Isolation And Biological Characteristics Of Mouse Adipose—derived Stem Cells

Adipose-derived stem cells (ASCs) are adult stem cells existed in fat tissue. In this study, the optimum isolation and culture system of ASCs in vitro was explored. ASCs were induced to differentiation into adipocytes, osteoblasts and sperm cells. The effect of reversine on the biological characteristics of ASCs was investigated.MTT assay, Immunohistochemical analysis and RT-PCR detection demonstrated that ASCs could keep committed biological characteristics until passages35. Doubling time of ASCs in Passage5,15and25was34.6h,37.8h and38.3h respectively. ASCs keeped on expressing surface marker such as CD29and CD44, did not express hemopoietic stem cell surface marker CD45. ASCs expressed specific genes CD29, CD34, CD90, Sca-1and c-Myc, did not express Sox-2and Oct-4. All those results suggested that the method used to isolate and culture ASCs in this experiment was appropriate and could maintain the stem cell characteristics of ASCs.ASCs were cultured in adipogenic differentiation medium to induce into adipose cells. Oil red O staining indicated lipid droplets in the sixth day. The induction rates of P5, P15and P25ASCs were78.3%±9.2%,73.9%±8.7%and67.0%±1.52%respectively. There is no significant difference in the induction rates of ASCs toward adipose cells between the different generations among P5, P15and P25. ASCs were cultured in osteoblast differentiation medium to induce into osteoblast cells. Alizarin red staining indicates calcium nodus in the eleventh day. The number of sained calcium nodules was count under100-fold magnification field of visionunder. The calcium nodus numbers of P5and P15ASCs were15±2.4and13±3.6respectively. There is no significant difference in the calcium nodus number between the different passages. P5ASCs were co-cultured with testis tissue to induce into sperm cells. RT-PCR results showed induced ASCs expressed spermatid specific genes Prmland Acr after10days.When the final concentration of reversine is over5μmol/L, it has obvious toxicityto ASCs. When the final concentration of reversine is1μmol/L, there is no influence to ASCs' proliferation rate, but the induction rate into adipose cells and osteoblast cells increases significantly.Our study built up isolation and culture system of mouse ASCs in vitro. ASCs could be cultured in our culture system for a long time and maitainec the stable biological characteristics. We proved that ASCs have the potential to differentiate into adipocytes, osteoblasts and sperm cells. We also comfirmed that reversine was able to improve the induction rate of ASCs into adipose cells and osteoblast cells, and could potentially be used as a useful small molecule to improve differentiation potential of ASCs.