Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull mice
2012; Elsevier BV; Volume: 40; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2012.01.015
ISSN1873-2399
AutoresSimon N. Robinson, Paul J. Simmons, Michael W. Thomas, Nathalie Brouard, Jeannie A. Javni, Suprita Trilok, Jae-Seung Shim, Hong Yang, David Steiner, William K. Decker, Dongxia Xing, Leonard D. Shultz, Barbara Savoldo, Gianpietro Dotti, Catherine M. Bollard, Leonard P. Miller, Richard E. Champlin, Elizabeth J. Shpall, Patrick A. Zweidler‐McKay,
Tópico(s)Immune Response and Inflammation
ResumoDelayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγnull (NSG) mice. By isolating fucosylated and nonfucosylated CD34+ cells from CB, we showed that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34+ cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting. Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγnull (NSG) mice. By isolating fucosylated and nonfucosylated CD34+ cells from CB, we showed that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34+ cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting. Cord blood (CB) has become an important source of hematopoietic support for cancer patients lacking human leukocyte antigen−matched donors. The ethnic diversity, relative ease of collection, ready availability, reduced incidence, and severity of graft-vs-host disease and tolerance of higher degrees of human leukocyte antigen disparity between donor and recipient, are positive attributes when compared with unrelated donor bone marrow (BM) [1Wagner J.E. Rosenthal J. Sweetman R. et al.Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease.Blood. 1996; 88: 795-802PubMed Google Scholar, 2Kurtzberg J. Laughlin M. Graham M.L. et al.Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients.N Engl J Med. 1996; 335: 157-166Crossref PubMed Scopus (978) Google Scholar, 3Gluckman E. Rocha V. Boyer-Chammard A. et al.Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group.N Engl J Med. 1997; 337: 373-381Crossref PubMed Scopus (1167) Google Scholar, 4Rocha V. Wagner Jr., J.E. Sobocinski K.A. et al.Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources.N Engl J Med. 2000; 342: 1846-1854Crossref PubMed Scopus (727) Google Scholar] or cytokine-mobilized peripheral blood (PB) [5Bensinger W.I. Clift R. Martin P. et al.Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation.Blood. 1996; 88: 2794-2800PubMed Google Scholar, 6Bensinger W.I. Weaver C.H. Appelbaum F.R. et al.Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor.Blood. 1995; 85: 1655-1658PubMed Google Scholar, 7Bensinger W.I. Martin P.J. Storer B. et al.Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers.N Engl J Med. 2001; 344: 175-181Crossref PubMed Scopus (825) Google Scholar]. However, significantly delayed neutrophil and platelet engraftment and elevated risk of graft failure are also associated with CB transplantation when compared with BM or PB transplantation [8McGlave P. Bartsch G. Anasetti C. et al.Unrelated donor marrow transplantation therapy for chronic myelogenous leukemia: initial experience of the National Marrow Donor Program.Blood. 1993; 81: 543-550PubMed Google Scholar, 9Kernan N.A. Bartsch G. Ash R.C. et al.Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program.N Engl J Med. 1993; 328: 593-602Crossref PubMed Scopus (779) Google Scholar, 10Schiller G. Feig S.A. Territo M. et al.Treatment of advanced acute leukaemia with allogeneic bone marrow transplantation from unrelated donors.Br J Haematol. 1994; 88: 72-78Crossref PubMed Scopus (50) Google Scholar, 11Szydlo R. Goldman J.M. Klein J.P. et al.Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.J Clin Oncol. 1997; 15: 1767-1777Crossref PubMed Scopus (392) Google Scholar, 12Berthou C. Legros-Maida S. Soulie A. et al.Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes.Blood. 1995; 85: 1540-1546PubMed Google Scholar, 13Majhail N.S. Mothukuri J.M. Brunstein C.G. Weisdorf D.J. Costs of hematopoietic cell transplantation: comparison of umbilical cord blood and matched related donor transplantation and the impact of posttransplant complications.Biol Blood Marrow Transplant. 2009; 15: 564-573Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. A critical part of engraftment is the recruitment of primitive hematopoietic progenitor cells (HPC) to the BM. This process is likely governed by a cascade of molecular interactions between members of the selectin, integrin, and CD44 superfamilies of adhesion molecules expressed by HPC and their various counter-receptors expressed by the microvasculature of the BM [14Frenette P.S. Subbarao S. Mazo I.B. von Andrian U.H. Wagner D.D. