Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Background: Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods: Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results: cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions: Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS

Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus

Mesenchymal progenitor cells play a vital role in bone regenerative medicine and tissue engineering strategies. To be clinically useful osteoprogenitors should be readily available with the potential to form bone matrix. While mesenchymal stromal cells from bone marrow have shown promise for tissue engineering, they are obtained in small numbers and there is risk of donor site morbidity. Osteogenic progenitor cells derived from dermal tissue may provide a more abundant and easily expandable source of cells. Bone turnover in vivo is regulated by mechanical forces, particularly oscillatory fluid shear stresses (FSS), and in vitro osteogenic progenitors have been shown to be regulated by mechanical stimuli. The aim of this study was to assess what effect osteogenic media and FSS, generated by a simple rocking platform, had on cell behaviour and matrix production in human progenitor dermal fibroblasts (HDFs) and the embryonic stem cell-derived mesenchymal progenitor cell line (hES-MP).Osteogenic media stimulated alkaline phosphatase activity (ALP) and calcium deposition in HDFs. The addition of FSS further enhanced ALP activity and mineralised matrix deposition in both progenitor cells cultured in osteogenic media. Both types of progenitor cell subjected to FSS showed increases in collagen secretion and apparent collagen organisation as imaged by second harmonic generation

Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment

Although there are many prospective targets in the tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSOC), pre-clinical testing is challenging, especially as there is limited information on the murine TME. Here, we characterize the TME of six orthotopic, transplantable syngeneic murine HGSOC lines established from genetic models and compare these to patient biopsies. We identify significant correlations between the transcriptome, host cell infiltrates, matrisome, vasculature, and tissue modulus of mouse and human TMEs, with several stromal and malignant targets in common. However, each model shows distinct differences and potential vulnerabilities that enabled us to test predictions about response to chemotherapy and an anti-IL-6 antibody. Using machine learning, the transcriptional profiles of the mouse tumors that differed in chemotherapy response are able to classify chemotherapy-sensitive and -refractory patient tumors. These models provide useful pre-clinical tools and may help identify subgroups of HGSOC patients who are most likely to respond to specific therapies