UC Santa Barbara

Heart disease is the leading cause of death in the US, and human cell models are needed to study how structure and function are related in heart health and disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are one promising cell model, but their immaturity (e.g., lower sarcomere alignment and contractility) relative to adult human cardiomyocytes (CMs) limits their physiological relevance. Controlling cellular shape via 2D protein micropatterning has been commonly used to enhance the maturity of single-cell hiPSC-CMs, but is often done via microcontact printing, which has low accuracy and reproducibility, or lift-off patterning, which is less commonly used because of the low-throughput and technical process for creating the pattern templates. In addition to limitations on protein patterning accuracy, these studies have failed to investigate the impact of two important features of native heart tissue: cell-cell contacts between neighboring CMs and the 3D microenvironment. In this work, we sought to scale up the lift-off protein patterning method and improve the single-cell hiPSC-CM model by developing platforms for: 1) dual protein patterning to imitate both CM-ECM and CM-CM interactions for single-cell hiPSC-CMs and 2) 3D microwells to create a 3D microenvironment for single-cell hiPSC-CMs. To scale up lift-off patterning, we created protein templates on a 4” wafer and then diced the wafer into individual templates. We showed that we could make at least 16 templates per wafer, and that the protein patterns made with this method were reproducible, had a shelf life of at least 6 months, and were compatible with pattern transfer to polyacrylamide (PA) hydrogels and subsequent culture of hiPSC-CMs. These results allow for the expanded use of lift-off patterning, which is a more accurate and precise method for patterning hiPSC-CMs. To achieve dual protein patterning, we used a two-step photomolecular adsorption process to create spatially-accurate protein patterns with laminin bodies (to replicate CM-ECM contacts) and N-cadherin end caps (to replicate CM-CM contacts). We cultured hiPSC-CMs on these dual protein patterns and studied their cell shape, subcellular structure, contractility, and force production. We found that dual protein patterning with N-cadherin end caps leads to greater cell area and increased contractility in the direction of sarcomere organization, but no differences in force production or sarcomere organization. To replicate the 3D microenvironment for single hiPSC-CMs, we utilized a PDMS double molding process to create a thin PDMS mold. The thin mold was incubated with Matrigel (an ECM protein cocktail) and then used when casting a polyacrylamide (PA) hydrogel, creating microwells in the PA hydrogel. We cultured hiPSC-CMs in the microwells and studied their size and subcellular structure. We found that the 3D-patterned hiPSC-CMs had greater cell height than 2D-patterned hiPSC-CMs, but interestingly no difference in cell volume. We also found that 3D-patterned hiPSC-CMs had greater myofibrillar content than 2D-patterned hiPSC-CMs, suggesting that they had more sarcomeres (subcellular force-producing units). Using these platforms, we have shown that we can improve hiPSC-CM structure and function through mimicking aspects of the microenvironment of mature human CMs. These platforms can be used to improve the hiPSC-CM model for future studies of heart function and disease.