Crosstalk of E-cadherin and small GTPase Rap1 coordinates the clonality of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) are pluripotent cells, capable of giving rise to all three germ layers while maintaining their ability to proliferate indefinitely in culture. However, little is known regarding the microenvironmental cues that govern hESC self-renewal, particularly the challenge of clonal propagation following single cell dissociation. Increasing evidence suggests that intracellular pathways that coordinate E-cadherin-mediated cell-cell and integrin-mediated cell-ECM adhesions, are indispensable for the maintenance and self-renewal of hESCs. I have demonstrated that a potential crosstalk between small GTPase Rap1 and E-cadherin coordinates the colony formation and self-renewal of hESCs. I demonstrate that Rap1 expression kinetically decreases following the dissociation-induced disruption of E-cadherin mediated cell-cell adhesion compared to adherent hESCs. Inhibition of Rap1 with GGTI-298 completely abolishes the colony formation and self-renewal capacity of dissociated hESCs, whereas ectopic expression of Rap1 augments colony formation and survival. Addition of a potential activator of Rap1, Bombesin, inhibited dissociation-induced loss of Rap1 in hESCs and subsequently enhanced their survival, clonal propagation and self-renewal. Given the considerable extracellular and intracellular activators of Rap1, this work may provide an intracellular target to improve hESCs maintenance and self-renewal and provide new insights into the mechanisms regulating clonality and self-renewal.