Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA.

Abstract

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor <i>ETV2</i> at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated 13 diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous <i>ETV2</i> were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Timely activation of <i>ETV2</i> was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.