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Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method

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

Active Publication Date: 2022-08-02
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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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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  • Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method
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  • Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method

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Embodiment Construction

[0016] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

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

[0018] Maintenance of HiPSCs

[0019] HiPSCs were routinely grown on Matrigel containing mTeSR1 medium. Passage with Versen3-5 every 3-5 days, cells need to be 80% confluent on 35mm culture plates to start differentiation.

[0020] Day 0:

[0021] Spreading: 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. The medium was aspirated, washed once with DPBS (without Ca2+ and Mg2+), and 4.5 ml of pre-warmed mTeSR1+ROCK inhibitor Y27632 (final concentration 10 μM) was added.

[0022] Digest seed plate: Aspirate mTeSR1 g...

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Abstract

The present invention relates to a method for inducing directional endothelial differentiation of human pluripotent stem cells, which specifically includes the following steps: 1. When the human induced pluripotent stem cells reach 80% confluence or more, use a differentiation medium containing 10-50ng / ml WNT3a to digest the pluripotent stem cells after digestion. 2. Use the differentiation medium containing 10-50ng / ml WNT3a and 10-30ng / ml BMP2 to induce differentiation of the cells; 3. Use the differentiation medium containing 10-30ng / ml BMP2, In step 2, the induced and differentiated cells are further induced to differentiate into mesoderm cells; 4. The cells are further induced to differentiate into endothelial cells by using a medium containing 10-15ng / ml FGF8. By improving the culture medium and related cytokines in the present invention, 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 caused by endothelial dysfunction, is one of the leading causes of death worldwide. Direct endothelial replacement therapy for CVD is hampered by the lack of methods to isolate sufficient numbers of functioning autologous endothelial cells (ECs) for transplantation, and our limited understanding of how endogenous ECs are dysfunctional in 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 or research to explo...

Claims

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Application Information

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Patent Type & Authority Patents(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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