Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Abstract

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.

High-throughput physical phenotyping of cell differentiation

“…For example, a recent study identified that the differentiation potential of mesenchymal stromal/stem cells is strongly dependent on their elastic and viscoelastic properties 5 . Similarly, it was shown that cell mechanical markers can be a promising alternative for predicting osteogenesis of differentiating stem cells 6 . Other work has demonstrated that physical changes are important for explaining natural phenomena such as leukocyte demargination 7 .…”

Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus

“…The epithelial-to-mesenchymal transition observed during LEC differentiation, coupled with the finding presented here of a corresponding stiffness increase, further supports the contention that cells with a mesenchymal morphology are stiffer than epithelial cells. In comparison, the mechanical changes are less clear for cell types that remain mesenchymal-like during differentiation, such as mesenchymal stem cell differentiation to osteoblast lineages (20,22,24).…”

“…Due to the nonnormal distribution of each biophysical parameter (ShapiroWilk W test, a ¼ 0.05), a bootstrapping ANOVA was performed using a custom MATLAB (The MathWorks, Natick, MA) code to discern statistically significant differences (heteroscedastic comparisons, 10,000 bootstrapping iterations, a ¼ 0.05), as previously described (20). Post hoc analyses were performed using bootstrapping Student's t-tests.…”

“…Recently, various cell mechanical properties, including stiffness (i.e., Young's modulus) and size, have been shown to distinguish stem cell phenotypes from differentiated cells at the single-cell level for various stem cell types (20)(21)(22)(23)(24). Changes in biomechanical properties during stem cell differentiation could enable microfluidic approaches for on-the-fly analysis and sorting of stem cells from differentiated cells.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…The cytoskeleton staining results showed that as osteogenic differentiation of hMSCs progressed, more and more stress fibers were replaced with a thinner actin network that was characteristic of mature osteoblasts, whereas there were no significant differences in the microtubule structure between the undifferentiated hMSC and osteoblasts. In 2014, Bongiorno et al [116] also used AFM to investigate the mechanical properties of hMSCs and human osteoblasts (hOBs). Gridded Petri dish was designed to facilitate the sequential measurement of live-cell stiffness and fluorescent protein biomarker expression at the single-cell level, enabling the direct correlation between mechanics and protein biomarkers of single cell.…”