BackgroundApparent Young’s modulus (AYM), which reflects the fundamental mechanical property of live cells measured by atomic force microscopy and is determined by substrate stiffness regulated cytoskeletal organization, has been investigated as potential indicators of cell fate in specific cell types. However, applying biophysical cues, such as modulating the substrate stiffness, to regulate AYM and thereby reflect and/or control stem cell lineage specificity for downstream applications, remains a primary challenge during in vitro stem cell expansion. Moreover, substrate stiffness could modulate cell heterogeneity in the single-cell stage and contribute to cell fate regulation, yet the indicative link between AYM and cell fate determination during in vitro dynamic cell expansion (from single-cell stage to multi-cell stage) has not been established.ResultsHere, we show that the AYM of cells changed dynamically during passaging and proliferation on substrates with different stiffness. Moreover, the same change in substrate stiffness caused different patterns of AYM change in epithelial and mesenchymal cell types. Embryonic stem cells and their derived progenitor cells exhibited distinguishing AYM changes in response to different substrate stiffness that had significant effects on their maintenance of pluripotency and/or lineage-specific characteristics. On substrates that were too rigid or too soft, fluctuations in AYM occurred during cell passaging and proliferation that led to a loss in lineage specificity. On a substrate with ‘optimal’ stiffness (i.e., 3.5 kPa), the AYM was maintained at a constant level that was consistent with the parental cells during passaging and proliferation and led to preservation of lineage specificity. The effects of substrate stiffness on AYM and downstream cell fate were correlated with intracellular cytoskeletal organization and nuclear/cytoplasmic localization of YAP.ConclusionsIn summary, this study suggests that optimal substrate stiffness regulated consistent AYM during passaging and proliferation reflects and contributes to hESCs and their derived progenitor cells lineage specificity maintenance, through the underlying mechanistic pathways of stiffness-induced cytoskeletal organization and the downstream YAP signaling. These findings highlighted the potential of AYM as an indicator to select suitable substrate stiffness for stem cell specificity maintenance during in vitro expansion for regenerative applications.