Preparation method for reprogramming somatic cells into neural stem cells by microRNA, and application of neural stem cells

A neural stem cell and reprogramming technology, which is applied in the fields of biotechnology and neurodevelopment, can solve the problems of inability to be applied clinically in time, clinical safety hazards, and teratomas

Active Publication Date: 2017-07-11
BINZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing indirect lineage transdifferentiation method involves the intervention of exogenous transcription factors, which may cause mutations caused by gene insertion errors, and may also cause the risk of teratoma, which has certain clinical safety risks
Moreover, the time for transdifferentiation is relatively long, and it cannot be applied in time to clinical practice.
[0005] Therefore, there is an urgent need in this field to develop a method for inducing somatic cell transdifferentiation into neural stem cells that does not require the intervention of exogenous transcription factors, to solve the problems of safety and low efficiency of directed differentiation, and as a new source of transplanted nerve cells, it has Important clinical application value

Method used

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  • Preparation method for reprogramming somatic cells into neural stem cells by microRNA, and application of neural stem cells
  • Preparation method for reprogramming somatic cells into neural stem cells by microRNA, and application of neural stem cells
  • Preparation method for reprogramming somatic cells into neural stem cells by microRNA, and application of neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1. Using reprogramming to obtain human neural stem cells from human adult foreskin fibroblasts

[0109] 1. Isolation and culture of skin fibroblasts:

[0110] The preparation method refers to the method of Ding et al. The foreskin discarded by circumcision was aseptically taken, placed in a petri dish filled with PBS solution, and washed twice to remove blood stains. Take it out and place it in a petri dish filled with PBS solution. Cut it into 1 mm3 fragments, transfer it to a centrifuge tube, add 0.25% trypsin, and place it in a 4°C refrigerator for overnight digestion. The next day, the culture medium containing fetal bovine serum was taken out and added to stop digestion and centrifugation to collect cells, and DMEF growth medium (high glucose DMEM, volume fraction 10% fetal bovine serum, 2mmol / L glutamine) was added to make a single cell suspension. Inoculate into culture flasks at a density of 4×104 cells / cm2, and incubate in an incubator at 37°C with a...

Embodiment 2

[0128] Example 2. Using reprogramming to obtain neural stem cells from mouse embryonic fibroblasts

[0129] 1. Isolation and cultivation of primary MEFs:

[0130] The preparation method refers to the method of McElroy SL et al. [12].

[0131] Pregnant mice of 13.5 days of pregnancy were killed by cervical dislocation and sterilized by immersing in 75% alcohol; the whole uterus was taken out, placed in a petri dish filled with PBS solution, washed three times to remove blood stains, transferred to a new petri dish, taken out For fetal mice, wash 3 times in a new petri dish; release the embryo, remove the head, tail, limbs and viscera of the embryo in turn, after washing 3 times, place the remaining body in a new petri dish, and cut the tissue with sterile scissors. Fully shred; add 0.25% trypsin-0.02% EDTA and gently pipette evenly, place in a 37°C carbon dioxide incubator and incubate for 10-20min, take it out every 5min and shake gently to make it fully digested; add fetal c...

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Abstract

The invention provides a method for induced transdifferentiation of somatic cells into neural stem cells, and an application of the neural stem cells, and concretely relates to neural stem cells with high yield, good pluripotency and good passage stability, formed through inducing human fibroblasts, epithelial cells, blood cells, adipocytes and other somatic cells in a normal physiological environment via a reprogramming technology by using microRNA-302, valproic acid, polybrene, epidermal growth factor and fibroblast growth factor. The method utilizes single microRNA to transfer the somatic cells without introducing an exogenous transcription factor, the clone forming time in the invention is much shorter than that in the prior art, and the neural stem cells are hopeful for developing therapeutic methods for drugs for treating degenerative neurological diseases (including spinal cord injury and other lesions), so the neural stem cells have a good clinical application prospect.

Description

technical field [0001] The invention belongs to the fields of biotechnology and neurodevelopment, in particular, the invention relates to a method for directly reprogramming and inducing transdifferentiation of somatic cells into neural stem cells and its application. Background technique [0002] With the increase of the aging society, the incidence of various neurodegenerative diseases remains high and has an upward trend. Neurodegenerative diseases and nervous system injuries (such as stroke, cerebral ischemia, spinal cord injury, etc.) are seriously threaten human health. Parkinson's disease, Alzheimer's disease, stroke and other diseases all have nerve cell loss and dysfunction due to the inability of differentiated mature nerve cells to divide and proliferate to compensate for damaged or dead cells. The pathological manifestations of diseases and injuries of the nervous system mostly involve the loss of nerve cells and defects in regeneration. Traditional medical and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85A61K35/30A61P25/00A61P25/28A61P25/16A61P25/14
CPCA61K35/30C12N5/0623C12N15/85C12N2501/115C12N2501/11C12N2500/38C12N2510/00C12N2506/1307C12N2501/999C12N2501/91C12N2800/107
Inventor 王跃嗣
Owner BINZHOU MEDICAL COLLEGE
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