WO2015057015A1 - Procédé de redifférenciation de cellules adultes en cellules souches pluripotentes induites au moyen d'un champ électromagnétique - Google Patents

Procédé de redifférenciation de cellules adultes en cellules souches pluripotentes induites au moyen d'un champ électromagnétique Download PDF

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WO2015057015A1
WO2015057015A1 PCT/KR2014/009792 KR2014009792W WO2015057015A1 WO 2015057015 A1 WO2015057015 A1 WO 2015057015A1 KR 2014009792 W KR2014009792 W KR 2014009792W WO 2015057015 A1 WO2015057015 A1 WO 2015057015A1
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cells
stem cells
pluripotent stem
induced pluripotent
adult
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김종필
박정극
백순봉
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동국대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • the present invention relates to a method for dedifferentiating adult cells using electromagnetic fields into induced pluripotent stem cells.
  • Stem cells are undifferentiated cells that can infinitely regenerate and differentiate into cells of all tissues of the body. Stem cell research is an important research area for research on the development of cellular medicines such as regenerative medicine, new drug development, the causes and treatment of human diseases, and the development of human bodies.
  • Pluripotent stem cells are cells up to 8 cells after fertilization of eggs and sperm. When these cells are separated and transplanted into the uterus, they can develop into one complete individual. Pluripotent stem cells originate from the inner cell mass located inside the blastocytes, which appear after 4-5 days of fertilization, and can occur with a variety of cells and tissues, but do not form new organisms. can not do it.
  • Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs in which they are contained. Embryonic stem cells of stem cells are made from the inner cell mass of embryos before implantation, can differentiate into more than 200 cells under appropriate circumstances, and can make whole organs (Nagy et al., Development, 110). : 815-821, 1990). However, embryonic stem cells as cell therapy have ethical problems that must be made by using eggs, and can be obtained only by destroying the embryos.
  • Induced pluripotent stem cells refer to cells having pluripotency by dedifferentiating differentiated cells, and are capable of differentiating into all types of cells of the body with the ability to self-renewal similarly to embryonic stem cells.
  • induced pluripotent stem cells have been reported to have very similar characteristics in embryonic stem cells, pluripotent stem cells, in terms of genetic and epigenetic properties and differentiation in gene expression and differentiation capacity (Takahashi and Yamanaka, Cell, 126: 663-676). , 2006).
  • intracellular introduction of dedifferentiation factors is most effective to date by means of intracellular delivery using viral vectors.
  • a technique that combines known waves into biology is a device for applying low-frequency energy of about 10 Hz or less to brain tissue.
  • Device public patent US20060205993
  • Zheng developed a technique (JP 2008-543388) to improve brain function by combining high frequency or multiple frequency components to give magnetic stimulation to the central nervous system.
  • Riken manufactures nerve cells by electropulsing embryonic stem cells.
  • Technology US200740065941 has been developed.
  • Gliner et al. Developed a technique for producing neurons by treating the cells with electric pulses (US20050075679).
  • the present inventors continue to study a technique for efficiently reprogramming cells to dedifferentiate them into induced pluripotent stem cells, and when culturing adult cells in an electromagnetic field having a specific frequency, the dedifferentiation efficiency is significantly high. By confirming that the present invention was completed.
  • the present invention also provides an induced pluripotent stem cell dedifferentiated by the above method.
  • the present invention provides a method for dedifferentiating adult cells into induced pluripotent stem cells, including culturing adult cells under electromagnetic fields.
  • the present invention also provides induced pluripotent stem cells dedifferentiated by the above method.
  • electromagnetic field refers to a phenomenon in which electromagnetic fields whose intensity changes periodically are propagated into space, and have the same meaning as electromagnetic waves, and the electromagnetic fields used in the present invention may include both pulse wave forms and continuous wave (sine wave) forms. have.
  • the frequency of the electromagnetic field may be 1 to 200 Hz, preferably 50 Hz, and the strength of the electromagnetic field may be 0.1 to 15 mT, preferably 0.5 to 2 mT.
  • stem cells are cells that have the ability to differentiate into various types of body tissues, that is, undifferentiated cells and can be classified into embryonic stem cells and adult stem cells.
