Cytochemistry
Immunocytochemistry for all surface markers was performed on live cells. Monoclonal antibodies VCAM, STRO-1, ICAM-1(CD54), CD105, CD29, and MF20 were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa, United States); CD73, CD44, and ALCAM(CD166) were from BD Biosciences Pharmingen (San Diego, California, United States). All other immunocytochemical analyses were performed after fixation in 4% paraformaldehyde and 0.15% picric acid, followed by permeabilization in 0.3% Triton X100. Polyclonal antibodies used were MyoD (Santa Cruz Biotechnology, Santa Cruz, California, United States) and nestin (gift from R. McKay); monoclonal antibodies were vimentin, alpha smooth muscle actin, fast-switch myosin, pan-cytokeratin (all from Sigma), and human nuclear antigen (Chemicon, Temecula, California, United States).
Alkaline phosphatase reaction was performed using a commercially available kit (Kit-86; Sigma) and the mineral was stained with silver nitrate according to the von Kossa method. Fat granules were visualized by Oil Red O staining solution (Sigma). Alcian Blue (Sigma) was used to detect extracellular matrix proteoglycans in chondrogenic cultures.