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WO2009157201A1 - Procédé et kit pour préparer des cellules ips - Google Patents

Procédé et kit pour préparer des cellules ips Download PDF

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Publication number
WO2009157201A1
WO2009157201A1 PCT/JP2009/002914 JP2009002914W WO2009157201A1 WO 2009157201 A1 WO2009157201 A1 WO 2009157201A1 JP 2009002914 W JP2009002914 W JP 2009002914W WO 2009157201 A1 WO2009157201 A1 WO 2009157201A1
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WO
WIPO (PCT)
Prior art keywords
cells
episomal vector
vector
group
nuclear reprogramming
Prior art date
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PCT/JP2009/002914
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English (en)
Inventor
Jun-Ichi Miyazaki
Fumi Tashiro
Satsuki Miyazaki
Eiji Yamato
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Osaka University
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Publication date
Application filed by Osaka University filed Critical Osaka University
Priority to JP2010550768A priority Critical patent/JP2011525794A/ja
Publication of WO2009157201A1 publication Critical patent/WO2009157201A1/fr

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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
    • C12N5/0696—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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • iPS cells can be isolated only based on observed morphological changes of cells, even without using, as a marker, gene expression specific to ES cells (nonpatent literature 5).
  • the team of Miguel Ramalho-Santos from University of California San Francisco demonstrated that iPS cells can be established by use of a lentivirus vector, a kind of a retrovirus vector, as well (nonpatent literature 6).
  • an object of the present invention is to provide a method for preparing iPS cells without use of such a vector that causes the integration of a foreign gene into the chromosomes of a host cell, for example, a retrovirus vector or a lentivirus vector, and a kit for preparing the same.
  • Fig. 1 is a diagrammatic view illustrating, in accordance with one of the examples, a method for preparing iPS cells by use of an episomal vector.
  • Fig. 2 shows the structure of pPyCAG-Oct3-IRES-Klf4-IRES-zeo.
  • Fig. 3 shows the structure of pPyCAG-Sox2-IRES-c-Myc-IRES-puro.
  • Fig. 4 is fluorescence microscope images showing immunostaining results of BMT10 cells with pPyCAG-Oct3-IRES-Klf4-IRES-zeo introduced therein.
  • FIG. 5 is fluorescence microscope images showing immunostaining results of BMT10 cells with pPyCAG-Sox2-IRES-c-Myc-IRES-puro introduced therein.
  • (a) is an immunostaining image for Sox2
  • (b) is an immunostaining image for c-Myc
  • (c) is a merged image of (a) and (b)
  • (d) is a merged image of a DAPI staining image on (c).
  • the nuclear reprogramming factor introduction step is to introduce, into somatic cells, an episomal vector into which the gene encoding a nuclear reprogramming factor is inserted in an expressible form.
  • the somatic cells are previously maintained in a suitable medium and a suitable culture condition according to the kind of the somatic cell to be used.
  • the method for introducing an episomal vector into somatic cells there can be used any method suitably selected from known transfection methods, depending on the episomal vector and the somatic cell to be used. Specific examples of the known transfection methods include the electroporation method, the calcium phosphate method, the lipofection method and the DEAE dextran method.
  • the replication factor introduction step when the replication factor introduction step is conducted in advance, only cells expected to express a replication factor (cells selected with the aid of a selection marker, such as a drug resistance marker) can be subjected to the nuclear reprogramming factor introduction step.
  • a selection marker such as a drug resistance marker
  • the nuclear reprogramming factor introduction step there is significantly higher probability of obtaining desired cells, that is, cells into which the episomal vector carrying the gene encoding a nuclear reprogramming factor is introduced.
  • the episomal vector carrying the gene encoding a nuclear reprogramming factor might drop off after introduced into cells. This is because immediately after introduced, this episomal vector can efficiently replicate with the aid of the replication factors that are already expressed in the cells.
