Andrew W. Holle

In situ mechanotransduction via vinculin regulates stem cell differentiation

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference‐mediated vinculin knockdown significantly decreased stiffness‐induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness‐mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site‐containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force‐sensitive adhesion protein can regulate stem cell fate. Stem Cells 2013;31:2467–2477

Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels

Significance Mechanobiology is receiving an increasing amount of focus, but the mechanics of cell-substrate behavior are often neglected in cell biology. As such, novel materials and systems that are simple to build and share in a nonengineering laboratory are sorely needed. We have fabricated gradient hydrogels with continuous linear gradients above and below the durotactic threshold, making it possible to pinpoint optimal stiffness values for a wide range of biological phenomena without the confounding effects of durotaxis. This system has the potential for wide adoption in the cell biology community because of its ease of fabrication, simple material ingredients, and wide gradient possibilities in a single well. The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell–matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.

Nanoscale and mechanical properties of the physiological cell–ECM microenvironment

Studying biological processes in vitro requires faithful and successful reconstitution of the in vivo extracellular matrix (ECM) microenvironment. However, the physiological basis behind in vitro studies is often forgotten or ignored. A number of diverse cell-ECM interactions have been characterized throughout the body and in disease, reflecting the heterogeneous nature of cell niches. Recently, a greater emphasis has been placed on characterizing both the chemical and physical characteristics of the ECM and subsequently mimicking these properties in the lab. Herein, we describe physiological measurement techniques and reported values for the three main physical aspects of the ECM: tissue stiffness, topography, and ligand presentation.

High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation

Human mesenchymal stem cells (hMSCs) receive differentiation cues from a number of stimuli, including extracellular matrix (ECM) stiffness. The pathways used to sense stiffness and other physical cues are just now being understood and include proteins within focal adhesions. To rapidly advance the pace of discovery for novel mechanosensitive proteins, we employed a combination of in silico and high throughput in vitro methods to analyze 47 different focal adhesion proteins for cryptic kinase binding sites. High content imaging of hMSCs treated with small interfering RNAs for the top 6 candidate proteins showed novel effects on both osteogenic and myogenic differentiation; Vinculin and SORBS1 were necessary for stiffness-mediated myogenic and osteogenic differentiation, respectively. Both of these proteins bound to MAPK1 (also known as ERK2), suggesting that it plays a context-specific role in mechanosensing for each lineage; validation for these sites was performed. This high throughput system, while specifically built to analyze stiffness-mediated stem cell differentiation, can be expanded to other physical cues to more broadly assess mechanical signaling and increase the pace of sensor discovery.

Cell–Extracellular Matrix Mechanobiology: Forceful Tools and Emerging Needs for Basic and Translational Research

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Engineered ECM Microenvironments and Their Regulation of Stem Cells

Stem cells possess numerous therapeutic benefits since it is possible to reproducibly control their ability to mature into different cell types even after prolonged culture in vitro; this ability makes stem cells well suited for tissue engineering and regenerative applications. Consequently, understanding stem cell differentiation is a crucial step for those applications. Regulating stem cell fate has traditionally relied on presenting small molecules such as growth factors and cytokines in developmentally appropriate ways, but such a view overlooks other important niche characteristics. Recently, extracellular matrix (ECM) properties have been shown to influence cellular behavior independent of chemical signals, and this has shifted the differentiation paradigm to include ECM properties, e.g. topography, stiffness, composition, porosity, and cell shape/size. Recent advances in bioengineering have enabled versatility in patterning cell types with controlled chemistries, geometries, and sizes. In this chapter, we detail the recent advances in nano- or microfabrication techniques, the biomechanical- and biophysical-driven stem cell differentiation, and the mechanism of how cells “feel” their ECM environment.

Focal Adhesion Mechanotransduction Regulates Stiffness-Directed Differentiation

Human mesenchymal stem cells (hMSCs) are capable of differentiating into mesodermal lineages, with their fate mirroring the tissue lineage possessing a matching in vivo stiffness. The precise mechanisms responsible for this mechanotransduction-induced change in fate are unknown beyond the requirement for force transmission from the extracellular niche through to the nucleus. As a result of cellular contraction, linker proteins connecting the cytoskeleton to the extracellular matrix (ECM) are exposed to differing levels of force and deform to different extents based on the adjacent ECM’s stiffness. Therefore, some of these linker proteins could act as ‘molecular strain gauges,’ as they have been shown to unfold in response to this force. The unfolding process could result in exposure of cryptic binding sites and induction of new signaling pathways. For example, talin exposes multiple vinculin binding sites under physiological force [1]. Vinculin binds at either end to talin and actin and is thought to change its conformation in conjunction with this force [2] similar to how a strain gauge works. Here we show that force-dependent changes in vinculin recruit MAPK1, inducing a signaling cascade that results in the expression of myogenic markers. Together these data suggest that specific proteins may act as ‘molecular strain gauges’ and play a role in mechanosensitive stem cell differentiation.Copyright