Lineage Tracing of RGS5-CreER-Labeled Cells in Long Bones During Homeostasis and Injury.

Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.

Osteoclasts Derive Predominantly from Bone Marrow-Resident CX3CR1+ Precursor Cells in Homeostasis, whereas Circulating CX3CR1+ Cells Contribute to Osteoclast Development during Fracture Repair.

Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX3CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX3CR1-EGFP/TRAP tdTomato mice, we found CX3CR1 expression in mononuclear cells, but not in multinucleated TRAP+ OC. However, CX3CR1-expressing cells generated TRAP+ OC on bone within 5 d in CX3CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato+ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX3CR1+ cells migrated to bone, lost expression of CX3CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX3CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX3CR1+ cells can become OC.

Engraftment of skeletal progenitor cells by bone-directed transplantation improves osteogenesis imperfecta murine bone phenotype.

Osteogenesis imperfecta (OI) is a genetic disorder most commonly caused by mutations associated with type I collagen, resulting in a defective collagen bone matrix. Current treatments for OI focus on pharmaceutical strategies to increase the amount of defective bone matrix, but do not address the underlying collagen defect. Introducing healthy donor stem cells that differentiate into osteoblasts producing normal collagen in OI patients has the potential to increase bone mass and correct the mutant collagen matrix. In this study, donor bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) expressing both αSMACreERT2/Ai9 progenitor reporter and osteoblast reporter Col2.3GFP were locally transplanted into the femur of OI murine (OIM) mice. One month post-transplantation, 18% of the endosteal surface was lined by donor Col2.3GFP expressing osteoblasts indicating robust engraftment. Long-term engraftment in the marrow was observed 3 and 6 months post-transplantation. The presence of Col1a2-expressing donor cell-derived cortical bone matrix was detected in transplanted OIM femurs. Local transplantation of BMSCs increased cortical thickness (+12%), the polar moment of inertia (+14%), bone strength (+30%), and stiffness (+30%) 3 months post-transplantation. Engrafted cells expressed progenitor markers CD51 and Sca-1 up to 3 months post-transplantation. Most importantly, 3 months post-transplantation donor cells maintained the ability to differentiate into Col2.3GFP+ osteoblasts in vitro, and in vivo following secondary transplantation into OIM animals. Locally transplanted BMSCs can improve cortical structure and strength, and persist as continued source of osteoblast progenitors in the OIM mouse for at least 6 months.

Perivascular osteoprogenitors are associated with transcortical channels of long bones.

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the ability to replenish mature osteoblasts and repair the skeleton. Using lineage tracing approaches, we identified a population of Dmp1+ cells that reside within cortical bone and are distinct from osteocytes. Our aims were to characterize this stromal population of transcortical perivascular cells (TPCs) in their resident niche and evaluate their osteogenic potential. To distinguish this population from osteoblasts/osteocytes, we crossed mice containing inducible DMP1CreERT2/Ai9 Tomato reporter (iDMP/T) with Col2.3GFP reporter (ColGFP), a marker of osteoblasts and osteocytes. We observed iDMP/T+;ColGFP- TPCs within cortical bone following tamoxifen injection. These cells were perivascular and located within transcortical channels. Ex vivo bone outgrowth cultures showed TPCs migrated out of the channels onto the plate and expressed stem cell markers such as Sca1, platelet derived growth factor receptor beta (PDGFRß), and leptin receptor. In a cortical bone transplantation model, TPCs migrate from their vascular niche within cortical bone and contribute to new osteoblast formation and bone tube closure. Treatment with intermittent parathyroid hormone increased TPC number and differentiation. TPCs were unable to differentiate into adipocytes in the presence of rosiglitazone in vitro or in vivo. Altogether, we have identified and characterized a novel stromal lineage-restricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand, and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation.

Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing.

Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.

Divergent effects of peripheral and global neuropeptide Y deletion.

OBJECTIVES: Neuropeptide Y (NPY) is involved in the coordination of bone mass and adiposity. However, multiple NPY sources exist and their individual contribution to the skeleton and adiposity not known. The objectives of our study were to evaluate the effects of peripheral mesenchymal derived NPY to the skeleton and adiposity and to compare them to the global NPYKO model. METHODS: To study the role of mesenchymal-derived NPY, we crossed conditional NPY (NPYfl/fl) mice with Prx1cre to generate PrxNPYKO mice. The bone phenotype was assessed using micro-CT. The skeletal phenotype of PrxNPYKO mice was subsequently compared to global NPYKO model. We evaluated body weight, adiposity and functionally assessed the feeding response of NPY neurons to determine whether central NPY signaling was altered by Prx1cre. RESULTS: We identified the increase in cortical parameters in PrxNPYKO mice with no changes to cancellous bone. This was the opposite phenotype to global NPYKO mice generated from the same conditional allele. Male NPYKOmice have increased adiposity, while PrxNPYKO mice showed no difference, demonstrating that local mesenchymal-derived NPY does not influence adiposity. CONCLUSION: NPY mediates both positive and negative effects on bone mass via separate regulatory pathways. Deletion of mesenchymal-derived NPY had a positive effect on bone mass.

