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Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling. ECs differentiated from human induced pluripotent stem cells (iPSCs) can serve as a viable alternative to primary ECs for these applications. This is because human iPSCs can proliferate indefinitely and have the potential to differentiate into a variety of somatic cell lines, providing a renewable source of patient-specific cells. Here, we present an optimized, highly reproducible method for the differentiation of human iPSCs toward vascular ECs. The protocol relies on the activation of the WNT signaling pathway and the use of growth factors and small molecules. The resulting iPSC-derived ECs can be cultured for multiple passages without losing their functionality and are suitable for both in vitro and in vivo studies.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient’s fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming., Induced pluripotent stem cells (iPSCs) have potential for regenerative medicine applications, but are generated with very low efficiency. Here, the authors show highly efficient reprogramming of human primary fibroblasts to iPSCs via the synergistic activity of synthetic modified mRNAs, mature miRNA mimics, and optimized culture methods.