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WO2015088153A1 - Composition contenant du sagunjatang comme principe actif pour favoriser la reprogrammation de cellules en cellules souches pluripotentes induites et procédé pour préparer des cellules souches pluripotentes induites l'utilisant - Google Patents

Composition contenant du sagunjatang comme principe actif pour favoriser la reprogrammation de cellules en cellules souches pluripotentes induites et procédé pour préparer des cellules souches pluripotentes induites l'utilisant Download PDF

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Publication number
WO2015088153A1
WO2015088153A1 PCT/KR2014/011124 KR2014011124W WO2015088153A1 WO 2015088153 A1 WO2015088153 A1 WO 2015088153A1 KR 2014011124 W KR2014011124 W KR 2014011124W WO 2015088153 A1 WO2015088153 A1 WO 2015088153A1
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WIPO (PCT)
Prior art keywords
cells
induced pluripotent
stem cells
pluripotent stem
reprogramming
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Ceased
Application number
PCT/KR2014/011124
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English (en)
Korean (ko)
Inventor
김기모
허덕림
방옥선
김노수
이준
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Korea Institute of Oriental Medicine KIOM
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Korea Institute of Oriental Medicine KIOM
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Publication of WO2015088153A1 publication Critical patent/WO2015088153A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

Definitions

  • composition for promoting reprogramming of cells into induced pluripotent stem cells including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same
  • the present invention was made by the task number C13040 in support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the task is the Korea Institute of Oriental Medicine, the research project name is “creative research project”, the research title is “medicinal herbs based pluripotent induction stem cells ( ips) Development of technology to enhance efficiency ", The lead institution is Korea Institute of Oriental Medicine.
  • the period of research is 2013.02.01 ⁇ 2013.11.30.
  • the present invention relates to a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient, and a method for producing induced pluripotent stem cells using the same.
  • iPSc induced pluripotent stem cells
  • Induced pluripotent progenitor cells refer to cells in which differentiated cells that did not have pluripotency were induced to have pluripotent capacity through artificial dedifferentiation process.
  • 1 Cell shape (isometric, large nucleus and phosphorus, small number) Cytoplasm) and growth rate (embryonic stem cell division time: 17 hr) are similar to embryonic stem cells, 2 gene expression and chromatin modification patterns are similar to embryonic stem cells, 3 pluripotent differentiation ability 4 Can form teratoma in immunodeficient mice, 5 form chimera mouse when inserted into blastocyst of mouse, 6 has germ line transmission of gene .
  • This dedifferentiation technique enables the patient-somatic cells to obtain autologous somatic cells in a relatively easy way, with little physical damage and discomfort to the patient, and thereby to produce induced pluripotent stem cells having characteristics such as human embryonic cells.
  • the company has evolved a strategy to establish a customized self-differentiating stem cell line, and it is recognized as an optimal solution to overcome the bioethics controversy and immunocompatibility problems that may occur when using human embryonic stem cells. It presents unlimited possibilities in the field of development.
  • these dedifferentiated pluripotent stem cells enable the development of immune immunosynthetic cell therapeutics for patients, and may cause differentiation into cells such as brain or heart, which are difficult to obtain cells, to identify the causes and treatment methods for brain diseases and heart diseases.
  • Sagunja-tang is a medicinal herb that consists of ginseng (Panax ginseng), bokyeong (Per ia cocos), baekrye (At ractylodi sj aponi ca) and licorice (Glycyrrhi za uralens is).
  • One object of the present invention is to provide a composition for promoting reprogramming into induced pluripotent stem cells (iPSc) of differentiated cells, including Sagunja-tang as an active ingredient.
  • iPSc induced pluripotent stem cells
  • Another object of the present invention is to provide a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
  • the present invention provides a composition for promoting reprogramming of differentiated cells into induced pluripotent stem cells (iPSc) comprising Sagunja-tang as an active ingredient.
  • the term "Sagunja-tang" means herbal medicines derived from ' Ginseng (Panax ginseng), Bokryeong (Peria cocos), Baekchul (Atractylodis japonica) and licorice (Glycyrrhiza uralensis) as a medicinal herb.
  • Sagunjatang of the present invention is preferably a hot water extract of ginseng, Bokyeong, Baekchul and Licorice.
  • hot water extraction is carried out with 1-2: 1-2: 1—2: 1- 2 weight ratio of ginseng, bokyeong, baekrye and licorice in 5 ⁇ 15 times of water and heated at 90 ⁇ 100 ° C for 1 ⁇ 4 hours. It can be done by.
  • the Sagunjatang may be commonly used with 'SGT-4'.
  • when treating the Sagunja-tang to differentiated cells introduced with a reprogramming induction factor it was found that the reprogramming to induced pluripotent cells can be promoted, and the recombination of the differentiated cells into induced pluripotent stem cells It was first identified that it can be used for promoting programming or reverse differentiation.
  • induced pluri otent stem cell refers to cell differentiation into differentiated somatic cells or cells with limited differentiation capacity. It is a cell that induces pluripotency, such as embryonic stem cells, which is returned to the cell stage before differentiation through injection of related genes and reprogramming. These induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, show similar cell shapes, gene and protein expression patterns are similar, and have the characteristics of differentiation in and out of the body. These induced pluripotent stem cells have the same pluripotency as human embryonic stem cells, but do not involve ethical problems such as human embryonic stem cells because they are derived from already differentiated somatic cells rather than obtained from female eggs.
  • reprogramming refers to a process by which differentiated cells can be restored or converted into a state having pluripotent.
  • the reprogramming includes any process of returning differentiated cells having a differentiation capacity of 0% to less than 100% to an undifferentiated state, preferably, differentiated cells having a differentiation capacity of 0% or more than 0%. It includes both restoring or converting partially differentiated cells having a differentiation capacity of less than 100% into cells having 100% differentiation ability.
