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Method for differentiation induction of myocardial cell from pluripotent stem cell

一种多能性干细胞、分化诱导的技术，应用在将多能性干细胞分化诱导成心肌细胞领域，能够解决心肌分化效果不明确、没有引起心肌分化诱导效果、没有抑制效果效果等问题

Inactive Publication Date: 2009-05-06
DAIICHI SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in our same study, no significant differentiation-promoting effect was found (Example 2), and the treatment of Wnt-3a to mouse ES cells by other research groups did not cause particularly significant myocardial differentiation-inducing effects (Non-Patent Document 25), or there is no report about the presence of an inhibitory effect (Non-Patent Document 29)
That is, the effect of activation of the canonical Wnt signaling pathway on the differentiation of pluripotent stem cells represented by ES cells on myocardial differentiation is not clear, and it cannot be considered that an optimal culture method for inducing myocardial differentiation has been established

Method used

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  • Method for differentiation induction of myocardial cell from pluripotent stem cell
  • Method for differentiation induction of myocardial cell from pluripotent stem cell
  • Method for differentiation induction of myocardial cell from pluripotent stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0250] Example 1: Research on the expression status of various Wnt genes during differentiation induction of ES cells (1)

[0251] Expression of various Wnt genes during differentiation induction of mouse ES cells was investigated. Mouse ES cells were used in Knockout-DMEM (Invitrogen) medium (hereinafter referred to as ESM) containing 20% ​​fetal calf serum, 2 mmol / L L-glutamine, and 0.1 mmol / L 2-mercaptoethanol Add 1000U / mL of LIF (ESGRO; Chemicon Corporation) to form a culture medium according to "Manipulating the Mouse Embryo: A Laboratory Manual" (Hogan et al. edited, Cold Spring Harbor Laboratory Press, 1994), "Embryonic Stem Cells: Methods and Protocols" (Turksen, Humana Press, 2002) et al. provided ES cells that maintained undifferentiated traits and were subcultured to the experiment. Hereinafter, ES cells subcultured under these conditions are referred to as ES cells subcultured under normal culture conditions. Furthermore, in the following experiments, D3 cells, R...

Embodiment 2

[0276] Example 2: ES cell-derived cardiomyocytes appear enhanced by recombinant Wnt protein treatment (1)

[0277] In the early stage of differentiation induction of ES cells, before the emergence of cardiomyocytes, the expression of various Wnt genes was found to increase instantaneously. Therefore, ES cells at this stage were treated with recombinant Wnt protein to study the induction effect of myocardial differentiation. The differentiation induction of ES cells was carried out in the same manner as in Example 1. In a part of the experimental group, commercially available recombinant WNT-1 protein (Peprotech), Wnt-3a protein (R&D systems company), or Wnt-5a protein (R&D systems company) culture medium. Hereinafter, the addition of recombinant proteins of canonical Wnts such as WNT-1 to the medium to act on ES cells is referred to as "Wnt treatment".

[0278] 【0094】

[0279] As an indicator of the differentiation and development of ES cells into cardiomyocytes, the occurre...

Embodiment 3

[0289] Example 3: The traits of ES cell-derived cardiomyocytes induced by Wnt treatment

[0290] As shown in Example 2, by performing Wnt treatment, the beating properties of EBs prepared from ES cells were significantly improved. In this beating EBs, it was confirmed that the beating cells were cardiomyocytes, so various cardiomyocytes were investigated. marker molecules for gene expression and protein production. ES cells were induced to differentiate in the same manner as in Example 2, and EBs were recovered on day 10 after differentiation induction to prepare cDNA. Real-time PCR quantitative reaction was carried out by TaqMan probe method. That is, using the above-mentioned cDNA (1 μL) as a template, TaqMan Universal PCR MasterMix (PE Applied Biosystems) was used to carry out according to the method described in the attached manual. TaqMan probes for detecting various genes were designed based on nucleotide sequence information of various genes using software for primer ...

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Abstract

The present invention provides a method for inducing differentiation of cardiomyocytes efficiently and selectively from stem cells. A method for inducing differentiation of cardiomyocytes from pluripotent stem cells, which comprises: (i) culturing the pluripotent stem cells in a culture medium containing no substance that promotes activation of the canonical Wnt signaling pathway during the time period between initiation of differentiation induction and 24 hours before the period of elevated canonical Wnt gene expression; and then (ii) culturing the pluripotent stem cells in a culture medium containing a substance that promotes activation of the canonical Wnt signaling pathway during a time period of 24 to 96 hours, starting from 24 to 0 hours before the period of elevated canonical Wnt gene expression.

Description

technical field [0001] 【0001】 [0002] The invention relates to a method for selectively and efficiently preparing cardiomyocytes from pluripotent stem cells such as ES cells. Background technique [0003] 【0002】 [0004] (1) Preparation of cardiomyocytes using pluripotent stem cells [0005] Generally speaking, cardiomyocytes actively undergo cell division while autonomously beating before birth, and lose their ability to divide immediately after birth, and there are only a very small number of undifferentiated stem cells or precursor cells, and their proliferation and differentiation capabilities are extremely low. When cardiomyocytes die due to various stresses such as myocardial infarction and myocarditis, the lost cardiomyocytes cannot be replaced. As a result, the remaining cardiomyocytes maintain cardiac function due to compensatory hypertrophy. If various stresses continue beyond the allowable range, further cardiomyocyte fatigue and death will be caused, showing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C07K14/47C12N5/06A61L27/00C12N15/09C12N5/077
CPCC12N2506/02C12N2501/415C12N5/0657C12N5/0602C12N5/00C12N5/0606
Inventor 小清水右一田中智文河岛佳代子门仓庆知
Owner DAIICHI SANKYO CO LTD
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