Spatial Patterning of Laminin and N-Cadherin for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Controlling cellular shape with protein micropatterning can mimic physiological morphologies and has been shown to improve reproducibility, enhancing our ability to collect statistics on single-cell behaviors. It has also advanced efforts in developing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a promising human model for studies of heart structure and function. hiPSC-CMs have key physiological differences from primary human cardiomyocytes (CMs), including lower sarcomere alignment and contractility, smaller area and lower aspect ratio, and lower force production. Protein micropatterning has been demonstrated to make hiPSC-CMs behave more like primary human CMs across these metrics. However, these micropatterned models typically use only extracellular matrix (ECM) proteins and have not investigated whether providing a protein associated with CM-CM interactions, such as N-cadherin, further enhances hiPSC-CM structure and function. Here, we developed a novel dual-protein patterning process to geometrically control single-cell CM placement on deformable hydrogels suitable for traction force microscopy (TFM). The patterns were comprised of rectangular laminin islands for attachment across the majority of the cell area, with N-cadherin "end-caps" imitating cell-cell interactions. We first photopatterned two proteins on a glass coverslip using a two-step process with photomolecular adsorption of proteins. After both photopatterning steps were complete, we transferred the pattern from the coverslip to a physiologically relevant [~]10-kPa polyacrylamide hydrogel. We seeded -actinin-tagged hiPSC-CMs on the dual-protein-patterned hydrogels and verified interaction between the hiPSC-CMs and the N-cadherin end-caps via immunofluorescent staining. We found hiPSC-CMs on dual-protein patterns have a higher cell area and contractility in the direction of sarcomere organization than those on laminin-only patterns, but no difference in sarcomere organization or force production. While N-cadherin modestly improves the single-cell patterned hiPSC-CM model, it is not sufficient to replicate the role of cell-cell contacts in CM development for in vitro hiPSC-CM systems.