Modulation of stem cell shape and fate B: mechanical modulation of cell shape and gene expression.

Condensation is a metamorphizing event for the mesenchymal stem cell. The balance of forces in the cell during condensation plays a key role in determining cell shape and cell fate. In the current study, we aim to elucidate the role of shape-changing deviatoric shear stresses and developmental context in modulation of gene transcription prior to cell commitment. We hypothesize that the magnitude and duration of exposure of multipotent embryonic stem cells to shear stress significantly affect activity of genes key to musculoskeletal development at the earliest stage of skeletogenesis--that is, mesenchymal condensation. To test this hypothesis, cells were exposed to 0.2 or 1 dyn/cm2 for 30 or 60 min, and real-time PCR was carried out to measure transcriptional profiles for markers of pre- (Runx2 and Msx2), peri- (ColIa1), and post- (Sox9 and ColIIa1) mesenchymal condensation, osteogenesis (Osx), and adipogenesis (Ppar-gamma2). Exposure of mesenchymal stem cells to shape-changing deviatoric stresses resulted in a significant upregulation of genes associated with pre- (Runx2), peri- (ColIa1, Sox9), and post-condensation (ColIIa1) events. In contrast, expression of terminal differentiation markers for chondrogenesis (AGC), adipogenesis (Ppar-gamma2), and osteogenesis (Osx) were not changed over baseline in response to shape-changing deviatoric shear stresses. In the preceding study, baseline expression of Sox9 and AGC was observed to increase six- and ninefold, respectively, over baseline density controls for cells allowed to proliferate to very high density (86,500 cells/cm2), indicative of chondrogenic lineage commitment; interestingly, exposure to deviatoric stress silenced this gene activity, reverting the cells to a pericondensation state. Further, interaction analyses indicated that duration of exposure to mechanical stress provides a more powerful stimulus for differentiation of multipotent cells than stress magnitude. In addition, the developmental context in which the cells are placed is a significant factor in modulation of gene activity important for pre-, peri-, and postmesenchymal condensation events. Within high-density cultures (35,000 cells/cm2) developmental context exerts a more significant effect on expression of the gene marking pre-condensation (Runx2) and early condensation events (ColIa1) than on expression of genes marking peri- and post-condensation events. In contrast, within very high-density cultures (86,500 cells/cm2), developmental context exerts a more profound influence on expression of genes marking peri- (ColIa1, Sox9) and post-condensation (ColIIa1) events than pre-condensation events. Taken together, these studies provide a first step for the engineering of mesenchymal condensations as templates for de novo production of tissue replacements.