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KMID : 0311120080490050819
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Yonsei Medical Journal
2008 Volume.49 No. 5 p.819 ~ p.827
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Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2
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Kim Yoon-Young
Moon Shin-Yong
Kim Hee-Sun
Ku Seung-Yup
Kim Seok-Hyun
Choi Young-Min
Oh Sun-Kyung
Jang Ji-Ho
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Abstract
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Purpose: Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs.
Materials and Methods: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed.
Results: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (αMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS.
Conclusion: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.
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KEYWORD
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Bone morphogenetic protein 2, cardiomyocytes, cell differentiation, embryoid bodies, embryonic stem cells, long-term
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