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Method of preparing dopaminergic neurons and/or dopaminergic neural precursor cells and modified stem cells used by method

A technology of neural precursor cells and pluripotent stem cells, applied in nervous system cells, artificially induced pluripotent cells, chemical instruments and methods, etc., can solve problems affecting the effect of symptom improvement

Inactive Publication Date: 2020-12-11
北京士马生物科技有限公司
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Problems solved by technology

[0005] Parkinson's disease is caused by the degeneration and death of dopaminergic neurons in the substantia nigra of the human brain. The approved drugs for Parkinson's disease are very limited, with annual sales of more than 2 billion US dollars, but they can only improve the symptoms of the disease to a limited extent. , and patients will gradually develop tolerance after taking the medicine for a long time, seriously affecting the improvement of symptoms

Method used

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  • Method of preparing dopaminergic neurons and/or dopaminergic neural precursor cells and modified stem cells used by method
  • Method of preparing dopaminergic neurons and/or dopaminergic neural precursor cells and modified stem cells used by method
  • Method of preparing dopaminergic neurons and/or dopaminergic neural precursor cells and modified stem cells used by method

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Embodiment 1

[0078] Example 1, preparation of transformed stem cells expressing Nurrl

[0079] 1. Overexpression of Nurr1 gene on human pluripotent stem cells and identification proves successful overexpression in human pluripotent stem cells

[0080] 1.1 Human pluripotent stem cell culture

[0081] Inoculate human pluripotent stem cells (20-60 passages) before transformation in E8 medium, at 37°C, 5% CO 2 Cultured in a constant temperature incubator, when the confluency was 60%-80%, digested with Dispase (1mg / ml, Gibco) and spread to 6-well culture plate, each passage density was 1:6, about 3-4 days long Subculture when reaching confluency.

[0082] 1.2 Overexpression of Nurr1 gene on human pluripotent stem cells to prepare modified stem cells expressing Nurr1

[0083] 293T cells were cultured in high-glucose DMEM+10% fetal calf serum. After 24 hr, the cells were completely attached and ready for transfection. Prepare 2M CaCl 2 solution, 2×HBS solution and laboratory water. Add 5ug ...

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Abstract

The invention discloses a method for preparing dopaminergic neurons and / or dopaminergic neural precursor cells and modified stem cells used by the method. The method comprises the following step that:the modified stem cells are induced and differentiated into the dopaminergic neurons and / or the dopaminergic neural precursor cells. The modified stem cells are in-vitro stem cells for expressing Nurr1 or expressing protein with the same function as the Nurr1. According to the method, Nurr1 genes are introduced into human pluripotent stem cells to obtain the modified stem cells, the Nurr1 genes are used for inducing the human pluripotent stem cells to differentiate towards a dopaminergic neuron subtype direction, and finally dopaminergic neural precursor cells and dopaminergic neurons are obtained. Compared with the prior method, the method can obviously enhance the efficiency of an inducing and differentiating process and obtaining of dopaminergic neuron subtype cells. The method can beused for treatment of diseases caused by dopaminergic neuron degeneration and / or damage, cell technology application, establishment of an in-vitro disease model, screening and development of drugs andthe like.

Description

technical field [0001] The present invention relates to a method for preparing dopaminergic neurons and / or dopaminergic neural precursor cells in the fields of genetic engineering, stem cells and regenerative medicine and the modified stem cells used therein. Background technique [0002] Nuclear receptor related factor 1 (nuclear receptor related factor 1, Nurr1) is a transcription factor and a member of the nuclear receptor gene superfamily orphan receptor subfamily. The transcription factor Nurr1 is mainly expressed in the midbrain and olfactory bulb regions of the brain. As a gene transcription regulatory protein, it participates in various physiological and pathological processes such as the development and survival of dopaminergic neurons in the midbrain, long-term memory, liver regeneration and inflammation. [0003] Pluripotent Stem Cells (Pluripotent Stem Cells) are a type of pluripotent cells with self-renewal and self-replication capabilities. Under certain condi...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/079C12N5/0793
CPCC07K14/47C12N5/0618C12N5/0619C12N2506/45
Inventor 李翔肖雄
Owner 北京士马生物科技有限公司
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