Background: 

During the growth of mouse embryonic stem cells within the three-dimensional tissue of embroid bodies (EBs), cells are subjected to mechanical strain. It is well known that many physiological processes depend on strain forces such as arteriogenesis, morphogenesis of the heart and development of the fetal lung. The aim of the present study is to investigate the meaning of mechanical strain for vasculogenesis of in vitro generated EBs.

Methods: 

For the experimental set-up 3-day-old EBs were plated on 6-well plates with a flexible membrane bottom, coated with collagen type I. The samples were categorised into two groups: control group and stretched group. The stretched groups were subjected to 10% mechanical strain by a computer controlled strain unit (FLEXERCELL) for 2 hours. Calcium was evaluated by Fluo-4 microfluorometry. Reactive oxygen species (ROS) were detected using the fluorescence indicator H₂DCF-DA, Nitric oxide (NO) was assessed my DAF-DA fluorescence.

Results: 

EBs subjected to mechanical strain display significantly increased vascularisation, as shown by staining for PECAM-1. Stimulation with mechanical strain elevated intracellular ROS and NO. Furthermore a calcium response was observed shortly after mechanical strain application. Treatment with the intracellular calcium chelator BAPTA-AM significantly reduced the effect of strain on vascularisation. Treatment with the NO synthase inhibitor L-NAME in either the absence or presence of the calcium chelator BAPTA-AM abolished the elevation of NO, suggesting that the mechanical strain-induced calcium response regulated NO generation. In summary our data demonstrate that calcium ROS and NO are involved in mechanical strain-induced vascularisation of mouse embryonic stem cells.