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

Abstract

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.

Inspiration from heart development: Biomimetic development of functional human cardiac organoids

“…The low contraction amplitude of cardiac organoids is possibly due to the increased extracellular matrix components (e.g., collagen) and interstitial cell types, resulting in a denser/stiffer construct that creates an increased load for cardiomyocyte contraction. This was supported by the improved contractile structure and gene profile in cardiac organoids despite low contraction amplitude, highlighting the importance of the multicellular and matrix environment for hiPSC-CM maturation and ventricular lineage specification seen by others (26, 81). It is worth noting that the observed relationship between cardiomyocyte function and changes in cellular/ECM components is equally important to cardiac development and maturation as it is to cardiac pathophysiology, such as in chronic cardiac fibrosis (82, 83).…”

Tbx20 controls the expression of the KCNH2 gene and of hERG channels

“…Tbx20, either WT or p.R311C, did not modify the voltage dependence of Kr channel activation (Table S3). APs were recorded in hiPSC-CMs that exhibited automatic activity (13). In cells infected with Tbx20 WT (n = 10), maximum diastolic potential and AP amplitude averaged −68.4 ± 1.9 and 98.7 ± 14.2 mV, respectively.…”

Skeletal Extracellular Matrix Supports Cardiac Differentiation of Embryonic Stem Cells: a Potential Scaffold for Engineered Cardiac Tissue

“…After transplantation, ideal ECT supposes to be paced by host heart. A recent study reported human iPS cell-derived CMs monolayer on biomatrix made of matrigel and PDMS are successively paced with different pacing frequencies from 0.7 Hz to 2.5 Hz [37]. In another report, the ECT composed with pig cECM and neonatal rat ventricular cells are response to different electrical stimulation from 1 to 5 Hz [10].…”