  • iPS cells show the same cell and colony morphology as that of ES cells. Specifically, the colony of iPS cells is formed in a round or elliptical shape like an upside-down bowl and has a clear rim, and an iPS cell has scant cytoplasm and unclear intercellular boundary. Therefore, cells within such a morphologically characteristic colony can be identified as iPS cells.
  • the episomal vector is prepared so as to carry the herpesvirus-derived thymidine kinase gene in addition to the gene encoding a replication factor and/or the gene encoding a nuclear reprogramming factor.
  • iPS cells which stop expressing the thymidine kinase due to loss of episomal vectors are allowed to selectively survive by addition of ganciclovir or aciclovir to medium after iPS cell selection.
  • a population consisting only of iPS cells without episomal vectors can be obtained.
  • the present inventors obtained a clonal mouse ES cell line which stably retains pMGD20neo as an episome (1.19 cell line), which was reported in the aforementioned publication of Gassmann et al., from Dr. Austin Smith (School of Biological Sciences, University of Edinburgh).
  • pMGD20neo was isolated from the ES cell line, amplified in Escherichia coli and purified for later use.
  • pPyCAG-IZ carrying the mouse polyomavirus replication origin (ori) and the zeocin-resistance gene (Genes Dev. 12: 2048-60, 1998) was used for construction of an episomal vector expressing Oct3/4 and Klf4.
  • pPyCAG-IP carrying the mouse polyomavirus replication origin (ori) and the puromycin resistance gene (Mol Cell Biol. 22: 1526-36, 2002) was used for construction of an episomal vector expressing Sox2 and c-Myc.
  • pPyCAG-IP and pPyCAG-IZ were obtained from Dr. Hitoshi Niwa, an author of the two aforementioned publications.
  • This Oct3/4-IRES-Klf4 fragment was inserted into pPyCAG-IZ between the CAG promoter and the IRES-zeo, resulting in pPyCAG-Oct3-IRES-Klf4-IRES-zeo (see Fig. 2).
  • a Sox2-IRES-c-Myc fragment was prepared. Namely, the Sox2 cDNA containing the translation initiation codon ATG and the stop codon was ligated upstream of another IRES excised from the pCITE-1 plasmid. Then, the c-Myc cDNA containing the stop codon was ligated so that ATG located immediately downstream of the IRES served as the translation initiation codon ATG for c-Myc.
  • BK virus-based episomal vector construction A DNA fragment containing the BK virus ori sequence and T antigen gene was excised from pRBK plasmid (Invitrogen) by ScaI digestion. A HindIII-KpnI DNA fragment including the MC1 promoter and the HSV Tk gene was excised from pMC1-tk plasmid (Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Vol. 2. Plainview, NY: Cold Spring Harbor Lab. Press; 1995. Pp. 217-253).
  • the iPS cells prepared according to the preparation method of the present invention have extremely low risk of cellular oncogenic transformation and of developing dysfunction, and therefore are highly useful for generation of tissues and organs used for regenerative medicine.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Transplantation (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé pour préparer des cellules iPS sans utiliser un vecteur qui cause l’intégration d’un gène étranger dans les chromosomes d’une cellule hôte, par exemple, un vecteur rétroviral ou un vecteur lentiviral, et un kit pour préparer celles-ci. Le procédé pour préparer des cellules iPS comprend une étape d’introduction de facteur de reprogrammation nucléaire qui met en œuvre l’introduction, dans des cellules somatiques, d’un vecteur épisomique dans lequel le gène codant pour un facteur de reprogrammation nucléaire est inséré sous une forme exprimable ; une étape de culture qui met en œuvre la culture de cellules somatiques dans lesquelles le vecteur épisomique est introduit ; et une étape de sélection qui met en œuvre la sélection de cellules iPS générées par reprogrammation des cellules somatiques.
PCT/JP2009/002914 2008-06-26 2009-06-25 Procédé et kit pour préparer des cellules ips WO2009157201A1 (fr)

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JP2010550768A JP2011525794A (ja) 2008-06-26 2009-06-25 ｉＰＳ細胞の製造方法および製造キット

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JP2008167555 2008-06-26
JP2008-167555 2008-06-26

