(117a) Extracellular Matrix Mechanics Affect Stem Cell Lineage in 3D by Controlling Integrin Binding

Stem cell therapies hold great clinical promise, but control of transplanted cell fate remains a significant challenge. (Mooney and Vandenburgh 2008). Material-based systems offer a promising means to program stem cells (Silva 2008), and in 2D cell culture, cell fate can be manipulated by changing either the biochemical composition or rigidity of the adhesion substrate (Kong 2005, Engler 2006, Klees 2005). However, the extent to which ECM mechanics affect stem cell fate in physiologically-relevant 3D micro-environments, and the biophysical mechanisms underlying mechanosensing are unclear. Here, we demonstrate that the rigidity of cell-encapsulating 3D biomaterials (RGD-modified alginate hydrogels) can change lineage specification in primary and clonally-derived mesenchymal stem cells (MSC). However, in contrast to 2D studies that suggest matrix rigidity directs stem cell fate by altering morphology (Engler 2006, McBeath 2004), ECM rigidity had very little effect on cell shape in these 3D cultures. Instead, matrix stiffness regulated integrin ligation by the adhesion ligand RGD in a biphasic manner, and matrices with optimal stiffness for integrin-RGD binding elicited the highest degree of osteogenic lineage specification in stem cells. Integrin-RGD binding correlated with cells' ability to reorganize adhesion ligands presented from the matrix, on the nanometer scale, via acto-myosin mediated traction forces. In 2D cell culture, MSC, like other cell types, used αV-integrins to ligate RGD when presented via surface adsorbed vitronectin, or from 2D RGD-modified hydrogel substrates. Strikingly, however, α5-integrins acted as RGD receptors in the same MSC when this adhesion ligand was presented, without PHSRN synergy sites, from a 3D, cell-encapsulating hydrogel α5-integrin-RGD bonds acted as 3D-mechanosensors, and inhibiting the formation of these bonds with function blocking antibodies diminished osteogenic lineage specification in MSC. Altogether, these data demonstrate that cells interpret changes in the physical properties and dimensionality of substrates as though they were chemical changes in adhesion ligand presentation. As cells themselves, by interplay between acto-myosin mediated traction forces and extracellular matrix mechanics, played a significant role in determining the structure of the cell-biomaterial interface, both in terms of the type and total number of bound integrins, this work suggests a paradigm to engineer living materials: namely, that cells can be harnessed as tools to process simple, scalable materials into complex structures that feedback to manipulate stem cell fate.

References: Engler et al. Cell 2006. Kong et al. Proc Natl Acad Sci USA 2005. Mooney and Vandenburgh Cell Stem Cell 2008. Silva et al. Proc Natl Acad Sci USA 2008. Klees et al. Mol Biol Cell 2005. McBeath et al. Dev Cell 2004.