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Spinal cord OLIG2<+> nerve progenitor cell induction and self-renewal culture system, induction method and application

A technology of neural progenitor cells and culture system, which is applied in the field of spinal cord OLIG2+ neural progenitor cells and maintains their stable self-renewal culture system, which can solve problems such as troubles and obstacles to basic research on cell therapy.

Active 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

[0006] However, at present, there is no way to obtain spinal cord OLIG2 that can stably self-renew in vitro. + Neural progenitor cells, and this undoubtedly brings certain obstacles and troubles to clinical transformation applications such as cell therapy and corresponding basic research

Method used

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  • Spinal cord OLIG2&lt;+&gt; nerve progenitor cell induction and self-renewal culture system, induction method and application
  • Spinal cord OLIG2&lt;+&gt; nerve progenitor cell induction and self-renewal culture system, induction method and application
  • Spinal cord OLIG2&lt;+&gt; nerve progenitor cell induction and self-renewal culture system, induction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Directed induction of human pluripotent stem cells to differentiate into spinal cord OLIG2 + neural progenitor cells and maintain their steady self-renewal

[0051] according to figure 1 , directed differentiation of human pluripotent stem cells into spinal cord OLIG2 + The neural progenitor cells are mainly divided into two steps: the first step is to induce the differentiation of human pluripotent stem cells to obtain spinal cord neural progenitor cells; the second step is to induce ventralization on the basis of the obtained spinal cord neural progenitor cells to obtain spinal cord progenitor cells. OLIG2 + neural progenitor cells.

[0052] 1. Directed induction of human pluripotent stem cells to differentiate into spinal cord neural progenitor cells

[0053] The induction and differentiation method of spinal cord neural progenitor cells is as follows: inoculate human pluripotent stem cells on a Matrigel-coated culture plate, and discard the human plur...

Embodiment 2

[0062] Example 2: Spinal cord OLIG2 + Neural progenitor cells differentiate into spinal cord motor neurons

[0063] Obtain the spinal cord OLIG2 of embodiment 1 gains + After neural progenitor cells are directed, the cells are directed to differentiate into mature spinal cord motor neurons. After induced differentiation, most of the cells have neuron morphology ( Figure 8 A). Immunofluorescent staining was performed on the obtained cells for identification, and the staining results showed that the cells were indeed neurons and had differentiated and matured ( Figure 8 B, C), while having the segmental positioning characteristics of the spinal cord ( Figure 8 C). In addition, this neuron expresses molecular markers of motor neurons and cholinergic neurons ( Figure 8 D, E).

[0064] The above results indicate that spinal cord OLIG2 + Neural progenitor cells can differentiate into mature spinal motor neurons in a relatively short period of time in vitro.

Embodiment 3

[0065] Example 3: Spinal cord OLIG2 + Neural progenitor cells differentiate into oligodendrocytes

[0066] Obtain the spinal cord OLIG2 of embodiment 1 gains + After neural progenitor cells, the cells are directed to differentiate into mature oligodendrocytes. After induction of differentiation, epithelial-like spinal cord OLIG2 + Neural progenitor cells gradually differentiated into bipolar or multipolar cells, and the cells at this stage were identified by immunofluorescence staining, and it was found that the cells expressed molecular markers of the oligodendrocyte lineage ( Figure 9 A-C), and the cells also have the ability to proliferate ( Figure 9 D), demonstrating that spinal cord OLIG2 + Neural progenitor cells and differentiate into oligodendrocyte precursor cells (OPCs). The precursor cells were planted on a laminin-coated culture plate to continue to induce their differentiation and maturation. After nearly 2 months of induction and differentiation, they were...

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Abstract

The invention relates to a spinal cord OLIG2<+> nerve progenitor cell induction and self-renewal culture system, an induction method and application. The culture system comprises a basic culture medium, an induced differentiation factor and an in-vitro maintenance factor, wherein the induced differentiation factor and the in-vitro maintenance factor are combined with the basic culture medium for use; the induced differentiation factor at least comprises a 0.01-50 microns of GSK3 inhibitor, a 0.01-500 microns of TGF beta inhibitor and a 0.01-500 microns of BMP inhibitor; and the in-vitro maintenance factor comprises a 0.01-50 microns of GSK3 inhibitor, a 0.01-500 microns of TGF beta inhibitor, 0.01-5000 ng / mL of Sonic Hedgehog, a 0.01-20 microns of Smoothened agonist, a 0.01-500ng / mL of LEGF family growth factor, a 0.01-500 ng / mL of FGF family growth factor, a 0.01-500 microns of BMP inhibitor, a 0.01-50 microns of JAK1 / 2 inhibitor, a 0.01-10000nM of Src kinase inhibitor and a 0.01-50 microns of RetinoicAcid.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a spinal cord OLIG2 capable of obtaining human pluripotent stem cells in vitro + The culture system, induction method and application of neural progenitor cells and maintaining their stable self-renewal. Background technique [0002] Human embryonic stem cells (hESCs) are human pluripotent stem cells (humanpluripotent stem cells, hPSCs) derived from the inner cell mass of embryos that can maintain stable self-renewal in vitro and retain multilineage differentiation potential. Theoretically, by simulating the developmental process of cells in vitro, hESCs can be induced to differentiate into any type of cells in the three germ layers of the human body. Because of this, directed induction of hESCs to obtain specific target cells has a very broad application prospect in disease cell therapy, drug screening and developmental biology research. [0003] During embryonic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797A61K35/30A61P25/00C12Q1/02
CPCC12N5/0623A61K35/30A61P25/00G01N33/5058C12N2501/724C12N2501/15C12N2501/155C12N2501/11C12N2501/115C12N2500/38C12N2501/727C12N2501/405C12N2506/45C12N2506/02C12N2533/90G01N2500/10Y02A50/30
Inventor 李文林张冠宇孙平新袁媛吕竺蔓
Owner THE NAVAL MEDICAL UNIV OF PLA
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