruz Cat# sc-29436, 44219
• siRNA and shRNA against human p53 can also be designed and synthesized using one of the aforementioned search
• shRNA against human p53 described in Science, 296, 550-553 (2002) and the like can be mentioned.
• the method using a cationic liposome is the most common and offers high transfer efficiency.
• transfection reagents such as Lipofectamine2000 and
• Oligofectamine for example, transfer reagents suitable for introduction of an siRNA, such as the GeneEraserTM siRNA transfection reagent (Stratagene) , are also commercially available.
• siRNA such as the GeneEraserTM siRNA transfection reagent (Stratagene)
• Vectors that express an siRNA are available in the tandem type and the stem loop (hairpin) type.
• the former is the type in which an expression cassette for a sense strand of an siRNA and an expression cassette for an antisense strand are ligated tandem, each strand being expressed in the cell and undergoing annealing to form a double-stranded siRNA (dsRNA) .
• dsRNA double-stranded siRNA
• the latter is the type in which an expression cassette for an shRNA is inserted into a vector, the shRNA being expressed in the cell and undergoing processing by a dicer to form a dsRNA.
• a polll system promoter for example, immediate-early promoter of CMV
• a polIII system promoter in order to allow the accurate transcription of short RNA.
• the polIII system promoter mouse and human U6-snRNA promoters, human Hl-RNase P RNA promoter, human valine-tRNA promoter and the like can be mentioned.
• a transcription termination signal a sequence of four or more T residues in succession is used.
• siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector, episomal vector, viral vector or the like.
• a plasmid vector episomal vector, viral vector or the like.
• nucleic acids that are nuclear reprogramming substances can be utilized preferably (viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai virus; animal cell expression plasmids, episomal vectors and the like) .
• viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai virus; animal cell expression plasmids, episomal vectors and the like
• the vector used can be chosen as appropriate according to the intended use of the GR cells differentiation-induced from the iPS cell obtained, as in the case of reprogramming genes. Because permanent functional inhibition of p53 potentially increases the risk of
• a vector capable of transiently expressing p53 and leaving the cell soon after establishment of iPS cells e.g., plasmid vector and the like
• a viral vector such as a retrovirus, a plasmid vector, an episomal vector and the like prepared on the basis of a commercially available plasmid (for example, pMKO.l-puro p53 shRNA2 : #10672, commercially
• transposon system can also be utilized as required.
• a vector that expresses an siRNA or shRNA against p53 with a somatic cell is achieved by introducing a plasmid vector, episomal vector or viral vector prepared as described above into the cell. Transfer of these genes can be achieved by the same technique as that described with respect to reprogramming genes.
• Other substances that inhibit the expression of the p53 gene include antisense nucleic acids and ribozymes against p53.
• the antisense nucleic acid may be a DNA or RNA or a
• the antisense nucleic acid is a DNA
• an RNA DNA hybrid formed by a target RNA and the antisense DNA is capable of being recognized by endogenous RNase H to cause selective degradation of the target RNA. Therefore, in the case of an antisense DNA to direct degradation by RNase H, the target sequence may be not only a sequence in p53 mRNA, but also a sequence in the intron region of the initial
• the length of the target region for the antisense nucleic acid is not
• the target region may be the entire sequence or a partial sequence of p53 mRNA, and may be a sequence of about 15 bases for the shortest, or of the entire sequence of the mRNA or initial transcription product for the longest.
• an oligonucleotide consisting of about 15-40 bases, particularly about 18 to 30 bases, is preferable. Positions of the target sequence include, but are not limited to, 5'- and 3'-UTR, vicinities of the start codon and the like.
• a ribozyme refers to an RNA possessing an enzyme activity to cleave a nucleic acid in the narrow sense, and is herein understood to be used as a concept encompassing DNA, as far as the ribozyme possesses sequence-specific nucleic acid cleavage activity.
• One of the most versatile ribozymes is a self- splicing RNA found in infectious RNAs such as viroid and virusoid, and the hammerhead type, the hairpin type and the like are known.
• the hammerhead type exhibits enzyme activity with about 40 bases in length, and it is possible to
• the target mRNA specifically cleave the target mRNA by making several bases at both ends adjoining to the hammerhead structure portion (about 10 bases in total) be a sequence complementary to the desired cleavage site of the mRNA.
• An antisense nucleic acid or a ribozyme can be any antisense nucleic acid or a ribozyme.
• nucleotide molecules that constitute them may also have the same modifications as those for siRNA, so as to increase the stability, specific activity and the like.
• the antisense nucleic acid or ribozyme can also be used in the form of a nucleic acid that encodes the same, as in the case of siRNA.
• the aforementioned functional inhibitor of p53 need to be brought into contact with a somatic cell in a way sufficient to inhibit the function of p53 in the step of somatic cell nuclear reprogramming.
• the nuclear reprogramming substance and the functional inhibitor of p53 may be brought into contact with the somatic cell simultaneously, or either one may be contacted in advance.
• the nuclear reprogramming substance is a nucleic acid that encodes a proteinous factor
• the functional inhibitor of p53 is a chemical inhibitor
• the former involves a given length of time lag from the transfection treatment to the mass-expression of the proteinous factor, whereas the latter is capable of rapidly inhibiting the function of p53.
• the chemical inhibitor of p53 can be added to the medium.
• nuclear reprogramming substance and the functional inhibitor of p53 are both used in the form of a viral vector, plasmid vector, episomal vector or the like, both may be introduced into the cell simultaneously.
• iPS cell establishment efficiency improvers include, but are not limited to, histone deacetylase (HDAC) inhibitors [e.g., low-molecular inhibitors such as valproic acid (VPA) (Nat. Biotechnol., 26(7): 795-797 (2008)),
• HDAC histone deacetylase
• nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool (registered
• G9a histone methyltransferase inhibitors e.g., low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)); nucleic acid-based expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (Santa Cruz
• L-calcium channel agonists e.g., Bayk8644
• UTF1 Cell Stem Cell, 3, 475-479 (2008)
• Wnt Signaling e.g., soluble Wnt3a
• 2i/LIF 2i/LIF [2i is an inhibitor of mitogen-activated protein kinase signaling and glycogen synthase kinase-3, PloS Biology, 6(10), 2237-2247
• nucleic acid-based expression inhibitors may be in the form of expression vectors harboring a DNA that encodes an siRNA or shRNA.
