Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the major MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating these cells are an indispensible stem cell population. Twist1+/− mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.

Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2+ pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1+ cells but not NG2+ cells exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from a NVB.

Tissue clearing technique enables visualization of opaque organs and tissues in 3-dimensions (3-D) by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissues that can be cleared with each individual protocol, which inevitably led to the presence of blind-spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, loss of endogenous fluorescence remains a major concern for solvent-based clearing methods. Here, we developed a polyethylene glycol (PEG)-associated solvent system (PEGASOS), which rendered nearly all types of tissues transparent and preserved endogenous fluorescence. Bones and teeth could be turned nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles, and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur, or knee joint in 3-D. In addition, we managed to image intact mouse brain at sub-cellular resolution and to trace individual neurons and axons over a long distance. We also visualized dorsal root ganglions directly through vertebrae. Finally, we revealed the distribution pattern of neural network in 3-D within the marrow space of long bone. These results suggest that the PEGASOS method is a useful tool for general biomedical research.

Leptin Receptor + (LepR + ) stromal cells in adult bone marrow are a critical source of growth factors, including Stem Cell Factor (SCF), for the maintenance of hematopoietic stem cells (HSCs) and early restricted progenitors 1 – 6 . LepR + cells are heterogeneous, including skeletal stem cells, osteogenic, and adipogenic progenitors 7 – 12 , though few markers have been available to distinguish these subsets or to compare their functions. Here we show expression of an osteogenic growth factor, Osteolectin 13 , 14 , distinguishes peri-arteriolar LepR + cells poised to undergo osteogenesis from peri-sinusoidal LepR + cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LepR + Osteolectin + cells are rapidly dividing, short-lived, osteogenic progenitors that increase in number after fracture and are depleted during aging. Deletion of Scf from adult Osteolectin + cells did not affect the maintenance of HSCs or most restricted progenitors but depleted common lymphoid progenitors (CLPs), impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar Osteolectin + cell maintenance required mechanical stimulation. Voluntary running increased, while hindlimb unloading decreased, the frequencies of peri-arteriolar Osteolectin + cells and CLPs. Deletion of the mechanosensitive ion channel, Piezo1 , from Osteolectin + cells depleted Osteolectin + cells and CLPs. A peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during aging.