Abstract
Summary
This study helps to elucidate how ColVI and Dcn within the pericellular matrix (PCM) of differentiating hMSCs directly impacts dynamic cytoskeletal response to load, and demonstrates an important role for the PCM in mechanotransduction during chondrogenesis.
Introduction
Mechanosignaling events in differentiating human mesenchymal stem cells (hMSCs) are dependent on their temporally changing micromechanical environment and their dynamic cytoskeleton. During chondrogenic differentiation, hMSCs develop a matrix composed of type VI collagen (ColVI) and proteoglycans such as decorin (Dcn). We have previously demonstrated that this developing PCM is important in cellular mechanotransduction. The aim of this study was to determine the functional roles of ColVI and Dcn in modulating load-induced changes in the organization of vimentin intermediate filaments (VIF), actin microfilaments (AM), and vinculin.
Methods
hMSCs were transduced with shRNA targeting either col6a1 (shColVI) or dcn (shDcn) and then cultured in 2% alginate beads for 14 days in chondrogenic media. GFP-transduced hMSCs were cultured in parallel. Cells with their intact PCM were isolated with 100mM sodium citrate, 30mM EDTA, and re-embedded in alginate discs. These hMSC-alginate constructs underwent unconfined compression at 0.1Hz for 1 hour from 0–10% strain. Free Swelling (FS) and loaded discs were fixed either immediately following (0hr) or 4 hours post-load. Discs were cryosectioned and fluorescently labeled for VIF, AM, or vinculin with DAPI counterstain. Confocal image-stacks were collected and corrected total cell fluorescence (CTCF) per area was calculated using ImageJ (NIH) to quantify cytoskeletal organization.
Results
VIF fluorescence showed a dynamic cytoskeletal response to load in non-infected and GFP-transduced controls with an increase in intensity. In both ColVI and Dcn knockdown groups, VIF exhibited higher fluorescence in FS, which then didn't significantly change following load, possibly due to its higher baseline concentration. AM showed a similar response to load in shColVI knockdown samples, with a significantly higher intensity in FS samples, which wasn't affected by loading and remained cortically localised in all samples. In shDcn samples, there was a significant increase in actin content immediately following load, similar to control. Vinculin staining showed significantly lower staining intensity in shColVI knockdown samples than non-infected and GFP-transduced samples at all timepoints, and exhibited a different trend in response to loading. Following loading, vinculin was diffusely stained across the cytoskeleton in all samples.
Discussion
This study demonstrates through targeted shRNA knockdown that the involvement of ColVI and Dcn in mechanosignaling events of differentiating hMSCs could be due to their interactions with and control over the dynamic cytoskeleton. Vimentin and actin have previously been shown to contribute to the viscoelastic mechanical properties of hMSCs and are important in the ability for cells to resist load. Our experiments indicate that specific components in the PCM can affect cytoskeletal dynamics, with knockdown samples lacking the significantly dynamic response to load seen in control samples. Actin cytoskeletal intensity and vinculin intensity show an inverse relationship to load. Since vinculin mediates interactions between actin cytoskeleton and integrins at focal adhesions, these data suggest that ColVI and Dcn regulation of cytoskeletal response may be governed more by the changing external micromechanical environment, rather than altered cell-matrix interactions. Further studies are needed to elucidate the roles of ColVI and Dcn in downstream intracellular mechanosignaling events of differentiating hMSCs.
