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  1. Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, a locked nucleic acid (LNA)/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincing evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation. 
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  2. Abstract

    Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

     
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