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.Proc Natl Acad Sci U S A. 1998; 95: 14423-14428Crossref PubMed Scopus (309) Google Scholar, 15Mazo I.B. Gutierrez-Ramos J.C. Frenette P.S. et al.Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.J Exp Med. 1998; 188: 465-474Crossref PubMed Scopus (364) Google Scholar, 16Katayama Y. Hidalgo A. Furie B.C. et al.PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin.Blood. 2003; 102: 2060-2067Crossref PubMed Scopus (151) Google Scholar, 17Papayannopoulou T. Craddock C. Nakamoto B. Priestley G.V. Wolf N.S. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.Proc Natl Acad Sci U S A. 1995; 92: 9647-9651Crossref PubMed Scopus (424) Google Scholar, 18Papayannopoulou T. Priestley G.V. Nakamoto B. Zafiropoulos V. Scott L.M. Molecular pathways in bone marrow homing: dominant role of alpha(4)beta(1) over beta(2)-integrins and selectins.Blood. 2001; 98: 2403-2411Crossref PubMed Scopus (164) Google Scholar, 19Vermeulen M. Le P.F. Gagnerault M.C. et al.Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells.Blood. 1998; 92: 894-900Crossref PubMed Google Scholar, 20Dimitroff 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. 2001; 153: 1277-1286Crossref PubMed Scopus (232) Google Scholar, 21Avigdor A. Goichberg P. Shivtiel S. et al.CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow.Blood. 2004; 103: 2981-2989Crossref PubMed Scopus (431) Google Scholar]. The initial step associated with homing is the rolling of HPC on the vascular endothelium of the hematopoietic microenvironment [14Frenette P.S. Subbarao S. Mazo I.B. von Andrian U.H. Wagner D.D. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.Proc Natl Acad Sci U S A. 1998; 95: 14423-14428Crossref PubMed Scopus (309) Google Scholar, 15Mazo I.B. Gutierrez-Ramos J.C. Frenette P.S. et al.Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.J Exp Med. 1998; 188: 465-474Crossref PubMed Scopus (364) Google Scholar, 16Katayama Y. Hidalgo A. Furie B.C. et al.PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin.Blood. 2003; 102: 2060-2067Crossref PubMed Scopus (151) Google Scholar, 17Papayannopoulou T. Craddock C. Nakamoto B. Priestley G.V. Wolf N.S. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.Proc Natl Acad Sci U S A. 1995; 92: 9647-9651Crossref PubMed Scopus (424) Google Scholar, 18Papayannopoulou T. Priestley G.V. Nakamoto B. Zafiropoulos V. Scott L.M. Molecular pathways in bone marrow homing: dominant role of alpha(4)beta(1) over beta(2)-integrins and selectins.Blood. 2001; 98: 2403-2411Crossref PubMed Scopus (164) Google Scholar, 19Vermeulen M. Le P.F. Gagnerault M.C. et al.Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells.Blood. 1998; 92: 894-900Crossref PubMed Google Scholar, 20Dimitroff 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. 2001; 153: 1277-1286Crossref PubMed Scopus (232) Google Scholar, 21Avigdor A. Goichberg P. Shivtiel S. et al.CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow.Blood. 2004; 103: 2981-2989Crossref PubMed Scopus (431) Google Scholar, 22Schweitzer K.M. Dräger A.M. van der Valk P. et al.Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues.Am J Pathol. 1996; 148: 165-175PubMed Google Scholar, 23McEver R.P. Moore K.L. Cummings R.D. Leukocyte trafficking mediated by selectin-carbohydrate interactions.J Biol Chem. 1995; 270: 11025-11028Crossref PubMed Scopus (584) Google Scholar, 24Peled A. Grabovsky V. Habler L. et al.The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.J Clin Invest. 1999; 104: 1199-1211Crossref PubMed Scopus (443) Google Scholar], an interaction mediated, at least in part, by P- and E-selectins constitutively expressed by the BM endothelium and selectin ligands expressed by the HPC, including P-selectin glycoprotein ligand (PSGL)-1 (CD162) [25Hidalgo A. Weiss L.A. Frenette P.S. Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally.J Clin Invest. 2002; 110: 559-569Crossref PubMed Scopus (109) Google Scholar]. The delayed engraftment and elevated risk of graft failure observed for CB recipients are due, at least in part, to low total nucleated cell [26Rubinstein P. Carrier C. Scaradavou A. et al.Outcomes among 562 recipients of placental-blood transplants from unrelated donors.N Engl J Med. 1998; 339: 1565-1577Crossref PubMed Scopus (1172) Google Scholar, 27Gluckman E. Rocha V. Arcese W. et al.Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice.Exp Hematol. 