Quiescent Bone Lining Cells Are a Major Source of Osteoblasts During Adulthood.

The in vivo origin of bone-producing osteoblasts is not fully defined. Skeletal stem cells, a population of mesenchymal stem cells resident in the bone marrow compartment, are thought to act as osteoprogenitors during growth and adulthood. Quiescent bone lining cells (BLCs) have been suggested as a population capable of activation into mature osteoblasts. These cells were defined by location and their morphology and studies addressing their significance have been hampered by their inaccessibility, and lack of markers that would allow for their identification and tracing. Using lineage tracing models, we have observed labeled osteoblasts at time points extending beyond the reported lifespan for this cell type, suggesting continuous reactivation of BLCs. BLCs also make a major contribution to bone formation after osteoblast ablation, which includes the ability to proliferate. In contrast, mesenchymal progenitors labeled by Gremlin1 or alpha smooth muscle actin do not contribute to bone formation in this setting. BLC activation is inhibited by glucocorticoids, which represent a well-established cause of osteoporosis. BLCs express cell surface markers characteristic of mesenchymal stem/progenitors that are largely absent in osteoblasts including Sca1 and Leptin Receptor. BLCs also show different gene expression profiles to osteoblasts, including elevated expression of Mmp13, and osteoclast regulators RANKL and macrophage colony stimulating factor, and retain osteogenic potential upon transplantation. Our findings provide evidence that bone lining cells represent a major source of osteoblasts during adulthood. Stem Cells 2016;34:2930-2942.

Inhibition of leukemia cell engraftment and disease progression in mice by osteoblasts.

The bone marrow niche is thought to act as a permissive microenvironment required for emergence or progression of hematologic cancers. We hypothesized that osteoblasts, components of the niche involved in hematopoietic stem cell (HSC) function, influence the fate of leukemic blasts. We show that osteoblast numbers decrease by 55% in myelodysplasia and acute myeloid leukemia patients. Further, genetic depletion of osteoblasts in mouse models of acute leukemia increased circulating blasts and tumor engraftment in the marrow and spleen leading to higher tumor burden and shorter survival. Myelopoiesis increased and was coupled with a reduction in B lymphopoiesis and compromised erythropoiesis, suggesting that hematopoietic lineage/progression was altered. Treatment of mice with acute myeloid or lymphoblastic leukemia with a pharmacologic inhibitor of the synthesis of duodenal serotonin, a hormone suppressing osteoblast numbers, inhibited loss of osteoblasts. Maintenance of the osteoblast pool restored normal marrow function, reduced tumor burden, and prolonged survival. Leukemia prevention was attributable to maintenance of osteoblast numbers because inhibition of serotonin receptors alone in leukemic blasts did not affect leukemia progression. These results suggest that osteoblasts play a fundamental role in propagating leukemia in the marrow and may be a therapeutic target to induce hostility of the niche to leukemia blasts.

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing.

Purpose of this study: To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. MATERIALS AND METHODS: Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks postoperatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. RESULTS: Three notable changes were apparent postoperatively. (1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. (2) The tendon proximal to the defect initially became hypercellular and disorganized. (3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. CONCLUSIONS: The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

Roles of parathyroid hormone (PTH) receptor and reactive oxygen species in hyperlipidemia-induced PTH resistance in preosteoblasts.

Bioactive lipids initiate inflammatory reactions leading to pathogenesis of atherosclerosis. Evidence shows that they also contribute to bone loss by inhibiting parathyroid hormone receptor (PTH1R) expression and differentiation of osteoblasts. We previously demonstrated that bone anabolic effects of PTH(1-34) are blunted in hyperlipidemic mice and that these PTH effects are restored by antioxidants. However, it is not clear which osteoblastic cell developmental stage is targeted by bioactive lipids. To investigate the effects of hyperlipidemia at the cellular level, hyperlipidemic Ldlr(-/-) mice were bred with Col3.6GFPtpz mice, in which preosteoblasts/osteoblasts carry a topaz fluorescent label, and with Col2.3GFPcyan mice, in which more mature osteoblasts/osteocytes carry a cyan fluorescent label. Histological analyses of trabecular bone surfaces in femoral as well as calvarial bones showed that intermittent PTH(1-34) increased fluorescence intensity in WT-Tpz mice, but not in Tpz-Ldlr(-/-) mice. In contrast, PTH(1-34) did not alter fluorescence intensity in femoral cortical envelopes of either WT-Cyan or Ldlr(-/-)-Cyan mice. To test the mechanism of PTH1R downregulation, preosteoblastic MC3T3-E1 cells were treated with bioactive lipids and the antioxidant Trolox. Results showed that inhibitory effects of PTH1R levels by bioactive lipids were rescued by pretreatment with Trolox. The inhibitory effects on expression of PTH1R as well as on PTH-induced osteoblastic genes were mimicked by xanthine/xanthine oxidase, a known generator of reactive oxygen species. These findings suggest an important role of the preosteoblastic development stage as the target and downregulation of PTH receptor expression mediated by intracellular oxidant stress as a mechanism in hyperlipidemia-induced PTH resistance.

Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model.

PURPOSE: Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. METHODS: Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. RESULTS: We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. CONCLUSION: We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

Endothelial to mesenchymal Notch signaling regulates skeletal repair.

We present a transcriptomic analysis that provides a better understanding of regulatory mechanisms within the healthy and injured periosteum. The focus of this work is on characterizing early events controlling bone healing during formation of periosteal callus on day 3 after fracture. Building on our previous findings showing that induced Notch1 signaling in osteoprogenitors leads to better healing, we compared samples in which the Notch 1 intracellular domain is overexpressed by periosteal stem/progenitor cells, with control intact and fractured periosteum. Molecular mechanisms and changes in skeletal stem/progenitor cells (SSPCs) and other cell populations within the callus, including hematopoietic lineages, were determined. Notably, Notch ligands were differentially expressed in endothelial and mesenchymal populations, with Dll4 restricted to endothelial cells, whereas Jag1 was expressed by mesenchymal populations. Targeted deletion of Dll4 in endothelial cells using Cdh5CreER resulted in negative effects on early fracture healing, while deletion in SSPCs using α-smooth muscle actin-CreER did not impact bone healing. Translating these observations into a clinically relevant model of bone healing revealed the beneficial effects of delivering Notch ligands alongside the osteogenic inducer, BMP2. These findings provide insights into the regulatory mechanisms within the healthy and injured periosteum, paving the way for novel translational approaches to bone healing.

Signaling pathways associated with Lgr6 to regulate osteogenesis.

Fracture management largely relies on the bone's inherent healing capabilities and, when necessary, surgical intervention. Currently, there are limited osteoinductive therapies to promote healing, making targeting skeletal stem/progenitor cells (SSPCs) a promising avenue for therapeutic development. A limiting factor for this approach is our incomplete understanding of the molecular mechanisms governing SSPCs' behavior. We have recently identified that the Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) is expressed in sub-populations of SSPCs, and is required for maintaining bone volume during adulthood and for proper fracture healing. Lgr family members (Lgr4-6) are markers of stem cell niches and play a role in tissue regeneration primarily by binding R-Spondin (Rspo1-4). This interaction promotes canonical Wnt (cWnt) signaling by stabilizing Frizzled receptors. Interestingly, our findings here indicate that Lgr6 may also influence cWnt-independent pathways. Remarkably, Lgr6 expression was enhanced during Bmp-mediated osteogenesis of both human and murine cells. Using biochemical approaches, RNA sequencing, and bioinformatic analysis of published single-cell data, we found that elements of BMP signaling, including its target gene, pSMAD, and gene ontology pathways, are downregulated in the absence of Lgr6. Our findings uncover a molecular interdependency between the Bmp pathway and Lgr6, offering new insights into osteogenesis and potential targets for enhancing fracture healing.

Hair follicle-resident progenitor cells are a major cellular contributor to heterotopic subcutaneous ossifications in a mouse model of Albright hereditary osteodystrophy.

Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). AHO is caused by heterozygous inactivation of GNAS, the gene that encodes the α-stimulatory subunit (Gαs) of G proteins. Previously, we had shown using our laboratory's AHO mouse model that SCOs develop around hair follicles (HFs). Here we show that SCO formation occurs due to inappropriate expansion and differentiation of HF-resident stem cells into osteoblasts. We also show in AHO patients and mice that Secreted Frizzled Related Protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice exacerbates SCO development. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.

In vivo fate mapping identifies mesenchymal progenitor cells.

Adult mesenchymal progenitor cells have enormous potential for use in regenerative medicine. However, the true identity of the progenitors in vivo and their progeny has not been precisely defined. We hypothesize that cells expressing a smooth muscle α-actin promoter (αSMA)-directed Cre transgene represent mesenchymal progenitors of adult bone tissue. By combining complementary colors in combination with transgenes activating at mature stages of the lineage, we characterized the phenotype and confirmed the ability of isolated αSMA(+) cells to progress from a progenitor to fully mature state. In vivo lineage tracing experiments using a new bone formation model confirmed the osteogenic phenotype of αSMA(+) cells. In vitro analysis of the in vivo-labeled SMA9(+) cells supported their differentiation potential into mesenchymal lineages. Using a fracture-healing model, αSMA9(+) cells served as a pool of fibrocartilage and skeletal progenitors. Confirmation of the transition of αSMA9(+) progenitor cells to mature osteoblasts during fracture healing was assessed by activation of bone-specific Col2.3emd transgene. Our findings provide a novel in vivo identification of defined population of mesenchymal progenitor cells with active role in bone remodeling and regeneration.

