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

ABSTRACT

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.