WO2018116511A1 - Method for producing pluripotent stem cells - Google Patents

Method for producing pluripotent stem cells Download PDF

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WO2018116511A1
WO2018116511A1 PCT/JP2017/026565 JP2017026565W WO2018116511A1 WO 2018116511 A1 WO2018116511 A1 WO 2018116511A1 JP 2017026565 W JP2017026565 W JP 2017026565W WO 2018116511 A1 WO2018116511 A1 WO 2018116511A1
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cells
pis
cell
pluripotent stem
dis
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尚史 塩見
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学校法人神戸女学院
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

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  • the present invention relates to a method for producing pluripotent stem cells.
  • Embryonic stem cells are cells that are made from an inner cell mass at the blastocyst stage and are pluripotent stem cells that can differentiate into various cells.
  • James Thomson of the University of California succeeded in producing human ES cells, and it has been expected to differentiate ES cells into various cells for use in regenerative medicine.
  • human ES cells it is necessary to use human fertilized eggs, and there are problems in bioethics for use in medicine. Therefore, at present, the use of ES cells for regenerative medicine has been postponed.
  • iPS cells pluripotent stem cells having functions equivalent to those of ES cells from somatic cells derived from mammary glands, and then succeeded in producing human iPS cells.
  • Non-Patent Document 1 Cell, 30, 861-872, 2007.
  • Human iPS cells can be obtained by introducing four genes (SOX2, OCT3 / 4, c-Myc, Klf-4 genes) into somatic cells by gene recombination and expressing them.
  • iPS cells have the same properties as ES cells and can be differentiated into various cells and organs, and since human embryos are not used, they are expected to be used for regenerative medicine.
  • Patent Document 1 JP 2008-283972 A
  • Patent Document 2 JP 2009-165480 A
  • Patent Document 3 JP-A-2014-000083
  • Patent Document 4 International Publication No. 2013/163296.
  • one of the problems with iPS cells is that the conversion rate from somatic cells to iPS cells is still low and it takes time to obtain them.
  • HLA antigens distinguish themselves from others by the HLA antigen presented on the cell surface.
  • iPS cells In order to use cell tissues and organs prepared with iPS cells for regenerative medicine, it is necessary to match the types of these HLA antigens.
  • the types of HLA antigens are very diverse, and the probability that the types of HLA antigens match is only one per thousand to 10,000.
  • the cell self and others are distinguished from those other than the HLA antigen, even if regenerative medicine is performed using iPS cells having the same HLA antigen type, the patient has an autoimmune reaction, and an immunosuppressive agent. Should continue to be administered. This imposes a heavy burden on the patient.
  • pluripotent stem cells can be produced from the patient's own cells. Feasibility is greatly enhanced. There are currently only two reports on methods for producing such cells.
  • STAP cells Non-patent Document 2: Nature, 505, 641-647, 2014, Patent Document 4.
  • STAP cells which are equivalent to iPS cells, can be produced by using black tea ingredients.
  • many verifications were made after that, and it was highly possible that STAP cells used ES cells themselves, and it was proved that the paper itself that showed the method for producing STAP cells was forgery or error.
  • Non-patent Document 3 Biochemical and Biophysical Research Communications, 472, 589-591, 2016
  • the headline that “STAP cells could be produced” was reported, and some reports reported incorrectly, but the cells obtained in this paper did not express iPS cell markers and were pluripotent stem cells. is not.
  • pluripotent stem cells having the same functions as iPS cells can be produced in a short time and with high efficiency without using genetic recombination, it is also possible to produce pluripotent stem cells using the patient's own cells. And the feasibility of regenerative medicine is greatly enhanced. However, as described above, no method for producing such pluripotent stem cells has been found so far.
  • the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing pluripotent stem cells capable of obtaining pluripotent stem cells having functions equivalent to iPS cells.
  • the present invention is as follows.
  • a method for producing pluripotent stem cells wherein pluripotent stem cells are obtained by culturing animal cells in a medium containing a polyamine or a DNA methylation inhibitor.
  • the polyamine is at least one selected from the group consisting of spermine, spermidine, putrescine, and acetylated products thereof, and two or more polymers of the polyamines. The manufacturing method as described.
  • the DNA methylation inhibitor is selected from siRNA, 5-aza-2′-deoxycytidine, sinefungin, zebralin, ethionine, acetylmethionine, and selenomethionine that inhibits expression of a gene encoding a DNA methylase.
  • the present inventor searched for a substance having a function of reinitializing cells using a large number of substances, and investigated the properties of the obtained cells.
  • polyamine has a function of inducing the expression of genes related to cell reprogramming.
  • polyamines consequently inhibit DNA methylation, and that it reinitializes the cells, and that similar cells can be obtained using DNA methylation inhibitors. I found it.
  • the differentiation induction of the cell by a polyamine or a DNA methylation inhibitor was advanced, it changed to the cell which has the marker protein of an ES cell or iPS cell, and discovered that the said cell turns into a pluripotent stem cell.
  • the pluripotent stem cells can be cultured in a state that retains pluripotency, and redifferentiate into various somatic cells such as endoderm, mesoderm, and ectoderm in a medium that differentiates into embryoid bodies. It has also been confirmed that the present invention has been completed.
  • pluripotent stem cells obtained using polyamine are referred to as PIS cells (Polyamine-Induced Stem cells), and pluripotent stem cells obtained using a DNA methylation inhibitor are referred to as DIS cells ( Sometimes called Demethylation-Induced Stem Cells).
  • the present invention it is possible to provide a method for producing pluripotent stem cells capable of obtaining pluripotent stem cells having functions equivalent to iPS cells.
  • the production method of the present invention is not only a completely new method, but the utility of the present invention is extremely high in that pluripotent stem cells are obtained from animal cells without using genetic recombination.
  • PIS cells and DIS cells have excellent characteristics that they can be obtained in 1 to 3 days, have a very high conversion efficiency into the cells, and can be easily separated. Since iPS cells not only take time and labor, but also have a low probability of obtaining iPS cells, PIS cells and DIS cells are highly inventive compared to the method of producing iPS cells.
  • FIG. 1 shows the state of change from lung-derived fibroblast TIG-1-20 to PIS cells.
  • FIG. 1A shows TIG-1-20 cells before induction with spermine.
  • FIG. 1B shows the changes that occur in TIG-1-20 cells after 6 hours induction with 40 ⁇ M spermine.
  • FIG. 1C shows PIS cells obtained by induction with 40 ⁇ M spermine for 36 hours.
  • FIG. 2 shows the expression level of mRNA measured by real-time PCR. The vertical axis represents the ratio (times) of the amount of mRNA in PIS cells to the amount of TIG-1-20 mRNA.
  • FIG. 3 shows PIS cells that have been subjected to activity staining or immunostaining with Human ES / iPS Cell Characterization Kit.
  • FIG. 4 shows a state in which PIS cells are induced into cardiomyocytes by Cardiomyocyte Differentiation Kit.
  • FIG. 4A shows a state where an EB is formed.
  • FIG. 4B shows how progenitor cardiomyocytes are formed from EB.
  • FIG. 4C shows a state of a cardiomyocyte having fine movement.
  • FIG. 5 shows the differentiation of mouse precursor white adipocyte cell line 3T3-L1 into PIS cells.
  • FIG. 5A shows the appearance of 3T3-L24 cells.
  • FIG. 5B shows a state of the formed EB.
  • FIG. 6 shows how mouse precursor white adipocytes C3H10T1 / 2 strain was differentiated into DIS cells.
  • FIG. 6A shows the state of DIS cells (DIS-SI cells).
  • FIG. 6B shows the state of the DIS cell (DIS-Aza cell).
  • C in FIG. 6 shows the state of early differentiation of blood cells obtained by differentiating DIS-Aza cells.
  • FIG. 6D shows the state of initial differentiation of the obtained neural stem cells and neural-like materials.
  • FIG. 7 shows how PIS cells were differentiated into various germ layers with high efficiency.
  • FIG. 7A shows a state of differentiation into ectoderm.
  • FIG. 7B shows a state of differentiation into mesoderm.
  • FIG. 7C shows a state of differentiation into endoderm.
  • FIG. 7A shows a state of differentiation into ectoderm.
  • FIG. 7B shows a state of differentiation into mesoderm.
  • FIG. 7C shows a state of differentiation into endoderm
  • FIG. 8 shows how PIS cells are differentiated into brown adipocytes.
  • FIG. 8A shows a state in which cells differentiated into brown adipocytes express UCP1.
  • FIG. 8B shows a state where cells differentiated into brown adipocytes accumulate fat.
  • FIG. 9 shows that cells obtained by differentiating mouse adipocytes 3T3-L24 into PIS cells differentiated into neural stem cells and mature neurons with high efficiency.
  • FIG. 9A shows a state of differentiation into neural stem cells.
  • B and C in FIG. 9 show how neural stem cells have differentiated into nerve cells
  • FIG. 9B shows how mature tumor cell marker gene Tubulin III is expressed.
  • FIG. 9C shows a state in which the marker gene MAP2 of mature neurons is expressed.
  • the present invention obtains pluripotent stem cells by adding polyamine or a DNA methylation inhibitor to a medium and culturing animal cells (until they have a similar form to ES cells or iPS cells).
  • a method for producing pluripotent stem cells is obtained.
  • the animal cell may be a normal animal cell that can be grown by culture.
  • the animal includes a human, preferably a mammal.
  • Mammals other than humans are not particularly limited, and examples include mice, rats, rabbits, and goats.
  • animals other than mammals include insects such as Drosophila.
  • animal cells include fibroblasts, epidermal cells, mammary cells, adipocytes, myoblasts, osteoblasts, hepatocytes, vascular endothelial cells, or precursor cells thereof.
  • animal cells include stem cells such as blood cell stem cells, mesenchymal stem cells, and neural stem cells.
  • human cells can be purchased from a storage organization (ATCC, JCRB, etc.) that sells human normal cells, purchased from commercial manufacturers that sell human normal cells, or cells from humans Can be obtained by taking out
  • a storage organization ATCC, JCRB, etc.
  • the tissue is obtained by sampling a part of the tissue using a tissue examination apparatus, partial excision of the tissue by surgery, liposuction, or the like. Thereafter, the cells are separated by an enzyme such as trypsin and cultured in a medium for animal cells.
  • the polyamine used in the present invention means a compound in which two or more primary amino groups or secondary amino groups are bonded to a linear aliphatic hydrocarbon in total.
  • polyamines examples include spermine (CAS registration number 71-44-3), spermidine (CAS registration number 124-20-9), putrescine (CAS registration number 110-60-1), and acetylated forms thereof. As well as two or more polymers of these polyamines.
  • acetylated polyamine examples include N-acetylputrescine, N-acetylspermidine, N-acetylspermine, diacetylputrescine, diacetylspermidine, and diacetylspermine.
  • Two or more polymers of polyamine can be obtained by chemical synthesis.
  • Polyamine is characterized by the presence of amino groups (—NH 2 group or —NH— group) at intervals of 3 to 4 carbon chains, acting on histones or chromosomes and inducing the expression of genes that initialize cells There is. Therefore, among polyamines other than the above (other polyamines existing in the body, other polyamines obtained by chemical synthesis, etc.), compounds having a structure similar to spermine, spermidine, putrescine, etc. are also used in the present invention. Can do. The ability to induce polyamine gene expression is strong in the order of spermine, spermidine, and putrescine, and the longer the length of the linear molecule, the greater the effect of inducing gene expression per molar concentration.
  • amino groups —NH 2 group or —NH— group
  • polyamine Since polyamine is water-soluble, an aqueous polyamine solution having a concentration of about 10 to 100 mM is prepared, sterilized with a 0.2 ⁇ m filter, and then added to the medium so as to have an appropriate concentration. it can.
  • DNA methylation inhibitor examples include the following two types.
  • siRNA that inhibits the expression of a gene encoding DNA methylase (DNMT).
  • the DNA methylase is an enzyme (DNMT1) that accurately transmits DNA methylation information from a parent cell to a daughter cell when replicating a cell, an enzyme (DNMT3) that methylates DNA of a daughter cell, and the like.
  • DNMT1-encoding gene or DNMT3-encoding gene corresponds to the DNMT-encoding gene.
  • siRNA from Qiagen is added according to the manual to methylate DNA. Can be efficiently inhibited.
  • a DNA methylation inhibitor is a methionine analog.
  • Methionine analogs inhibit the methylation reaction by inhibiting the synthesis of adenosylmethionine, which is the reaction substrate for the methylation reaction.
  • methionine analogs examples include 5-aza-2'-deoxycytidine (5-Aza-dc, CAS registration number 23533-33-5), sinefungin (CAS registration number 58944-73-3), and zebralin (CAS registration). No. 360-10-6), ethionine (CAS registration number 13073-35-3), acetylmethionine (CAS registration number 567-82-7), and selenomethionine (CAS registration number 3211-76-5). Since these methionine-like substances are water-soluble, prepare an aqueous solution with a concentration of about 10-100 mM, sterilize the aqueous solution with a 0.2 ⁇ m filter, and add it to the medium to an appropriate concentration. Can be used.
  • PIS cells pluripotent stem cells
  • DIS cells pluripotent stem cells
  • DNA methylation inhibitors DNA methylation inhibitors
  • PIS cells pluripotent stem cells
  • DIS cells pluripotent stem cells
  • DNA methylation inhibitors DNA methylation inhibitors
  • a normal medium used for cell growth to a flask, dish or plate for cell culture, seeding the cells, and culturing in a CO 2 incubator at a normal culture temperature (about 37 ° C.) Allow cells to grow on bottom of flask.
  • a normal medium used for the growth of human cells for example, a minimum essential medium (MEM) or a medium for iPS cells can be used. In the case of a 25 cm 2 culture flask, 100,000 to 100 It is preferable to seed about 10,000 cells.
  • MEM minimum essential medium
  • iPS cells In the case of a 25 cm 2 culture flask, 100,000 to 100 It is preferable to seed about 10,000 cells.
  • This cell is a pluripotent stem cell PIS cell or DIS cell.
  • polyamines or DNA methylation inhibitors should be added at the lowest concentration at which changes to PIS cells or DIS cells occur, so as not to cause apoptosis as much as possible.
  • concentration of polyamine or DNA methylation inhibitor and the culture time depend on the cell type and the number of cells. For this reason, it is desirable to confirm the minimum concentration at which polyamine or DNA methylation inhibitor is added to PIS cells or DIS cells by optical microscope observation by preliminary experiments.
  • these cells can be changed to PIS cells by culturing in a medium supplemented with about 10 ⁇ M to 100 ⁇ M of spermine for 1 to 2 days.
