The role of soluble factors in determining embryonic stem cell fate has been well studied. A growing body of research indicates mechanical cues are equally important to chemical signals in eliciting cellular response, especially during the early developmental stages of embryos. Here, we seek to control substrate stiffness and exposure to mechanical cues in order to gain a better understanding of how mechanotransduction directs mouse embryonic stem cell (mESC) fate. A magnetic twisting cytometer (MTC) was used to apply oscillating force to mES cells through fibronectin (FN) coated magnetic microbeads. The ligand coated magnetic beads were allowed to bind to undifferentiated mES cells plated on 0.6 kPa polyacrylamide gels prior to loading. The cells used were stably transfected with green fluorescent protein (GFP) bound to oct3/4, an important indicator of differentiation. These cells were imaged during and after stress application to record the intensity of GFP expression. Previous research has shown force application through the synthetic ligand RGD results in cell spreading and downregulation of oct3/4. The use of fibronectin, a natural ligand, more closely replicates conditions in vivo and transmits force through different receptors than RGD. As expected, cells stressed with fibronectin-coated beads differentiated on a similar timescale to RGD experiments. Unexpectedly, cells did not spread after force application.In order to gain a better understanding of GFP downregulation dynamics, mES cells were photobleached and treated with increasing concentrations of retinoic acid (RA), a chemical known to direct pluripotent cells towards a partially differentiated neural fate. The goal was to bleach GFP expression down to a baseline level and then compare the relative magnitude of chemical and mechanical effects on the production or downregulation of oct3/4. Low to medium concentrations of RA kept oct3/4 expression at or below baseline as expected, with higher concentrations becoming cytotoxic. This RA result could not be compared to an equivalent mechanical test, as the combination of bleaching and stress application was too stressful for cells. However, these data reinforce the idea that chemical and mechanical cues are of similar importance in determining the fate of mESC and that more ligands should be explored for force application.
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Cyclic loading of fibronectin coated beads induces differentiation in mouse embryonic stem cells
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