WO2017022981A9 - 합성 펩타이드를 이용한 유도만능줄기세포의 제조방법 - Google Patents
합성 펩타이드를 이용한 유도만능줄기세포의 제조방법 Download PDFInfo
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- WO2017022981A9 WO2017022981A9 PCT/KR2016/007857 KR2016007857W WO2017022981A9 WO 2017022981 A9 WO2017022981 A9 WO 2017022981A9 KR 2016007857 W KR2016007857 W KR 2016007857W WO 2017022981 A9 WO2017022981 A9 WO 2017022981A9
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- cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0056—Xeno-free medium
Definitions
- the present invention relates to a method for producing induced pluripotent stem cells using a synthetic peptide, and more particularly, a peptide that inhibits the activity of NF- ⁇ B and promotes mesenchymal-Epithelial transition (MET). It relates to a method for producing induced pluripotent stem cells.
- a synthetic peptide and more particularly, a peptide that inhibits the activity of NF- ⁇ B and promotes mesenchymal-Epithelial transition (MET). It relates to a method for producing induced pluripotent stem cells.
- MET mesenchymal-Epithelial transition
- Stem cells are undifferentiated cells capable of infinitely self-renewing and differentiating into cells of all tissues of the body. They are used for regenerative medicine, the development of new medicines for cell therapy, the causes and treatment of human diseases. It is an important object of research. Embryonic stem cells can differentiate into various cells and make whole organs, but there are ethical problems and immune rejection reactions that require the use of an egg as a cell therapy.
- somatic cells which have been differentiated in 2006, during in vitro culture, induces differentiation-induced pluripotent stem cells with autologous regeneration and pluripotency, which are characteristic of embryonic stem cells.
- the technology for producing cells was the first in the world to be succeeded by Yamanaka's team at Kyoto University, Japan (Takahashi K et al., Cell 126 (4): 663-676, 2006; Takahashi K et al., Cell 131 (5). ): 861-72, 2007).
- Reverse differentiation refers to a state in which already differentiated cells return to their initial undifferentiated state.
- iPSC refers to cells that have acquired pluripotency similar to embryonic stem cells capable of giving artificially stimulated externally differentiated somatic cells to cells of all organs forming our bodies. .
- dedifferentiation factors are most effective with the use of viruses, but the use of viruses for the production of dedifferentiated pluripotent stem cells for therapeutic purposes has the potential risk, and the randomization of intracellular genomes is very stable.
- problems such as mutations in genes.
- xenopathogens such as fetal bovine serum (FBS) and mouse embryonic fibroblasts (MEF), which are animal-derived feeder cells, are needed in the process of inducing differentiation.
- FBS fetal bovine serum
- MEF mouse embryonic fibroblasts
- the ultimate purpose of dedifferentiated pluripotent stem cells is to produce tissue that can be implanted into the human body, there is a limit to the clinical application due to the above risk.
- NF- ⁇ B promotes Epithelial-Mesenchymal Transition (EMT) (Huber MA et al., J Clin Invest. 114 (4): 569-81, 2004), and mesenchyme.
- EMT Epithelial-Mesenchymal Transition
- MET Mesenchymal-Epithelial Transition
- the present inventors have made efforts to increase the efficiency of induction of dedifferentiation of iPSCs in order to develop cell therapeutics that are safe for clinical application.
- the present inventors have found a functional peptide that inhibits the activity of intracellular NF- ⁇ B protein.
- Excavation and confirmed that the peptide induces mesenchymal-Epithelial transition (MET) to promote differentiation into pluripotent stem cells (iPSC), and completed the present invention.
- MET mesenchymal-Epithelial transition
- An object of the present invention is to provide a composition for promoting reverse differentiation into induced pluripotent stem cells from differentiated cells containing the peptide represented by the amino acid sequence of the general formula (I) as an active ingredient.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- Another object of the invention is the step of (a) introducing a differentiation inducer into the differentiated cells; And (b) treating and incubating a peptide having the amino acid sequence represented by the general formula I in a cell into which the reverse differentiation inducer has been introduced, thereby preparing a reverse differentiation induced pluripotent stem cell from the differentiated cells. .