  • Such reprogramming may be performed by a reprogramming factor.
  • reprogramming inducer 1 refers to a substance that, when processed or expressed in differentiated cells, induces iPSc-specific gene expression and induces induced pluripotent stem cells having pluripotency. Induction of reprogramming Factors may be included in the present invention as long as the substance induces reprogramming of differentiated cells, and examples thereof may include 0ct3 / 4, sox-2, c-Myc, and Kl f-4. It can select according to the kind of cell and is not limited to the above example.
  • Differentiated cells into which the reprogramming inducer may be introduced may be cells derived from various animals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, preferably humans It may be a cell derived from, but is not limited to the differentiated cells that can be differentiated into induced pluripotent stem cells.
  • Such differentiated cells may be somatic cells.
  • Such a somatic cell is a cell constituting an adult means a cell having limited differentiation and self-producing ability, and the kind thereof is not particularly limited, but may be, for example, fibroblast.
  • 0ct3 / 4, c-Myc, Kl f4 and sox-2 were introduced into human fibroblasts, and oct4, sox-2 and Nanog expression was confirmed in the introduced cells.
  • the results showed that the pluripotent stem cell marker protein was expressed (FIG. 2).
  • four groups of fibroblasts into which four genes were introduced were treated with four-fold ethanol, induced pluripotent stem cell marker genes Nanog, sox-2, and oct4.
  • the present invention provides a method for promoting reprogramming of differentiated cells into induced pluripotent stem cells, comprising the step of treating Sagunja-tang to differentiated cells into which reprogramming factors have been introduced.
  • the reprogramming factor, differentiated cells, Sagunja-tang, induced pluripotent cells and reprogramming are as described above.
  • the method specifically includes the steps of: (a) introducing a reprogramming factor into differentiated cells; And (b) treating Sagunja-tang with the differentiated cells into which the reprogramming factor is introduced.
  • the method of introducing a reprogramming factor into the differentiated cells can be used without limitation the method of providing a substance to the cells commonly used in the art, according to the type of reprogramming inducer, differentiation of the reprogramming inducer
  • the method of administering to the culture of the cells, or the method of directly injecting the reprogramming inducer into the differentiated cells can be used, wherein the reprogramming inducer used is packaged transfected with a viral vector inserted the gene of the factor Viruses obtained from cells, plasmid vectors containing the corresponding inducer genes (e.g. epi some vectors), messenger RNAs produced by in vitro transcrt ion, or proteins produced in various cell lines It can be used in the form of.
  • the method of directly injecting the reprogramming inducer into the differentiated cells may be used by selecting any method known in the art, but is not limited thereto, and may include, but are not limited to, micro crosting method, electroporation method ( El ectroporat ion, particle injection method, direct muscle injection method, insulator and transposon method may be appropriately selected and applied. -... —
  • the differentiated cells into which the reprogramming factor is introduced may be cultured according to appropriate media and culture conditions known in the art, and may be treated by adding to Sagunjatanol medium during such culture.
  • culture conditions for inducing reprogramming hES (human embryonic stem cell) culture conditions may be used, but are not particularly limited thereto.
  • Sagunjatang of the present invention effectively enhance the efficiency of induced pluripotent stem cells, can be effectively used in the field of production of induced pluripotent stem cells.
  • Figure 1 shows the iPSc manufacturing process of the present invention.
  • Figure 2 shows the results of confirming the expression level of oct4, Sox-2 and Nanog in human fibroblasts introduced with 0ct3 / 4, sox-2, lf4 and Myc by immunocytochemical analysis.
  • FIG. 3 is a diagram illustrating the expression of iPSc marker genes Nanog, sox-2, oct4 and Klf4 by treating Sagunja-tang with human fibroblasts into which 0ct3 / 4, sox-2, Klf4 and Myc were introduced.
  • FIG. 4 is a diagram showing the treatment of Sagunja-tang in human fibroblasts into which 0ct3 / 4, sox-2, lf4 and Myc were introduced, and the degree of induction into iPSc was confirmed by AP lkaline phosphatase staining.
  • HFF Human foreskin-derived fibroblasts
  • Episomal vector purchased from addgene for iPSc induction in subculture of human HFF cells (pCXLE-0ct3 / 4, pCXLE-Sox-2, pCXLE-K.lf4, pCXLE-Myc, hereinafter referred to as 'OSK') After transforming each of the bacteria to grow, episomal DNA was isolated from each of the measured concentrations and stored at -70 ° C.
  • Immunostaining was performed on gelatin-coated four well chamber slides and was specifically performed as follows.
  • cells induced by 0SKM were fixed with 4% paraformaldehyde, washed with PBS, and then permeabilized (in PBS / 0.2% BSA, 0.1% Triton X-100), and blocking with 4% BSA at room temperature. 1 hour at. After blocking, antibodies bound to oc, Sox-2 or Nanog, respectively, were stained by diluting at a ratio of 1: 100 in PBS / 0.2% BSA. After washing, the secondary antibody was stained with FITC- or Alexa 488, 594-conjugated secondary antibody at room temperature for 1 hour, and then contrast-stained (act in) and observed under a microscope.
  • the group which introduced 0SKM (iPSc) and Sagunjatang here As a result, as shown in FIG. 3, the group which introduced 0SKM (iPSc) and Sagunjatang here.
  • the treated group of the present invention iPSC + SGT-4) showed the result of effectively expressing all markers of iPSc.
  • AP staining was purchased from System Bisciences and performed in the prescribed order. Specifically, the cells treated with SGT-4 by concentration at the same conditions as those transfected with 0SKM were incubated for 20 days, and then washed with PBS buffer, followed by citrate-acetone-formaldehyde (cit rate-acetone-formaldehyde). After fixing for 2 minutes), the AP staining solution was treated in the dark for 15 minutes, and the stained cells were observed under a microscope. As a result, as shown in Fig. 4, when treated with Sagunjatang of the present invention to fibroblasts transfected with 0SKM, the concentration-dependent AP stained colony number (purple: undifferentiated cells; colorless: differentiated cells) increased.