2004; 32: 397-407Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar] and CD34+ cell [28Wagner J.E. Barker J.N. Defor T.E. et al.Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival.Blood. 2002; 100: 1611-1618Crossref PubMed Scopus (47) Google Scholar] dose transplanted. However, there is also evidence that CB CD34+ cells possess a defect in homing to BM [29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar, 30Hidalgo 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. 2005; 105: 567-575Crossref PubMed Scopus (43) Google Scholar]. Appropriate carbohydrate modification (fucosylation) of selectin ligands appears critical for the rolling of primitive HPC on P- and E-selectins expressed by the hematopoietic microvasculature [23McEver R.P. Moore K.L. Cummings R.D. Leukocyte trafficking mediated by selectin-carbohydrate interactions.J Biol Chem. 1995; 270: 11025-11028Crossref PubMed Scopus (584) Google Scholar, 29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar, 30Hidalgo 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. 2005; 105: 567-575Crossref PubMed Scopus (43) Google Scholar]. CB CD34+ cells interact poorly with selectins when compared with CD34+ cells from PB or BM. A correlation between the level of CB CD34+ fucosylation and binding to P- and E-selectins exists with more heavily fucosylated CD34+ cells exhibiting a greater affinity for P- and E-selectins [29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar]. Consistent with this observation, we demonstrate that fucosylated rather than nonfucosylated CD34+ cells are responsible for engraftment in the NSG mouse model [31Shultz L.D. Lyons B.L. Burzenski L.M. et al.Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.J Immunol. 2005; 174: 6477-6489PubMed Google Scholar]. These observations provide a rationale for increasing the level of CD34+ cell surface fucosylation with the goal of improving CB engraftment. Levels of cell surface fucosylation can be increased by ex vivo treatment with α1-3 fucosyltransferase (FT)-VI [29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar, 30Hidalgo 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. 2005; 105: 567-575Crossref PubMed Scopus (43) Google Scholar, 32Kobzdej 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. 2002; 100: 4485-4494Crossref PubMed Scopus (44) Google Scholar]. Increased levels of fucosylation augment the level of P- and E-selectin binding of CD34+ cells and increase the levels of rolling on P- or E-selectin in vitro [29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar] and BM microvasculature in vivo [30Hidalgo 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. 2005; 105: 567-575Crossref PubMed Scopus (43) Google Scholar]. However, the impact of ex vivo fucosylation on hematopoietic engraftment of CB remains unclear, with different groups reporting opposing findings [29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar, 30Hidalgo 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. 2005; 105: 567-575Crossref PubMed Scopus (43) Google Scholar]. A correlation between the level of fucosylation of engrafting hematopoietic cells and their interaction with selectins expressed by the hematopoietic microvasculature is consistent with a potentially key role for fucosylation in recruitment to the BM. Ultimately, a better understanding of the deficiencies in homing of CB CD34+ cells will be necessary to enable the development of more effective CB transplantation techniques. Here we used the NSG mouse model to further explore the impact of fucosylation on the rate and magnitude of CB engraftment, with the goal of improving engraftment after CB transplantation in the clinic. Samples were provided under University of Texas MD Anderson Cancer Center Institutional Review Board−approved protocols. All animal work was performed under University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee−approved protocols. Unless otherwise stated, CB CD34+ cells were selected by magnetic-activated cell sorting (MACS) according to manufacturer’s instructions (CD34 Reagent; Miltenyi Biotec, Auburn, CA, USA). CB CD34+ cells were incubated at 106 cells/mL for 30 minutes at room temperature with 1 mM GDP β-fucose (EMD Biosciences, San Diego, CA, USA), 1 mM MnCl2 in 1 mL Hank’s Buffered Saline Solution (HBSS) containing 1% human serum albumin (HSA; Baxter Healthcare Corp., Westlake Village, CA, USA) and 94 mU/mL FT-VI (America Stem Cell, Inc., Carlsbad, CA, USA). After incubation, cells were washed in HBSS containing 1% HSA. Levels of fucosylation were determined by flow cytometry. Fucosylation is characterized by the presence of sialyl Lewis X residues that are revealed by flow cytometry using antibody HECA-452 (BD Biosciences, San Jose, CA, USA) against cutaneous lymphocyte antigen (CLA). CLA shares a carbohydrate domain with the sialyl Lewis X antigen [23McEver R.P. Moore K.L. Cummings R.D. Leukocyte trafficking mediated by selectin-carbohydrate interactions.J Biol Chem. 1995; 270: 11025-11028Crossref PubMed Scopus (584) Google Scholar, 29Xia 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. 