Background: Generation of large quantities of endothelial cells is highly desirable for vascular research, for the\ntreatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically\ndefined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by\ngoing through an MESP1 mesoderm progenitor stage.\nMethods: Mesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using\nan MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles\nof MESP1+ and MESP1âË?â?? cells and identified new signaling pathways that may promote endothelial differentiation.\nWe also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial\ncell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the\nrepairing potential of endothelial-primed MESP1+ cells.\nResults: MESP1+ mesoderm progenitors, but not MESP1âË?â?? cells, have strong endothelial differentiation potential. Global\ngene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched\nin MESP1+ cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition\nof S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche\nand priming with VEGF and bFGF, MESP1+ cells markedly upregulated genes related to vessel development and\nregeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1+\ncells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed\nMESP1+ cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia.\nConclusions: Our study demonstrates that combining MESP1+ mesoderm progenitor cells with tissue-engineered\n3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the\nproduction of endothelial cells in vitro and augment their regenerative power in vivo.
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