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

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Abstract

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).

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Abbreviations

α-MEM:

Alpha minimum essential medium,

bMSC:

Bone marrow mesenchymal stem cell

BSA:

Bovine serum albumin

CS:

Chondroitin sulphate

DAPI:

4′,6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle’s Medium

DMMB:

Dimethylmethylene blue

DS:

Dermatan sulphate

ECM:

Extracellular matrix

FBS:

Foetal bovine serum

GAG:

Glycosaminoglycan

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

Hep:

Heparin

HRP:

Horseradish peroxidase

HS:

Heparan sulphate

ITS:

Insulin–transferrin–sodium selenite

KS:

Keratan sulphate

mAb:

Monoclonal antibody

MTT:

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

PG:

Proteoglycan

PNPX:

Para-nitro-phenyl- β-xyloside

PVDF:

Polyvinylidene fluoride

TGF-β:

Transforming growth factor-β

References

  • Barbero A, Ploegert S, Heberer M, Martin I (2003) Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes. Arthritis Rheum 48:1315–1325

    Article  PubMed  CAS  Google Scholar 

  • Bastiaansen-Jenniskens YM, Koevoet W, Feijt C, Bos PK, Verhaar JA, Van Osch GJ, DeGroot J (2009a) Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair. J Tissue Eng Regen Med 3:117–123

    Article  PubMed  CAS  Google Scholar 

  • Bastiaansen-Jenniskens YM, Koevoet W, Jansen KM, Verhaar JA, DeGroot J, VanOsch GJ (2009b) Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production. Biochem Biophys Res Commun 379:222–226

    Article  PubMed  CAS  Google Scholar 

  • Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling KW, Sham MH, Koopman P, Tam PP, Cheah KS (1997) SOX9 directly regulates the type-II collagen gene. Nat Genet 16:174–178

    Article  PubMed  CAS  Google Scholar 

  • Carey DJ (1991) Biological functions of proteoglycans: use of specific inhibitors of proteoglycan synthesis. Mol Cell Biochem 104:21–28

    Article  PubMed  CAS  Google Scholar 

  • Caterson B (2012) Fell-Muir lecture: chondroitin sulphate glycosaminoglycans: fun for some and confusion for others. Int J Exp Pathol 93:1–10

    Article  PubMed  CAS  Google Scholar 

  • Caterson B, Griffin J, Mahmoodian F, Sorrell JM (1990) Monoclonal antibodies against chondroitin sulphate isomers: their use as probes for investigating proteoglycan metabolism. Biochem Soc Trans 18:820–823

    PubMed  CAS  Google Scholar 

  • Chen WC, Yao CL, Chu IM, Wei YH (2010) Compare the effects of chondrogenesis by culture of human mesenchymal stem cells with various type of the chondroitin sulfate C. J Biosci Bioeng 111:226–231

    Article  PubMed  Google Scholar 

  • Choi S, Kim Y, Park H, Han IO, Chung E, Lee SY, Kim YB, Lee JW, Oh ES, Yi JY (2009) Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin alpha2. Biochem Biophys Res Commun 384:231–235

    Article  PubMed  CAS  Google Scholar 

  • Deepa SS, Yamada S, Zako M, Goldberger O, Sugahara K (2004) Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor. J Biol Chem 279:37368–37376

    Article  PubMed  CAS  Google Scholar 

  • Delise AM, Tuan RS (2002) Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro. Dev Dyn 225:195–204

    Article  PubMed  CAS  Google Scholar 

  • Edwards GO, Coakley WT, Ralphs JR, Archer CW (2010) Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap. Eur Cell Mater 19:1–12

    PubMed  CAS  Google Scholar 

  • Farndale RW, Sayers CA, Barrett AJ (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9:247–248

    Article  PubMed  CAS  Google Scholar 

  • Frescaline G, Bouderlique T, Huynh MB, Papy-Garcia D, Courty J, Albanese P (2012) Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells. Stem Cell Res 8:180–192

    Article  PubMed  CAS  Google Scholar 

  • Gao L, McBeath R, Chen CS (2010) Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin. Stem Cells 28:564–572

    PubMed  CAS  Google Scholar 

  • Gibson KD, Segen BJ, Audhya TK (1977) The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors. Biochem J 162:217–233

    PubMed  CAS  Google Scholar 

  • Gould RP, Day A, Wolpert L (1972) Mesenchymal condensation and cell contact in early morphogenesis of the chick limb. Exp Cell Res 72:325–336

    Article  PubMed  CAS  Google Scholar 

  • Groot CG, Thesingh CW, te Pas M, Moskalewski S (1987) Influence of beta-D-xyloside on growth and histological aspect of long bones in chicken embryos. Teratology 35:447–454

