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A method of inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets

A technology of stem cells and inducing differentiation, applied in the field of cell biology, can solve the problems of induced transdifferentiation of cells and low feasibility of transdifferentiation of similar cells, achieve high amplification efficiency, good multi-blast differentiation potential, convenient and accurate The effect of operation and system quality control

Active Publication Date: 2020-09-08
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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  • A method of inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets
  • A method of inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets
  • A method of inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0240] 1. Isolation of skin fibroblasts

[0241] 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 DMEM.

[0242] 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.

[0243] 2. Activation of skin fibroblasts

[0244] 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

[0252] 1. The separation of skin fibroblasts is the same as in Example 1.

[0253] 2. When starting transdifferentiation (Day0), completely replace the basal culture medium with the following culture medium, culture for 6-12 days, at 37°C, 5% CO 2 environment to grow cells. The culture solution is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMEM medium (Gibco)+forskolin (2μM~20μM)+Repsox (2~15uM)+CHIR99021(1μM~10μM)+VPA(0.5mM~1.5mM)+TTNPB(3μM~8μM)+AM580(0.03~0.08μM)+EPZ004777(3~8μM)+Go6983(1~15μM) +Y-27632(3~15μM)+L-Ascorbinacid 2-phosphate(0.15~0.25mM), 10% fetal bovine serum in this culture system can also be replaced by a serum substitute (invitrogen) at a concentration of 10%-20% ; 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can not be used.

[0254] 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 feed...

Embodiment 3

[0258] 1. The separation of skin fibroblasts is the same as in Example 1.

[0259] 2. Activation of skin fibroblasts

[0260] 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 DMEM 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.

[0261] 3. Orientation induction of skin fibroblasts

[0262] After the treatment in the second step above, completely replace it with the second-stage culture medium ...

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Abstract

The invention discloses a method for inducing differentiated cells to prepare mesenchymal stem cells and a combination of regulatory targets. The method prepares mesenchymal stem cells by directional induction of differentiated cells, and the directional induction includes inhibiting the signaling pathway of TGF-β, inhibiting the activity of PKC, activating the signaling pathway of WNT / β-catenin and activating the signaling pathway of cAMP . The present invention induces differentiated cells into mesenchymal stem cells by regulating the corresponding signaling pathways and / or enzyme activities in stages, and the present invention can reprogram differentiated cells by regulating the corresponding targets in stages by using a combination of small molecule compounds For mesenchymal stem cells, each step can achieve precise quality control, which is convenient for standardized operation and large-scale production. The method provided by the present invention has a wide range of sources of donors, and the patient himself can be used as a donor, and the mesenchymal stem cells required for basic research, clinical treatment or tissue engineering production can be obtained in a relatively short period of 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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Patent Type & Authority Patents(China)
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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