Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-β1

Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor-β1 (TGF-β1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-β1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-β1-induced protein ig-h3 (BGH3) protein levels by TGF-β1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-β1, but calponin 3 was not significantly regulated by mechanical strain or TGF-β1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-β1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-β1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms

Notch and Senescence.

Cellular senescence, previously thought of as an autonomous tumour suppressor mechanism, is emerging as a phenotype and effector present throughout the life of an organism from embryogenesis to senile decline. Senescent cells have powerful non-autonomous effects upon multiple players within their microenvironment mainly through their secretory phenotype. How senescent cells co-ordinate numerous, sometimes functionally contrasting outputs through their secretome had previously been unclear. The Notch pathway, originally identified for its involvement in Drosophila wing development, has more recently been found to underpin diverse effects in human cancer. Here we discuss recent findings that suggest that Notch is intimately involved in the development of senescence and how it acts to co-ordinate the composition and functional effects of the senescence secretome. We also highlight the complex physical and functional interplay between Notch and p53, critical to both senescence and cancer. Understanding the interplay between Notch, p53 and senescence could allow us develop the therapeutics of the future for cancer and ageing

NOTCH1 mediates a switch between two distinct secretomes during senescence

Senescence, a persistent form of cell-cycle arrest, is often associated with a diverse secretome, which provides complex functionality for senescent cells within the tissue microenvironment. We show that oncogene-induced senescence is accompanied by a dynamic fluctuation of NOTCH1 activity, which drives a TGF-β-rich secretome, while suppressing the senescence-associated pro-inflammatory secretome through inhibition of C/EBPβ. NOTCH1 and NOTCH1-driven TGF-β contribute to 'lateral induction of senescence' through a juxtacrine NOTCH-JAG1 pathway. In addition, NOTCH1 inhibition during senescence facilitates upregulation of pro-inflammatory cytokines, promoting lymphocyte recruitment and senescence surveillance in vivo. As enforced activation of NOTCH1 signalling confers a near mutually exclusive secretory profile compared with typical senescence, our data collectively indicate that the dynamic alteration of NOTCH1 activity during senescence dictates a functional balance between these two distinct secretomes: one representing TGF-β and the other pro-inflammatory cytokines, highlighting that NOTCH1 is a temporospatial controller of secretome composition.This work was supported by the University of Cambridge, Cancer Research UK and Hutchison Whampoa. The M.N. laboratory is supported by Cancer Research UK Cambridge Institute Core Grant (C14303/A17197). M.H. was supported by CRUK Translational Medicine Research Fellowship and CRUK Clinician Scientist Fellowship (C52489/A19924). This work was also supported by a Wellcome Trust PRF (WT101835) to P.J.L., a Wellcome Trust Senior Fellowship to M.P.W. (108070/Z/15/Z), a Wellcome Trust Training Fellowship to N.J.M. (093964/Z/10/Z), and a Wellcome Trust Intermediate Fellowship (097162/Z/11/Z) to S.S. L.Z. was funded by the German Research Foundation (DFG; grants FOR2314 and SFB685), the Gottfried Wilhelm Leibniz Program, the European Research Council (projects ‘CholangioConcept’), the German Ministry for Education and Research (BMBF) (eMed-Multiscale HCC), the German Universities Excellence Initiative (third funding line: ‘future concept’), the German Center for Translational Cancer Research (DKTK) and the German–Israeli Cooperation in Cancer Research (DKFZ–MOST).This is the author accepted manuscript. The final version is available from Nature Publishing Group at http://dx.doi.org/10.1038/ncb3397