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Method for inducing directional endothelial differentiation of human pluripotent stem cells

A technique for inducing differentiation of pluripotent stem cells, applied in the field of directional differentiation of induced human pluripotent stem cells, can solve the problems of limited application, low differentiation efficiency, large cell heterogeneity, etc., and achieve high endothelial induction efficiency

Active Publication Date: 2021-06-18
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of the current endothelial differentiation protocols have low differentiation efficiency and large heterogeneity of differentiated cells, which limits the application of HiPSCs-derived endothelial cells to a certain extent.

Method used

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  • Method for inducing directional endothelial differentiation of human pluripotent stem cells
  • Method for inducing directional endothelial differentiation of human pluripotent stem cells
  • Method for inducing directional endothelial differentiation of human pluripotent stem cells

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Embodiment Construction

[0016] The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

[0017] A method for inducing directed endothelial differentiation of human pluripotent stem cells, comprising the following steps:

[0018] Maintenance of HiPSCs

[0019] HiPSCs are routinely cultured on Matrigel containing mTeSR1 medium. Passaging with Versen3-5 every 3-5 days, the cells need to be 80% confluent on a 35mm culture plate to start differentiation.

[0020] Day 0:

[0021] Laying gel: Matrigel was thawed on ice, diluted 1:100 with growth factor-free DMEM medium, spread on a 60mm culture plate, and incubated at 37°C for 0.5h. Aspirate the medium, wash once with DPBS (without Ca2+ and Mg2+), and add 4.5 ml of pre-warmed mTeSR1+ROCK inhibitor Y27632 (final concentration 10 μM).

[0022] Digest the see...

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Abstract

The invention relates to a method for inducing directional endothelial differentiation of human pluripotent stem cells. The method specifically comprises the following steps: 1, when the human pluripotent stem cells reach 80% fusion or above, induced differentiation is performed on digested stem cells by using a differential medium containing 10-50ng / ml of WNT3a; 2, induced differentiation is further performed on the cells by using a differentiation medium containing 10-50ng / ml of WNT3a and 10-30ng / ml of BMP2; 3, induced differentiation is further performed on the induced differentiated cells in the step 2 by using a differential medium containing 10-30ng / ml of BMP2 into mesodermal cells; and 4, induced differentiation is further performed on the cells by using a culture medium containing 10-15ng / ml of FGF8 into endothelial cells. According to the invention, by improving the mediums and related cell factors, the experimental scheme can have higher endothelial induction efficiency.

Description

technical field [0001] The invention relates to the field of directional differentiation of induced human pluripotent stem cells, in particular to a method for inducing directional endothelial differentiation of human pluripotent stem cells. Background technique [0002] Cardiovascular disease (CVD), often triggered by endothelial dysfunction, is one of the leading causes of death in the world. Direct treatment of CVD through endothelial replacement has been hampered by the lack of methods to isolate sufficient numbers of transplanted functional autologous endothelial cells (ECs) and our limited understanding of how endogenous ECs become dysfunctional during the development of various cardiovascular diseases . Innovations in human induced pluripotent stem cells (HiPSCs), cells reprogrammed from somatic cells to a pluripotent state similar to embryonic stem cells (ESCs), can generate large numbers of patient-specific ECs that can be used for transplantation, drug screening, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/069C12N2506/45C12N2501/165C12N2501/115C12N2501/155
Inventor 董念国谢明辉程敏乔韡华曹红严格
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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