Histochemistry and Cell Biology

, Volume 139, Issue 1, pp 59–74 | Cite as

The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells

  • Siyuan Li
  • Anthony J. HayesEmail author
  • Bruce Caterson
  • Clare E. Hughes
Original Paper


Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).


Mesenchymal stem cells Chondrogenesis Beta-xyloside Chondroitin/dermatan sulphate Glycosaminoglycans Proteoglycan 



Alpha minimum essential medium,


Bone marrow mesenchymal stem cell


Bovine serum albumin


Chondroitin sulphate




Dulbecco’s Modified Eagle’s Medium


Dimethylmethylene blue


Dermatan sulphate


Extracellular matrix


Foetal bovine serum




Glyceraldehyde 3-phosphate dehydrogenase




Horseradish peroxidase


Heparan sulphate


Insulin–transferrin–sodium selenite


Keratan sulphate


Monoclonal antibody


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide




Para-nitro-phenyl- β-xyloside


Polyvinylidene fluoride


Transforming growth factor-β



Thanks to Mr Marc Isaacs for the provision of technical support in confocal laser scanning microscopy. This research was supported by Arthritis Research UK funding (# 18331).

Supplementary material

418_2012_1017_MOESM1_ESM.jpg (4.1 mb)
Supplemental Fig. 1 DMMB analysis of sGAG detected in culture medium and cell lysate over the 4 week culture period. Data has been normalised to total protein levels. A. sGAG in culture medium. Note significant increase in sGAG detected in culture medium in response to PNPX treatment. B. sGAG in cell lysate. There was no significant differences in the levels of sGAG detected in cell lysates between control and PNPX-treated cultures. ** p≤0.01. (JPEG 4173 kb)
418_2012_1017_MOESM2_ESM.jpg (3.2 mb)
Supplemental Fig. 2 Chondrogenic marker gene expression of bMSCs cultured in basal versus chondrogenic medium after 1 week of culture. Total RNA was extracted and SOX-9, aggrecan and collagen type II gene expression were determined by real-time PCR. N.B. the levels of gene expression for SOX9, aggrecan and collagen type II are significantly higher in chondrogenic growth medium relative to basal medium. ** p≤0.01; *** p≤0.001. (JPEG 3233 kb)
418_2012_1017_MOESM3_ESM.jpg (6 mb)
Supplemental Fig. 3 Distribution analysis of the native chondroitin/dermatan sulphate sulphation motif epitopes recognised by mAbs 6C3 and 7D4 with the focal adhesion protein vinculin. Bovine bone marrow stem cells were cultured as monolayer in chondrogenic medium for 3 days. Focal adhesions were labelled with a monoclonal anti-vinculin antibody (hVIN-1; Sigma Aldrich; green label), whereas CS/DS sulphation motif epitopes were labelled with mAbs 6C3 and 7D4 (red). Cell nuclei were counterstained with DAPI (blue). Note that 6C3 labelling is very closely associated with focal contacts the cells make with their underlying substrate, but does not co-localise with vinculin. 7D4 labelling is much more widespread over the cell surface but, like 6C3, also localises prominently between vinculin-rich focal contacts. Scale bar represents 50μm. (JPEG 6116 kb)
418_2012_1017_MOESM4_ESM.jpg (4.2 mb)
Supplemental Fig. 4 Type II collagen gene expression of bMSCs harvested from PNPX exchange experiment. MSCs were cultured for 2 weeks in chondrogenic growth conditions +/- PNPX. PNPX was then either added (C1/2+P3/4) or withdrawn (P1/2+C3/4) for the remaining 2 weeks of culture and the levels of collagen type II gene expression quantified by qPCR at the 3 and 4 week time points. These were compared with cells cultured continuously over 3 or 4 week periods in chondrogenic medium with or without PNPX (PNPX and chon, respectively). Addition or removal of PNPX at the two week time point had no significant effect on the gene expression levels of collagen type II at 3 or 4 weeks, suggestive that PNPX-induced inhibition of normal GAG chain attachment disrupts early stages of chondrogenesis. * p≤0.05; ** p≤0.01; *** p≤0.001. (JPEG 4335 kb)
418_2012_1017_MOESM5_ESM.jpg (5.3 mb)
Supplemental Fig. 5 Type II collagen protein expression of bMSCs harvested from PNPX exchange experiment. MSCs were cultured for 2 weeks in chondrogenic growth conditions +/- PNPX. PNPX was then either added (C1/2+P3/4) or withdrawn (P1/2+C3/4) for the remaining 2 weeks of culture and the levels of collagen type II gene protein quantified by Western blot analysis at the 3 and 4 week time points. These were compared with cells cultured continuously over 3 or 4 week periods in chondrogenic medium with or without PNPX (PNPX and chon, respectively). Addition of PNPX at the 2 week time point caused a slight, but significant reduction in collagen type II protein expression at 3 and 4 weeks. Removal of PNPX at 2 weeks had no effect on collagen type II protein expression at 3 and 4 weeks. ** p≤0.01; *** p≤0.001. (JPEG 5427 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Siyuan Li
    • 1
    • 2
  • Anthony J. Hayes
    • 2
    Email author
  • Bruce Caterson
    • 2
  • Clare E. Hughes
    • 2
  1. 1.Key Laboratory of Environment and Genes Related to DiseasesXi’an Jiaotong University, Ministry of EducationXi’anChina
  2. 2.Connective Tissue Biology Laboratories, Division of Pathophysiology and Repair, School of BiosciencesCardiff UniversityCardiffUK

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