  • Embryonic stem cells are undifferentiated cells that have differentiation ability, but have not yet differentiated, and mean cells with pluripotency capable of differentiating into various tissue cells if appropriate conditions are set in such an undifferentiated state. In its broadest sense, it also includes embryoid bodies derived from embryonic stem cells.
  • Adult stem cell means a cell having a limited differentiation capacity that cannot differentiate into all tissues but can differentiate into each target organ.
  • induced pluripotent stem cells refers to cells having pluripotency by dedifferentiating differentiated cells, and have all the types of cells of the body with the ability to self-renewal similarly to embryonic stem cells. It is characterized by being capable of differentiation and is also referred to as "dedifferentiated stem cells”. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have similar cell morphology, similar gene and protein expression patterns, pluripotency in vitro and in vivo, and teratoma. When formed and inserted into the blastocyst of the mouse, chimera mice are formed and germline transmission of the gene is possible.
  • adult cell refers to a cell derived from an adult born and alive, as opposed to embryonic cells.
  • the genetic background of the adult cells used in the present invention is not limited, but canine, feline, boar, bovine, deer and animal, giraffe and animal, pelican, camel, hippopotamus, horse and animal, May be derived from one or more selected from the group consisting of veins, rhinos, animals, weasels, rabbits, rodents and primates.
  • the adult cells may be autologous, allogeneic or xenogeneic, and limit their types such as fat cells, fibroblasts, fibroblasts, muscle cells, heart cells, blood cells, bone marrow cells, germ cells, etc. I never do that.
  • Dedifferentiation in the present invention refers to an epigenetic retrograde process that allows partial or final differentiated cells to return to an undifferentiated state, such as pluripotency or pluripotency, to allow formation of new differentiated tissue.
  • This reverse differentiation is possible because epigenetic changes in the cell genome are not fixed but are reversible processes that can be erased and re-formed.
  • Reverse differentiation also called “reprogramming,” relates to the process of changing the genetic and expressive profile of partially or finally differentiated cells to be similar to that of embryonic stem cells. For example, such changes include changes in the methylation pattern, changes in the expression rate of stem cell genes, and the like.
  • Artificial dedifferentiation process is performed by the use of virus-mediated or non-viral vectors with retroviruses and lentiviruses, introduction of non-virus-mediated dedifferentiation factors using protein and cell extracts, or by stem cell extracts, compounds, etc. Although it includes a reverse differentiation process, it is preferable to use a lentiviral in the present invention.
  • Reverse differentiation factors in the present invention are Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28, preferably Oct4, Sox2, Klf4 and C-Myc, but is not limited thereto.
  • the step of delivering dedifferentiation inducers into the somatic cells is essential.
  • Oct4, Sox2, Klf4 and retroviruses are used as transporters.
  • Genes encoding c-Myc's dedifferentiation inducers are transferred to somatic cells (Takahashi. K. et al, Cell, 131: 861-872, 2007), or using lentiviruses as transporters, Oct4, Sox2, Klf4 and Genes encoding c-Myc dedifferentiation inducers can be transferred to somatic cells.
  • Sox family genes are known to play an important role in maintaining pluripotency, similar to Oct4. But while the Oct4 gene is only involved in pluripotent stem cells, the Sox family of genes is also associated with pluripotent stem cells or pluripotent stem cells.
  • the SRY-type high mobility group box 2 (Sox2) transcription factor is the only Sox family protein that plays an important role in maintaining pluripotency of embryonic stem cells (Avilion et al., Genes Dev, 17: 126-140, 2003). Inhibition of Sox-2 expression in mouse embryonic stem cells, as in Oct4, induces differentiation (Ivanova et al., Nature, 442: 533-538, 2006).
  • Sox-2 binding sites found in the promoter regions of several Sox2 subgenes often exist adjacent to Oct4 and Nanog binding sites (Boyer et al., Cell, 122: 947-956, 2005). Therefore, the interaction between Sox2 transcription factor and Oct4 transcription factor is expected to provide a basic framework for induced pluripotent stem cells to maintain their undifferentiated state and to characterize embryonic stem cells (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18). : 467-473, 2007).
