Stem Cell Factor(SCF)Stimulates Osteogenic Progress Of Adipose-derived Stem Cells By Inhibiting AKT/mTOR Signaling Pathway

Objective: Mesenchymal stem cells(MSCs) play an important role in cell therapy and regenerative medicine. And human adipose-derived stem cells(ASCs) are a member of MSCs. Their characteristics of lower donor morbidity, more plasticity, higher yield and etc.,make them become a reliable source of seed cells in bone constructive progress. Thereby, it will be better to treat bone injuries when the osteogenic differentiation ability of ASCs was improved. In this study, we attempted to elucidate the mechanisms of role of stem cell factor(SCF) to improve ASCs osteogenesis.Methods: We performed type I collagenase digestion method to obtain ASCs from adipose tissue, then identified them with oil red O staining to inspect their adipogenic ability and alizarin red staining to inspect their osteogenic ability. Flow cytometry analysis identified ASCs surface markers. MTT assay was taken to detect the proliferation of ASCs by SCF.The role of SCF for different osteogenic stages of ASCs were analyzed by quantitative real-time reverse transcription polymerase chain reaction(RT-q PCR), alizarin red staining and alkaline phosphatase(ALP) staining. The levels of AKT, mammalian target of Rapamycin(m TOR) were investigated in the ASCs when treated with SCF. The RT-q PCR,western blot, alizarin red staining and ALP staining were used to analyze the mechanisms in ASCs osteogenic progress with SCF treatment.Results: Cells obtained from adipose tissue adherent in the plastic dishes and they had the abilities to undergo differentiations of adipogenesis and osteogenesis. The obtained cells positive expressed of CD105, CD73 and CD90 and negative expressed CD45, CD34,CD14 and HLA-DR. SCF promoted the proliferation of ASCs in a dose-dependent situation especially in 10ng/ml. SCF stimulated the differentiation of ASCs especially for the osteogenic part. Moreover, SCF also down-regulated the “stemness” genes oct4/nanog/sox2/bmi1 to enhance ASCs differentiations. Besides, we found SCF promoted osteogenic differentiation of ASCs in the early stage by up-regulating the expression of osteogenic makers like opn/runx2/alp. We also found that the p-AKT and p-m TOR were inhibited by SCF in the process of ASCs osteogenesis. However, SCF stimulated the osteogenic ability of ASCs and enhanced by LY294002, the inhibitor of AKT. Meanwhile,the osteogenic ability of ASCs was improved by Rapamycin, which is the inhibitor of m TOR, and it also coordinated with SCF to stimulate the osteogenesis of ASCs.Conclusion: Our results demonstrated that SCF stimulated ASCs differentiated into osteoblasts and its effect can be enhanced by AKT/m TOR inhibitions, Rapamycin and LY294002, which offered a potential new therapeutic strategy for bone reconstruction therapy.