Vitaminc Promotes Differentiation Of Mouse Induced Pluripotent Stem Cells Into Cardiomyocyte In Vitro

Objective: To investigate the effect of VitaminC on the cardiomyocytes differentiation of mouse induced pluriptent stem cells(miPSc).Methods: 1.miPSc were recovered and plated on the prepared MEF feeder layer, we proved the totipotency of miPSc from morphology, Oct4 staining and alkaline phosphatase activity. Embryoid bodies (EBs) were generated from miPSc in liquid suspension and hanging drop culture. In hanging drop culture, the suspension was placed onto plastic petri dishes to allow MEF attachment. Then, the suspension was drawn out and cultured as hanging drops, we compared the quantity of rule EBs after different attach times. In suspension culture, we observed the effect of different cell concentration on number of rule EB formation. At the same time, we viewed the EB development in vitro.2. EBs formed from miPSc were induced by differentiation medium containing various concentration of vitaminC. Furthermore, the differentiated cultures were dissociated and enriched by Percoll density centrifugation, cardiomyocytes makers such as cTnT andα-actin were detected.Results: 1.miPSc were plated onto a feeder layer of MEFs. They exhibited a typical ESC-like morphology, growing in an undifferentiated state as compact domed colonies with well-defined edges. Colonies produced green fluorescence (Oct4-GFP +) under a fluorescence microscope. Alkaline phosphatase activity was assessed with the cells staining blue.2. In hanging drop culture, we used four different velocity adherent time 15min,30min,60min and 90min to remove the MEF, 60min was the best time, there was a significant difference compared with other groups (P<0.05).3. In suspension culture, we adjust cell concentration into 103/ml, 104/ml, 105/ml and 106/ml. From the second day to the fifth day, rule EBs numbers were counted under microscope. In the fourth day, we find most EBs in each group. Compared with other groups, when cell concentration was 104/ml, there were 171.67±3.51 EBs, the largest number of rule EBs (P<0.05).4. The best concentration of vitaminC to promote the cardiac differentiation of miPSc is 10^-4mol/L. Beating area were found in 57.00±3.20% of EBs There was significant difference compared with the control group 5.13±0.55% (P<0.01). Special cardiac proteinsα-actin and cTnT were detected. 5. The differentiated cultures could be dissociated and enriched by Percoll density centrifugation to four levels , in the 3^rd and 4^th levels ,it gave a population containing 26±1% and 60±2% cardiomyocytes, but in the 1^st and 2^nd, there were only 3±1% and 5±1% cardiomyocytes were found (P<0.05). The enriched population was showed appropriate expression of cardiomyocyte markers.Conclusion: 1.VitaminC could redound differentiation of miPSc to cardiomyocytes.2.The differentiated cultures could be dissociated and enriched by Percoll density centrifugation.