Document Type : Original Article
EMBRYOLOGY DEPARTMENT, ROYAN INSTITUTE,TEHRAN, IRAN
Introduction: Embryonic stem (ES) cells are pluripotent cells that derived from inner cell mass (ICM) of blastocyst stage and are able to differentiate in vitro into different cells. Purpose: This study was initiated to evaluate the effect of basic fibroblast growth factor (bFGF) on differentiation of ES cells into cardiomyocytes among with their response to cardiac drugs.
Methods: The mouse embryonic stem cells (Royan B1) were cultured as 800 cells per 20μl medium. After two days ES cells in each drop aggregated to form embryoid bodies (EBs). EBs were transferred to bacterial dishes for 5 days. In the first two days, cells were treated with 10ng/ml bFGF. After 5 days EBs were plated in 24-well plates and cardiomyocytes were differentiated. Beating rate of EBs were recorded in control and bFGF groups daily. The chronotropic effects of isoprenaline and phenylephrine on ES cells–derived cardiomyocytes at 3th, 7th and 14th days after plating were investigated. The expression of cardiac specific genes included: α-MHC(Myosin Heavy Chain), β-MHC, MLC-2V(Myosin Light Chain), β –tubulin , ANF(Atrial Natriuretic Factor) and oct-4 in control and bFGF groups with RT-PCR at 7+14 day was also assayed. Moreover immunostaining was done by staining the cardiomyocytes with anti α-actinin.
Results: Our results showed that Compared to bFGF, the beating rate was higher on EBs of the control group, when no drugs was used and treatment of cardiomyocytes with isoprenaline and phenylephrine increased the rate of beating in both groups, and in early stage of development, this enhancement rate was significantly more in bFGF group than control (p<0.035 and p<0.019 , respectively). As RT-PCR showed, cardiomyocytes on 7+14 day expressed α-MHC, β-MHC, MLC-2V, β -tubulin and ANF in bFGF and control groups but there was no difference between these groups on this day. Immunostaining showed presence of cardiac-specific proteins and their spatial organization.
Conclusion: These results showed that although bFGF probably exerts its effects on the expression or function of α-adrenergic and β-adrenergic receptors , but individually it has no influential effect on cardiogenesis.