EphrinB2-Fc induces forward signaling in MSCs. (A) Donor and patient MSCs were starved overnight and stimulated with clustered ephrinB2-Fc (4 μg/mL) for 20 minutes before being assayed for phosphorylated EphB4 using ELISA. Note induction of EphB4 phosphorylation by ephrinB2 in donor and patient MSCs. (B) Donor and patient MSCs cultured in osteogenic media were treated with Fc or clustered ephrinB2-Fc (4 μg/mL) for 5 days and then subjected to immunohistochemical staining for osteocalcin. Note that, although ephrinB2-Fc increased expression of osteocalcin in both types of MSCs, the effect of the chimeric protein on osteoblast differentiation (mineralization) was more profound in donor MSCs. (C) Accumulation of calcium as determined by von Kossa staining (brown) in MSCs treated with Fc or ephrinB2-Fc for 10 days. Note increased calcium deposition by ephrinB2-Fc in both types of MSCs and stronger staining in donor MSCs than patient MSCs. Similar results were obtained in MSCs from 3 donors and patients with MM.