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.

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.

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.

Hematopoietic and stromal DMP1-Cre labeled cells form a unique niche in the bone marrow.

Skeletogenesis and hematopoiesis are interdependent. Niches form between cells of both lineages where microenvironmental cues support specific lineage commitment. Because of the complex topography of bone marrow (BM), the identity and function of cells within specialized niches has not been fully elucidated. Dentin Matrix Protein 1 (DMP1)-Cre mice have been utilized in bone studies as mature osteoblasts and osteocytes express DMP1. DMP1 has been identified in CXCL12+ cells and an undefined CD45+ population. We crossed DMP1-Cre with Ai9 reporter mice and analyzed the tdTomato+ (tdT+) population in BM and secondary hematopoietic organs. CD45+tdT+ express myeloid markers including CD11b and are established early in ontogeny. CD45+tdT+ cells phagocytose, respond to LPS and are radioresistant. Depletion of macrophages caused a significant decrease in tdT+CD11b+ myeloid populations. A subset of CD45+tdT+ cells may be erythroid island macrophages (EIM) which are depleted after G-CSF treatment. tdT+CXCL12+ cells are in direct contact with F4/80 macrophages, express RANKL and form a niche with B220+ B cells. A population of resident cells within the thymus are tdT+ and express myeloid markers and RANKL. In conclusion, in addition to targeting osteoblast/osteocytes, DMP1-Cre labels unique cell populations of macrophage and stromal cells within BM and thymus niches and expresses key microenvironmental factors.

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.

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.

Nanofiber matrix formulations for the delivery of Exendin-4 for tendon regeneration: In vitro and in vivo assessment.

Tendon and ligament injuries are the most common musculoskeletal injuries, which not only impact the quality of life but result in a massive economic burden. Surgical interventions for tendon/ligament injuries utilize biological and/or engineered grafts to reconstruct damaged tissue, but these have limitations. Engineered matrices confer superior physicochemical properties over biological grafts but lack desirable bioactivity to promote tissue healing. While incorporating drugs can enhance bioactivity, large matrix surface areas and hydrophobicity can lead to uncontrolled burst release and/or incomplete release due to binding. To overcome these limitations, we evaluated the delivery of a peptide growth factor (exendin-4; Ex-4) using an enhanced nanofiber matrix in a tendon injury model. To overcome drug surface binding due to matrix hydrophobicity of poly(caprolactone) (PCL)-which would be expected to enhance cell-material interactions-we blended PCL and cellulose acetate (CA) and electrospun nanofiber matrices with fiber diameters ranging from 600 to 1000 nm. To avoid burst release and protect the drug, we encapsulated Ex-4 in the open lumen of halloysite nanotubes (HNTs), sealed the HNT tube endings with a polymer blend, and mixed Ex-4-loaded HNTs into the polymer mixture before electrospinning. This reduced burst release from ∼75% to ∼40%, but did not alter matrix morphology, fiber diameter, or tensile properties. We evaluated the bioactivity of the Ex-4 nanofiber formulation by culturing human mesenchymal stem cells (hMSCs) on matrix surfaces for 21 days and measuring tenogenic differentiation, compared with nanofiber matrices in basal media alone. Strikingly, we observed that Ex-4 nanofiber matrices accelerated the hMSC proliferation rate and elevated levels of sulfated glycosaminoglycan, tendon-related genes (Scx, Mkx, and Tnmd), and ECM-related genes (Col-I, Col-III, and Dcn), compared to control. We then assessed the safety and efficacy of Ex-4 nanofiber matrices in a full-thickness rat Achilles tendon defect with histology, marker expression, functional walking track analysis, and mechanical testing. Our analysis confirmed that Ex-4 nanofiber matrices enhanced tendon healing and reduced fibrocartilage formation versus nanofiber matrices alone. These findings implicate Ex-4 as a potentially valuable tool for tendon tissue engineering.

PDGF inhibits BMP2-induced bone healing.

