Application of Ascl1 in induction of transdifferentiation of astrocytes into functional neurons

Astrocytes, a functional technology for use in biotechnology and cell therapy

Active Publication Date: 2016-05-04
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is currently no method or pathway that can t

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  • Application of Ascl1 in induction of transdifferentiation of astrocytes into functional neurons
  • Application of Ascl1 in induction of transdifferentiation of astrocytes into functional neurons
  • Application of Ascl1 in induction of transdifferentiation of astrocytes into functional neurons

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Experimental program
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preparation example Construction

[0116] The preparation of astrocytes refers to "Preparation of separate astroglia and oligodendroglial cells 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]For the immunochromogenicity of cultured cells, refer to "Direct conversion off ibroblaststofunctional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). The immunochromogenic combination of tissue sections combined with in situ hybridization and immunochromogenic double labeling experiments refer to published methods conduct. The primary antibodies used for immunochromogenicity include: mouseanti-GFAP (Millipore, 1:1,000), rabbi...

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. The packaging of lentivirus refers 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 promoter (2....

Embodiment 2A

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

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Abstract

The invention provides an application of Ascl1 in induction of transdifferentiation of astrocytes into functional neurons, and concretely provides a use of an achaete-scute complex homolog-like 1 (Ascl1) gene or a protein or a promoter thereof in preparation of a medicinal composition for inducing astrocytes into functional neurons, and/or in preparation of a medicinal composition for treating nervous system diseases. The application is hopeful to become an effective method for in vitro culture of neurons and generation of new neurons through stimulation in human bodies, so the Ascl1 can be widely applied in treatment of the nervous system diseases, such as neurodegenerative diseases and central nervous trumtic diseases.

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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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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