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A culture system for induced pluripotent stem cells

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of culture system of induced pluripotent stem cells, can solve the problems of differences in molecular expression markers, unsatisfactory effects, low induction efficiency, etc., and achieve good gene modification and low cost , the effect of improving the induction efficiency

Active Publication Date: 2018-06-26
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, the first porcine iPS cell line was established, and then it was replicated in multiple laboratories, but the molecular expression markers were somewhat different, especially for SSEA1 and SSEA4, but it was disappointing that the expression of foreign genes was to maintain pluripotency Necessary for status, although chimera experiments have been reported to be possible, so far have not been replicated by other laboratories
However, the effect is not ideal. In view of the above problems, the present invention explores and optimizes the induced culture system first, and can obtain porcine iPS with high efficiency, and these porcine iPS are more inclined to mouse-like clones, and can be passaged by single cells. Higher developmental potential and better transgenic efficiency
Since cells from mice, monkeys and humans also suffer from low induction efficiency, there is currently a need for a medium capable of inducing pluripotent stem cells and improving their induction efficiency

Method used

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  • A culture system for induced pluripotent stem cells
  • A culture system for induced pluripotent stem cells
  • A culture system for induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] The preparation of embodiment 1 LBX liquid culture medium

[0096] Prepare LBX liquid medium A-E according to the components and proportioning provided in Table 1-3, the steps of the medium are as follows:

[0097] 1) According to the total volume of preparation, calculate the amount of various components required;

[0098] 2) directly dissolve the water-soluble components in the components in sterilized deionized water, and stir evenly;

[0099] 3) Dissolving other components in a small amount of DMSO; wherein, the amount of DMSO used does not exceed 0.5% of the total volume;

[0100] 4) Mix the two obtained in step 2) and step 3) evenly; dilute to the total volume; filter with a 0.22 micron filter.

[0101] The composition and ratio of medium A and B of table 1,

[0102] Unless otherwise specified, the concentration unit of each component in Table 1 is mg / l

[0103]

[0104]

[0105]

[0106]

[0107] Table 2 Composition and ratio of medium C and D ...

Embodiment 2

[0116] Example 2 Using the medium of the present invention to induce pluripotent stem cells

[0117] Specific implementation steps:

[0118] Prepare porcine fibroblasts (purchased from the Institute of Zoology, Chinese Academy of Sciences) one day in advance, press 2*10 4 / cm 2 Inoculated on 10% DMEM medium.

[0119] One day later, the medium was replaced with 10% DMEM without sp, and then the virus packaged with the four factors Klf4, Sox2, Nanoge, and Oct4 was added to the pre-inoculated porcine fibroblasts at the same time, and 8 μg / ml was added at the same time Polybrene (increases infection efficiency).

[0120] The induction steps of the ips cells are as follows:

[0121] Preliminary preparation: the cells that need to be induced are divided into 2*10 4 / cm 2 Inoculated on 10% DMEM medium containing SP.

[0122] Day 0: Change the medium and replace it with 10% DMEM without sp, then add the virus packaged with four factors Klf4, Sox2, Nanoge, and Oct4 to the pre-...

Embodiment 3

[0129] Example 3 Comparison of induced pluripotent stem cells using the medium of the present invention and KOSR medium

[0130] Use medium E and KOSE medium in Example 1 respectively to induce porcine pluripotent stem cells according to the method of Example 2; and analyze

[0131] Wherein, the KOSR medium is a conventional medium for culturing stem cells, the KOSR medium in this embodiment;

[0132] KOSR culture medium is prepared from the following finished products: 78% KODMEM+20%KOSR+1%SP+1%L-glu+1%NEAA+0.1%β-ME, the above reagents are all purchased from life technology company:

[0133] Wherein, the proportioning is a volume percentage, and the item numbers or other information of each component are as shown in Table 4:

[0134] Table 4

[0135]

[0136]

[0137] Using Wang, J., et al., Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells. Genomics Proteomics Bioinformatics. 11(2013): 304-311 and Zh...

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Abstract

The invention provides a medium for inducing pluripotent stem cells. The medium is an LBX liquid medium, and the stem cells are porcine stem cells, mouse stem cells, monkey stem cells or human stem cells. The invention also provides a method for inducing the pluripotent stem cells by using the medium. The medium for well improving the induction efficiency of induction pluripotency is invented for the first time. The cost of the medium is low. Induced pluripotent stem cells induced in the invention are more likely to be in a mouse-like state, and single cell passage can be realized, so large-scale amplification and clinic application are benefited.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture system of induced pluripotent stem cells. Specifically, the invention relates to a culture system of induced pluripotent stem cells, a preparation method and application thereof. Background technique [0002] The establishment of mouse embryonic stem cells (embryonic stem cells, ES) and human embryonic stem cells has created a new field of biology and medicine. The ability of embryonic stem cells to self-renew and differentiate into various germ layer cells provides a powerful tool for studying individual development and cell differentiation. tool, and has broad application prospects in the fields of regenerative medicine and tissue engineering, and will soon be applied to cell therapy and disease model research, which can provide tissue regeneration replacement cells for diseases that humans cannot repair themselves (Parkinson, diabetes, etc.) . [0003] The establishment o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
Inventor 周琪顾奇郝捷王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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