SPECIAL FOCUS Cardiomyogenic Differentiation of Embryoid Bodies Is Promoted by Rotary Orbital Suspension Culture Carolyn Y. Sargent, B.S., 1 Geoffrey Y. Berguig, B.S., 1 and Todd C. McDevitt, Ph.D. 1,2 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 (a-MHC and MLC-2v) was increased within EBs cultured in rotary suspension conditions. Rotary orbital culture also yielded a greater percentage of EBs that were immunoreactive for a-sarcomeric actin protein compared to static suspension, and augmented the average percentage of a-sarcomeric actin–positive cells detected via flow cytometry. These results demonstrate that rotary orbital suspension culture enhances endogenous cardiomyo- genesis of EBs and therefore could benefit the development of regenerative cardiac therapies. Introduction M yocardial infarction due to an ischemic episode results in significant death of heart muscle cells (car- diomyocytes) that support normal cardiac contractility. Ma- ture cardiomyocytes are incapable of significant (if any) proliferation; thus, adult mammals fail to regenerate func- tional myocardial tissue in response to tissue injury or in- sult. 1–3 Severe myocardial ischemia initiates a cascade of cellular and molecular remodeling events within the heart, such as protease activity, extracellular matrix synthesis and degradation, chamber dilatation, thinning of the ventricular wall, and ultimately fibrotic scar formation. 4,5 Structural changes to the geometry of the heart, along with a significant decrease in the number of contractile muscle cells, lead to decreased cardiac output volume and eventually culminates in heart failure and mortality. 4–8 The emphasis of most cur- rent clinical therapies to treat myocardial infarction is on revascularization strategies to restore normal blood flow, protect residual cells, and slow the normal progression of heart failure. However, current clinical treatment options do not adequately address the need to replace contractile car- diomyocytes that are lost due to cardiac disease or injury. Tissue engineering and regenerative cellular therapies ca- pable of replacing cardiomyocytes to injured myocardium offer a promising approach to restore functional tissue; however, an appropriate cell source should have the potential to produce large yields of cardiomyocytes capable of functionally incor- porating within the host tissue. Attempts thus far to employ cell therapies to achieve these rigorous criteria have been minimally successful. Skeletal myoblasts and bone marrow– derived cells transplanted within the infarcted heart have eli- cited small improvements in cardiac function, but have not generated significant amounts of integrated muscle tissue. 9–14 The identification and recent characterization of a putative cardiac stem cell population within the heart represents a novel cell source for cardiac regenerative therapies, but inefficient isolation and cultivation methods for these cells currently limit their therapeutic utility. 15–18 In contrast to these other cell types, embryonic stem cells (ESCs) are capable of significant expan- sion in vitro and inherently retain the ability to differentiate into definitive contractile cardiomyocytes that can survive and electromechanically couple with host cells of the heart. 19–22 However, before ESC-derived cardiomyocytes can be realisti- cally implemented as a viable cell therapy, efficient methods of differentiating ESCs to cardiomyocytes using scalable 1 The Wallace H. Coulter Department of Biomedical Engineering and 2 The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology=Emory University, Atlanta, Georgia. TISSUE ENGINEERING: Part A Volume 15, Number 2, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0145 331