Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers.

Rationale Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) offer a powerful in-vitro tool to investigate disease mechanisms and to perform patient-specific drug screening. To date electrophysiological analysis of iPSC-CMs has been limited to single cell recordings or low resolution microelectrode array mapping of small cardiomyocyte aggregates. A new method of generating and optically mapping impulse propagation of large human iPSC-CM cardiac monolayers is needed. Objective Our first aim was to develop an imaging platform with versatility for multi-parameter electrophysiological mapping of cardiac preparations, including human iPSC-CM monolayers. Our second aim was to create large electrically coupled human iPSC-CM monolayers for simultaneous action potential and calcium wave propagation measurements. Methods and Results A fluorescence imaging platform based on electronically-controlled light-emitting-diode (LED) illumination, a multi-band emission filter and single camera sensor was developed and utilized to monitor simultaneously action potential and intracellular calcium wave propagation in cardiac preparations. Multiple large diameter (≥1cm) electrically coupled human cardiac monolayers were then generated that propagated action potentials and calcium waves at velocities similar to those commonly observed in rodent cardiac monolayers. Conclusions The multi-parametric imaging system presented here offers a scalable enabling technology to measure simultaneously action potential and intracellular calcium wave amplitude and dynamics of cardiac monolayers. The advent of large-scale production of human iPSC-CMs makes it possible now to generate sufficient numbers of uniform cardiac monolayers that can be utilized for the study of arrhythmia mechanisms and offers advantages over commonly used rodent models.