60/494, 317 filed August 11, 2003, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Number RO1 DK-56638 awarded by the National Institutes of Health. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION Hematopoietic progenitor cells (HPCs) can be transplanted to the bone marrow (BM) of another individual following a simple intravenous infusion. The ability of HPCs to home to the BM was first demonstrated by Jacobson et al. , who showed that shielding of the spleen allowed mice to recover from a lethal dose of radiation. Jacobson et al. (1949) J. Lab. Clin. Med. 34: 1538-1543. This study and others established that pluripotent stem cells present in the spleen or infused exogenously could repopulate distant hematopoietic organs. Lorenz et al. (1951) J.

Natl. Cancer Inst. 12: 197-201; Barnes et al. (1956) Br. Med. J. 2: 626-627. This feature is the basis for the clinical use of BM transplantation in treatment of hematologic malignancies. Thomas (1995) Perspect. Biol. Med. 38: 230-237.

However, despite an advanced knowledge of the mechanisms that allow the migration of mature leukocytes into inflamed tissues, the mechanisms of human HPC homing to the BM are still poorly understood.

The migration of leukocytes to sites of inflammation is initiated by labile but critical adhesive interactions (attachment and rolling) that are largely mediated by the selectin family and its glycoconjugated ligands. Two members of the selectin family, P-and E-selectins, are expressed on endothelial cells. Frenette et al. (1996) N. Eng. J.

Med. 335: 43-45; Kansas (1996) Blood 88: 3259-3287. P-selectin is stored in granules of endothelial cells and platelets and is rapidly translocated to the cell surface after stimulation with various secretagogues. E-selectin expression is induced by endotoxin or inflammatory cytokines. Although the E-selectin gene is silent in cultured endothelial cells, low levels of E-selectin are expressed in most tissues in vivo and regulate leukocyte homeostasis together with endothelial P-selectin.

Frenette et al. (1996) Cell 84: 563-574. Mice lacking both P-and E-selectins display severe leukocytosis and can develop spontaneous skin infections. Bullard et al.

(1996) J. Exp. Med. 183 : 2329-2336. The main ligand for P-selectin, P-selectin glycoprotein ligand-l (PSGL-1), is a disulfide-bonded homodimeric mucin-like glycoprotein expressed on leukocytes, platelets, and CD34+ cells. Moore et al. (1992) J. Cell Biol. 118: 445-456; Frenette et al. (2000) J. Exp. Med. 191: 1413-1432; Zannettino et al. (1995) Blood 85: 3466-3477; Tracey et al. (1996) Exp. Hematol.

24: 1494-1500. PSGL-1 requires specific posttranslational modifications in order to be functional. These include sialylation and fucosylation of O-linked sugars, as well as sulfation of tyrosine residues present in the N-terminus of the protein. Moore et al.

(1998) Leuk. Lymphoma 29: 1-15. While P-and E-selectin are critical for myeloid cell rolling in venules of the systemic circulation, the leukocyte selectin (L-selectin) mediates lymphocyte rolling in specialized venules of secondary lymphoid organs.

The close interaction between leukocytes and endothelial cells during the rolling step allows the activation of leukocyte integrins triggered by chemokines present on the surface of the endothelium. This leads to the firm adhesion and migration of leukocytes through the vessel wall into the extravascular space. Springer (1995) Annu. Rev. Physio. 57: 827-872. Whether a similar paradigm is applicable for immature hematopoietic precursors is not known. Studies in mice suggest that the recruitment of HPCs into the BM requires multiple adhesion pathways, including endothelial selectins and VCAM-1. Papayannopoulous et al. (1995) Proc. Natl. Acad.

Sci. USA 92: 9647-9651 ; Frenette et al. (1998) Proc. Natl. Acad. Sci. USA 95 : 14423- 14428.

The study of primitive human hematopoiesis in vivo has been facilitated by the generation of NOD/LtSz-scid/scid mice (hereafter referred to as NOD/SCID mice) that have multiple defects in innate and adaptive immunologic functions.

Shultz et al. (1995) J. Immunol. 154: 180-191. The BM of NOD/SCID mice can be repopulated by human HPCs (CD34+ cells) after sublethal doses of radiation. Dick et al. (1997) Stem Cells 15 (Suppl. 1): 199-207.