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- the present invention provides a composition for promoting reverse differentiation into induced pluripotent stem cells from differentiated cells containing the peptide represented by the amino acid sequence of the general formula (I) as an active ingredient.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- the invention also comprises the steps of (a) introducing a differentiation inducer into differentiated cells; And (b) treating and incubating the peptide into which the reverse differentiation inducer is introduced, the peptide represented by the amino acid sequence of the general formula I below.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- Figure 1 is treated with functional peptides (P1, P2, P3) and control peptides (C1, C2) in human dermal fibroblasts introduced into the differentiation factor at 24 hours intervals for 10 days, alkaline force Colony staining was confirmed by Alkaline phosphatase staining (AP).
- P1, P2, P3 functional peptides
- C1, C2 control peptides
- FIG. 2 is a result of confirming Oct4 expression of an embryonic stem cell (ES cell) marker through immunofluorescence (IF) after treatment of the peptide in the same manner as in FIG. 1.
- ES cell embryonic stem cell
- IF immunofluorescence
- FIG. 3 shows the degree of mesenchymal-Epithelial transition (MET), which is a process of dedifferentiating somatic cells differentiated through flow cytometry (FACS) after treating peptides in the same manner as in FIG. 1.
- MET mesenchymal-Epithelial transition
- FACS flow cytometry
- a functional peptide that inhibits the activity of NF- ⁇ B protein is identified, and the peptide inhibits epithelial-mesenchymal transition (EMT) and further mesenchymal-epithelial metastasis (Mesenchymal-Epithelial). Induced transition, MET) to promote dedifferentiation.
- EMT epithelial-mesenchymal transition
- Mesenchymal-Epithelial mesenchymal-Epithelial metastasis
- the present invention relates to a composition for promoting reverse differentiation into induced pluripotent stem cells from differentiated cells containing the peptide represented by the amino acid sequence of the general formula (I) as an active ingredient at a consistent point.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- the present invention relates to a novel use of a peptide represented by the amino acid sequence of the general formula (I) below for promoting reverse differentiation from differentiated cells into induced pluripotent stem cells.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- the present invention provides a method for preparing a cell including: (a) introducing a differentiation inducer into differentiated cells; And (b) treating and incubating a peptide into which a reverse differentiation inducer is introduced, the peptide represented by the amino acid sequence of the general formula (I) below.
- X 1 and X 2 are cysteine (C: cysteine) or methionine (M: methionine).
- the peptide is preferably any one selected from the group consisting of SEQ ID NOs: 1 to 3, but is not limited thereto.
- P1 peptide GKCSTRGRKCCRRKK (SEQ ID NO: 1)
- P3 Cectide GKCSTRGRKMCRRKK (SEQ ID NO: 3)
- the peptide is preferably 0.01 to 100 ⁇ M concentration, more preferably 10 ⁇ M, but is not limited thereto.
- the peptide is preferably treated for 10 days at intervals of 24 hours, but is not limited thereto.
- the differentiated cells are preferably somatic cells or progenitor cells, preferably derived from humans, but are not limited thereto.
- Somatic cell of the present invention refers to human dermal fibroblasts having mesenchymal characteristics.
- EMEM Eagle's Minimum Essential Medium, ATCC 30-2003
- Essential 8 Medium Gibco, A15169-01
- the reverse differentiation inducer is preferably at least one selected from the group consisting of Oct3 / 4, Sox2, c-Myc, Klf4 and Lin28, but is not limited thereto.
- Oct3 / 4, Sox2, Klf4, c-Myc or Lin28 reprogramming genes refer to genes that can reprogram differentiated cells, in particular Oct4, Sox2, Klf4 and c-Myc are Yamanaka factors It is called.
