Resolving mesenchymal stem cells heterogeneity via single-cell analysis of matrix directed differentiation towards soft versus stiff lineages

Mesenchymal stem cells (MSCs) form a heterogeneous population of multipotent progenitor cells that contribute to tissue remodeling, repair, and homeostasis. While differentiation of MSCs towards soft and stiff tissue lineages is directed by matrix mechanics, single cells differ by their matrix-sensing potential, which in turn affects their differentiation capacity. Human MSCs were cultured on soft and stiff matrices that mimicked fat and precalcified bone and were exposed to a bi-potential adipogenic/osteogenic induction medium. To study lineage specification, we obtained single-cell transcriptomes of thousands of MSCs at naïve, matrix-conditioned and early differentiating states using droplet-based single-cell RNA (scRNA) profiling. While adipogenesis was favored on soft matrices and osteogenesis on stiff matrices, scRNA transcriptomes revealed matrix directed lineage differentiation only in a fraction of cells — cell clustered into distinctive subpopulations defined by matrix priming and lineage specification. Diffusion pseudotime reconstruction of naïve states, matrix-priming, and lineage specification histories revealed a cell-fate decision-making bifurcation towards fat and bone fates. Adipogenesis was retarded on stiff matrices, and soft matrices activated chondrogenic markers in osteogenic cells. Differential gene expression screening between matrix-sensitive cells that differentiated in parallel with matrix mechanics and matrix-insensitive cells that exclusively express stress-associated surface markers revealed specific cytoskeletal proteins whose signaling functions we validated via knockdown and overexpression assays. Our work provides a dynamic mapping of MSCs subpopulations characterized by multilineage differentiation capacity associated with cytoskeletal proteins that mediate mechanical signaling.