Over the last decade, human induced pluripotent stem cell (h-iPSC)–derived endothelial cells (h-iECs) have become a valuable tool in cardiovascular research, offering a broad range of translational and clinical applications. However, current differentiation protocols remain largely inefficient and lack reliability, thus hampering the expansion of this technology. We examined standard protocols to differentiate h-iPSCs into h-iECs and identified that a critical source of inconsistency resides in the nonuniform activation of the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. To overcome this limitation, we developed a method that entails the precise delivery of modified mRNA (modRNA) encoding ETV2 into mesodermal intermediates. Our protocol achieved transient and precise activation of exogenous ETV2 activity throughout the entire mesodermal population. As a result, all h-iPSC lines tested differentiated into h-iECs with exceedingly high efficiency (>90%) and reproducibility. Importantly, we validated that the resulting h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. In contrast, we showed that protocols that solely relied on endogenous ETV2 were less efficient and notably inconsistent. Furthermore, we showed that the delivery of exogenous ETV2 directly into h-iPSCs also yielded high differentiation efficiency. However, we demonstrated that bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and incapable of forming functional vessels in vivo. In summary, we present an approach to differentiate h-iPSCs into h-iECs with high efficiency and reproducibility, irrespective of the cellular source from which the h-iPSC clones originate. We demonstrated that timely activation of ETV2 at the mesodermal stage is critical to achieving consistency and functional competence. Our results provide a means to generate h-iECs for vascular therapies and research effectively.

Dr. Wang received his B.E. in Biomedical Engineering from Southeast University, China, in 2010. He went on to earn his Ph.D. in Biomedical Engineering from Peking University, China, in 2016. Dr. Wang’s doctoral research focused on the hierarchical micropattern scaffold and transplantation of pancreatic islets for treating diabetes. From 2016 to 2021, he was trained as a postdoctoral fellow at Boston Children’s Hospital/Harvard Medical School in the Department of Cardiac Surgery. His research led to the development of a highly efficient protocol for differentiation of human stem cells into vascular endothelial cells. In 2021, he joined the Department of Physiology and Pathophysiology at Peking University as an Assistant Professor. Dr. Wang is directing the Laboratory of Stem Cells and Tissue Engineering, where the research is focused on bioengineering of stem cell derived vascular organoids for the disease modeling and therapeutic purposes. His research work has been published in prestigious journals including Sci. Adv (2020), Adv. Mater (2017) and Biomaterials (2016, 2017). Dr. Wang is the recipient of Vascular Biology Grant (2020). He was invited as a guest editor for JoVE and he is serving in the Editorial Board of Data in Brief and Military Medical Research.