• reprogramming substances SV40 Large T antigen and the like, for example, can also be encompassed in the scope of iPS cell establishment efficiency improvers because they are factors not essential but auxiliary for somatic cell nuclear
• reprogramming is dealt with as an overall event resulting from contact of a nuclear reprogramming substance and an iPS cell establishment efficiency improver with a somatic cell;
• the other iPS cell establishment efficiency improvers may be brought into contact with the somatic cells simultaneously with the nuclear reprogramming ' substance, and either one may be contacted in advance, as far as the efficiency of iPS cell establishment from a somatic cell improves significantly compared with the efficiency obtained in the absence of the substance.
• the iPS cell establishment efficiency improver can be contacted with a somatic cell at the same timing as that described above with respect to functional inhibitors of p53, according to the properties of the improver.
• hypoxic conditions means that the oxygen concentration in the ambient atmosphere during cell culture is significantly lower than that in the air. Specifically, such conditions include lower oxygen concentrations than the oxygen concentrations in the ambient atmosphere of 5-10% CO 2 /95-90% air, which is commonly used for ordinary cell culture; for example, oxygen concentrations of 18% or less in the ambient atmosphere are applicable.
• the oxygen concentration in the ambient atmosphere of 5-10% CO 2 /95-90% air, which is commonly used for ordinary cell culture; for example, oxygen concentrations of 18% or less in the ambient atmosphere are applicable.
• the oxygen concentration in the ambient atmosphere of 5-10% CO 2 /95-90% air, which is commonly used for ordinary cell culture; for example, oxygen concentrations of 18% or less in the ambient atmosphere are applicable.
• the oxygen concentration in the ambient atmosphere of 5-10% CO 2 /95-90% air, which is commonly used for ordinary cell culture; for example, oxygen concentrations of 18% or less in the ambient atmosphere are applicable.
• the oxygen concentration in the ambient atmosphere of 5-10% CO 2 /95-90% air, which
• concentration in the ambient atmosphere is 15% or less (e.g., 14% or less, 13% or less, 12% or less, 11% or less and the like), 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less and the like), or 5% or less (e.g., 4% or less, 3% or less, 2% or less and the like) .
• the oxygen concentration in the ambient atmosphere is preferably 0.1% or more (e.g., 0.2% or more, 0.3% or more, 0.4% or more and the like), 0.5% or more (e.g., 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more and the like), or 1% or more (e.g., 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more and the like).
• C0 2 incubators are commercially available from a number of manufacturers of equipment (e.g., C0 2 incubators for hypoxic culture
• the timing of beginning cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells compared with that obtained at a normal oxygen concentration (20%) .
• the starting time may be before or after contact of nuclear reprogramming substances with a somatic cell, and may be at the same time as the contact.
• cell culture under hypoxic conditions be begun just after contacting a nuclear reprogramming substance with a somatic cell, or after a given time (e.g., 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8 or 9) days) following the contact.
• the duration of cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells
• iPS cell candidate colonies are selected with drug resistance as an indicator, it is
• a normal oxygen concentration be restored from hypoxic conditions by the start of drug selection.
• the preferred starting time and duration of cell culture under hypoxic conditions also vary depending on the choice of nuclear reprogramming substances used, the efficiency of establishment of iPS cells under conditions involving a normal oxygen concentration, and other factors.
• the cell can be cultured under conditions suitable for the cultivation of, for example, ES cells.
• the cultivation is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a
• fibroblast growth factor bFGF
• SCF stem cell factor
• the cells are cultured in the co-presence of mouse embryonic fibroblasts treated with radiation or an antibiotic to terminate their cell division, as feeder cells.
• the STO cell line ATCC CRL-1503
• other lines of mouse embryonic fibroblasts are commonly used as feeders.
• SNL cells SNL76/7 STO cells;
• ECACC 07032801 [McMahon, A. P. & Bradley, A., Cell 62, 1073- 1085 (1990)], which are prepared by stably incorporating the neomycin resistance gene and the LIF gene into STO cells, and the like are commonly used.
• MEF primary culture
• Mitomycin C-treated MEFs are commercially available from Millipore Company and ReproCELL Company. Co-culture with these feeder cells may be started before contact of the nuclear reprogramming substance, at the time of the contact, or after the contact (e.g., 1-10 days later) .
• a candidate colony of iPS cells can be selected by a method with drug resistance and reporter activity as
• a colony positive for drug resistance and/or reporter activity is selected using a recombinant somatic cell wherein a drug resistance gene and/or a reporter gene is targeted to the locus of a gene highly expressed specifically in pluripotent cells (e.g., Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4) .
• pluripotent cells e.g., Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4
• Examples of such recombinant somatic cells include MEFs from a mouse having the ⁇ -geo gene (which encodes a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase) knocked-in to the Fbxl5 locus [Takahashi & Yamanaka, Cell, 126, 663-676 (2006) ] , MEFs from a transgenic mouse having the green fluorescent protein (GFP) gene and the puromycin resistance gene integrated in the Nanog locus [Okita et al., Nature, 448, 313-317 (2007)] and the like.
• GFP green fluorescent protein
• the GR cells of the present invention highly express the Oct3/4 gene (Oct4- positive) , it is more preferable to use cells wherein a reporter gene that encodes a visualizing protein such as GFP or RFP is knocked in to the ,Oct3/4 locus, in the method with reporter activity as an index.
• a reporter gene that encodes a visualizing protein such as GFP or RFP is knocked in to the ,Oct3/4 locus, in the method with reporter activity as an index.
• examples of the latter method based on visual examination of morphology include the method described by Takahashi et al. in Cell, 131, 861-872 (2007) .
• the method using reporter cells is convenient and efficient, it is desirable from the viewpoint of safety that colonies be selected by visual examination when the GR cells differentiation-induced from iPS cells are
• the identity of the cells of the selected colony as iPS cells can be confirmed by positive responses to an Oct 4 (or Nanog) reporter (puromycin resistance, GFP positivity and the like) , as well as by the formation of a visible ES cell-like colony, as described above.
• Oct 4 or Nanog reporter
• Examples of available methods of generating ES cells include, but are not limited to, methods in which a mammalian inner cell mass in the blastocyst stage is cultured [see, for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)], methods in which an early embryo prepared by somatic cell nuclear transfer is cultured [Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Akira
• ES cells are available from specified organizations, and commercial products may be purchased.
• the human ES cells KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.
• somatic cell nuclear transplantation When using somatic cell nuclear transplantation, the choice of somatic cell and the source of somatic cells to be collected are the same as those in the above-described case of iPS cells.