Utilization of transgenic models in the evaluation of osteogenic differentiation of embryonic stem cells.

Previous studies reported that embryonic stem cells (ESCs) can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteo-inductive conditions. It is probable that osteogenic differentiation requires that ESCs undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. Based on our previous studies indicating that adult mesenchymal progenitor cells express α-smooth muscle actin (αSMA), we have generated ESCs from transgenic mice in which an αSMA promoter directs the expression of red fluorescent protein (RFP) to mesenchymal progenitor cells. To track the transition of ESC-derived MSCs into mature osteoblasts, we have utilized a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP). Following osteogenic induction in ESCs, we have observed expression of alkaline phosphatase (ALP) and subsequent mineralization as detected by von Kossa staining. After 1 week of osteogenic induction, ESCs begin to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP positive cells did not show activation of the Col2.3GFP promoter, even after 7 weeks of osteogenic differentiation in vitro. In contrast, Col2.3GFP expression was detected in vivo, in mineralized areas following teratoma formation. Our results indicate that detection of ALP activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. Our study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of ESCs into the osteoblast lineage.

AcanCreER lacks specificity to chondrocytes and targets periosteal progenitors in the fractured callus.

Aggrecan (Acan) is a large proteoglycan molecule constituting the extracellular matrix of cartilage, secreted by chondrocytes. To specifically target the chondrocyte lineage, researchers have widely used the AcanCreER mouse model. Evaluation of specificity and efficiency of recombination, requires Cre animals to be crossed with reporter mice. In order to accurately interpret data from Cre models, it is imperative to consider A) the amount of recombination occurring in cells/tissues that are not intended for targeting (i.e., non-specific expression), B) the efficiency of Cre recombination, which can depend on dose and duration of tamoxifen treatment, and C) the activation of CreER without tamoxifen induction, known as "Cre leakage." Using a highly sensitive reporter mouse (Ai9, tdTomato), we performed a comprehensive analysis of the AcanCreER system. Surprisingly, we observed expression in cells within the periosteum. These cells expand at a stage when chondrocytes are not yet present within the forming callus tissue (Acan/Ai9+ cells). In pulse-chase experiments, we confirmed that fibroblastic Acan/Ai9+ cells within the periosteum can directly give rise to osteoblasts. Our results show that Acan/Ai9+ is not specific for the chondrocyte lineage in the fracture callus or with the tibial holes. The expression of AcanCreER in periosteal progenitor cells complicates the interpretation of studies evaluating the transition of chondrocytes to osteoblasts (termed transdifferentiation). Awareness of these issues and the limitations of the system will lead to better data interpretation.

CD51 labels periosteal injury-responsive osteoprogenitors.

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1-CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1-CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51- cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1-CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation.

Inhibition of CGRP signaling impairs fracture healing in mice.

Calcitonin gene-related peptide (CGRP) is a neuropeptide produced by sensory nerves and functions as a pain sensor. It acts by binding to the calcitonin-like receptor (CLR, protein; Calcrl, gene). CGRP inhibition has been recently introduced as therapeutic treatment of migraine-associated pain. Previous studies have shown that CGRP stimulates bone formation. The aim of our study is to determine whether the inhibition of CGRP signaling negatively impacted fracture healing. Using α-smooth muscle actin (αSMA) Cre animals crossed with Ai9 reporter mice, we showed that CGRP-expressing nerves are near αSMA + cells in the periosteum. In vitro experiments revealed that periosteal cells express Calcrl and receptor activity modifying protein 1; and CGRP stimulation increased periosteal cell proliferation. Using a tamoxifen-inducible model αSMACre/CLRfl/fl , we targeted the deletion of CLR to periosteal progenitor cells and examined fracture healing. Microcomputed tomography of fractured femurs showed a reduction in bone mass in αSMACre+/CLRfl/fl female mice relative to controls and callus volume in males. Pharmacological CGRP-CLR inhibition was achieved by subcutaneous delivery of customized pellets with small molecule inhibitor olcegepant (BIBN-4096) at a dose of 10 µg/day. BIBN-4096-treated C57BL/6J mice had a higher latency toward thermal nociception than placebo-treated mice, indicating impaired sensory function through CGRP inhibition. CGRP inhibition also resulted in reduced callus volume, bone mass, and bone strength compared to placebo controls. These results indicate that inhibiting CGRP by deleting CLR or by using BIBN-4096, contributes to delayed bone healing.