Figure 1

Differentiation of MK cells and suppression of bone resorption by MK CM. (a) K562 cells were incubated with phorbol 12-myristate 13-acetate (PMA) for the indicated times. (b) Cells from mouse fetal liver were differentiated with thrombopoietin (TPO) for the indicated days. Arrows indicate proplatelet-bearing megakaryocytes (MKs). Cell morphology was observed by microscope, and MK differentiation was detected by Wright-Giemsa staining at 3 and 4 days, respectively. Scale bars, 100 μm. (c) DNA polyploid content was analyzed by flow cytometry. K562 and primary cells were treated with PMA or TPO for 3 days or 4 days, respectively. The percentage of cells in each ploidy (2 N, 4 N, and ≥8 N) was shown. (d) Differentiation rates of K562 and primary cells. Mature MKs were scored by counting larger than 25 µm in diameter and extensive multinuclearity. (e) A resorption pit formation assay of mouse bone marrow macrophages (BMMϕ) cultured with M-CSF and RANKL to form osteoclasts in the presence or absence of 10% (v/v) conditioned media (CM) of MKs and pro-megakaryocytes (pro-MKs) on a dentine disc for the indicated times. MKs were derived from K562 cells. Resorbed areas were quantified as percentages of the total area. Data are presented as mean ± SEM. *P < 0.05 vs. non-conditioned media (non-CM, α-MEM media) or primary cells; ^#P < 0.05 vs. pro-MK CM. NS, not significant.