• EG cells can be induced by isolating primordial germ cells by a conventional method, and culturing the cells in the presence of LIF, bFGF and SCF.
• mGS cells can be prepared from testicular cells according to a method described in WO
• Multipotent adult progenitor cells can be isolated from the bone marrow according to a method described in J. Clin. Invest. 109:337-346 (2002).
• Any medium useful for animal cell culture can be used as the basal medium for differentiation induction of GR cells.
• Examples include the Neurobasal medium, Neural Progenitor
• Basal medium Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, a-MEM medium, DMEM medium, DMEM/F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. More preferably, the basal medium is the Neurobasal medium.
• These media may contain a serum [e.g., fetal calf serum (FCS) , human serum and the like] or not.
• FCS fetal calf serum
• a serum substitute additive e.g., Knockout Serum Replacement (KSR) (produced by KSR
• addition concentration can be chosen as appropriate over the range of 0 to 20%; preferably, a serum-free or low-serum (e.g., 0 to 5%, preferably 0 to 2%) medium can be used.
• a serum-free or low-serum (e.g., 0 to 5%, preferably 0 to 2%) medium can be used.
• albumins such as bovine serum albumin (BSA) and human serum albumin (HSA) and the like
• reducing agents e.g., 2-mercaptoethanol and the like
• growth factors e.g., insulin, bFGF, HGF, FGF9 and the like
• stem cell differentiation suppressants e.g., LIF, Wnt, TGF- ⁇ and the like
• iron sources e.g., transferrin and the like
• minerals e.g., sodium selenite
• amino acids e.g., non-essential amino acids such as glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, and tyrosine
• vitamins e.g., choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid
• one or more factors selected from among the medium additives listed in Table 1. are used as added to any one of the basal media described above. Those skilled in the art are able to add these factors at
• concentrations (final concentrations.) shown in the "Medium component" panel in Fig. 27 may be chosen.
• Insulin-Transferrin-Selenium Supplement (Invitrogen)
• BMP4 bone morphogenetic protein 4
• GDNF neurotrophic factor
• the pluripotent stem cells are cultured in the presence of three factors, i.e., B P4 and two growth factors selected from among GDNF, EGF and SCF, more preferably in the presence of the four factors BMP4, GDNF, EGF and SCF.
• B P4 three factors
• BMP4 two growth factors selected from among GDNF, EGF and SCF, more preferably in the presence of the four factors BMP4, GDNF, EGF and SCF.
• differentiation induction medium or cells that produce the factors may be used as feeder cells and co-cultured with the pluripotent stem cells.
• the BMP4, GDNF, EGF and SCF used may be derived from an optionally chosen mammal (e.g. , humans, mice, monkeys, pigs, rats, dogs and the like, preferably humans or mice)
• an optionally chosen mammal e.g. , humans, mice, monkeys, pigs, rats, dogs and the like, preferably humans or mice
• one derived from the same species as the pluripotent stem cells.
• human GR cells when human GR cells are used for therapeutic purposes in humans, it is desirable that the human GR cells be induced under conditions wherein no heterologous components are present (xeno-free) ; therefore, human-derived proteins are preferably used as these growth factors.
• these growth factors may be ones isolated/purified from cells [e.g., nerve cells, glial cells, hepatocytes, splenocytes, pancreatic ⁇ cells, myelocytes, mesangial cells, Langerhans' cells, epidermal cells, epithelial cells,
• cells e.g., nerve cells, glial cells, hepatocytes, splenocytes, pancreatic ⁇ cells, myelocytes, mesangial cells, Langerhans' cells, epidermal cells, epithelial cells,
• endothelial cells smooth muscle cells, fibroblasts,
• fibrocytes e.g., myocytes, adipocytes, immune cells (e.g.,
• T cells T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes
• B cells natural killer cells
• mast cells neutrophils, basophils, eosinophils, monocytes
• megakaryocytes synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cells or interstitial cells, or corresponding precursor cells, stem cells or cancer cells thereof, and the like] of a mammal that produces the same (e.g., humans, mice, monkeys, pigs, rats, dogs and the like) or any tissues where such cells are present [e.g., brain, parts of brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum) , spinal cord,
• brain parts of brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum) , spinal cord,
• hypophysis stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscles (e.g., smooth muscle, skeletal muscle) , lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood,
• muscles e.g., smooth muscle, skeletal muscle
• lung gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood,
• adipose tissues e.g., white adipose tissue, brown adipose tissue
• adipose tissues e.g., white adipose tissue, brown adipose tissue
• methods based on differences in solubility, such as salting-out and solvent precipitation e.g., methods based on differences in solubility, such as salting-out and solvent precipitation; methods based mainly on differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS- polyacrylamide gel electrophoresis; methods based on
• growth factors may also be proteins synthesized chemically or
• synthesized biochemically in a cell-free translation system they are preferably recombinant proteins produced by a
• transformant incorporating a nucleic acid that encodes one of these proteins.
• a host cell e.g., animal cells, insect cells, Bacillus subtilis, yeast, Escherichia coli and the like
• culture the resulting transformant e.g., culture the resulting transformant, and isolate/purify the desired recombinant protein from the resulting culture supernatant using the above-described techniques of protein separation and
• differentiation of the GR cells of the present invention may be a protein having an amino acid sequence different from the amino acid sequence encoded by one of the above-described cDNA sequences, as far as it possesses the capability of inducing the differentiation of GR cells from pluripotent stem cells by being combined with other factors.
• the BMP4, GDNF, EGF or SCF used to induce the differentiation of GR cells means a protein that comprises:
• homology means a ratio (%) of identical amino acid residues and similar amino acid residues to all
• a similar amino acid means an amino acid having similar physicochemical properties
• amino acids classified under the same group such as aromatic amino acids (Phe, Trp, Tyr) , aliphatic amino acids (Ala, Leu, lie, Val) , polar amino acids (Gin, Asn) , basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp) , amino acids having a hydroxyl group (Ser, Thr) and amino acids having a small side-chain (Gly, Ala, Ser, Thr, Met) .
• differentiation of GR cells in (a) above is more preferably a protein comprising an amino acid sequence having an identity of about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, to the amino acid sequence encoded by the cDNA sequences shown by one of the aforementioned NCBI accession numbers.