- the dedifferentiation factor is prepared by dividing the three different episomal vectors and expressing p53 shRNA together (pCXLE-hOCT3 / 4-shp53, pCXLE-hSK, pCXLE-hUL). Increased cell establishment efficiency (Okita K et al., Nat Methods. 8 (5): 409-12, 2011).
- a method for delivering a retrodifferentiation factor to differentiated cells may be administered by injecting a retrodifferentiation inducer into a culture of differentiated cells, by direct infusion, or by transfection with a viral vector containing a retrodifferentiation factor. It is preferable to use a method of infecting cells differentiated with viruses obtained from packaging cells, but is not limited thereto. It is preferable to use microinjection, electroporation, insulator, particle bombardment, etc. as a method of directly injecting the dedifferentiation factor into differentiated cells, but is not limited thereto. It is not.
- induction of differentiation factor by electroporation using a nucleofector (Amaxa, US / Nucleofector, Electroporation Gene Transfer, Lonza) device of Lonza, somatic cells into which the differentiation inducer was introduced is 3 ⁇ in a culture dish. It is preferable to treat the peptide after stabilizing the medium every day for 5 days.
- a nucleofector Amaxa, US / Nucleofector, Electroporation Gene Transfer, Lonza
- the present invention can be used as a cell therapy by preparing a differentiated induced pluripotent stem cells under xenopathogen free or feeder cell free conditions.
- a Vitronectin Recombinant Human protein VTN-N
- VTN-N Vitronectin Recombinant Human protein
- Treatment of the functional peptides while culturing somatic cells into which the retrodifferentiation factor is introduced is carried out in a culture plate coated with Vitronectin protein, may also promote the efficiency of dedifferentiation into induced pluripotent stem cells.
- the peptide may be treated directly in a cell culture medium or mixed with a biomaterial for culture, and the biomaterial refers to a synthetic polymer or a natural polymer.
- the synthetic polymer is preferably poloxamer, polyethylene glycol and polypropylene glycol
- the natural polymer is preferably vitronectin, collagen, gelatin, alginic acid, chondroitin sulfate, fibronectin, and extracellular matrix protein. It is not limited.
- a natural polymer, vitronectin was used.
- Such a biomaterial is preferably used by coating a culture vessel, but is not limited thereto.
- the present invention provides a culture biomaterial that can replace the mouse embryonic fibroblasts (MEF), which is an animal-derived feeder cells used in the conventional iPSC culture (iPSC) By using it, it is possible to exclude xenopathogens generated by supplying various factors from MEF supporting cells to induced pluripotent stem cells. Therefore, the production of iPSCs using peptides of the present invention will improve both xenopathogen and efficiency of inducing differentiation to be considered when using undifferentiated pluripotent stem cells as cell therapy. Can be.
- MEF mouse embryonic fibroblasts
- iPSC animal-derived feeder cells used in the conventional iPSC culture
- the functional peptide may be characterized by inhibiting NF- ⁇ B activity by inhibiting nuclear transfer of NF- ⁇ B protein in somatic cells into which the differentiation factor is introduced and inhibiting NF- ⁇ B signaling mechanism.
- E MT epithelial-mesenchymal transition
- telomere reverse transcriptase hERT
- SSEA-4 human telomerase reverse transcriptase
- MET mesenchymal-epithelial transition
- the present invention can improve the efficiency of induction of dedifferentiation, and induced pluripotent stem cells prepared by this method are pluripotent stem cells in an undifferentiated state that normally display their characteristics as pluripotent stem cells. Therefore, it is very useful to effectively manufacture a pluripotent stem cell that can be clinically applied ultimately, and to develop a mass culture system that can secure pluripotent stem cell resources that can be clinically applied.
