Supplementary Material for: Differentiation of Multipotent Adult Germline Stem Cells Derived from Mouse Testis into Functional Endothelial Cells

Pluripotent stem cells hold great promise for the treatment of cardiovascular disease. We previously described multipotent adult germline stem cells (maGSCs) from mouse testis with differentiation potential similar to embryonic stem cells. The aim of this work was to differentiate maGSCs into functional endothelial cells and to study their potential for vasculogenesis. MaGSCs were cocultivated with OP9 stromal cells to induce differentiation into cardiovascular progenitors, i.e. fetal liver kinase 1-positive (Flk-1+) cells. Five days later, Flk-1+ cells were separated using fluorescence-activated cell sorting, followed by cultivation on collagen type IV under endothelial differentiation conditions. At different time points, maGSC-derived endothelial-like cells were characterized using RT-PCR, flow cytometry, immunofluorescence and functional assays. Cultivation of Flk-1+ cells resulted in the progressive upregulation of endothelial cell markers, including VE-cadherin, von Willebrand factor and endothelial nitric oxide synthase. Moreover, Flk-1+ maGSC-derived endothelial-like cells were able to branch and form networks in vitro and promoted functional blood vessel formation in vivo. Importantly, Flk-1+ cells retained their potential to proliferate and could be continuously expanded, while the ability of contact inhibition was preserved. Thus, maGSCs may provide a useful source of endothelial-like cells to study the basic mechanisms of vasculogenesis or endothelial differentiation.