• the BMP4, GDNF, EGF or SCF used to induce the differentiation of GR cells in (b) above can preferably a protein comprising (i) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids deleted from the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (ii) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids added to the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (iii) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids inserted into the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (iv) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids substituted by other amino acids in the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI acces
• the position of the insertion, deletion or substitution is not particularly limited, as far as the differentiation induction activity for the GR cells is retained.
• another protein of the BMP family for example, BMP8b, BMP2, BMP7 and the like, may be used in place of, or in addition to, BMP4 when inducing differentiation from pluripotent stem cells to GR cells.
• BMP4 and BMP8b are used in combination.
• the BMP4 concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the BMP4 concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less. When another protein of the BMP family is used in combination with BMP4, it is preferable that the BMPs be added to obtain a total concentration in the aforementioned range.
• the GDNF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the GDNF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less. When a GDNF equivalent is used in combination with GDNF, it is preferable that the GDNF and the equivalent be added to obtain a total concentration in the aforementioned range.
• the EGF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the EGF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• the SCF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more.
• the SCF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• the components (a) and (b) may be provided separately from the above-described basal medium or the medium comprising the basal medium and the above-described medium additives added thereto, and used as added to the medium just before use.
• these components may be provided in the form of a differentiation induction medium already containing the components, as far as they do not adversely affect the
• At least one factor, preferably all the factors used, out of the factors used in inducing the differentiation of the GR cells of the present invention are supplied from feeder cells, rather than added to the medium.
• the feeder cells may be mammalian cells that produce these growth factors by nature, it is more preferable to use recombinant cells that are introduced with genes encoding the growth factors and overexpress the growth factors.
• a plurality of kinds of cells that express different growth factors can be used in combination, it is preferable to use one kind of cells that express all the growth factors to be supplied from the feeders.
• Cells that can be used as the host include, but are not limited to, cells that have traditionally been suitably used as feeder cells. For example, mouse
• STO cell line ATCC CRL-1503
• SNL cells which are prepared by stably incorporating the neomycin resistance gene and the LIF gene into STO cells (SNL76/7 STO cells; ECACC 07032801) and the like are also preferable. It is also preferable to use cells of the same species as the pluripotent stem cells as feeders. For example, when GR cells are to be induced from human
• pluripotent stem cells human dermal fibroblasts (HDF) , human dental pulp stem cells and the like can be used as feeders.
• HDF human dermal fibroblasts
• M15-BMP4 cells [Proc. Natl. Acad. Sci. USA, 100: 11457- 11462 (2003)], for example, can be used as the feeder cells.
• one or more growth factors selected from among GDNF, EGF and SCF are also to be supplied from feeder cells, it is also possible to use transformant cells obtained by introducing an expression vector harboring nucleic acids that encode the one or more growth factors selected from among GDNF, EGF and SCF, for example, into 15-BMP4 cells.
• a viral vector harboring the nucleic acids that encode GDNF, EGF and SCF is introduced into M15-BMP4 cells to create cells that overexpress the four growth factors BMP4 , GDNF, EGF and SCF (M15-4GF) .
• Cultivation for inducing differentiation from pluripotent stem cells to GR cells can, for example, be performed as described below.
• Any culture vessel for cell culture can be used in this step of differentiation induction.
• Such culture vessels can be used in this step of differentiation induction.
• the culture vessel can be non-cell-adhesive (weakly cell-adhesive) or cell-adhesive, depending on the method of cultivation
• a cell-adhesive culture vessel is a culture vessel whose surface is coated with a cell support substrate to improve adhesion of cells (pluripotent stem cells or feeder cells) .
• cell support substrates include, for example, collagen, gelatin, Matrigel, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
• Pluripotent stem cells are seeded to the above-described culture vessel to obtain a cell density of, for example, about 0.5 to 50*10 4 cells/cm 2 , preferably about 1 to ⁇ ⁇ ⁇ 4 cells/cm 2 , and cultured, for example, in a C0 2 incubator under an
• the cultivation may be performed by suspension culture or adhesion culture, and preferably by suspension culture.
• at least one factor of (a) BMP4 and (b) one or more growth factors selected from among GDNF, EGF and SCF (differentiation induction growth factors of the present invention) are supplied from feeder cells, the pluripotent stem cells and feeder cells are mixed in a ratio of 1:10 to 10:1, preferably 1:5 to 5:1, more preferably 1:2 to 2:1, seeded to the culture vessel, and subjected to suspension culture.
• the pluripotent stem cells may be seeded to the culture vessel to which feeder cells have been seeded in advance, and co-cultured. Even when all of the differentiation induction growth factors of the present invention are used as added to the medium, the pluripotent stem cells may be cultured in the presence of feeder cells.
• useful feeder cells include ordinary MEF [e.g., STO cell line (ATCC CRL-1503) and the like], SNL cells (SNL76/7 STO cells; ECACC 07032801), HDF, dental pulp stem cells and the like treated with radiations or an antibiotic to terminate their cell division.
• pluripotent stem cells Differentiation from pluripotent stem cells to GR cells can be confirmed with an Oct4-positive and Vasa-positive response as an index.
• pluripotent stem cells incorporating a reporter gene for visualization when using pluripotent stem cells incorporating a reporter gene for visualization
• Oct4-positive Vasa-positive (Oct4 + /Vasa + ) cells can be sorted and isolated from other undifferentiated cells (Oct4 + /Vasa ⁇ ) and cells that have differentiated into somatic cells (Oct4 ⁇ ) by flow cytometry (FACS) .
• FACS flow cytometry
• suspension culture is performed using feeder cells, suspended cell masses containing the feeder cells are formed; therefore, for example, trypsin/EDTA, collagenase and the like are added to dissociate the cell masses, and the resulting cell
• suspension is seeded to, and incubated on, a culture vessel coated with a cell support substrate, after which suspended cells are recovered, whereby the feeder cells can be separated and removed.
• pluripotent stem cells wherein a
• the detection can be' achieved by analyzing the phenotype of the surface antigen of the cells using a cell sorter and antibodies against an undifferentiated cell surface marker corresponding to the expression of Oct4 and against a germ line cell surface marker corresponding to the expression of the Vasa protein or Vasa.
• undifferentiated cell surface markers include SSEA-1, Forssman antigen, ⁇ - and ⁇ -integrin and the like
• germline cell surface markers include EpCAM, CD9, EE2, c-kit and the like.
• the expression of Oct4 and other transcription factors may also be examined as required.
• the present invention also provides iPS cell-derived Oct4-positive Vasa-positive germline stem cells, i.e., GR cells, obtained by the method described above.
