Single-cell Mechanobiology: Identifying Functional Markers that Mediate Matrix Signals to Direct Mesenchymal Stem Cell Differentiation

Mesenchymal stem cells (MSCs) form a heterogeneous population of multipotent progenitor cells that can be derived from several solid tissues including bone marrow, and that play an important clinical role by contributing to tissue regeneration and by delivering immunomodulatory effects. MSCs’ adipogenic and osteogenic differentiation is a mechanobiological model system to study matrix-directed cell-fate decision making. The heterogeneous nature of primary cells, specifically of MSCs, makes it difficult to obtain an understanding of the response of cell differentiation to mechanical triggers based on bulk experiments. To obtain insight into the heterogeneous nature of MSCs, we employed single-cell transcriptional profiling methods to study the bi-potential matrix-directed differentiation of human MSCs. Following three days of culture on elastic matrices that mimic fat and pre-calcified bone, we identified a subpopulation of highly mechanosensitive cells that further committed lineage according to matrix elasticity when cultured in a bi-potential differentiation medium. By performing a genome-wide screen, we detected a small set of cytoskeletal proteins and signaling factors that were associated with matrix-directed differentiation only in the mechanosensitivie subpopulation. Overexpression and knockdown of selected markers revealed novel mediators of cellular mechanotransduction that are associated with established signal transduction pathways. Identifying cell surface markers of mechanosensitive MSCs will allow sorting of cells with high clinical potential in cell-based therapeutics.