  • a medium to which about 5 to 10 nM of siRNA that inhibits the expression of a gene encoding DNMT, a medium to which about 20 to 80 ⁇ M of 5-Aza-dc is added, or ethionine If the cells are cultured in a medium supplemented with about 1 to 10 mM for 1 to 3 days, these cells can be changed to DIS cells.
  • PIS cells or DIS cells obtained by culturing in a medium containing the polyamine or the siRNA can be cultured in a medium containing the polyamine or the siRNA for about 1 to 4 days.
  • the survival rate gradually decreases due to apoptosis, when the change to PIS cells was confirmed by light microscopy, the culture medium containing suspended PIS cells or DIS cells was collected within 0 to 1 day, and the normal rate PIS cells are preferably collected by centrifugation at 800-2000 rpm for about 3 minutes.
  • an inhibitor for preventing a decrease in the survival rate of PIS cells may be added to the medium during the culture.
  • a Rock inhibitor and an apoptosis inhibitor can be used as an inhibitor for preventing a decrease in survival rate.
  • the Rock inhibitor include Y-27632, thiazobibin, SB431542, PD0325901
  • examples of the apoptosis inhibitor include a p53 inhibitor, a Bax inhibitor, and a caspase inhibitor.
  • PIS cells or DIS cells collected by centrifugation are differentiated from cells at a very high rate, redifferentiation directly into endoderm, ectoderm, and mesoderm via the embryoid body (EB) Can do.
  • EB embryoid body
  • a medium for acquiring and retaining pluripotent traits of PIS cells or DIS cells for example, in the case of human cells, Cellartis (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.), mTeSR1 ( Many commercially available media such as Veritas) can be used.
  • mTeSR1 Many commercially available media such as Veritas
  • PIS cells or DIS cell aggregates When PIS cells or DIS cell aggregates are cultured in these media, they adhere weakly (or float). The cells are suspended by pipetting (an operation in which the culture medium is aspirated and suspended by repeated pipetting), the medium and cells are collected, the cells are collected by centrifugation, and then transferred to a new medium. Since the original somatic cells that have not differentiated into PIS cells or DIS cells adhere firmly, they are not detached by pipetting, and only PIS cell aggregates or DIS cell aggregates can be recovered.
  • the aggregate of PIS cells or DIS cells is made dispersed with Tryp LE Select (Gibco), 0.25% trypsin solution (Gibco), Accutase-Solution (Funakoshi), etc., and Cellaritis (registered trademark) is used.
  • Tryp LE Select Gibco
  • 0.25% trypsin solution Gibco
  • Accutase-Solution Funakoshi
  • Cellaritis registered trademark
  • a PIS cell or a DIS cell has a trait equivalent to that of an iPS cell indicates that expression of genetic markers such as SOX2, Oct3 / 4, c-Myc specific to iPS cells, expression of alkaline phosphatase activity, or SSEA-4 Can be confirmed by examining the expression of surface antigen markers such as TRA1-60, TRA1-81, etc.
  • RT-PCR or real-time PCR can be used to confirm the increase in gene expression, iPS cell-specific enzyme or surface antigen
  • immunostaining using antibodies, Western blotting or FACS may be used.
  • Differentiation induction from PIS cells or DIS cells into various cells can be performed by the same method as the induction method used for iPS cells.
  • PIS cells or DIS cells differentiate into endoderm, mesoderm, ectoderm, etc. through Embryoid body (EB), and then into various somatic cells such as cardiomyocytes, adipocytes, and nerve cells. Differentiate. In the differentiation of cells, the formation efficiency of PIS cells or DIS cells is increased by forming uniform EBs similarly to iPS cells.
  • EB Embryoid body
  • a uniform EB can be obtained by using an embryoid body forming plate Agrowell (Veritas), Lipidure (registered trademark) -coated plate (NOF Corporation), EZSPHERE (AGC Techno Glass), or the like.
  • differentiation into various cells can be performed in exactly the same medium as in the case of iPS cells.
  • reagents that can induce differentiation into various cells There are many commercially available reagents that can induce differentiation into various cells, and their use is also an effective means.
  • differentiation into definitive endoderm (ED) is Cellaritis (registered trademark) Definitive Endoderm ChiPSC18 (Takara Bio Inc.)
  • differentiation into cardiomyocytes and hepatocytes is PSdif-Cardio Cardiomyocyte Differentiation Kit (Funakoshi Co., Ltd.), Cellartis ( (Registered trademark) iPS Cell to Hepatocyte Differentiation System (Takara Bio Inc.)
  • SETMdiff Neural Induced Medium is used to efficiently differentiate cells from PIS cells or DIS cells. Can do.
  • Human lung-derived fibroblasts TIG-1-20 were obtained from the JCRB cell bank (JCRB0501 strain). Human fibroblast TIG-1-20 (number of divisions: 33 times, 100,000) was cultured in a 25 cm 2 culture flask containing 6 mL of minimal medium (MEM) for 1 day to allow the cells to adhere. Those added with 10 mM putrescine, 200 ⁇ M spermidine, or 40 ⁇ M spermine and those not added (control) were cultured at 37 ° C. in a CO 2 incubator for 1 day.
  • MEM minimal medium
  • FIG. 1A The results when culturing with spermine added is shown in FIG.
  • Fine particles appear in the cells of fibroblasts TIG-1-20 (FIG. 1A) after 6 hours (FIG. 1B), and when the culture is continued, the cells peel off from the bottom of the flask and become ES cells. It changed to a close cell (FIG. 1C).
  • the cells (PIS cells) suspended in the medium are collected by centrifugation (800 rpm, 3 minutes), and Cellartis (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.) is collected. ).
  • a PBS (+) diluted solution of DEF-CS COAT-1 in a 24-well plate was treated for 1 hour, and then PIS cells were suspended in DEF-CS Basal Medium to which 6 mL of a medium additive was added, and 2 mL each. The solution was dispensed into 3 wells and cultured at 37 ° C. for 2 days. The cells were detached by pipetting, and PIS cells were collected by centrifugation (800 rpm, 3 minutes).
  • the obtained cells were extracted with mRNA and synthesized cDNA using RNeasy Lipid Tissue Mini Kit and QuantiTech Reverse Transcription Kit (Qiagen), and using real-time PCR primers and real-time PCR (Qiagen), The expression levels of TERT, DMNT1, SOX2, and OCT3 / 4 mRNA were examined.
  • the results of real-time PCR are shown in FIG.
  • the DNMT1 gene is a gene encoding a methyltransferase that plays a role in transmitting methylation information of parent cell DNA to daughter cells during DNA replication.
  • FIG. 2 in PIS cells, it was found that DNMT1 expression was completely suppressed, and initialization of DNA methylation occurred.
  • the expression of a telomerase-encoding gene (TERT gene) that extends telomeres was significantly increased. In other words, telomere elongation (initialization) was shortened due to aging.
  • SOX2 and OCT3 / 4 genes which are ES cell marker genes and induce cell reprogramming, increased. As described above, it was demonstrated that polyamine induces cell reprogramming.
  • PIS cells were cultured using Cellaris (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.) in exactly the same manner as used in Example 1 to obtain PIS cells with stable characteristics. After weakly adhering PIS cells in the medium were suspended by pipetting, the cells were collected by centrifugation at 1,500 rpm for 4 minutes.
  • the obtained PIS cells were confirmed for alkaline phosphatase activity and three surface antigens (SSEA-4, TRA-1-60, TRA-1-81) by Human ES / iPS Cell Characterization Kit (Applied Stem Cell). went.
  • IPS cells are also characterized by expressing alkaline phosphatase.
  • SSEA-4, TRA-1-60, and TRA-1-81 are iPS cell marker genes and are proteins that are specifically expressed in iPS cells.
  • PIS cells were placed in a 0.2 mL sample tube, fixed, permeabilized, and blocked in a suspended cell state, and then the primary antibody and the secondary antibody were bound.
  • the cells labeled with the secondary antibody were mixed with the mount solution 1: 1 to prepare a slide glass, and the fluorescence was confirmed with a fluorescence microscope (EVOS, EVOS FL Auto). The results are shown in FIG.
  • FIG. 3 is a diagram in which chromosomes are fluorescently stained, and is a diagram of activity staining or immunostaining with a fluorescent substance. If there is fluorescence, it is shown in gray in the picture of FIG. As is clear from FIG. 3, strong alkaline phosphatase activity and strong expression of SSEA-4, TRA-1-60 and TRA-1-81 antigens were confirmed. As described above, it was proved that PIS cells expressed a protein specific to iPS cells.
  • Mouse preadipocyte cell line 3T3-L1 (FIG. 5A) was obtained from EDCC.
  • the 3T3-L1 strain was cultured in a 25 cm 2 culture flask containing 6 mL of DMEM (Dulbecco's modified Eagle Medium) for 1 day to allow the cells to adhere.
  • the one supplemented with 1 mM spermine was cultured at 37 ° C. for 3 days in a CO 2 incubator.
  • ES cell-like cells (PIS-L1 cells) could be obtained in the same manner as in Example 1, and the PIS-L1 cells formed EBs as shown in FIG. 5B.
  • the method for producing PIS cells can be applied not only to human fibroblasts but also to various cells of various species such as mouse adipocytes.
  • Example 5 Demonstration that a DNA methylation inhibitor has the ability to reprogram cells and changes to DIS cells
  • SiRNAs (catalog numbers SI000189910, SI00982338 and SI00165382) that inhibit the expression of genes encoding Qiagen DNA methylases (DNMT1, DNMT3A and DNMT3B) were obtained.
  • mouse adipocyte-derived strain C3H10T1 / 2 (EC90110523-F0) was obtained from ECACC.
  • the cells were cultured in DMEM for 1 day using a 24-well plate and the cells were allowed to attach to the bottom of each well of the plate.
  • Gene silencing by siRNA was performed using RNAi Human / Mouse Starter Kit (Qiagen).
  • the mouse adipocyte-derived strain C3H10T1 / 2 was changed to a DIS cell (DIS-SI cell), but the mouse adipocyte-derived strain C3H10T1 / 2 that was not changed was attached to the cell bottom surface. As such, only DIS-SI cells could be recovered from the medium.
  • DIS-Aza cells were subjected to initial differentiation using ES-Cut Hematopoietic Differentiation Kit with Cytokine (Veritas, catalog number ST-03160), which is a differentiation medium from mouse ES cells to hematopoietic cells. .
  • Cytokine (Veritas, catalog number ST-03160)
  • initial differentiation into hematopoietic cells was confirmed as shown in FIG.
  • DIS-Aza cells were differentiated into neurons using a differentiation medium for neural progenitor cells (STEMdiff Neural Induction Medium, Veritas).
  • FIG. 6D DIS-Aza cells differentiated into neural stem cells and nerve-like cells.
  • a DNA methylation inhibitor it has the ability to initialize cells to pluripotent stem cells, as in the case of polyamines, and it is possible to produce DIS cells that differentiate into various cells.
  • somatic cells can be converted into PIS cells with high efficiency
  • the obtained PIS cells can be differentiated into ectoderm, mesoderm or endoderm with high efficiency
  • Human lung-derived fibroblasts TIG-1-20 (number of divisions 30-35) were cultured in a 25 cm 2 culture flask for 1 day using 6 mL of minimal medium (MEM) and attached to the bottom of the flask I let you.
  • MEM minimal medium
  • 20 ⁇ M Y-27632 and 5 ⁇ M thiazobibin, which are a Rock inhibitor, and 60 ⁇ M p53 inhibitor (Cyclic pifthrin- ⁇ -hydrobide) and 30 ⁇ M Bax inhibitor (Bax Inhibitor Peptide Peptide) are apoptosis inhibitors. was added and cultured for 8 hours, and these four types of inhibitors were sufficiently taken into the cells.
  • the PIS cells were then differentiated into ectoderm, mesoderm or endoderm using Stem XVivo Ectoderm Kit, Stem XVivo Mesoderm Kit, Stem XVivo Enderdom Kit (R & D Systems).
  • the differentiated cells were obtained by using anti-human Oct2 goat antibody, anti-human Brachyury goat antibody and anti-human SOX17 goat antibody included in these kits as primary antibodies, and anti-goat IgG rabbits bound with Alexa Fluor (registered trademark) 555. Immunofluorescence staining was performed using the antibody as a secondary antibody. As a result, as shown in FIGS. 7A to 7C, it was confirmed that most cells were efficiently (90% or more) and differentiated into ectoderm, mesoderm or endoderm.
  • PIS cells were induced into mesoderm and differentiated into brown adipocytes using PS-dif BA Brown Adiposite Difference Kit (Veritas). The obtained cells were immunostained using an anti-UCP1 rabbit antibody (Biosis: bs192R). Moreover, 0.1 mM oleic acid was added and cultured. As a result, the differentiated cells were not only brown, but also expressed UCP1 as shown in FIG. 8A, and had the property of accumulating fat as shown in FIG. 8B. From this, it was confirmed that PIS cells were differentiated into brown adipocytes. Moreover, the differentiation efficiency of the surviving cells into brown adipocytes was 90% or more, and it was confirmed that differentiation was possible with very high efficiency.
  • Example 7 Confirmation that PIS cells can be produced from adipocytes and can be differentiated with high efficiency
  • Mouse adipocyte 3T3-L24 (N. Shiomi et al. (2011) JBiSE 4, 684) was cultured in a 25 cm 2 culture flask for 1 day using 6 mL of DMEM and attached to the bottom of the flask.
  • a Rock inhibitor 20 ⁇ M Y-27632 and 5 ⁇ M thiazobibin, and an apoptosis inhibitor, 60 ⁇ M p53 inhibitor (Cyclic pifthrin- ⁇ -hydrobide) and 30 ⁇ M Bax inhibitor (Bax Inhibitor Peptide)
  • 60 ⁇ M p53 inhibitor Cyclic pifthrin- ⁇ -hydrobide
  • Bax inhibitor Bax Inhibitor Peptide
  • StemSure (registered trademark) 0.1 w ⁇ v% gelatin solution (Wako Pure Chemical Industries, Ltd.) coated in each well of a 24-well culture plate, neurodifferentiation medium NDiff (registered trademark) 227 (Takara Bio Inc.) 2 mL of company: Y40002) was added, and PIS cells were cultured for 3 days to induce neural stem cells. Furthermore, these cells were differentiated into nerve cells and glial cells using Neurocult (registered trademark) Differentiation Medium (Veritas).
  • the obtained nervous system cells were immunostained using an anti- ⁇ -tubulin III rabbit antibody and a MAP2 rabbit antibody as a primary antibody, and a Cy3-conjugated anti-rabbit goat antibody as a secondary antibody.
  • FIG. 9A As shown in FIG. 9A, almost 100% of the cells differentiated into neural stem cells.