• GR cells iPS cell-derived Oct4-positive Vasa-positive germline stem cells
• a cell line permitting long-time cultivation induced from spermatogonial stem cells (e.g., WO 2004/092357 and the like) to the testis of the mouse.
• GS cells a cell line permitting long-time cultivation induced from spermatogonial stem cells
• germline stem cells capable of causing spermiogenesis could not be established from iPS cells when using these conventional culturing conditions; iPS cell-derived germline stem cells were obtained for the first time by using the method of differentiation induction of the present invention.
• BMP4 BMP4 and (b) one or more growth factors selected from among GDNF, EGF and SCF are added to the medium, or supplied from feeder cells.
• the present invention also provides an inducer of differentiation from pluripotent stem cells to Oct4-positive Vasa-positive germline stem cells (GR cells) , comprising (a) BMP4 and (b) one or more growth factors selected from among GDNF, EGF and SCF in combination.
• the GR cell differentiation inducer of the present invention comprises two factors or more of GDNF, EGF and SCF, more preferably all of the three factors.
• growth factors may be provided in solution in water or an appropriate buffer solution, and may be provided as a lyophilized powder and dissolved in an appropriate solvent just before use.
• These components may stand as separate reagents to form a kit, and may be provided as a single reagent comprising a blend of two or more, as far as they do not adversely affect each other.
• the GR cell differentiation inducer of the present invention can further comprise physiologically acceptable carriers, excipients, antiseptics, stabilizers, binders, solubilizers, nonionic surfactants, buffering agents,
• differentiation inducer of the present invention comprises cells that produce (a) B P4 and (b) one or more growth factors selected from among GDNF, EGF and SCF.
• the cells include recombinant cells that overexpress these growth factors, obtained by introducing an expression vector
• cells as feeder cells, are co- cultured with pluripotent stem cells, it is preferable to use cells that have traditionally been used as feeder cells in the relevant technical field, as the host cells. Specifically, the cells described above can be mentioned.
• Cells that produce (a) BMP4 and (b) one or more growth factors selected from among GDNF, EGF and SCF can be provided in a state suspended at an appropriate cell density in an appropriate medium (e.g., the above-described basal media and the like) , or in a state preserved under freezing by a conventional method.
• an appropriate medium e.g., the above-described basal media and the like
• the present invention also provides a GR cell
• differentiation induction medium comprising any one of the above-described basal media or a medium prepared by
• the differentiation induction medium may be provided as supplemented with the differentiation inducer, and the differentiation inducer may stand as a separate reagent to form a kit to be added to the medium just before use.
• the thus-obtained Oct4-positive Vasa-positive germline stem cells can be cultured in the presence of GDNF, EGF, SCF and basic fibroblast growth factor (bFGF) for a long time, while maintaining the Oct4-positive Vasa-positive property. Accordingly, the present invention also provides a method of expanding GR cells, comprising culturing the GR cells in the presence of GDNF, EGF, SCF and bFGF.
• the GR cells used for the method of cell maintenance and expansion of the present invention are not subject to limitations with regard to derivation, as far as they are Oct4-positive Vasa-positive cells destined to become germline cells that are capable of survive for a long time without forming tumors and without being eliminated when transplanted to the testis, the GR cells used are preferably those derived from pluripotent stem cells, more preferably those induced by the above-described method of differentiation induction of the present invention.
• the GR cells may be provided as an isolated and purified homogenous population of Oct4-positive Vasa-positive cells, and may be provided as a heterogenous population of cells wherein 0ct4-negative or Vasa-negative cells are also present (hereinafter,
• heterogenous cell populations containing GR cells are included in the scope of GR cells unless otherwise stated) .
• the basal medium and optionally chosen medium additives used in the method of the present invention for maintaining and expanding GR cells are the same as those used in the above-described method of inducing differentiation from pluripotent stem cells to GR cells. As stated below, however, the method of the present invention for maintaining and expanding GR cells is performed in the presence of SCF and bFGF, so that the combined use of LIF can induce the
• one or more factors selected from among the medium additives listed in Table 1 are used as added to the basal medium. Those skilled in the art are able to add these factors at appropriate concentrations to. the basal medium; for example, the concentrations (final
• GR cells are cultured in the presence of GDNF, EGF, SCF and bFGF.
• growth factors may be used as added to one of the above-described media to obtain a differentiation induction medium, or cells that produce the factors may be used as feeder cells and co-cultured with the pluripotent stem cells, the factors are preferably used as added to the medium.
• the GDNF, EGF, SCF and bFGF used may be derived from an optionally chosen mammal (e.g., humans, mice, monkeys, pigs, rats, dogs and the like, preferably humans or mice)
• an optionally chosen mammal e.g., humans, mice, monkeys, pigs, rats, dogs and the like, preferably humans or mice
• one derived from the same species as the GR cells it is preferable to use one derived from the same species as the GR cells.
• human GR cells when human GR cells are used for therapeutic purposes in humans, it is desirable that the human GR cells be induced under conditions wherein no heterologous components are present (xeno-free) ; therefore, human-derived proteins are preferably used as these growth factors.
• these growth factors may be ones isolated/purified from cells of a mammal that produces the same (for example, humans, mice, monkeys, pigs, rats, dogs and the like) or any tissues where such cells are present and the like by a method of protein separation and purification known per se.
• the growth factors may also be proteins synthesized chemically or synthesized biochemically in a cell-free translation system, they are preferably recombinant proteins produced by a
• Recombinant proteins of GDNF, EGF and SCF can be prepared by the above-described method.
• Information on the human and mouse cDNA sequences of bFGF is available with reference to the NCBI accession numbers NM_002006 and
• NM_008006 those skilled in the art are able to isolate a cDNA of bFGF by a conventional method on the basis of the
• Recombinant proteins of GDNF, EGF, SCF and bFGF are commercially available.
• the GDNF, EGF, SCF or bFGF used to maintain and expand the GR cells of the present invention may be a protein having an amino acid sequence different from the amino acid sequence encoded by one of the aforementioned cDNA sequences, as far as it supports the capability of the GR cells of self-replication while maintaining their differentiated state by being combined with other factors.
• the GDNF, EGF, SCF or bFGF used to maintain and expand the GR cells means a protein that comprises:
• the GDNF, EGF, SCF or bFGF used to maintain and expand the GR cells in (b) above can preferably a protein comprising (i) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids deleted from the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (ii) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids added to the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (iii) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids inserted into the amino acid sequence encoded by the cDNA sequence shown by one of the aforementioned NCBI accession numbers, (iv) an amino acid sequence having 1 to 10, more preferably one to several (5, 4, 3, or 2) amino acids
• the position of the insertion, deletion or substitution is not particularly limited, as far as the maintenance and expansion activity for the GR cells is retained.
