In Vitro Development of iPSC-derived Osteoblast and Osteoclast Co-cultures for 3D Modeling of Bone Regeneration

Abstract

Understanding the interaction between osteoblasts and osteoclasts is essential to accurately model human bone regeneration. However, most existing co-culture models rely on primary or animal-derived cells, which limits their reproducibility and human-specific relevance. To address this, this thesis establishes a fully human 3D in vitro model using iPSC-derived osteoblasts and osteoclasts seeded on triphasic calcium phosphate scaffolds. This offers a scalable and physiologically relevant platfrom for bone research in human cell lines. Osteoblasts were generated using a monolayer approach involving mesoderm specification and mesenchymal differentiation, while osteoclast precursors were derived through embryoid body formation and myeloid induction. Two macrophage conditions were compared, with or without IL-3 supplementation, to investigate their effect on osteoclastogenesis. Multinucleated, TRAP-positive osteoclasts formed only in the IL-3 condition, although the low expression of late-stage markers may indicate incomplete maturation. Co-culture with macrophages slightly upregulated early osteogenic genes but suppressed late-stage markers, likely due to inflammatory signaling. Additionally, OPG/RANKL ratio and cytokine profiles revealed that IL-3 modulated both osteoclast development and osteoblast behavior. Despite limitations, such as qPCR inconsistencies, GFP-related staining interference, and limited maturation, this model demonstrates the feasibility of using iPSCs to replicate key aspects of human bone remodeling in vitro. It highlights IL-3 as a key modulator of osteoclastogenesis and demonstrates the potential of a reproducible and human-specific model for studying bone cell crosstalk. Future work should extend culture duration, incorporate functional assays, and further explore the molecular mechanisms underlying IL-3-mediated osteoclastogenesis.