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Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof

A technology of transforming growth factor and pluripotent stem cells, applied in the field of culture of induced pluripotent stem cells, can solve the problem of low efficiency of iPS induction

Inactive Publication Date: 2010-03-31
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to overcome the above-mentioned problem of extremely low efficiency of inducing iPS, the present invention provides the application and method of a type I transforming growth factor receptor (ALK5) inhibitor in producing induced pluripotent stem cells (iPS)

Method used

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  • Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof
  • Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof
  • Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Stem cell medium supplemented with ALK5 inhibitor can promote the formation of SKOM iPS

[0059] A. The virus of the four factors (SKOM) was mixed 1:1:1:1 (each 1ml) and infected into a total of 20,000 OG2 mouse embryonic fibroblasts (MEFs) in one well of a 12-well plate, And at 37 degrees, 5% CO 2 Cultured in mES medium (DMEM supplemented with 15% fetal bovine serum, 1000 U / ml leukemia inhibitory factor (LIF), penicillin / streptomycin, L-glutamine and non-essential amino acids). From day 3, add ALK5 inhibitor (A-83-01: 0.5 μM; LY-364947: 0.8 μM; SB-431542: 2 μM); on day 6, cells were digested with trypsin, counted, and inoculated 3000 Cells were pre-seeded with feeder cells (cultured in mouse embryonic fibroblasts treated with mitomycin (10 μg / ml)) in a 6 cm dish, and continued to be cultured with the addition of ALK5 inhibitor or solvent control; in Fourteen days later, alkaline phosphatase staining was performed and the number of AP-positive clones was co...

Embodiment 2

[0063] Example 2: Adding ALK5 inhibitor stem cell medium to promote the formation of SKO iPS

[0064] A. Infect a total of 20,000 mouse embryonic fibroblasts (MEFs) in a well of a 12-well plate after the viruses of the three factors (SKO) were mixed 1:1:1 (each 1ml), and at 37 degrees, 5% CO 2 cultured in mES medium. From day 3 onwards, ALK5 inhibitor or solvent control was added; on day 9, cells were digested with trypsin, counted, and 6000 cells were inoculated on feeder layer cells (cultured with mitomycin (10 μg / ml ) treated mouse embryonic fibroblasts) in a 6 cm dish, continue to use the addition of ALK5 inhibitor or solvent control to culture; count the number of Oct3 / 4-GFP positive clones under a fluorescent microscope on the 25th day, and carry out Alkaline phosphatase staining and counting the number of AP positive clones. Such as figure 2 As shown in a, compared with the control group, the addition of ALK5 inhibitors can increase the number of SKO-induced AP-pos...

Embodiment 3

[0066] Example 3: Identification experiment of pluripotency of induced pluripotent stem cell (iPS) cell line obtained by adding ALK5 inhibitor

[0067] A. As mentioned above, fibroblasts were infected with 3 factors, and then they were cultured in mES medium supplemented with ALK5 inhibitors. On day 25 after infection, representative ones were selected according to the clone morphology and fluorescence expression After several generations of stable passage, a uniform iPS cell line was formed.

[0068] Such as Figure 3a As shown, the left panel is a normal embryonic stem cell, and the right panel: the above-mentioned iPS cell line. These cell lines are morphologically very similar to embryonic stem cells and strongly express the green fluorescence of Oct3 / 4-GFP.

[0069] B. Before RNA purification, feeder cells were removed and uploaded on 0.2% gelatin for 2 passages. Total RNA was extracted using Trizol (Takara) reagent according to the manufacturer's instructions. Carry ...

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Abstract

The invention discloses application of an I type transforming growth factor receptor (ALK5) inhibitor in producing induced multi-potent stem cells (iPS) and a method thereof. The invention applies a culture medium containing the I type transforming growth factor receptor inhibitor to the producing induced multi-potent stem cells. The method comprises the following steps: inducing cDNA of multi-potent stem cell factors to mouse embryonic fibroblasts; culturing the mouse embryonic fibroblasts treated in the step (1) by using the culture medium added with the ALK5 inhibitor; and identifying clone of induced multi-potent stem cells. The I type transforming growth factor receptor (ALK5) inhibitor of the invention can efficiently produce induced multi-potent stem cells (iPS); and the method forefficiently inducing iPS cells has significance for promoting the medical application of the iPS, establishing a disease model, transplanting treatment, and developing safe iPS technology.

Description

technical field [0001] The invention relates to a culture method for induced pluripotent stem cells, in particular to the application of a type I transforming growth factor receptor inhibitor in producing induced pluripotent stem cells and the method thereof. Background technique [0002] The application prospect of hES cell (human embryonic stem cells) research is mainly transplantation therapy. In the field of tissue engineering, hES cells are used as seed cells to provide a large number of materials for clinical cell, tissue or organ transplantation therapy. Specific tissue cell types can be obtained by controlling the hES cell differentiation culture environment and transfecting key molecular genes that can promote the directed differentiation of ES cells. The use of such cells in transplantation therapy will bring new hope to the treatment of diabetes, Parkinson's disease, spinal cord injury, leukemia, myocardial injury, kidney failure, liver cirrhosis and other disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
Inventor 裴端卿米盖尔·埃斯特班梁佳良李荣辉
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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