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Method for inducing differentiated cells to prepare mesenchymal stem cells and combination for regulating targets

A technology of mesenchymal stem cells and induction of differentiation, applied in the field of cell biology, can solve the problems of induced transdifferentiation of cells and the low feasibility of transdifferentiation of similar cells, and achieve high amplification efficiency, good multi-layered multi-directional differentiation potential, and convenient and accurate Operational and Systemic Quality Control Effects

Active Publication Date: 2018-05-22
YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Since there are about 25% genetic differences between humans and mice, it is not feasible to apply the above-mentioned successful patent application technology scheme in mouse cell experiments to human cells of the same kind; on the other hand, due to the induction of similar cells The specific theoretical basis and technical means of transdifferentiation to obtain different target cells are not the same. The applicant used the technical solutions reported above to repeat the experiments, but failed to successfully apply the transdifferentiation technical solutions applied to mouse cells to similar human cells. transdifferentiation, and failed to induce transdifferentiation of human differentiated cells into mesenchymal stem cells

Method used

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  • Method for inducing differentiated cells to prepare mesenchymal stem cells and combination for regulating targets
  • Method for inducing differentiated cells to prepare mesenchymal stem cells and combination for regulating targets
  • Method for inducing differentiated cells to prepare mesenchymal stem cells and combination for regulating targets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] 1. Isolation of skin fibroblasts

[0242] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0243] 1.2 Subsequent passage of cells to a large number of expansion, the number of cell generations between the 6th and 12th passages is used for the induction of transdifferentiation into mesenchymal stem cells. The day before initiation of differentiation (Day-1), inoculate cell density at 1-2.5×10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.

[0244] 2. Activation of skin fibroblasts

[0245] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and cultivate the cells for 4-6 days. The first-stage culture medium refers to: 10% fetal ...

Embodiment 2

[0253] 1. The separation of skin fibroblasts is the same as in Example 1.

[0254] 2. When transdifferentiation is started (Day0), completely replace the basal culture medium with the following culture medium, and culture the cells for 4-6 days, and the culture time is 6-12 days, at 37°C, 5% CO 2 environment to grow cells. The second stage culture solution is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMDM ​​medium (Gibco)+forskolin (2μM~ ( 1~15μM)+Y-27632(3~15μM)+L-Ascorbin acid 2-phosphate(0.15~0.25mM), 10% fetal bovine serum in this culture system can also be replaced by serum substitute (invitrogen) with 10%- A concentration of 20% was substituted; 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0255] 3. Then replace it with the following culture medium for 3-8 days, and culture the cells at 37°C and 5% CO2 environment. The medium for the feeding stage is: BMP4 (10-20ng / mL)+...

Embodiment 3

[0259] 1. The separation of skin fibroblasts is the same as in Example 1.

[0260] 2. Activation of skin fibroblasts

[0261] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and culture the cells for 4 to 6 days. The first-stage culture medium refers to: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin ( Sigma) + 100μg / ml streptomycin (Sigma) + high glucose DMDM ​​medium (Gibco) + forskolin (2μM ~ 25μM) + Repsox (2 ~ 15uM) + CHIR99021 (1μM ~ 10μM) + VPA (0.5mM ~ 1.5mM ), 10% fetal bovine serum in this culture system can also be replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100U / ml penicillin (Sigma) and 100μg / ml streptomycin (Sigma) can not be used . at 37°C, 5% CO 2 environment to grow cells.

[0262] 3. Orientation induction of skin fibroblasts

[0263] After the treatment in the second step above, completely replace it with the second-stage culture mediu...

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Abstract

The invention discloses a method for inducing differentiated cells to prepare mesenchymal stem cells and a combination for regulating targets. The method comprises that the differentiated cells are subjected to directional induction to prepare the mesenchymal stem cells; directional induction comprises inhibition of a TGF-beta signal pathway, inhibition of the activity of PKC, activation of a WNT / beta-catenin signal pathway and activation of a cAMP signal pathway. The differentiated cells are induced to the mesenchymal stem cells by phased regulation of the corresponding signal pathways and / orenzyme activity. The differentiated cells can be reprogramed into the mesenchymal stem cells by using the small-molecular compound combination for phased regulation of corresponding targets, all steps can be subjected to precise quality control, and standardization operation and large-scale production are convenient. The method provided by the invention has wide donor sources, a patient himself can be used as a donor, and the mesenchymal stem cells needed for basic research, clinical treatment or tissue engineering production can be obtained in relatively short time.

Description

technical field [0001] The invention relates to the fields of cell biology, tissue engineering and regenerative medicine, in particular to a method for inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets. Background technique [0002] Mesenchymal stem cells are a type of adult stem cells with multi-directional differentiation potential, which widely exist in human bone marrow, fat, and peripheral blood. Compared with embryonic stem cells or iPS cells, mesenchymal stem cells have higher safety and stability With low immunogenicity, it has been relatively mature in clinical research or clinical treatment of bone and joint injuries, tumors, liver cirrhosis, diabetes, degenerative diseases, nerve damage, Alzheimer's disease and lupus erythematosus, and has huge potential However, there are limitations such as scarcity, limited sources, difficulty in enrichment, complicated acquisition process, restrictions on the health status ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/071
CPCC12N5/06C12N2501/01C12N2501/115C12N2501/135C12N2500/38C12N2506/09C12N2506/1307C12N2506/13C12N2506/11C12N2501/73C12N2501/999C12N2501/415C12N2501/155C12N2501/15C12N5/0663C12N2501/727
Inventor 胡敏李燕皎
Owner YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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