JOURNAL ARTICLE
RESEARCH SUPPORT, NON-U.S. GOV'T
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Effect of bone morphogenetic protein-4 on cardiac differentiation from mouse embryonic stem cells in serum-free and low-serum media.

In spite of previous reports, the precise role of bone morphogenetic proteins (BMPs) on cardiomyocyte differentiation, especially in the absence or presence of minimum amount of serum in culture medium is still unclear. So, the aim of the present study was to investigate the effect of BMP-4 on mouse embryonic stem cells (ESCs)-derived cardiomyocyte differentiation in serum-free and low-serum media. The mouse ESCs differentiation to cardiomyocytes was induced by embryoid bodies' (EBs') development through hanging drop, suspension and plating stages. Different models of differentiation were designed according to addition of fetal bovine serum (FBS) or knockout serum replacement (KoSR) to the medium of three stages. 10 ng/ml BMP-4 was added throughout the suspension period. Up to 30 days after plating, contraction and beating frequency were monitored and evaluated daily. The growth characteristics of cardiomyocytes were assessed by cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR). In the complete absence of serum, neither control nor BMP-4 treated groups resulted in cardiac differentiation. Addition of FBS to hanging drop stage resulted in the appearance of beating cardiac clusters in some BMP-4 treated EBs. In the best designed differentiation model in which only hanging drop and the first 24 h of plating stage was carried out at the presence of FBS, the BMP-4 treatment resulted in cardiac differentiation in EBs characterized by positive immunostaining for the applied antibodies, chronotropic response to the cardioactive drugs and cardiac-specific genes expression at different developmental stages. These cardiomyocytes showed immature myofibrils and numerous intercellular junctions. In conclusion, BMP-4 is unable to induce cardiomyocyte differentiation from mouse ESCs in serum-free models, and at least small amount of FBS in hanging drop stage is necessary. Furthermore, serum factors are not strictly necessary after the initial activation, but they do favor a better differentiation of cardiomyocytes.

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