  • c-Myc is a carcinogenic gene that performs a variety of intracellular functions such as cell growth, differentiation, proliferation, cell death and transformation into cancer cells. It has also been identified as a subgene of LIF (Leukemia Inhibitory Factor) / STAT3 and Wnt signaling mechanisms, which are key mechanisms for maintaining pluripotency (Sears et al., Genes Dev, 14: 2501-2514, 2000).
  • LIF Leukemia Inhibitory Factor
  • STAT3 Wnt signaling mechanisms
  • c-Myc not only binds to Myc recognition sites in the genome, but also changes the chromatin structure to help Oct4 and Sox2 bind well to target genes (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18: 467-473, 2007).
  • Klf4 is involved in growth inhibition and regulates the cell cycle. Recent studies have shown that, similar to c-Myc, it acts as a sub-gene of STAT3 in embryonic stem cells and inhibits the differentiation of mouse embryonic stem cells by maintaining Oct4 expression upon overexpression (Li et al., Blood, 105: 635-637, 2005).
  • Nanog is an embryo-specific gene that, like Oct4 and Sox2, is required for maintaining pluripotency of embryonic stem cells.
  • LIN28 is also an mRNA binding protein expressed in embryonic stem cells and embryonic tumor cells and is known to be involved in differentiation and proliferation (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007 ).
  • Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 genes are referred to as "reprogramming-inducing genes," which are genes that can reprogram differentiated cells. I mean.
  • Oct4, Sox2, Klf4 and c-Myc are called Yamanaka factors.
  • the adult cells can be cultured in FBS (Fetal Bovine serum) and penicillin / streptomycin-added medium.
  • FBS Fetal Bovine serum
  • the medium may be DMEM (Dulbecco's Modified Eagle Medium), but is not limited thereto. 5-15% (v / v) Fetal Bovine serum (FBS), preferably 10% FBS, 0.1-5% (v / v) penicillin / streptomycin, preferably 1% penicillin / streptomycin Adult cells can be cultured in the medium to which the medium is added, and the culture can be cultured for 1 to 10 days, preferably 5 days.
  • FBS Fetal Bovine serum
  • penicillin / streptomycin preferably 1% penicillin / streptomycin
  • adult cells are adult cells derived from a mouse, in a medium containing FBS (Fetal Bovine serum), non-essential amino acids, penicillin / streptomycin, glutamine, -mercaptoethanol and leukemia inhibitory factor (LIF) Adult cells can be further cultured.
  • FBS Fetal Bovine serum
  • non-essential amino acids penicillin / streptomycin
  • glutamine glutamine
  • -mercaptoethanol leukemia inhibitory factor (LIF)
  • LIF leukemia inhibitory factor
  • the medium for further culture may be DMEM (Dulbecco ⁇ s Modified Eagle Medium), 5-20% (v / v) of FBS (Fetal Bovine serum), preferably 15% of FBS, 0.1-5% (v / v) non-essential amino acids, preferably 1% non-essential amino acids, 0.1-5% (v / v) penicillin / streptomycin, preferably 1% penicillin / streptomycin, 0.1-5% (v / v), preferably 1% (v / v) glutamine (100 mM to 500 mM, preferably 200 mM), 0.1 to 5% (v / v), preferably 1% (v / v) medium containing -mercaptoethanol of v) and Leukemia Inhibitory factor (LIF) of 10 -6 to 10 -5 % (v / v), preferably 4 10 -6 % (v / v)
  • LIF Leukemia Inhibitory factor
  • DF12 culture medium may be used as a medium for further culture, and 10-30% (v / v), preferably 20% (v / v) serum substitute ( serum replacer), 0.1% to 5% (v / v), preferably 1% (v / v) non-essential amino acids, 0.1% to 5% (v / v), preferably 1% penicillin / strepto Mycin, a mixture of 0.1 to 5% (v / v), preferably 1% (v / v) of glutamine and -mercaptoethanol and 10 -5 to 10 -4 % (v / v), preferably 1.4
  • Adult cells may be additionally cultured in a medium containing 10 ⁇ 5 (v / v)% bFGF (bovine fibroblast growth factor), and the culturing may be performed for 20 to 40 days, preferably 30 days.
  • bFGF bovine fibroblast growth factor
  • Induced pluripotent stem cells differentiated according to the method of the present invention have a high expression level of dedifferentiation factor, and the promoter region of dedifferentiation factor is demethylated.
