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Bone is regarded as one of few tissues that heals without fibrous scar. The outer layer of the periosteum is covered with fibrous tissue, whose function in bone formation is unknown. We herein developed a system to distinguish the fate of fibrous-layer periosteal cells (FL-PCs) from the skeletal stem/progenitor cells (SSPCs) in the cambium-layer periosteum and bone marrow in mice. We showed that FL-PCs did not participate in steady-state osteogenesis, but formed the main body of fibrocartilaginous callus during fracture healing. Moreover, FL-PCs invaded the cambium-layer periosteum and bone marrow after fracture, forming neo-SSPCs that continued to maintain the healed bones throughout adulthood. The FL-PC-derived neo-SSPCs expressed lower levels of osteogenic signature genes and displayed lower osteogenic differentiation activity than the preexisting SSPCs. Consistent with this, healed bones were thinner and formed more slowly than normal bones. Thus, the fibrous periosteum becomes the cellular origin of bones after fracture and alters bone properties permanently.

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The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison with the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis and hematopoiesis. Our data also suggest dynamic inter- and intra-compartment interactions, as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

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Vertebrate lonesome kinase (Vlk) is a secreted tyrosine kinase important for normal skeletogenesis during embryonic development. Vlk null mice (Vlk-/- ) are born with severe craniofacial and limb skeletal defects and die shortly after birth. We used a conditional deletion model to remove Vlk in limb bud mesenchyme (Vlk-Prx1 cKO) to assess the specific requirement for Vlk expression by skeletal progenitor cells during endochondral ossification, and an inducible global deletion model (Vlk-Ubq iKO) to address the role of Vlk during fracture repair. Deletion of Vlk with Prx1-Cre recapitulated the limb skeletal phenotype of the Vlk-/- mice and enabled us to study the postnatal skeleton as Vlk-Prx1 cKO mice survived to adulthood. In Vlk-Prx1 cKO adult mice, limbs remained shorter with decreased trabecular and cortical bone volumes. Both Vlk-Prx1 cKO and Vlk-Ubq iKO mice had a delayed fracture repair response but eventually formed bridging calluses. Furthermore, levels of phosphorylated osteopontin (OPN) were decreased in tibias of Vlk-Ubq iKO, establishing OPN as a Vlk substrate in bone. In summary, our data indicate that Vlk produced by skeletal progenitor cells influences the timing and extent of chondrogenesis during endochondral bone formation and fracture repair. © 2022 American Society for Bone and Mineral Research (ASBMR).

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Hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR+ adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT+ cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT+ cells gave rise to 20% of LEPR+ stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT+ cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1+ fetal bone marrow stromal cells, but not LEPR+ adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT+ skeletal progenitors as an important source of bones formed during development.

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The periosteum is critical for bone repair and contains skeletal stem cells (SSCs), but these cells are still poorly characterized. In the bone marrow, cells expressing the Nes-GFP transgene have been described to be SSCs. Here, we investigated whether Nes-GFP expression also typifies SSCs in the periosteum. We show that in adult mice, Nes-GFP cells are present in the periosteum and localize closely to blood vessels, but periosteal Nes-GFP cells express SSC and progenitor markers differently compared to Nes-GFP cells in the bone marrow. Periosteal Nes-GFP cells show in vitro clonogenicity and tri-lineage differentiation potential and they can form bone in vivo. Shortly after fracture, they start to proliferate and they contribute to the osteoblast pool during the repair process. However, periosteal Nes-GFP cells are not slow dividing nor self-renewing in vivo. These results indicate that in adult mice, periosteal Nes-GFP expressing cells are skeletal progenitors rather than true SSCs, and they participate in the fracture healing process.

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Defects of the craniofacial skeleton arise as a direct result of trauma, diseases, oncological resection, or congenital anomalies. Current treatment options are limited, highlighting the importance for developing new strategies to restore form, function, and aesthetics of missing or damaged bone in the face and the cranium. For optimal reconstruction, the goal is to replace "like with like." With the inherent challenges of existing options, there is a clear need to develop alternative strategies to reconstruct the craniofacial skeleton. The success of mesenchymal stem cell-based approaches has been hampered by high heterogeneity of transplanted cell populations with inconsistent preclinical and clinical trial outcomes. Here, we discuss the novel characterization and isolation of mouse skeletal stem cell (SSC) populations and their response to injury, systemic disease, and how their re-activation in vivo can contribute to tissue regeneration. These studies led to the characterization of human SSCs which are able to self-renew, give rise to increasingly fate restricted progenitors, and differentiate into bone, cartilage, and bone marrow stroma, all on the clonal level in vivo without prior in vitro culture. SSCs hold great potential for implementation in craniofacial bone tissue engineering and regenerative medicine. As we begin to better understand the diversity and the nature of skeletal stem and progenitor cells, there is a tangible future whereby a subset of human adult SSCs can be readily purified from bone or activated in situ with broad potential applications in craniofacial tissue engineering.

