Analysis of ephrinB2-induced effects on the migration and spreading of human thymus smooth muscle cells (HTSMCs) as well as the organization of porcine aortic endothelial cells (PAECs) and HTSMCs in 3-dimensional coculture spheroids. (A) The length of sprouts originating from the spheroids was reduced on treatment with dimeric ephrinB2 (**P < .01 vs control, representing one of 3 experiments with similar results; scale bar represents 200 μm). This effect could be blocked by simultaneous stimulation with dimeric ephrinB2 (2 μg/mL) and its receptor EphB4 (6 μg/mL). (B) Spreading of HTSMCs originating from spheroids (⬆), which were seeded on top of a confluent monolayer of PAECs, was inhibited if the smooth muscle cells were surrounded by PAECs overexpressing ephrinB2 or ΔephrinB2 (**P < .01, *P < .05 vs PAEC-mock, n = 3; scale bar represents 200 μm). (C) Mock, ephrinB2, ΔephrinB2, or EphB4-transfected PAECs were cocultured with HTSMCs in 3-dimensional spheroids. Overexpression of ephrinB2 or ΔephrinB2 in PAECs (PAEC-ephrinB2) enhanced the segregation of both cell types (***P < .001, **P < .01 vs PAEC-mock; n = 3; bottom panel shows CD31-stained cross sections of a paraffin-embedded PAEC-mock/HTSMC (left) and a PAEC-ephrinB2/HTSMC (right) coculture spheroid; scale bars represent 100 μm).