Consistent apparent Young’s modulus of human embryonic stem cells and derived cell types stabilized by substrate stiffness regulation promotes lineage specificity maintenance

Abstract

Background Apparent 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. Results Here, 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. Conclusions In 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.

Spatial cell fate manipulation of human pluripotent stem cells by controlling the microenvironment using photocurable hydrogel

“…S4B), probably due to the preferred endoderm stiffness range. Similar with previous report(Guo et al, 2020; Keung et al, 2012) and our RNA-seq results (Fig. S2).…”

Mechanobiology: A landscape for reinterpreting stem cell heterogeneity and regenerative potential in diseased tissues

“…In particular, stem cell culturing on extremely rigid or soft substrates resulted in Young’s modulus fluctuations and loss in lineage specificity throughout cell passaging and proliferation. 10 The same study revealed that stem cell growing onto defined substrate stiffness around 3.5 kPa ensued in a constant level Young’s modulus consistent with preservation of lineage specificity during cell proliferation throughout multiple passaging. Adding further complexity to the role of mechanobiology in stem cell heterogeneity, asymmetry in integrin-mediated mechanical forces between the periphery and interior of a mesenchymal stem cell (MSC) population can spatially direct osteogenic-adipogenic 12 , 13 , 14 or chondrogenic-myogenic 15 differentiation decisions.…”

“… 8 Growing evidence shows that stem cell fate can be predicted by cellular mechanical properties. 9 , 10 , 11 The apparent Young’s modulus, which estimates the substrates stiffness and cytoskeletal organization in living cells, as measured by atomic force microscopy (AFM), was found to change dynamically during stem cell passaging and proliferation on substrates with different stiffness. 10 This, in turn, resulted in significant difference in the maintenance of pluripotency and/or lineage-specific characteristics in embryonic stem cells and their derived progenitor cells.…”

“… 9 , 10 , 11 The apparent Young’s modulus, which estimates the substrates stiffness and cytoskeletal organization in living cells, as measured by atomic force microscopy (AFM), was found to change dynamically during stem cell passaging and proliferation on substrates with different stiffness. 10 This, in turn, resulted in significant difference in the maintenance of pluripotency and/or lineage-specific characteristics in embryonic stem cells and their derived progenitor cells. In particular, stem cell culturing on extremely rigid or soft substrates resulted in Young’s modulus fluctuations and loss in lineage specificity throughout cell passaging and proliferation.…”

Synthetic liver fibrotic niche extracts achieve in vitro hepatoblasts phenotype enhancement and expansion

“…In addition to the biochemical components, we also preliminarily investigated the effects of hypoxia and substrate stiffness on HBs culture. It was found that hypoxia with 5% oxygen ( Figure S10 ) and an appropriate substrate Young’s modulus (3.5 kPa) simulating the tissue mechanics of early-stage hepatic fibrosis ( Guo et al., 2020 ) were conducive to the maintenance of phenotype and expansion in HBs, which shed light on the involvement of multifaceted biochemical and biophysical cues in future exploration for niche optimization.…”