  • FIGS. 9B and 9C it was confirmed that almost 100% of the cells further differentiated from the neural stem cells were mature neurons.
  • the method for producing pluripotent stem cells of the present invention is much simpler, faster and more efficient than iPS production. Therefore, it can be used for various treatments in the field of regenerative medicine, and its industrial utility value is extremely high.

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Abstract

Provided is a method for producing pluripotent stem cells whereby pluripotent stem cells having a function comparable to iPS cells can be obtained. The method for producing pluripotent stem cells comprises culturing animal cells in a medium containing a polyamine or a DNA methylation inhibitor and thus acquiring the pluripotent stem cells.

Description

多能性幹細胞の製造方法Method for producing pluripotent stem cells
　本発明は、多能性幹細胞の製造方法に関する。 The present invention relates to a method for producing pluripotent stem cells.
　胚性幹細胞（ＥＳ細胞）は、胚盤胞期の内部細胞塊から作られる細胞であり、様々な細胞に分化できる多能性幹細胞である。１９９８年にカリフォルニア大学のジェームズ・トムソンらが、ヒトのＥＳ細胞の作製に成功したことをきっかけに、ＥＳ細胞を様々な細胞に分化させ、再生医療に利用することが期待されてきた。しかし、ヒトのＥＳ細胞を作製するにはヒトの受精卵を用いる必要があり、医療への利用は生命倫理上問題がある。そのため、現在ではＥＳ細胞を再生医療に利用することは見送られている。 Embryonic stem cells (ES cells) are cells that are made from an inner cell mass at the blastocyst stage and are pluripotent stem cells that can differentiate into various cells. In 1998, James Thomson of the University of California succeeded in producing human ES cells, and it has been expected to differentiate ES cells into various cells for use in regenerative medicine. However, in order to produce human ES cells, it is necessary to use human fertilized eggs, and there are problems in bioethics for use in medicine. Therefore, at present, the use of ES cells for regenerative medicine has been postponed.
　これに対して、山中らは乳腺由来の体細胞から、ＥＳ細胞と同等の機能を有する多能性幹細胞（ｉＰＳ細胞）の作製にマウスで成功し、その後、ヒトのｉＰＳ細胞の作製にも成功している（非特許文献１：Cell、３０巻、８６１頁－８７２頁、２００７年）。ヒトのｉＰＳ細胞は、４つの遺伝子（ＳＯＸ２、ＯＣＴ３／４、ｃ－Ｍｙｃ、Ｋｌｆ－４遺伝子）を、遺伝子組み換えにより同時に体細胞に導入して発現させることにより得られる。ｉＰＳ細胞はＥＳ細胞と同様の性質を有して、様々な細胞や臓器に分化できる上に、ヒト胚を使用しないことから、再生医療への利用が期待されている。しかし、ｉＰＳ細胞は、遺伝子操作により得るため、体細胞あたりのｉＰＳ細胞の変換率は非常に低い。そのため、現在までｉＰＳ細胞の作製方法と選別方法に様々な改良が加えられてきた（特許文献１：特開２００８－２８３９７２号公報、特許文献２：特開２００９－１６５４８０号公報、特許文献３：特開２０１４－００００８３号公報、特許文献４：国際公開第２０１３／１６３２９６号）。しかしながら、依然として体細胞からｉＰＳ細胞への変換率は低く、取得までに時間がかかることがｉＰＳ細胞の問題点の１つである。 In contrast, Yamanaka et al. Succeeded in producing pluripotent stem cells (iPS cells) having functions equivalent to those of ES cells from somatic cells derived from mammary glands, and then succeeded in producing human iPS cells. (Non-Patent Document 1: Cell, 30, 861-872, 2007). Human iPS cells can be obtained by introducing four genes (SOX2, OCT3 / 4, c-Myc, Klf-4 genes) into somatic cells by gene recombination and expressing them. iPS cells have the same properties as ES cells and can be differentiated into various cells and organs, and since human embryos are not used, they are expected to be used for regenerative medicine. However, since iPS cells are obtained by genetic manipulation, the conversion rate of iPS cells per somatic cell is very low. For this reason, various improvements have been made to the iPS cell production and selection methods until now (Patent Document 1: JP 2008-283972 A, Patent Document 2: JP 2009-165480 A, Patent Document 3: JP-A-2014-000083, Patent Document 4: International Publication No. 2013/163296). However, one of the problems with iPS cells is that the conversion rate from somatic cells to iPS cells is still low and it takes time to obtain them.
　さらに、細胞は細胞表面に提示したＨＬＡ抗原により、自己と他人を区別している。ｉＰＳ細胞で作製した細胞組織や臓器を再生医療に利用するためには、このＨＬＡ抗原の型を一致させる必要がある。言い換えれば、ｉＰＳ細胞による再生医療では、すべてのＨＬＡ型を有するｉＰＳ細胞群を準備し、それをすべての細胞や臓器に分化したものを前もって準備しておく必要がある。しかし、ＨＬＡ抗原の型は非常に多岐にわたっており、ＨＬＡ抗原の型が一致する確率は千人から１万人に一人にすぎない。ＨＬＡ型を有するｉＰＳ細胞群とそこから分化した細胞群を準備することは不可能ではないが、現実的には極めて困難である。さらに、細胞の自己と他人の区別は、ＨＬＡ抗原以外でも行われているため、ＨＬＡ抗原型の一致したｉＰＳ細胞により、再生医療を行ったとしても、患者は自己免疫反応が生じ、免疫抑制剤を投与し続ける必要がある。このことは、患者にとって大きい負担を強いることになる。 Furthermore, cells distinguish themselves from others by the HLA antigen presented on the cell surface. In order to use cell tissues and organs prepared with iPS cells for regenerative medicine, it is necessary to match the types of these HLA antigens. In other words, in regenerative medicine using iPS cells, it is necessary to prepare a group of iPS cells having all HLA types, and to prepare those differentiated into all cells and organs in advance. However, the types of HLA antigens are very diverse, and the probability that the types of HLA antigens match is only one per thousand to 10,000. Although it is not impossible to prepare an iPS cell group having an HLA type and a cell group differentiated therefrom, it is actually extremely difficult. Furthermore, since the cell self and others are distinguished from those other than the HLA antigen, even if regenerative medicine is performed using iPS cells having the same HLA antigen type, the patient has an autoimmune reaction, and an immunosuppressive agent. Should continue to be administered. This imposes a heavy burden on the patient.
　上記の２つの問題は、「患者から直接細胞を取り出し、それを再生医療に使う」ことにより解決できる。しかし、ｉＰＳ細胞では、（１）４つの遺伝子を遺伝子操作により導入し選別するため、作製の時間と手間がかかる、（２）ｉＰＳ細胞を得る確率は低く再生医療に十分な細胞を確保するまでに時間がかかる、（３）遺伝子を導入するために得られたｉＰＳ細胞の安全確認に時間がかかる、（４）ひとり当たりに膨大な労力が必要になり高額の医療費が発生するなどの理由により、患者自身の細胞を再生医療に利用することは極めて困難である。 The above two problems can be solved by “removing cells directly from a patient and using them for regenerative medicine”. However, in iPS cells, (1) it takes four hours to introduce and select four genes by genetic manipulation, which takes time and labor for preparation. (2) The probability of obtaining iPS cells is low, and sufficient cells for regenerative medicine are secured. (3) It takes time to confirm the safety of iPS cells obtained to introduce a gene, (4) A huge amount of labor is required per person, and high medical costs are incurred. Therefore, it is extremely difficult to use the patient's own cells for regenerative medicine.
　一方、遺伝子組換えを用いることなく、短時間、高効率で多能性幹細胞を作製する方法を開発できれば、患者自身の細胞から、多能性幹細胞を作製することが可能になり、再生医療の実現性は非常に高まる。このような細胞の作製法に関する報告は、現在２つしか報告されていない。 On the other hand, if a method for producing pluripotent stem cells with high efficiency can be developed in a short time without using genetic recombination, pluripotent stem cells can be produced from the patient's own cells. Feasibility is greatly enhanced. There are currently only two reports on methods for producing such cells.
　その１つは、ＳＴＡＰ細胞である（非特許文献２：Nature、５０５巻、６４１頁－６４７頁、２０１４年、特許文献４）。理化学研究所の小保方らのグループは、紅茶の成分を使うことでｉＰＳ細胞と同等の細胞であるＳＴＡＰ細胞を作製できることを発見した。しかし、その後に数多くの検証がなされ、ＳＴＡＰ細胞は、ＥＳ細胞自身を用いた可能性が高く、ＳＴＡＰ細胞の作製方法を提示した論文そのものが、捏造または誤りであることが立証された。 One of them is STAP cells (Non-patent Document 2: Nature, 505, 641-647, 2014, Patent Document 4). The group of Kobokata et al. Of RIKEN discovered that STAP cells, which are equivalent to iPS cells, can be produced by using black tea ingredients. However, many verifications were made after that, and it was highly possible that STAP cells used ES cells themselves, and it was proved that the paper itself that showed the method for producing STAP cells was forgery or error.
　もう１つは、２０１６年にＫｉｍらにより報告された研究（非特許文献３：Biochemical　and　Biophysical　Research　Communications、４７２巻、５８９頁－５９１頁、２０１６年）であり、これが最新の研究である。彼らは癌細胞に刺激を与え、生き残った細胞がｉＰＳ細胞に近い細胞であることを発見した。この論文は、「ＳＴＡＰ細胞を作製できた」という見出しで、一部の報道が誤って報道したが、当該論文で得られた細胞は、ｉＰＳ細胞マーカーを発現しておらず、多能性幹細胞ではない。論文の著者自身の結論も「多能性幹細胞は得られなかった」と結論している。 The other is a study reported by Kim et al. In 2016 (Non-patent Document 3: Biochemical and Biophysical Research Communications, 472, 589-591, 2016), which is the latest research. They stimulated cancer cells and found that the surviving cells were close to iPS cells. In this paper, the headline that “STAP cells could be produced” was reported, and some reports reported incorrectly, but the cells obtained in this paper did not express iPS cell markers and were pluripotent stem cells. is not. The author's own conclusion also concluded that “pluripotent stem cells could not be obtained”.
　すなわち、２０１６年現在においても、化合物で刺激することにより多能性幹細胞を誘導する方法は知られていない。 That is, as of 2016, there is no known method for inducing pluripotent stem cells by stimulation with a compound.
特開２００８－２８３９７２号公報JP 2008-283972 A 特開２００９－１６５４８０号公報JP 2009-165480 A 特開２０１４－００００８３号公報JP 2014-000083 A 国際公開第２０１３／１６３２９６号International Publication No. 2013/163296
　遺伝子組換えを用いることなく、短時間、高効率でｉＰＳ細胞と同等の機能を有する多能性幹細胞を製造できれば、患者自身の細胞を使って、多能性幹細胞を製造することも可能であり、再生医療の実現性は非常に高まる。しかしながら、上述のとおり、そのような多能性幹細胞の製造方法は、これまで全く見出されていない。 If pluripotent stem cells having the same functions as iPS cells can be produced in a short time and with high efficiency without using genetic recombination, it is also possible to produce pluripotent stem cells using the patient's own cells. And the feasibility of regenerative medicine is greatly enhanced. However, as described above, no method for producing such pluripotent stem cells has been found so far.
　本発明は、上記の課題に鑑みてなされたものであり、ｉＰＳ細胞と同等の機能を有する多能性幹細胞を得ることのできる、多能性幹細胞の製造方法を提供することを目的とする。 The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing pluripotent stem cells capable of obtaining pluripotent stem cells having functions equivalent to iPS cells.
　本発明は、以下のとおりである。
［１］　ポリアミン、または、ＤＮＡメチル化阻害物質を含む培地中で、動物細胞を培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法。
［２］　前記ポリアミンは、スペルミン、スペルミジン、プトレシン、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体、からなる群から選択される少なくとも１種である、［１］に記載の製造方法。
［３］　前記ＤＮＡメチル化阻害物質は、ＤＮＡメチル化酵素をコードする遺伝子の発現を阻害するｓｉＲＮＡ、５－アザ－２’－デオキシシチジン、シネフンギン、ゼブラリン、エチオニン、アセチルメチオニン、および、セレノメチオニンからなる群から選択される少なくとも１種である、［１］または［２］に記載の製造方法。 The present invention is as follows.
[1] A method for producing pluripotent stem cells, wherein pluripotent stem cells are obtained by culturing animal cells in a medium containing a polyamine or a DNA methylation inhibitor.
[2] The polyamine is at least one selected from the group consisting of spermine, spermidine, putrescine, and acetylated products thereof, and two or more polymers of the polyamines. The manufacturing method as described.
[3] The DNA methylation inhibitor is selected from siRNA, 5-aza-2′-deoxycytidine, sinefungin, zebralin, ethionine, acetylmethionine, and selenomethionine that inhibits expression of a gene encoding a DNA methylase. The production method according to [1] or [2], which is at least one selected from the group consisting of:
　本発明者は、細胞を初期化する働きのある物質について、数多くの物質を用いて探索し、得られた細胞の性質を調べた。本発明者の鋭意努力の結果、ポリアミンに、細胞の初期化に関する遺伝子群の発現を誘導する働きがあることを発見した。さらに、本発明者は、ポリアミンが結果的にＤＮＡのメチル化を阻害し、それが細胞を初期化していることを発見し、ＤＮＡメチル化阻害物質を用いても同様の細胞を得られることを見出した。また、ポリアミン、または、ＤＮＡメチル化阻害剤による細胞の分化誘導を進めると、ＥＳ細胞またはｉＰＳ細胞のマーカータンパク質を有する細胞に変化し、当該細胞が多能性幹細胞になることを発見した。 The present inventor searched for a substance having a function of reinitializing cells using a large number of substances, and investigated the properties of the obtained cells. As a result of the inventor's diligent efforts, it was discovered that polyamine has a function of inducing the expression of genes related to cell reprogramming. Furthermore, the present inventor has discovered that polyamines consequently inhibit DNA methylation, and that it reinitializes the cells, and that similar cells can be obtained using DNA methylation inhibitors. I found it. Moreover, when the differentiation induction of the cell by a polyamine or a DNA methylation inhibitor was advanced, it changed to the cell which has the marker protein of an ES cell or iPS cell, and discovered that the said cell turns into a pluripotent stem cell.
　それだけでなく、当該多能性幹細胞は、多能性を保持した状態で培養可能であり、胚様体に分化させる培地で、内胚葉、中胚葉、外胚葉などの様々な体細胞に再分化することも確認でき、本発明を完成するに至った。 In addition, the pluripotent stem cells can be cultured in a state that retains pluripotency, and redifferentiate into various somatic cells such as endoderm, mesoderm, and ectoderm in a medium that differentiates into embryoid bodies. It has also been confirmed that the present invention has been completed.
