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System for inducing nerve stem cell differentiation and inducing method thereof

A technology of neural stem cells and cells, which is applied in the fields of biotechnology and cell biology, and can solve problems such as the limitation of the developmental potential of neural stem cells

Inactive Publication Date: 2011-05-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the neural stem cells induced by RA have limited developmental potential and can only form limited neural cell types.

Method used

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  • System for inducing nerve stem cell differentiation and inducing method thereof
  • System for inducing nerve stem cell differentiation and inducing method thereof
  • System for inducing nerve stem cell differentiation and inducing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P19

[0166] Example 1P19 cells are equivalent to epiblast cells in early embryos

[0167] It has been reported that P19 cells are similar to epiblast cells in early embryos in terms of developmental period. In order to further confirm this statement, the inventors used RT-PCR, immunostaining and in situ hybridization experiments to detect the expression of molecular markers of inner cell mass and epiblast in ES cells and P19 cells.

[0168] RT-PCR results see figure 1 a. The results showed that P19 cells expressed epiblast molecular marker FGF5, but did not express inner cell mass (Inner cell mass) molecular markers FGF4, Gbx2, Rex1, CRTR-1; on the contrary, ES expressed inner cell mass The molecular markers FGF4, Gbx2, Rex1 and CRTR-1 were expressed, but the molecular marker FGF5 of the epiblast was not expressed.

[0169] The results of immunostaining for FGF4 and FGF5 and in situ hybridization for Gbx2 are consistent with the results of RT-PCR, see figure 1 b.

[0170] Alth...

Embodiment 2N2B27

[0171] Example 2 N2B27 can induce P19 cells to differentiate into a high proportion of neural stem cells

[0172] P19 cells were suspended and cultured in N2B27 serum-free medium for 4 days (P19NB / Agg4d). After 4 days, a large number of cell clusters could be seen suspended in the culture medium. The cell clusters were carefully collected by natural sedimentation, fixed, and frozen sectioned to investigate the expression of stem cell-specific molecular marker Oct4 and neural stem cell-specific molecular marker Sox in P19 cell clusters induced by N2B27 for 4 days .

[0173] It was found that uninduced P19 cells were pluripotent and all cells were Oct4 + sox + , cells with stem cell properties. After 4 days of suspension culture in serum-containing medium, the cell mass without RA treatment (P19Agg4d) no longer expressed Sox, but continued to express the molecular marker Oct4 of pluripotent cells; induced by RA (P19RA / Agg4d) or N2B27 (P19NB / Agg4d) The 4-day-old cell pellet n...

Embodiment 3

[0178] The neural induction of embodiment 3N2B27 is not accompanied by the induction of mesoderm and endoderm

[0179] Neural induction in serum-containing medium is often accompanied by the emergence of mesoderm and endoderm cells. To test whether this is also the case for neural induction in N2B27 medium, the inventors detected the expression of molecular markers of the three germ layers by RT-PCR.

[0180] It was found that the expression of Sox1, 2, 3, the molecular markers of neuroectoderm, was up-regulated after N2B27 induction (NB / Agg). It is especially noteworthy that the expression of Sox1 was greatly up-regulated on the third day of induction, which is consistent with the expression of neural stem cells induced by N2B27. Consistent results began to appear on Day 3. Mesoderm molecular markers Brachyury and Goosecoid were only expressed at low levels during N2B27 induction, while endoderm molecular markers HNF3β and GATA6 were almost undetectable; these molecular mark...

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Abstract

The invention discloses a method for preparing neural stem cells, which comprises a step of inductively culturing P19 cells in N2B27 culture medium to obtain a cell group containing neural stem cells above 94%. The method can culture the P19 cells in an environment without blood serum and retinoic acid, thus suppressing the interference of the blood serum with complex components and preventing the obtained neural stem cells from being transformed posteriorly by retinoic acid. Moreover, the method can directly transform pluripotent stem cells to neutral stem cells without resulting in selective cell apoptosis. The neutral stem cells obtained by the invention have anterior neutral plate characteristics and totipotency, and can well simulate the neurogenesis process in body. Accordingly, theneural stem cells can be used as the research model for analyzing neural induction and neural differentiation process from epiblast to neuroderm, thus providing an ideal path for researching development of embryo after nidation.

Description

technical field [0001] The invention belongs to the field of biotechnology and cell biology, and more particularly, the invention relates to a method for inducing differentiation of neural stem cells and neural stem cells obtained by the method. Background technique [0002] Early embryonic development in mice is a complex process. In early pregnancy (E2.5-E4.5), the mouse embryo consists of trophectoderm (Trophectoderm) and inner cell mass (Inner cell mass, ICM). Along with development, ICM cells undergo recombination to form another group of multipotential epithelial-like cells, the epiblast (E4.5-E6.5). Epiblast cells go through gastrulation to form three germ layers (E6.5-E7.5) of the mouse embryo, namely, ectoderm (Ectoderm), mesoderm (Mesoderm) and endoderm (Endoderm). The embryo then develops into the organogenesis stage (E7.5-E12.5). During this period, the nervous system was the first to develop. The anterior ectoderm derived the neural plate structure, which the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/0797
Inventor 景乃禾夏彩虹
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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