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Medium for direct differentiation of pluripotent stem cell-derived mesenchymal stem cell, method for preparing mesenchymal stem cell by using same, and mesenchymal stem cell prepared thereby

A technology of pluripotent stem cells and mesenchymal stem cells, which is applied in the field of direct differentiation of pluripotent stem cells, can solve the problems of affecting differentiation efficiency, unstable differentiated cell properties, uneven drug effects, etc., and achieves the effect of simple preparation process and high yield

Pending Publication Date: 2021-06-18
车比奥泰有限公司
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Problems solved by technology

In addition, in the direct differentiation method without embryoid bodies, there are also cases where differentiation is induced by treatment with SB431542, one of the TGF-β inhibitors, but it has been reported that in most cases, at a high concentration of 10 μM or more Requires a drug treatment period of more than 11 days
Therefore, it takes a long time for mesenchymal stem cells prepared according to the currently reported methods to exhibit high content of mesoderm properties
In addition, even if embryoid bodies of uniform size and shape are formed for use, the effect of the treated drug may be uneven due to the difference in the microenvironment in the cells constituting the inside and outside of the embryoid body, so there may be effects that affect the differentiation efficiency and Technical instability issues with differentiated cell properties
[0003] Accordingly, although it is a biological therapeutic agent prepared based on the proliferation phenomenon of cells, no preventive or supplementary measures are taken during its preparation to prevent the risk of cellular senescence and mutations that may be caused by the proliferation phenomenon, so it is necessary to solve this problem. technical solution to the problem

Method used

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  • Medium for direct differentiation of pluripotent stem cell-derived mesenchymal stem cell, method for preparing mesenchymal stem cell by using same, and mesenchymal stem cell prepared thereby
  • Medium for direct differentiation of pluripotent stem cell-derived mesenchymal stem cell, method for preparing mesenchymal stem cell by using same, and mesenchymal stem cell prepared thereby
  • Medium for direct differentiation of pluripotent stem cell-derived mesenchymal stem cell, method for preparing mesenchymal stem cell by using same, and mesenchymal stem cell prepared thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, direct differentiation of human embryonic stem cells to mesenchymal stem cells

[0077] 1.1. Culture of human embryonic stem cells

[0078] To culture human embryonic stem cells without supporting cells, proceed as follows.

[0079] Specifically, CTS Cellstart, a cell adhesion function enhancer composed only of components of human origin TM at 2.0cm 2 Surface area 4-well tissue culture dishes (Tissue culture dish) were treated at 4°C at a concentration of 160 μl / well for 24 hours. Thereafter, remove the remaining CTSCellstart from the tissue culture dish (Tissue culture dish) at room temperature TM , and then the mTeSR as embryonic stem cell culture medium TM (StemCells, USA) the culture solution was placed in a tissue culture dish at a concentration of 500 μL / well, and then placed at 37°C in 5% CO 2 incubator.

[0080] Next, human embryonic stem cells (CHA-hES NT 18, CHA University) proliferating on Sertoli cells were broken into small pieces ( sma...

experiment example

[0097] Experimental example, analysis of characteristics of mesenchymal stem cells derived from human embryonic stem cells

[0098] 1. Analysis of cell morphology

[0099] In order to analyze the cell morphology characteristics of the mesenchymal stem cells obtained in Example 1 and Comparative Example 1, under a phase-contrast microscope (Nikon TE-2000), by forming The cell proliferation form (vortex shape) to confirm whether it is proliferating, the results are shown in figure 2 and image 3 .

[0100] figure 2 It is a photograph of cell morphology characteristics of human embryonic stem cell-derived mesenchymal stem cells according to a specific embodiment.

[0101] image 3 It is a photograph of cell morphology characteristics of human embryonic stem cell-derived mesenchymal stem cells by the embryoid body formation system according to Comparative Example.

[0102] like figure 2 As shown, it can be confirmed that the human embryonic stem cell-derived mesenchymal ...

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Abstract

The present invention relates to a medium for direct differentiation of embryonic stem cell-derived mesenchymal stem cells, a method for preparing mesenchymal stem cells by using same, mesenchymal stem cells prepared thereby, and a cell therapy product comprising the same mesenchymal stem cells. In a medium composition and a method according to an embodiment, mesenchymal stem cells can be prepared at high yield within a short period of time. In addition, the method is simple in preparation procedure because of the absence of an embryoid body formation step and allows homogeneous cells to be prepared, thus advantageously providing a cell therapy product within a reduced period of time, compared to conventional methods.

Description

technical field [0001] The present invention relates to a medium for directly differentiating pluripotent stem cells, such as embryonic stem cell-derived mesenchymal stem cells, a method for preparing mesenchymal stem cells therefrom, mesenchymal stem cells prepared thereby, and cell therapy agents comprising the same. Background technique [0002] As a prior art for producing mesenchymal stem cells using human embryonic stem cells, there is a production method in which a TGF-β inhibitor is treated for 14 days after embryoid bodies are formed. The TGF-β inhibitor treatment was carried out in order to easily secure mesoderm cells by inhibiting endoderm cell differentiation of embryoid bodies. In addition, in TGF-β inhibitors generally used in preparation of mesenchymal stem cells from human embryonic stem cells, the treatment amount of SB431542 is 10 μM or more. Among TGF-β inhibitors, treatment with 1 μM or more of SB431542 has the effect of inhibiting the activity of trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28
CPCA61K35/28C12N5/0668C12N2501/727C12N2501/15C12N2506/02C12N2501/115C12N2501/73C12N2502/1323C12N2513/00C12N5/0662C12N2500/32C12N2506/45C12N2500/30C12N2501/734C12N2527/00C12N2533/00
Inventor 李东律李定恩郑寿敬朴卿顺朴志勋
Owner 车比奥泰有限公司
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