　以下、本明細書において、ポリアミンを用いて得られた多能性幹細胞をＰＩＳ細胞（Polyamine-Induced　Stem　cells）と呼び、ＤＮＡメチル化阻害剤を用いて得られた多能性幹細胞をＤＩＳ細胞（Demethylation-Induced　Stem　Cells）と呼ぶ場合がある。 Hereinafter, in this specification, pluripotent stem cells obtained using polyamine are referred to as PIS cells (Polyamine-Induced Stem cells), and pluripotent stem cells obtained using a DNA methylation inhibitor are referred to as DIS cells ( Sometimes called Demethylation-Induced Stem Cells).
　本発明によれば、ｉＰＳ細胞と同等の機能を有する多能性幹細胞を得ることのできる、多能性幹細胞の製造方法を提供することができる。なお、本発明の製造方法は、全く新しい方法であるだけでなく、遺伝子組換えを用いることなく、動物細胞から多能性幹細胞を得た点において、本発明の有用性は極めて高い。 According to the present invention, it is possible to provide a method for producing pluripotent stem cells capable of obtaining pluripotent stem cells having functions equivalent to iPS cells. In addition, the production method of the present invention is not only a completely new method, but the utility of the present invention is extremely high in that pluripotent stem cells are obtained from animal cells without using genetic recombination.
　さらに、ＰＩＳ細胞およびＤＩＳ細胞では、１～３日で得られる上に、当該細胞への転換効率も非常に高く、細胞の分離も容易という優れた特徴がある。ｉＰＳ細胞は、時間と手間がかかるだけでなく、ｉＰＳ細胞を得る確率は低いことから、ＰＩＳ細胞およびＤＩＳ細胞はｉＰＳ細胞の作製方法と比較して、進歩性が極めて高い。 Furthermore, PIS cells and DIS cells have excellent characteristics that they can be obtained in 1 to 3 days, have a very high conversion efficiency into the cells, and can be easily separated. Since iPS cells not only take time and labor, but also have a low probability of obtaining iPS cells, PIS cells and DIS cells are highly inventive compared to the method of producing iPS cells.
　また、患者から取得した細胞をｉＰＳ細胞に分化して再生医療に利用することは、既に記載の理由により困難であり、そのことが再生医療の実用化における大きな障害となっている。本発明のＰＩＳ細胞またはＤＩＳ細胞を作製する方法では、さまざまな細胞を短時間且つ高効率でＰＩＳ細胞またはＤＩＳ細胞に変換できる。このため、患者の一部の組織または脂肪組織等を採取すれば、再生医療に十分なＰＩＳ細胞またはＤＩＳ細胞の細胞量を短期間で確保することができる。即ち、ＰＩＳ細胞またはＤＩＳ細胞を作製する方法を利用すれば、患者自身の細胞を用いて短期間で再生医療を行うことが可能になり、治療後に免疫抑制剤を使用する必要がなく、理想的な再生医療を行うことが可能である。ｉＰＳ細胞と比較した場合、この点において本発明の進歩性および有効性は極めて高い。 Also, it is difficult to differentiate cells obtained from patients into iPS cells and use them in regenerative medicine for the reasons already described, and this is a major obstacle in the practical application of regenerative medicine. In the method for producing PIS cells or DIS cells of the present invention, various cells can be converted into PIS cells or DIS cells in a short time and with high efficiency. For this reason, if a part of a patient's tissue or adipose tissue is collected, a sufficient amount of PIS cells or DIS cells for regenerative medicine can be secured in a short period of time. That is, if a method for producing PIS cells or DIS cells is used, regenerative medicine can be performed in a short period of time using the patient's own cells, and it is not necessary to use an immunosuppressant after treatment, which is ideal. Regenerative medicine can be performed. Compared with iPS cells, the inventive step and effectiveness of the present invention are extremely high in this respect.
図１は、肺由来線維芽細胞ＴＩＧ－１－２０からＰＩＳ細胞への変化の様子を示す。図１のＡは、スペルミンで誘導前のＴＩＧ－１－２０細胞を示す。図１のＢは、４０μＭのスペルミンで６時間誘導後にＴＩＧ－１－２０細胞に起こる変化の様子を示す。図１のＣは、４０μＭのスペルミンで３６時間誘導することにより得られたＰＩＳ細胞を示す。FIG. 1 shows the state of change from lung-derived fibroblast TIG-1-20 to PIS cells. FIG. 1A shows TIG-1-20 cells before induction with spermine. FIG. 1B shows the changes that occur in TIG-1-20 cells after 6 hours induction with 40 μM spermine. FIG. 1C shows PIS cells obtained by induction with 40 μM spermine for 36 hours. 図２は、リアルタイムＰＣＲにより測定したｍＲＮＡの発現量を示す。縦軸は、ＴＩＧ－１－２０のｍＲＮＡ量に対するＰＩＳ細胞のｍＲＮＡ量の比率（倍）を示す。FIG. 2 shows the expression level of mRNA measured by real-time PCR. The vertical axis represents the ratio (times) of the amount of mRNA in PIS cells to the amount of TIG-1-20 mRNA. 図３は、Human　ES/iPS　Cell　Characterization　Kitにより活性染色または免疫染色を施したＰＩＳ細胞を示す。左からアルカリホスファターゼの活性染色、ＳＳＥＡ４、ＴＲＡ－１－６０、ＴＲＡ－１－８１に対する抗体を用いた蛍光免疫染色。灰色に見える部分が蛍光部位を示す。FIG. 3 shows PIS cells that have been subjected to activity staining or immunostaining with Human ES / iPS Cell Characterization Kit. From the left, alkaline phosphatase activity staining, fluorescent immunostaining using antibodies against SSEA4, TRA-1-60, TRA-1-81. The part that appears gray indicates the fluorescent site. 図４は、Cardiomyocyte　Differentiation　KitによりＰＩＳ細胞を心筋細胞に誘導した様子を示す。図４のＡは、ＥＢを形成している様子を示す。図４のＢは、ＥＢから前駆心筋細胞が形成される様子を示す。図４のＣは、微細動のある心筋細胞の様子を示す。FIG. 4 shows a state in which PIS cells are induced into cardiomyocytes by Cardiomyocyte Differentiation Kit. FIG. 4A shows a state where an EB is formed. FIG. 4B shows how progenitor cardiomyocytes are formed from EB. FIG. 4C shows a state of a cardiomyocyte having fine movement. 図５は、マウス前駆白色脂肪細胞株３Ｔ３－Ｌ１をＰＩＳ細胞に分化した様子を示す。図５のＡは、３Ｔ３－Ｌ２４細胞の様子を示す。図５のＢは、形成されたＥＢの様子を示す。FIG. 5 shows the differentiation of mouse precursor white adipocyte cell line 3T3-L1 into PIS cells. FIG. 5A shows the appearance of 3T3-L24 cells. FIG. 5B shows a state of the formed EB. 図６は、マウス前駆白色脂肪細胞Ｃ３Ｈ１０Ｔ１／２株をＤＩＳ細胞に分化した様子を示す。図６のＡは、ＤＩＳ細胞（ＤＩＳ－ＳＩ細胞）の様子を示す。図６のＢは、ＤＩＳ細胞（ＤＩＳ－Ａｚａ細胞）の様子を示す。図６のＣは、ＤＩＳ－Ａｚａ細胞を分化して得られた血球系細胞の初期分化の様子を示す。図６のＤは、得られた神経幹細胞と神経様物資の初期分化の様子を示す。FIG. 6 shows how mouse precursor white adipocytes C3H10T1 / 2 strain was differentiated into DIS cells. FIG. 6A shows the state of DIS cells (DIS-SI cells). FIG. 6B shows the state of the DIS cell (DIS-Aza cell). C in FIG. 6 shows the state of early differentiation of blood cells obtained by differentiating DIS-Aza cells. FIG. 6D shows the state of initial differentiation of the obtained neural stem cells and neural-like materials. 図７は、ＰＩＳ細胞をさまざまな胚葉に高効率で分化させた様子を示す。図７のＡは、外胚葉に分化した様子を示す。図７のＢは、中胚葉に分化した様子を示す。図７のＣは、内胚葉に分化した様子を示す。FIG. 7 shows how PIS cells were differentiated into various germ layers with high efficiency. FIG. 7A shows a state of differentiation into ectoderm. FIG. 7B shows a state of differentiation into mesoderm. FIG. 7C shows a state of differentiation into endoderm. 図８は、ＰＩＳ細胞を褐色脂肪細胞に分化した様子を示す。図８のＡは、褐色脂肪細胞に分化した細胞がＵＣＰ１を発現している様子を示す。図８のＢは、褐色脂肪細胞に分化した細胞が脂肪を蓄積している様子を示す。FIG. 8 shows how PIS cells are differentiated into brown adipocytes. FIG. 8A shows a state in which cells differentiated into brown adipocytes express UCP1. FIG. 8B shows a state where cells differentiated into brown adipocytes accumulate fat. 図９は、マウス脂肪細胞３Ｔ３－Ｌ２４をＰＩＳ細胞に分化した細胞が、高効率で神経幹細胞および成熟神経細胞に分化した様子を示す。図９のＡは、神経幹細胞に分化した様子を示す。図９のＢとＣは、神経幹細胞が神経細胞に分化した様子を示し、図９のＢは成熟神経細胞のマーカー遺伝子TubulinIIIを発現している様子を示す。図９のＣは成熟神経細胞のマーカー遺伝子ＭＡＰ２を発現している様子を示す。FIG. 9 shows that cells obtained by differentiating mouse adipocytes 3T3-L24 into PIS cells differentiated into neural stem cells and mature neurons with high efficiency. FIG. 9A shows a state of differentiation into neural stem cells. B and C in FIG. 9 show how neural stem cells have differentiated into nerve cells, and FIG. 9B shows how mature tumor cell marker gene Tubulin III is expressed. FIG. 9C shows a state in which the marker gene MAP2 of mature neurons is expressed.
　本発明は、培地に、ポリアミン、または、ＤＮＡメチル化阻害物質を添加し、動物細胞を（ＥＳ細胞またはｉＰＳ細胞と類似の形態を有するまで）培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法である。 The present invention obtains pluripotent stem cells by adding polyamine or a DNA methylation inhibitor to a medium and culturing animal cells (until they have a similar form to ES cells or iPS cells). A method for producing pluripotent stem cells.
　なお、動物細胞とは、培養により増殖可能で正常な動物の細胞であればよい。ここで、動物はヒトを含み、好ましくは哺乳動物である。ヒト以外の哺乳動物としては、特に限定されないが、例えば、マウス、ラット、ウサギ、ヤギなどが挙げられる。哺乳動物以外の動物としては、例えば、ショウジョウバエなどの昆虫類などが挙げられる。 The animal cell may be a normal animal cell that can be grown by culture. Here, the animal includes a human, preferably a mammal. Mammals other than humans are not particularly limited, and examples include mice, rats, rabbits, and goats. Examples of animals other than mammals include insects such as Drosophila.
　動物細胞としては、例えば、線維芽細胞、表皮細胞、乳腺細胞、脂肪細胞、筋芽細胞、骨芽細胞、肝細胞、血管内皮細胞、または、それらの前駆細胞などが挙げられる。他の動物細胞としては、例えば、血球系幹細胞、間葉系幹細胞、神経幹細胞などの幹細胞が挙げられる。 Examples of animal cells include fibroblasts, epidermal cells, mammary cells, adipocytes, myoblasts, osteoblasts, hepatocytes, vascular endothelial cells, or precursor cells thereof. Examples of other animal cells include stem cells such as blood cell stem cells, mesenchymal stem cells, and neural stem cells.
　例えば、ヒトの細胞は、ヒト正常細胞の分譲を行っている保存機関（ＡＴＣＣ、ＪＣＲＢなど）から分譲を受けるか、ヒト正常細胞の販売を行う市販のメーカーから購入するか、または、ヒトから細胞を取り出すことにより取得できる。例えば、ヒトなどの動物の組織から細胞を取り出して用いる場合は、組織検査の装置を用いた組織の一部のサンプリング、手術等による組織の部分的切除、脂肪吸引等の方法により、組織を得た後、トリプシン等の酵素により細胞を分離し、動物細胞用の培地で培養する。 For example, human cells can be purchased from a storage organization (ATCC, JCRB, etc.) that sells human normal cells, purchased from commercial manufacturers that sell human normal cells, or cells from humans Can be obtained by taking out For example, when cells are extracted from a tissue of an animal such as a human and used, the tissue is obtained by sampling a part of the tissue using a tissue examination apparatus, partial excision of the tissue by surgery, liposuction, or the like. Thereafter, the cells are separated by an enzyme such as trypsin and cultured in a medium for animal cells.
　（ポリアミン）
　本発明に用いられるポリアミン（polyamine）は、直鎖脂肪族炭化水素に第一級アミノ基あるいは第二級アミノ基が合計で２つ以上結合した化合物を意味する。 (Polyamine)
The polyamine used in the present invention means a compound in which two or more primary amino groups or secondary amino groups are bonded to a linear aliphatic hydrocarbon in total.
　ポリアミンとしては、例えば、スペルミン（CAS登録番号71-44-3）、スペルミジン（CAS登録番号124-20-9）、プトレシン（CAS登録番号110-60-1）、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体が挙げられる。 Examples of polyamines include spermine (CAS registration number 71-44-3), spermidine (CAS registration number 124-20-9), putrescine (CAS registration number 110-60-1), and acetylated forms thereof. As well as two or more polymers of these polyamines.
　ポリアミンのアセチル化体（アセチル化ポリアミン）としては、例えば、Ｎ－アセチルプトレシン、Ｎ－アセチルスペルミジン、Ｎ－アセチルスペルミン、ジアセチルプトレシン、ジアセチルスペルミジン、ジアセチルスペルミンが挙げられる。なお、ポリアミンの２つ以上の重合体は、化学合成により得ることができる。 Examples of the acetylated polyamine (acetylated polyamine) include N-acetylputrescine, N-acetylspermidine, N-acetylspermine, diacetylputrescine, diacetylspermidine, and diacetylspermine. Two or more polymers of polyamine can be obtained by chemical synthesis.