Bone regeneration depends on a pool of bone/cartilage stem/progenitor cells and signaling mechanisms regulating their differentiation. Using in vitro approach, we have shown that PDGF signaling through PDGFRß inhibits BMP2-induced osteogenesis, and significantly attenuates expression of BMP2 target genes. We evaluated outcomes of treatment with two anabolic agents, PDGF and BMP2 using different bone healing models. Targeted deletion of PDGFRß in αSMA osteoprogenitors, led to increased callus bone mass, resulting in improved biomechanical properties of fractures. In critical size bone defects BMP2 treatment increased proportion of osteoprogenitors, while the combined treatment of PDGF BB with BMP2 decreased progenitor number at the injury site. BMP2 treatment induced significant bone formation and increased number of osteoblasts, while in contrast combined treatment with PDGF BB decreased osteoblast numbers. This is in vivo study showing that PDGF inhibits BMP2-induced osteogenesis, but inhibiting PDGF signaling early in healing process does not improve BMP2-induced bone healing.

Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing.

Despite the remarkable regenerative capacity of skeletal tissues, nonunion of bone and failure of fractures to heal properly presents a significant clinical concern. Stem and progenitor cells are present in bone and become activated following injury; thus, elucidating mechanisms that promote adult stem cell-mediated healing is important. Wnt-associated adult stem marker Lgr6 is implicated in the regeneration of tissues with well-defined stem cell niches in stem cell-reliant organs. Here, we demonstrate that Lgr6 is dynamically expressed in osteoprogenitors in response to fracture injury. We used an Lgr6-null mouse model and found that Lgr6 expression is necessary for maintaining bone volume and efficient postnatal bone regeneration in adult mice. Skeletal progenitors isolated from Lgr6-null mice have reduced colony-forming potential and reduced osteogenic differentiation capacity due to attenuated cWnt signaling. Lgr6-null mice consist of a lower proportion of self-renewing stem cells. In response to fracture injury, Lgr6-null mice have a deficiency in the proliferation of periosteal progenitors and reduced ALP activity. Further, analysis of the bone regeneration phase and remodeling phase of fracture healing in Lgr6-null mice showed impaired endochondral ossification and decreased mineralization. We propose that in contrast to not being required for successful skeletal development, Lgr6-positive cells have a direct role in endochondral bone repair.

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.

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.

Vitamin C epigenetically controls osteogenesis and bone mineralization.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

Natural Polymer-Based Micronanostructured Scaffolds for Bone Tissue Engineering.

Although bone tissue allografts and autografts aremoften used as a regenerative tissue during the bone healing, their availability, donor site morbidity, and immune response to grafted tissue are limiting factors their more common usage. Tissue engineered implants, such as acellular or cellular polymeric structures, can be an alternative solution. A variety of scaffold fabrication techniques including electrospinning, particulate leaching, particle sintering, and more recently 3D printing have been used to create scaffolds with interconnected pores and mechanical properties for tissue regeneration. Simply combining particle sintering and molecular self-assembly to create porous microstructures with imbued nanofibers to produce micronanostructures for tissue regeneration applications. Natural polymers like polysaccharides, proteins and peptides of plant or animal origin have gained significant attention due to their assured biocompatibility in tissue regeneration. However, majority of these polymers are water soluble and structures derived from them are in the form of hydrogels and require additional stabilization via cross-linking. For bone healing applications scaffolds are required to be strong, and support attachment, proliferation and differentiation of osteoprogenitors into osteoblasts. Our ongoing work utilizes plant polysaccharide cellulose derivatives and collagen to create mechanically stable and bioactive micronanostructured scaffold for bone tissue engineering. Scaffold microstructure is essentially solvent sintered cellulose acetate (CA) microspheres in the form of a negative template for trabecular bone with defined pore and mechanical properties. Collagen nanostructures are imbued into the 3D environment of CA scaffolds using collagen molecular self-assembly principles. The resultant CA-collagen micronanostructures provide the benefits of combined polymers and serve as an alternative material platform to many FDA approved polyesters. Our ongoing studies and published work confirm improved osteoprogenitor adhesion, proliferation, migration, differentiation, extracellular matrix (ECM) secretion in promoting bone healing. In this chapter we will provide a detailed protocol on the creation of micronanostructured CA-collagen scaffolds and their characterization for bone tissue engineering using human mesenchymal stem cells.

