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Neural stem cell capable of self-renewing, preparation method and application thereof

A technology of neural stem cells and cells, applied in the fields of cytology and neurobiology, can solve problems such as high tumorigenicity and clinical application threats

Active Publication Date: 2014-11-19
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of NSC can self-replicate and proliferate in large quantities in vitro, and still has multi-lineage differentiation potential after transplantation into the human body, but the clinical application of such cells is threatened by transgenic manipulation, and such cells are often highly tumorigenic

Method used

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  • Neural stem cell capable of self-renewing, preparation method and application thereof
  • Neural stem cell capable of self-renewing, preparation method and application thereof
  • Neural stem cell capable of self-renewing, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Isolation and culture of primary mouse neural stem cells

[0043] 1) Obtaining mouse fetuses and primary culture of brain tissue

[0044] Mice pregnant for more than 5 days were killed by neck dislocation, the abdominal cavity was opened under aseptic conditions, the uterus was taken out, and washed three times with PBS containing antibiotics. The fetus is removed from the womb, the torso is removed, and the brain tissue is carefully peeled away under a microscope. The stripped brain tissue was digested with 0.25% trypsin for 2 minutes, after stopping with serum, pipetting several times, centrifuged at 1000 rpm for 5 minutes, poured off the supernatant, washed twice with N2B27+CHIR99021 (3mM)+Y27632 (10nmol / L) culture solution After that time, add the pre-warmed culture medium in 5% CO 2 , 37 ℃ incubator culture.

[0045] 2) Passage and purification of mouse neural stem cells

[0046] The above-mentioned primary cells can be digested and subcultured after...

Embodiment 2

[0049] Example 2: Directed Neural Stem Cell Differentiation from Embryonic Stem Cells

[0050] 1) The step of directing mouse embryonic stem cells to NSC

[0051] Digest mES cells (mouse 46C embryonic stem cells, donated by Professor Ying Qilong, University of California, USA) with 0.25% trypsin into a single-cell suspension, add serum to stop centrifugation, and use directional neural differentiation medium (and primary culture medium) same ingredients) resuspended. Before the resuspended cells are transferred to the culture plate, the culture plate should be coated with 50 μg / ml Laminin for more than 2 hours to help the cells adhere to the wall. The cell seeding density was 1.0×10 per ml 4 with 1.0×10 5 Between individual cells, the cells adhere to the wall and grow in a single layer. There is a clear boundary between the cells. If the cells are too dense, the cells proliferate too fast and will soon fill the culture dish. The cell density is too low, which is not conduci...

Embodiment 3

[0054] Embodiment 3: NSC cell subcutaneous injection tumor formation experiment in nude mice

[0055] NSC cells were digested with trypsin, dispersed into single cells and resuspended in 0.3ml of PBS. The cell suspension was injected subcutaneously in the armpit of male Balb / c nude mice. After 2 weeks, teratomas could be seen in the armpit of nude mice injected with ES cells. The tumor matures after 30-50 days. Mature teratomas were isolated and fixed in 10% neutral formalin, embedded in paraffin for routine histological sectioning, HE staining, and photomicrographing. No tumor was found in the group injected with NSC cells.

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Abstract

The invention provides a neural stem cell (NSC) strain. The NSC strain can be isolated from animal brain tissues from different species. Embryonic stem cells (ES), induced pluripotent stem cells and other stem cells can be differentiated into the NSC. The NSC strain is characterized by quick proliferating and stable passaging; prolonged expressing biomarker proteins of Oct4 and Sox1; high efficient being differentiated into a plurality of neurons and glial cells. The invention further provides an establishing method and an application for the NSC.

Description

technical field [0001] The present invention relates to the fields of cytology and neurobiology, in particular, the present invention relates to a neural stem cell, its preparation method and its application. Background technique [0002] Neural stem cell (NSC) is a kind of mother cell with division potential and self-renewal ability, which can produce various cells of nerve tissue through asymmetric division. According to the timing of differentiation potential, it is divided into: neural tube epithelial cells, radial glial neurons and neuroblasts mainly exist in the embryonic period. Radial glial neurons mainly exist in juveniles, which can divide to produce themselves and produce neuronal precursor cells or glial cells at the same time; neuroblasts mainly exist in adult animals, which can produce neural precursor cells, neurons and glial cells. Various glial cells. According to the classification of parts, it can be mainly divided into two categories: neural crest stem ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797A61K35/30A61P9/00A61P9/10A61P25/00A61P25/16A61P25/28
Inventor 钱其军彭欣荣高海霞刘韬李琳芳吴孟超
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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