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Application of ascl1 in inducing transdifferentiation of astrocytes into functional neurons

A technology of astrocytes, neurons, used in biotechnology and cell therapy

Active Publication Date: 2020-08-18
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no method or pathway that can transdifferentiate normal astrocytes into functional neurons

Method used

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  • Application of ascl1 in inducing transdifferentiation of astrocytes into functional neurons
  • Application of ascl1 in inducing transdifferentiation of astrocytes into functional neurons
  • Application of ascl1 in inducing transdifferentiation of astrocytes into functional neurons

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0116] The preparation of astrocytes refers to "Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue" (McCarthy, K.D. & de Vellis, J.J. Cell Biol. 85, 890-902 (1980)). The dorsal midbrain of postnatal day 5-7 mice or adult mice was removed and digested with 0.25% trypsin for 15 min. The blown cells were cultured in DMEM / F12 solution containing 10% serum for 7-9 days. After the oligodendrocytes are removed by shaking, the obtained cells are astrocytes.

[0117] immunochromogenic

[0118]The immunochromogenicity of cultured cells refers to "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). Color double-labeling experiments were carried out according to published methods. The primary antibodies used for immunochromogenicity include: mouse anti-GFAP (Millipore, 1:1,000), rabbit-GFAP (DAKO, 1:1,000), mouse anti-Tuj1 (Covance, 1:500), mouse anti-Map2 (Sigma, 1...

Embodiment 1

[0124] Example 1 plasmid construction and virus infection

[0125] The cDNA of mouse Ascl1 gene was cloned into lentiviral expression vector FUGW-IRES-EGFP to obtain FUGW-Ascl1. Replacement of GFP in the FUGW-Ascl1 plasmid with tdTomato yielded FUW-Ascl1-tdTomato. The empty lentiviral expression vector FUGW and FUW-tdTomato were used as controls respectively. For the packaging of lentiviral vectors, refer to the literature "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, I.M. Nat. Protoc. 1, 241-245 (2006)).

[0126] Add lentivirus after 24 hours of plate culture of astrocytes, and replace the medium after 24 hours of infection: DMEM / F12, B27, Glutamax and penicillin / streptomycin. After 6-7 days of infection, brain-derived neurotrophic factor (BDNF; PeproTech, 20 ng / ml) was added to the medium every three days.

[0127] To make the GFAP-AAV vector, the CMV promoter in the AAV-FLEX-Arch-GFP plasmid (Addgene) was replaced with the hGFAP ...

Embodiment 2

[0128] Example 2 Ascl1 will in vitro Transdifferentiation of dorsal midbrain astrocytes into neurons

[0129] Firstly, astrocytes from the dorsal midbrain of postnatal day 5-7 (P5-P7) mice were isolated and purified. The properties of these glial cells were verified by examining molecular markers of different cell types ( figure 1 ). Most of the cells express GFAP and S100β, the marker molecules of astrocytes, a small amount of cells express O4 and CNPase, the marker molecules of oligodendrocytes, and a small number of cells express NG2, the marker molecules of NG2 glial cells, which are not detected Expression of neuron marker Tuj1 and stem cell markers Sox2 and Oct4.

[0130] result

[0131] 2.1 Transformed astrocytes express mature neuron marker molecules

[0132] 10 days after astrocytes were transfected with the control lentiviral vector FUGW, astrocytes did not express the neuron marker molecule Tuj1( figure 2 a), still maintaining the morphology of glial cells w...

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Abstract

Uses of an achaete-scute complex homolog-like 1 (Ascl1) gene or a protein thereof or an accelerator thereof: (i) use in preparing a pharmaceutical composition for inducing astrocytes to become functional neuron cells; and / or (ii) use in preparing a pharmaceutical composition for treating neurological disorders such as neurodegenerative pathologies, central nervous system trauma, and so on.

Description

technical field [0001] The invention belongs to the field of biotechnology and cell therapy, in particular, the invention relates to a method for inducing transdifferentiation of astrocytes into functional neuron cells and its application. Background technique [0002] Many transcription factors and chromatin epigenetic modification processes play very important roles in maintaining the identity stability of differentiated cells. However, studies of induced pluripotent stem cells (iPS cells) have shown that differentiated cells are not irreversibly locked in their mature state, but can be dedifferentiated by selective overexpression of specific transcription factors. [0003] Recent findings have found that specific transcription factors can directly induce fibroblasts into functional neurons, further suggesting that non-neuronal cells can be directly transdifferentiated into neurons. For example, astrocytes in the cerebral cortex of postnatal mice can be transdifferentiate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K38/17A61K45/00A61K35/30A61P25/00C12N15/85C12N5/10C12N5/0793C12Q1/68G01N33/68
CPCA61K38/17A61K45/00A61K48/00C12N5/10C12N15/63C12Q1/02C12Q1/68
Inventor 程乐平章晓辉刘月光缪庆龙袁嘉成
Owner CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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