• a GDNF equivalent described in WO 2004/092357 may be used in place of, or in addition to, GDNF, when maintaining and expanding GR cells.
• the GDNF concentration is, for example, about 0.1 ng/ml or more,
• the GDNF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• a GDNF equivalent is used in combination, it is preferable that the GDNF and the equivalent be added to obtain a total concentration in the aforementioned range.
• the EGF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the EGF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• the SGF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the SCF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• the bFGF concentration is, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more. Also, the bFGF concentration is, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• these components may be provided separately from the above-described basal medium or the medium comprising the basal medium and the above-described medium additives which are optionally chosen components added thereto, and used as added to the medium just before use, or may be provided as a GR cell maintenance and expansion medium in a form already contained in the medium, as far as they do not adversely affect the stability and the like of the growth factors and other components of the medium.
• At least one factor out of the factors used in the maintenance and expansion of GR cells of the present invention can be supplied from feeder cells, rather than added to the medium.
• the feeder cells may be mammalian cells that produce these growth factors by nature, it is more preferable to use recombinant cells that are introduced with genes encoding the growth factors and overexpress the growth factors.
• a plurality of kinds of cells that express different growth factors can be used in combination, it is preferable to use one kind of cells that express all the growth factors to be supplied from the feeders.
• Cells that can be used as the host are the same cells as those described above with respect to the differentiation induction of GR cells.
• the recombinant cells can be produced by the same techniques as those
• the GR cells be cultured in the presence of, in addition to the aforementioned four factors (GDNF, EGF, SCF and bFGF) , one or more medium additives selected from among other growth factors such as HGF and FGF9 and interleukins such as IL-2.
• Each of growth factors such as HGF and FGF9 can be added to the medium at concentrations of, for example, about 0.1 ng/ml or more, preferably about 0.5 ng/ml or more, more preferably about 1 ng/ml or more, particularly preferably about 5 ng/ml or more, and at, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• Interleukins such as IL-2 can be added to the medium at concentrations of, for example, about 0.01 ng/ml or more, preferably about 0.1 ng/ml or more, more preferably about 0.5 ng/ml or more, particularly preferably about 1 ng/ml or more, and at, for example, about 100 ng/ml or less, preferably about 50 ng/ml or less, more preferably about 30 ng/ml or less, particularly preferably about 20 ng/ml or less.
• testosterone and the like and/or a derivative thereof (e.g., forskolin) .
• these components can be added to the medium, as far as they support the maintenance and expansion of the GR cells and do not adversely affect the survival of the cells; the components can be added to the medium at concentrations of, for example, about 0.001 ⁇ or more, preferably 0.01 ⁇ or more, more preferably 0.05 ⁇ or more, and at about 1000 ⁇ or less, preferably about 500 ⁇ or less, more preferably about 100 ⁇ or less.
• one or more factors selected from among the medium additives listed in Table 1 and one or more factors selected from among the medium additives listed in Table 2 are used as added to any one of the above- described basal media.
• Those skilled in the art are able to add these factors at appropriate concentrations to the basal medium; for example, the concentrations (final concentrations) shown in Fig. 27 may be chosen.
• GR cells Maintenance and expansion culture of GR cells can, for example, be performed as described below.
• Any culture vessel for cell culture can be used in this step of cultivation.
• Such culture vessels include, for example, the same culture vessels as those used in the step of GR cell differentiation induction.
• the culture vessel can be non-cell- adhesive (weakly cell-adhesive) or cell-adhesive, depending on the method of cultivation (suspension culture or adhesion culture) .
• a cell-adhesive culture vessel is a culture vessel whose surface is coated with a cell support substrate to improve adhesion of cells (pluripotent stem cells or feeder cells) .
• Such cell support substrata include, for example, collagen, gelatin, Matrigel, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
• the GR cells are seeded to the above-described culture vessel to obtain a cell density of, for example, about 0.5 to 50*10 4 cells/cm 2 , preferably about 1 to 10*10 4 cells/cm 2 , and cultured, for example, in a C0 2 incubator under an atmosphere of about 1% to about 10%, preferably about 2% to about 5%, C0 2 concentration at about 30°C to about 40°C, preferably about 37°C.
• GDNF GDNF
• EGF EGF
• SCF bFGF
• the feeder cells are seeded to the culture vessel to obtain a cell density of, for example, about
• feeder cells include ordinary MEFs [e.g., STO cell line (ATCC CRL-1503) and the like], SNL cells (SNL76/7 STO cells; ECACC 07032801), HDF, dental pulp stem cells and the like treated with radiations or an antibiotic to terminate their cell division.
• STO cell line ATCC CRL-1503
• SNL cells SNL76/7 STO cells
• HDF dental pulp stem cells
• dental pulp stem cells treated with radiations or an antibiotic to terminate their cell division.
• GR cells are provided as a heterogenous cell population containing Oct4-negative or Vasa-negative cells
• homogenous GR cells can be obtained by separating and
• the GR cells can be separated by the same method as that used to separate the GR cells induced from pluripotent stem cells.
• the cell population induced from pluripotent stem cells by the method of GR cell differentiation induction of the present invention contains not only GR cells, but also Oct4- positive Vasa-negative undifferentiated cells.
• the separated undifferentiated cells were cultured under the above-described GR cell maintenance and expansion conditions, they were stably expanded while maintaining the Oct4-positive Vasa-negative differentiated state; therefore, this
• Gsp cells undifferentiated cell line was named Gsp cells.
• the present invention also provides Gsp cells, which are novel pluripotent stem cells induced in the process of GR cell differentiation induction, and are stably
• the Gsp cells like the mGS cells, which have been discovered in the course of establishing the GS cells, are expected to find a broad range of applications as a substitute for known pluripotent stem cells such as ES cells and iPS cells.
• the present invention also provides an expansion support agent for Oct4- positive Vasa-positive germline stem cells (GR cells) ,
• Gsp cells Oct4-positive Vasa-negative pluripotent stem cells
• expansion support agent also serves as an expansion support agent for Gsp cells.
• Each of the factor GDNF, EGF, SCF, and bFGF may be provided in solution in water or an appropriate buffer solution, and may be provided as a lyophilized powder and dissolved in an appropriate solvent just before use.
