Osteoclast-like formation of adipofibroblast/PBMNC co-cultures.

<p>(<b>A-D</b>) the osteoclast regulating potential of the adipofibroblast derived from MSC-derived adipocyte cultures was assessed by TRAP staining. After 15 days of co-culture with peripheral blood mononuclear cells (PBMNC) and was compared to (<b>C</b>) positive controls where PBMNCs cultures were given 50 ng/ml of M-CSF and 60 ng/ml RANKL in DMEM:RPMI (1/1) medium and (<b>D</b>) untreated controls where PBMNC were cultured in the same mixed basal medium only. (<b>C</b>) Multinucleated cells formed after treatment with M-CSF and RANKL for 15 days showing a distinct osteoclast like cell phenotype including multi-nucleation, TRAP positivity and a large diameter. Positive osteoclasts-like cells were numerous in (<b>A</b>) co-cultures with adipofibroblasts and (<b>C</b>) positive controls (<i>black arrows</i>) while scarce in (<b>D</b>) co-cultures with dermal fibroblasts. (<b>A</b>) Multi-nucleated TRAP positive osteoclast-like cells formed in ‘adipofibroblast’ PBMNC co-cultures after 15 days in culture. Osteoclast-like cell formation was comparable to that of the (<b>B</b>) positive controls while no significant formation of multi-nucleated TRAP-positive osteoclast-like cell formation was seen in (<b>B</b>) dermal fibroblast and PBMNC co-cultures. Scale bar in panel A = 200 µm for panels A-B and 300 µm for panels C-D. <i>n</i> = <i>3–4 donors for both MSC and PBMNC donors respectively</i>.