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Abstract

La présente invention concerne une composition contenant du sagunjatang comme principe actif pour favoriser la reprogrammation de cellules différenciées en cellules souches pluripotentes induites (CSPi) et un procédé pour préparer les cellules souches pluripotentes induites l'utilisant. Selon la présente invention, le sagunjatang améliore favorise efficacement l'efficacité de cellules souches pluripotentes induites et peut donc être utilisé efficacement dans le domaine de la préparation de cellules souches pluripotentes induites.
PCT/KR2014/011124 2013-12-10 2014-11-19 Composition contenant du sagunjatang comme principe actif pour favoriser la reprogrammation de cellules en cellules souches pluripotentes induites et procédé pour préparer des cellules souches pluripotentes induites l'utilisant Ceased WO2015088153A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020130153461A KR102070967B1 (ko) 2013-12-10 2013-12-10 사군자탕을 유효성분으로 포함하는, 세포의 유도만능줄기세포로의 리프로그래밍 촉진용 조성물 및 이를 이용한 유도만능줄기세포의 제조방법
KR10-2013-0153461 2013-12-10

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WO2015088153A1 true WO2015088153A1 (fr) 2015-06-18

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100440863B1 (ko) * 2001-05-24 2004-07-19 한국원자력연구소 조혈기능 증진 및 방사선 방호용 생약추출물
KR20050035906A (ko) * 2003-10-07 2005-04-20 롯데제과주식회사 항스트레스 및 뇌기능 개선 효과를 가지는 가미사군자탕
US20090068742A1 (en) * 2005-12-13 2009-03-12 Shinya Yamanaka Nuclear Reprogramming Factor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100440863B1 (ko) * 2001-05-24 2004-07-19 한국원자력연구소 조혈기능 증진 및 방사선 방호용 생약추출물
KR20050035906A (ko) * 2003-10-07 2005-04-20 롯데제과주식회사 항스트레스 및 뇌기능 개선 효과를 가지는 가미사군자탕
US20090068742A1 (en) * 2005-12-13 2009-03-12 Shinya Yamanaka Nuclear Reprogramming Factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GIM, GI MO: "Induced pluripotent stem cell -based medicine prescribed promote efficiency", KOREAN MEDICINE STORY, vol. 71, October 2013 (2013-10-01) *
LEE, HO-YEON G ET AL.: "Samultang, sagunjatang, palmultang comparative study of pharmacological activity sipjeon daebotang", JOURNAL OF NEUROPSYCHIATRY, vol. 21, no. 4, December 2010 (2010-12-01), pages 41 - 51 *

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KR102070967B1 (ko) 2020-01-29
KR20150067678A (ko) 2015-06-18

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