Clinically, human HPCs are obtained from three different sources: BM, mobilized peripheral blood (mPB), and cord blood (CB). CB-derived progenitors represent an especially promising source of human hematopoietic stem cells because they are widely available, easy to harvest and their use is associated with reduced graft-versus-host disease. Rocha et al. (2001) Blood 97: 2962-2971. However, transplantation using CB cells has been restricted mostly to children due to the limited number of cells available in the placenta. Rubinstein et al. (1998) N. Engl. J. Med.

339: 1565-1577. Further, significant delays in platelet and myeloid engraftment have been reported in CB transplantation. Rubenstein et al. , supra.; Barker et al. (2001) Blood 97-2957-2961. Further, CB cells migrate to the BM less efficiently than stem cells from other sources.

Accordingly, there is a need in the field to understand the molecular mechanisms mediating the interactions of CB-derived HPCs with the BM microvasculature, and thereby to improve therapies using CB derived HPCs.

SUMMARY OF THE INVENTION The present invention is directed to cord blood hematopoietic progenitor cells in which selectin ligands have increased fucosylation relative to native cord blood hematopoietic progenitor cells. The invention also provides a method of making such cells. Cord blood hematopoietic cells in which selectin ligands have increased fucosylation exhibit a statistically significant increase in P-selectin binding relative to native cord blood hematopoietic cells.

In another embodiment, the invention provides cord blood hematopoietic progenitor cells that have enhanced ability to home to and engraft in bone marrow relative to native cord blood hematopoietic progenitor cells, and a method of making such cells.

The present invention further provides a method of increasing the ability of cord blood hematopoietic progenitor cells to home to and engraft in bone marrow.

In another embodiment the present invention provides a method of bone marrow transplantation utilizing fucosylated cord blood hematopoietic progenitor cells. The methods of the present invention are useful for the treatment of diseases and disorders including anemia, immune deficiencies, cancer, genetic disorders of hematopoiesis, inherited storage diseases, thalassemia and sickle cell disease. In particular, the present method is useful for the treatment of lymphoblastic leukemia, myelogenous leukemia, Hodgkin's lymphom, non-Hodgkin's lymphom, myelodysplastic syndrome, sickle cell anemia, plastic anemia and thalassemia.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a bar graph depicting human CD34+ cell rolling and arrest in collecting venules and sinusoids of the BM in NOD/SCID mice. CFSE-labeled human CD34+ cells purified from mPB were injected into NOD/SCID mice, and the fractions of cells rolling or arrested in collecting venules or sinusoids were determined by IVM analysis of videotapes. Bars represent mean SEM. The differences between Cv and sinusoids for the number of rolling and arrested cells are not significant (P = 0.14 and 0.6, respectively). n = 7 different human donors and the same number of mice.

Fig. 2 demonstrates the role of P-and E-selectins and VCAM-1 in the initial interactions of human CD34+ cells with BM microvessels. Fluorescently labeled human CD34+ cells purified from mPB were injected into P/E/-or wild-type mice backcrossed into the NOD/SCID background. Mice were injected via carotid catheter with control rat IgG (or no Ab in two experiments) and then with labeled CD34+ cells.

After 15 minutes of recording, fluorescent cells were completely cleared from the circulation. The same mouse was then injected with anti-VCAM-1 and a second bolus of fluorescent CD34+ cells. Horizontal bars represent mean values. Each dot represents a single experiment before (filled squares) or after anti-VCAM-1 injection (open circles). *P = 0.05 by paired Student t test; * *P = 0. 003 by unpaired Student t test compared with IgG group in wild-type animals.

Figs. 3a-3e demonstrate the role of P-and E-selectins in the homing of human CD34+ cells to the BM of NOD/SCID mice. Sublethally irradiated NOD/SCID or P/E/-NOD/SCID mice were injected intravenously with 9 X 107 human MNCs from mPB samples. Cells were allowed to home for 2 hours, and the femoral BM was harvested. (a) Double staining of the transplanted human MNCs for human CD34 and CD45. (b) BM cells recovered from an uninjected control mouse stained for human CD34 and CD45. (c) Wild-type NOD/SCID mouse transplanted with human MNCs.

(d) P/E/~ NOD/SCID mouse transplanted with human MNCs. (e) Number of human CD34+ cells homed per femur in transplanted wildtype NOD/SCID and P/E-/- NOD/SCID mice. The percentage of gated CD34+/CD45+ human cells (upper region in the scattergrams in c-d) in the recipient BM was determined by FACS analysis, and the number of homed cells per femur was calculated based on the femoral cellularity. The number of homed human CD34+ cells is drastically reduced in P/E-/- NOD/SCID mice. n = 6 mice injected with MNCs from two different human donors.

