The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow hematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors prior to Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C-X-C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2-creER, as well as Sox9-creER and aggrecan (Acan)-creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals.

Summary Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types including chondrocytes, osteoblasts and marrow stromal cells. The emerging model postulates a distinct type of skeletal stem cells closely associated with the growth plate 1 - 4 , a special cartilaginous tissue playing critical roles in bone elongation 5 . The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP) that interacts with Indian hedgehog (Ihh) released from the hypertrophic zone 6 - 10 , while providing a source of other chondrocytes 11 . However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show that skeletal stem cells are formed among PTHrP + chondrocytes within the resting zone of the postnatal growth plate. PTHrP + chondrocytes expressed a panel of markers for skeletal stem/progenitor cells and uniquely possessed the properties as skeletal stem cells in cultured conditions. Cell lineage analysis revealed that PTHrP + resting chondrocytes continued to form columnar chondrocytes long term, which underwent hypertrophy and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone, which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh), provided instructive cues to maintain cell fates of PTHrP + resting chondrocytes. Our findings unravel a unique somatic stem cell type that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niche are specified postnatally and maintained throughout tissue growth by a tight feedback regulation system.

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Celllineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER + perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. β-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration.Therefore, quiescent Cxcl12-creER + BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.