Graduate Student Kyoto University Kyoto, United States
Abstract: Vascular endothelial cells (ECs) derived from human pluripotent stem cells (hPSCs) are critical for constructing vascularized tissues in regenerative medicine. However, conventional protocols for differentiating hPSCs into ECs yield only about 70% purity. Here, we present a highly efficient differentiation protocol that consistently produces ECs from human embryonic stem cells (hESCs), eliminating the need for intermediate cell sorting. In our approach, hESCs were first exposed to a WNT inhibitor, bone morphogenetic protein, and fibroblast growth factor 2 to induce mesodermal differentiation. The resulting cells were then cultured with vascular endothelial growth factor (VEGF), a γ-secretase inhibitor, and a cell-permeable adenylyl cyclase activator to generate endothelial progenitor cells, which were subsequently matured into ECs in VEGF-supplemented medium. Flow cytometric analysis revealed 90% of the differentiated cells expressed EC markers, CD31 and CD144, comparable to the 97% expression observed in human umbilical vein endothelial cells (HUVECs). The hESC-derived ECs also displayed the characteristic sprouting morphology typical of ECs when cultured on gel substrates. RNA sequencing identified 2,607 differentially upregulated genes relative to HUVECs, with gene set enrichment analysis highlighting “Angiogenesis” as the top hallmark pathway, including Thrombomodulin and Muscle segment homeobox 1. Collectively, these findings demonstrate that our protocol robustly generates functional ECs from hESCs, providing a promising tool for tissue engineering applications that require vascularization.
Funding Source: The Japan Society for the Promotion of Science (JSPS; 21H01728, 22J22729 and 24H00797)