　ポリアミンは、炭素鎖３～４の間隔でアミノ基（－ＮＨ２基または－ＮＨ－基）が存在することで、ヒストンまたは染色体に作用し、細胞を初期化する遺伝子群の発現を誘導する特徴がある。したがって、上記以外のポリアミン（生体内に存在する他のポリアミン、化学合成により得られた他のポリアミンなど）のうち、スペルミン、スペルミジン、プトレシン等と類似の構造を有する化合物も、本発明に用いることができる。なお、ポリアミンの遺伝子発現を誘導する能力は、スペルミン、スペルミジン、プトレシンの順に強く、直鎖分子の長さが長いほど、モル濃度当たりの遺伝子の発現を誘導する効果は大きい。 Polyamine is characterized by the presence of amino groups (—NH 2 group or —NH— group) at intervals of 3 to 4 carbon chains, acting on histones or chromosomes and inducing the expression of genes that initialize cells There is. Therefore, among polyamines other than the above (other polyamines existing in the body, other polyamines obtained by chemical synthesis, etc.), compounds having a structure similar to spermine, spermidine, putrescine, etc. are also used in the present invention. Can do. The ability to induce polyamine gene expression is strong in the order of spermine, spermidine, and putrescine, and the longer the length of the linear molecule, the greater the effect of inducing gene expression per molar concentration.
　なお、ポリアミンは水溶性のため、１０～１００ｍＭ程度の濃度のポリアミン水溶液を作製し、０．２μｍの濾過フィルターにより滅菌処理した後、適切な濃度になるように培地に添加して使用することができる。 Since polyamine is water-soluble, an aqueous polyamine solution having a concentration of about 10 to 100 mM is prepared, sterilized with a 0.2 μm filter, and then added to the medium so as to have an appropriate concentration. it can.
　（ＤＮＡメチル化阻害物質）
　本発明に用いられるＤＮＡメチル化阻害物質としては、例えば、以下の２種類が挙げられる。 (DNA methylation inhibitor)
Examples of the DNA methylation inhibitor used in the present invention include the following two types.
　ＤＮＡメチル化阻害物質の１種としては、ＤＮＡメチル化酵素をコードする遺伝子（ＤＮＭＴ）の発現を阻害するｓｉＲＮＡが挙げられる。ＤＮＡメチル化酵素とは、細胞を複製する際に親細胞から娘細胞にＤＮＡのメチル化情報を正確に伝える酵素（ＤＮＭＴ１）、娘細胞のＤＮＡをメチル化する酵素（ＤＮＭＴ３）などである。ｓｉＲＮＡとは、特定の遺伝子のｍＲＮＡと相補する短いＲＮＡであり、該特定の遺伝子の発現を阻害することができる。ヒトやマウスでは、ＤＮＭＴ１コード遺伝子またはＤＮＭＴ３コード遺伝子が、ＤＮＭＴをコードする遺伝子に該当し、例えば、それらの発現を阻害するｓｉＲＮＡとして、キアゲン社のｓｉＲＮＡをマニュアルに従って添加することにより、ＤＮＡのメチル化を効率よく阻害することができる。 An example of a DNA methylation inhibitor is siRNA that inhibits the expression of a gene encoding DNA methylase (DNMT). The DNA methylase is an enzyme (DNMT1) that accurately transmits DNA methylation information from a parent cell to a daughter cell when replicating a cell, an enzyme (DNMT3) that methylates DNA of a daughter cell, and the like. siRNA is short RNA complementary to mRNA of a specific gene, and can inhibit the expression of the specific gene. In humans and mice, the DNMT1-encoding gene or DNMT3-encoding gene corresponds to the DNMT-encoding gene. For example, as a siRNA that inhibits their expression, siRNA from Qiagen is added according to the manual to methylate DNA. Can be efficiently inhibited.
　ＤＮＡメチル化阻害物質の他の１種としては、メチオニン類似物質が挙げられる。メチオニン類似物質は、メチル化反応の反応基質であるアデノシルメチオニンの合成を阻害することにより、メチル化反応を阻害する。 Another example of a DNA methylation inhibitor is a methionine analog. Methionine analogs inhibit the methylation reaction by inhibiting the synthesis of adenosylmethionine, which is the reaction substrate for the methylation reaction.
　メチオニン類似物質としては、例えば、５－アザ－２’－デオキシシチジン（5-Aza-dc，CAS登録番号2353-33-5）、シネフンギン（CAS登録番号58944-73-3）、ゼブラリン（CAS登録番号360-10-6）、エチオニン（CAS登録番号13073-35-3）、アセチルメチオニン（CAS登録番号567-82-7）、セレノメチオニン（CAS登録番号3211-76-5）が挙げられる。これらのメチオニン類似物質は、水溶性であるため、１０－１００ｍＭ程度の濃度の水溶液を調製し、該水溶液を０．２μｍの濾過フィルターにより滅菌処理した後、適切な濃度になるように培地に添加して使用することができる。 Examples of methionine analogs include 5-aza-2'-deoxycytidine (5-Aza-dc, CAS registration number 23533-33-5), sinefungin (CAS registration number 58944-73-3), and zebralin (CAS registration). No. 360-10-6), ethionine (CAS registration number 13073-35-3), acetylmethionine (CAS registration number 567-82-7), and selenomethionine (CAS registration number 3211-76-5). Since these methionine-like substances are water-soluble, prepare an aqueous solution with a concentration of about 10-100 mM, sterilize the aqueous solution with a 0.2 μm filter, and add it to the medium to an appropriate concentration. Can be used.
　ポリアミンによる多能性幹細胞（ＰＩＳ細胞）、または、ＤＮＡメチル化阻害物質による多能性幹細胞（ＤＩＳ細胞）への誘導は、動物細胞をポリアミンまたはＤＮＡメチル化阻害物質の在下で培養することにより実施できる。細胞培養用のフラスコ、デッィシュあるはプレートに、細胞の増殖で使用される通常の培地を添加し、細胞を播種し、通常の培養温度（３７℃程度）のＣＯ２インキュベーターで培養することにより、細胞をフラスコ底面で増殖している状態にする。ヒト細胞の増殖で使用される通常の培地として、例えば、最少培地（Minimum　Essential　medium：ＭＥＭ）やｉＰＳ細胞用の培地などを利用することができ、２５ｃｍ２の培養フラスコの場合、１０万～１００万個程度の細胞を播種することが好ましい。 Induction of pluripotent stem cells (PIS cells) by polyamines or pluripotent stem cells (DIS cells) by DNA methylation inhibitors is carried out by culturing animal cells in the presence of polyamines or DNA methylation inhibitors. it can. By adding a normal medium used for cell growth to a flask, dish or plate for cell culture, seeding the cells, and culturing in a CO 2 incubator at a normal culture temperature (about 37 ° C.) Allow cells to grow on bottom of flask. As a normal medium used for the growth of human cells, for example, a minimum essential medium (MEM) or a medium for iPS cells can be used. In the case of a 25 cm 2 culture flask, 100,000 to 100 It is preferable to seed about 10,000 cells.
　フラスコ底面に細胞が付着して細胞の増殖が開始した後、適切な濃度のポリアミン水溶液、または、ＤＮＡメチル化阻害物質の水溶液を添加し、さらに通常の培養温度（３７℃程度）のＣＯ２インキュベーターで１～４日間程度培養する。当該操作により、細胞内には無数の小さな球体が生じ、フラスコ底面に付着していた細胞が浮遊し、小さな球体の入った球状の細胞となって、ＥＳ細胞またはｉＰＳ細胞と類似の形態に変化する。この細胞が、多能性幹細胞のＰＩＳ細胞またはＤＩＳ細胞である。 After cells have adhered to the bottom of the flask and cell growth has started, an appropriate concentration of polyamine aqueous solution or DNA methylation inhibitor aqueous solution is added, and a CO 2 incubator at a normal culture temperature (about 37 ° C.) is added. Incubate for about 1 to 4 days. By this operation, innumerable small spheres are generated in the cells, the cells attached to the bottom of the flask float, become spherical cells containing small spheres, and change into a shape similar to ES cells or iPS cells. To do. This cell is a pluripotent stem cell PIS cell or DIS cell.
　ポリアミンまたはＤＮＡメチル化阻害物質の添加量が少ない場合、ＰＩＳ細胞またはＤＩＳ細胞への変化が起こらず、添加量が多い場合、細胞のアポトーシス（細胞死）が誘導される。そのため、ポリアミンまたはＤＮＡメチル化阻害物質は、ＰＩＳ細胞またはＤＩＳ細胞への変化が起こる最低濃度で添加し、可能な限りアポトーシスを起こさないようにする必要がある。ポリアミンまたはＤＮＡメチル化阻害物質の添加濃度と培養時間は、細胞種と細胞数に依存する。そのため、ポリアミンまたはＤＮＡメチル化阻害物質の添加濃度は、光学顕微鏡観察により、ＰＩＳ細胞またはＤＩＳ細胞に変化することを確認できる最少濃度を、予備実験により確認することが望ましい。 When the addition amount of the polyamine or DNA methylation inhibitor is small, no change to PIS cells or DIS cells occurs, and when the addition amount is large, cell apoptosis (cell death) is induced. Therefore, polyamines or DNA methylation inhibitors should be added at the lowest concentration at which changes to PIS cells or DIS cells occur, so as not to cause apoptosis as much as possible. The concentration of polyamine or DNA methylation inhibitor and the culture time depend on the cell type and the number of cells. For this reason, it is desirable to confirm the minimum concentration at which polyamine or DNA methylation inhibitor is added to PIS cells or DIS cells by optical microscope observation by preliminary experiments.
　例えば、１０～１００万個の正常な線維芽細胞、脂肪細胞の場合、スペルミンを１０μＭ～１００μＭ程度添加した培地で１～２日間培養すれば、それらの細胞をＰＩＳ細胞に変化させることができる。さらに、上記の繊維芽細胞等に対しては、ＤＮＭＴをコードする遺伝子の発現を阻害するｓｉＲＮＡを５～１０ｎＭ程度添加した培地、５－Ａｚａ－ｄｃを２０～８０μＭ程度添加した培地、または、エチオニンを１～１０ｍＭ程度添加した培地で、１～３日間培養すれば、それらの細胞をＤＩＳ細胞に変化させることができる。 For example, in the case of 100,000 to 1,000,000 normal fibroblasts and adipocytes, these cells can be changed to PIS cells by culturing in a medium supplemented with about 10 μM to 100 μM of spermine for 1 to 2 days. Furthermore, for the fibroblasts described above, a medium to which about 5 to 10 nM of siRNA that inhibits the expression of a gene encoding DNMT, a medium to which about 20 to 80 μM of 5-Aza-dc is added, or ethionine If the cells are cultured in a medium supplemented with about 1 to 10 mM for 1 to 3 days, these cells can be changed to DIS cells.
　上記ポリアミンまたは上記ｓｉＲＮＡを含有する培地で培養することにより得られたＰＩＳ細胞またはＤＩＳ細胞は、上記ポリアミンまたは上記ｓｉＲＮＡを含有する培地で１～４日間程度培養できる。しかし、アポトーシスにより、次第に生存率が低下するため、光学顕微鏡によりＰＩＳ細胞への変化を確認すると、０～１日以内に、浮遊したＰＩＳ細胞またはＤＩＳ細胞を含む培養液を回収し、通常の速度（８００～２０００ｒｐｍで３分間程度）で遠心分離を行うことにより、ＰＩＳ細胞を集めることが好ましい。 PIS cells or DIS cells obtained by culturing in a medium containing the polyamine or the siRNA can be cultured in a medium containing the polyamine or the siRNA for about 1 to 4 days. However, since the survival rate gradually decreases due to apoptosis, when the change to PIS cells was confirmed by light microscopy, the culture medium containing suspended PIS cells or DIS cells was collected within 0 to 1 day, and the normal rate PIS cells are preferably collected by centrifugation at 800-2000 rpm for about 3 minutes.
　また、培地には、培養の際に、ＰＩＳ細胞の生存率の低下を防ぐための阻害剤を添加してもよい。生存率の低下を防ぐための阻害剤としては、例えば、Ｒｏｃｋ阻害剤およびアポトーシス阻害剤が使用できる。Ｒｏｃｋ阻害剤としては、例えば、Ｙ－２７６３２、チアゾビビン、ＳＢ４３１５４２、ＰＤ０３２５９０１、アポトーシス阻害剤としては、例えば、ｐ５３阻害剤、Ｂａｘ阻害剤、カスパーゼ阻害剤が挙げられる。 In addition, an inhibitor for preventing a decrease in the survival rate of PIS cells may be added to the medium during the culture. As an inhibitor for preventing a decrease in survival rate, for example, a Rock inhibitor and an apoptosis inhibitor can be used. Examples of the Rock inhibitor include Y-27632, thiazobibin, SB431542, PD0325901, and examples of the apoptosis inhibitor include a p53 inhibitor, a Bax inhibitor, and a caspase inhibitor.
　一方、ＤＮＡメチル化阻害物質として、５－アザ－２’－デオキシシチジン、シネフンギン、ゼブラリン、エチオニン、アセチルメチオニン、セレノメチオニンを用いた場合、ＤＮＡのメチル化だけでなく様々なメチル化を阻害し、強い細胞毒性を有する。したがって、細胞の浮遊が起こるとすぐに、浮遊したＤＩＳ細胞を含む培養液を回収し、通常の速度（８００～２０００ｒｐｍで３分間程度）で遠心分離を行うことによりＤＩＳ細胞を集めることが好ましい。 On the other hand, when 5-aza-2'-deoxycytidine, sinefungin, zebularine, ethionine, acetylmethionine, selenomethionine is used as a DNA methylation inhibitor, it inhibits not only DNA methylation but also various methylation, Has strong cytotoxicity. Therefore, as soon as cell floating occurs, it is preferable to collect the DIS cells by collecting the culture medium containing the suspended DIS cells and centrifuging at a normal speed (800 to 2000 rpm for about 3 minutes).
　遠心分離により回収したＰＩＳ細胞またはＤＩＳ細胞は、細胞からの分化が非常に高い割合で起こるため、胚様体（ＥＢ）を経由した内胚葉、外胚葉、中胚葉への再分化を直接行うことができる。しかしながら、再生医療に使う場合、「ＥＳ細胞またはｉＰＳ細胞と同等の形質を安定して発現させ、且つＰＩＳ細胞またはＤＩＳ細胞のみを選別する」必要がある。この場合は、ＥＳ細胞またはｉＰＳ細胞様の多能性の形質を獲得し且つ保持するための培地に添加することが好ましい。ＰＩＳ細胞またはＤＩＳ細胞の多能性の形質を獲得し且つ保持するための培地として、例えば、ヒトの細胞の場合、Ｃｅｌｌａｒｔｉｓ（登録商標）　ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）、ｍＴｅＳＲ１（ベリタス社）など多くの市販の培地を用いることができる。また、当該培地での培養において、ＰＩＳ細胞の生存率の低下を防ぐためには、Ｒｏｃｋ阻害剤Ｙ－２７６３２、チアゾビビン、ＳＢ４３１５４２、ＰＤ０３２５９０１を添加することも可能である。 Since PIS cells or DIS cells collected by centrifugation are differentiated from cells at a very high rate, redifferentiation directly into endoderm, ectoderm, and mesoderm via the embryoid body (EB) Can do. However, when it is used for regenerative medicine, it is necessary to “stablely express traits equivalent to those of ES cells or iPS cells and select only PIS cells or DIS cells”. In this case, it is preferable to add it to a medium for acquiring and maintaining ES cell or iPS cell-like pluripotent traits. As a medium for acquiring and retaining pluripotent traits of PIS cells or DIS cells, for example, in the case of human cells, Cellartis (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.), mTeSR1 ( Many commercially available media such as Veritas) can be used. In order to prevent a decrease in the survival rate of the PIS cells in the culture in the medium, it is also possible to add a Rock inhibitor Y-27632, thiazobibin, SB431542, PD0325901.
