Tae-Hoon Lee

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.

Osteoporosis is a major skeletal disorder characterized by reduced bone strength and increased risk of fractures. Excessive osteoclast-mediated bone resorption is a primary cause of this condition, underscoring the need for effective anti-resorptive therapies. N -phenyl-methylsulfonamido-acetamide (PMSA) compounds have been previously identified as potential anti-resorptive agents that inhibit osteoclastogenesis. In this study, ribonucleic acid (RNA)-sequencing and proteomic analyses identified calcineurin (CaN) as a potential target of PMSA implicated in osteoclast differentiation. PMSA bound to CaN and suppressed its phosphatase activity, which is essential for the activation and translocation of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis. PMSA treatment resulted in altered NFATc1-related signaling and increased phospho-NFATc1 levels in osteoclasts. Overall, these findings suggest that PMSA may inhibit CaN activity during osteoclast differentiation, positioning it as a promising therapeutic candidate for osteoporosis.

Keap1 interacts with Nrf2 by assisting in its ubiquitination and subsequent proteolysis. By preventing ROS accumulation during RANKL-induced osteoclastogenesis, Nrf2 activation can prevent the differentiation of osteoclasts. Additionally, inhibiting the Keap1-Nrf2 PPI can be an effective strategy for triggering Nrf2 to regulate oxidative stress. Structure-based virtual screening was performed to discover a potentially novel Keap1-Nrf2 PPI inhibitor wherein KCB-F06 was identified. The inhibitory effects of KCB-F06 on osteoclastogenesis were investigated in vitro through TRAP staining and bone resorption assays. An ovariectomy-induced osteoporosis mouse model was applied to evaluate KCB-F06’s therapeutic effects in vivo. Lastly, the underlying mechanisms were explored using real-time PCR, Western blotting, and co-IP assays. KCB-F06 was discovered as a novel Keap1-Nrf2 PPI inhibitor. As a result, the expression of antioxidants (HO-1 and NQO1) was suppressed, hence reducing ROS accumulation during osteoclastogenesis. Subsequently, this caused the inactivation of RANKL-induced IKB/NF-kB signaling. This eventually led to the downregulation of osteoclast-specific proteins including NFATc1, which is an essential transcription factor for osteoclastogenesis. These results demonstrated that Nrf2 activation in osteoclasts is a valuable tool for osteoclastic bone loss management. In addition, KCB-F06 presents as an alternative candidate for treating osteoclast-related bone diseases and as a novel small molecule that can serve as a model for further Keap1-NRF2 PPI inhibitor development.

To discover a potent candidate for suppressing mature osteoclasts formation in vitro using a TRAP staining assay. A series of PMSA derivatives were synthesized and evaluated for their bioactivity in our current study. Our results showed that PMSA derivative 11 exhibited the most promising bioactivity, with an IC₅₀ value of 322.9 nM, which was ∼15-fold better than PMSA-3-Ac in suppressing osteoclastogenesis in vitro. Additionally, 11 blocked the formation of F-action belts and bone resorption in a concentration-dependent manner. Mechanistically, 11 decreased the expression of genes required for osteoclastogenesis by blocking NFATc1 translocation from the cytoplasm to nucleus. Furthermore, 11 demonstrated a therapeutic inhibitory effect on the differentiation of human iPSC-derived primary osteoclasts. In vivo investigation showed that 11 prevented excessive osteoclastogenesis-mediated bone loss in ovariectomized osteoporosis mimic mice. These findings highlighted the therapeutic potential of 11 as a lead compound for anti-osteoporosis by targeting NFATc1 translocation.