High‐Throughput Multiparametric Quantification of Mechanics Driven Heterogeneity in Mesenchymal Stromal Cell Population

Abstract Mesenchymal stromal or stem cells (MSCs) are one of the most promising candidates for a myriad of cell therapy applications. Despite showing promise in numerous preclinical and clinical studies, MSC‐based therapy is not yet a reality for regenerative medicine due to its suboptimal outcome at the clinical endpoint. The mechanical environment is a critical determinant of MSC gene expression and function. This study reports that MSC population becomes phenotypically heterogenous and commits to an unwanted osteoprogenitor pathway when it experiences an abnormal mechanically stiff environment, compared to its native softer environment. A method is developed to measure the heterogeneity using nuclear shape, chromatin state, and CD73 marker. Heterogeneity is shown to be associated with a larger spread in the nuclear shape parameters and a smaller spread in the chromatin openness. Subsequently, intervention strategies are investigated to create a more homogeneous MSC population. Culturing MSCs on soft surfaces or inhibiting actomyosin on stiff surfaces can make them more homogeneous, while inhibiting YAP, Runx2, and actin polymerization helps maintain but does not fully homogenize them. This study offers insights for cell and tissue engineers, aiding in the design of optimal conditions and materials for MSC culture, ultimately enhancing their therapeutic potential.