Spatiotemporal cell junction assembly in human iPSC-CM models of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that causes life-threatening arrhythmias and myocardial dysfunction. Pathogenic variants in Plakophilin-2 (PKP2), a desmosome component within specialized cardiac cell junctions, cause the majority of ACM cases. However, the molecular mechanisms by which PKP2 variants induce disease phenotypes remain unclear. Here we built bioengineered platforms using genetically modified human induced pluripotent stem cell-derived cardiomyocytes to model the early spatiotemporal process of cardiomyocyte junction assembly in vitro. Heterozygosity for truncating variant PKP2R413X reduced Wnt/ß-catenin signaling, impaired myofibrillogenesis, delayed mechanical coupling, and reduced calcium wave velocity in engineered tissues. These abnormalities were ameliorated by SB216763, which activated Wnt/ß-catenin signaling, improved cytoskeletal organization, restored cell junction integrity in cell pairs, and improved calcium wave velocity in engineered tissues. Together, these findings highlight the therapeutic potential of modulating Wnt/ß-catenin signaling in a human model of ACM.

Fattening chips: hypertrophy, feeding, and fasting of human white adipocytes in vitro.

Adipose is a distributed organ that performs vital endocrine and energy homeostatic functions. Hypertrophy of white adipocytes is a primary mode of both adaptive and maladaptive weight gain in animals and predicts metabolic syndrome independent of obesity. Due to the failure of conventional culture to recapitulate adipocyte hypertrophy, technology for production of adult-size adipocytes would enable applications such as in vitro testing of weight loss therapeutics. To model adaptive adipocyte hypertrophy in vitro, we designed and built fat-on-a-chip using fiber networks inspired by extracellular matrix in adipose tissue. Fiber networks extended the lifespan of differentiated adipocytes, enabling growth to adult sizes. By micropatterning preadipocytes in a native cytoarchitecture and by adjusting cell-to-cell spacing, rates of hypertrophy were controlled independent of culture time or differentiation efficiency. In vitro hypertrophy followed a nonlinear, nonexponential growth model similar to human development and elicited transcriptomic changes that increased overall similarity with primary tissue. Cells on the chip responded to simulated meals and starvation, which potentiated some adipocyte endocrine and metabolic functions. To test the utility of the platform for therapeutic development, transcriptional network analysis was performed, and retinoic acid receptors were identified as candidate drug targets. Regulation by retinoid signaling was suggested further by pharmacological modulation, where activation accelerated and inhibition slowed hypertrophy. Altogether, this work presents technology for mature adipocyte engineering, addresses the regulation of cell growth, and informs broader applications for synthetic adipose in pharmaceutical development, regenerative medicine, and cellular agriculture.