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Method for inducing differentiation of pluripotent stem cells into somatic cells

A technology for pluripotent stem cells and differentiation induction, which is applied in the field of differentiation induction from pluripotent stem cells to somatic cells, and can solve problems such as unreported extracellular matrix

Pending Publication Date: 2019-06-25
OSAKA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has not been reported that the engineered laminin described in Patent Document 1 is used as an extracellular matrix at the time of induction of differentiation

Method used

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  • Method for inducing differentiation of pluripotent stem cells into somatic cells
  • Method for inducing differentiation of pluripotent stem cells into somatic cells
  • Method for inducing differentiation of pluripotent stem cells into somatic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] [Example 1: Verification of the usefulness of a conjugate of laminin E8 linked to the mucilin domain 1 in inducing differentiation of human iPS cells into cardiomyocytes]

[0090]

[0091] (1) Laminin E8 and conjugates of laminin E8 and strandrin domain 1

[0092] Using human laminin 511E8 (hereinafter referred to as "LN511E8"), a conjugate (hereinafter referred to as "P-511E8 (D1 a-C)"), human laminin 421E8 (hereinafter referred to as "LN421E8"), and a conjugate in which domain 1 of human trechanin was fused to the C-terminus of the α4 chain of LN421E8 (hereinafter referred to as " P-421E8 (D1; a-C)"). LN511E8 was produced according to the method of Ido et al. (HiroyukiIdo et al., J. Biol. Chem., 282, 11144-11154, 2007) (refer to the examples of WO2014 / 199754A1). P-511E8 (D1; a-C) is the same molecule as "Plus#5 laminin E8" of the Example of WO2014 / 199754A1, and its production method is described in WO2014 / 199754A1. LN421E8 was prepared by changing α5 chain E8 to ...

Embodiment 2

[0124] [Example 2: Verification of the usefulness of a conjugate of laminin E8 and mucilin domain 1 in inducing differentiation of human iPS cells into skeletal muscle cells (Part 1)]

[0125]

[0126] (1) Laminin E8 and conjugates of laminin E8 and strandrin domain 1

[0127] LN421E8, P-421E8 (D1; a-C) and P-511E8 (D1; a-C) were used. These are the same as those used in Example 1.

[0128] (2) hiPS cells

[0129] The hiPS cell line 201B7 was used. The 201B7 strain was subjected to the same recombination using the CRISPR / CAS9 system according to a conventional method, so that the tdTomato sequence was linked to the 5' side of the start codon of the MYF5 locus, and an iPS cell line expressing tdTomato in combination with the expression of MYF5 was created. (MYF5-tdTomato C3 iPSCs, hereinafter referred to as "MYF5-tdTomato"). It was confirmed that MYF5-tdTomato had undergone genetic recombination only at the monoallelic gene.

[0130] (3) Pre-cultivation of hiPS cells

...

Embodiment 3

[0158] [Example 3: Verification of the usefulness of a conjugate of laminin E8 and mucilin domain 1 in inducing differentiation of human iPS cells into skeletal muscle cells (Part 2)]

[0159]

[0160] (1) Laminin E8 and conjugates of laminin E8 and strandrin domain 1

[0161]In addition to LN421E8, P-421E8 (D1; a-C) and P-511E8 (D1; a-C) used in Example 2, domain 1 of human trechanin was fused to human laminin 111E8 (hereinafter "LN111E8"). ”) and the C-terminus of the α1 chain of LN111E8 (hereinafter “P-111E8 (D1; a-C)”), which fused domain 1 of human trechanin to human laminin 211E8 (hereinafter “ LN211E8") and the C-terminus of the α2 chain of LN211E8 (hereinafter "P-211E8 (D1; a-C)"), in which domain 1 of human trechanin was fused to human laminin 332E8 (hereinafter "LN332E8") and the C-terminus of the α3 chain of LN332E8 (hereinafter "P-332E8 (D1; a-C)"), in which domain 1 of human trechanin was fused to human laminin 411E8 ( Conjugate (hereinafter "LN411E8") and the...

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Abstract

Provided is a method for inducing differentiation of pluripotent stem cells into somatic cells using a medium containing heparin binding growth factor, characterized by comprising contacting startingcells with a conjugate in which a laminin E8 fragment is bound to a fragment containing the growth factor-binding site of heparan sulfate proteoglycan. According to the present invention, differentiation of pluripotent stem cells into arbitrary somatic cells can be induced at a high efficiency.

Description

technical field [0001] The present invention relates to a method for inducing differentiation from pluripotent stem cells to somatic cells, and more specifically, to a conjugate using a laminin fragment and a fragment containing a growth factor binding site of heparan sulfate proteoglycan (conjugate) A method of inducing the differentiation of pluripotent stem cells into somatic cells. Background technique [0002] The application of human pluripotent stem cells such as human ES cells and human iPS cells in regenerative medicine is attracting worldwide attention. In order to apply human pluripotent stem cells to regenerative medicine, it is necessary to develop a technique for stably differentiating these stem cells into somatic cells with high efficiency. It is considered that in the selective differentiation induction from human pluripotent stem cells to arbitrary somatic cells, the selection of the growth factors added to the medium and the extracellular matrix as a scaf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/34A61K35/545A61P9/00A61P21/00A61P43/00
CPCA61P9/04A61P21/00C12N5/0657C12N5/0658C12N5/069C12N2506/45C12N2501/165C12N2500/25C12N2501/415C12N2501/727C12N2501/155C12N2501/105C12N2533/52C12N2533/50C12N2533/90C12N2501/115C12N2501/12C12N2501/16C12N2501/91C12N2501/998A61P43/00A61K35/34A61K35/545C12N5/10A61P9/00
Inventor 关口清俊戎富美樱井英俊赵明明斋藤润
Owner OSAKA UNIV
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