Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone

Nature Medicine volume 14, pages 181–187 (2008)Cite this article

Abstract

The capacity to direct migration ('homing') of blood-borne cells to a predetermined anatomic compartment is vital to stem cell–based tissue engineering and other adoptive cellular therapies. Although multipotent mesenchymal stromal cells (MSCs, also termed 'mesenchymal stem cells') hold the potential for curing generalized skeletal diseases, their clinical effectiveness is constrained by the poor osteotropism of infused MSCs (refs. 1–3). Cellular recruitment to bone occurs within specialized marrow vessels that constitutively express vascular E-selectin4,5, a lectin that recognizes sialofucosylated determinants on its various ligands. We show here that human MSCs do not express E-selectin ligands, but express a CD44 glycoform bearing α-2,3-sialyl modifications. Using an α-1,3-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, we converted the native CD44 glycoform on MSCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL)6, which conferred potent E-selectin binding without effects on cell viability or multipotency. Real-time intravital microscopy in immunocompromised (NOD/SCID) mice showed that intravenously infused HCELL+ MSCs infiltrated marrow within hours of infusion, with ensuing rare foci of endosteally localized cells and human osteoid generation. These findings establish that the HCELL glycoform of CD44 confers tropism to bone and unveil a readily translatable roadmap for programming cellular trafficking by chemical engineering of glycans on a distinct membrane glycoprotein.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: MSCs lack E-selectin ligands but express CD44 reactive with SACK-1 mAb, which recognizes a sialic acid–dependent epitope displayed on a CD44-specific N-glycan substitution.
Figure 2: FTVI treatment of MSCs elaborates sialofucosylations on N-linked glycans of CD44, resulting in HCELL expression.
Figure 3: HCELL+ MSCs have markedly enhanced shear-resistant adhesive interactions with endothelial E-selectin under defined shear stress conditions.
Figure 4: Human MSC homing to mouse bone marrow.

Similar content being viewed by others

References

  1. Horwitz, E.M. et al. Isolated allogeneic bone marrow–derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc. Natl. Acad. Sci. USA 99, 8932–8937 (2002).

    Article  CAS  Google Scholar 

  2. Jin-Xiang, F., Xiaofeng, S., Jun-Chuan, Q., Yan, G. & Xue-Guang, Z. Homing efficiency and hematopoietic reconstitution of bone marrow–derived stroma cells expanded by recombinant human macrophage-colony stimulating factor in vitro. Exp. Hematol. 32, 1204–1211 (2004).

    Article  Google Scholar 

  3. Gao, J., Dennis, J.E., Muzic, R.F., Lundberg, M. & Caplan, A.I. The dynamic in vivo distribution of bone marrow–derived mesenchymal stem cells after infusion. Cells Tissues Organs 169, 12–20 (2001).

    Article  CAS  Google Scholar 

  4. Sipkins, D.A. et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973 (2005).

    Article  CAS  Google Scholar 

  5. Schweitzer, K.M. et al. Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am. J. Pathol. 148, 165–175 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Dimitroff, C.J., Lee, J.Y., Rafii, S., Fuhlbrigge, R.C. & Sackstein, R. CD44 is a major E-selectin ligand on human hematopoietic progenitor cells. J. Cell Biol. 153, 1277–1286 (2001).

    Article  CAS  Google Scholar 

  7. Mauney, J.R., Volloch, V. & Kaplan, D.L. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng. 11, 787–802 (2005).

    Article  CAS  Google Scholar 

  8. Mangi, A.A. et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat. Med. 9, 1195–1201 (2003).

    Article  CAS  Google Scholar 

  9. Pittenger, M.F. & Martin, B.J. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ. Res. 95, 9–20 (2004).

    Article  CAS  Google Scholar 

  10. Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49 (2002).

    Article  CAS  Google Scholar 

  11. Sackstein, R. The lymphocyte homing receptors: gatekeepers of the multistep paradigm. Curr. Opin. Hematol. 12, 444–450 (2005).

    Article  Google Scholar 

  12. Polley, M.J. et al. CD62 and endothelial cell–leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewis x. Proc. Natl. Acad. Sci. USA 88, 6224–6228 (1991).

    Article  CAS  Google Scholar 

  13. Lapidot, T., Dar, A. & Kollet, O. How do stem cells find their way home? Blood 106, 1901–1910 (2005).

    Article  CAS  Google Scholar 

  14. Dimitroff, C.J., Lee, J.Y., Fuhlbrigge, R.C. & Sackstein, R. A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells. Proc. Natl. Acad. Sci. USA 97, 13841–13846 (2000).

    Article  CAS  Google Scholar 

  15. Sackstein, R. & Dimitroff, C.J. A hematopoietic cell L-selectin ligand that is distinct from PSGL-1 and displays N-glycan–dependent binding activity. Blood 96, 2765–2774 (2000).

    CAS  PubMed  Google Scholar 

  16. Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    Article  CAS  Google Scholar 

  17. Kobzdej, M.M., Leppanen, A., Ramachandran, V., Cummings, R.D. & McEver, R.P. Discordant expression of selectin ligands and sialyl Lewis x–related epitopes on murine myeloid cells. Blood 100, 4485–4494 (2002).

    Article  CAS  Google Scholar 

  18. Xia, L., McDaniel, J.M., Yago, T., Doeden, A. & McEver, R.P. Surface fucosylation of human cord blood cells augments binding to P-selectin and E-selectin and enhances engraftment in bone marrow. Blood 104, 3091–3096 (2004).