　これらの培地でＰＩＳ細胞またはＤＩＳ細胞の集合体を培養すると、弱く付着（または浮遊）する。ピペッティング（ピペットにより培養液を吸引、排出を繰りかえして懸濁する操作）により当該細胞を浮遊させ、培地と細胞を回収し、遠心分離により細胞を集めた後、新しい培地に移す操作を行う。ＰＩＳ細胞またはＤＩＳ細胞に分化していないもとの体細胞は、強固に付着するため、ピペッティングでは剥がれず、ＰＩＳ細胞の集合体またはＤＩＳ細胞の集合体のみを回収できる。 When PIS cells or DIS cell aggregates are cultured in these media, they adhere weakly (or float). The cells are suspended by pipetting (an operation in which the culture medium is aspirated and suspended by repeated pipetting), the medium and cells are collected, the cells are collected by centrifugation, and then transferred to a new medium. Since the original somatic cells that have not differentiated into PIS cells or DIS cells adhere firmly, they are not detached by pipetting, and only PIS cell aggregates or DIS cell aggregates can be recovered.
　さらに、ＰＩＳ細胞またはＤＩＳ細胞の集合体を、Ｔｒｙｐ　ＬＥ　Ｓｅｌｅｃｔ（Ｇｉｂｃｏ）、０．２５％トリプシン溶液（Ｇｉｂｃｏ）、Ａｃｃｕｔａｓｅ－Ｓｏｌｕｔｉｏｎ（フナコシ社）等によって、細胞が分散した状態にし、Ｃｅｌｌａｒｔｉｓ（登録商標）　ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）で培養した場合、個々のＰＩＳ細胞を培養器の底面に弱く付着した状態で増殖させることができる。 Further, the aggregate of PIS cells or DIS cells is made dispersed with Tryp LE Select (Gibco), 0.25% trypsin solution (Gibco), Accutase-Solution (Funakoshi), etc., and Cellaritis (registered trademark) is used. ) When cultured in DEF-CS 500 Culture System (Takara Bio Inc.), individual PIS cells can be grown in a state of weakly adhering to the bottom of the incubator.
　Ｒｏｃｋ阻害剤およびアポトーシス阻害剤を添加して培養した場合、細胞にわずかな収縮が見られるが、細胞の浮遊は起こらない。この場合、Ｔｒｙｐ　ＬＥ　Ｓｅｌｅｃｔ、０．２５％トリプシン、Ａｃｃｕｔａｓｅ－Ｓｏｌｕｔｉｏｎ等により細胞を剥離させ、細胞をＰＢＳ等で洗浄後、ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｔｅｍ等のｉＰＳ細胞用の培地で１－３日間培養すればよい。こうして得られたＰＩＳ細胞は、ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　ＳｙｔｅｍあるいはＳｔｅｍＳｕｒｅ（登録商標）　ゼラチン溶液（和光純薬（株））等のゼラチン溶液でプレートをコートすることで、付着培養することができる。これにより、細胞の障害が修復され、アポトーシスを起こさず、且つ、高効率でＰＩＳ細胞を得ることができる。 When cultured with the addition of a Rock inhibitor and an apoptosis inhibitor, slight contraction of the cells is observed, but no cell floating occurs. In this case, the cells are detached with Tryp LE Select, 0.25% trypsin, Accutrate-Solution, etc., washed with PBS, etc., and cultured in a medium for iPS cells such as DEF-CS 500 Culture System for 1-3 days. do it. The PIS cells thus obtained can be attached and cultured by coating the plate with a gelatin solution such as DEF-CS 500 Culture System or StemSure (registered trademark) gelatin solution (Wako Pure Chemical Industries, Ltd.). Thereby, cell damage is repaired, apoptosis does not occur, and PIS cells can be obtained with high efficiency.
　ＰＩＳ細胞またはＤＩＳ細胞がｉＰＳ細胞と同等の形質を有することは、ｉＰＳ細胞に特異的なＳＯＸ２、Ｏｃｔ３／４、ｃ－Ｍｙｃなどの遺伝子マーカーの発現、アルカリホスファターゼ活性の発現、または、ＳＳＥＡ－４、ＴＲＡ１－６０、ＴＲＡ１－８１などの表面抗原マーカーの発現等を調べることにより確認でき、遺伝子の発現上昇の確認にはＲＴ－ＰＣＲまたはリアルタイムＰＣＲを、ｉＰＳ細胞に特異的な酵素または表面抗原の確認には、抗体を用いた免疫染色、ウエスタンブロッティングまたはＦＡＣＳを利用すればよい。 The fact that a PIS cell or a DIS cell has a trait equivalent to that of an iPS cell indicates that expression of genetic markers such as SOX2, Oct3 / 4, c-Myc specific to iPS cells, expression of alkaline phosphatase activity, or SSEA-4 Can be confirmed by examining the expression of surface antigen markers such as TRA1-60, TRA1-81, etc. RT-PCR or real-time PCR can be used to confirm the increase in gene expression, iPS cell-specific enzyme or surface antigen For confirmation, immunostaining using antibodies, Western blotting or FACS may be used.
　ＰＩＳ細胞またはＤＩＳ細胞から様々な細胞への分化誘導は、ｉＰＳ細胞で使用される誘導方法と同様の方法により行うことができる。 Differentiation induction from PIS cells or DIS cells into various cells can be performed by the same method as the induction method used for iPS cells.
　ＰＩＳ細胞またはＤＩＳ細胞は、ｉＰＳ細胞と同様に、Ｅｍｂｒｙｏｉｄ　ｂｏｄｙ（ＥＢ）を経て、内胚葉、中胚葉、外胚葉などに分化し、その後、心筋細胞、脂肪細胞、神経細胞など様々な体細胞に分化できる。細胞の分化において、ｉＰＳ細胞と同様に、均一なＥＢを形成することで、ＰＩＳ細胞またはＤＩＳ細胞の分化効率が高まる。 Like iPS cells, PIS cells or DIS cells differentiate into endoderm, mesoderm, ectoderm, etc. through Embryoid body (EB), and then into various somatic cells such as cardiomyocytes, adipocytes, and nerve cells. Differentiate. In the differentiation of cells, the formation efficiency of PIS cells or DIS cells is increased by forming uniform EBs similarly to iPS cells.
　均一なＥＢの形成には、数多くの市販のプレートが存在し、それら利用することも有効な手段である。例えば、胚様体形成プレートＡｇｒｏｗｅｌｌ（ベリタス社）、リピジュア（登録商標）－コートプレート（日油株式会社）、ＥＺＳＰＨＥＲＥ（ＡＧＣテクノグラス）などを用いることで均一なＥＢを得ることができる。 There are a number of commercially available plates for the formation of uniform EB, and their use is also an effective means. For example, a uniform EB can be obtained by using an embryoid body forming plate Agrowell (Veritas), Lipidure (registered trademark) -coated plate (NOF Corporation), EZSPHERE (AGC Techno Glass), or the like.
　また、様々な細胞への分化もｉＰＳ細胞の場合と全く同じ培地で行うことができる。様々な細胞への分化誘導を行える市販の試薬が数多く存在し、それら利用することも有効な手段である。例えば、胚体内胚葉（ＥＤ）への分化はＣｅｌｌａｒｔｉｓ（登録商標）　Definitive　Endoderm　ChiPSC18（タカラバイオ株式会社）、心筋細胞や肝細胞への分化はPSdif-Cardio　Cardiomyocyte　Differentiation　Kit（フナコシ株式会社）、Ｃｅｌｌａｒｔｉｓ（登録商標）　iPS　Cell　to　Hepatocyte　Differentiation　System（タカラバイオ株式会社）、神経細胞への分化には、SETMdiff　Neural　Induced　Medium（ベリタス社）を用いることにより、ＰＩＳ細胞またはＤＩＳ細胞から効率よく細胞を分化することができる。 Also, differentiation into various cells can be performed in exactly the same medium as in the case of iPS cells. There are many commercially available reagents that can induce differentiation into various cells, and their use is also an effective means. For example, differentiation into definitive endoderm (ED) is Cellaritis (registered trademark) Definitive Endoderm ChiPSC18 (Takara Bio Inc.), and differentiation into cardiomyocytes and hepatocytes is PSdif-Cardio Cardiomyocyte Differentiation Kit (Funakoshi Co., Ltd.), Cellartis ( (Registered trademark) iPS Cell to Hepatocyte Differentiation System (Takara Bio Inc.) For differentiation into neurons, SETMdiff Neural Induced Medium (Veritas) is used to efficiently differentiate cells from PIS cells or DIS cells. Can do.
　本発明を以下の実施例によりさらに詳細に説明する。しかしながら、以下の実施例は本発明の例示であって、本発明を限定することを意図するものではない。 The present invention will be described in further detail with reference to the following examples. However, the following examples are illustrative of the present invention and are not intended to limit the present invention.
実施例１：ポリアミンに細胞を初期化する能力があることの証明 Example 1: Demonstration of the ability of polyamines to initialize cells
　この実験は、ポリアミンが、初期化する能力があることを証明するために実施した。 This experiment was conducted to prove that polyamines have the ability to initialize.
　ヒト肺由来の線維芽細胞ＴＩＧ－１－２０をＪＣＲＢ細胞バンク（ＪＣＲＢ０５０１株）より入手した。ヒト線維芽細胞ＴＩＧ－１－２０（分裂回数３３回、１０万個）を、６ｍＬの最少培地（ＭＥＭ）を含む２５ｃｍ２の培養フラスコで、１日間培養し、細胞を付着させた。１０ｍＭのプトレシン、２００μＭのスペルミジン、または４０μＭのスペルミンを添加したものと、それらを添加していないもの（コントロール）とを、３７℃で１日間ＣＯ２インキュベーター中で培養した。 Human lung-derived fibroblasts TIG-1-20 were obtained from the JCRB cell bank (JCRB0501 strain). Human fibroblast TIG-1-20 (number of divisions: 33 times, 100,000) was cultured in a 25 cm 2 culture flask containing 6 mL of minimal medium (MEM) for 1 day to allow the cells to adhere. Those added with 10 mM putrescine, 200 μM spermidine, or 40 μM spermine and those not added (control) were cultured at 37 ° C. in a CO 2 incubator for 1 day.
　スペルミンを添加して培養したときの結果を、図１に示した。繊維芽細胞ＴＩＧ－１－２０（図１Ａ）の細胞内に６時間後には細かな粒子上のものが出現し（図１Ｂ）、その後培養を続けると、細胞がフラスコ底部からはがれてＥＳ細胞に近い細胞（図１Ｃ）に変化した。 The results when culturing with spermine added is shown in FIG. Fine particles appear in the cells of fibroblasts TIG-1-20 (FIG. 1A) after 6 hours (FIG. 1B), and when the culture is continued, the cells peel off from the bottom of the flask and become ES cells. It changed to a close cell (FIG. 1C).
　細胞が剥がれてから１２時間後、培地中に懸濁した当該細胞（ＰＩＳ細胞）を遠心分離（８００ｒｐｍ、３分間）により回収し、Ｃｅｌｌａｒｔｉｓ（登録商標）　ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて培養した。培養は、２４穴のプレートにＤＥＦ－ＣＳ　ＣＯＡＴ－１のＰＢＳ（＋）希釈液を１時間処理した後、ＰＩＳ細胞を６ｍＬの培地添加剤を添加したＤＥＦ－ＣＳ　Ｂａｓａｌ　Ｍｅｄｉｕｍに懸濁し、２ｍＬずつ３ウェルに分注して、３７℃で２日間培養した。細胞をピペッティングにより剥がし、遠心分離（８００ｒｐｍ、３分間）によりＰＩＳ細胞を回収した。得られた細胞は、RNeasy　Lipid　Tissue　Mini　KitとQuantiTech　Reverse　Transcription　Kit　（キアゲン社）を用いて、ｍＲＮＡの抽出とｃＤＮＡの合成を行い、リアルタイムＰＣＲ用のプライマーとリアルタイムＰＣＲ（キアゲン社）を用いて、ＴＥＲＴ、ＤＭＮＴ１、ＳＯＸ２、ＯＣＴ３／４のｍＲＮＡの発現量を調べた。 Twelve hours after the cells are detached, the cells (PIS cells) suspended in the medium are collected by centrifugation (800 rpm, 3 minutes), and Cellartis (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.) is collected. ). For the culture, a PBS (+) diluted solution of DEF-CS COAT-1 in a 24-well plate was treated for 1 hour, and then PIS cells were suspended in DEF-CS Basal Medium to which 6 mL of a medium additive was added, and 2 mL each. The solution was dispensed into 3 wells and cultured at 37 ° C. for 2 days. The cells were detached by pipetting, and PIS cells were collected by centrifugation (800 rpm, 3 minutes). The obtained cells were extracted with mRNA and synthesized cDNA using RNeasy Lipid Tissue Mini Kit and QuantiTech Reverse Transcription Kit (Qiagen), and using real-time PCR primers and real-time PCR (Qiagen), The expression levels of TERT, DMNT1, SOX2, and OCT3 / 4 mRNA were examined.
　リアルタイムＰＣＲの結果を図２に示した。ＤＮＭＴ１遺伝子は、ＤＮＡ複製の際に、親細胞ＤＮＡのメチル化情報を娘細胞に伝える役割のメチル化転移酵素をコードする遺伝子である。図２に示すように、ＰＩＳ細胞では、ＤＮＭＴ１の発現が完全に抑制されており、ＤＮＡのメチル化の初期化が起こることがわかった。また、テロメアを伸長するテロメラーゼをコードする遺伝子（ＴＥＲＴ遺伝子）の発現が著しく増加した。つまり、老化により短くなったテロメア伸長（初期化）が起こっていた。さらに、ＥＳ細胞のマーカー遺伝子であり細胞の初期化を誘導するＳＯＸ２とＯＣＴ３／４遺伝子の発現量が増加していた。以上のように、ポリアミンは、細胞の初期化を誘導することが立証された。 The results of real-time PCR are shown in FIG. The DNMT1 gene is a gene encoding a methyltransferase that plays a role in transmitting methylation information of parent cell DNA to daughter cells during DNA replication. As shown in FIG. 2, in PIS cells, it was found that DNMT1 expression was completely suppressed, and initialization of DNA methylation occurred. In addition, the expression of a telomerase-encoding gene (TERT gene) that extends telomeres was significantly increased. In other words, telomere elongation (initialization) was shortened due to aging. Furthermore, the expression levels of SOX2 and OCT3 / 4 genes, which are ES cell marker genes and induce cell reprogramming, increased. As described above, it was demonstrated that polyamine induces cell reprogramming.