2004; 104: 3091-3096Crossref PubMed Scopus (151) Google Scholar, 33Xia L. Sperandio M. Yago T. et al.P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow.J Clin Invest. 2002; 109: 939-950Crossref PubMed Scopus (219) Google Scholar]. Briefly, cells were stained with combinations of fluorochrome-conjugated anti-CD34, anti-CD45 and anti-CLA (HECA 452) antibodies (all from BD Biosciences) or with isotype controls. Antibody staining was revealed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and analysis performed using CELLQuest Pro software (Becton Dickinson). MACS isolated CD34+ cells from multiple CB units were pooled and stained with HECA-452 to reveal sialyl Lewis X residues indicative of fucosylation. Cells were resuspended in buffer containing 0.6 μg/mL 4′,6-diamidino-2-phenylindole (Invitrogen) to allow exclusion of dead cells and separated by fluorescence-activated cell sorting, using a FACSAria II (BD Bioscience, San Jose, CA, USA) fitted with a 100-μm nozzle and Blue Diode 488, HeNe 633, and UV 355 lasers, into the following fractions: (1) total CD34+, containing both fucosylated (HECA+) and unfucosylated (HECA−) CD34+ cells; CD34+HECA+, containing only fucosylated CD34+ cells, and (3) CD34+HECA−, containing only unfucosylated CD34+ cells. Recipient NSG mice were sublethally irradiated (320 cGy using a 137Cs source delivered at approximately 300 cGy/min; J. L. Shepherd and Associates Mark I-25 Irradiator, San Fernando, CA, USA) and sufficient cells collected to provide an intravenous dose of >104 total CD34+, CD34+HECA+, or CD34+HECA− cells/mouse (five mice per group). Human engraftment was evaluated by flow cytometric analysis of BM and spleens of mice >6 weeks after transplantation using human CD45 and mouse CD45 antibodies (BD Biosciences), a FACSCalibur flow cytometer (Becton Dickinson), and CELLQuest Pro software (Becton Dickinson). The human chronic myelogenous leukemia cell line K562 produces solid tumor masses in the liver, kidney, and other organs, rather than engrafting to BM [34Matsuura N. Puzon-McLaughlin W. Irie A. Morikawa Y. Kakudo K. Takada Y. Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.Am J Pathol. 1996; 148: 55-61PubMed Google Scholar]. Although CB CD34+ cells express PSGL-1 [16Katayama Y. Hidalgo A. Furie B.C. et al.PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin.Blood. 2003; 102: 2060-2067Crossref PubMed Scopus (151) Google Scholar, 35Winkler I.G. Snapp K.R. Simmons P.J. Levesque J.P. Adhesion to E-selectin promotes growth inhibition and apoptosis of human and murine hematopoietic progenitor cells independent of PSGL-1.Blood. 2004; 103: 1685-1692Crossref PubMed Scopus (33) Google Scholar, 36Greenberg A.W. Kerr W.G. Hammer D.A. Relationship between selectin-mediated rolling of hematopoietic stem and progenitor cells and progression in hematopoietic development.Blood. 2000; 95: 478-486Crossref PubMed Google Scholar], K562 cells do not (upper panel). K562 cells expressing PSGL-1 (K562/PSGL-1) were generated by electroporation (Cell-Porator; Gibco BRL, Carlsbad, CA, USA) with pcDNA3.1 (Invitrogen) containing the full complementary DNA for human PSGL-1 and a neomycin resistance cassette. Cells were then selected at low density in medium containing G418 (1 mg/mL), stained with anti-human PSGL-1 antibody KPL-1 (BD Biosciences) and isolated by fluorescence-activated cell sorting. To measure BM homing, K562/PSGL-1 and parental K562 lines were labeled with carboxyfluorescein succinimidyl ester (CFSE). Cells (106/mL) were incubated for 10 minutes at 37°C in 10 μM CFSE (Molecular Probes Invitrogen, Eugene, OR, USA), washed, incubated in medium for 1 hour at 37°C to allow efflux of unbound stain and cells treated ± FT-VI. The following treatment groups were generated: untreated K562, FT-VI−treated K562, untreated K562/PSGL-1, and FT-VI−treated K562/PSGL-1. Cells were injected intravenously into sublethally irradiated NSG recipients at 5 × 106 cells/mouse (three mice