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A rapid and direct method for inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells

A technology of neuroepithelial cells and embryonic stem cells, applied in animal cells, nervous system cells, vertebrate cells, etc.

Active Publication Date: 2015-11-04
南通大学技术转移中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there is no method to rapidly and directly induce mouse embryonic stem cells to differentiate into neuroepithelial cells with self-renewal and proliferation capabilities

Method used

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  • A rapid and direct method for inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells
  • A rapid and direct method for inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells
  • A rapid and direct method for inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Rapid and direct induction of mouse embryonic stem cells to differentiate into neuroepithelial cells

[0080] The purpose of the experiment: mouse embryonic stem cells differentiate into neuroepithelial cells with high efficiency and the generated neuroepithelial cells can carry out self-replication and long-term proliferation. Mouse embryonic stem cells (mESCs) were purchased from Shanghai Stance Biotechnology Co., Ltd., strain M1.

[0081] First, 24-well plates (Corning) were coated with Matrigel (BD). After cell counting, mouse embryonic stem cells (mESCs) were plated at 1x10 3 The number per well was seeded on a cell culture plate. Add 400 μl of culture medium to each well, and change the medium every other day. After 3-5 days, the cells grow into clump-like clones, and then start induction, which is divided into 3 stages.

[0082] 1) The first stage: the medium of the adherent cultured mouse embryonic stem cell cluster clones was replaced with the fir...

Embodiment 2

[0089] Example 2 Immunological identification of mouse neuroepithelial cells

[0090] The first to third generation of neuroepithelial cells obtained in Example 1 were subjected to immunofluorescence detection, and the specific steps included fixation, membrane rupture, and blocking solution for blocking for 1 hour, and then adding primary antibodies at the ratio of Nestin (Beyotime) 1:10 , Sox2 (CST) 1:200, Sox1 (R&D) 1:10, Pax6 (Abcam) 1:20, CD133 (Bioss) 1:100 or Sox9 (Millipore) 1:5000 overnight at 4°C, after washing with PBS, add a Anti-appropriate fluorescent secondary antibody FITC (ImmunoResearch), CY3 (ImmunoResearch) was incubated at room temperature for 2 hours. Finally, after washing with PBS, the cells were stained with Hoechst or DAPI and mounted with a mounting agent. Detection was performed under a fluorescence microscope (Zeiss) and a scanning laser confocal imaging system (Leica). Immunofluorescent staining of NE revealed expression of the neural progenitor...

Embodiment 3

[0091] Example 3 Identification of polar structure of neuroepithelial cells and neural rosette-shaped specific markers The neuroepithelial cells obtained in Example 1 were subjected to cell immunofluorescence staining, the operation method was the same as in Example 2, and the primary antibodies used were respectively PLZF (Santa Cruz) 1:50, ZO-1 (Santa Cruz) 1:50, SOX2 (CST) 1:200, PAX6 (Abcam) 1:200, the results show that the neuroepithelial cells obtained in Example 1 show nuclear expression transcription Factors PLZF and DACH1, and the expression of neuroepithelial polarity marker ZO-1 was also found (such as Figure 6 As shown, A. NE-specific transcription factor DACH1, also expresses neural precursor marker Nestin. B. NE can also be stained by the neural precursor transcription factor Sox2. C.NE can express the polarity marker ZO-1, indicating the polarity characteristics of neuroepithelial cells, and can also express the neural precursor marker Pax6. D.NE expresses it...

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Abstract

The invention discloses a method for rapidly and directly directionally inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells, by adding an induction medium containing dorsomorphin and noggin, SB431542 and CHIR99021 to the adherent cultured mouse embryonic stem cell pellet clones to induce mouse Embryonic stem cells form rosette-shaped neurospheres; then add the second-stage induction medium containing bFGF to form suspended neurospheres; finally digest the neurospheres into single cells, and then use the third-stage induction medium to form adherent cultures Neuroepithelial cells with longer self-proliferation and renewal. The invention abandons the traditional EB forming method, shortens the differentiation time, and the differentiation efficiency reaches more than 95%. The invention can quickly induce NE cells to replace NSC cells to treat central system damage, and has high clinical applicability.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a composition for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells, especially a method for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells using the composition, and A method for further directing neuroepithelial cells to differentiate into neuron cells or glial cells. Background technique [0002] Neural stem cells (NSCs) are a kind of mother cells with division potential and self-renewal ability, which exist in adult brain tissue as a kind of stem cells. Mainly distributed in the ependyma, subventricular zone, striatum, hippocampal dentate gyrus and other regions, it is a kind of pluripotent cell that can self-renew and differentiate into a variety of neural cells in the adult nervous system. neurons, astrocytes, and oligodendrocytes. The discovery of neural stem ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079C12N5/0793
Inventor 卞菁郑娇
Owner 南通大学技术转移中心有限公司
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