Characterization of monocyte-derived cultured OCs. (A) Schematic diagram of OC characterization studies. Top arrow: fractionated CD14⁺ monocytes were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Cultured OCs were analyzed after 3 weeks. Middle arrow: cell fusion assay was carried out by culturing CD14⁺ monocytes labeled with either red or green lipophilic dyes at a 1:1 ratio. Bottom arrow: bone resorption assay was performed after CD14⁺ monocytes were cultured for 3 weeks in the OC culture assay on top of a bone mineral–coated surface. All experiments were performed in duplicate. Average values are depicted. (B) Morphological analysis of cultured OCs. (i) Representative phase-contrast images of mature OCs derived from CD14⁺ monocytes of healthy donors (Ctrl; n = 3) and MF patients (n = 12). Compared with Ctrl OCs, MF OCs are smaller and rounder, with almost no protrusions. (ii) Quantitative analysis of OC diameter. Dots represent average values for 500 cells from each patient. Bars denote median. (C) Assessment of OC nuclei. (i) Representative images of Ctrl’s (n = 3) and MF patients’ (n = 12) OCs stained with F-actin dye, anti-TRAP antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Compared with normal OCs, MF OCs are characterized by fewer nuclei and abnormal F-actin ring (arrowheads) formation. (ii) Quantitation of cultured OCs shows a reduced number of nuclei in MF OCs. Dots represent average values for 500 cells from each patient. Bars denote median. (D) Clonal analysis of MF OCs. (i) Allele burdens of JAK2^V617F (n = 15) and CALR exon 9 mutation (n = 6) in low-density cells (LDC) and OCs cultured from MF patients carrying a JAK2^V617F or CALR exon 9 mutation. As shown, OCs carried a higher allele mutant burden than circulating LDC. Dashed lines denote samples with a decrease in allele burden. (ii) Fluorescence in situ hybridization analyses confirm that MF OCs are derived from the neoplastic clone. Shown are representative images (×100 magnification) of cultured OCs from MF patients with 13q and 20q deletions (n = 4). Individual chromosomes were stained using 1, 16, 13q, and 20q probes, and nuclei were counterstained using DAPI. Nuclei of MF OCs lack 1 long arm of chromosome 13 or 20 (nuclei showing only 1 red dot). Chromosomes 16 and 1 were used as internal controls (2 green dots). (E) Cell fusion capacity of OC-forming monocytes. (i) Representative images of OCs cultured from 2 populations of CD14⁺ monocytes from Ctrl (n = 5) and MF patients (n = 12). Membranes of live cells were stained using lipophilic dyes PKH26 and PKH67. A smaller number of cells harboring both dyes (yellow), indicating decreased cell fusion, is seen in MF patient–derived cells. (ii) Quantitation of cells with colocalization signals. Horizontal lines denote median. (F) Bone mineral resorption capacity of cultured OCs. (i) Representative images of OCs grown on top of bone mineral–coated surface from Ctrl (n = 4) and MF patients (n = 8). Cultured MF OCs exhibit a significant decrease in the total resorbed area (white). (ii) Quantitation of the resorbed mineral-coated surface areas. Horizontal lines denote median. No γ correction was applied. Bars, 200 µm.