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Culture medium for induced pluripotent stem cells and application of culture medium

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of pluripotent stem cell culture medium, can solve problems such as gaps

Active Publication Date: 2014-12-10
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The early iPSC induction efficiency is only 0.01-0.5%, which is far from the reprogramming efficiency based on somatic cell nuclear transfer. Therefore, how to improve the iPSC induction efficiency is one of the hot research topics in the iPSC research field

Method used

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  • Culture medium for induced pluripotent stem cells and application of culture medium
  • Culture medium for induced pluripotent stem cells and application of culture medium
  • Culture medium for induced pluripotent stem cells and application of culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The preparation of embodiment 1 culture medium

[0056] KOSR medium is a conventional medium for culturing stem cells, and the KOSR medium in this example was purchased from Invitrogen. The formula is shown in Table 1

[0057] Table 1 Formula of KOSR medium

[0058]

[0059] Dissolve ginsenoside monomer Rb1, ginsenoside monomer Rb3, and ginsenoside monomer Rd in a small amount of DMSO respectively to form a certain concentrated storage solution, and then add a corresponding volume of concentrated storage solution to the above-mentioned KOSR medium as required to make ginseng The concentration gradients of saponin monomer Rb1, ginsenoside monomer Rb3 and ginsenoside monomer Rd were 1 μM, 5 μM, 25 μM, 50 μM, 75 μM, and 100 μM. The DMSO control group is to add DMSO equal to the volume of the ginsenoside monomer concentrated storage solution to the above KOSR medium; the positive control group is the KOSR medium containing 50 μg / ml Vc.

Embodiment 2

[0060] Example 2 Induction and Culture of Mouse iPS Cells

[0061] The inventors of the present application prepared culture media containing ginsenoside monomer Rb1, ginsenoside monomer Rb3, and ginsenoside monomer Rd respectively, and the concentrations of ginsenoside in the culture medium were 1 μM, 5 μM, 25 μM, 50 μM, and 75 μM, respectively. , 100 μM.

[0062] In the following experimental procedures, except for the above medium, the rest are the same.

[0063] The negative control is KOSR medium containing equal volume of DMSO; the positive control is KOSR medium containing 50ug / ml Vc.

[0064] Mouse ips cells were induced using the culture medium prepared in Example 1, wherein MEF culture fluid (10% DMEM) was also used, and its formula is shown in Table 1:

[0065] Table 1 MEF culture medium

[0066]

[0067] Described method comprises the following steps:

[0068] First, pluripotency-related genes (Klf4, Sox2, Nanoge and Oct4) were introduced into the donor ce...

Embodiment 3

[0081] Example 3 Effect of Adding Ginsenoside Monomer Culture Solution on iPS Induction

[0082]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 Zhao, X.Y., et al., iPS cells produce viable mice through tetraploid complementation.Nature, 2009.461(7260):p.86-90. The method described observes and identifies the state of cells in the induction process, specifically including:

[0083] 2.1 Alkaline phosphatase (AP) staining method

[0084] The cells induced by the ginsenoside monomer culture solution with a final concentration of 25 μM were selected, and the cells in the culture plate were fixed on the 10th day, and after AP staining, the number of clones was determined by counting the number of colored clones.

[0085] For details, please refer to the instructions of the BCIP / NBT alkaline phosphatase staining kit.

[0086] The res...

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Abstract

The invention provides a culture medium for culturing induced pluripotent stem cells. The culture medium contains a ginsenoside monomer Rb1, a ginsenoside monomer Rb3 and / or a ginsenoside monomer Rd. The invention also provides a method for inducing pluripotent stem cells. The method comprises the following steps: (1) introducing pluripotent-related genes into donor cells; (2) preparing the donor cells in which the pluripotent-related genes are introduced into single-cell suspension, and culturing the donor cells (in which the pluripotent-related genes are introduced in the step (1)) by using the culture medium provided by the invention; and (3) identifying cloning of the pluripotent stem cells. In consideration of the application bottleneck of ips to clinical test at present, the ginsenoside monomer Rb1, the ginsenoside monomer Rb3 and the ginsenoside monomer Rd are discovered, the ips induction efficiency can be obviously improved, cell senescence can be obviously delayed, cell proliferation can be promoted, and far-reaching influence is generated in the entire field of stem cell research and new drug research and development.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the use of ginsenosides, in particular to the use of a pluripotent stem cell culture medium containing ginsenoside monomers in improving cell reprogramming efficiency, delaying cell aging and promoting cell proliferation. technical background [0002] Chinese herbal medicine provides a broad source of medicine and plays an important role in traditional medicine. Some traditional Chinese medicines have significant curative effects on brain development and immunity, but the specific molecular mechanisms are still unclear, including targets, signaling pathways, etc. In traditional Chinese medicine, ginseng can enhance memory, improve eyesight and calm the mind. Analysis found that ginseng contains a specific active substance - ginsenoside (ginsenoside, GS). Ginsenosides have similar basic chemical structures and can be roughly classified into dammarane type and oleanane type. Due to the...

Claims

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Application Information

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IPC IPC(8): C12N5/0735A61K31/704A61P39/06A61P35/00A23L1/30A23L33/10
Inventor 周琪王秀杰顾奇郝捷韩伟方刘鑫王磊王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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