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 ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated thirteen diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Importantly, timely activation of ETV2 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.

The hemogenic endothelium: a critical source for the generation of PSC-derived hematopoietic stem and progenitor cells

“…Inducible overexpression of ETV2 in human iPSC-derived, mesodermal-primed cells resulted in an almost pure population of cells with a vascular endothelial immunophenotype (CD144 + /CD73 + ) [135]. Similarly, transient expression of exogenous ETV2 by modified and stabilized mRNA efficiently generated functional endothelial cells with the ability to form perfused vascular networks in vivo [136]. However, overexpression of ETV2 alone was not described to robustly induce hemogenic potential.…”

Vascularized organoids on a chip: strategies for engineering organoids with functional vasculature

“…Through temporal delivery of modified mRNA encoding ETV2 at an intermediate mesodermal stage of differentiation, Wang et al achieved exceedingly high efficiency (>90%) in differentiating 13 different human iPSC lines to functional ECs. 48 Cakir et al were the first to take advantage of this technique to vascularize organoids. By incorporating 20% of iPSCs that were transduced with a doxycycline (Dox) inducible ETV2 gene in the initial 3D aggregates, they successfully induced the development of vascular structures within organoids following traditional cerebral organoid culture protocol.…”

mRNA-Enhanced Cell Therapy and Cardiovascular Regeneration

“…Furthermore, mRNA can be used to accelerate differentiation of iPSCs to the desired derivative. For example, mRNA encoding ETV2 has been used to generate iPSC-derived ECs with high (90%) efficiency [ 35 ].…”