We were able to show a significant difference in cell stiffness between human fibroblasts, hASCs, hASC-iPSCs, fibroblast-iPSCs, and hESCs. These findings have substantial implications in the cell mechanics of uni-, multi-, and pluri-potent cells, and create the development of a possible biomarker for stem cells. Elastic modulus, E, was used as the measure of stiffness, with higher moduli corresponding with stiffer materials. Elastic modulus describes the stiffness of a material, in this case the cell, and its resistance to elastic deformation. Elastic deformation occurs when a material returns to its original shape immediately upon removal of the applied stress; in pure viscous behavior, deformation is delayed in response to an applied load and the material does not completely recover its original shape. Cells have characteristics of both, a property called viscoelasticity, in which an initial elastic response to a load is followed by viscous, time-dependent deformation. Viscoelasticity is a characteristic of an amorphous material (the cell) and describes the movement of the cell components with the cytoplasm. To determine the elastic moduli of these cells, AFM nanoindentation readings were taken within the elastic deformation range of the cell.
Cell stiffness increased in the following order: hASC-iPSCs, hESCs, fibroblast-iPSCs, fibroblasts, and hASCs. The relatively lower stiffness of the reprogrammed iPSCs may be due to active remodeling of multiple components of the cells internal structure, such as the filaments of the cytoskeleton-actin filaments, intermediate filaments, and microtubules. The overall apparent compliance of the pluripotent cells may denote the cells' unrestrained ability to assume several cell types and the associated material properties. The relatively larger stiffness of the fibroblast cell is expected, as the fibroblast commonly composes the main cell type of connective tissues, which provide considerable structural support in many parts of the body. An interesting finding here was that hASCs, which are multipotent cells, were stiffer than the differentiated fibroblast cell. This may be due to the fact that hASCs are relatively smaller cells than fibroblasts (widths of ~15 vs. ~30
μm, respectively), which increases the actin filament density in the hASCs compared to the fibroblast, and in turn perhaps increasing the stiffness.
Using the AFM as an instrument to read cell stiffness is a powerful tool, though there are factors that have to be taken into consideration when using such a technique on cells. One important element is adhesion of the AFM probe tip to the cell itself. Inaccurate stiffness readings would result when adhesion forces dominated during probing. One possible reason for this phenomenon is the presence of excess Matrigel atop the cells, creating a sticky layer. If adhesion appeared to be a problem during readings, those results were discarded and new cells were seeded and re-probed.
Stem cells have the unique characteristic of preferentially forming colonies. To obtain consistent cell stiffness readings between cell types, the traditionally noncolony forming fibroblasts and hASCs were plated into a cloning ring to induce high-density, colony-like seeding. Although there was a difference in the cell density achieved in these noncolonizing cells (4
for both hASCs and human fibroblasts, versus 7
for hESCs and both iPS cell types), this difference was not substantial enough to account for the large, significant difference in elastic modulus between cell types. Fibroblasts are inherently larger cells than the hESCs, iPSCs, and hASCs (~30
μm in width and ~70
μm in length for fibroblasts, compared to ~15
μm in diameter for the stem cells studied here). This affects the maximum density attainable with the pseudo-colonies of fibroblasts, hence the lower number of cells per given area in comparison.
To investigate the extent of the affect of boundary conditions, cells at the center and at the periphery of the colonies were probed for cell stiffness. In the hASC pseudo-colony and iPSC colony, cells at the edge of the colonies exhibit slightly lower elastic moduli. This may indicate that the cells in the center of the colonies receive some structural support from neighboring cells and their corresponding cytoskeleton organization, as opposed to cells at the edge that only receive support from a few sides (seen during actin staining).
In the current highly interdisciplinary research environment, an AFM is not an unlikely piece of equipment for any researcher. Tools such as the AFM may provide an informative method for simplifying various measures in stem cell and, overall, biological research. Finally, the mechanobiological properties of stem cells may lead to additional strategies to promote either dedifferentiation during reprogramming, or to differentiate stem cells for cell-based regenerative medicine.