M-CSF priming enhances the efficiency and reproducibility of hiPSC-derived osteoclast differentiation and pharmacological responsiveness to anti-resorptive agents.

Human induced pluripotent stem cell (hiPSC)-derived osteoclasts offer a potentially scalable platform for studying osteoclast biology and anti-resorptive pharmacology. However, differentiation efficiency and inter-line reproducibility remain limited. To address this limitation, we investigated whether cytokine priming during the monocyte-to-macrophage transition is an important determinant of efficient and reproducible osteoclast differentiation from hiPSCs across diverse genetic backgrounds. Monocytes derived from three independent hiPSC lines were primed briefly with M-CSF, GM-CSF, or no cytokine, followed by osteoclast differentiation with M-CSF plus RANKL. Among these conditions, M-CSF priming reproducibly increased osteoclast differentiation efficiency compared with GM-CSF priming or no priming, yielding > 70% CD51/CD61⁺ cells across hiPSC lines derived from distinct tissue sources. Osteoclast maturation and function were then assessed by multinucleation, TRAP staining, and bone resorption assays, which further showed that M-CSF priming enhanced osteoclast maturation and resorptive function across the tested hiPSC lines. We also performed extracellular flux-based measurements of OCR/ECAR and ATP production rates to examine metabolic changes associated with osteoclast maturation. This analysis showed that an early rise in OXPHOS-derived ATP production preceded peak induction of mature osteoclast genes, suggesting that early oxidative metabolism may support subsequent fusion and functional maturation. Finally, we assessed the pharmacological responsiveness of hiPSC-derived osteoclasts using two clinically used bisphosphonates, alendronate and zoledronate, both of which elicited dose-dependent anti-osteoclastic effects. Collectively, these results indicate that brief M-CSF priming enhances the efficiency and inter-line reproducibility of hiPSC-derived osteoclast differentiation, while early oxidative metabolism temporally precedes osteoclast gene induction. This improved reproducibility and functional maturation may allow hiPSC-derived osteoclasts to support mechanistic studies of osteoclastogenesis and the evaluation of pharmacological responses to osteoclast-targeted agents.