Mapping the mechanome of live stem cells using a novel method to measure local strain fields in situ at the fluid-cell interface.

During mesenchymal condensation, the initial step of skeletogenesis, transduction of minute mechanical forces to the nucleus is associated with up or down-regulation of genes, ultimately resulting in formation of the skeletal template and appropriate cell lineage commitment. The summation of these biophysical cues affects the cell's shape and fate. Here, we predict and measure surface strain, in live stem cells, in response to controlled delivery of stresses, providing a platform to direct short-term structure--function relationships and long-term fate decisions. We measure local strains on stem cell surfaces using fluorescent microbeads coated with Concanavalin A. During delivery of controlled mechanical stresses, 4-Dimensional (x,y,z,t) displacements of the bound beads are measured as surface strains using confocal microscopy and image reconstruction. Similarly, micro-particle image velocimetry (µ-piv) is used to track flow fields with fluorescent microspheres. The measured flow velocity gradient is used to calculate stress imparted by fluid drag at the surface of the cell. We compare strain measured on cell surfaces with those predicted computationally using parametric estimates of the cell's elastic and shear modulus. Finally, cross-correlating stress--strain data to measures of gene transcription marking lineage commitment enables us to create stress--strain--fate maps, for live stem cells in situ. The studies show significant correlations between live stem cell stress--strain relationships and lineage commitment. The method presented here provides a novel means to probe the live stem cell's mechanome, enabling mechanistic studies of the role of mechanics in lineage commitment as it unfolds.

Modulation of stem cell shape and fate A: the role of density and seeding protocol on nucleus shape and gene expression.

Mesenchymal stem cell shape and fate are intrinsic manifestations of form and function at the cellular level. We hypothesize that cell seeding density and initial seeding protocol influence stem cell shape and fate. Nucleus shape and early (within days of seeding) expression of genes typical for pre-, peri-, and postcondensation events were compared between groups of cells after seeding at or proliferating to target density (low density [LD], 16,500 cells/cm2; high density [HD], 35,000 cells/cm2; very high density [VHD], 86,500 cells/cm2). Significant differences in nuclear shape could be attributed to seeding protocol in the VHD group, where nuclei from cells that proliferated to VHD were significantly rounder than nuclei from cells seeded at target VHD. Furthermore, cells that proliferated to VHD exhibited significantly rounder nuclei than nuclei from all other cell density and seeding protocol groups. In contrast, nuclei from cells that were seeded at the VHD were flatter than nuclei from cells of all other groups. Furthermore, the significant rounding of nuclei in the cells that proliferated to VHD was accompanied by a two-, six-, and ninefold increase from baseline in Runx2, Sox9, and Aggrecan (AGC) expression, markers indicative of precondensation, peri-, and post-condensation events, respectively. None of the other groups showed significant changes in gene expression over baseline. Finally, seeding at target density results in greater overlap of cells compared to groups in which cells proliferate to target density, conferring increased thickness to multicellular culture aggregates seeded at target density. These data suggest that seeding protocols can be exploited to modulate mesenchymal stem cell shape and early gene expression typical for condensation events in development, which occur over an approximately 12-h period at E11.5 in the mouse limb bud. Follow-on studies will delineate longer-term effects of density and seeding protocol on modulation of stem cell fate and cell assembly to form tissues.

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.