Pharmaceutical Research

, Volume 28, Issue 6, pp 1385–1394 | Cite as

The Effect of Human Bone Marrow Stroma-Derived Heparan Sulfate on the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells

  • Diah S. Bramono
  • David A. Rider
  • Sadasivam Murali
  • Victor Nurcombe
  • Simon M. Cool
Research Paper



In order to address cell dose limitations associated with the use of cord blood hematopoietic stem cell (HSC) transplantation, we explored the effect of bone marrow stroma-derived heparan sulfate (HS) on the ex vivo expansion of HSCs.


Heparan sulfate was isolated and purified from the conditioned media of human bone marrow stromal cells and used for the expansion of cord blood-derived CD34+ cells in the presence of a cocktail of cytokines.


The number of myeloid lineage-committed progenitor cells was increased at low dosage of HS as illustrated by an increase in the total number of colony-forming cells (CFC) and colonies of erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) precursors. Notably, the stroma-derived HS did not alter the growth of CD34+ HSCs or negatively affect the levels of various HSC phenotypic markers after expansion.


This study shows that HS secreted into solution by stromal cells has the capacity to support hematopoietic cytokines in the maintenance and expansion of HSCs. The incorporation of stroma-derived HS as a reagent may improve the efficacy of cord blood HSC transplantation by enhancing the number of committed cells and accelerating the rate of engraftment.


cord blood glycosaminoglycan hematopoietic stem cell heparan sulfate stem cell expansion 


