Extracellular Matrix–Mediated Maturation of Human Pluripotent Stem Cell–Derived Cardiac Monolayer Structure and Electrophysiological Function

TLDR

Human pluripotent stem cell–derived cardiomyocyte monolayers are currently immature, limiting their usefulness for research and cardiac regeneration. The study aimed to identify an extracellular matrix that promotes significant electrophysiological and structural maturation of these monolayers. Maturation was achieved by culturing cells on PDMS coverslips for one week, which activates integrin signaling and focal adhesion kinase pathways. The optimal ECM doubled impulse propagation velocity to ~44 cm/s, produced mature action potentials, induced hypertrophic growth, upregulated key sarcolemmal and myofilament markers, and demonstrated that integrin signaling and focal adhesion kinase activation are essential for this maturation.

Abstract

Human pluripotent stem cell-derived cardiomyocytes (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 electric impulse propagation velocity and immature action potential profiles.Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; 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 extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin 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 activation prevented structural maturation.Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.