Human Atrial Extracellular Matrix Drives Differentiation Of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Toward An Atrial Phenotype

Extracellular matrix (ECM) can directly modulate cell proliferation, migration and differentiation by mediating diverse growth factors and signaling interactions. Protocols for cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) that recapitulate cardiac development frequently result in a mixed cardiac cell population dominated overwhelmingly by ventricular-like cells. Utilizing the inherent biological capabilities of decellularized ECM (dECM) from human myocardium, we developed a method for committing human iPSCs to an atrial-like cell phenotype. We employed a modified decellularization method to generate small particles (125-500 μm) of human atrial and ventricular dECM. The particles presented a fractal dimension (1.63 and 1.71) that suggested self-similarity across particle sizes of both atrial and ventricular dECM. Quantifications of DNA (3.37±0.50 and 2.77±0.62% of cadaveric), GAG (0.44±0.08 and 0.59±0.13 μg/mg), and SDS (2.46±1.20 and 2.91±2.53 μg/mg) validated the absence of difference of atrial and ventricular dECM. Proteomic profiling revealed dECM chamber-specific clustered populations. Ventricular and atrial dECM segregated into ventricular and atrial parts based on component 1 (19.5%) and component 2 (13.9%). A total of 14% of atrial proteins were matrisome atrial-related and 13% of ventricle proteins were matrisome ventricular-related. Myocytes differentiated in the presence of atrial dECM showed similar differentiation efficiency (66.6±10.2 vs 65.5±12.7% of cTNT) and, importantly, increased atrial markers, as confirmed by qPCR (SLP and COUPF-I) and flow cytometry (43.5%±12.7% vs 23.9%±10.8% of MLC2a) in comparison to control. We observed an increase in atrial cells (38.4% vs 14.8%) by action potential duration (APD), with statistical differences in cAPD10 (57.1±20.2 vs 104.4±48.7 ms) and cAPD20 (76.2±22 vs 126±47.4 ms). Altogether, we demonstrate that human atrial ECM retains cues to drive cardiac differentiation to an atrial fate, doubling the number of atrial cells with a functional atrial phenotype. These findings are a critical step toward generating sufficient quantities of atrial cells, which can be used for chamber-specific cardiac disease modeling and drug development.