• These components may stand as separate reagents to form a kit, and may be provided as a single reagent comprising a blend of two or more, as far as they do not adversely affect each other.
• the GR cell expansion support agent of the present invention can further comprise physiologically acceptable carriers, excipients, antiseptics, stabilizers, binders, solubilizers, nonionic surfactants, buffering agents,
• the GR cell expansion support agent of the present invention is combined with one or more, preferably two or more, factors, more preferably all the three factors, selected from among HGF, IL-2 and FGF9, in addition to the four factors GDNF, EGF, SCF and bFGF.
• factors preferably all the three factors, selected from among HGF, IL-2 and FGF9, in addition to the four factors GDNF, EGF, SCF and bFGF.
• reagents to form a kit may be provided as a single
• reagent comprising a blend of two or more, as far as they do not adversely affect each other.
• the present invention also provides a GR cell maintenance and expansion medium comprising any one of the above-described basal media or a medium prepared by supplementing the basal medium with the above-described medium additives which are optionally chosen components, and the above-described GR cell expansion support agent of the present invention added thereto.
• the maintenance and expansion medium may be provided as
• expansion support agent may be provided as a separate reagent as a component of a kit to be added to the medium just before use .
• the present invention still also provides a method of allowing an infertile animal to form sperms by transplanting GR cells obtained by the above-described method of
• the subject animal is preferably a human.
• subject animals include mice, rats, guinea pigs, hamsters, gerbils, rabbits, dogs, monkeys and the like that have traditionally been in common use as laboratory animals.
• farm animals e.g., bovines, horses, pigs, sheep, goat and the like
• companion animals e.g., dogs, cats and the like
• Transplantation of GR cells to the testis can be achieved by, for example, using the GR cells of the present invention in place of GS cells, by the methods described in WO
• invention is used for infertility treatment, it is
• the GR cells to be transplanted be induced from the recipient animal individual.
• the germline stem cells To produce sperms derived from a patient, the germline stem cells
• iPS cells are faulty in that the establishment efficiency is low, when they are intended to be generated by protein transfer or virus-free gene transfer in view of safety concerns. For this reason, provided that the patient's own spermatogonial stem cells are collectable, it is sometimes advantageous to use GS cells induced from spermatogonial stem cells. However, if it is impossible to collect spermatogonial stem cells, or if
• GR cells prepared by a method of the present invention from iPS cells that are readily inducible from skin cells and the like. Therefore, when the method of
• spermiogenesis of the present invention is used for
• preferred recipients of the GR cells include infertile animals from which spermatogonial stem cells are difficult to collect.
• GR cells differentiation- induced from iPS cells or mGS cells induced from a hereditary disease patient cannot be used as they are for transplantation. For this reason, it is necessary to genetically manipulate pluripotent stem cells induced from the patient or GR cells differentiation-induced from the pluripotent stem cells by a method of the present invention to replace the mutant gene with a normal gene, or by other treatment, and to transplant the resulting GR cells to the patient.
• sperms derived from the patient, but having the normal gene function, it is possible to prevent the patient's mutant gene from descending to .his or her posterity to avoid the development of the, hereditary disease in the offspring.
• testis of another individual to allow the individual to form sperms derived from the pluripotent stem cells, in the event that the excellent ancestry ceases.
• the excellent ancestry could be maintained by reproduction using the individual.
• the present invention also provides a therapeutic agent for male infertility comprising GR cells obtained by the above-described method of differentiation induction or GR cells maintained by the above-described method of maintenance and expansion.
• the GR cells of the present invention are produced as a parenteral preparation such as an injection, suspension, drip infusion and the like by being blended with a pharmaceutically acceptable carrier by a conventional means or otherwise.
• Pharmaceutically acceptable carriers that can be contained in the parenteral preparation include, for example, aqueous solutions for injection, such as physiological saline and isotonic solutions containing glucose or another auxiliary drug (e.g.; D-sorbitol, D-mannitol, sodium chloride and the like) .
• the agent of the present invention may be formulated with, for example, a buffering agent (e.g., phosphate buffer solution, sodium acetate buffer solution) , a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride and the like), a stabilizer (e.g., human serum albumin, polyethylene glycol and the like) , a preservative, an antioxidant and the like.
• a buffering agent e.g., phosphate buffer solution, sodium acetate buffer solution
• a soothing agent e.g., benzalkonium chloride, procaine hydrochloride and the like
• a stabilizer e.g., human
• GR cells are suspended in the above- described aqueous liquid to obtain a cell density of about
• the therapeutic agent for male infertility of the present invention may be provided in a state preserved under freezing conditions in common use for freezing preservation of stem cells, and thawed just before use.
• serum or a substitute therefor an organic solvent (e.g., DMSO) and the like may further be contained.
• the concentration of serum or substitute therefor is not particularly limited, and 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, and can be 0% to about 50% (v/v) , preferably about 5% to about 20% (v/v) .
• the preparation thus obtained is stable and less toxic, it can be safely administered to mammals such as humans.
• the method of administration is not particularly limited, the preparation is preferably, administered by
• the therapeutic agent for infertility of the present invention can be administered, for example, at about 1.0*10 5 to about 1*10 7 cells per dose based on GR cells, once or multiple times (e.g., 2 to 10 times) at intervals of about 1 to 2 weeks, for a human infertility patient.
• Example 1 Establishment of iPS cells
• the Oct3/4 (Oct4) and mouse vasa genes are known to be expressed specifically in undifferentiated cells and germ cell lineages, respectively.
• This mouse line was created by spontaneous mating of a Tg mouse generated by micro-in ecting a reporter plasmid DNA comprising the expression control region of the mouse Oct4 gene and the GFP gene ligated thereto into a
• the retroviruses used for the reprogramming were prepared by separately introducing retroviral expression vectors (pMXs ⁇ Oct3/4, pMXs-Sox2, pMXs-Klf4, pMXs-Nanog) to Plat-E cells
• each expression vector was added, and the medium was allowed to stand at room temperature for 15 minutes, after which they were added to a Plat-E cell culture broth.
• the Plat- E cell culture supernatant was replaced with a fresh supply of the medium.
