Biochemical and Biophysical Research Communications
Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes
Section snippets
Materials and methods
Cell culture and reagents. The ES cell line ST-1 was used in the present study; this cell line was originally established from the blastocyst of a BALB/c mouse strain, and it was transmitted through germline in the chimeric mice. ES cells were grown on mitomycin C-inactivated feeder layer of primary cultures of mouse embryonic fibroblasts to maintain them in an undifferentiated state in Dulbecco’s modified Eagle’s medium (DMEM: Invitrogen, Carlsbad, CA) containing 20% fetal bovine serum (FBS:
Effect of A2-P on cardiac differentiation
ST-1 cells are pluripotent ES cells with a typical morphology. The initiation of cardiac differentiation was indicated by EB formation. The EBs adhered to the plates and continued to proliferate and then differentiated into beating cardiomyocytes. We first examined the effect of A2-P on the differentiation of ES cells into cardiomyocytes. On day 3 of the A2-P (1–100 μM) treatment, the percentage of EBs containing beating areas showed a significant increase in a dose-dependent manner (Fig. 1A).
Discussion
The major findings of this study are (1) A2-P, a long-acting ascorbic acid, significantly enhanced cardiac differentiation of ES cells, and this effect of A2-P was observed when A2-P was added during the early phase of EB culture; (2) treatment with the antioxidant NAC failed to mimic the effect of ascorbic acid on cardiac differentiation; (3) two distinct inhibitors of collagen synthesis, i.e., AzC and CIS, significantly inhibited A2-P-enhanced cardiac differentiation. The findings obtained
Acknowledgments
We thank Ryu-Ichiro Hata (Kanagawa Dental College) for helpful suggestion and Tomoko Hamaji, Junko Yano, and Kazuko Misawa for excellent technical assistance. This study was supported by research grants from the Ministry of Education, Science and Culture, the Ministry of Health, Labor and Welfare (#16590667 to M.T., and #16390220 to U.I.), and Mitsubishi Pharma Research Foundation (to M.T.).
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