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 13 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. 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.

Development and Application of Endothelial Cells Derived From Pluripotent Stem Cells in Microphysiological Systems Models

“…They concluded that the robust and transient expression of ETV2 was sufficient to generate ECs independent of endogenous VEGFR2 and ETV2 expression. These ECs function adequately in comparison with ECFCs and ECs generated from a previous protocol ( 43 ).…”

“…Recently, Wang et al used chemically modified RNA (modRNA) to endogenously express ETV2 during the mesodermal stage of iPSC differentiation, resulting in highly efficient EC production ( 43 ). After inducing mesodermal progenitors by the addition of CHIR, modRNA was transfected into mesodermal cells via lipofection or electroporation to deliver ETV2.…”

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity

“…Drug testing using heterogeneous cell populations may lead to an inaccurate conclusion, especially if it is based on ensemble measurements ( Altschuler and Wu, 2010 ). To tackle this issue, a variety of strategies can be employed, such as optimization of the differentiation protocol ( Wang et al, 2020 ), purification using genetically engineered reporter cell lines ( Hu et al, 2018 ), or magnetic-activated cell sorting (MACS) based on the desired surface marker ( Li et al, 2017 ).…”

“…Genetically identical to the donors, iPSCs hold the promise to recapitulate individual predisposition to various risks ( Kitani et al, 2019 ; Lam et al, 2019 ). Furthermore, recent advances in iPSC differentiation strategies now enable the efficient generation of all the major cell lineages of the human cardiovascular system, including cardiomyocytes (CMs) ( Lian et al, 2012 ; Burridge et al, 2014 ), endothelial cells (ECs) ( Lian et al, 2014 ; Olmer et al, 2018 ; Williams and Wu, 2019 ; Wang et al, 2020 ), smooth muscle cells (SMCs) ( Cheung et al, 2012 ; Patsch et al, 2015 ), and cardiac fibroblasts (CFs) ( Zhang et al, 2019a ). These cells functionally and structurally resemble their counterpart primary cells.…”

Modeling Precision Cardio-Oncology: Using Human-Induced Pluripotent Stem Cells for Risk Stratification and Prevention

“…A myriad of researchers also use fluorescence-activated cell sorting (FACS) or flow cytometry to enumerate and separate cell types. Many functional assessments are in use to further characterize cells and cultures, including assays for shear stress, nitric oxide production, tube formation, vascular networks, and perfusion [ 60 ].…”