Method for inducing mouse embryonic stem cells to differentiate toward nerve cells

A mouse embryo, inducing differentiation technology, applied in embryonic cells, animal cells, germ cells, etc., can solve the problems of difficult control, complicated operation methods, low differentiation ratio, etc., to achieve stable induction effect, simple experimental method, and experimental cycle. short effect

Inactive Publication Date: 2010-11-24
中国医科大学
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used specific inducer is retinoic acid, but its disadvantage is that mESCs need to be cultured first to form embryoid bodies, and because embryoid bodies contain cells of three germ layers, it is not conducive to the analysis and study of the regulation of neural cell differentiation, and The use of inducers may also interfere with the pattern of neurogenesis and the homogeneity of neurons
At present, it is difficult to control the differentiation process of mouse embryonic stem cells in the direction of neural cells in vitro. The experimental operation method is complicated, and the differentiation rate is as low as 50% to 70%.
Stem cells and other undifferentiated or poorly differentiated cells still exist, seriously affecting subsequent research and application

Method used

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  • Method for inducing mouse embryonic stem cells to differentiate toward nerve cells
  • Method for inducing mouse embryonic stem cells to differentiate toward nerve cells
  • Method for inducing mouse embryonic stem cells to differentiate toward nerve cells

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Experimental program
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Effect test

Embodiment 1

[0026] The method for culturing and inducing differentiation of mouse embryonic stem cells of the present invention comprises the following steps:

[0027] 1. Preparation of feeder layer cells

[0028] Use PMEF as the feeder layer, aseptically take 13.5-day pregnant mouse embryos, remove the head, tail, limbs and viscera, cut into pieces, digest with 0.25% trypsin-EDTA for 30 minutes, apply PMEF culture medium, at 37 ° C, with a volume fraction of 5% CO 2 Cultivate in the environment, cultivate for 8 days, use the second generation PMEF with good proliferation state, add 10mg / L mitomycin C, act for 2.5 hours, wash thoroughly and inoculate in a 75ml culture bottle, the inoculation density is 1.0×10 8 L -1 ;

[0029] 2. Culture of mESCs

[0030] mESCs at 1.0 × 10 8 L -1 Inoculate on the feeder layer cells at a density of 1:4, change the medium every 24 to 48 hours, and passage at a ratio of 1:4 every 3 days, see figure 1 ; when the cells reach 80% confluence, use 0.25% tr...

Embodiment 2

[0034] The method for culturing and inducing differentiation of mouse embryonic stem cells of the present invention comprises the following steps:

[0035] 1. Preparation of feeder layer cells

[0036] Use PMEF as the feeder layer, aseptically take 13.5-day pregnant mouse embryos, remove the head, tail, limbs and viscera, cut them into pieces, digest them with 0.125% trypsin-EDTA for 30 minutes, apply PMEF culture medium, at 37 ° C, with a volume fraction of 5% CO 2 Cultivate in the environment, cultivate for 10 days, use the PMEF with good proliferation status of the 4th generation, add 10mg / L mitomycin C, act for 2.5 hours, wash thoroughly and inoculate in a 75ml culture bottle, the inoculation density is 1.0×10 8 L -1 ;

[0037] 2. mESCs culture

[0038] mESCs at 1.0 × 10 8 L -1 Inoculate on the feeder layer cells at a density of 48 hours, replace the culture medium, discard the non-adherent cells, and change the medium every 4 days according to the growth of the cells....

Embodiment 3

[0042] The method for culturing and inducing differentiation of mouse embryonic stem cells of the present invention comprises the following steps:

[0043] 1. Optimization of induction differentiation conditions of the present invention

[0044] A 6-well culture plate was used for induction, and only one condition was changed each time, and the remaining conditions were kept unchanged. The three experimental conditions of the sequential induction method were optimized: (1) The concentration gradient of fetal bovine serum in the culture medium used for the first inoculation during induction: ①The serum concentration was 10%. ②The serum concentration is 12.5%. ③ serum concentration of 15%. (2) Density gradient of the first inoculation of mESCs during induction: ① inoculation density 1.0×10 10 L -1 . ②Inoculation density 1.0×10 9 L -1 . ③Inoculation density 1.0×10 8 L -1 . ④Inoculation density 1.0×10 7 L -1 . (3) Time gradient for complete replacement with serum-fre...

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Abstract

The invention relates to a method for inducing mouse embryonic stem cells (mESCs) to differentiate toward nerve cells. Primary mouse embryonic fibroblasts (PMEF) are used as a feeder layer. The method comprises the following steps of: inoculating the mESCs to the feeder layer, inoculating the mESCs to mixed solution of stem cells and mESCs culture liquid, and culturing nerve stem cell culture solution; and gradually changing the culture into the culture of nerve cell culture solution by completely using serum-free added alkali fibroblast growth factors, wherein the optimal serum concentration of the mixed solution for primary inoculation is 12.5 percent, the optimal inoculation density of primary mESCs inoculation is 1.0*108<-1>, and the time of completely changing the serum-free culture solution is the 5th day. By induction of the method, the mouse stem cells can be differentiated into the nerve cells in vitro. The method has the advantages of short experimental period, simple and convenient experimental process and stable and efficiency inducing results, can quickly finish a cell culture period, and is favorable for subsequent experiments.

Description

technical field [0001] The invention relates to a method for differentiating embryonic stem cells into nerve cells, in particular to a method for inducing mouse embryonic stem cells to differentiate into nerve cells. Background technique [0002] Embryonic stem cells are derived from the inner cell mass of the blastocyst, which can self-replicate for a long time in vitro, and have the pluripotency to differentiate into three embryonic cells: inner, middle and outer. Many studies have shown that mouse embryonic stem cells (mESCs) can be induced to differentiate into neurons in vitro. [0003] At present, the induction methods at home and abroad can be roughly divided into two categories: genetic modification and extragenic induction. Gene modification is to transfect a nerve-specific gene into embryonic stem cells (Embryonic stem cells, ESCs), and then obtain nerve cells by screening reporter genes or marker genes. Extragenic induction is to induce the differentiation of em...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 庞希宁顾文佳李晓丰刘朝阳施萍
Owner 中国医科大学
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