Figure 1. Human Megakaryocytes express hyaluronan (HA) and HAS isoenzymes necessary for its biosynthesis.
A) Real Time qPCR analysis of HAS1, HAS2 and HAS3 expression in cord blood derived CD34+ HSCs and differentiated Mks. Results are expressed as fold increase of HAS isoenzymes expression in Mks with respect to CD34+ HSCs. β2 microglobulin gene expression was used for the comparative concentration analysis. At least three independent experiments were performed. n.d. = not detectable. B) Western blotting analysis of HAS3 expression in human fibroblasts and differentiated Mks. C) Western blotting analysis of HAS3 expression in differentiated Mks and human peripheral blood platelets. D) Densitometry analysis of HAS3 levels in Mks and peripheral blood platelets. β-actin was used as internal control of protein loading. At least three independent experiments were performed. E) Immunofluorescence staining of HAS-3 (red) (middle panel) in TPO differentiated CD61+ (green) (left panel) Mks. 1×105 Mks at day 13 of culture were cytospun onto poly-L-lysine coated glass coverslips and processed for immunofluorescence. Composite of images is shown on the right panel. Scale Bar=20μm. F) Immunofluorescence staining of hyaluronan (red) (middle panel) in TPO differentiated CD61+ (green) (left panel) Mks. Composite of images are shown on the right panel. 1×105 Mks at day 13 of culture were cytospun, fixed and permeabilized or not with Triton X-100 to demonstrate the intracellular localization of HA. Cells were stained with Hyaluronan Binding Protein (HABP) and CD61 antibody, followed by Alexa Fluor 594 coniugated streptavidin and Alexa Fluor 488 conjugated antibody, respectively. Treatment with hyaluronidase was performed before Mks staining to confirm the presence of intracellular HA. Scale Bar=10μm. Hoechst 33258 was used to highlight nuclei (blue).