    Article  PubMed  CAS  Google Scholar 

  • Hall BK, Miyake T (2000) All for one and one for all: condensations and the initiation of skeletal development. BioEssays 22:138–147

    Article  PubMed  CAS  Google Scholar 

  • Hardingham T (2010) Cell- and tissue-based approaches for cartilage repair. Altern Lab Anim 38(Suppl 1):35–39

    PubMed  CAS  Google Scholar 

  • Hardingham TE, Oldershaw RA, Tew SR (2006) Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat 209:469–480

    Article  PubMed  CAS  Google Scholar 

  • Hayes AJ, Tudor D, Nowell MA, Caterson B, Hughes CE (2008) Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells. J Histochem Cytochem 56:125–138

    Article  PubMed  CAS  Google Scholar 

  • Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E (1994) Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 302(Pt 2):527–534

    PubMed  CAS  Google Scholar 

  • Hwang SG, Yu SS, Lee SW, Chun JS (2005) Wnt-3a regulates chondrocyte differentiation via c-Jun/AP-1 pathway. FEBS Lett 579:4837–4842

    Article  PubMed  CAS  Google Scholar 

  • Hwang NS, Kim MS, Sampattavanich S, Baek JH, Zhang Z, Elisseeff J (2006) Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem Cells 24:284–291

    Article  PubMed  CAS  Google Scholar 

  • Ibold Y, Lubke C, Pelz S, Augst H, Kaps C, Ringe J, Sittinger M (2009) Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures. Tissue Cell 41:249–256

    Article  PubMed  CAS  Google Scholar 

  • Janners MY, Searls RL (1970) Changes in rate of cellular proliferation during the differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Dev Biol 23:136–165

    Article  PubMed  CAS  Google Scholar 

  • Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238:265–272

    Article  PubMed  CAS  Google Scholar 

  • Li S, Duance VC, Blain EJ (2008) Zonal variations in cytoskeletal element organization, mRNA and protein expression in the intervertebral disc. J Anat 213:725–732

    Article  PubMed  CAS  Google Scholar 

  • Li F, Shi W, Capurro M, Filmus J (2011) Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J Cell Biol 192:691–704

    Article  PubMed  CAS  Google Scholar 

  • Little CB, Hughes CE, Curtis CL, Janusz MJ, Bohne R, Wang-Weigand S, Taiwo YO, Mitchell PG, Otterness IG, Flannery CR, Caterson B (2002) Matrix metalloproteinases are involved in C-terminal interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. Matrix Biol 21:271–288

    Article  PubMed  CAS  Google Scholar 

  • Lohmander LS, Hascall VC, Caplan AI (1979) Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures. J Biol Chem 254:10551–10561

    PubMed  CAS  Google Scholar 

  • Malavaki C, Mizumoto S, Karamanos N, Sugahara K (2008) Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease. Connect Tissue Res 49:133–139

    Article  PubMed  CAS  Google Scholar 

  • Mitchell D, Hardingham T (1982) The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans. Biochem J 202:387–395

    PubMed  CAS  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  PubMed  CAS  Google Scholar 

  • Nadanaka S, Kinouchi H, Taniguchi-Morita K, Tamura J, Kitagawa H (2011) Down-regulation of chondroitin 4-O-sulfotransferase-1 by Wnt signaling triggers diffusion of Wnt-3a. J Biol Chem 286:4199–4208

    Article  PubMed  CAS  Google Scholar 

  • Oohira A, Nogami H, Nakanishi Y (1981) Abnormal overgrowth of chick embryos treated with p-nitrophenyl beta-D-xyloside at early stages of development. J Embryol Exp Morphol 61:221–232

    PubMed  CAS  Google Scholar 

  • Park JS, Yang HJ, Woo DG, Yang HN, Na K, Park KH (2010) Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long-term release of TGF-beta 3 complexed with chondroitin sulfate. J Biomed Mater Res A 92:806–816

    PubMed  Google Scholar 

  • Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74

    Article  PubMed  CAS  Google Scholar 

  • Richter W (2009) Mesenchymal stem cells and cartilage in situ regeneration. J Intern Med 266:390–405

    Article  PubMed  CAS  Google Scholar 

  • Robinson JA, Robinson HC (1981) Control of chondroitin sulphate biosynthesis. beta-D-Xylopyranosides as substrates for UDP-galactose: D-xylose transferase from embryonic-chicken cartilage. Biochem J 194:839–846

    PubMed  CAS  Google Scholar 

  • Robinson HC, Brett MJ, Tralaggan PJ, Lowther DA, Okayama M (1975) The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage. Biochem J 148:25–34

    PubMed  CAS  Google Scholar 

  • Rodgers KD, San Antonio JD, Jacenko O (2008) Heparan sulfate proteoglycans: a GAGgle of skeletal-hematopoietic regulators. Dev Dyn 237:2622–2642

