The Study Of Mesenchymal Cells And Sca-1～+ Cells From Murine Fetal Liver Differentiating Into Muscle-like Cells In Vitro And In Vivo.

Objectives: To investigate potentials of mouse fetal liver mesenchymal cells and Sca-1~+ cells differentiating into muscle-like cells in vitro and in vivo.Methods: The fetal livers from 14.5-day-old mouse were taken, which were prepared into single cell suspension. The cells were cultured in DMEM/HEPES/F12 medium supplemented with 10%FCS and adherent cells were acquired after discarding nonadherent cells. The 3rd passage cells were induced by 10μmol/l 5-azacytidine and 1mg/1 amphotericin B for 24 hours. The characteristics of treated cells were assayed by immunocytochemistry staining analysis (the second antibody is labeled with desmin and a-Actin) and semi-quantitative RT-PCR method (the mRNA are Myf5 and myogenin, while p-actin is as internal control) in different times after induction. In order to detect the differentiating capacity of mesenchymal cells from fetal liver in vivo, the female murine model of muscle injury was made with focal injection of cardiotoxin at 8μg/g of body weight in the left quadriceps femoris. After 24 hours the male mesenchymal cells were injected into the left quadriceps femoris.of the model mice. The number of cells is 1 X 106in volume of 0.2-0.3ml per mouse. The model mice were feed in the sanitary house. Two months later, the mice were killed and the left quadriceps femoris were took out to be made tissue slices for detecting of Fluorescence in situ hybridization (FISH) and immunochemistry, the positive cells with Y chromosome in skeletal muscle were dectected.In order to detect the differentiating capacity of Sca-l+cells from fetal liver into skeletal muscle and cardiomyocyte in vivo, the Sca-1+ cells from the murine livers of 14.5 days were separated with Magnetic cell sorting (MACS) technology. 2x103 of Sca-l + cells from the male and transplanted through the tail vein into female mice of 8-12 weeks irradiated with the lethally dosage of y ray from Co60 source (8~10Gy) .Two months later, the mice were killed and their left quadriceps femoris and heart were taken out to make tissue slides detected with FISH and immunochemistry so as to find the positive cells with Y chromosome in skeletal muscle and cardiomyocyte.Results: After induced by 5-azacytidine and amphotericin B, the 3rd passage mesenchymal cells exhibited skeletal muscle-like morphology, and expressed skeletal muscle specific proteins such as Desmin and a-Actin. Untreated cells didn't change their morphology, and didn't express skeletal muscle markers. Semi-quantitative RT-PCR showed that Myf5 and myogenin mRNA was detectable and the their expression changed in different times.After 2 months of mesenchymal cells location transplantation, the positive cells with Y-chromosome in female skeletal muscle was found and phenotype characteristics of these cells was Desmin+/Flt-1~-/CD45-/F4/80-. The cells with Y chromosome in the skeletal and cardiac muscle of female mice transplanted with Sca-1+ cells were also found. The phenotype characteristics of these cells was Desmin+/Flt-1-/CD45-/F4/80" in cardiac muscle, while Dystrophin+/Flt-1-/CD45-/F4/80" in skeletal muscle.Conclusions: These results indicate that mesemchymal cells from murine fetal livers can differentiate into skeletal muscle-like cells after induction in vitro, respectively, these cells also can take part in the repairing of the muscles when they were transplanted and localized in the injured muscles site caused by cardiotoxin. The Sca-1+ cells can differentiate into skeletal muscle and cardiac muscle -like cells after transplanted into female mice irradiated with the lethally dosage of V ray.Mesenchymal cells and Sca-1+ cells from murine fetal liver have some potentials of differentiating into muscle-like cells in vitro and in vivo.