Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

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Abstract

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.

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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