Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof

A technology of neural stem cells and small molecule compounds, which is applied in the fields of biotechnology and neurodevelopment, and can solve problems such as foreign gene intervention and clinical safety hazards

Active Publication Date: 2015-09-09
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the methods of extracting neural stem cells from brain tissue and differentiating embryonic stem cells and induced pluripotent stem cells into neural stem cells have been matured. In addition, the methods of inducing the transdifferentiation of somatic cells into neural stem cells with different factor combinations are also becoming more and more perfect; but the existing The transdifferentiation method involves the intervention of exogenous genes, which has great clinical safety risks

Method used

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  • Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof
  • Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof
  • Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1 Screening Compound Combinations for Inducing Somatic Cells to Produce Neural Stem Cells under Normal Physiological Hypoxic Conditions

[0140] 1.1 Screening the clones formed by VPCR combination cultured somatic cells under normal physiological hypoxia

[0141] Through a large number of composition screening, it is basically determined that the combination of VPCR (VPA, CHIR99021, Repsox and Parnate) at 3% or 5% O 2 Treat mouse fibroblasts under the condition of about 10 days to produce dense clones, but under the condition of 21% O2, no dense clones can be produced ( figure 1 A). About 40 compact clones can emerge from 200,000 starting cells. The induction efficiency of intermediate clones was at 5% O 2 relative to 3%O 2 The condition is slightly higher, therefore, 5% O is used in subsequent induction experiments 2 cultivation conditions.

[0142] 1.2 AP staining of cell clones in 1.1

[0143] Alkaline phosphatase AP staining of these intermediate clones...

Embodiment 2V

[0148] Example 2 Detection of characteristics of VCR-treated cell clones

[0149] 2.1 Expression of Sox2 under different oxygen pressure

[0150] Sox2 expression could not be effectively induced under normoxic conditions or with other compound combinations lacking Repsox, CHIR99021 or VPA ( figure 2 C and 2D)

[0151] 2.2 Expression of different neural stem cell-related genes in cell clones

[0152] Such as image 3 As shown in B, the mouse fibroblasts were treated with VCR under normal physiological hypoxic conditions, and it was found that the expression of Sox2 was significantly increased on the 5th day, reached the peak on the 10th day, and fell slightly on the 15th day; while Oct4 and Nanog The expression of was only slightly increased on the 10th day.

[0153] Conclusion: Small molecular compound combination VCR can be used in 5% O 2 Normal Physiological Hypoxic Conditions Facilitate Transdifferentiation of Mouse Embryonic Fibroblasts to Intermediate Dense Clones. ...

Embodiment 3

[0154] Example 3 Neural stem cell differentiation of VCR-treated cell clones

[0155] 3.1 Morphological observation of cultured clones

[0156] Under normal physiological hypoxic conditions, the cells treated with the VCR combination for about 10 days were digested and re-plated, and cultured in the neural stem cell medium containing heparin, epidermal growth factor EGF, and basic fibroblast growth factor bFGF. After about 7-10 days, neural stem cell-like bipolar morphology appeared in the cultured cells ( image 3 C).

[0157] 3.2 Neural stem cell-specific gene detection

[0158] Neural stem cell marker genes Nestin, Sox2 and Pax6 can be detected by immunofluorescence staining ( Figure 4 A). Further, reverse transcription polymerase chain reaction detected that the expression levels of neural stem cell-specific genes including Sox2, Pax6, Blbp, Ascl1 and Brn2 were also enhanced ( image 3 D, ciNPCp1).

[0159] Conclusion: Neural stem cell-like cells appeared in the cul...

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Abstract

Provided are a method for inducing the transdifferentiation of somatic cells into neural stem cells and application for same. Using a combination of a histone deacetylase (HDAC) inhibitor, a glycogen synthase kinase (GSK-3) inhibitor, and a transforming growth factor β (TGF-β) signal pathway inhibitor, in a low-oxygen normal physiological environment, induce somatic cells such as fibroblasts and epithelial cells to form into neural stem cells having good pluripotency and passage stability.

Description

technical field [0001] The invention belongs to the fields of biotechnology and neurodevelopment, and specifically relates to a method for inducing somatic cells to transdifferentiate into neural stem cells and its application. Background technique [0002] Terminally differentiated cells are considered to be a class of cells that have specific functions and phenotypes and have lost the potential for further development. However, earlier studies have found that nuclei from terminally differentiated cells can be used to clone animals, and in vitro cell fusion can also lead to reprogramming of cell lineages, suggesting that epigenetic modifications during development are reversible. A large number of recent studies have found that the combination of specific transcription factors can not only induce somatic cells to dedifferentiate into pluripotent stem cells through reprogramming, but also directly transdifferentiate into specific somatic cells of other lineages, thus providi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/071C12N5/0797A61K35/30A61P25/00
CPCA61K35/30A61K35/33A61P25/00C12N5/0619C12N2501/065C12N2501/15C12N2501/727C12N2506/1307G01N33/502G01N33/5026
Inventor 裴钢赵简程林胡文祥裘斌龙
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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