  • the dedifferentiated induced pluripotent stem cells when transplanted into mice, pluripotency is shown. Therefore, the dedifferentiated induced pluripotent stem cells can be usefully used for cell therapy and research in regenerative medicine.
  • a cell therapy product refers to a cell autologous, allogenic, or xenogenic cell in vitro to proliferate or otherwise restore the tissue and function of the cell. It is a medicine used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the biological characteristics of the cell by the method.
  • the field of cellular therapeutics in which the induced pluripotent stem cells induced by the method of the present invention can be used is not limited. For example, tumors, neurodegenerative diseases, immune diseases, nervous system diseases, physical organ damage, nerve damage, diabetes , Cerebrovascular disease, spinal cord injury, osteoblastic metastasis, hematopoietic dysfunction, osteoarthritis, leukocytosis, and the like.
  • the method for reverse differentiation into induced pluripotent stem cells using the electromagnetic field according to the present invention has an excellent efficiency of dedifferentiating adult cells into induced pluripotent stem cells, and can easily obtain induced pluripotent stem cells.
  • the induced pluripotent stem cells prepared by the above method have a high expression level of dedifferentiation factor, and when transplanted into mice, pluripotency is shown. Therefore, the dedifferentiated induced pluripotent stem cells can be usefully used for cell therapy and research in regenerative medicine.
  • Figure 1 shows the GFP-positive induced pluripotent stem cell colonies when 50 Hz electromagnetic wave treatment after introducing the differentiation factors (Oct4, Sox2, c-Myc, Klf4) into the fibroblasts of Oct4-GFP Knock-in mouse It is also.
  • differentiation factors Oct4, Sox2, c-Myc, Klf4
  • Figure 2 shows the GFP-positive cell population using FACS, when 50 Hz electromagnetic wave is treated after introducing the differentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice This is the figure analyzed.
  • Figure 3 shows the introduction of dedifferentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice, followed by time-dependent pluripotency marker gene (Oct4, Sox2 and Nanog) is a diagram showing the change in mRNA expression over time.
  • Figure 4 shows the differentiation factor (Oct4, Sox2, c-Myc, Klf4) introduced into fibroblasts of Oct4-GFP Knock-in mouse, and then treated with 50 Hz electromagnetic wave, the pluripotency marker gene (by immunostaining method) Oct4, Sox2, Nanog) is a diagram confirming the expression at the protein level.
  • FIG. 5 shows the propensity for methylation of Oct4 and Nanog promoters when 50 Hz electromagnetic wave was introduced after the introduction of reverse differentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice. It is also.
  • FIG. 6 is a diagram confirming the pluripotency of induced pluripotent stem cells through the generation of chimeric mice when injected mouse-induced pluripotent stem cells prepared through the electromagnetic field of the present invention in immunodeficient mice.
  • FIG. 7 is a diagram confirming the generation of human induced pluripotent stem cells when the electromagnetic field is treated to human fibroblasts.
  • FIG. 8 is a diagram showing mRNA expression changes of pluripotency marker genes with time when electromagnetic fields were treated to human fibroblasts.
  • FIG. 9 is a diagram confirming the expression of the pluripotency marker gene protein level through immunostaining, when the electromagnetic field is treated to human fibroblasts.
  • FIG. 10 is a diagram confirming the pluripotency by analyzing the teratoma ability when the human induced pluripotent stem cells prepared through the electromagnetic field of the present invention injected into the immunodeficiency mouse.
  • a lentivirus into which four reverse differentiation factors (Oct4 / Sox2 / c-Myc / Klf4) were introduced was introduced. Treated. Thereafter, an electromagnetic wave device generating an electromagnetic field (50 Hz, 0.5 to 2 mT) is mounted in the incubator, and the culture dish is placed in the electromagnetic field, and the ratio of 10% (v / v) of FBS, 1% (v / v) to DMEM is placed. Essential amino acids, 1% (v / v) penicillin / streptomycin, 1% (v / v) 200 mM glutamine,
  • Example 1-1 The induced pluripotent stem cells prepared in Example 1-1 were passaged.
  • mouse embryonic fibroblasts embryonic stage
  • mitomycin C 10 g / ml
  • CO 2 incubator maintained at 37 days prior to passage one day.
