Table 1.
Primers used for standard, semi-quantitative and quantitative RT-PCR.
Fig 1.
Time course and efficiency of osteoclastogenesis from BALB/c-derived ER-Hoxb8 SCs.
(A) The time course of OC differentiation from ER-Hoxb8 SCs (24,000 cells per cm2) is illustrated by quantification of TRAP activity in supernatants isolated between d1 and d7. No signs of TRAP activity are detectable in supernatants until d4. Enzyme activity starts to develop at d5 of differentiation and reaches its maximum at d6. Data are shown as the mean ± SD, n = 3. (B) SCs were inoculated with indicated cell counts. TRAP activity of supernatants was measured at d5 of M-CSF/sRANKL-induced OC differentiation. Data are displayed as the mean ± SD, n = 4. (C, D) Representative bright-field microscopy images of TRAP-stained ER-Hoxb8-derived OCs at d5 of cultivation in the presence of M-CSF and sRANKL. Remarkable differences in cell morphology and multi-nucleation can be seen between wells of intermediary (C) and high cell density (D) inoculations. Scale bars = 100 μm.
Fig 2.
Comparison of OC differentiation from ER-Hoxb8 SCs with conventional OC progenitor sources.
(A) Representative microscopy images of OCs derived from ER-Hoxb8 SCs (BALB/c WT), primary BMMs (BALB/c WT) or RAW 264.7 macrophages after formalin fixation and TRAP staining. Scale bars = 150 μm. (B) Examples of bright-field microscopy images of mature OCs after Cytospin® centrifugation and modified Wright-Giemsa staining (DiffQuik®). Scale bars = 50 μm. (C) Graphic illustration of percentage of TRAP-positive cells with indicated number of nuclei per cell. OCs derived from RAW 264.7 cells show higher percentage of TRAP-positive cells with 7 or more nuclei. Data are displayed as the mean ± SD, n = 4 (4 different 24-wells).
Fig 3.
Comparison of osteoclastogenesis using ER-Hoxb8 cells from different WT mouse strains.
(A) ER-Hoxb8 OC progenitors (16,000 cells per cm2) obtained from BM cells of C57BL/6J, BALB/c and C3H/HeJ WT mice were subjected to osteoclastogenesis with M-CSF and sRANKL. TRAP activity of supernatants was measured at d5 of differentiation. Data are displayed as the mean ± SD, n = 3 (three differentiation experiments from identical batches of ER-Hoxb8 SCs). Significant differences in average TRAP activity compared to C57/BL6J-derived OC values are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images of formalin-fixed and TRAP-stained ER-Hoxb8-derived OCs of indicated WT origin. Representative multi-nucleated and TRAP-positive OCs are shown at lower (left column; scale bars = 200 μm) and higher magnification (right column; scale bars = 100 μm).
Fig 4.
Effect of inhibitory and stimulatory cytokines on OC differentiation of primary BMs or ER-Hoxb8 SCs.
(A) BALB/c BMMs or ER-Hoxb8 OC precursor cells of BALB/c and IL-4R KO origin (23,500 cells per cm2) were cultured with 1) M-CSF alone (“MФ”), 2) M-CSF and sRANKL (“OC”), or 3) the additional supplementation of indicated cytokines (GM-CSF: 10 ng/ml, remaining cytokines: 20 ng/ml). TRAP activity of supernatants was measured and compared to control differentiation (“OC”). Data are presented as the mean ± SD, n = 4. Significant differences of average TRAP activity compared to respective control values (“OC”) are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images illustrating morphology and TRAP staining of formalin-fixed cells after treatment with inhibitory and stimulatory modulators during OC differentiation. M-CSF-treated BMMs and Hoxb8 SCs as well as IL-4-treated BALB/c ER-Hoxb8 and BMMs do not show signs of TRAP staining. IL-4R KO cells are not sensitive to IL-4 and thus show normal OC differentiation potential. Scale bars = 150 μm.
Fig 5.
C3H/HeJ ER-Hoxb8-derived OCs show different stages of F-actin ring formation.