    Article  PubMed  CAS  Google Scholar 

  • Roughley PJ, White RJ, Magny MC, Liu J, Pearce RH, Mort JS (1993) Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem J 295:421–426

    PubMed  CAS  Google Scholar 

  • Sekiya I, Colter DC, Prockop DJ (2001) BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 284:411–418

    Article  PubMed  CAS  Google Scholar 

  • Sekiya I, Vuoristo JT, Larson BL, Prockop DJ (2002) In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA 99:4397–4402

    Article  PubMed  CAS  Google Scholar 

  • Sobue M, Habuchi H, Ito K, Yonekura H, Oguri K, Sakurai K, Kamohara S, Ueno Y, Noyori R, Suzuki S (1987) beta-D-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo. Biochem J 241:591–601

    PubMed  CAS  Google Scholar 

  • Sorrell JM, Carrino DA, Caplan AI (1993) Structural domains in chondroitin sulfate identified by anti-chondroitin sulfate monoclonal antibodies. Immunosequencing of chondroitin sulfates. Matrix 13:351–361

    Article  PubMed  CAS  Google Scholar 

  • Sorrell JM, Carrino DA, Caplan AI (1996) Regulated expression of chondroitin sulfates at sites of epithelial-mesenchymal interaction: spatio-temporal patterning identified with anti-chondroitin sulfate monoclonal antibodies. Int J Dev Neurosci 14:233–248

    Article  CAS  Google Scholar 

  • Ueno M, Yamada S, Zako M, Bernfield M, Sugahara K (2001) Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence. J Biol Chem 276:29134–29140

    Article  PubMed  CAS  Google Scholar 

  • Uygun BE, Stojsih SE, Matthew HW (2009) Effects of immobilized glycosaminoglycans on the proliferation and differentiation of mesenchymal stem cells. Tissue Eng Part A 15:3499–3512

    Article  PubMed  CAS  Google Scholar 

  • Vainio S, Jalkanen M, Bernfield M, Saxen L (1992) Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney. Dev Biol 152:221–232

    Article  PubMed  CAS  Google Scholar 

  • Varghese S, Hwang NS, Canver AC, Theprungsirikul P, Lin DW, Elisseeff J (2008) Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells. Matrix Biol 27:12–21

    Article  PubMed  CAS  Google Scholar 

  • Wada N, Javidan Y, Nelson S, Carney TJ, Kelsh RN, Schilling TF (2005) Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull. Development 132:3977–3988

    Article  PubMed  CAS  Google Scholar 

  • Watanabe Y, Takeuchi K, Higa Onaga S, Sato M, Tsujita M, Abe M, Natsume R, Li M, Furuichi T, Saeki M, Izumikawa T, Hasegawa A, Yokoyama M, Ikegawa S, Sakimura K, Amizuka N, Kitagawa H, Igarashi M (2010) Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development. Biochem J 432:47–55

    Article  PubMed  CAS  Google Scholar 

  • Williams GM, Sah RL (2011) In vitro modulation of cartilage shape plasticity by biochemical regulation of matrix remodeling. Tissue Eng Part A 17:17–23

    Article  PubMed  CAS  Google Scholar 

  • Wolf F, Candrian C, Wendt D, Farhadi J, Heberer M, Martin I, Barbero A (2008) Cartilage tissue engineering using pre-aggregated human articular chondrocytes. Eur Cell Mater 16:92–99

    PubMed  CAS  Google Scholar 

  • Worster AA, Brower-Toland BD, Fortier LA, Bent SJ, Williams J, Nixon AJ (2001) Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix. J Orthop Res 19:738–749

    Article  PubMed  CAS  Google Scholar 

  • Yoo JU, Barthel TS, Nishimura K, Solchaga L, Caplan AI, Goldberg VM, Johnstone B (1998) The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 80:1745–1757

    PubMed  CAS  Google Scholar 

  • Young MF, Bi Y, Ameye L, Chen XD (2002a) Biglycan knockout mice: new models for musculoskeletal diseases. Glycoconj J 19:257–262

    Article  PubMed  CAS  Google Scholar 

  • Young RD, Vaughan-Thomas A, Wardale RJ, Duance VC (2002b) Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee. Osteoarthr Cartil 10:420–428

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann B (1984) Assembly and disassembly of gap junctions during mesenchymal cell condensation and early chondrogenesis in limb buds of mouse embryos. J Anat 138(Pt 2):351–363

    PubMed  Google Scholar 

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Acknowledgments

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).

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Correspondence to Anthony J. Hayes.

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418_2012_1017_MOESM1_ESM.jpg

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

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

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

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

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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Li, S., Hayes, A.J., Caterson, B. et al. The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells. Histochem Cell Biol 139, 59–74 (2013). https://doi.org/10.1007/s00418-012-1017-1

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