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Most bones in mammals display a limited capacity for natural large-scale repair. The ribs are a notable exception, yet the source of their remarkable regenerative ability remains unknown. Here, we identify a Sox9-expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones. Deletion of the obligate Hedgehog co-receptor, Smoothened, in Sox9-expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration. In contrast to its role in development, Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury. Instead, Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties. This type of callus cell may be critical for bridging large bone injuries. Thus despite contributing to only a subset of callus cells, Sox9-positive progenitors play a major role in orchestrating large-scale bone regeneration. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.

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Four and a half LIM domain 2 (FHL2) is a multifunctional scaffolding protein of well-known function regulating cell signalling cascades and gene transcription in cancer tissues. However, its function in embryonic systems is poorly characterized. Here, we show that Fhl2 is involved in the differentiation of connective tissues of developing limb autopod. We show that Fhl2 exhibits spatially restricted and temporally dynamic expression around the tendons of developing digits, interphalangeal joint capsules, and fibrous peridigital tissue. Immunolabelling analysis of the skeletal progenitors identified a predominant, but not exclusive, cytoplasmic distribution of FHL2 being associated with focal adhesions and actin cytoskeleton. In the course of chondrogenic differentiation of cultures of limb skeletal progenitors, the expression of Fhl2 is down-regulated. Furthermore, cultures of skeletal progenitors overexpressing Fhl2 take on a predominant fibrogenic appearance. Both gain-of-function and loss-of-function experiments in the micromass culture assays revealed a positive transcriptional influence of Fhl2 in the expression of fibrogenic markers including Scleraxis, Tenomodulin, Tenascin C, ßig-h3, and Tgif1. We further show that the expression of Fhl2 is positively regulated by profibrogenic signals including Tgfß2, all-trans-retinoic acid, and canonical Wnt signalling molecules and negatively regulated by prochondrogenic factors of the bone morphogenetic protein family. Expression of Fhl2 is also regulated negatively in immobilized limbs, but this influence appears to be mediated by other connective tissue markers, such as Tgfßs and Scleraxis.

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INTRODUCTION: The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9+ cell type is retained in the adult periosteum. We hypothesized that Sox9+ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair. METHODS: To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9+ cells in Sox9CreErt2:Td-Tomato mice. Intact femora were analyzed with immunostaining and RNA sequencing to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive, Sox9-descendent cells in the adult femur. To assess the role of Td-tomato+cells in the fracture healing process, mice underwent a closed mid-diaphyseal femoral fracture. Fractured hind limbs were analyzed by X-ray, histology and immuno-staining at 3, 9 or 56days post-fracture. RESULTS: In the intact adult mouse femur, Td-Tomato-labeled cells were observed in the primary spongiosa, periosteum and endosteum. RNA sequencing showed that Td-Tomato positive periosteal cells were co-enriched for Sox9 transcripts, and mRNAs for osteoblast and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the early stages of bone repair (day 3 post-op) contributing to the fracture repair process by differentiating into chondrocytes, osteoblasts and osteocytes. CONCLUSION: A Sox9+ skeletal progenitor population resides in the adult periosteum. Fate tracing studies show that descendants of the Sox9+ periosteal progenitors give rise to chondrocytes, osteoblasts and mature cortical osteocytes in repair of the fractured femur. To our knowledge this is the first report of a reparative Sox9+ progenitor population in the periosteum of the adult long bone. Taken together with developmental studies, our data suggest a broad role for Sox9+ osteochondroprogenitors in development and repair of the mammalian skeleton.

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In tetrapods the digit pattern has evolved to adapt to distinct locomotive strategies. The number of digits varies between species or even between hindlimb and forelimb within the same species. These facts illustrate the plasticity of embryonic limb autopods. Sox9 is a precocious marker of skeletal differentiation of limb mesenchymal cells. Its pattern of expression in the developing limb has been widely studied and reflects the activity of signaling cascades responsible for skeletogenesis. In this assay we stress previously overlooked differences in the pattern of expression of Sox9 in limbs of avian, mouse and turtle embryos which may reflect signaling differences associated with distinct limb skeletal morphologies observed in these species. Furthermore, we show that Sox9 gene expression is higher and maintained in the interdigital region in species with webbed digits in comparison with free digit animals.

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A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.

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