Matrix-driven formation of mesenchymal stem cell–extracellular matrix microtissues on soft alginate hydrogels

Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell–matrix and cell–cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G′ ⩽ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine–glycine–aspartic acid cell–adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell–cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.