Use of long-term cultured embryoid bodies may enhance cardiomyocyte differentiation by BMP2.
OBJECTIVE
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.


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 (aMHC). 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.


CONCLUSIONS
We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.
Kim
Yoon Young
YY
Institute of Reproductive Medicine and Population, Medical Research Center, Seoul, Korea.
Ku
Seung-Yup
SY
Jang
Jiho
J
Oh
Sun Kyung
SK
Kim
Hee Sun
HS
Kim
Seok Hyun
SH
Choi
Young Min
YM
Moon
Shin Yong
SY
D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects; D015398:Signal Transduction
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2