Isolation and Biological Characterization of Muscle-Derived Stem Cells from Sheep Skeletal Muscle

The Objective of this study was to investigate the isolation, cultivation, identification and differentiation potential of Muscle-derived stem cells (MDSCs) from sheep skeletal muscle and provide an experimental evidence for clinical application. The skeletal muscle tissue were studied from fetal sheep, and the stepwise digestion by XI collagenase and trypsin was used to isolate MDSCs and were purified by differential attachment technique. Cell morphology were observed using an inverted microscope, and MDSCs proliferation pattern were determined through growth curve analyses. MDSCs were identified by immunofluorescence and RT-PCR, and the immunofluorescence antibody invoved Sca-1, CD34, CD144, Desmin and CD45 which are the makers of MDSCs. Finally, MDSCs were induced into Adipocytes, osteoblasts, chondrocytes and neuron-like cells by the optimized inducing medium. After the two-step digestion and differential attachment technique, high purity MDSCs were successfully cultured. MDSCs were identified by round or short spindle cell, and gathered into clusters; The average doubling time and clone efficiency of the cells were 35.5h and 47.2%, respectively. The MDSCs makers, such as Sca1, CD34, CD144 and Desmin, were positive expressed by immunofluorescence and RT-PCR detection, and CD45 was negative expressed. MDSCs were successfully induced into Adipocytes, osteoblasts, chondrocytes, myoblasts and neuron-like cells under the optimized inducing medium. To conclude, in vitro high purity MDSCs have multiple differentiation potential, and can play an important role in muscle tissue repair, and provide seed cells for tissue engineering research and clinical applications.