Intrinsic extracellular matrix properties regulate stem cell differentiation
Introduction
Stem cell differentiation has traditionally employed cocktails of various growth factors, but differentiation has recently become increasingly linked to mechanobiological concepts such as cell-generated physical forces. These forces originate from myosin bundles sliding along actin filaments and are transmitted to the extracellular matrix (ECM), a three-dimensional fibrillar protein scaffold which surrounds and anchors cells. Transduction of these signals employs a vast array of adhesive proteins that assemble together to mechanically link the extracellular and intracellular worlds. Cells rely on ECM resistance much in the way that an electrical current requires the wire's internal resistance to permit the movement of charge: cells pull against matrix and “feel” the resistance to deformation by the adjacent environment, which provides feedback via mechanically-sensitive proteins along this connection. Though the molecular details require further exploration, the intention of this review is to provide insight into what, where, and when a stem cell senses its ECM cues, how they induce differentiation, and how such cues can be incorporated into new, three-dimensional scaffolds to treat disease.
A sound understanding of the interplay between development and ECM expression provides a starting point for tackling stem cell differentiation. ECM is composed predominantly of three major structural proteins, i.e. collagen, fibronectin, and laminin, as well as a host of elastin, fibrillin, tenascin, glycosaminoglycans, and proteoglycans. Each of these has a very specific distribution and assembly pattern in the body (Hay, 1991) that could contribute to certain developmental processes, especially given that the onset of matrix expression coincides with the late morula stage (Eyal-Giladi, 1995) just before the emergence of pluripotent embryonic stem cells (ESCs) and the first cell movements. For example, the matrix protein fibronectin regulates cell migration during the rearrangement process called gastrulation (Darribere and Schwarzbauer, 2000), which occurs just as stem cell fate is being decided and the body plan and axis is being established. Moreover, interactions between specific germ layers and matrix proteins, e.g. mesendoderm morphogenesis and the synergy site of fibronectin (Davidson et al., 2002), also suggest a link between intrinsic matrix properties and differentiation. On the other hand, adult stem cells, such as bone-marrow-derived mesenchymal stem cells (MSCs), are multipotent meaning that they can mature into a specific subset of cells. Though they may not have temporally-changing matrix as with ESCs, they are known to migrate from the marrow to a variety of tissues with different matrix compositions (Pittenger et al., 1999), providing good cause to examine their susceptibility to matrix variation. Both cell types sit in contrast with lineage-specific progenitor cells, which can only become a single cell type and thus may behave more closely to that adult cell type; therefore, we will limit our discussion to stem cells with multi-lineage potential.
For both embryonic and adult stem cells, ECM properties, including (1) structural (Dalby et al., 2007; McBeath et al., 2004), (2) biochemical (Helen et al., unpublished; Ott et al., 2008), and (3) mechanical (Engler et al., 2006; McBeath et al., 2004; Ruiz and Chen, 2008) cues, develop in concert. Here we will present a discussion of several of these mechanisms and how they regulate differentiation. Stem cells are surprisingly sensitive in vitro to these stimuli, especially with respect to how these cues change cell-generated forces or how forces influence the presentation of these properties. We will discuss how new materials have enabled the separation of these variables, which are often coupled matrix parameters in the body. Our review will only focus on stem cells, and we would refer the reader to the rich literature regarding cell–ECM interactions and the signaling that it induces in mature cells for a broader treatment of the subject (see (Butcher et al., 2009; Geiger et al., 2001; Nelson and Bissell, 2006; Schwartz, 2001; Schwartz and DeSimone, 2008; Vogel and Sheetz, 2006) among others), including many of the reviews within this special issue.
Section snippets
Matrix structure and organization
In vivo, matrix is first secreted by cells and subsequently assembled into a fibrillar network by their integrins which attach to the cell-binding regions of matrix, e.g. the Arginine–Glycine–Aspartic acid (or RGD) on fibronectin. Upon binding, clusters containing multiple bound integrins are pulled apart such that matrix proteins are unwound to reveal self-association domains, permitting homotypic binding, e.g. fibronectin–fibronectin (Mao and Schwarzbauer, 2005). In the absence of certain
Matrix chemistry
Biochemical regulation of stem cell fate is not limited to soluble ligands; the composition of matrix can regulate cells via differential integrin binding to adhesion sites – such as those shown in Fig. 1A—demonstrating a second mechanism to alter cell fate: ECM composition. For example, acellular tissue explants, having a distinct matrix composition, can interact with a specific subset of integrins on the MSC surface and direct a portion of the cell population to become beating cardiomyocytes
Mechanical properties of the matrix
Many observations in modern cell biology have been performed on rigid glass coverslips, often coated with a thin layer of ECM as in Fig. 1B. However, such thin coatings do not recapitulate the normal mechanical environment of most cell types (Engler et al., 2004c) and can lead to de-differentiation or loss of function in cells (Engler et al., 2008). The correct mechanical properties of a cell niche vary as much as 300-fold from soft brain tissue (0.1 kiloPascal or kPa; a unit of elasticity, E)
Designing matrix for the third dimension: microenvironment in context
Most of these ECM-based stimuli mentioned above were studied individually, and any given cue, whether growth factors or ECM structure, elasticity, or composition, does not appear to be sufficient to promote full differentiation relative to mature adult cells but results only in precursors to those cell types. Thus, MSCs grown on a gel of muscle elasticity, EM, become spindle-shaped and express only early muscle markers (Fig. 2A (Engler et al., 2006; Rowlands et al., 2008)); they cannot fuse
Designing for the future
Stem cells are exquisitely sensitive to the intrinsic properties of their extracellular matrix, and as illustrated here, one of the key differentiation mediators is the mechanical interaction between the cell and its matrix. These mechanobiological relationships and the cell pathways they modulate need to be better understood in order to manipulate stem cell differentiation for in vitro and clinical applications. In order to properly separate these variables and understand their individual
Conflict of interest statement
None
Acknowledgements
The authors would like to thank Nikolaj Gadegaard for the image of the PMMA semi-disordered substrate in Fig. 1. The authors would also like to acknowledge Jennifer Edwards and Fergal O’Brien (Department of Anatomy, Royal College of Surgeons, Ireland) for the confocal images of cells in large and small diameter porous scaffolds in Fig. 4, respectively. The authors additionally acknowledge funding from AHA 0865150F (to AJE), UCSD-RI-324G-ENGLER (to AJE), and the Foreign and Commonwealth Office
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