Culture medium for inducing totipotent stem cells of mice and induction method

A technology for inducing culture medium and stem cells, which is applied in the field of cell culture, can solve the problems of incomplete consistency, low overall efficiency, lack of induction and maintenance culture methods of totipotent-like stem cells, etc., and achieve the effect of low cost and simple operation

Active Publication Date: 2022-05-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two ways to obtain totipotent-like cells: one is to increase the proportion of 2CLC in mESC by overexpressing some key factors such as Dux, but the cells obtained by this method have the following defects: 1. The overexpression of key factors only It can increase the proportion of 2CLC in mESC, but the overall efficiency is still relatively low; 2. The obtained cells are in a transient state, which cannot be maintained stably for a long time; The blastomeres are not fully consistent, so the currently widely used 2CLC may be an intermediate or immature state
Therefore, there is still a lack of induction and maintenance culture methods for totipotent-like stem cells that are highly similar to 2C

Method used

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  • Culture medium for inducing totipotent stem cells of mice and induction method
  • Culture medium for inducing totipotent stem cells of mice and induction method
  • Culture medium for inducing totipotent stem cells of mice and induction method

Examples

Experimental program
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Embodiment 1

[0054] Embodiment 1: in vitro induction of mouse embryonic stem cells (mESC) into totipotent-like stem cells (TLSC)

[0055] (1) The following culture medium is used in the present embodiment:

[0056] mESC medium: 2iL medium; includes the following concentrations of ingredients:

[0057] The formula of the medium is: 1% N2 supplement, 2% B27 supplement, 1× non-essential amino acid solution, 2mM L-glutamine, 50-100 μg / ml are added to DMEM / F12 and Neurobasal medium with a volume of 1:1 Vitamin C (also known as L-ascorbic acid), 50-110 μM 2-mercaptoethanol, 5-20 ng / ml LIF, 0.5-2 μM PD0325901, 0.5-5 μM CHIR99021; or

[0058] The formula of the medium is: KnockOut TM Add 10-20% KnockOut to DMEM / F-12 medium TM Serum replacement, 1× non-essential amino acid solution, 1~5mM L-glutamine, 50~100μg / ml vitamin C (also known as L-ascorbic acid), 10~100μg / ml bovine serum albumin, 50~110μM 2-mercapto Ethanol and 5-20ng / ml LIF.

[0059] Totipotency Induction Medium: KnockOut TM DMEM / ...

Embodiment 2

[0068] Example 2: In vitro induction of blastomeres in mouse two-cell embryos into totipotent-like stem cells

[0069] (1) The following culture medium is used in the present embodiment:

[0070] Totipotency Induction Medium: KnockOut TM DMEM / F-12 medium was added with the following concentrations of components:

[0071] 20% Knock Out TM Serum replacement, 1× non-essential amino acid solution, 2mM L-glutamine, 100μg / ml vitamin C (also known as L-ascorbic acid), 50μg / ml bovine serum albumin, 100μM 2-mercaptoethanol, 2μM SGC0946, 3μM AS8351, 10ng / ml IL-6, 10ng / ml sIL-6Rα;

[0072] Totipotency Maintenance Medium: KnockOut TM DMEM / F-12 medium was added with the following concentrations of components:

[0073] 20% KnockOut serum replacement, 1× non-essential amino acid solution, 2mM L-glutamine, 100μg / ml vitamin C (also known as L-ascorbic acid), 50μg / ml bovine serum albumin, 100μM 2-mercaptoethanol, 2μM SGC0946, 2μM A366, 3 μM AS8351, 10 ng / ml IL-6, 10 ng / ml sIL-6Rα.

[0...

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Abstract

The invention provides a culture medium for inducing totipotent stem cells of mice and an induction method. The culture medium for inducing the mouse totipotent-like stem cells comprises an induction culture and maintenance culture medium and a maintenance culture medium. The invention provides a method for inducing mESC into totipotent-like stem cells in vitro by utilizing the heterogeneity characteristic that the mESC contains low-proportion mouse two-cell embryonic-like cells (2C-like cells, 2CLC). According to the method, a special culture medium is used for inducing cells to reach a totipotent state, so that the cells are more similar to in-vivo 2C embryos in transcriptome and epigenetics, have the capability of differentiating to form embryos and extraembryonic tissues (placentas, yolk sacs and the like), and are totipotent stem cells. The method provided by the invention is simple to operate and low in cost, and provides a basis for research on early embryonic development of mice and construction of disease models of various tissues or cells.

Description

technical field [0001] The application belongs to the technical field of cell culture, and in particular relates to a culture medium and an induction method for inducing mouse totipotent-like stem cells. Background technique [0002] Due to their self-renewal and multi-directional differentiation potential, stem cells have attracted much attention in research in various fields such as life sciences, biology, and medicine. Stem cells are the seed cells for the regeneration of tissues and organs, and are an important prerequisite for the practice of regenerative medicine. Among them, totipotent stem cells have stronger developmental potential than any other type of cells, which can produce embryos and extraembryonic tissues (placenta, yolk sac, etc.) , and eventually form a complete biological individual in a highly ordered manner, which is a special type of stem cell that plays a pivotal role. [0003] Mouse zygotes and 2C stage cells are totipotent cells, but they are limit...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/074
CPCC12N5/0606C12N5/0696C12N2500/32C12N2500/38C12N2500/44C12N2501/2306C12N2501/50C12N2501/72C12N2501/998C12N2501/71C12N2506/02
Inventor 王继厂杨明珠余汉文梁诗琪喻秀
Owner SUN YAT SEN UNIV
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