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

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.

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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.

References

  1. 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).

    CAS  Article  Google Scholar 

  2. 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. 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).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 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. 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).

    CAS  Article  Google Scholar 

  7. 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).

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

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

    Article  Google Scholar 

  12. 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).

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 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).

    CAS  Article  Google Scholar 

  15. 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. 16

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

    CAS  Article  Google Scholar 

  17. 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).

    CAS  Article  Google Scholar 

  18. 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).

    CAS  Article  Google Scholar 

  19. 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).

    CAS  Article  Google Scholar 

  20. 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).

    CAS  Article  Google Scholar 

  21. 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).

    CAS  Article  Google Scholar 

  22. 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).

    CAS  Article  Google Scholar 

  23. 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).

    CAS  Article  Google Scholar 

  24. 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).

    CAS  Article  Google Scholar 

  25. 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).

    CAS  Article  Google Scholar 

  26. 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).

    CAS  Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 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).

    CAS  Article  Google Scholar 

  29. 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. 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).

    CAS  Article  Google Scholar 

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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.

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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)

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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

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