Maintenance of undifferentiated state of human induced pluripotent stem cells through cytoskeleton-driven force acting to secreted fibronectin on a dendrimer-immobilized surface

Abstract

Understanding of the fundamental mechanisms that govern adhesive properties of human induced pluripotent stem cells (hiPSCs) to culture environments provides surface design strategies for maintaining their undifferentiated state during cell expansion. Polyamidoamine dendrimer surface with first-generation (G1) with dendron structure was used for co-cultures of hiPSCs and SNL feeder cells that formed tightly packed compact hiPSC colonies, similar to those on a conventional gelatin-coated surface. hiPSCs passaged up to 10 times on the G1 surface maintained their undifferentiated state. Immunostaining and reverse transcriptase PCR analysis of fibronectin showed that the secreted fibronectin matrix from feeder cells on the G1 surface contributed to hiPSC attachment. Compared with cells on the gelatin-coated surface, F-actin and paxillin immunostaining revealed a well-organized network of actin stress fibers and focal adhesion formation at cell-substrate sites in hiPSC colonies on the G1 surface. E-cadherin expression levels on these surfaces were almost same, but paxillin and Rac1 expression levels on the G1 surface were significantly higher than those on the gelatin-coated surface. Zyxin showed prominent expression on the G1 surface at sites of focal adhesion and cell-cell contact in colonies, whereas zyxin expression on the gelatin-coated surface was not observed in regions of cell-cell contact. These findings indicate that transduction of mechanical stimuli through actin polymerization at sites of focal adhesion and cell-cell contact results in maintenance of undifferentiated hiPSC colonies on G1 surface. The G1 surface enables a substrate design based on the mechanical cues in the microenvironment from feeder cells to expand undifferentiated hiPSCs in long-term culture.