Visualization of F-actin ring and podosome structures in permeabilized (Triton™ X-100) OCs was enabled by interaction of FITC-conjugated phalloidin (green) with F-actin. Nuclei were counterstained with DAPI (blue). Representative fluorescence microscopy images of F-actin structures observed in ER-Hoxb8-derived OCs show examples of podosome cluster, podosome ring and podosome belt in cells differentiated on uncoated glass coverslips. A representative example of fluorescence microscopy images of mature F-actin ring structures in OCs which were differentiated on 100 mm dishes (300,000 cells) and subsequently plated on CaP-coated glass coverslips is shown in the lowest panel. Scale bars = 50 μm.
Fig 6.
Resorption activity of C3H/HeJ ER-Hoxb8-derived OCs on CaP-coated cell culture plates.
(A) Representative bright-field microscopy images of TRAP-stained OCs (seeding density of SCs: 20,000 cells per cm2) after 5 d of direct differentiation on CaP-coated plates show intracellular as well as longitudinal tracks of extracellular TRAP enzyme activity. Scale bars = 100 μm. (B) Representative images of TRAP-stained OCs on AgNO3-colored CaP-coated cell culture plates surrounded by areas without CaP indicating actively resorbing OCs. OCs were differentiated on uncoated dishes for 5 d, plated on CaP-coated 24-wells and incubated for a further 48 h. Scale bars = 100 μm. (C) Examples of microscopy images at 4x (left column; scale bars = 200 μm) and 20x magnification (right column; scale bars = 100 μm) showing resorption activity of ER-Hoxb8-derived OCs on CaP-coated cell culture plates after AgNO3 and UV treatment. Resorption is visualized as white spots lacking CaP and remaining unstained despite application of AgNO3.
Fig 7.
Resorption activity of ER-Hoxb8-derived OCs compared to conventional OCs.
(A) Representative microscopy images of pit formation assays performed with dentin discs and mature OCs from indicated sources. Resorption pits were visualized after removal of cells and toluidine blue staining. RAW 264.7 macrophages (“MΦ”) were used as negative control. Scale bars = 400 μm. (B) Microscopy images of mature OCs re-plated in CaP-coated cell culture plates after formalin-fixation and TRAP plus AgNO3 staining. Scale bars = 100 μm. (C) Representative examples of merged and inverted overviews of 24-well cell culture plates obtained from 7x7 individual microscopic images at 20x magnification. Resorption areas are visible as black spots. Scale bars = 1 mm.
Fig 8.
Gene expression profiles of ER-Hoxb8-derived OCs and ER-Hoxb8 SCs.
(A) Relative mRNA expression of selected OC or SC marker genes probed with cDNA originating from C57BL/6J ER-Hoxb8 SCs compared to cDNAs of ER-Hoxb8-derived OCs and IL-4-treated OC differentiations. Expression was determined by Mouse Gene 2.0 ST Array from Affimetrix. (B) qRT-PCR analyses of indicated OC and SC marker genes illustrate OC differentiation. RNAs were isolated from ER-Hoxb8 cells of indicated WT or IL-4R KO origin after 5 d under OC differentiation conditions (20,000 cells per cm2). Gene expression of respective SC cultures served as reference control. Data are mean ± SD of technical duplicates. Each sample was pooled together from three 24-wells. Housekeeping gene Hprt was used for normalization of samples. (C) qRT-PCR analyses of indicated OC and SC marker genes were performed with samples isolated from BALB/c cells after 3–7 d under OC differentiation conditions. Gene expression of respective SC culture served as reference control. Data are mean ± SD of technical duplicates. Samples were pooled together from three 24-wells. Expression of Hprt was used for normalization. Note log scale on y axis (A-C).
Fig 9.
RT-PCR analyses of gene expression in ER-Hoxb8-derived OCs and ER-Hoxb8 SCs.
Agarose gels of RT-PCR analyses showing expression of selected OC and SC marker genes in BALB/c- or IL-4R KO-derived SCs, OCs or IL-4-treated OC differentiation. Time course of gene expression between d3 and d7 of OC differentiation is illustrated in BALB/c-derived cells. Gene expression of OC marker genes is maximal at d5 of differentiation and decreases again at d6 and d7. Uniform Hprt expression is shown as housekeeping control. Plasmid DNAs served as positive controls (+); water was used as negative control (-).