  • the next day 5000-1000 induced pluripotent stem cells were treated with trypsinase, and then cultured on the fibroblasts.
  • pluripotency marker genes Oct4, Sox2, Nanog
  • FIG. 3 results of confirming the expression at the protein level of pluripotency marker genes (Oct4, Sox2, Nanog) through the immunostaining method is shown in FIG.
  • FIG. 3 results of demethylation of the promoter region of Oct4, Nanog of the induced pluripotent stem cells of the present invention is shown in FIG.
  • FIG. 3 results of confirming the pluripotency of the induced pluripotent stem cells of the present invention is shown in FIG.
  • pluripotency marker genes Oct4, Sox2, Nanog
  • Human fibroblasts were cultivated to treat lentiviral in which the differentiation factor (Oct4 / Sox2 / c-Myc / Klf4) was introduced, an electromagnetic wave device was placed in the incubator, a culture plate was placed in the electromagnetic field, and 10% of DMEM (v / v) incubated for 5 days in a culture medium containing FBS and 1% (v / v) penicillin / streptomycin (incubation medium every 2 days), then 50 ml (v / v) in 200 ml of DF12 medium.
  • the differentiation factor Oct4 / Sox2 / c-Myc / Klf4
  • Example 2-1 For passage culture of the induced pluripotent stem cells prepared in Example 2-1, after culturing feeder cells in a 6-well plate coated with 0.2% gelatin one day before passage, the induced pluripotent stem cells were fed the next day. Cultured on phase.
  • mRNA was obtained by a conventional method, followed by quantitative PCR of pluripotency marker genes after cDNA synthesis, and the results thereof are shown in FIG. 8.
  • the results of confirming the expression of the pluripotency marker at the protein level through the immunostaining method is shown in FIG.
  • the results of confirming the pluripotency through the analysis of the teratoma ability when the human induced pluripotent stem cells of the present invention injected into the immunodeficiency mouse is shown in FIG.
  • teratoma is By confirming that the three-germ layer is generated, it was confirmed that the induced pluripotent stem cells of the present invention have pluripotency.

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Abstract

La présente invention concerne un procédé de redifférenciation de cellules adultes en cellules souches pluripotentes induites au moyen d'un champ électromagnétique. Selon la présente invention, ledit procédé de redifférenciation en cellules souches pluripotentes induites au moyen d'un champ électromagnétique présente une excellente efficacité pour la redifférenciation de cellules adultes en cellules souches pluripotentes induites, et des cellules souches pluripotentes induites peuvent être facilement obtenues. En outre, les cellules souches pluripotentes induites préparées par ledit procédé présentent un grand nombre d'expressions de facteur de redifférenciation, et lorsque les cellules souches sont greffées dans une souris la pluripotence est visible. Ainsi, les cellules souches plutipotentes induites redifférenciées peuvent être appliquées utilement au développement d'un agent thérapeutique cellulaire et à des études dans le domaine de la médecine régénérative.
PCT/KR2014/009792 2013-10-18 2014-10-17 Procédé de redifférenciation de cellules adultes en cellules souches pluripotentes induites au moyen d'un champ électromagnétique WO2015057015A1 (fr)

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US11118161B2 (en) * 2015-10-26 2021-09-14 Dongguk University Industry-Academy Cooperation Foundation Method for direct transdifferentiation reprogramming into neurons using electromagnetic-induced metal nanoparticles

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KR102128032B1 (ko) * 2018-10-15 2020-06-30 동국대학교 산학협력단 전자기파 반응성 프로모터를 이용하여 타겟 유전자를 과발현시키는 방법
KR20240022421A (ko) * 2022-08-10 2024-02-20 동국대학교 산학협력단 전자기파에 반응하는 유전자 프로모터 및 이의 활용

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US20110275157A1 (en) * 2010-05-10 2011-11-10 Korea University Research And Business Foundation COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Bmi1 AND LOW MOLECULAR WEIGHT SUBSTANCE, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME
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US11118161B2 (en) * 2015-10-26 2021-09-14 Dongguk University Industry-Academy Cooperation Foundation Method for direct transdifferentiation reprogramming into neurons using electromagnetic-induced metal nanoparticles

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