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METHOD FOR PRODUCING PLURIPOTENT STEM CELLS TO BE DIFFERENTIATED INTO CARDIOMYOCYTES

Foreign code F200010079
File No. 5885
Posted date May 15, 2020
Country WIPO
International application number 2018JP038729
International publication number WO 2019078278
Date of international filing Oct 17, 2018
Date of international publication Apr 25, 2019
Priority data
  • P2017-202029 (Oct 18, 2017) JP
Title METHOD FOR PRODUCING PLURIPOTENT STEM CELLS TO BE DIFFERENTIATED INTO CARDIOMYOCYTES
Abstract Provided is a method for producing pluripotent stem cells to be differentiated into cardiomyocytes, said method comprising: (1) culturing pluripotent stem cells on a weakly cell-adhesive substrate so as to form a dome-shaped colony; and (2) acquiring cells from the dome-shaped colony. Also provided is a method for producing a cardiomyocyte population, said method comprising inducing the differentiation of a population of the pluripotent stem cells acquired by the above method into cardiomyocytes.
Outline of related art and contending technology BACKGROUND ART
In recent years, the pluripotent stem cells (for example, embryonic pluripotent stem cells (ES cells), an induced pluripotent stem cell (iPS cell) ) and research, regenerative medicine, drug discovery, drug safety test a variety of applications are expected to be applied to. For example, of the iPS cells such as human pluripotent stem cells induced from the myocardial cells, cultured myocardial tissue piece is obtained, and evaluate the toxicity of the drug or implant is used to assess the kinetics expected.
Such applications, high ability of pluripotency and differentiation of pluripotent stem cell lines can be used for uniform is required. However, by the ES cell lines, different pluripotent and differentiated have been reported (Non-Patent Document 1-3). On the other hand, when the inducing iPS cells, is initialized to a large number of cells were introduced into the gene for initializing, the resulting cell population has different properties of a hetero-cell or a mixture. In addition, cell lines established as the characteristics of the iPS cell lines differ from each other.
IPS cell lines of the existing efficiency of the method for inducing differentiation, depending on the cell of interest, for example, liver cells, lung cells such as differentiation Bronchoalveolar rate is low, the function of the derived cell is insufficient. On the other hand, myocardial cell differentiation induction method and has been developed (Patent Document 1 and 2, 5 and Non-Patent Document 4), the problem of purity substantially been solved. However, induced pluripotent stem cells of the mature cardiomyocytes differentiated from a low degree, for different cardiac muscle cells in vivo, drug evaluation and application of the regenerative medicine is not to be achieved.
In this way, cells differentiated from a pluripotent stem cell, in particular differentiated to develop a technique to obtain cardiac muscle cells is. In this context, the pluripotent stem cells in order to improve culture method, a fiber diameter of the nanofibers on the order of nanometers can be used as the culture substrate has been proposed (Patent Document 3, Non-Patent Document 6).
Scope of claims (In Japanese)[請求項1]
 心筋細胞に分化させるための多能性幹細胞の製造方法であって、
(1)弱い細胞接着性を有する基板上で多能性幹細胞を培養し、ドーム状のコロニーを形成させること、
(2)ドーム状のコロニーから細胞を得ること、
の各段階を含む方法。

[請求項2]
 基板が、ヒドロゲルまたはナノファイバーを含む、請求項1に記載の方法。

[請求項3]
 基板が、マトリゲル、フィブロネクチン、ビトロネクチンおよびラミニンから選択されるヒドロゲルを含む、請求項2に記載の方法。

[請求項4]
 基板が、約0.01~1.0μg/cm 2のマトリゲル、約0.01~1.0μg/cm 2のフィブロネクチン、約0.01~1.0μg/cm 2のビトロネクチンおよび約0.01~0.1μg/cm 2のラミニンから選択されるヒドロゲルを含む、請求項1~3のいずれかに記載の方法。

[請求項5]
 基板がゼラチンナノファイバーを含む、請求項1または2に記載の方法。

[請求項6]
 ゼラチンナノファイバーの密度が約3~5μg/cm 2である、請求項5に記載の方法。

[請求項7]
 ドーム状のコロニーが、50μm以上の高さのコロニーである、請求項1~6のいずれかに記載の方法。

[請求項8]
 段階(1)において、1個の多能性幹細胞に由来するコロニーを形成させる、請求項1~7のいずれかに記載の方法。

[請求項9]
 請求項1~8のいずれかに記載の方法により得られる多能性幹細胞を含む、心筋細胞を製造するための組成物。

[請求項10]
 請求項1~8のいずれかに記載の方法により得られる多能性幹細胞の集団を心筋細胞分化誘導することを含む、心筋細胞集団の製造方法。

[請求項11]
 請求項10に記載の方法により製造される心筋細胞集団。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KYOTO UNIVERSITY
  • Inventor
  • LIU, Li
  • YU, Leqian
  • LI, Junjun
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 DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW 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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