Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate

Citation: Tse JR, Engler AJ ( Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate Justin R. Tse 0 Adam J. Engler 0 Nic D. Leipzig, The University of Akron, United States of America 0 Department of Bioengineering, University of California San Diego , La Jolla, California , United States of America Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ,8.761.5 kPa/mm, or normal tissue variation, e.g., myocardium ,0.660.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.060.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior. - Funding: This work was supported by the National Institutes of Health (1DP02OD006460), American Heart Association (0865150F), and National Science Foundation (0754718). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In their niche, cells are presented with an array of complex biophysical and biochemical signals from the surrounding extracellular matrix (ECM) [1,2,3]. The Youngs modulus, E, often referred to in a biological context simply as elasticity or stiffness, is an intrinsic ECM characteristic that has a profound effect on cell spreading, morphology, and function [4,5,6,7]. In particular, stem cells show lineage-specific differentiation when cultured on substrates matching the stiffness corresponding to native tissue; neural stem cells become either neural or glial lineages depending on matrix elasticity [8], pre-osteoblasts most efficiently form calcified deposits when cultured on optimally stiff substrates [9], and multipotent mesenchymal stem cells (MSCs) [10] become neurogenic, myogenic, and osteogenic when cultured on substrates mimicking neural, muscle, and bone stiffness environments, respectively [11,12], by regulating their cell tension [11,13]. However these studies utilize polymer systems that have static parameters while their native counterparts reside in a dynamic environment in which elasticity may change spatially and/or temporally. For example, epicardial stiffness increases approximately 3-fold during development [14] while myocardium post-infarction forms a fibrotic scar that is 3- to 4-fold more stiff than surrounding muscle [15]. Elasticity also varies naturally at interfaces, e.g. hard, calcified bones are connected to soft cartilage [11,16]. As MSCs egress from bone marrow and hone to these interfaces or migrate through tissue [17], they may encounter such stiffness gradient(s), and it is not clear whether the MSC response to these stimuli is to remain in place and differentiate, as with static materials [11,12], or migrate in response to the stiffness gradient as with fibroblasts [18]. Several methods have developed in vitro elasticity gradients starting with polymerizing adjacent solutions of differing polymer concentrations to obtain a gradient at the solution interface [18]. More complex methods have employed microfluidic devices [19] or photolithographically-patterned photoactivated initiators [19,20, 21,22] to generate monomer and/or crosslinking density gradients. A hallmark of these studies is the observation that most somatic cells, e.g. fibroblasts, endothelial cells, and vascular smooth muscle cells [18,19,20,21,23], migrate in response to stiffness gradients in a process called durotaxis, with specific exceptions for cells originating from highly stratified structures [22]. However gradient strength, i.e. the degree of stiffness change per length, for these studies is typically in a pathological rather than physiological range [15]. A notable exception has shown that somatic cell migration is dependent on gradient strength, though the shallowest gradient 10 kPa/mm was still within a pathological range [24]. While some somatic cells may durotax in physiological gradients [20], each mature cell type exhibits lineage specific behavior within a physiologically relevant stiffness range [4,6,25]. On the other hand, undifferentiated MSCs lack such a preference and are in fact programmed by these surroundings [11,12,13]. Since much of their migration is likely to occur through tissue with physiological rather than pathological gradient(s) before reaching the site in need of regeneration, perhaps a more fundamental question is whether they durotax when presented with a physiological stiffness gradient ,1 kPa/ mm in the absence of other stimuli, e.g. soluble growth factor gradients which could induce chemotaxis. To better understand the role this potential signal could play in MSC fate, we cultured MSCs on a photopolymerized polyacrylamide (PA) hydrogel of varying stiffness and provide the first evidence that MSCs indeed appear to undergo durotaxis rather than remain stationary. Morphological and lineage marker assessment indicates that MSCs, even within shallow durotactic gradients, migrate to stiffer matrix and then differentiate into a more contractile cell, though this behavior is complicated by some degree of memory of the previously soft environment from which they migrated. Surface Characterization of Gradient Hydrogels A photomask with a radial grayscale pattern was used to create a crosslinking gradient in a 10% acrylamide/0.3% bis-acrylamide hydrogel via selective activation of the photoinitiator Irgacure 2959 (Fig. 1A [26]). The elastic modulus with respect to distance from the edge to center of the hydrogels was measured by atomic force microscopy (AFM) and found to have a range of 1 to 14 kPa (Fig. 1B). Data was found to have a gradient strength of 1.060.1 kPa/mm. Such a gradient is within the physiological range of natural cardiac tissue variations, e.g. 0.660.9 kPa/mm, and considerably less than the pathophysiological range of infarct cardiac tissue, e.g. 8.761.5 kPa/mm, as previously measured [15]. To permit cell attachment, both gradient and static hydrogels were covalently functionalized with type I collagen using SulfoSANPAH, which showed relatively uniform attachment via antibody staining when observed in the XZ cross-section by confo (...truncated)