実施例２：ＰＩＳ細胞がｉＰＳ細胞と同等の形質を有していることの証明 Example 2: Demonstration that PIS cells have traits equivalent to iPS cells
　この実験は、ＰＩＳ細胞が、ｉＰＳ細胞に特異的なタンパク質を発現していることを証明するために行った。 This experiment was performed to prove that PIS cells express a protein specific to iPS cells.
　実施例１で用いた方法と全く同じ方法によりＰＩＳ細胞を、Ｃｅｌｌａｒｔｉｓ（登録商標）　ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて培養し、形質の安定したＰＩＳ細胞を得た。当該培地中で、弱く付着したＰＩＳ細胞をピペティングにより懸濁した後、１，５００ｒｐｍで４分間遠心分離して細胞を集めた。 PIS cells were cultured using Cellaris (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.) in exactly the same manner as used in Example 1 to obtain PIS cells with stable characteristics. After weakly adhering PIS cells in the medium were suspended by pipetting, the cells were collected by centrifugation at 1,500 rpm for 4 minutes.
　得られたＰＩＳ細胞は、Human　ES/iPS　Cell　Characterization　Kit　（Applied　Stem　Cell社）により、アルカリホスファターゼ活性と３つの表面抗原（ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１）の確認を行った。また、ｉＰＳ細胞は、アルカリホスファターゼを発現する特徴がある。さらに、ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１は、ｉＰＳ細胞のマーカー遺伝子であり、ｉＰＳ細胞に特異的に発現するタンパク質である。 The obtained PIS cells were confirmed for alkaline phosphatase activity and three surface antigens (SSEA-4, TRA-1-60, TRA-1-81) by Human ES / iPS Cell Characterization Kit (Applied Stem Cell). went. IPS cells are also characterized by expressing alkaline phosphatase. Furthermore, SSEA-4, TRA-1-60, and TRA-1-81 are iPS cell marker genes and are proteins that are specifically expressed in iPS cells.
　ＰＩＳ細胞を０．２ｍＬ用のサンプルチューブに入れ、懸濁細胞の状態で固定化処理、透過処理、ブロッキングをした後、１次抗体および２次抗体を結合させた。２次抗体で標識した細胞は、マウント液と１：１で混合してスライドグラスを作製し、蛍光顕微鏡（ＥＶＯＳ社、ＥＶＯＳ　ＦＬ　Ａｕｔｏ）により蛍光を確認した。結果を図３に示した。 PIS cells were placed in a 0.2 mL sample tube, fixed, permeabilized, and blocked in a suspended cell state, and then the primary antibody and the secondary antibody were bound. The cells labeled with the secondary antibody were mixed with the mount solution 1: 1 to prepare a slide glass, and the fluorescence was confirmed with a fluorescence microscope (EVOS, EVOS FL Auto). The results are shown in FIG.
　図３は、染色体を蛍光染色した図であり、蛍光物質により、活性染色または免疫染色の図である。蛍光がある場合、図３の写真では灰色に示される。図３から明らかなように、アルカリホスファターゼ活性の強い活性およびＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１抗原の強い発現を確認した。以上のように、ＰＩＳ細胞はｉＰＳ細胞に特異的なタンパク質を発現していることを証明できた。 FIG. 3 is a diagram in which chromosomes are fluorescently stained, and is a diagram of activity staining or immunostaining with a fluorescent substance. If there is fluorescence, it is shown in gray in the picture of FIG. As is clear from FIG. 3, strong alkaline phosphatase activity and strong expression of SSEA-4, TRA-1-60 and TRA-1-81 antigens were confirmed. As described above, it was proved that PIS cells expressed a protein specific to iPS cells.
実施例３：ＰＩＳ細胞が様々な細胞に分化する能力を保持していることの確認 Example 3: Confirmation that PIS cells retain the ability to differentiate into various cells
　この実験は、ＰＩＳ細胞が、胚様体を形成し、様々な細胞に再分化できる能力があることを証明するために行った。 This experiment was conducted to prove that PIS cells have the ability to form embryoid bodies and redifferentiate into various cells.
　実施例２において得られたＰＩＳ細胞を用いて、心筋細胞に分化させる実験を行った。PSdif-Cardio　Cardiomyocyte　Differentiation　Kit（フナコシ株式会社）を用い、当該キットが提供するマニュアルに従って、培養を行った。心筋細胞に分化誘導した結果を図４に示した。ＰＩＳ細胞は、ＥＢを形成し（図４Ａ）、前駆心筋細胞（図４Ｂ）に変化し、最終的には微細な振動をする心筋細胞（図４Ｃ）に変化した。以上のように、ＰＩＳ細胞は、胚葉体および体細胞に分化する能力を有する多能性幹細胞であることを証明できた。 Experiment using PIS cells obtained in Example 2 to differentiate into cardiomyocytes was performed. Culture was performed using PSdif-Cardio Cardiomyocyte Differentiation Kit (Funakoshi Co., Ltd.) according to the manual provided by the kit. The results of differentiation induction into cardiomyocytes are shown in FIG. PIS cells formed EBs (FIG. 4A), changed to progenitor cardiomyocytes (FIG. 4B), and finally changed to cardiomyocytes with fine oscillations (FIG. 4C). As described above, PIS cells were proved to be pluripotent stem cells having the ability to differentiate into embryoid bodies and somatic cells.
実施例４：ヒト線維芽細胞以外の細胞でもＰＩＳ細胞を作製できることの確認 Example 4: Confirmation that PIS cells can be produced using cells other than human fibroblasts
　この実験は、ヒト線維芽細胞以外の細胞から、ＰＩＳ細胞を作製できることを確認するために行った。 This experiment was conducted to confirm that PIS cells can be produced from cells other than human fibroblasts.
　マウス前駆脂肪細胞株３Ｔ３－Ｌ１（図５Ａ）をＥＤＣＣより入手した。３Ｔ３－Ｌ１株を６ｍＬのＤＭＥＭ（Dulbecco's　modified　Eagle　Medium）を含む２５ｃｍ２の培養フラスコで１日間培養し、細胞を付着させた。１ｍＭのスペルミンを添加したものを、３７℃で３日間ＣＯ２インキュベーター中で培養した。その結果、実施例１と同様に、ＥＳ細胞様の細胞（ＰＩＳ－Ｌ１細胞）を得ることができ、図５のＢに示すようにＰＩＳ－Ｌ１細胞は、ＥＢを形成した。以上のように、ＰＩＳ細胞の作製方法は、ヒト線維芽細胞だけでなく、マウス脂肪細胞など様々な種の様々な細胞に適用できる方法であることを証明できた。 Mouse preadipocyte cell line 3T3-L1 (FIG. 5A) was obtained from EDCC. The 3T3-L1 strain was cultured in a 25 cm 2 culture flask containing 6 mL of DMEM (Dulbecco's modified Eagle Medium) for 1 day to allow the cells to adhere. The one supplemented with 1 mM spermine was cultured at 37 ° C. for 3 days in a CO 2 incubator. As a result, ES cell-like cells (PIS-L1 cells) could be obtained in the same manner as in Example 1, and the PIS-L1 cells formed EBs as shown in FIG. 5B. As described above, it was proved that the method for producing PIS cells can be applied not only to human fibroblasts but also to various cells of various species such as mouse adipocytes.
実施例５：ＤＮＡメチル化阻害物質に細胞を初期化する能力があり、ＤＩＳ細胞に変化することの証明 Example 5: Demonstration that a DNA methylation inhibitor has the ability to reprogram cells and changes to DIS cells
　この実験は、ＤＮＡメチル化阻害剤に、細胞をＥＳ細胞の状態まで初期化する能力があり、ＤＩＳ細胞を作製できることを証明するために実施した。 This experiment was conducted in order to prove that the DNA methylation inhibitor has the ability to initialize cells to the state of ES cells and can produce DIS cells.
　キアゲン社のＤＮＡメチル化酵素（ＤＮＭＴ１、ＤＮＭＴ３ＡおよびＤＮＭＴ３Ｂ）をコードする遺伝子の発現を阻害するｓｉＲＮＡ（カタログ番号SI000189910、SI00982338およびSI00165382）を入手した。また、マウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２（EC90110523-F0）をＥＣＡＣＣより入手した。２４穴のプレートを用いて、ＤＭＥＭ中で該細胞を１日間培養し、細胞をプレートの各ウェルの底面に付着させた。ｓｉＲＮＡによる遺伝子のサイレンシングは、RNAi　Human/Mouse　Starter　Kit（キアゲン社）を用いて行った。これら３つのｓｉＲＮＡ（５ｎＭ）を含む２００μＬのＤＭＥＭ（血清を含まない）に、６μＬのHyperfect　Transfer　Reagentを加えてｓｉＲＮＡの沈澱物を形成した後、１日間培養した。その後、２００μＬのＬＩＦ（Ｗａｋｏ（株）　カタログ番号129-05601）を添加したＥＳ細胞用無血清培地（ＤＳファーマバイオ社）と交換しさらに３日間培養した。その結果、図６のＡに示すように、マウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２は、ＤＩＳ細胞（ＤＩＳ－ＳＩ細胞）に変化したが、変化しないマウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２は細胞底面に付着したままであり、ＤＩＳ－ＳＩ細胞のみを培地から回収できた。 SiRNAs (catalog numbers SI000189910, SI00982338 and SI00165382) that inhibit the expression of genes encoding Qiagen DNA methylases (DNMT1, DNMT3A and DNMT3B) were obtained. In addition, mouse adipocyte-derived strain C3H10T1 / 2 (EC90110523-F0) was obtained from ECACC. The cells were cultured in DMEM for 1 day using a 24-well plate and the cells were allowed to attach to the bottom of each well of the plate. Gene silencing by siRNA was performed using RNAi Human / Mouse Starter Kit (Qiagen). 6 μL of Hyperfect Transfer Reagent was added to 200 μL of DMEM (serum-free) containing these three siRNAs (5 nM) to form a siRNA precipitate, followed by culturing for 1 day. Thereafter, the medium was replaced with a serum-free medium for ES cells (DS PharmaBio) supplemented with 200 μL of LIF (Wako Co., Ltd. catalog number 129-05601) and further cultured for 3 days. As a result, as shown in FIG. 6A, the mouse adipocyte-derived strain C3H10T1 / 2 was changed to a DIS cell (DIS-SI cell), but the mouse adipocyte-derived strain C3H10T1 / 2 that was not changed was attached to the cell bottom surface. As such, only DIS-SI cells could be recovered from the medium.
　５－Ａｚａ－ｄｃについても同様の実験を行った。Ｃ３Ｈ１０Ｔ１／２株を５００ｍＬのＤＭＥＭを含む２５ｃｍ２の培養フラスコで１日間培養し、細胞を付着させた４０μＭの５－Ａｚａ－ｄｃを添加したＤＭＥＭで培養した後、６ｍＬのＬＩＦ（Ｗａｋｏ（株）　カタログ番号129-05601）と４０μＭの５－Ａｚａ－ｄｃを添加したＥＳ細胞用無血清培地（ＤＳファーマバイオ社）と交換しさらに３日間培養した。その結果、図６のＢに示すように、前駆脂肪細胞の３Ｔ３－Ｌ１は、ＤＩＳ細胞（ＤＩＳ－Ａｚａ細胞と呼ぶ）に変化したが３Ｔ３－Ｌ１細胞は細胞底面に付着したままであり、ＤＩＳ－Ａｚａ細胞のみを培地から回収できた。 A similar experiment was conducted for 5-Aza-dc. C3H10T1 / 2 strain was cultured for 1 day in a 25 cm 2 culture flask containing 500 mL of DMEM, cultured in DMEM supplemented with 40 μM 5-Aza-dc to which cells were attached, and then 6 mL of LIF (Wako Corp.). The medium was replaced with a serum-free medium for ES cells (DS PharmaBio) supplemented with catalog number 129-05601) and 40 μM 5-Aza-dc, and further cultured for 3 days. As a result, as shown in FIG. 6B, the preadipocyte 3T3-L1 was changed to a DIS cell (referred to as DIS-Aza cell), but the 3T3-L1 cell remained attached to the cell bottom. -Only Aza cells could be recovered from the medium.
　さらに、得られたＤＩＳ－Ａｚａ細胞をマウスＥＳ細胞から造血系細胞への分化培地であるES-Cut　Hematopoietic　Differentiation　Kit　with　Cytokine（ベリタス社、カタログ番号ST-03160）を用いて、初期分化を行った。その結果、図６のＣに示すように血球系細胞への初期分化を確認した。また、神経前駆細胞への分化培地（STEMdiff　Neural　Induction　Medium、ベリタス社）を用いて、ＤＩＳ－Ａｚａ細胞を神経細胞に分化させた。図６のＤに示すように、ＤＩＳ－Ａｚａ細胞は、神経幹細胞および神経様細胞に分化した。以上のように、ＤＮＡメチル化阻害物質を用いてもポリアミンと同様に、細胞を多能性幹細胞に初期化する能力があり、様々な細胞に分化するＤＩＳ細胞を作製できることを証明できた。 Furthermore, the obtained DIS-Aza cells were subjected to initial differentiation using ES-Cut Hematopoietic Differentiation Kit with Cytokine (Veritas, catalog number ST-03160), which is a differentiation medium from mouse ES cells to hematopoietic cells. . As a result, initial differentiation into hematopoietic cells was confirmed as shown in FIG. Further, DIS-Aza cells were differentiated into neurons using a differentiation medium for neural progenitor cells (STEMdiff Neural Induction Medium, Veritas). As shown in FIG. 6D, DIS-Aza cells differentiated into neural stem cells and nerve-like cells. As described above, it was proved that, even if a DNA methylation inhibitor is used, it has the ability to initialize cells to pluripotent stem cells, as in the case of polyamines, and it is possible to produce DIS cells that differentiate into various cells.
