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Due to the restricted potential of the heart to regenerate its damaged region, stem cell therapy is a promising treatment modality for myocardial infarction. It has been shown that incubation of bone marrow-derived stromal cells (BMSCs) with initial steps of cardiac differentiation in vitro, can have a significant effect on their therapeutic potential to treat myocardial infarction. Based on these well-established principals we were encouraged to study the direct co-culture of rat BMSCs with neonatal mouse almost pure cardiomyocytes (APCs) and cardiac niche cells (CNCs) in static 2D and microfluidic cell culture systems. Our results showed that the difference regarding the beating rate in isolated APCs and CNCs in both 2D and the microfluidic system was not statistically significant for 30 days. No beat rate could be observed in induced BMSCs in all groups during experiment time. Except for BMSCs cultured alone in both experimental culture conditions, data obtained from Real-time PCR analysis showed that differentiated BMSCs in all co-cultured groups expressed GATA4, Nkx2.5, CX43, cTnI, cTnT, and ß-MHC during 4 weeks. BMSCs demonstrated a higher expression of these cardiac factors in microfluidic chips than those co-cultured in 24 well plates. Moreover, immunocytochemistry (ICC), also revealed the GATA4 expression in differentiated BMSCs in all co-cultured groups. It was found that, when combined with shear stress, co-culture with cardiomyocyte can differentiate BMSCs significantly toward cardiomyocyte rather than co-culture alone. [ABSTRACT FROM AUTHOR]