In Vitro Mesenchymal Stem Cell Differentiation Landscape

We employed a tissue engineered system designed to recapitulate mesenchymal stem cell differentiation and performed single-cell profiling to identify differentiation states. We constructed the Mesenchymal Tissue Landscape (MTL) using single-cell RNA-sequencing (scRNA-seq) data from multiple mesenchymal lineages (osteogenic and adipogenic) at various differentiation stages. Human primary bone marrow-derived MSCs were differentiated on polyacrylamide-based hydrogels of different stiffnesses (low = 1.5 kPa, high = 25-40 kPa) corresponding to the stiffnesses of the related mesenchymal tissues (adipose ~ 1-2 kPa, osteoid ~ 25-40 kPa). Upon adding differentiation induction medium, cells were collected at specified time points until terminal differentiation.