    Article  CAS  Google Scholar 

  19. Hidalgo, A. & Frenette, P.S. Enforced fucosylation of neonatal CD34+ cells generates selectin ligands that enhance the initial interactions with microvessels but not homing to bone marrow. Blood 105, 567–575 (2005).

    Article  CAS  Google Scholar 

  20. Murray, B.W., Takayama, S., Schultz, J. & Wong, C.H. Mechanism and specificity of human α-1,3-fucosyltransferase V. Biochemistry 35, 11183–11195 (1996).

    Article  CAS  Google Scholar 

  21. Schrantz, N. et al. Manganese induces apoptosis of human B cells: caspase-dependent cell death blocked by bcl-2. Cell Death Differ. 6, 445–453 (1999).

    Article  CAS  Google Scholar 

  22. de Bruyn, K.M., Rangarajan, S., Reedquist, K.A., Figdor, C.G. & Bos, J.L. The small GTPase Rap1 is required for Mn2+- and antibody-induced LFA-1– and VLA-4–mediated cell adhesion. J. Biol. Chem. 277, 29468–29476 (2002).

    Article  CAS  Google Scholar 

  23. Alon, R. et al. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128, 1243–1253 (1995).

    Article  CAS  Google Scholar 

  24. Chigaev, A. et al. Real time analysis of the affinity regulation of α4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn2+ or antibody activation. J. Biol. Chem. 276, 48670–48678 (2001).

    Article  CAS  Google Scholar 

  25. Takamatsu, Y., Simmons, P.J. & Levesque, J.P. Dual control by divalent cations and mitogenic cytokines of α4β1 and α5β1 integrin avidity expressed by human hemopoietic cells. Cell Adhes. Commun. 5, 349–366 (1998).

    Article  CAS  Google Scholar 

  26. Mazo, I.B., Quackenbush, E.J., Lowe, J.B. & von Andrian, U.H. Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow. Blood 99, 4182–4191 (2002).

    Article  CAS  Google Scholar 

  27. Mohler, W., Millard, A.C. & Campagnola, P.J. Second harmonic generation imaging of endogenous structural proteins. Methods 29, 97–109 (2003).

    Article  CAS  Google Scholar 

  28. Hauschka, P.V., Lian, J.B. & Gallop, P.M. Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc. Natl. Acad. Sci. USA 72, 3925–3929 (1975).

    Article  CAS  Google Scholar 

  29. Yao, L. et al. Divergent inducible expression of P-selectin and E-selectin in mice and primates. Blood 94, 3820–3828 (1999).

    CAS  PubMed  Google Scholar 

  30. Mocco, J. et al. HuEP5C7 as a humanized monoclonal anti-E/P-selectin neurovascular protective strategy in a blinded placebo-controlled trial of nonhuman primate stroke. Circ. Res. 91, 907–914 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C.A. Knoblauch, L. Liu and J.Y. Lee for assistance in manuscript preparation and for skilled technical support, as well as P.V. Hauschka for helpful discussion of the data. We are grateful to the staff of the Cell Processing Laboratory of the Bone Marrow Transplantation Unit at the Massachusetts General Hospital and the Cell Manipulation Core Facility of Dana Farber Cancer Center for their assistance in procuring the bone marrow harvest filter sets. This effort was supported by National Institutes of Health grants RO1 HL73714 (R.S.), RO1 HL60528 (R.S.) and Massachusetts General Hospital Wellman Center Advanced Microscopy startup fund (C.P.L.). This report is dedicated to the memory of Dr. Harvey R. Colten.

Author information

Authors and Affiliations

  1. Departments of Dermatology and Medicine, Brigham and Women's Hospital and Harvard Skin Disease Research Center, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Robert Sackstein, Jasmeen S Merzaban, Derek W Cain & Nilesh M Dagia

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Robert Sackstein

  3. Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Joel A Spencer & Charles P Lin

  4. Research Specialties, Sigma-Aldrich, Buchs, CH-9470, Switzerland

    Roland Wohlgemuth

Authors
  1. Robert Sackstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jasmeen S Merzaban
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Derek W Cain
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nilesh M Dagia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joel A Spencer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles P Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Roland Wohlgemuth
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.S. conceived the study and reagents, created hybridomas, developed the SACK-1 mAb and the conditions for surface fucosylation of live cells, performed experiments and supervised all research, wrote the manuscript and funded the research; J.S.M., D.W.C. and N.M.D. performed cell culture, biochemical studies and adhesion assays; J.A.S. and C.P.L. performed intravital microscopy and C.P.L. partially funded the research; R.W. synthesized fucosyltransferase.

Corresponding author

Correspondence to Robert Sackstein.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–6 and Supplementary Methods (PDF 5151 kb)

Supplementary Movie 1

This video segment shows a marrow sinusoidal endothelial bed within the first minute after injection of FTVI-treated, HCELL-expressing MSCs (bright cells). Note evident rolling interactions and firm adherence of MSCs onto marrow sinusoidal endothelium. (MP4 3000 kb)

Supplementary Movie 2

This video segment shows a marrow sinusoidal endothelial bed withing the first minute after injection of FTVI-Sialidase MSCs. Compared to HCELL+ MSCs shown in Video 1, FTVI-Sialidase MSCs show minimal binding interactions with marrow sinusoidal endothelium. (MP4 3100 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sackstein, R., Merzaban, J., Cain, D. et al. Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med 14, 181–187 (2008). https://doi.org/10.1038/nm1703

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1703

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing