Long-Term Expandable Periosteal Organoids Reconstruct a Functional Regenerative Niche That Support Hematopoietic Stem Cell Maintenance and Bone Repair.

The periosteum plays a central role in skeletal regeneration by serving as a reservoir of skeletal stem cells and a dynamic regenerative niche. However, in vitro models that faithfully preserve its cellular complexity, long-term stability, and functional capacity remain limited, restricting mechanistic studies and translational applications. Here, we establish a long-term expandable periosteal organoid system that can be stably propagated for over 20 passages while preserving cellular heterogeneity, multilineage differentiation potential, and genomic integrity. Single-cell transcriptomic analysis reveals sustained maintenance of core periosteal lineages and regenerative signaling programs over extended culture. Functionally, these organoids reconstruct a niche-like microenvironment that supports hematopoietic stem cell (HSC) maintenance, preserves stem cell fitness, and attenuates aging-associated functional decline under prolonged ex vivo conditions. Upon transplantation, periosteal organoids robustly engraft and actively participate in tissue repair, promoting vascularized bone formation and reconstruction of marrow-like structures across multiple defect models. Mechanistically, we identify ZFP575 as a 3D niche-dependent regulator required for maintaining in vivo osteogenic competence. Together, our findings establish periosteal organoids as a physiologically relevant and functionally competent regenerative platform, providing a versatile system for modeling skeletal repair and stem cell-niche interactions.