Differentiation of endothelial cells derived from mouse embryoid bodies: A possible in vitro vasculogenesis model

Mouse embryonic stem cells (mES cells), which are pluripotent and self-renewal cells, are derived from the inner cell mass of mouse blastocysts. The objective of this study was to construct more efficient mES cell-derived embryoid bodies (EBs) for use as a vasculogenesis model and as an in vitro vascular toxicity testing model. EBs were formed for 3 days using hanging drop cultures and plated on gelatin-coated plates in endothelial growth medium-2 (EGM-2) to promote vascular development. The differentiation of mES cell-derived EBs was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry within 7 days after plating EBs. The mRNA and protein expressions of vascular endothelial growth factor receptors-2 (FLK-1), platelet endothelial cell adhesion molecule (PECAM), and vascular endothelial-cadherin (VE-cadherin) were observed in differentiated mES cells. When placed in matrigel, mES cell-derived endothelial like cells formed networks similar to vascular structures. mES cells were also exposed to 5-fluorouracil (5-FU), a strong inhibitor of vessel formation, and its cytotoxicity was determined using MTT assays. The inhibitory concentrations (IC50) of 5-FU for mES cells and C166 cells were 0.72 μM and 1.04 μM, respectively. These results demonstrate that mES cells can be used to study vasculogenesis and for cytotoxicity screening.