Mouse embryonic stem cell (mESC) differentiation into embryoid bodies (EBs) and confrontation culture with tumor spheroids. (a) R1 mES cells grown as colonies on feeder layers of mouse embryonic fibroblasts (MEFs) in the presence of leukemia inhibitory factor (LIF). (b) Suspension growth of mESCs on ultra-low attachment (ULA) 96-well round-bottomed plates allows spontaneous differentiation into EBs. Representative images of EBs are shown. Scale bar: 500 μm. (c) EB growth curve, determined by automated imaging analysis on a Celigo cytometer, (n = 12 EBs/timepoint). Values are mean ± SD. (d) CD34 staining of day 12 EBs shows endothelial differentiation (brown staining). Scale bar: 100 μm (left) or 50 μm (right). (e) Confrontation culture of green fluorescent protein (GFP)-transduced U-87 MG tumor spheroid (TS) and EB is represented in a cartoon on the left: a single TS and EB are cocultured in each well of a ULA 96-well round-bottomed plate. Representative images are shown at t0 and 55 h. Images were obtained on a Celigo cytometer. Scale bar: 500 μm. (f) Selected frames of a timelapse study show mutual invasion and coalescence of the two organoids. Images were obtained on an inverted microscope. Scale bar: 500 μm.