Isolation And Identification Of Adipose-Derived Stem Cells In Mouse And Its Induced Myogenic Diffrentiation

Here in this study Adipose-derived Stem Cells (ADSCs) were isolated from inguinal fat pad of4weeks female ICR mice by the method of collagenase I digestion. ADSCs were cultured in vitro and then passaged. We observed their morphological changes, drew cell growth curve, did chromosome analysis and detected cell cycle. After that we detected the transcription factors by RT-PCR, and the surface markers CD29, CD31, CD44, CD45by flow cytometric analysis. It showed that ADSCs cultured in vitro had a fibroblast-like morphology, proliferated stably with a fast confluent potential, Different passages of cells experienced the growth incubation phase, the exponential growth phase and the plateau phase, the growing state of ADSCs were normal. Cell surface markers CD29expression rate was95.51%,CD44expression was31.63%, CD31expression was20.22%, CD45expression was negative. RT-PCR results showed the detection of the transcription factors of ADSCs, ADSCs expressed little Oct-4and C-kit, while expression of Sea-1was a lot. ADSCs could be induced to adipocytes by induced adipogenic medium, they showed blue color after Sudan black staining and expressed gene PPARy2which was an important regulatory factor in adipocytes differentiation. When they were cultured in osteogenic induction system, they could become osteoblasts. They showed red color after Alizarin red staining, and the expression of gene Osteocalcin was positive.Primary skeletal satellite cells could be isolated by the method of combined digestion of collagenase and trypsin. It took much shorter time then other methods like tissue culture. The immunofuluoresence staining displayed its expression of Cy3-labeled Desmin antibody and FITC-labeled Myod antibody. RT-PCR results showed that mouse skeletal muscle satellite cells express myogenic determination factors like MyoD, Myogenin and Myf5while MyoD expression was the highest. Different passages of skeletal satellite cells expressed different levels of myogenic determination factors, the amount difference of MyoD expression between freshly isolated and the second passage of cells were not significant, but the difference of MyoG was significant, difference of Myf5was very significant. It told us that the skeletal muscle satellite cells in the growing passages rapidly differentiated into mature myoblasts in a very short time and lost the proliferation and differentiation ability.The ADSCs were cultured in the induced medium with different concentrations of Demethylating agent5-azacytidine (5-Aza). Then we could find the optimal culture condition and had the detection of myogenic differentiation. Immunofluorescence staining results showed that the directed differentiation ability of ADSCs to muscle cells was very strong. The optimal conditioned medium which added10μmol·L-15-Aza and10%horse serum in the basic medium had better inducing effects. Part of the adjacent cells after induction began to merge to form myotube-like cells. Expression of muscle-specific antibody anti-MyoD and anti-Desmin were positive. RT-PCR results of5-Aza alone induced group,5-Aza with horse serum (HS) synergistically induced group, negative control Adipose-derived mesenchymal stem cells (ADSCs) group and positive control group skeletal muscle satellite cells (SCs) showed the expression levels of myogenic determination factors MyoD, Myogenin and Myf5were different among the groups, which said the different myogenic differentiation ability to myoblasts.In this study we got the method of isolating and culturing mouse ADSCs. The way to get derived materials of cells were easy and it also brought impurities cells rarely. The separation method was simple. ADSCs had a strong growth and proliferation ability, they could remain grow stably after several passages, proliferate fast with high rate of adherence when cultured in vitro. And we can use demethylating agent5-Aza and optimal synergistic induction system in a relatively short period of time to induce ADSCs to differentiate into myoblast-like cells, wich had a strong ability of expression of symbolic myogenic protein MyoD and muscle skeleton protein Desmin. So they could be used as the seed cell source for muscle tissue engineering in vitro.