Isolation, Culture And Induced Differentiation Research Of Fetal Bovine Skeletal Muscle Mesenchymal Stem Cells In Vitro

Mesenchymal stem cells which possess the fetures of amplification ability,self-renewal and pluripotent. Initially, MSCs were discovered in the bone marrow, subsequently, they were able to be extracted from amniotic membrane, amniotic fluid, peripheral blood, muscle, and other tissues. However, current researches concentrated in human, rats and mice bone marrow, little is known on fetal bovine mesenchymal stem cells. Our study focused on isolation, culture, identification and induced differentiation of fetal bovine skeletal muscle mesenchymal stem cells.The results are as follows:1、 In this study, we isolated and purified fetal bovine MSCs from skeletal muscle using collagenase IV. Fetal bovine skeletal muscle MSCs germinated vigorously in a typical fibroblastic morphology under microscope, most of them developed in the elongated fusiform or irregular triangle.The cytoplasm were plump and left a stereo impression. As the cellls inflated the whole petri dish, they exhibited the swirling or flame-shaped trend.The cells were subcultured to as many as passage 25 and were cryopreserved for 75 vials in all.2、 Immunofluorescence were adopted to identify the MSCs specific cell surface markers. The results showed that cells were CD29, CD44 and CD106 positive while CD34 was negative.This meet MSCs suface markers.3、 In order to detect the MSCs specific cell surface markers in vitro cultured type P3, P8, P13, RT-PCR were used.The results showed that cells expressed CD29, CD44, CD73, CD90 and CD106, and the expression of CD34 and CD45 was negative.This meet MSCs suface markers.4、 Fetal bovine skeletal muscle derived MSCs differentiated into adipocytes, which were have lipid droplets with pale yellow, shine, after being induced by dexamethasone, IBMX, insulin and indomethacin. Red lipid droplets were showed by oil red O staining, and RT-PCR detection of PPAR-γ、C/EBP δ、FABP4 was positive after induction. The results showed that the fetal bovine skeletal muscle derived MSCs still obtain the ability to generate mesoderm cell types.5、 The MSCs originated from Fetal bovine skeletal muscle derived MSCs differentiated into myoblast, after being induced by horse serumof 5%, dexamethasone, hydrocortisone. Immunofluorescence and RT-PCR were adopted to identify myoblast expressed fast skeletal myosin. The results showed that the fetal bovine skeletal muscle derived MSCs still obtained the pluripotency to turn into mesoderm cell types.These results confirmed that the fetal bovine skeletal muscle mesenchymal stem cells were suitable for isolation and culture in vitro.The MSCs were still possessing strong biological characteristics such as self-renewal, proliferation and differentiation after passage cultured repeatedly. This study established a method and processes of culture and identification of bovine fetal bovine skeletal muscle MSCs.