Method for differentiating human embryonic stem cell into nerve cells

A technology of human embryonic stem cells and nerve cells, which is applied in the fields of developmental biology, cell biology and cell therapy, can solve problems such as uneven distribution, complicated operating procedures, and many types of cells, and achieve a short induction cycle and uniform cell differentiation High, easy-to-operate effect

Inactive Publication Date: 2012-07-11
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has many disadvantages. First, the structure of the three germ layers is complex, and there are many types of cells and uneven distribution; second, the operation procedures are complicated. In the early stage, embryoid bodies need to be produced, and the target cells must be screened in the later stage.

Method used

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  • Method for differentiating human embryonic stem cell into nerve cells
  • Method for differentiating human embryonic stem cell into nerve cells
  • Method for differentiating human embryonic stem cell into nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Induction of neural cell differentiation

[0050] (1) Separation and purification of human embryonic stem cells;

[0051] Inoculate human embryonic stem cells onto mouse fibroblast feeder cells in 0.1% gelatin-coated culture well plates, in human embryonic stem cell culture medium, 37°C, 5% CO 2 , cultured under 100% humidity for 6 days to obtain a cell mixture of mouse fibroblasts and expanded human embryonic stem cells; the obtained cell mixture was digested in the digestion solution for 5 minutes at 37°C, and the suspension was obtained and placed in a human In culture flasks coated with 1% gelatin in embryonic stem cell culture medium, culture at 37°C for 1.5 hours, the mouse fibroblast feeder layer cells will adhere to the wall, while most of the human embryonic stem cells are still suspended in the culture medium, collect the culture medium Centrifuge, and discard the supernatant to obtain amplified human embryonic stem cell monomers; the digestion sol...

Embodiment 2

[0056] Example 2: Detection of nerve cells

[0057] 1. Immunohistochemistry

[0058] step

[0059] (1) Add 3 ml of nerve cells obtained by the method in Example 1 to preheat to 35° C., and rinse the cells with Duchenne’s phosphate buffered solution (DPBS) twice, each time for 3 minutes;

[0060] (2) Add 3ml of 4% paraformaldehyde fixative, fix at room temperature for 30min;

[0061] (3) Rinse with DPBS 3 times, 3 minutes each time. Incubate with DPBS containing 1% detergent (Triton X-100) at 37°C for 30 minutes.

[0062] (4) Add 3ml of DPBS (blocking solution) containing 5% goat serum, and act for 30min at room temperature.

[0063] (5) After aspirating and discarding the blocking solution with a pipette tip, add 1:500 diluted mouse anti-human β-tublin III monoclonal antibody (Millipore Company). 4°C overnight (more than 18h).

[0064] (6) Discard the primary antibody reaction solution with a pipette tip, rinse with DPBS 3 times, 5 min each time.

[0065] (7) Add goat a...

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Abstract

The invention discloses a method for inducing and differentiating a human embryonic stem cell into nerve cells. The method comprises the following steps of: separating and purifying a human embryonic stem cell to obtain human embryonic stem cell monomers; and inoculating the human embryonic stem cell monomers according to the cell density of (1-5)*104 / cm<2>, and performing single-layer adherence induced culturing, wherein in the induced culturing, nerve cells are obtained by pre-inducing the human embryonic stem cell with a sodium butyrate and an inducting culture medium I and performing differentiated induced culturing with an inducing culture medium. According to the method, the defects of high nerve cell heterogeneous degree, low differentiating efficiency, inconvenience for observing and operating in real time, need of a subsequent sorting process and the like existing in the conventional differentiating method can be overcome; the method has the advantages of easiness and convenience for operating, short inducting period (10-12) days, high nerve cell uniformity (the ratio is over 85 percent), and the like; and according to electrophysiologic monitoring, the nerve cells have convulsion discharging functions.

Description

(1) Technical field [0001] The invention relates to the fields of developmental biology, cell biology and cell therapy, and more specifically relates to a technology for directed differentiation of human embryonic stem cells. The invention provides a new method for inducing human embryonic stem cells into nerve cells in vitro. (2) Background technology [0002] Cell transplantation to treat nervous system diseases is a hot field of biomedical research today. Although cell therapy has made great progress, the pace of its clinical application is limited due to factors such as cell sources and donor shortages, and it also brings ethical concerns. moral debate. Embryonic stem cells are highly undifferentiated cells that can be cultured in vitro. They have pluripotency and can be induced to differentiate into various tissue cells. Inducing embryonic stem cells to differentiate into specific types of nerve cells can not only solve the above problems, but also make donor cells mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0735
Inventor 黄华荣杨晓娟张遵义张铭
Owner HANGZHOU NORMAL UNIVERSITY
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