Direct cell conversion-based method for differentiation of neural stem cells into astrocytes

A technology of astrocytes and neural stem cells, applied in nervous system cells, biochemical equipment and methods, animal cells, etc. Problems such as low differentiation time efficiency

Pending Publication Date: 2021-09-24
KOREA UNIV RES & BUSINESS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since astrocytes differentiated by conventional methods contain other cells besides pure astrocytes, there is a problem of low astrocyte differentiation efficiency and purity (Korean Patent Publication No. 10-2014-0071512)
Furthermore, the conventional method requires a long differentiation time of 180 days or more and is inefficient
Differentiation using other cytokines or small molecule compounds is being investigated as a solution to these problems posed by conventional methods, but a definitive differentiation method has not yet been established

Method used

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  • Direct cell conversion-based method for differentiation of neural stem cells into astrocytes
  • Direct cell conversion-based method for differentiation of neural stem cells into astrocytes
  • Direct cell conversion-based method for differentiation of neural stem cells into astrocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Astrocyte Differentiation Medium

[0093] Prepare 1x10 in a 60mm Petri dish 5 Personal neural stem cells (see Korean Patent No. 1816103) were cultured the next day in neurobasal medium containing DMEM / F12, N2, B27, bFGF, and EGF. Then, the culture solution was changed every 2 to 3 days. On day 5, cells were removed using Accutase and seeded on Petri dishes 1x10 6 cells were cultured in suspension. At this point, the seeded NSCs aggregated together rather than attaching to the bottom of the plate, forming pellets. The medium was replaced with fresh medium every 2 days, and after about 7 days, the spheres had reached a size visible to the naked eye. The plate was first coated with PLO / FN and then the spheres were placed on it.

[0094] The next day, the medium was replaced with a medium for differentiation into astrocytes. A medium containing CNTF (5 ng / ml), bFGF (8 ng / ml) and BMP4 (10 ng / ml) in a basal medium containing DMEM / F12, N2 and B27 was used for ...

Embodiment 2

[0096] Example 2: Validation of Characterization of Differentiated Astrocytes

[0097] Neural stem cells were differentiated into astrocytes by the method using the differentiation medium of Example 1, and it was determined whether the differentiated astrocytes exhibited general characteristics.

[0098] First, immunocytochemistry (ICC) was performed to determine whether astrocytes expressed astrocyte markers. Differentiated cells were fixed overnight in 4% paraformaldehyde (PFA) in the refrigerator. Cells were washed twice with PBS buffer and then treated with 0.1% triton X-100 for 10 minutes to create an environment for efficient staining (permeabilization step). After blocking with 10% NDS (normal donkey serum) for one hour, the primary anti-GFAP antibody was diluted 1:400 in 2% NDS to stain the cells, and the cells were treated with the resulting antibody at 4°C for one day. The next day, the cells were reacted with the secondary antibody for one hour at room temperature...

Embodiment 3

[0103] Example 3: Validation of Maturation of Differentiated Astrocytes

[0104] Astrocytes must mature to be able to perform their primary functions, such as supporting the maintenance of neuronal function. To verify this maturation, it was determined whether ion channels were localized in the astrocyte membrane, or whether glutamate uptake was being performed appropriately.

[0105] 3-1: Detection of ion channel markers

[0106] The presence of ion channels in astrocytes was used as a criterion for determining whether astrocytes were mature enough to perform various functions. Therefore, aquaporin 4 (AQP4), Kir4.1, and Twik-1, which are ion channel markers present in astrocytes, were detected by PCR. The results showed that channels that could exchange ions were formed in differentiated astrocytes (Fig. 2A). AQP4, Kir4.1 and Twik-1 were not detected in negative control HDFs (human dermal fibroblasts), but the expression of AQP4, Kir4.1 and Twik-1 was detected in differe...

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Abstract

The present invention relates to a method for efficiently differentiating neural stem cells into astrocytes and, more particularly, to a cell conversion-based method for more efficiently differentiating human neural stem cells into astrocytes that exhibit immune response suppression ability within a short period of time. Unlike a conventional method, the method for differentiating neural stem cells into astrocytes by using a differentiation medium containing a combination of several cytokines, according to the present invention, involves a shortened differentiation time and has excellent differentiation efficiency, and the differentiated astrocytes exhibit immune response suppression ability, and thus can be useful as an agent for treating various brain diseases such as degenerative neurological diseases.

Description

technical field [0001] The present invention relates to a method for efficiently differentiating neural stem cells into astrocytes, and more particularly to a method for more efficiently differentiating human neural stem cells into cells exhibiting immune response suppression based on cell transformation in a short period of time. Competence of the astrocyte approach. Background technique [0002] The world has long been actively researching ways to treat diseases caused by neuronal cell death, such as neurodegenerative diseases such as Huntington's disease, Parkinson's disease and Alzheimer's disease. Typically, research is underway to develop a method of differentiating stem cells into neurons to replace damaged neurons. Due to the inherent characteristics of stem cells, it is difficult to differentiate stem cells into desired cells, so this method is not widely used. No innovative method has yet been developed. [0003] Most neurodegenerative diseases are primarily cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCA61P29/00A61K35/30C12N5/0622C12N2506/08C12N2501/115C12N2501/11C12N2501/155C12N2501/13
Inventor 洪性会张娥荣崔京雅
Owner KOREA UNIV RES & BUSINESS FOUND
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