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Restoring extracellular matrix synthesis in senescent stem cells.

ABSTRACT: Collagen type III (COL3) is one of the 3 major collagens in the body, and loss of expression or mutations in the COL3 gene have been associated with the onset of vascular diseases such the Ehlers-Danlos syndrome. Previous work reported a significant reduction of COL3 in tissues such as skin and vessels with aging. In agreement, we found that COL3 was significantly reduced in senescent human mesenchymal stem cells and myofibroblasts derived from patients with Hutchinson-Gilford progeria syndrome, a premature aging syndrome. Most notably, we discovered that ectopic expression of the embryonic transcription factor Nanog homeobox (NANOG) restored COL3 expression by restoring the activity of the TGF-β pathway that was impaired in senescent cells. RNA sequencing analysis showed that genes associated with the activation of the TGF-β pathway were up-regulated, whereas negative regulators of the pathway were down-regulated upon NANOG expression. Chromatin immunoprecipitation sequencing and immunoprecipitation experiments revealed that NANOG bound to the mothers against decapentaplegic (SMAD)2 and SMAD3 promoters, in agreement with increased expression and phosphorylation levels of both proteins. Using chemical inhibition, short hairpin RNA knockdown, and gain of function approaches, we established that both SMAD2 and SMAD3 were necessary to mediate the effects of NANOG, but SMAD3 overexpression was also sufficient for COL3 production. In summary, NANOG restored production of COL3, which was impaired by cellular aging, suggesting novel strategies to restore the impaired extracellular matrix production and biomechanical function of aged tissues, with potential implications for regenerative medicine and anti-aging treatments.-Rong, N., Mistriotis, P., Wang, X., Tseropoulos, G., Rajabian, N., Zhang, Y., Wang, J., Liu, S., Andreadis, S. T. Restoring extracellular matrix synthesis in senescent stem cells.

SUBMITTER: Rong N

PROVIDER: S-EPMC6766659 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. STATEMENT OF SIGNIFICANCE: Our study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown. We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap. This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

Project description:BackgroundMesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA).MethodsExosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1?) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks.ResultsWe found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1?. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model.ConclusionsThe exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development.

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Restoring extracellular matrix synthesis in senescent stem cells.

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