Method of non-exogenous induction of pluripotent stem cell to be GABA (Gamma Amino Acid Butyric Acid) neuron and application

A human pluripotent stem cell and neuron technology, applied in animal cells, nervous system cells, nervous system diseases, etc., can solve the problems of low differentiation efficiency, complicated culture medium, and the inability to exclude the interference of exogenous factors in the differentiation process.

Inactive Publication Date: 2015-08-26
NANJING MEDICAL UNIV
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Problems solved by technology

However, there are still some defects in the existing differentiation methods, including the complexity of the culture medium, the interference of external factors cannot be ruled out during the differentiation process, and the differentiation efficiency is not high.

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  • Method of non-exogenous induction of pluripotent stem cell to be GABA (Gamma Amino Acid Butyric Acid) neuron and application
  • Method of non-exogenous induction of pluripotent stem cell to be GABA (Gamma Amino Acid Butyric Acid) neuron and application
  • Method of non-exogenous induction of pluripotent stem cell to be GABA (Gamma Amino Acid Butyric Acid) neuron and application

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Embodiment 1

[0034] In the culture method of this embodiment, human pluripotent stem cells cultured without a feeder layer are mainly digested, suspended, attached to the wall, resuspended and other processes to obtain GABA neurons with high purity.

[0035] This embodiment includes the following steps:

[0036] (1) culturing human pluripotent stem cells in a feeder-free system;

[0037] (2) After digesting the human pluripotent stem cells, make a cell ball, and change the culture medium into a certain proportion of neural induction differentiation solution;

[0038] (3) After culturing with neural induction differentiation medium for 6±1 days, provide cell attachment conditions to allow cell attachment growth;

[0039] (4) On day 10±1, continuous induction was performed with Sonic hedgehog (SHH) or the small molecule compound Purmophine;

[0040] (5) After 6 days of sonic hedgehog or Purmophine induction, the cells (Rosette) with neural tube structure were blown up and resuspended;

[...

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Abstract

The invention discloses a method of non-exogenous induction of a pluripotent stem cell to be a GABA neuron. The method includes the following steps of 1 cultivating a pluripotent stem cell in a Matrigel or a Vitronectin matrigel system to the density of 60 % -80 %; 2 digesting the pluripotent stem cell obtained in step 1 with Dispase or EDTA (Ethylene Diamine Tetraacetic Acid), differentiating the suspension culture through neural induction on that day, and recording the day as the 0 day; 3 adding neural induction differentiation solution for further cultivation when a spherical cell formed by the pluripotent stem cell can be observed; 4 performing adherence on the seventh day; 5 adding SHH or a small molecule compound Purmophine on the tenth day for continuous induction; 6 blowing off a formed neural tube structure on the sixteenth day and continuing to cultivate with the neural induction solution; 7 obtaining the GABA neuron on the twenty-second day. The method of non-exogenous induction of the pluripotent stem cell to be the GABA neuron can significantly improve cultivation solution complication and low differentiation rate and eliminate the interference of exogenous factors.

Description

technical field [0001] The invention relates to a method and application for efficiently inducing human pluripotent stem cells into GABA neurons without exogenous substances, and belongs to the technical field of biomedicine. Background technique [0002] In recent years, with the aggravation of the aging society, neurodegenerative diseases have gradually become the focus of attention. Due to the development of human induced pluripotent stem cell technology and the preliminary results obtained in animal experiments, the application of this technology in the treatment of central nervous system diseases has shown broad prospects. [0003] Conventional wisdom holds that adult mammalian neurons cannot regenerate once damaged. This view has greatly limited the treatment of many neurodegenerative diseases. Although medicine and operation have made progress, but still can not obtain satisfactory curative effect. At present, due to the rapid development of stem cell technology, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793A61K35/30A61P25/28A61P25/16A61P25/08
Inventor 刘妍朱东亚袁方方恺恒徐敏
Owner NANJING MEDICAL UNIV
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