UC Merced

A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, endothelial cells (EC) can exhibit a number of distinct functional subphenotypes like arterial or venous EC, as well as angiogenic tip and stalk EC. This study focused on directing the differentiation of endothelial cells subphenotypes from mouse and human embryonic stem cells (ESC), as well as, human induced pluripotent stem (iPS) cells in vitro using a staged and chemically-defined methodology. Using these methods, we discovered and characterized highly angiogenic tip/stalk-containing EC emerging as distinct from less proliferative and less migratory phalanx EC, and examined our ability to direct specification of these subphenotypes. We found that both tip/stalk-containing and phalanx-containing sub-populations were more than 80% VE-cad+ without FACS purification. These EC exhibited distinct mRNA gene expression profiles, surface marker expression, and sprouting capacity in a fibrin gel assay. The tip/stalk EC are Flt4+/Dll4+/Flt-1-/Notch-1- reflecting a migratory more VEGF responsive phenotype indicative of tip cell surface expression pattern. Phalanx ECs are more homogeneous and less responsive to VEGF signaling – comprising of higher levels of Flt-1 and Notch-1. Human stem cell derived EC required additional purification step and treatment with TGFβR1 inhibitor, small molecule SB431542 in order to maintain stability of expanded EC populations. In our attempts at directing the differentiation of EC subpopulations, many of the cell populations did not survive. Of the surviving cells, signaling from PIGF and BMP treatments exhibited the greatest potential for directing stem cells toward tip-like EC and stalk-like EC, respectively. Additionally, immobilized rhDll4 induced quiescence and high VE-cad+/CD31+ expression in the EC consistent with a phalanx-like EC subphenotype. The ability to generate these functionally distinct vascular ESC-EC in vitro is an important development in regenerative medicine.