Fig. 1

Cell culture characterization. Phase contrast microscopy of 6-day BM-MNCs in selective culture conditions showed adherent cells characterized by distinctive MPC morphology with round fried egg-shape and sporadic polar elongation (a). Flow cytometry showed homogeneous expression of MPC-related markers CD31, CD18, and CD11c and lack of MSC markers STRO-1, CD73, and CD90 (b). Immunofluorescence revealed F-actin typical podosome-like distribution (red) and intense positive stain for nestin (green) (c). Nuclei visualization was performed by DAPI staining (blue). No difference in nestin expression (red) was found between MPCs (d.1) and HUVECs (d.2) while von Willebrand factor (vWF) was detected in HUVECs only (green) (d). One-week MPC mesengenic differentiation (P1-MSCs) produced mixed cultures of flattened elongated multibranched cells with residual highly rifrangent MPCs. Subculturing P1-MSCs for a further week in MesenPRO® RS Medium led to monomorphic cultures of confluent fibroblastoid spindle-shaped cells (P2-MSCs) (e). Flow cytometry revealed a standard MSC phenotype for P2-MSCs (f). Immunofluorescence showed F-actin reorganization in stress fibers (red) and loss of nestin, occasionally expressed by few residual multibranched cells (green) (g). P2-MSCs were also able to differentiate selectively into adipocytes, as revealed by intracellular lipid droplet accumulation (red in h.1) or osteocytes featuring intense extracellular calcium deposition (green in h.2) (h). HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell (Color figure online)