- P1 peptide (GKCSTRGRKCCRRKK: SEQ ID NO: 1) was synthesized from the C terminus by F-moc solid phase chemical synthesis using a synthesis apparatus. In other words, it was synthesized using Rink resin (0.075 mmol / g, 100-200 mesh, 1% DVB crosslinking) combined with Fmoc- (9-Fluorenylmethoxycarbonyl) as a blocking group, and 50 mg of Rink Amide in the synthesizer. After swelling the resin with DMF after adding MBHA resin, 20% piperidine / DMF solution was used to remove the Fmoc-group.
- 0.5M amino acid solution (solvent: DMF), 1.0M DIPEA (solvent: DMF & NMP), and 0.5M HBTU (solvent: DMF) were added in sequence from the terminal C to 1, 2 hours under nitrogen stream. Reacted for a while. Each time the deprotection and coupling steps were completed, the process was washed twice with DMF and methanol. After coupling the last amino acid (coupling) deprotection (deprotection) to remove the Fmoc-group.
- P1 peptide GKCSTRGRKCCRRKK (SEQ ID NO: 1)
- P2 peptide in which the C-terminal fifth cysteine of the P1 peptide (SEQ ID NO: 1) was substituted with methionine
- P3 peptide in which the C-terminal six cysteine of the P1 peptide (SEQ ID NO: 1) was substituted by the methionine No. 3 was synthesized by F-moc solid-phase chemical synthesis using a synthesizer.
- P2 Cectide GKCSTRGRKC M RRKK (SEQ ID NO: 2)
- P3 Cectide GKCSTRGRK M CRRKK (SEQ ID NO: 3)
- the dedifferentiation factors are prepared by dividing into three different episomal vectors according to a known method and expressing p53 shRNA together (pCXLE-hOCT3 / 4-shp53, pCXLE-hSK, pCXLE-hUL) to establish dedifferentiated stem cells. Increased efficiency (Okita K et al., Nat Methods. 8 (5): 409-12, 2011). Cells into which the differentiation factor was introduced were incubated in EMEM (Eagle's minimal essential medium, ATCC) medium, and the medium was replaced at 24 hour intervals until the cell confluency became 50%.
- EMEM Eagle's minimal essential medium, ATCC
- Naphthol / Fast Red Violet dye was added to the wells and stored at room temperature in a dark space for at least 20 minutes, followed by DPBS (Dulbecco's phosphate). The dye was washed with buffered saline) and the number of colonies stained under a microscope was checked.
- dedifferentiation factor was introduced into the cells by electroporataion into human dermal fibroblasts and dispensed into 6-well coated with Vitronectin. It was. Then, the expression level of Oct4, an embryonic stem cell marker, was compared by using immunofluorescence (IF) technique.
- IF immunofluorescence
- nucleus dye (Hoechst 33342, blue) was treated at room temperature for 10 minutes and washed with buffer solution (PBS) for confocal scanning microscope (IX 70, Olympus Co, Tokyo, Japan). ), The colonies with the clearest fluorescence in the wells were photographed.
- dedifferentiation factor was introduced into the cells by electroporataion into human dermal fibroblasts and dispensed into 6-well coated with Vitronectin. It was. After 10 days of peptide stabilized cells, cells were collected in a 15 ml Falcon tube using a scraper and centrifuged at 1500 rpm for 3 minutes. After washing with buffer (PBS), 1.5 ⁇ 10 5 cells are transferred to each tube using cold buffer (cold PBS).
- THY1 10 ⁇ g / ml concentration of THY1 (cat: sc-59398) and EPCAM (cat: ab20160) antibodies were diluted in 3% BSA / PBS solution and reacted at 4 ° C for 30 minutes, followed by 3 minutes at 1500 rpm for 3 minutes. Centrifuged twice. After washing with cold buffer (cold PBS), it was transferred to a round tube and analyzed by flow cytometry (FACS). Prior to measuring samples, THY1 used human dermal fibroblasts and EPCAM used human mammary epithelial cells (HMEC) to fix the control baseline.