*P = 0.0008.

Fig. 4 is a graph providing a comparative analysis of the interactions of CD34+ cells with BM microvessels in NOD/SCID mice. Fluorescently labeled CD34+ cells from steady-state human BM, mPB, or CB were injected into NOD/SCID mice, and the numbers of cells interacting (rolling or arrested) with the BM microvasculature were determined by analysis of video recordings from fluorescence intravital microscopy experiments. n = 7-10 mice and human donors. *P < 0. 02 compared with BM and mPB. * *P = 0. 03.

Figs. 5a-5d demonstrate binding of soluble P-selectin and PSGL-1 expression on CB and mPB CD34+ cells. (a) MNCs obtained from CB or mPB samples were stained for CD34 and P-selectin-IgG binding in the presence or absence of 5 mM EDTA. Data represent geometric mean of fluorescence SEM of P-selectin-IgG binding on gated CD34+ cells. n = 8 separate experiments from different donors. *P = 0.008. (b) Scattergram profiles of CB and mPB MNCs stained for CD34+ cells and P-selectin-IgG in the presence or absence of 5 mM EDTA. In the presence of divalent cations, virtually all mPB CD34+ cells bind P-selectin-IgG, whereas two distinct subpopulations are present among CB CD34+ cells: one that binds P-selectin- IgG normally (~70%) and one that does not bind soluble P-selectin (-30%). (c) Expression of PSGL-1 in CB and mPB CD34+ cells. (d) PSGL-1 is the sole P-selectin ligand on CB and mPB CD34+ cells. MNCs were preincubated with 2 llg of function- blocking (KPL1) or nonblocking (PSL-275) anti-PSGL-1 before staining to detect P- selectin-IgG chimera binding and CD34. The numbers indicate the percentage of CD34+ cells in each quadrant. The scattergrams represent one of at least five replicates.

Figs 6a and 6b are graphs demonstrating the role of P-selectin and PSGL-1 in CD34+ cell interactions with BM microvessels. Fluorescently labeled CD34+ cells from mPB (a) or CB (b) were preincubated with anti-PSGL-1 (black bars) or isotype matched control Ab and injected into wild-type or E-/-NOD/SCID mice that had been treated with anti-P-selectin (white bars) or isotype matched control Ab (gray bars).

The number of cells interacting with the BM microvasculature was determined by analysis of video recordings from fluorescence intravital microscopy experiments. *P < 0.02 compared with control Ab; **P = 0.06 compared with control Ab; #P < 0.02 compared with mPB control Ab group shown in a.

Figs. 7a and 7b demonstrate that engraftment of CB CD34+ cells in NOD/SCID mouse BM is not compromised. CD34+ cells isolated from CB or healthy mPB donors were transplanted into sublethally irradiated (350-375 cGy) NOD/SCID animals or NOD/SCID mice deficient in ß2 microglobulin (NOD/SCID-p2mnull).

Four weeks after transplantation, mice were sacrificed to assess the levels of human- derived CD45+ cells (a) or colony-forming progenitors (b) in their BM. There is a trend toward higher engraftment of CB-derived CD34+ cells (P = 0.07 for CD45+ and P = 0.1 for CFC content). Each circle in a represents an individual mouse.

Figs. 8a-d provide scattergram profiles showing binding of P-and E-selectin to cord blood CD34+ cells (8a, b), and showing that CD34+ cells that do not bind selectins lack the HECA 452 epitope.

Fig. 9 presents graphs showing that cells that do not bind selectin and lack the HECA 452 epitope are also unable to bind E-selectin. The upper panel shows the strategy to separate CD34+ cells on the basis of selectin binding.

Fig. 10a shows RT-PCR profiles of enzymes involved in posttranslational modifications of selectin ligands. Fig. 1 Ob is a bar graph depicting fucosyl transferase activity in populations of CD34+ cells that bind P-selectin compared to cells that do not bind P-selectin.

Fig. 11 provides histograms demonstrating selectin binding activity in cells treated with FTV1 and in untreated cells.

Figs. 12a and b are bar graphs demonstrating that FTV1 treatment does not affect the activity of the chemokine receptor CXCR4 or the integrin receptor VCAM- 1.