per group). Mice were euthanized approximately 16 hours after injection, femora were removed, and marrow suspensions prepared for flow cytometric assessment of the levels of CFSE-positive (human K562) cells present, relative to marrow suspensions from control (uninjected) mice. 4′,6-Diamidino-2-phenylindole (0.6 μg/mL; Invitrogen) was added to identify nonviable cells and >500,000 viable events acquired (LSRII flow cytometer; Becton Dickinson) and analyzed (CELLQuest Pro software; Becton Dickinson). CD34+ cells were selected from a pool of fresh CB units using the Isolex Magnetic Cell Selection System (Baxter Healthcare Corp.) according to manufacturer’s instructions. After labeling with CFSE and allowing efflux of unbound stain, cells were divided across five treatment groups. Groups 2 to 4 were not treated with FT-VI, but assessed the impact of the following buffer components: group 2, HBSS containing 0.5% HSA (HBSS/HSA); group 3, HBSS/HSA with 1 mM βDFucose; and group 4, HBSS/HSA with 1 mM βDFucose + 1 mM manganese (Mn2+). Groups 5 and 6 were treated with FT-VI ± Mn2+: group 5, HBSS/HSA with 1 mM βDFucose + FT-VI and group 6, HBSS/HSA with 1 mM βDFucose + 1 mM Mn2+ + FT-VI. After incubation for 30 minutes at room temperature, cells were washed and numbers determined. Ex vivo fucosylation reaction was confirmed by flow cytometry using the HECA-452 antibody. Sublethally irradiated NSG recipients (n = 3/group) received 3 × 105 CFSE-labeled CB CD34+ cells intravenously. Group 1 received no cells and provided negative background data. Mice were euthanized approximately 18 to 20 hours after injection, femora were removed and BM cell suspensions stained with APC-conjugated anti-huCD34 and PE-Cy 7−conjugated anti-human CD38 or isotype controls (BD Bioscience) to determine whether CFSE+ cells homing to the BM were skewed toward more primitive (CD34+38−) or more mature (CD34+38+) HPC. 4′,6-Diamidino-2-phenylindole was used to exclude dead cells and data acquired using an LSRII flow cytometer (Becton Dickinson). A mean of >106 viable events were acquired for each sample and analyzed using Diva Software (Becton Dickinson). Primitive HPC with the capacity to engraft immunodeficient mice represent a small proportion of total CD34+ cells. Although as many CB CD34+ cells were infused per mouse as was practically possible in the CB CD34+ ± FT-VI experiment described here, and large data files were acquired during flow cytometry, concerns were raised that the rarity of human events might not provide sufficient power to accurately detect differences in BM homing as a result of treatment with FT-VI. To address this concern, a parallel experimental approach was adopted. The content of human myeloid (colony-forming unit granulocyte-macrophage [CFU-GM]), erythroid (burst-forming unit erythroid [BFU-E]), and multipotent (colony-forming unit granulocyte-erythrocyte-monocyte-megakaryocyte [CFU-GEMM]) clonogenic progenitors in the BM of NSG mice was measured at early time points (7, 14, and 21 days) after transplantation of purified CD34+ cells ± FT-VI. We reasoned that treatments resulting in enhanced numbers of human clonogenic HPC at these early times after CD34+ cell infusion would reflect increased recruitment of primitive HPC to the BM. The ability of these CFU assays to detect only human HPC would be confirmed by the absence of colonies in cultures performed with BM from sublethally irradiated, nontransplanted NSG mice. Marrow cells were plated in triplicate 1-mL cultures in 35-mm plates in medium comprising 0.9% methyl cellulose in Iscove’s modified Dulbecco’s medium supplemented with 30% fetal bovine serum and 3 mM l-glutamine. Growth of human CFU was stimulated by the addition of recombinant human interleukin 3 (IL-3; 10 ng/mL), human granulocyte-macrophage colony stimulating factor (GM-CSF; 10 ng/mL), human stem cell factor (SCF; 50 ng/mL) (all from R&D Systems, Minneapolis, MN, USA) and human erythropoietin (4 U/mL) (Epoetin alfa; Amgen, Inc., Thousand Oaks, CA, USA). After 14 days of incubation at 37°C in a 5% CO2, fully humidified atmosphere, colonies were scored using an inverted microscope. CB CD34+ cells were divided into two fractions, treated ± FT-VI, washed and equivalent numbers of fucosylated or untreated cells injected into groups of sublethally irradiated NSG mice (n = 5 mice/group). Fucosylation reactions were initially performed using 10 mM Mn2+, however, this dose was associated with significant hematotoxicity. A signif
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