- HMEC human mammary epithelial cells
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Abstract
Description
Claims (17)
- 하기 일반식 I의 아미노산 서열로 표시되는 펩타이드를 유효성분으로 함유하는 분화된 세포에서 유도만능줄기세포로의 역분화 촉진용 조성물:일반식 IGKCSTRGRKX1X2RRKK상기 X1 및 X2는 시스테인(C: cysteine) 또는 메티오닌(M: methionine)이다.
- 제1항에 있어서, 상기 펩타이드는 서열번호 1 내지 3으로 구성된 군에서 선택된 어느 하나인 것을 특징으로 하는 유도만능줄기세포로의 역분화 촉진용 조성물.
- 제1항에 있어서, 상기 분화된 세포는 체세포 또는 전구세포인 것을 특징으로 하는 유도만능줄기세포로의 역분화 촉진용 조성물.
- 제1항에 있어서, 상기 분화된 세포는 인간 유래인 것을 특징으로 하는 유도만능줄기세포로의 역분화 촉진용 조성물.
- 다음 단계를 포함하는 분화된 세포로부터 역분화 유도만능줄기세포의 제조방법:(a) 분화된 세포에 역분화 유도인자를 도입하는 단계; 및(b) 역분화 유도인자가 도입된 세포에 하기 일반식 I의 아미노산 서열로 표시되는 펩타이드를 처리하여 배양하는 단계;일반식 IGKCSTRGRKX1X2RRKK상기 X1 및 X2는 시스테인(C: cysteine) 또는 메티오닌(M: methionine)이다.
- 제5항에 있어서, 상기 펩타이드는 서열번호 1 내지 3으로 구성된 군에서 선택된 어느 하나인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 펩타이드는 0.01~100μM 농도인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 분화된 세포는 체세포 또는 전구세포인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 분화된 세포는 인간 유래인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 무-이종감염(xenopathogen free) 또는 무-지지세포(feeder cell free) 조건인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 역분화 유도인자는 Oct3/4, Sox2, c-Myc, Klf4 및 Lin28로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 펩타이드는 NF-κB 활성을 억제하는 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 펩타이드는 중간엽세포-상피세포 전이(Mesenchymal-Epithelial Transition, MET)를 촉진하는 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제5항에 있어서, 상기 (b) 단계는 펩타이드를 배양 배지에 직접 처리하거나, 배양용 생체재료와 혼합하여 처리하는 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제14항에 있어서, 상기 생체재료는 합성고분자 또는 천연고분자인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제15항에 있어서, 상기 합성고분자는 폴록사머, 폴리에틸렌 글리콘 및 폴르프로필렌 글리콜로 구성된 군에서 선택된 어느 하나인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
- 제15항에 있어서, 상기 천연고분자는 비트로넥틴, 콜라겐, 젤라틴, 알긴산, 황산 콘드로이틴, 피브로넥틴 및 세포외기질 단백질로 구성된 군에서 선택된 어느 하나인 것을 특징으로 하는 역분화 유도만능줄기세포의 제조방법.
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KR101529634B1 (ko) | 2013-08-28 | 2015-06-30 | 서울대학교산학협력단 | 역분화 유도를 위한 세포투과성 융합 단백질 및 그 용도 |
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2015
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2016
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- 2016-07-19 US US15/514,337 patent/US10066212B2/en active Active
- 2016-07-19 JP JP2017543696A patent/JP6446566B2/ja active Active
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EP3345999A1 (en) | 2018-07-11 |
JP6446566B2 (ja) | 2018-12-26 |
WO2017022981A1 (ko) | 2017-02-09 |
JP2017533731A (ja) | 2017-11-16 |
US20170275594A1 (en) | 2017-09-28 |
EP3345999B1 (en) | 2023-10-18 |
KR20170017385A (ko) | 2017-02-15 |
US10066212B2 (en) | 2018-09-04 |
KR101906557B1 (ko) | 2018-10-11 |
EP3345999A4 (en) | 2019-01-16 |
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