• the culture supernatant was recovered and filtered through a 0.45 um sterile filter (Whatman), polybrene (Nacalai Tesque) was added to obtain a final
• Fibroblasts were isolated from a fetus (13.5 days after fertilization) of the above-described Oct4-GFP/Mvh-RFP Tg mouse. These MEFs were seeded to 6-well culture plates (Falcon) coated with 0.1% gelatin (Sigma) at 1 x 10 5 cells per well. The culture broth used was DMEM/10% FCS, and the fibroblasts were cultured at 37°C, 5% C0 2 . The following day, each retrovirus liquid was added to cause overnight infection for gene transfer of the four genes Oct3/4, Sox2, Klf4 and Nanog, or the three genes Oct3/4, Sox2 and Klf4.
• the cells were cultured using an ES cell culture medium supplemented with LIF [DMEM (Nacalai Tesque) supplemented with 15% fetal bovine serum, 2 mM L-glutamine (Invitrogen), 100 ⁇ non-essential amino acids (Invitrogen), 100 ⁇ 2-mercaptoethanol (Invitrogen), 50 U/mL penicillin (Invitrogen) and 50 ⁇ g/mL streptomycin (Invitrogen)].
• LIF fetal bovine serum
• Invitrogen 100 ⁇ non-essential amino acids
• Invitrogen 100 ⁇ 2-mercaptoethanol
• 50 U/mL penicillin Invitrogen
• streptomycin Invitrogen
• iPS clone was examined for the expression of undifferentiation markers by RT-PCR analysis using the Rever Tra Ace kit (Takara) . The results are shown in Fig. 3. Every iPS clone exhibited the expression of undifferentiation markers at levels comparable to those in ES cells and Nanog- iPS [Nature, 448, 313-317 (2007)].
• a teratoma was formed according to the method described in Cell, 126, 663-676 (2006) . Specifically, 1 x 10 6 iPS cells were subcutaneously injected to an immunodeficient mouse; 4 weeks later, a teratoma was isolated. The teratoma was shredded, and fixed in PBS(-) containing 4% formaldehyde. Paraffin-embedded tissue was sliced and stained with
• the tumor was composed of a plurality of kinds of cells, with neural tissue, epidermal tissue, muscular tissue, cartilage tissue, adipose tissue, and gut-like
• epithelial tissue noted, demonstrating the multipotency of the established iPS cells.
• Example 2 Establishment of differentiation maintenance medium for germline stem cells
• EBs embryoids
• DME /10% FCS 6-well weakly cell-adhesive culture plates
• M15- BMP4 cells which were used as differentiation support cells in PNAS, 100, 11457-11462 (2003) , in order to establish culturing conditions for inducing the differentiation of iPS cells into germ cells.
• GDNF glial cell-derived neurotrophic factor
• mSCF membrane- binding stem cell factor
• EGF epithelial cell growth factor
• RT-PCR confirmed that the expression of the transferred GDNF, mSCF, and EGF, in addition to BMP4,
• composition is shown in Fig. 27.
• Neurobasal medium Invitrogen
• lx B-27 Supplement Invitrogen
• 1 x Penicillin- Streptomycin-Glutamine Invitrogen
• 5 mg/mL Bovine Albumin MP Biomedicals
• StemPro Supplement (Invitrogen) were added to the above- described culture broth (final concentrations are shown; see the "Supplement" panel in Fig. 27) .
• iPS cells and M15-4GF cells were mixed in the same culture broth as the above, but not supplemented with growth factors (Supplement) , to obtain a cell density of 5*10 5 cells per mL for each cell type, and seeded to 6-well weakly cell-adhesive culture plates. With medium exchanges every two days, suspension culture was performed; differentiation of Mvh-RFP-positive cells in cell masses was confirmed.
• EK cells which represent an Oct4-GFP-negative / Mvh-RFP-positive germ celllike cell line that had been differentiation-induced and
• Undifferentiated iPS cells or GR cells were seeded onto mitomycin C-treated MEF feeder cells that had been seeded to
• tumor cells or undifferentiated iPS cells are subcutaneously transplanted to an immunodeficient nu/nu mouse, a tumor is formed at the transplantation site. Meanwhile, it is known that tumorigenesis is not noted in the
• GS cells which represent a line of spermatogonial stem cells.
• GR cells and Gsp cells were transplanted to immunodeficient mice, which were then examined for tumorigenesis . As a result, it was found that when Gsp cells were transplanted, they cancerated,
• the iPS cells used for the differentiation induction had the exogenous genes transferred thereto by means of a retrovirus, some of the cells obtained might have been immortalized due to reactivation of the retrovirus.
• GR cells were found to be Oct4-GFP-positive, and to undergo suppression at the protein level, despite confirmation by RT-PCR of the occurrence of the transcription of the endogenous Oct4 gene. Also observed in the GR cells was increased expression of the proteins of Nanog, ECAT1, vh, and Dnmt3L.
• the DNA methylation status was analyzed by bisulphite genomic sequencing.
• the results are shown in Fig. 24.
• EK cells the methylation control regions of the female imprinted genes Igf2r, SNRPN, and Litl are in the demethylated state, whereas the male imprinted gene Hi9 is in a highly methylated state; this demonstrates the acquisition of an imprint status of the male type.
• GR cells the methylation control regions of the female imprinted genes exhibited a somatic cell pattern, demonstrating that the elimination of genomic imprinting, which is to be induced in the fetal genital primordium, has not occurred.
• the GR cells represent a cell line that reflects the characteristics of primordial germ cells in the later stage of migration (just before or after penetration into the fetal genital primordium) .
• GR cells were transplanted to the testis of a W/Wv mouse, which is a model of infertility developed by
• GR cells were suspended in DMEM/10% FCS at 3*10 7 cells/mL, and injected into the
• the cells were maintained in the testis for a long time of 4 months, with no evidence of tumorigenesis, demonstrating the identity of the GR cells as cells destined to become germline cells.
• the finding differed from the normal spermiogenesis profile.
• TUNEL staining demonstrated the induction of apoptosis-based cell death in the lumens where mature spermatids after meiosis are normally present.
• Example 4 Differentiation induction from ES cells to Oct4- GFP-positive / Mvh-RFP-positive cells
• the methods of GR cell differentiation induction and maintenance and expansion of the present invention make it possible to induce germline stem cells capable of
• spermiogenesis from iPS cells that can easily be generated from somatic cells such as skin cells, and to maintain and expand the same induced, and are therefore particularly useful in infertility treatment and gene therapy for patients with non-obstructive azoospermia, a condition that poses the problem of difficulty and/or risk in collecting spermatogonial stem cells.
• the GR cells generated according to the present invention are of high utility for the purpose of conserving animal varieties with favorable characters in the field of breeding.

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