Fig. 5

Morphology assessment of cells following osteoclast differentiation using CLSM. A–H Representative CLSM images of hematopoietic cells from a PBMC-derived iPSC line A–D or a fibroblast-derived iPSC line (E–H) that had been differentiated either according to an embryoid body-based (EB) (A, B, E, F) or a monolayer-based (MB) protocol (C, D, G, H) were further subjected to osteoclast differentiation and stained for Cathepsin K (turquoise), F-actin (red) and counterstained with DAPI nuclear stain (blue). Cells differentiated according to the EB protocol showed large multinucleated polykaryons (solid arrows in A, E) which also demonstrated Cathepsin K expression (arrow tips in B, F). MB differentiated cells on the other hand showed a low number of cells with up to 5 nuclei (solid arrow in D) in the PBMC-derived iPSC line and cells with a stellar-like morphology in the fibroblast-iPSC line (chevron arrows in H). A limited number of cells expressing Cathepsin K can be seen in both groups (arrow heads in D, H). Mononuclear cells with some degree of Cathepsin K expression can be seen throughout all groups (empty arrows in A, D, E, H). I-K Image quantitation shows a significant difference in osteoclast number (3 or more nuclei) between the EB and MB protocols (I). No significant differences were observed in osteoclast size or number of nuclei when the EB protocol was used with the different iPSC lines (J, K). Scale bars: A, C, E, G = 100 µm, B, D, F, H = 25 µm. Statistics are based on ANOVA followed by Tukey’s multiple comparison post hoc test (I n = 3 well replicates, J, K n = 50 analyzed cells, **p < 0.01, ***p < 0.001)