Fig. 7

Assessment and quantification of the mineral resorption activity of osteoclasts. A–H Following hematopoietic differentiation of PBMC-derived iPSCs A–D or fibroblast-derived iPSCs (E–H), either according to an embryoid body based (EB) A, B, E, F or a monolayer-based (MB) protocol (C, D, G, H), hematopoietic cells were matured with M-CSF and differentiated into OCs with RANKL on calcium-phosphate coated wells. Osteoclasts differentiated from PBMC-derived iPSCs using the EB protocol showed clearly visible resorption pits on tiled full-well images acquired with a widefield microscope in phase-contrast mode A in comparison with undifferentiated negative controls (B). Similarly, osteoclasts from fibroblast-derived iPSCs differentiated with the EB protocol also showed clearly visible pits, albeit the total resorption area appeared much larger E than that of the negative control (F). In comparison, cells differentiated according to the MB protocol did not show visible resorption pits for either cell line C, G when compared to negative controls (D, H). Scale bar = 1 mm. I Image quantification demonstrates comparable resorption levels of PBMC-derived iPSC osteoclasts differentiated according to the EB protocol to osteoclasts differentiated from primary CD34⁺ PBMCs. The highest level of mineral resorption was observed in osteoclasts differentiated from the fibroblast-derived iPSC line using the EB protocol. Quantification confirms the absence of mineral resorption in cells differentiated according to the MB protocol for either iPSC line. Statistics are based on ANOVA followed by Tukey’s multiple comparison post hoc test (n = 3 well replicates, ****p < 0.0001)