Binary Colloidal Crystals Drive Spheroid Formation and Accelerate Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The development of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides significant advances to cell therapy, disease modeling, and drug screening applications. However, the current differentiation protocol is inefficient in mimicking biophysical and biochemical characteristics of cardiac niche. Hence, immature cardiomyocytes are often generated. In this study, hiPSC-CMs were generated on a new family of substrates called monolayer binary colloidal crystals (BCCs). Four BCCs were fabricated with different sizes (2 or 5 or 0.4 or 0.2 μm) and materials [Si or polystyrene (PS) or poly(methyl methacrylate)] abbreviated as 2PS, 5PS, 2PM, and 5PM. BCCs have complex surface micro-/nanotopographies and heterogeneous chemistries which are important modulators in microenvironments in vitro. The results showed that hiPSCs formed adhered spheroids with strong pluripotent markers ( Oct4, Nanog, and Sox2) on PM surfaces compared to PS and flat surfaces. After 30-day differentiation, hiPSC-CMs on PM surfaces showed markedly improved myofibril ultrastructures, Ca2+ handling, and electrophysiological properties, indicating that more mature hiPSC-CMs were generated. hiPSC-CMs generated on 5PM are more similar to adult heart tissue compared to other surfaces in terms of genes ( ACTC1, TNNT2, RYR2, SERCA2a, SCN5a, KCNJ2, CACNA1c, ITGB1, GJA1, MYH6, and MYH7) and protein (ssTnI and cTnI) expressions. We further demonstrated that 5PM surfaces facilitated cadherin switching (from E- to N-) during cardiac differentiation and mature N-cadherin expression, which were positively correlated with the cardiogensis markers ( GATA4, MEF2c, and NKX2.5). This study illuminated that a tailored surface nanotopography was beneficial in hiPSC culture and in situ cardiac differentiation. This one-step approach and BCCs can be a next-generation tool for hiPSC expansion and CM differentiation.