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011119942A1 (fr) * 2010-03-25 2011-09-29 Vistagen Therapeutics, Inc. Induction de cellules ips en utilisant des vecteurs épisomiques transitoires
US8048675B1 (en) 2010-05-12 2011-11-01 Ipierian, Inc. Integration-free human induced pluripotent stem cells from blood
WO2011152798A1 (fr) * 2010-06-02 2011-12-08 Agency For Science, Technology And Research Procédé d'induction de pluripotence dans cellules somatiques humaines par prdm14 ou nfrkb
WO2012060473A1 (fr) * 2010-11-04 2012-05-10 Kyoto University Procédé pour l'établissement efficace de cellules souches pluripotentes induites
WO2013022022A1 (fr) * 2011-08-08 2013-02-14 国立大学法人京都大学 Procédé pour l'établissement efficace de cellules souches pluripotentes artificielles
WO2013137491A1 (fr) 2012-03-15 2013-09-19 国立大学法人京都大学 Procédé pour produire un mélange de cellules cardiaques et vasculaires à partir de cellules souches pluripotentes artificielles
WO2013140927A1 (fr) 2012-03-21 2013-09-26 国立大学法人京都大学 Procédé de criblage d'agents thérapeutique et/ou prophylactique pour la maladie d'alzheimer
WO2013151186A1 (fr) 2012-04-06 2013-10-10 国立大学法人京都大学 Méthode d'induction de cellules produisant de l'érythropoïétine
JP2014501114A (ja) * 2010-12-31 2014-01-20 ウニヴェルズィテート・フューア・ボーデンクルトゥーア・ウィーン 人工多能性幹細胞および分化した細胞を生成する方法
US8765470B2 (en) 2010-08-04 2014-07-01 Cellular Dynamics International, Inc. Reprogramming immortalized B-cells to induced pluripotent stem cells
JP2014520551A (ja) * 2011-07-11 2014-08-25 セルラー ダイナミクス インターナショナル， インコーポレイテッド 細胞のリプログラミング方法およびゲノムの改変方法
JP2014523244A (ja) * 2011-07-08 2014-09-11 シルバイオテック エス．アー． 哺乳動物静止細胞を可逆的に不死化する遺伝子導入系
WO2015083582A1 (fr) 2013-12-02 2015-06-11 国立大学法人京都大学 Agent prophylactique et thérapeutique pour maladies fgfr3, et son procédé de dépistage
JP2015213522A (ja) * 2008-10-24 2015-12-03 ウイスコンシン アラムニ リサーチ ファンデーション 非ウイルス性再プログラムによる多能性幹細胞
US9644184B2 (en) 2008-06-04 2017-05-09 Cellular Dynamics International, Inc. Methods for the production of IPS cells using Epstein-Barr (EBV)-based reprogramming vectors
WO2021117886A1 (fr) 2019-12-12 2021-06-17 国立大学法人千葉大学 Préparation lyophilisée contenant des mégacaryocytes et des plaquettes
WO2022075384A1 (fr) 2020-10-07 2022-04-14 マイキャン・テクノロジーズ株式会社 Cellules infectées par un coronavirus et procédé pour les préparer

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JP2023515672A (ja) * 2020-03-02 2023-04-13 テナヤ セラピューティクス， インコーポレイテッド 心筋細胞発現マイクロｒｎａによる遺伝子ベクター制御

