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WO2018116511A1 - Procédé de production de cellules souches pluripotentes - Google Patents

Procédé de production de cellules souches pluripotentes 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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Kobe College
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Kobe College
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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

L'invention concerne un procédé de production de cellules souches pluripotentes permettant d'obtenir des cellules souches pluripotentes présentant une fonction comparable à des cellules SPi. Le procédé de production de cellules souches pluripotentes consiste à cultiver des cellules animales dans un milieu contenant une polyamine ou un inhibiteur de méthylation de l'ADN et à acquérir ainsi les cellules souches pluripotentes.
PCT/JP2017/026565 2016-12-19 2017-07-21 Procédé de production de cellules souches pluripotentes Ceased WO2018116511A1 (fr)

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JP2022504764A (ja) * 2018-10-11 2022-01-13 ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ 細胞培養のための組成物および方法

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JP2011050379A (ja) * 2009-08-07 2011-03-17 Kyoto Univ イヌｉＰＳ細胞及びその製造方法
WO2013100140A1 (fr) * 2011-12-29 2013-07-04 国立大学法人 京都大学 Induction de cellules souches pluripotentes à partir de cellules germinales

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JP2011050379A (ja) * 2009-08-07 2011-03-17 Kyoto Univ イヌｉＰＳ細胞及びその製造方法
WO2013100140A1 (fr) * 2011-12-29 2013-07-04 国立大学法人 京都大学 Induction de cellules souches pluripotentes à partir de cellules germinales

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022504764A (ja) * 2018-10-11 2022-01-13 ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ 細胞培養のための組成物および方法
JP7635119B2 (ja) 2018-10-11 2025-02-25 ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ 細胞培養のための組成物および方法

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