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Preparing method of iPS cell and medium for preparing iPS cell

A technology for pluripotent stem cells and culture medium, which is applied in the preparation of induced pluripotent stem cell culture medium and the field of induced pluripotent stem cell preparation, can solve the problems of low induction efficiency of iPS cells, obstacles to the development and application of iPS cells, etc. Good totipotency effect

Inactive Publication Date: 2012-07-11
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods will lead to lower induction efficiency of iPS cells. At present, the formation efficiency of iPS cells is generally lower than 1%, which poses a huge obstacle to the development and application of iPS cells.

Method used

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  • Preparing method of iPS cell and medium for preparing iPS cell
  • Preparing method of iPS cell and medium for preparing iPS cell
  • Preparing method of iPS cell and medium for preparing iPS cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 : Stem cell medium supplemented with lithium salt can promote the formation of iPS induced by four factors

[0060] a. Mix the virus with four factors (Oct4, Sox2, Klf4, c-Myc) in equal volumes, infect a total of 180,000 OG2 mouse embryonic fibroblasts in one well of a 6-well plate, and incubate at 37°C for 5 %CO 2cultured in DMEM supplemented with 10% fetal bovine serum. The day when the virus was added was regarded as day 0. On the second day, the cells were digested and resuspended in mES medium, and planted in a 96-well plate pre-filled with feeder cells (radiation-treated mouse embryonic fibroblasts). There were 5000 cells in the well, and the mES medium supplemented with different concentrations of LiCl (0.6mM, 1.2mM, 2.5mM, 5mM, 10mM, 20mM and 40mM) was used on the 3rd day, and the mES medium supplemented with different concentrations of LiCl ( 0.6mM, 1.2mM, 2.5mM, 5mM, 10mM, 20mM and 40mM) KSR medium, and then changed to KSR medium for culture on t...

Embodiment 2

[0063] Example 2 : Stem cell medium supplemented with lithium salt can promote the formation of iPS induced by three factors

[0064] a. Mix the viruses of the three factors (Oct4, Sox2, Klf4) in equal volumes, infect a total of 180,000 OG2 mouse embryonic fibroblasts in one well of a 6-well plate, and incubate at 37°C, 5% CO 2 cultured in DMEM supplemented with 10% fetal bovine serum. The day when the virus was added was regarded as day 0. On the second day, the cells were digested and resuspended in mES medium, and planted in a 96-well plate pre-filled with feeder cells (radiation-treated mouse embryonic fibroblasts). There were 5000 cells in the well, and the mES medium supplemented with different concentrations of LiCl (0.6mM, 1.2mM, 2.5mM, 5mM, 10mM, 20mM and 40mM) was used on the 3rd day, and the mES medium supplemented with different concentrations of LiCl ( 0.6mM, 1.2mM, 2.5mM, 5mM, 10mM, 20mM and 40mM) KSR medium, and then changed to KSR medium for culture on the 8...

Embodiment 3

[0067] Example 3 : The iPS cell line obtained by adding lithium salt is pluripotent

[0068] a. As above, mouse embryonic fibroblasts were infected with 4 factors (Oct4, Sox2, Klf4, c-Myc) or 3 factors (Oct4, Sox2, Klf4), cultured in stem cell medium supplemented with LiCl, after infection After 14 days (4 factors) or 20 days (3 factors), representative clone groups were selected according to the clone morphology and fluorescence expression, and a uniform iPS cell line was formed after passage.

[0069] b. Observing the morphology of the selected iPS cell lines and staining for stem cell-specific proteins. Such as Figure 5 As shown, the iPS cell line has a characteristic morphology similar to embryonic stem cells, strongly expresses the green fluorescence of Oct-GFP, and is positive for alkaline phosphatase (AP). Immunofluorescent staining of iPS cells using antibodies against stem cell-specific proteins showed that both iPS cell lines expressed stem cell-specific protein...

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Abstract

The invention provides a preparing method of an iPS cell, which includes the following steps: step 1, introducing one or many stem cell pluripotent sex factors into a somatic cell; step 2, adopting the medium added with lithium to cultivate the somatic cell introduced with the stem cell pluripotent sex factors in the step 1; and step 3, identifying the cloning of the iPS cell. Furthermore, the invention also provides a medium for preparing the iPS cell, wherein the cell further includes lithium. The lithium-contained medium is applied to efficiently generating the iPS cell. In the invention, the lithium can improve the generation efficiency of the mouse iPS cell to be 5 to 60 times. The efficient iPS cell inducing method is highly significant in improving the iPS technical safety and applying the iPS cell to the field of regeneration medicine.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing induced pluripotent stem cells and a culture medium for preparing induced pluripotent stem cells. Background technique [0002] Embryonic stem cells are derived from the inner cell mass of early embryos, which can self-renew, maintain pluripotency and have the ability to differentiate into three germ layer cells in vitro. With the successful establishment of mouse embryonic stem cells in 1981 and human embryonic stem cells in 1998 [1, 2], the research on regenerative medicine really kicked off. The application prospect of embryonic stem cells is mainly used for transplantation therapy. Using embryonic stem cells as the starting cells, through in vitro culture and directed differentiation, can provide a large number of tissue and organ transplantation materials for clinical treatment. Through the research on the self-renewal and directed differentiation mechanism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N2501/603C12N15/85C12N5/0696C01D15/06C01D15/08C12N2501/604C01D15/04C12N2510/00C12N2500/12C01D15/10C12N2501/602C12N2501/606C12N5/0607C12N5/10
Inventor 谢欣王荃
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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