Cardiomyogenic Differentiation of Embryoid Bodies Is Promoted by Rotary Orbital Suspension Culture

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, including cardiomyocytes, which may be used for regenerative cardiac cell therapies. ESCs are commonly differentiated via cell aggregates known as embryoid bodies (EBs), but current cardiomyogenic differentiation methods, such as formation via hanging drops, yield relatively small numbers of EBs and differentiated cells. On the other hand, batch culture methods, like static suspension, yield increased numbers of EBs and cells, but typically exhibit less overall cardiomyogenic differentiation. The objective of this study was to determine if rotary orbital suspension culture, which produces EBs resembling hanging drops, was capable of enhancing cardiomyogenic differentiation compared to static suspension culture. Similar to hanging drops, rotary suspension culture significantly increased the proportion of spontaneously contracting EBs compared to static suspension culture. The gene expression of mesoderm (Brachyury-T) and cardiac transcription factors (Gata4, Nkx2.5, and Mef2c), as well as sarcomeric muscle proteins (alpha-MHC and MLC-2 nu) was increased within EBs cultured in rotary suspension conditions. Rotary orbital culture also yielded a greater percentage of EBs that were immunoreactive for alpha-sarcomeric actin protein compared to static suspension, and augmented the average percentage of alpha-sarcomeric actin-positive cells detected via flow cytometry. These results demonstrate that rotary orbital suspension culture enhances endogenous cardiomyogenesis of EBs and therefore could benefit the development of regenerative cardiac therapies.