Abstract
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.
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Abbreviations
- BMD:
-
Bone mineral density
- BMP-4:
-
Bone morphogenetic protein 4
- cDNA:
-
Complementary DNA
- DAPI:
-
4′,6-diamidino-2-phenylindole
- EB:
-
Embryoid body
- ECAR:
-
Extracellular acidification rate
- ELISA:
-
Enzyme-linked immunosorbent assay
- FCCP:
-
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
- GM-CSF:
-
Granulocyte–macrophage colony-stimulating factor
- hiPSC:
-
Human induced pluripotent stem cell
- hiPSC-OC:
-
HiPSC-derived osteoclast
- hPBMC:
-
Human peripheral blood mononuclear cell
- hsRANKL:
-
Human soluble receptor activator of nuclear factor-κB ligand
- M-CSF:
-
Macrophage colony-stimulating factor
- MMP9:
-
Matrix metalloproteinase 9
- OCR:
-
Oxygen consumption rate
- OXPHOS:
-
Oxidative phosphorylation
- P/S:
-
Penicillin/streptomycin
- PFA:
-
Paraformaldehyde
- qRT-PCR:
-
Quantitative reverse transcription polymerase chain reaction
- SCF:
-
Stem cell factor
- TRAP:
-
Tartrate-resistant acid phosphatase
- VEGF:
-
Vascular endothelial growth factor
Funding
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF)& funded by the Korean government (MSIT) (No. RS-2023-00261905, 2022M3A9E4017151). This work was also supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT)(RS-2025-00516170).
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The authors declare no competing interests.
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The human induced pluripotent stem cell (hiPSC) lines used in this study were obtained from the National Stem Cell Bank of Korea. This original source confirmed that patient informed consent and Institutional Review Board (IRB) approval were obtained for the original collection of human cells and derivation of these hiPSC lines (IRB No. 2013-06EXP-06-R and 2014-10CON-04–1 C-A). The derivation and characterization of these hiPSC lines have been reported previously28–30. The authors used these hiPSC lines without access to any donor-identifying information, in accordance with the Bank’s distribution policies and applicable regulations.
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Jeong, J., Choi, Y., Sa, Y. et al. M-CSF priming enhances the efficiency and reproducibility of hiPSC-derived osteoclast differentiation and pharmacological responsiveness to anti-resorptive agents. Sci Rep (2026). https://doi.org/10.1038/s41598-026-52457-z
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DOI: https://doi.org/10.1038/s41598-026-52457-z