  1. 1.
    Rocha V, Gluckman E. Clinical use of umbilical cord blood hematopoietic stem cells. Biol Blood Marrow Transplant. 2006;12(1 Suppl 1):34–41.PubMedCrossRefGoogle Scholar
  2. 2.
    Rocha V, Gluckman E. Improving outcomes of cord blood transplantation: HLA matching, cell dose and other graft- and transplantation-related factors. Br J Haematol. 2009;147(2):262–74.PubMedCrossRefGoogle Scholar
  3. 3.
    Gratwohl A, Baldomero H, Schwendener A, et al. The EBMT activity survey 2008 impact of team size, team density and new trends. Bone Marrow Transplant. 2010:1–8. doi:10.1038/bmt.2010.69
  4. 4.
    Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL, Bernstein ID. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat Med. 2010;16(2):232–6.PubMedCrossRefGoogle Scholar
  5. 5.
    da Silva CL, Goncalves R, Crapnell KB, Cabral JMS, Zanjani ED, Almeida-Porada G. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells. Exp Hematol. 2005;33(7):828–35.PubMedCrossRefGoogle Scholar
  6. 6.
    De Angeli S, Di Liddo R, Buoro S, Toniolo L, Conconi MT, Belloni AS, et al. New immortalized human stromal cell lines enhancing in vitro expansion of cord blood hematopoietic stem cells. Int J Mol Med. 2004;13(3):363–71.PubMedGoogle Scholar
  7. 7.
    Gupta P, McCarthy JB, Verfaillie CM. Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells. Blood. 1996;87(8):3229–36.PubMedGoogle Scholar
  8. 8.
    Kadereit S, Deeds LS, Haynesworth SE, Koc ON, Kozik MM, Szekely E, et al. Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(−) early progenitors cultured over human MSCs as a feeder layer. Stem Cells. 2002;20(6):573–82.PubMedCrossRefGoogle Scholar
  9. 9.
    Kobune M, Kawano Y, Kato J, Ito Y, Chiba H, Nakamura K, et al. Expansion of CD34(+) cells on telomerized human stromal cells without losing erythroid-differentiation potential in a serum-free condition. Int J Hematol. 2005;81(1):18–25.PubMedCrossRefGoogle Scholar
  10. 10.
    Dexter TM. Stromal cell associated haemopoiesis. J Cell Physiol Suppl. 1982;1:87–94.PubMedCrossRefGoogle Scholar
  11. 11.
    Roberts R, Gallagher J, Spooncer E, Allen TD, Bloomfield F, Dexter TM. Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature. 1988;332(6162):376–8.PubMedCrossRefGoogle Scholar
  12. 12.
    Roecklein BA, Torokstorb B. Functionally distinct human marrow stromal cell-lines immortalized by transduction with the human papilloma-virus E6/E7 genes. Blood. 1995;85(4):997–1005.PubMedGoogle Scholar
  13. 13.
    Coombe DR. Biological implications of glycosaminoglycan interactions with haemopoietic cytokines. Immunol Cell Biol. 2008;86(7):598–607.PubMedCrossRefGoogle Scholar
  14. 14.
    Gordon MY, Riley GP, Watt SM, Greaves MF. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature. 1987;326(6111):403–5.PubMedCrossRefGoogle Scholar
  15. 15.
    Bishop JR, Schuksz M, Esko JD. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature. 2007;446(7139):1030–7.PubMedCrossRefGoogle Scholar
  16. 16.
    Esko JD, Linhardt RJ. Proteins that bind sulfated glycosaminoglycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. La Jolla: Cold Spring Harbor Laboratory Press; 2008.Google Scholar
  17. 17.
    Nurcombe V, Goh FJ, Haupt LM, Murali S, Cool SM. Temporal and functional changes in glycosaminoglycan expression during osteogenesis. J Mol Histol. 2007;38:469–81.PubMedCrossRefGoogle Scholar
  18. 18.
    Haupt LM, Murali S, Mun FK, Teplyuk N, Mei LF, Stein GS, et al. The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis. J Cell Physiol. 2009;220(3):780–91.PubMedCrossRefGoogle Scholar
  19. 19.
    Dombrowski C, Song SJ, Chuan P, Lim X, Susanto E, Sawyer AA, et al. Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells. Stem Cells Dev. 2009;18(4):661–70.PubMedCrossRefGoogle Scholar
  20. 20.
    Murali S, Manton KJ, Tjong V, Su XD, Haupt LM, Cool SM, et al. Purification and characterization of heparan sulfate from human primary osteoblasts. J Cell Biochem. 2009;108(5):1132–42.PubMedCrossRefGoogle Scholar
  21. 21.
    Mayani H, Lansdorp PM. Thy-l expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood. 1994;83(9):2410–7.PubMedGoogle Scholar
  22. 22.
    Lewinsohn DM, Nagler A, Ginzton N, Greenberg P, Butcher EC. Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule. Blood. 1990;75(3):589–95.PubMedGoogle Scholar
  23. 23.
    Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science. 1999;283(5403):845–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Schofield R. The relationship between the spleen colony-forming cell and the haematopoietic stem cell. Blood Cells. 1978;4:7–25.PubMedGoogle Scholar
  25. 25.
    Wight TN, Kinsella MG, Keating A, Singer JW. Proteoglycans in human long-term bone marrow cultures—biochemical and ultrastructural analyses. Blood. 1986;67(5):1333–43.PubMedGoogle Scholar
  26. 26.
    Brickman YG, Ford MD, Small DH, Bartlett PF, Nurcombe V. Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. J Biol Chem. 1995;270(42):24941–8.PubMedCrossRefGoogle Scholar
  27. 27.
    Li W, Johnson DJD, Esmon CT, Huntington JA. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol. 2004;11(9):857–62.PubMedCrossRefGoogle Scholar
  28. 28.
    Ostrovsky O, Berman B, Gallagher J, Mulloy B, Fernig DG, Delehedde M, et al. Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes. J Biol Chem. 2002;277(4):2444–53.PubMedCrossRefGoogle Scholar
  29. 29.
    Luikart SD, Maniglia CA, Furcht LT, McCarthy JB, Oegema Jr TR. A heparan sulfate-containing fraction of bone marrow stroma induces maturation of HL-60 cells in vitro. Cancer Res. 1990;50(12):3781–5.PubMedGoogle Scholar
  30. 30.
    Sanderson RD, Turnbull JE, Gallagher JT, Lander AD. Fine structure of heparan sulfate regulates syndecan-1 function and cell behavior. J Biol Chem. 1994;269(18):13100–6.PubMedGoogle Scholar
  31. 31.
    Takada T, Katagiri T, Ifuku M, Morimura N, Kobayashi M, Hasegawa K, et al. Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins. J Biol Chem. 2003;278(44):43229–35.PubMedCrossRefGoogle Scholar
  32. 32.
    Gupta P, Oegema TR, Brazil JJ, Dudek AZ, Slungaard A, Verfaillie CM. Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche. Blood. 1998;92(12):4641–51.PubMedGoogle Scholar
  33. 33.
    Gupta P, Oegema Jr TR, Brazil JJ, Dudek AZ, Slungaard A, Verfaillie CM. Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins. Blood. 2000;95(1):147–55.PubMedGoogle Scholar
  34. 34.
    D’Arenna G, Musto P, Cascavilla N, Di Giorgio G, Zendoli F, Carotenuto M. Human umbilical cord blood: immunophenotypic heterogeneity of CD34+ hematopoietic progenitor cells. Haematologica. 1996;81(5):404–9.Google Scholar
  35. 35.
    Krause DS, Fackler MJ, Civin CI, May WS. CD34: structure, biology, and clinical utility. Blood. 1996;87(1):1–13.PubMedGoogle Scholar
  36. 36.
    Broudy VC. Stem cell factor and hematopoiesis. Blood. 1997;90(4):1345–64.PubMedGoogle Scholar
  37. 37.
    Shah AJ, Smogorzewska EM, Hannum C, Crooks GM. Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38 cells and maintains progenitor cells in vitro. Blood. 1996;87(9):3563–70.PubMedGoogle Scholar
  38. 38.
    Ramsfjell V, Borge OJ, Cui L, Jacobsen SEW. Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+CD38) human bone marrow progenitor cells. J Immunol. 1997;158(11):5169–77.PubMedGoogle Scholar
  39. 39.
    de Lima M, McMannis J, Gee A, Komanduri K, Couriel D, Andersson BS, et al. Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant. 2008;41(9):771–8.PubMedCrossRefGoogle Scholar
  40. 40.
    Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System. Blood. 2003;101(12):5061–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB, et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant. 2002;8(7):368–76.PubMedCrossRefGoogle Scholar
  42. 42.
    Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM, Rubinstein P. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood. 2000;96(8):2717–22.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Diah S. Bramono
    • 1
  • David A. Rider
    • 1
  • Sadasivam Murali
    • 1
  • Victor Nurcombe
    • 1
  • Simon M. Cool
    • 1
    • 2
  1. 1.Stem Cells and Tissue Repair Group, Institute of Medical BiologyA*STAR (Agency for Science, Technology and Research)SingaporeSingapore
  2. 2.Department of Orthopaedic Surgery Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore

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