(805b) Nitric Oxide-Generating Hydrogels for Driving Maturation of Stem Cell-Derived Cardiomyocytes

The increasing prevalence of heart disease in the United States has necessitated development of novel cardiac regeneration therapies to augment existing treatments. Pluripotent stem cells present one option as a cell source capable of creating physiologically-consistent cardiomyocytes; however, contemporary methods for differentiating stem cells are often limited by the creation of cardiac cells with immature electrophysiological phenotypes. Nitric oxide (NO) is small signaling molecule important in directing many physiological processes including pluripotent stem cell differentiation; while previous studies have examined the effects of soluble NO-donors on the protein and genetic phenotype of stem cell-derived cardiomyocytes, the electrophysiological effects of NO addition have not been evaluated. Poly(ethylene glycol) (PEG)-coupled NO donors have been shown to provide localized NO delivery up to 5 months.  Having previously demonstrated that pluripotent stem cells can be encapsulated in PEG-based hydrogel microspheres and differentiated, this study examines the ability of NO to enhance cardiac differentiation. NO-generating PEG ligands can be covalently coupled into the PEG-based microspheres at the time of stem cell encapsulation. In this study, populations of embryoid bodies (EBs) were exposed to either the soluble NO-donor S-nitrosocysteine or NO synthase inhibitor (L-NAME); spontaneous contractile activity was observed and quantified over a period of 18 days. Assessment of stem cell-derived cardiomyocyte phenotype was carried out via calcium transient analysis, immunostaining, and quantitative PCR. EBs treated with the NO-donor exhibited significantly higher percentages of spontaneous contraction and contraction frequency at later time points (days 12 and 14) than control EBs. Repolarization times were reduced in NO-treated EBs, resulting in a higher maximum capture rate in this group. The results of this study will be utilized in establishing a platform for improving the electrophysiological maturity and homogeneity of stem cells when encapsulated in PEG microspheres for differentiation in large-scale culture environments.