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KAJI K. ET AL.: "Virus-free induction of pluripotency and subsequent excision of reprogramming factors.", NATURE, vol. 458, April 2009 (2009-04-01), pages 771 - 775 *
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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9644184B2 (en) 2008-06-04 2017-05-09 Cellular Dynamics International, Inc. Methods for the production of IPS cells using Epstein-Barr (EBV)-based reprogramming vectors
JP2021094040A (ja) * 2008-10-24 2021-06-24 ウイスコンシン アラムニ リサーチ ファンデーション 非ウイルス性再プログラムによる多能性幹細胞
JP2015213522A (ja) * 2008-10-24 2015-12-03 ウイスコンシン アラムニ リサーチ ファンデーション 非ウイルス性再プログラムによる多能性幹細胞
JP2018174945A (ja) * 2008-10-24 2018-11-15 ウイスコンシン アラムニ リサーチ ファンデーション 非ウイルス性再プログラムによる多能性幹細胞
JP7165228B2 (ja) 2008-10-24 2022-11-02 ウイスコンシン アラムニ リサーチ ファンデーション 非ウイルス性再プログラムによる多能性幹細胞
WO2011119942A1 (fr) * 2010-03-25 2011-09-29 Vistagen Therapeutics, Inc. Induction de cellules ips en utilisant des vecteurs épisomiques transitoires
US8048675B1 (en) 2010-05-12 2011-11-01 Ipierian, Inc. Integration-free human induced pluripotent stem cells from blood
WO2011152798A1 (fr) * 2010-06-02 2011-12-08 Agency For Science, Technology And Research Procédé d'induction de pluripotence dans cellules somatiques humaines par prdm14 ou nfrkb
US9234180B2 (en) 2010-06-02 2016-01-12 Agency For Science, Technology And Research Method for inducing pluripotency in human somatic cells with PRDM14 or NFRKB
EP2576766A4 (fr) * 2010-06-02 2015-03-18 Agency Science Tech & Res Procédé d'induction de pluripotence dans cellules somatiques humaines par prdm14 ou nfrkb
US8765470B2 (en) 2010-08-04 2014-07-01 Cellular Dynamics International, Inc. Reprogramming immortalized B-cells to induced pluripotent stem cells
WO2012060473A1 (fr) * 2010-11-04 2012-05-10 Kyoto University Procédé pour l'établissement efficace de cellules souches pluripotentes induites
JP2013544069A (ja) * 2010-11-04 2013-12-12 国立大学法人京都大学 効率的な人工多能性幹細胞の樹立方法
US9637732B2 (en) 2010-11-04 2017-05-02 Kyoto University Method of efficiently establishing induced pluripotent stem cells
US9926532B2 (en) 2010-12-31 2018-03-27 Guangzhou Institute Of Biomedicine And Health Method of generating induced pluripotent stem cells and differentiated cells
JP2014501114A (ja) * 2010-12-31 2014-01-20 ウニヴェルズィテート・フューア・ボーデンクルトゥーア・ウィーン 人工多能性幹細胞および分化した細胞を生成する方法
JP2014523244A (ja) * 2011-07-08 2014-09-11 シルバイオテック エス．アー． 哺乳動物静止細胞を可逆的に不死化する遺伝子導入系
JP2014520551A (ja) * 2011-07-11 2014-08-25 セルラー ダイナミクス インターナショナル， インコーポレイテッド 細胞のリプログラミング方法およびゲノムの改変方法
WO2013022022A1 (fr) * 2011-08-08 2013-02-14 国立大学法人京都大学 Procédé pour l'établissement efficace de cellules souches pluripotentes artificielles
WO2013137491A1 (fr) 2012-03-15 2013-09-19 国立大学法人京都大学 Procédé pour produire un mélange de cellules cardiaques et vasculaires à partir de cellules souches pluripotentes artificielles
WO2013140927A1 (fr) 2012-03-21 2013-09-26 国立大学法人京都大学 Procédé de criblage d'agents thérapeutique et/ou prophylactique pour la maladie d'alzheimer
WO2013151186A1 (fr) 2012-04-06 2013-10-10 国立大学法人京都大学 Méthode d'induction de cellules produisant de l'érythropoïétine
WO2015083582A1 (fr) 2013-12-02 2015-06-11 国立大学法人京都大学 Agent prophylactique et thérapeutique pour maladies fgfr3, et son procédé de dépistage
EP3513789A2 (fr) 2013-12-02 2019-07-24 Kyoto University Agents prophylactiques et thérapeutiques pour maladies fgfr3
WO2021117886A1 (fr) 2019-12-12 2021-06-17 国立大学法人千葉大学 Préparation lyophilisée contenant des mégacaryocytes et des plaquettes
WO2022075384A1 (fr) 2020-10-07 2022-04-14 マイキャン・テクノロジーズ株式会社 Cellules infectées par un coronavirus et procédé pour les préparer

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