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METHOD FOR PRODUCING CANINE iPS CELLS meetings

Foreign code F170008953
File No. (S2015-1659-N0)
Posted date Feb 16, 2017
Country WIPO
International application number 2016JP068194
International publication number WO 2016204298
Date of international filing Jun 19, 2016
Date of international publication Dec 22, 2016
Priority data
  • P2015-123775 (Jun 19, 2015) JP
Title METHOD FOR PRODUCING CANINE iPS CELLS meetings
Abstract [Problem] To produce, by using a liquid culture having an extremely simple composition, a canine iPS cell line that can be subcultured stably for a long period of time.
[Solution] The present invention obtains canine iPS cells by culturing canine somatic cells having introduced therein four transcription factors of OCT3/4, SOX2, KLF4, and C-MYC that are nuclear reprogramming factors by means of a drug-inducible vector that can control expression of the transcription factors through usage of, for example, doxycycline, in a liquid culture containing basic fibroblast growth factor (bFGF) but not containing a pluripotency maintaining factor (not including basic fibroblast growth factor) such as LIF, and further preferably not containing a differentiation suppressor such as MEK suppressor and GSK3β suppressor.
Outline of related art and contending technology BACKGROUND ART
Dogs, rodent such as a mouse or a rat compared to close to physiological conditions in addition to the human, large size, service life is also long, can be administered in a relatively large amount of a drug, is also suited to a long term experiment. In addition, living in the human and in the same environment and the human disease model animals as it is more . Further, canine genetic diseases found in more than half of the same gene mutation similar to human disease, canine genomic sequence was elucidated from the fact that, as a disease model for the demand of the dogs may be further extended.
So far, the fabrication of canine iPS cells but various report as an example, in these reports, a basal medium Ham's F-12 DMEM/F12(Dulbecco's Modified Eagle medium medium were mixed with each other by equal amount) to, such as KSR of serum such as FBS or serum replacement, further LIF on the differentiation of a reducing agent such as have been used as the culture medium. For example, in Patent Document 1, basal MAPKK (mitogen-activated protein kinase kinase) inhibitor and PD0325901 inhibitor ALK (activin receptor-like kinase) and A-83-01 GSK (glycogen synthase kinase) inhibitor HDAC and LIF (leukemia inhibitory factor) and CHIR99021 (histone deacetylase) inhibitor valproic acid mixed with a culture medium is used. In addition, in Patent Document 2, basal medium and serum substitute KSR LIF and bFGF (basic fibroblast growth factor) a mixture of a culture medium is used. Non-patent document 1 is further, and basal bFGF and LIF KSR, further GSK3 β (glycogen, synthase, kinase 3 β) inhibitor, MEK (ERK-MAP kinase pathway blockers) suppressor, TGF-β antagonist (beta growth factor mutant form) a mixture of a culture medium is used.
On the other hand, in human iPS cells and mouse iPS cells, without using bFGF, neural cell culture medium is a basal DMEM/F12 supplement N2 (trade name) used in a mixture of neurobasal medium and B27 medium supplement (trade name) of the culture media mixture to mixing LIF, MEK inhibitor, medium GSK3 β inhibitor used (non-patent document 2, 3). In these documents, the resulting iPS colonies to an enzyme treatment can be a single-cell can be obtained, the single culture of the cells can be about 50 and margin is.
Further, in non-patent document 4 and 5, undifferentiated mouse iPS cells than similar to having a three-dimensional colony forming ability when it is reported that, by enzymatic passaging method cannot be resistant to, if the amount of each inhibitor was added to the medium cannot be maintained its form. In addition, a long period of time may not be from the culture, the strains of these iPS cells has not been considered.
To date, such as LIF or maintain pluripotent differentiation SK3 β inhibitors such as inhibitors on the differentiation factors MEK without using factors, cell growth-promoting factor such as bFGF in the presence of only, dogs from body cells to produce iPS cells, iPS cells can be subcultured over a long period has been no report of produced.
