Bioengineering the Extracellular Matrix to Enhance the Structural Maturity and Predictivity of iPSC Cardiomyocytes in Vitro

Stem cells hold great promise for improving the predictive power of preclinical in vitro drug screens and the translation of new technologies from bench to the bedside. However, traditional in vitro cultureware often fails to recapitulate the complex organization and interplay of cells and the extracellular matrix (ECM), failing to mimic critical in vivo phenotypes. Here, we present a novel biomimetic substrate with submicron topographies that is capable of mimicking the mechanical and structural cues of the ECM while benefiting from high-precision, easy-to-reproduce, and scalable photolithography-based fabrication techniques. We incorporate the substrate into SLAS/ANSI-format microplates for use in standard industry endpoint assays and high-throughput automated platforms. Additionally, this substrate is capable of providing an active mechanical loading environment to the monolayer of cells for more physiological in vitro conditioning. Here, we focus on how structural alignment can enhance development at the cellular level and improve the expression of different genes at the molecular level for improved maturity of human induced pluripotent stem cells (hiPSCs). We demonstrate that hiPSC-derived cardiomyocytes (hiPSC-CMs) grown on this biomimetic surface exhibits in vivo-like myofibril alignment, sarcomere spacing and width, and expression of cardiomyocyte specific protein isoforms that are present only in mature myocytes. Metabolic assays that measure oxygen consumption rate (OCR) show that biomimetically patterned cells exhibit higher maximum respiratory capacity and lower metabolic stress when compared to unpatterned hiPSC-CMs.  We also evaluated the electrophysiological response of these cells through patch clamp and MEA platforms demonstrating more adult-like action potentials. Furthermore, we tested the effect of these maturational stimuli with respect to the detection of the arrhythmogenicity of known cardiotoxicants. Our data show that these cues can impart these cells with the ability to properly classify previously misidentified compounds, demonstrating that cell maturity plays a critical role in assay fidelity. Lastly, we confirm the utility of our approach by showing phenotypic enhancement in other adherent mammalian cell types. We conclude that our approach is a reliable and scalable method for re-creating specific aspects of the ECM to enhance the development and maturation of stem cell-derived cardiomyocyte models.