Abstract
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.
Highlights
After just 11.5 days in the womb, before the first twitch of skeletal muscle, cells of the developing mouse limb bud experience a life-changing event. This transformative event is the first step in skeletogenesis
[4] the spatial and temporal unfolding, mechanotransduction mechanisms, as well as the plasticity of cell fate determination have yet to be elucidated, in part due to the challenge of controlling the applied stresses while measuring cell scale strains in situ and in live cells. This challenge provided the impetus for our current study, where we developed methods to probe the stem cell’s ‘‘mechanome,’’ enabling for the first time to our knowledge the elucidation of structure - function relationships and unfolding lineage commitment in live model embryonic mesenchymal stem cells
Cells A model mesenchymal stem cell (MSC) line, derived from the mesenchyme of murine embryos (C3H/10T1/2 cell line, CCL226; ATCC, Manassas, VA), was used; these cells do not show the phenotypic drift that we observed previously in primary murine cells derived from the mesoderm at the time of condensation [1]
Summary
After just 11.5 days in the womb, before the first twitch of skeletal muscle, cells of the developing mouse limb bud experience a life-changing event. Though the exact timing of mesenchymal condensation varies between vertebrate species, prior to condensation, every cell in the developing mesoderm shares common DNA and a common, undifferentiated phenotype, rendering multipotency. Prior to this time in development, diffusive transport is efficient to insure cell viability in the tiny limb template. The subsequent exposure of stem cells to spatially and temporally varying biophysical and chemical signals guides the cells to specialize their structure for prevailing function, or to commit to a specific lineage. In this way, ‘‘form emerges from function in the stem cell’s mechan[o-chemo-biolog]ical world’’ [1,3]
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