実施例６：高効率でＰＩＳ細胞を作製できることの確認 Example 6: Confirmation that PIS cells can be produced with high efficiency
　この実験は、（１）体細胞を高効率でＰＩＳ細胞にでき、（２）得られたＰＩＳ細胞を高効率で外胚葉、中胚葉または内胚葉に分化することが可能であり、（３）高効率で様々な細胞に分化できることを証明するために実施した。 In this experiment, (1) somatic cells can be converted into PIS cells with high efficiency, (2) the obtained PIS cells can be differentiated into ectoderm, mesoderm or endoderm with high efficiency, and (3) This was carried out to prove that the cells can be differentiated into various cells with high efficiency.
　ヒト肺由来の線維芽細胞ＴＩＧ－１－２０（分裂回数３０－３５回）を、６ｍLの最少培地（ＭＥＭ）を用いて、２５ｃｍ２の培養フラスコ中で1日間培養し、フラスコの底面に付着させた。培養フラスコに、Ｒｏｃｋ阻害剤である２０μＭのＹ－２７６３２および５μＭのチアゾビビンと、アポトーシス阻害剤である６０μＭのｐ５３阻害剤（Ｃｙｃｌｉｃ　ｐｉｆｔｈｒｉｎ―α―ｈｙｄｒｏｂｔｏｍｉｄｅ）および３０μＭのＢａｘ阻害剤（Ｂａｘ　Ｉｎｈｉｂｉｔｏｒ　Ｐｅｐｔｉｄｅ　Ｖ５）とを添加して８時間培養し、この４種類の阻害剤を十分に細胞内に取り込ませた。その後、１００ｍＭのスペルミン溶液を添加して、２４時間培養した。培養した細胞は、阻害剤の影響によりＲｏｃｋの活性化とアポトーシスとが抑制されるためにやや縮んだ状態になるが、それ以外には変化が見られなかった。また、Ｈｏｅｃｈｓｔ　３３３４２で核染色した場合も、アポトーシス時に起こる染色体の断片化は見られなかった。 Human lung-derived fibroblasts TIG-1-20 (number of divisions 30-35) were cultured in a 25 cm 2 culture flask for 1 day using 6 mL of minimal medium (MEM) and attached to the bottom of the flask I let you. In a culture flask, 20 μM Y-27632 and 5 μM thiazobibin, which are a Rock inhibitor, and 60 μM p53 inhibitor (Cyclic pifthrin-α-hydrobide) and 30 μM Bax inhibitor (Bax Inhibitor Peptide Peptide) are apoptosis inhibitors. Was added and cultured for 8 hours, and these four types of inhibitors were sufficiently taken into the cells. Thereafter, 100 mM spermine solution was added and cultured for 24 hours. The cultured cells were slightly contracted due to the inhibition of Rock activation and apoptosis due to the influence of the inhibitor, but no other changes were observed. In addition, even when nuclear staining was performed with Hoechst 33342, chromosome fragmentation that occurred during apoptosis was not observed.
　培養フラスコに、Ｔｒｙｐ－ＬＥ　Ｓｅｌｅｃｔ溶液（Ｇｉｂｃｏ）０．８ｍＬを添加して細胞をはがし、ＰＢＳ溶液１０ｍＬに入れた後、遠心分離（８００ｒｐｍ、３分間）により細胞を回収した。上澄みを取り除いた後、ＰＢＳ溶液１０ｍＬを用いて同様の操作を行った。得られた細胞を、ＤＥＦ－ＣＳ　５００　Ｃｕｌｔｕｒｅ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて２日間培養し、細胞の修復を行うと同時に、安定したＰＩＳ細胞を得た。その後、このＰＩＳ細胞を、Ｓｔｅｍ　ＸＶｉｖｏ　Ｅｃｔｏｄｅｒｍ　Ｋｉｔ、Ｓｔｅｍ　ＸＶｉｖｏ　Ｍｅｓｏｄｅｒｍ　Ｋｉｔ、Ｓｔｅｍ　ＸＶｉｖｏ　Ｅｎｄｏｄｅｒｍ　Ｋｉｔ　（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）を用いて、外胚葉、中胚葉または内胚葉に分化させた。分化した細胞は、これらのキットに含まれている抗ヒトＯｃｔ２ヤギ抗体、抗ヒトＢｒａｃｈｙｕｒｙヤギ抗体および抗ヒトＳＯＸ１７ヤギ抗体を一次抗体として用い、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）　５５５を結合した抗ヤギＩｇＧウサギ抗体を二次抗体として用いて、蛍光免疫染色を行った。その結果、図７のＡ～Ｃに示すように、ほとんどの細胞が効率良く（９０％以上）、外胚葉、中胚葉または内胚葉に分化していることを確認した。 To the culture flask, 0.8 mL of Tryp-LE Select solution (Gibco) was added to peel off the cells. After putting in 10 mL of PBS solution, the cells were collected by centrifugation (800 rpm, 3 minutes). After removing the supernatant, the same operation was performed using 10 mL of PBS solution. The obtained cells were cultured for 2 days using DEF-CS 500 Culture System (Takara Bio Inc.) to repair the cells, and at the same time, stable PIS cells were obtained. The PIS cells were then differentiated into ectoderm, mesoderm or endoderm using Stem XVivo Ectoderm Kit, Stem XVivo Mesoderm Kit, Stem XVivo Enderdom Kit (R & D Systems). The differentiated cells were obtained by using anti-human Oct2 goat antibody, anti-human Brachyury goat antibody and anti-human SOX17 goat antibody included in these kits as primary antibodies, and anti-goat IgG rabbits bound with Alexa Fluor (registered trademark) 555. Immunofluorescence staining was performed using the antibody as a secondary antibody. As a result, as shown in FIGS. 7A to 7C, it was confirmed that most cells were efficiently (90% or more) and differentiated into ectoderm, mesoderm or endoderm.
　さらに、ＰＩＳ細胞に対し、ＰＳ－ｄｉｆ　ＢＡ　Ｂｒｏｗｎ　Ａｄｉｐｏｃｙｔｅ　Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　Ｋｉｔ（ベリタス社）を用いて、中胚葉への誘導および褐色脂肪細胞への分化を行った。得られた細胞は、抗ＵＣＰ１ウサギ抗体（Ｂｉｏｓｉｓ社：ｂｓ１９２Ｒ）を用いて免疫染色した。また、０．１ｍＭのオレイン酸を添加して培養した。その結果、分化した細胞は、褐色を呈するだけでなく、図８のＡに示すようにＵＣＰ１を発現しており、図８のＢに示すように脂肪を蓄積する性質があった。このことから、ＰＩＳ細胞が褐色脂肪細胞に分化していることを確認した。また、生存した細胞の褐色脂肪細胞へ分化効率は９０％以上であり、非常に高効率で分化できることを確認した。 Furthermore, PIS cells were induced into mesoderm and differentiated into brown adipocytes using PS-dif BA Brown Adiposite Difference Kit (Veritas). The obtained cells were immunostained using an anti-UCP1 rabbit antibody (Biosis: bs192R). Moreover, 0.1 mM oleic acid was added and cultured. As a result, the differentiated cells were not only brown, but also expressed UCP1 as shown in FIG. 8A, and had the property of accumulating fat as shown in FIG. 8B. From this, it was confirmed that PIS cells were differentiated into brown adipocytes. Moreover, the differentiation efficiency of the surviving cells into brown adipocytes was 90% or more, and it was confirmed that differentiation was possible with very high efficiency.
実施例７：脂肪細胞からＰＩＳ細胞を作製でき、高効率で分化できることの確認 Example 7: Confirmation that PIS cells can be produced from adipocytes and can be differentiated with high efficiency
　この実験は、体内に多量に存在し、且つ脂肪吸引等で多量に得られる脂肪細胞から、効率よくＰＩＳ細胞を作製および分化させることが可能であることを証明するために実施した。 This experiment was conducted in order to prove that PIS cells can be efficiently produced and differentiated from adipocytes present in large amounts in the body and obtained in large amounts by liposuction or the like.
　マウス脂肪細胞３Ｔ３－Ｌ２４（N.Shiomi　et　al.(2011)　JBiSE　4,　684）を、６ｍＬのＤＭＥＭを用いて、２５ｃｍ２の培養フラスコで１日間培養し、フラスコの底面に付着させた。培養フラスコに、Ｒｏｃｋ阻害剤である２０μＭのＹ－２７６３２および５μＭのチアゾビビンと、アポトーシス阻害剤である６０μＭのｐ５３阻害剤（Ｃｙｃｌｉｃ　ｐｉｆｔｈｒｉｎ－α－ｈｙｄｒｏｂｔｏｍｉｄｅ）および３０μＭのＢａｘ阻害剤（Ｂａｘ　Ｉｎｈｉｂｉｔｏｒ　Ｐｅｐｔｉｄｅ　Ｖ５）と、を添加して８時間培養し、この４種類の阻害剤を十分に細胞内に取り込ませた。その後、１００ｍＭのスペルミン溶液を添加して、２日間培養した。Ｔｒｙｐ－ＬＥ　Ｓｅｌｅｃｔ溶液（Ｇｉｂｃｏ）１ｍＬを添加して細胞をはがし、ＰＢＳ溶液１０ｍＬに入れた後、遠心分離（８００ｒｐｍ、３分間）により細胞を回収した。上澄みを取り除いた後、ＰＢＳ溶液１０ｍＬを用いて同様の操作を行った。さらに、得られた細胞は、ＳｔｅｍＭｅｄｉｕｍ（登録商標）マウスＥＳ細胞用無血清培地（ＤＳファーマバイオメディカル株式会社）を用いて２日間培養し、細胞の修復を行うと同時に、形質の安定したＰＩＳ細胞を得た。 Mouse adipocyte 3T3-L24 (N. Shiomi et al. (2011) JBiSE 4, 684) was cultured in a 25 cm 2 culture flask for 1 day using 6 mL of DMEM and attached to the bottom of the flask. In a culture flask, a Rock inhibitor, 20 μM Y-27632 and 5 μM thiazobibin, and an apoptosis inhibitor, 60 μM p53 inhibitor (Cyclic pifthrin-α-hydrobide) and 30 μM Bax inhibitor (Bax Inhibitor Peptide) Were added and cultured for 8 hours, and these four types of inhibitors were sufficiently taken into the cells. Thereafter, 100 mM spermine solution was added and cultured for 2 days. The cells were detached by adding 1 mL of Tryp-LE Select solution (Gibco), put into 10 mL of PBS solution, and then collected by centrifugation (800 rpm, 3 minutes). After removing the supernatant, the same operation was performed using 10 mL of PBS solution. Furthermore, the obtained cells were cultured for 2 days using a serum-free medium for Stemdium (registered trademark) mouse ES cells (DS Pharma Biomedical Co., Ltd.) to repair the cells, and at the same time, stable PIS cells Got.
　ＳｔｅｍＳｕｒｅ（登録商標）　０．１ｗ・ｖ％ゼラチン溶液（和光純薬工業株式会社）を用いてコーティングされた２４ウェルの培養プレートの各ウェルに、神経分化培地ＮＤｉｆｆ（登録商標）　２２７（タカラバイオ株式会社：Ｙ４０００２）を２ｍＬ入れ、ＰＩＳ細胞を３日間培養して神経幹細胞に誘導した。さらに、これらの細胞を、Ｎｅｕｒｏｃｕｌｔ（登録商標）　Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　Ｍｅｄｉｕｍ　（ベリタス社）を用いて神経細胞やグリア細胞に分化させた。得られた神経系の細胞に対し、抗β―チューブリンIIIウサギ抗体とＭＡＰ２ウサギ抗体を一次抗体、Ｃｙ３結合抗ウサギヤギ抗体を二次抗体として、免疫染色を行った。その結果、図９のＡに示すように、ほぼ１００％の細胞が神経幹細胞に分化した。図９のＢとＣに示すように、さらに神経幹細胞から分化した細胞も、ほぼ１００％が成熟神経細胞であることを確認した。 StemSure (registered trademark) 0.1 w · v% gelatin solution (Wako Pure Chemical Industries, Ltd.) coated in each well of a 24-well culture plate, neurodifferentiation medium NDiff (registered trademark) 227 (Takara Bio Inc.) 2 mL of company: Y40002) was added, and PIS cells were cultured for 3 days to induce neural stem cells. Furthermore, these cells were differentiated into nerve cells and glial cells using Neurocult (registered trademark) Differentiation Medium (Veritas). The obtained nervous system cells were immunostained using an anti-β-tubulin III rabbit antibody and a MAP2 rabbit antibody as a primary antibody, and a Cy3-conjugated anti-rabbit goat antibody as a secondary antibody. As a result, as shown in FIG. 9A, almost 100% of the cells differentiated into neural stem cells. As shown in FIGS. 9B and 9C, it was confirmed that almost 100% of the cells further differentiated from the neural stem cells were mature neurons.
　今回開示された実施形態および実施例はすべての点で例示であって制限的なものではないと考えられるべきである。本発明の範囲は上記した説明ではなくて請求の範囲によって示され、請求の範囲と均等の意味および範囲内でのすべての変更が含まれることが意図される。 The embodiments and examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.
　本発明の多能性幹細胞の製造方法は、ｉＰＳの作製よりも、はるかに簡便、迅速かつ高効率である。したがって、再生医療の分野の様々な治療に利用可能であり、産業上の利用価値は極めて高い。 The method for producing pluripotent stem cells of the present invention is much simpler, faster and more efficient than iPS production. Therefore, it can be used for various treatments in the field of regenerative medicine, and its industrial utility value is extremely high.

Claims (3)

  1. 　ポリアミン、または、ＤＮＡメチル化阻害物質を含む培地中で、動物細胞を培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法。 A method for producing pluripotent stem cells, wherein pluripotent stem cells are obtained by culturing animal cells in a medium containing polyamine or a DNA methylation inhibitor.
  2. 　前記ポリアミンは、スペルミン、スペルミジン、プトレシン、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体、からなる群から選択される少なくとも１種である、請求項１に記載の製造方法。 2. The production according to claim 1, wherein the polyamine is at least one selected from the group consisting of spermine, spermidine, putrescine, and acetylated products thereof, and two or more polymers of the polyamines. Method.
  3. 　前記ＤＮＡメチル化阻害物質は、ＤＮＡメチル化酵素をコードする遺伝子の発現を阻害するｓｉＲＮＡ、５－アザ－２’－デオキシシチジン、シネフンギン、ゼブラリン、エチオニン、アセチルメチオニン、および、セレノメチオニンからなる群から選択される少なくとも１種である、請求項１または２に記載の製造方法。 The DNA methylation inhibitor is selected from the group consisting of siRNA, 5-aza-2′-deoxycytidine, cinefungin, zebralin, ethionine, acetylmethionine, and selenomethionine that inhibits the expression of a gene encoding a DNA methylase. The production method according to claim 1, wherein the production method is at least one selected.
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