CD13 is a critical regulator of cell-cell fusion in osteoclastogenesis.

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell-cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Tmem100- and Acta2-Lineage Cells Contribute to Implant Osseointegration in a Mouse Model.

Metal implants are commonly used in orthopedic surgery. The mechanical stability and longevity of implants depend on adequate bone deposition along the implant surface. The cellular and molecular mechanisms underlying peri-implant bone formation (ie, osseointegration) are incompletely understood. Herein, our goal was to determine the specific bone marrow stromal cell populations that contribute to bone formation around metal implants. To do this, we utilized a mouse tibial implant model that is clinically representative of human joint replacement procedures. Using a lineage-tracing approach, we found that both Acta2.creERT2 and Tmem100.creERT2 lineage cells are involved in peri-implant bone formation, and Pdgfra- and Ly6a/Sca1-expressing stromal cells (PαS cells) are highly enriched in both lineages. Single-cell RNA-seq analysis indicated that PαS cells are quiescent in uninjured bone tissue; however, they express markers of proliferation and osteogenic differentiation shortly after implantation surgery. Our findings indicate that PαS cells are mobilized to repair bone tissue and participate in implant osseointegration after surgery. Biologic therapies targeting PαS cells might improve osseointegration in patients undergoing orthopedic procedures. © 2021 American Society for Bone and Mineral Research (ASBMR).

Heterogeneity of murine periosteum progenitors involved in fracture healing.

The periosteum is the major source of cells involved in fracture healing. We sought to characterize progenitor cells and their contribution to bone fracture healing. The periosteum is highly enriched with progenitor cells, including Sca1+ cells, fibroblast colony-forming units, and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that alpha smooth muscle actin (αSMA) identifies long-term, slow-cycling, self-renewing osteochondroprogenitors in the adult periosteum that are functionally important for bone formation during fracture healing. In addition, Col2.3CreER-labeled osteoblast cells contribute around 10% of osteoblasts but no chondrocytes in fracture calluses. Most periosteal osteochondroprogenitors following fracture can be targeted by αSMACreER. Previously identified skeletal stem cell populations were common in periosteum but contained high proportions of mature osteoblasts. We have demonstrated that the periosteum is highly enriched with skeletal progenitor cells, and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

Diabetes impairs periosteal progenitor regenerative potential.

Diabetics are at increased risk for fracture, and experience severely impaired skeletal healing characterized by delayed union or nonunion of the bone. The periosteum harbors osteochondral progenitors that can differentiate into chondrocytes and osteoblasts, and this connective tissue layer is required for efficient fracture healing. While bone marrow-derived stromal cells have been studied extensively in the context of diabetic skeletal repair and osteogenesis, the effect of diabetes on the periosteum and its ability to contribute to bone regeneration has not yet been explicitly evaluated. Within this study, we utilized an established murine model of type I diabetes to evaluate periosteal cell differentiation capacity, proliferation, and availability under the effect of a diabetic environment. Periosteal cells from diabetic mice were deficient in osteogenic differentiation ability in vitro, and diabetic mice had reduced periosteal populations of mesenchymal progenitors with a corresponding reduction in proliferation capacity following injury. Additionally, fracture callus mineralization and mature osteoblast activity during periosteum-mediated healing was impaired in diabetic mice compared to controls. We propose that the effect of diabetes on periosteal progenitors and their ability to aid in skeletal repair directly impairs fracture healing.

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.

Origin and Lineage Plasticity of Endogenous Lacrimal Gland Epithelial Stem/Progenitor Cells.

The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future.