Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα s , Gα i/o , Gα q/11 , and Gα 12/13 ) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα s mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gα s led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood.DesignThe impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment.ResultsWe show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants.ConclusionCombining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer.

BACKGROUND CONTEXT Spine pain and the disability associated with it are epidemic in the United States. According to the National Center for Health Statistics, more than 650,000 spinal fusion surgeries are performed annually in the United States, and yet there is a failure rate of 15%–40% when standard methods employing current commercial bone substitutes are used. Autologous bone graft is the gold standard in terms of fusion success, but the morbidity associated with the procedure and the limitations in the availability of sufficient material have limited its use in the majority of cases. A freely available and immunologically compatible bone mimetic with the properties of live tissue is likely to substantially improve the outcome of spine fusion procedures without the disadvantages of autologous bone graft. PURPOSE This study aimed to compare a live human bone tissue analog with autologous bone grafting in an immunocompromised rat model of posterolateral fusion. DESIGN/SETTING This is an in vitro and in vivo preclinical study of a novel human stem cell–derived construct for efficacy in posterolateral lumbar spine fusion. METHODS Osteogenically enhanced human mesenchymal stem cells (OEhMSCs) were generated by exposure to conditions that activate the early stages of osteogenesis. Immunologic characteristics of OEhMSCs were evaluated in vitro. The secreted extracellular matrix from OEhMSCs was deposited on a clinical-grade gelatin sponge, resulting in bioconditioned gelatin sponge (BGS). Bioconditioned gelatin sponge was used alone, with live OEhMSCs (BGS+OEhMSCs), or with whole human bone marrow (BGS+hBM). Efficacy for spine fusion was determined by an institutionally approved animal model using 53 nude rats. RESULTS Bioconditioned gelatin sponge with live OEhMSCs did not cause cytotoxicity when incubated with immunologically mismatched lymphocytes, and OEhMSCs inhibited lymphocyte expansion in mixed lymphocyte assays. Bioconditioned gelatin sponge with live OEhMSC and BGS+hBM constructs induced profound bone growth at fusion sites in vivo, with a comparable rate of fusion with syngeneic bone graft (negative [0 of 10], BGS alone [0 of 10], bone graft [7 of 10], BGS+OEhMSC [10 of 15], and BGS+hBM [8 of 8]). CONCLUSIONS Collectively, these studies demonstrate that BGS+OEhMSC constructs possess low immunogenicity and drive vertebral fusion with efficiency matching syngeneic bone graft in rodents. We also demonstrate that BGS serves as a promising scaffold for spine fusion when combined with hBM.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal calcium-permeable cation channel that is highly expressed in cartilage and is sensitive to a variety of extracellular stimuli. The expression of this channel has been associated with the process of chondrogenesis in adult stem cells as well as several cell lines. Here, we used a chondrogenic reporter (Col2a1-GFP) in murine induced pluripotent stem cells (iPSCs) to examine the hypothesis that TRPV4 serves as both a marker and a regulator of chondrogenesis. Over 21 days of chondrogenesis, iPSCs showed significant increases in Trpv4 expression along with the standard chondrogenic gene markers Sox9, Acan, and Col2a1, particularly in the green fluorescent protein positive (GFP+) chondroprogenitor subpopulation. Increased gene expression for Trpv4 was also reflected by the presence of TRPV4 protein and functional Ca2+ signaling. Daily activation of TRPV4 using the specific agonist GSK1016790A resulted in significant increases in cartilaginous matrix production. An improved understanding of the role of TRPV4 in chondrogenesis may provide new insights into the development of new therapeutic approaches for diseases of cartilage, such as osteoarthritis, or channelopathies and hereditary disorders that affect cartilage during development. Harnessing the role of TRPV4 in chondrogenesis may also provide a novel approach for accelerating stem cell differentiation in functional tissue engineering of cartilage replacements for joint repair.