Nuclear mechanotransduction in stem cells.

In development and in homeostatic maintenance of tissues, stem cells and progenitor cells are constantly subjected to forces. These forces can lead to significant changes in gene expression and function of stem cells, mediating self-renewal, lineage specification, and even loss of function. One of the ways that has been proposed to mediate these functional changes in stem cells is nuclear mechanotransduction - the process by which forces are converted to signals in the nucleus. The purpose of this review is to discuss the means by which mechanical signals are transduced into the nucleus, through the linker of nucleoskeleton and cytoskeleton (LINC) complex and other nuclear envelope transmembrane (NET) proteins, which connect the cytoskeleton to the nucleus. We discuss how LINC/NETs confers tissue-specific mechanosensitivity to cells and further elucidate how LINC/NETs acts as a control center for nuclear mechanical signals, regulating both gene expression and chromatin organization. Throughout, we primarily focus on stem cell-specific examples, notwithstanding that this is a nascent field. We conclude by highlighting open questions and pointing the way to enhanced research efforts to understand the role nuclear mechanotransduction plays in cell fate choice

Embryonic stem cells become mechanoresponsive upon exit from ground state of pluripotency

Stem cell fate decisions are driven by a broad array of signals, both chemical and mechanical. Although much progress has been made in our understanding of the impact of chemical signals on cell fate choice, much less is known about the role and influence of mechanical signalling, particularly in embryonic stem (ES) cells. Many studies use substrates with different stiffness to study mechanical signalling, but changing substrate stiffness can induce secondary effects which are difficult to disentangle from the direct effects of forces/mechanical signals. To probe the direct impact of mechanical stress on cells, we developed an adaptable cell substrate stretcher to exert specific, reproducible forces on cells. Using this device to test the response of ES cells to tensile strain, we found that cells experienced a transient influx of calcium followed by an upregulation of the so-called immediate and early genes. On longer time scales, however, ES cells in ground state conditions were largely insensitive to mechanical stress. Nonetheless, as ES cells exited the ground state, their susceptibility to mechanical signals increased, resulting in broad transcriptional changes. Our findings suggest that exit from ground state of pluripotency is unaffected by mechanical signals, but that these signals could become important during the next stage of lineage specification. A better understanding of this process could improve our understanding of cell fate choice in early development and improve protocols for differentiation guided by mechanical cues.This work was financially supported by the Engineering and Physical Sciences Research Council, the Medical Research Council, the Wellcome Trust and the European Research Council. K.J.C. is a University Research Fellow of the Royal Society, which also provided financial support for this work

Implications of Cellular Mechanical Memory in Bioengineering

The ability to maintain and differentiate cells in vitro is critical to many advances in the field of bioengineering. However, on traditional, stiff (E approximate to GPa) culture substrates, cells are subjected to sustained mechanical stress that can lead to phenotypic changes. Such changes may remain even after transferring the cells to another scaffold or engrafting them in vivo and bias the outcomes of the biological investigation or clinical treatment. This persistence-or mechanical memory-was initially observed for sustained myofibroblast activation of pulmonary fibroblasts after culturing them on stiff (E approximate to 100 kPa) substrates. Aspects of mechanical memory have now been described in many in vitro contexts. In this Review, we discuss the stiffness-induced effectors of mechanical memory: structural changes in the cytoskeleton and activity of transcription factors and epigenetic modifiers. We then focus on how mechanical memory impacts cell expansion and tissue regeneration outcomes in bioengineering applications relying on prolonged 2D plastic culture, such as stem cell therapies and disease models. We propose that alternatives to traditional cell culture substrates can be used to mitigate or erase mechanical memory and improve the efficiency of downstream cell-based bioengineering applications.ISSN:2373-987

Cell Shape Dynamics Reveal Balance of Elasticity and Contractility in Peripheral Arcs

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension

Microsurgery-aided in-situ force probing reveals extensibility and viscoelastic properties of individual stress fibers

Actin-myosin filament bundles (stress fibers) are critical for tension generation and cell shape, but their mechanical properties are difficult to access. Here we propose a novel approach to probe individual peripheral stress fibers in living cells through a microsurgically generated opening in the cytoplasm. By applying large deformations with a soft cantilever we were able to fully characterize the mechanical response of the fibers and evaluate their tension, extensibility, elastic and viscous properties

Protein Isolation from 3D Hydrogel Scaffolds

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels. Basic Protocol 2: Isolating proteins from collagen hydrogels. Basic Protocol 3: Isolating proteins from alginate hydrogels. Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel. Support Protocol: Isolating protein and RNA simultaneously from the same samples.ISSN:2691-129

Continuous Production of Acoustically Patterned Cells Within Hydrogel Fibers for Musculoskeletal Tissue Engineering

Many mammalian tissues have a specific cellular arrangement that enables their unique function. For example, parallel alignment of myofibers enables uniaxial muscle contraction. To engineer structured tissues ex vivo, it is critical to recapitulate this cellular arrangement. Conventional 3D encapsulation often fails to recapitulate this complexity, motivating the need for advanced patterning approaches. In this work, an acoustofluidic device to continuously pattern mammalian cells within hydrogel fibers is engineered. Contactless acoustofluidic forces are used to control the spacing between parallel lines of cells. To enable continuous extrusion of cell-laden hydrogel fibers, a low friction Teflon tube is integrated into the device. A photopolymerizable hydrogel allows triggering gelation externally with light once the cells are under the influence of the acoustic field, setting the patterned cells within the hydrogel fiber. Using this device, the muscle progenitor cells (myoblasts) within the hydrogel are patterned in parallel lines to mimic the structure of skeletal muscle. The increased formation of myotubes and spontaneous twitching of the myotubes in patterned samples are observed. This approach combining continuous fabrication with the tunability of acoustofluidics can create complex 3D tissues to engineer skeletal muscles as well as tendons, ligaments, vascular networks, or combinations thereof in the future.ISSN:1616-3028ISSN:1616-301