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Small-molecular compound combination for differentiated cell reprogramming, reagent kit and application of small-molecular compound combination

A small molecule compound and reprogramming technology, which is applied in the field of small molecule compound combination for cell reprogramming, can solve the problems of low feasibility of transdifferentiation of similar cells, induced transdifferentiation of cells, etc.

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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  • Small-molecular compound combination for differentiated cell reprogramming, reagent kit and application of small-molecular compound combination
  • Small-molecular compound combination for differentiated cell reprogramming, reagent kit and application of small-molecular compound combination
  • Small-molecular compound combination for differentiated cell reprogramming, reagent kit and application of small-molecular compound combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0239] 1. Isolation of skin fibroblasts

[0240] 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.

[0241] 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 1-2.5x10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.

[0242] 2. Activation of skin fibroblasts

[0243] 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...

Embodiment 2

[0251] 1. The separation of skin fibroblasts is the same as in Example 1.

[0252] 2. When starting the transdifferentiation (Day0), completely replace the basal culture medium with the following culture medium, 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)+Y27362(3~15μM)+L-Ascorbin acid 2-phosphate(0.15~0.25m M), 10% fetal bovine serum in this culture system can also be replaced by serum substitute (invitrogen) at 10%-20 The concentration of % is substituted; 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted.

[0253]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 culture medium for the feeding stage is: BMP4 (10-20ng / mL...

Embodiment 3

[0257] 1. The separation of skin fibroblasts is the same as in Example 1.

[0258] 2. Activation of skin fibroblasts

[0259] 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.

[0260] 3. Orientation induction of skin fibroblasts

[0261] 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 small-molecular compound combination for differentiated cell reprogramming, and a reagent kit and an application of the small-molecular compound combination. The small-molecular compound combination includes the following components: a TGF-beta inhibitor, a WNT / beta-catenin agonist, a cAMP agonist and a PKC inhibitor; furthermore, the small-molecular compound combinationalso includes at least one of an RAR agonist, a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, ascorbic acid, a JNK inhibitor, an ROCK inhibitor and a lysine deacetylase inhibitor.Differentiated cells can be reprogrammed into mesenchymal stem cells by phased induction with the small-molecular compound combination, 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 present invention relates to multiple fields of basic medicine or clinical medicine, such as cell biology, pharmacy, tissue engineering, regenerative medicine, etc., and particularly relates to a small molecule compound combination, kit and application for differentiated cell reprogramming. 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, comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N5/0655C12N5/0656C12N5/0662C12N2501/415C12N2501/15C12N2501/73C12N2501/999C12N2506/1346C12N2501/01
Inventor 胡敏李燕皎
Owner YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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