Optimizing mesoderm progenitor selection and three-dimensional microniche culture allows highly efficient endothelial differentiation and ischemic tissue repair from human pluripotent stem cells.

Background: Generation of large quantities of endothelial cells is highly desirable for vascular research, for the treatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically defined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by going through an MESP1 mesoderm progenitor stage.

Methods: Mesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using an MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles of MESP1 and MESP1 cells and identified new signaling pathways that may promote endothelial differentiation. We also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial cell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the repairing potential of endothelial-primed MESP1 cells.

Results: MESP1 mesoderm progenitors, but not MESP1 cells, have strong endothelial differentiation potential. Global gene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched in MESP1 cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1 cells markedly upregulated genes related to vessel development and regeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1 cells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed MESP1 cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia.

Conclusions: Our study demonstrates that combining MESP1 mesoderm progenitor cells with tissue-engineered 3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the production of endothelial cells in vitro and augment their regenerative power in vivo.