Anisotropic high throughput culture of human induced pluripotent stem cell-derived cardiomyocytes leads to cellular features of enhanced physiological relevance

Cardiac liability is an important cause of drug failure in late stages of clinical trials, as well as in the removal from the market. Part of these failures have been attributed to the deployment of non-human and non-cardiac systems in cardiac safety assessments. More recently human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) have increasingly become a complementary tool for the study of acute cardiac toxicity and safety, providing a human and tissue-specific model to deconvolute potential cardiac liabilities. However, hiPSC-CMs typically display an abnormal sub-cellular structural organization and unsatisfactory electro physiological maturity. When cultured in standard cell cultureware, hiPSC-CMs often display undefined or disarrayed sarcomeric organization. Here we describe and characterize a novel micro-engineered high-density screening platform for hiPSC-CMs that emulates correct cardiac muscle fiber organization through passive self-alignment, generating anisotropic cultures. In agreement with previous reports utilizing anisotropic cardiomyocte cultures, the platform employed here improved sarcomeric organization, as seen by readily identifiable, correctly patterned myofibrils along the cell body, while nuclear size and shape were significantly changed. Contraction patterns of hiPSC-CM preparations were observed to become markedly unidirectional in this platform. Increased gene expression of RYR2, ATP2A2, and pln, key components of cardiomyocyte calcium handling pathways, which are crucial for cardiac physiology, were also observed. The expression levels of cardiac ion channel genes such as CACNAC1C, SCN5A, KCNE1, KCNQ1 as well as cardiac cell junction components GJA1, GJA5 and DSP also showed an increase. High throughput kinetic fluorescence analysis of calcium flux in hiPSC-CMs indicated that the anisotropic hiPSC-CM cultures presented significant changes in cardiomyocyte physiology, highlighting that anisotropic hiPSC-CM cultures may present an attractive platform for cardiac safety and toxicity studies. In summary, high throughput anisotropic hiPSC-CM cultures displayed several physiologically-relevant changes which may have a significant impact in the investigation of cardiac liabilities of developing drugs.

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