Substrate stiffness orchestrates epithelial cellular heterogeneity with controlled proliferative pattern via E-cadherin/β-catenin mechanotransduction.

UNLABELLED: Epithelial cellular heterogeneity has been observed in pathological tissues with abnormal matrix stiffness and cells cultured on rigid substrates. However, it remains unclear how matrix stiffness influences cellular heterogeneity formation in multi-cellular population. Here, we demonstrated that cellular heterogeneity regulated by substrate stiffness is evident starting from the initial single-cell stage (indicated by cellular Young's modulus and morphology) until the resulting multi-cellular stage (indicated by cellular functions) through distinguished proliferative patterns. Epithelial cells on soft substrate proliferated in a neighbor-dependent manner with stronger E-cadherin expression and more homogeneous E-cadherin/β-catenin localization compared to those on coverslips, which resulted in reduced heterogeneity in downstream cellular functions of the multi-cellular population. In particular, decreased heterogeneity in human embryonic stem cells upon expansion and endodermal induction was achieved on soft substrate. Overall, our work provides new insights on mechanotransduction during epithelial proliferation which regulates the formation of cellular heterogeneity and potentially provides a highly efficient approach to regulate stem cell fate by fine-tuning substrate stiffness.

STATEMENT OF SIGNIFICANCE: This study demonstrates that cellular heterogeneity regulated by substrate stiffness is evident starting from the initial single-cell stage until the resulting multi-cellular stage through distinguished proliferative patterns. During this process, E-cadherin/β-catenin mechanotransduction is found to play important role in substrate stiffness-regulated epithelial cellular heterogeneity formation. In particular, decreased heterogeneity in human embryonic stem cells upon expansion and endodermal induction is achieved on soft substrate. Hence, we believe that this work not only provides new insights on mechanotransduction of E-cadherin/β-catenin which regulates the formation of cellular heterogeneity during proliferation, but also potentially provides a highly efficient approach to regulate stem cell fate by fine-tuning substrate stiffness.