In-vitro induction and long-term culture system, induced culture method and application of forebrain neural stem cells

A neural stem cell and culture system technology, applied in the field of biomedical engineering, can solve the problem of inability to maintain forebrain neural stem cells for a long time

Pending Publication Date: 2021-04-09
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is still a lack of effective means in the field to stably maintain the molecular phenotype and self-renewal of forebrain neural stem cells in vitro, especially the long-term maintenance of in vitro forebrain neural stem cells cannot be achieved under culture conditions with defined chemical components

Method used

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  • In-vitro induction and long-term culture system, induced culture method and application of forebrain neural stem cells
  • In-vitro induction and long-term culture system, induced culture method and application of forebrain neural stem cells
  • In-vitro induction and long-term culture system, induced culture method and application of forebrain neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Induced acquisition and long-term culture of forebrain neural stem cells in vitro and their molecular phenotype and functional testing

[0049] 1. Acquisition of forebrain neural stem cells

[0050] The surface of the cell culture dish was pre-coated with DMEM / F12 medium containing 1% matrigel (Corning, Cat#356231), and stored at 4°C overnight. Human pluripotent stem cells (H1 cell line) were inoculated at a density of 10%, and cultured in an induction medium of forebrain neural stem cells.

[0051] The optimal composition of the medium is as follows: basal medium DMEM / F12, 0.5×N2 (Thermo Fisher Scientific, Cat#17502048), 0.5×B27 (Thermo Fisher Scientific, Cat#17504044), 60 μg / mL 2-phospho-vitamin C ( Sigma-Aldrich, Cat#A8960), 1% penicillin / streptomycin, and the above medium is named as basal medium or BM. Add 1 μM BMP receptor inhibitor DMH1 (Selleck, S714602), 0.1 μM Porcupine inhibitor LGK974 (Selleck, S714302), 2 μM TGFβ receptor inhibitor SB431542 (Se...

Embodiment 2

[0058] Example 2: Long-term cultured forebrain neural stem cells in vitro can be differentiated into forebrain cortical neurons

[0059] 1. Monolayer differentiation: the surface of the cell culture dish was pre-coated with DMEM / F12 medium containing 1% Matrigel, and stored at 4°C overnight. The long-term cultured forebrain neural stem cells were digested and centrifuged, resuspended with neuron differentiation medium and inoculated on the pre-coated culture support, the medium was replaced every 1-3 days, and cultured for 2 to 3 weeks; neurosphere differentiation: The long-term cultured forebrain neural stem cells were digested and centrifuged, resuspended with neuron differentiation medium and inoculated in a low-adsorption six-well culture plate, the cells converged into balls, the medium was replaced every 1-3 days, and cultured for more than 1 month .

[0060] The optimal composition of the medium is as follows: 1×B27 supplement, 1% penicillin / streptomycin, 60 μg / mL 2-ph...

Embodiment 3

[0063] Example 3: Long-term cultured forebrain neural stem cells in vitro can be applied to transplantation

[0064] The forebrain neural stem cells obtained by the method consistent with Example 1 were cultured in vitro. According to 1*10 5 Forebrain neural stem cells were passaged to a six-well plate, and EGFP lentivirus (Shanghai Heyuan Biotechnology, clone number GL107) was added to the medium after 24 hours, and a blank control group was set at the same time (there was no virus in the medium) After 8-10 hours, replace with virus-free SCDEF medium. After 48 hours, the fluorescence positive rate of the cells was observed under an inverted microscope. Because the virus-transfected cells will have puromycin (puromycin, puro) resistance, puro (Selleck, S741706) was added to select for 2-3 days, and the cells without puro resistance will die. EGFP-labeled cell lines can then be obtained. Digest with TrypLE trypsin (Gibco, Lot: 2152550) at 37°C for 1 minute before transplant...

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Abstract

The invention provides an in-vitro induction and long-term culture system, an induced culture method and application of forebrain neural stem cells. The in-vitro induction and long-term culture system comprises a basal culture medium, as well as an induced differentiation factor and an in-vitro culture factor which are combined with the basal culture medium for use, wherein the basal culture medium comprises a DMEM / F12 liquid basal culture medium, a 0-5*B27 additive, a 0-5*N2 additive, 1% penicillin / streptomycin and 0.01-500 mu g / mL 2-phosphoric acid- vitamin C; the induced differentiation factor comprises 0.01-500 mu M of a TGF[beta] inhibitor, 0.01-10000 mu M of a BMP inhibitor and 0.01-50 mu M of a Porcupine inhibitor; and the in-vitro culture factor comprises 0.01-10000 nM of an Src kinase inhibitor, 0.01-500 mu M of a TGF[beta] inhibitor, 0.01-50 mu M of a GSK3 inhibitor, 0.01-500 ng / mL of an EGF family growth factor and 0.01-500 ng / mL of an FGF family growth factor. By using the culture system, the pluripotent stem cells can be induced in vitro to be differentiated into the forebrain neural stem cells and cultured in vitro for a long time, and the stable molecular phenotype and differentiation potential of the forebrain neural stem cells are maintained.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a system and method for inducing human pluripotent stem cells to differentiate into forebrain neural stem cells in vitro and maintaining the long-term culture of forebrain neural stem cells. Background technique [0002] Neurodegenerative diseases are a class of diseases that occur in the nervous system, causing damage or dysfunction of neurons and their attached dendrites, axons, and synapses, as well as glial cells throughout the nervous system. The incidence of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis has risen dramatically in recent years. At present, there are more than 3 million people in my country suffering from Parkinson's disease, more than 200,000 people have ALS, and it is reported that the number of people with Alzheimer's disease has exceeded 10 million. With the ...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/071C12N5/0793C12Q1/02A61K35/30A61P25/00
CPCC12N5/0623C12N5/0619G01N33/5058G01N33/5073A61K35/30A61P25/00C12N2500/38C12N2501/15C12N2501/155C12N2501/727C12N2501/724C12N2501/11C12N2501/115C12N2501/415C12N2533/90C12N2506/45C12N2503/02G01N2500/10Y02A50/30
Inventor 李文林袁媛李铁军章越凡虞欣璐
Owner THE NAVAL MEDICAL UNIV OF PLA
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