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Method for Inducing Growth Factors to Generate Neural Precursor Cells

A technology of neural precursor cells and growth factors, applied in nervous system cells, biochemical equipment and methods, animal cells, etc., can solve problems such as tumorigenicity, gene insertion mutation, unfavorable cell transdifferentiation technology application, etc., to achieve Reduce tumorigenicity, improve the effect of low-efficiency, high-efficiency cell transdifferentiation methods

Active Publication Date: 2020-10-09
TONGJI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can obtain high gene transduction efficiency, because the viral sequence is integrated into the genome of the cell, it will lead to gene insertion mutations and even tumorigenicity, so this potentially dangerous gene introduction method, Obviously not conducive to the application of cell transdifferentiation technology in the field of regenerative medicine

Method used

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  • Method for Inducing Growth Factors to Generate Neural Precursor Cells
  • Method for Inducing Growth Factors to Generate Neural Precursor Cells
  • Method for Inducing Growth Factors to Generate Neural Precursor Cells

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Common mouse embryonic fibroblasts were prepared and cultured in embryonic fibroblast medium. Thereafter, cells were seeded in 24-well cell culture plates at a density of 150,000 cells / well, using induction starter medium. During the first 7 days of induction, the cells were gently blown once a day in order to suspend the aggregated cells and form a better cell sphere. After 7 days, the cells were no longer blown and blown, so that the cells rapidly expanded from the adhering clumps, and regional proliferation of the cells could be observed. 14 days after the initiation of induction, the cells in each well of the 24-well culture plate were digested with trypsin / EDTA (Invitrogen), spread into a corresponding 35mm cell culture dish, and the culture system was replaced with fresh neural stem cells at this time Medium. After continuing to culture for 7 days, it can be observed that the adherent cells will proliferate regionally, and many neuroepithelial-like cells crawl o...

Embodiment 2

[0045] Nestin-GFP mouse embryonic fibroblasts were prepared and cultured in embryonic fibroblast medium.

[0046] (1) Cells were planted in a 24-well cell culture plate at a density of 150,000 cells / well, and the culture system was replaced with fresh induction starting medium. During the first 7 days of induction, the cells were gently blown once a day in order to suspend the aggregated cells and form a better cell sphere. After 7 days, the cells were no longer blown and blown, so that the cells rapidly expanded from the adhering clumps, and regional proliferation of the cells could be observed. 14 days after the initiation of induction, the cells in each well of the 24-well culture plate were digested with trypsin / EDTA (Invitrogen), spread into a corresponding 35mm cell culture dish, and the culture system was replaced with fresh neural stem cells at this time Medium. After continuing to culture for 7 days, it can be observed that the adherent cells will proliferate region...

Embodiment 3

[0049] Molecular and functional identification of the neural precursor cells obtained by the growth factor induction method obtained in Examples 1 and 2:

[0050] (1) extract the RNA of the neural progenitor cell that embodiment 1 and 2 obtain, and carry out real-time quantitative PCR detection, find that cell 1# and 2# have highly expressed neural progenitor cell molecular markers such as Nestin, Pax6 and Sox2, but not The original fibroblast markers Thy1 and Col3a1 were expressed again, which proved that the neural precursor cells obtained in Examples 1 and 2 did have the characteristics of neural precursor cells. Figure 5 shown.

[0051] (2) Extract the RNA of the neural precursor cells obtained in Examples 1 and 2, and perform gene expression profiling analysis on cells 1# and 2# through the next-generation sequencing technology-RNA-seq, which proves that these cells are derived from global gene expression The angle is very similar to the obtained neural progenitor cells...

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Abstract

The invention relates to a method for generating neural progenitor cells through induction by growth factors. The method comprises the following steps of A, regulating and controlling cell upstream and downstream signal channels by adding specific growth factors into a basic culture medium, wherein cell factors including LIF (Leukaemia Inhibitory Factors), B27 (not containing vitamin A), heparin,FGF-2 and EGF are used; and through the combination of the factors, mammal body cells are dedifferentiated to a partial reprogramming state; B, then using passage-maintaining growth factors N2, B27 (containing vitamin A), FGF2, EGF and the like of neurological cells for continuously dedifferentiating the part of cells in the reprogramming state into functional neural progenitor cells; and C, further culturing the neural progenitor cells obtained after the induction into a neural stem cell culture medium for further culture (amplification and storage can be performed). The method provided by the invention has the advantages that on one hand, the immunogenicity and the exogenous gene insertion of a conventional viral mediated induction method are avoided; the potential tumorigenicity is alsoreduced by using the growth factor; and on the other hand, the problem of low efficiency in the existing small molecular compound induction method is also greatly solved.

Description

technical field [0001] The invention relates to a method for inducing and obtaining functional neural precursor cells from mammalian somatic cells in vitro, especially a method for generating neural precursor cells induced by growth factors. Background technique [0002] As a country with a large population in the world, and facing increasingly severe population aging, the existing medical and surgical treatment methods in my country are no longer sufficient to meet the increasing number of organ defects, failures, and functional problems caused by trauma, disease, aging, and genetic lesions in our country. barriers and other clinical needs. Therefore, the research on stem cells and regenerative medicine has received widespread attention from all walks of life including many scientific research institutes and biotechnology companies. [0003] Stem cells generally refer to primitive undifferentiated cells that have the potential to differentiate into one or more types of cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0618C12N2501/11C12N2501/115C12N2501/235C12N2501/91
Inventor 高绍荣高睿陈嘉瑜张林凤臧茹歌刘文强陈珺
Owner TONGJI UNIV
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