Characterization of MSCs. (A) MSCs (passage 4) displayed a fibroblast-like morphology in culture and were (B) EGFP⁺ as shown by fluorescence microscopy. (C) Flow cytometry analysis of enriched MSCs (passage 5) proved typical expression of surface markers; note the lack of the hematopoietic cell markers CD45 and CD11b. (D) Osteogenic differentiation of MSCs (passage 10) in vitro led to formation of aggregates and trabecular structures (inset, 17 days); Ca²⁺ deposition was demonstrated by von Kossa staining. (E) In vitro differentiation of MSCs (passage 7) into adipocytes. The accumulation of lipid droplets in vacuoles (inset, 2 weeks in culture) was confirmed by staining with oil red O. (F) Chondrogenic differentiation of MSCs (passage 10) in vitro was determined using combined Alcian blue/nuclear fast red staining. Bar represents 180 μm (panels A,B), 860 μm (panel D), 600 μm (panel D inset), 100 μm (panel E), 300 μm (panel E inset), and 550 μm (panel F). See “Materials and Methods; Image acquisition and preparation” for microscopy details.