Abstract
Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.
Highlights
In comparison to other glycosaminoglycans, hyaluronan (HA) is a relatively simple molecule: it is non-sulfated, unbranched and not linked to any proteoglycan core protein
HA occurs in pericellular coats which were observed around different cell types like chondrocytes, fibroblasts [3] and mesenchymal stem cells [4]
Since it is known that only hyaluronan synthases (HASes) localized in the plasma membrane are active [28], these results indicate that the HAS-eGFP fusion proteins are correctly transported to the plasma membrane
Summary
In comparison to other glycosaminoglycans, hyaluronan (HA) is a relatively simple molecule: it is non-sulfated, unbranched and not linked to any proteoglycan core protein. In various cell types one of the earliest steps of adhesion is mediated by the pericellular HA coat, which binds the cell to the surface but still keeping the cell membrane a few micrometers away from it [12,14]. The thin hyaluronan coat of epithelial cells mediates rapid adhesion to the surface [14,19,20]. The Pericellular Coat on HAS-eGFP Overexpressing hMSCs For the visualization of the membrane-bound, pericellular HA, monolayer cultured HAS-eGFP overexpressing SCP1 and control cells were fixed and incubated with biotinylated HA-binding complex, followed by staining with Alexa Fluor 647-conjugated streptavidin. All three SCP1-HAS-eGFP cell lines oversecrete HA, they express the transgene proteins in their active form.
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