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Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts

A fibroblast and osteoblast technology, applied in the field of cell biology and tissue engineering, can solve the problems of unfavorable cell differentiation, side effects of surrounding healthy tissues, high cost of recombinant protein, etc., achieve obvious induction effect, short induction process and stable performance Effect

Active Publication Date: 2015-11-18
YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression products of these genes are secreted proteins, and their expression and secretion regulation is complex
Since they can cause ectopic bone formation through paracrine effects on surrounding non-osseous tissues, long-term sustained high expression in transdifferentiated cells may lead to side effects on healthy tissues surrounding the transplantation site
Moreover, due to the high cost of recombinant proteins and their unstable properties for a long time, it is not conducive to large-scale cell differentiation production.

Method used

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  • Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts
  • Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts
  • Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. After obtaining positive human skin fibroblasts infected by lentivirus RUNX2 (two days after virus infection), replace the culture medium

[0031] For TraA, all mediums were changed every other day. The culture was continued for 3 days. The formula of TraA is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+10ng / mlbFGF+10nM dexamethasone;

[0032]2. TraB culture medium and TraA culture medium were cultured for three days, and the medium was completely changed, and the TraB culture medium was replaced. The formula of TraB is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+9 μ M CHIR99021+10 μ M Forskolin+10 nM dexamethasone Methasone;TraB was continuously cultured for 10 days, and the medium was completely changed every other day.

[0033] 3. TraC culture medium, after 10 days of culture in the previous stage, ...

Embodiment 2

[0042] 1. After obtaining positive human skin fibroblasts infected by lentivirus RUNX2 (two days after virus infection), replace the culture medium TraA, and change all the medium every other day. The cultivation was continued for 5 days. The formula of TraA is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+10ng / mlbFGF+10nM dexamethasone;

[0043] 2. TraB culture medium and TraA culture medium were cultured for three days, and the medium was completely changed, and the TraB culture medium was replaced. The formula of TraB is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+9 μ M CHIR99021+10 μ M Forskolin+10 nM dexamethasone Methasone;TraB was continuously cultured for 18 days, and the medium was completely changed every other day.

[0044] 3. TraC culture medium, after 10 days of culture in the previous stage, complete...

Embodiment 3

[0053] 1. After obtaining positive human skin fibroblasts infected by lentivirus RUNX2 (two days after virus infection), replace the culture medium TraA, and change all the medium every other day. The culture was continued for 3 days. The formula of TraA is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+10ng / mlbFGF+10nM dexamethasone;

[0054] 2. TraB culture medium and TraA culture medium were cultured for three days, and the medium was completely changed, and the TraB culture medium was replaced. The formula of TraB is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high glucose DMDM ​​medium (Gibco)+9 μ M CHIR99021+10 μ M Forskolin+10 nM dexamethasone Methasone;TraB were continuously cultured for 30 days, and the medium was completely changed every other day. Followed by staining.

[0055] 3. Identification of Alizarin Red Staining

[0056] ① Aspir...

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Abstract

According to the present invention, based on the exogenous introduction method of bone tissue cell specific expressing transcription factor Runx2 into human skin fibroblasts, a set of culture system for promoting differentiation of fibroblasts at various stages is developed so as to effectively promote transformation into osteoblasts, and the prevent invention belongs to the field of cell biology and tissue engineering. The system comprises three sets of culture liquids, wherein the culture liquids respectively act on three key stages in the transdifferentiation process, the high survival rate and the high proliferation of the RUNX2 positive cells are maintained in the stage a, the expression of the RUNX2 within a specific time is regulated in the stage b, and the transformation efficiency from the cells to the osteoblasts is improved and the transformation time is shortened after the cells are re-programmed so as to achieve the transdifferentiation of the human skin fibroblasts to the osteoblasts. According to the present invention, the three sets of the culture liquids are matched, and the respective acting time can be flexibly regulated according to the final use purpose of the cells so as to culture the large-scale inductive osteoblasts; the culture system is used for cell therapies of bone metabolic diseases, fractures and the like; and the pharmaceutical composition of the present invention can further be used for development and research and production of the tissue engineering bonds, provides convenience for basic research, and provides prospects for clinical applications.

Description

technical field [0001] The present invention relates to an induction culture system acting on the reprogramming of human skin fibroblasts expressing Runx2 into osteoblasts, especially a combination of small molecule compounds and hormones, which is a set of methods to promote the transformation of human skin fibroblasts into osteoblasts. Differentiated culture systems. It belongs to the field of cell biology and tissue engineering. Background technique [0002] Bone tissue engineering technology integrates various bioactive factors into or uses a three-dimensional scaffold with a special structure to inoculate seed cells, induces seed cells from various sources to differentiate in the direction of osteogenesis, and forms tissue-engineered composite bone to replace traditional bone tissue engineering. material for bone repair. According to different sources, seed cells mainly include late-differentiated osteoblasts, osteoblast lines, bone marrow mixed cells or purified mese...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 胡敏李燕皎
Owner YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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