Asset Details

  • Description:
  • HA mediated CD44‐EGFR interaction drives ESC‐smooth muscle cell (SMC) differentiation. (A): FACS analysis showing expression of HA cell surface receptor CD44. The percentages of positive cells were calculated based on the isotype controls (gray plot). Only 24.8% of ESCs expressed CD44 (i) whereas following differentiation, esSMC expressed 85.4% (ii). Treatment with 4MU (0.5 mmol/l) during differentiation restores CD44 levels back to that of undifferentiated ESCs (iii). During differentiation of ESCs the addition of either exogenous LMW‐HA (iv) or HMW‐HA (v) treatment (200 μg/ml) did not alter CD44 expression. FACS data representative for at least three independent experiments with similar results are shown. (B): ESCs were seeded on collagen IV‐coated plates (5 μg/ml) and cultured in DM for the indicated times. RT‐qPCR analysis shows augmented gene expression of CD44 over the course of ESC‐SMC differentiation. (C): To further examine the role of CD44, ESCs were transfected with CD44 siRNA or scrambled oligonucleotide control (scramble) for 48 hours prior to further culture in DM for 72 hours. RT‐qPCR shows a significant attenuation in SMC markers, α‐SMA, calponin and sm22‐α. (D): ESCs were transfected with CD44 siRNA or scrambled oligonucleotide control (scramble) for 48 hours prior to further differentiation for 72 hours in DM alone (−) or stimulation with exogenous LMW‐HA (L) or HMW‐HA (H) (200 μg/ml). Western blot analysis demonstrated that knockdown of CD44 suppressed basal and HA‐mediated ERK1/2 activation. (E): ESCs were preincubated with an anti‐CD44 antibody (5 μg/ml) or IgG control then differentiated in the presence of either LMW‐HA (L) or HMW‐HA (H) (200 μg/ml) for 120 m. Western blot analysis demonstrates that HA mediated pERK1/2 is retarded in the presence of anti‐CD44 antibody. (F‐H): For all experiments samples were IP with anti‐CD44 antibody, followed by immunoblotting with anti‐EGFR antibody. Western blot analysis of the precipitated complexes revealed marked binding of CD44 with EGFR during the course of differentiation (d2‐d8) compared to undifferentiated ESCs (ESC) (F). CD44‐EGFR co‐localisation of esSMCs (ctl) was enhanced in the presence of HMW‐HA but not LMW‐HA (G). ESCs were infected with an adenovirus for HAS2 overexpression (HAS2 Ad) or a null adenovirus (Null) for 48 hours then differentiated toward SMCs in the absence or presence of the ERK1/2 inhibitor PD98059 (10 μmol/l). HAS2 overexpression resulted in enhanced CD44 and EGFR association which was unaltered by ERK1/2 inhibition (H). All blots shown are representative of at least three separate experiments, graphs are shown as mean ± SEM of at least three independent experiments. Analyses based on Student's t test. *, p < .05, versus scramble, or one‐way ANOVA, followed by Dunnett's Multiple Comparison Test. ≠, p < .01, versus ESC. Abbreviations: 4MU, 4‐methylumbelliferone; DM, differentiation medium; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; HA, hyaluronan; HMW‐HA, high molecular weight‐hyaluronan; LMW‐HA, low molecular weight‐hyaluronan; IP, immunoprecipitated.
  • License:
  • CC-BY
  • Rights Holder:
  • Creative Commons by John Wiley & Sons
  • License Rights Holder:
  • © 2016 AlphaMed Press
  • Asset Type:
  • Image
  • Asset Subtype:
  • Image Orientation:
  • Landscape
  • Image Dimensions:
  • 1270 x 1236
  • Image File Size:
  • 482 KB
  • Creator:
  • Russell M.L. Simpson, Xuechong Hong, Mei Mei Wong, Eirini Karamariti, Shirin Issa Bhaloo, Derek Warren, Wei Kong, Yanhua Hu, Qingbo Xu
  • Credit:
  • Simpson, R. M. L., Hong, X., Wong, M. M., Karamariti, E., Bhaloo, S. I., Warren, D., Kong, W., Hu, Y., & Xu, Q. (2016). Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage. STEM CELLS, 34(5), 1225-1238..
  • Collection:
  • Keywords:
  • Restrictions:
  • This image was published the terms of the Creative Commons Attribution (CC BY) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Permission is not required for this kind of use.
  • Property Release:
  • No
  • Model Release:
  • No
  • Purchasable:
  • Yes
  • Sensitive Materials:
  • No
  • Article Authors:
  • Russell M.L. Simpson, Xuechong Hong, Mei Mei Wong, Eirini Karamariti, Shirin Issa Bhaloo, Derek Warren, Wei Kong, Yanhua Hu, Qingbo Xu
  • Article Copyright Year:
  • 2016
  • Publication Volume:
  • 34
  • Publication Issue:
  • 5
  • Publication Date:
  • 05/01/2016
  • DOI:

Click on image to enlarge

Would you like to download this asset? Tell us more and we can generate a quote, license and downloadable file.

Add to Cart