Substrate elasticity modulates the responsiveness of mesenchymal stem cells to commitment cues.

Fate choices of stem cells are regulated in response to a complex array of biochemical and physical signals from their microenvironmental niche. Whereas the molecular composition and the role of mechanical niche cues have been extensively studied, relatively little is known about how both effectors act in concert to modulate stem cell fate. Here we utilized a recently developed artificial niche microarray platform to investigate whether the stiffness of a cell culture substrate influences how niche signaling factors exert their role on adipogenic differentiation of human mesenchymal stem cells (hMSC). We found that substrate stiffness imposes a strictly non-overlapping range of differentiation, highlighting the dominance of physical over the biochemical factors. At a given stiffness, a significant protein-dependent effect on adipogenic differentiation was observed. Furthermore, we show that synergistic interactions between proteins can also be driven by the substrate stiffness. Our results thus highlight the importance of considering the mechanical properties of a target tissue when investigating biochemical niche signals in vitro.

3D niche microarrays for systems-level analyses of cell fate.

The behaviour of mammalian cells in a tissue is governed by the three-dimensional (3D) microenvironment and involves a dynamic interplay between biochemical and mechanical signals provided by the extracellular matrix (ECM), cell-cell interactions and soluble factors. The complexity of the microenvironment and the context-dependent cell responses that arise from these interactions have posed a major challenge to understanding the underlying regulatory mechanisms. Here we develop an experimental paradigm to dissect the role of various interacting factors by simultaneously synthesizing more than 1,000 unique microenvironments with robotic nanolitre liquid-dispensing technology and by probing their effects on cell fate. Using this novel 3D microarray platform, we assess the combined effects of matrix elasticity, proteolytic degradability and three distinct classes of signalling proteins on mouse embryonic stem cells, unveiling a comprehensive map of interactions involved in regulating self-renewal. This approach is broadly applicable to gain a systems-level understanding of multifactorial 3D cell-matrix interactions.