Stem cell mechanoadaptation - Part A - Effect of microtubule stabilization and volume changing stresses on cytoskeletal remodeling

Here we report on the first part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling, which underpins the evolution of stem cell shape and fate, and the emergence of tissue structure and function. In Part A of these studies, we first develop protocols to stabilize microtubules exogenously using paclitaxel (PAX) in a standardized model murine embryonic stem cell line (C3H/10T1/2) to maximize comparability with previous published studies. We then probe native and microtubule stabilized stem cells’ capacity to adapt to volume changing stresses effected by seeding at increasing cell densities, which emulates local compression and tissue template formation during development.

Within the concentration range 1 – 100 nM, microtubule stabilized stem cells maintain viability and reduce proliferation. PAX-stabilization of microtubules is associated with increased cell volume as well as flattening of the cell and nucleus. Compared to control cells, microtubule stabilized cells exhibit thick, bundled microtubules and highly aligned, thicker and longer F-actin fibers, corresponding to an increase in the Young’s Modulus of the cell. Both F-actin and microtubule concentration increase with increasing PAX concentration, whereby the increase in F-actin is more prominent in the basal region of the cell. The corresponding increase in microtubule is observed more globally across the apical and basal region of the cell.

Seeding at increasing target densities induces local compression on cells. This increase in local compression modulates cell volume and concomitant increases in F-actin and microtubule concentration to a greater degree than microtubule stabilization via PAX. Cells seeded at high density (HD) exhibit higher bulk modulus than corresponding cells seeded at low density (LD). These data demonstrate the capacity of stem cells to adapt to an interplay of mechanical and chemical cues, i.e. respective compression and exogenous microtubule stabilization; the resulting cytoskeletal remodeling manifests as evolution of mechanical properties relevant to development of multicellular tissue constructs.

Significance statement

Elucidation of mechanisms by which stem cells adapt across length and time scales may prove enabling for the development of regenerative medicine therapies and devices that emulate natural processes. Dynamic cytoskeletal remodeling underpins the emergence of structure-function relationships at the tissue length scale. Here we stabilized the tubulin cytoskeleton exogenously using paclitaxel (PAX), a microtubule depolymerization inhibitor. We probed stem cell mechanoadaptation by seeding at increasing density to introduce local compression to cells. Changes in cytoskeletal architecture and concentration of F-actin and tubulin per cell occurred in a PAX concentration-dependent manner. Compression from increasing seeding density modulated this PAX-induced cytoskeletal remodeling and mechanical properties of the multicellular constructs. Hence, mechanical cues counterbalance concentration-dependent effects of exogenous chemical microtubule stabilization.