Scope of claims (In Japanese)[請求項1]
イヌの体細胞からイヌiPS細胞株を作製する方法であって、
イヌ体細胞に核初期化因子を導入する工程と、
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養をする工程を含む方法。
[請求項2]
前記培養液は、分化抑制因子を含まない培養液である請求項1に記載の方法。
[請求項3]
前記培養液は、N2B27培地を基本培地とする請求項1又は2に記載の方法。
[請求項4]
イヌの体細胞からイヌiPS細胞株を作製する方法であって、
イヌ体細胞に核初期化因子を導入する工程と、
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子のみを含むN2B27培地を用いて培養をする工程を含む方法。
[請求項5]
前記核初期化因子は次の3つの遺伝子:OCT3/4、SOX2、KLF 4であるか、当該3つの遺伝子にさらにC-MYCを含む4つの遺伝子である請求項1~4の何れか1項に記載の方法。
[請求項6]
前記核初期化因子の発現を、薬剤の添加により制御できる薬剤誘導性ベクターを用いて、前記核初期化因子をイヌ体細胞に導入する請求項1~5の何れか1項に記載の方法。
[請求項7]
フィーダー細胞の存在下又はフィーダー細胞の非存在下にあるマトリゲル上で、前記核初期化因子が導入されたイヌ体細胞を培養する請求項1~6の何れか1項に記載の方法。
[請求項8]
請求項1~7の何れか1項に記載の方法で作成されたイヌiPS細胞。
[請求項9]
イヌ体細胞に核初期化因子を導入する工程と、
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養して、イヌiPS細胞株を得る工程と、
培養で得られた細胞を分化誘導する工程を含む、イヌ体細胞の製造方法。
[請求項10]
前記イヌ体細胞は、間葉系幹細胞である請求項9に記載のイヌ体細胞の製造方法。
[請求項11]
前記イヌ体細胞は、骨芽細胞又は神経幹細胞である請求項9に記載のイヌ体細胞の製造方法。
[請求項12]
内因性のOCT3/4、KLF4、SOX2の少なくとも1つの遺伝子、好ましくは全ての遺伝子とNANOG遺伝子の発現が見られ、かつ分化多能性を有する生体から分離されたイヌ体細胞由来の細胞。
[請求項13]
さらに、GBX2の遺伝子の発現が見られる請求項12に記載のイヌ体細胞由来の細胞。
[請求項14]
ドキシサイクリンの存在下で、外因性遺伝子のOCT3/4、KLF4、SOX2の少なくとも1つ、好ましくは全ての遺伝子の発現、さらにC-MYCが導入された場合にはこれら4つの遺伝子の発現が見られる請求項12又は13に記載の細胞。
[請求項15]
酵素処理により単一細胞に分割可能である請求項12~14の何れか1項に記載の細胞。
[請求項16]
請求項12~15の何れか1項に記載の細胞から分化誘導されたイヌ体細胞。
[請求項17]
請求項12~15の何れか1項に記載の細胞から分化誘導された間葉系幹細胞。
[請求項18]
請求項12~15の何れか1項に記載の細胞から分化誘導された骨芽細胞又は神経幹細胞。
[請求項19]
請求項12~15の何れか1項に記載の細胞を分化誘導して、イヌ体細胞を作製する方法。
[請求項20]
請求項12~15の何れか1項に記載の細胞を分化誘導して、間葉系幹細胞を作製する方法。
[請求項21]
請求項12~15の何れか1稿に記載の細胞を分化誘導して、骨芽細胞又は神経幹細胞を作製する方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • OSAKA PREFECTURE UNIVERSITY PUBLIC CORPORATION
  • Inventor
  • INABA Toshio
  • NISHIMURA Toshiya
  • TANAKA Erina
  • HATOYA Shingo
  • SUGIURA Kikuya
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG

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