Expression of mature osteoclast markers. Mature osteoclasts derived from hESCs express F-actin rings (A-C) and VNR (E-G). H1-derived hematopoietic precursors from day 21 cultures were seeded at 105 cells/96-mm well onto dentine slices in the presence of M-CSF and RANKL and stained 14 days later with TRITC-phalloidin (A-C) or by immunohistochemistry for VNR with the 23C6 antibody (E-G). F-actin rings (white arrows, panels A-C), ruffled borders (white arrowheads, panels B-C), and strong VNR expression (black arrows, panels E-G) are present in actively resorbing, multinucleated osteoclasts. Resorption trails are clearly visible in panels E-G (*). Images were acquired with a Zeiss Axioskop2 Plus microscope equipped with epifluorescence using plan-Neofluar objectives and an Axiocam camera. Auto adjustments were performed with Adobe Photoshop software. Laser confocal microscopy demonstrates the coexpression of β3 integrin (green), F-actin rings (red), and cathepsin K (blue) in H1-dervied (H) and MSC-iPS1–derived (I) osteoclasts. F-actin ring–positive cells were never detected in cultures lacking RANKL (D). Original magnifications: ×10 (A,D); ×20 (B,C,E,F); and ×40 (G).