Fig. 13 is a bar graph showing the percentage of rolling and arrested CD34+ cells after injection in NOD/SCID mice.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a population of cord blood hematopoietic progenitor cells which have increased fucosylation of selectin ligands relative to native cord blood hematopoietic progenitor cells. The term hematopoietic progenitor cells, or HPCs, as used herein includes hematopoietic progenitor and stem cells. It has been discovered in accordance with the present invention that such populations of cord blood cells having increased fucosylation exhibit increased interactions with the bone marrow microvasculature, and thus increased homing and enhanced bone marrow engraftment. Such cells are thus useful for bone marrow transplantation.

Cord blood hematopoietic progenitor cells may be obtained from human umbilical cord blood. Methods for obtaining umbilical cord blood are know in the art and disclosed for example in U. S. Patent Nos. 6, 461, 645 ; 6,179, 919 ; and 6, 102, S71, the disclosures of which are incorporated herein by reference. Generally, cord blood is collected from the cut end of the umbilical cord after normal full term obstetrical delivery into a sterile collection container with an anticoagulant.

Cord blood may be enriched for hematopoietic progenitor cells by methods known to those of ordinary skill in the art, including for example physical and immunological cell separation techniques. The progenitor cells are present in the low density fraction of the cord blood, and thus in a preferred embodiment the low density fraction is first separated, for example by known density separation procedures. In a preferred embodiment, low density mononuclear cells are collected after centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, N. J. ) having a density of 1. 077gm/cm3.

The low density fraction may be further enriched for HPCs by positive or negative immunological selection using antibodies that recognize cell surface determinants. For example, the CD34 antigen is a marker for early hematopoietic cells and thus antibodies that recognize CD34 antigen may be used to isolate HPCs.

In a preferred embodiment, CD34+ cells are isolated from the low density mononuclear cell fraction, for example by using a commercially available kit such as the mini MACS kit (Miltenyi) or the Easy Sep kit (Stem Cell Technologies, Vancouver, Canada).

Such separation methods yield populations having a purity of isolated CD34+ cells ranging from 70% to 95%, which purities are suitable for use in accordance with the present invention. The isolated HPCs may be maintained in culture conditions known in the art to be suitable for maintaining and expanding the primitive stem cell fraction and the P-selectin binding and non-binding populations in cord blood cells.

Suitable culture conditions are disclosed for example by Ueda et al. (2000) J. Clin.

Invest. 105: 1013-1021, the disclosure of which is incorporated by reference herein.

It has been discovered in accordance with the present invention that a subset of the population of cord blood derived CD34+ cells does not bind P-selectin and E- selectin, and further that this subset exhibits deficient fucosylation. It has further been discovered that treatment of the cord blood derived CD34+ cells with a fucosyltransferase provides a population of cells which have more fucosylated selectin ligands than an untreated population of cells. Further, more cells in the treated population are capable of binding to P selectin and E-selectin relative to the untreated population.

Accordingly, the method of making the population of cord blood HPCs which have increased fucosylation comprises obtaining a population of cord blood HPCs as described hereinabove, and treating such cells with a fucosyltransferase under conditions whereby unfucosylated selectin ligands on such cells are fucosylated. The fucosyltransferase may be a native or recombinant enzyme. Recombinant fucosyltransfease is preferred. In a preferred embodiment the fucosyltransferase is an al, 3-fucosyltransferase. The al, 3-fucosyltransferase VII (FTVII) expressed in hematopoietic cells may be used. In a preferred embodiment the fucosyltransferase is recombinant al, 3-fucosyltransferase VI (FTVI), which is not natively expressed in hematopoietic cells, but has a broader range of substrates than FTVII. Recombinant FTVI is commercially available.

Suitable conditions for fucosylation comprise a buffer, a fucose source and time and temperature sufficient for fucosylation. Such conditions can be determined by those of ordinary skill in the art. A preferred fucose source is guanosine 5'- diphospho-L-fucose (GDP-fucose), which is commercially available. Conditions further include the presence of cofactors for the fucosyltransferase such as manganese.

For example, cord blood HPCs may be treated with fucosyltransferase as follows. From about one-half million to five million freshly purified cord blood derived CD34+ cells are washed in a buffer such as Hanks Balanced Salt Solution (HBSS) buffered with 20mm HEPES, pH 7.4. The cells are then incubated in HBSS/0. 1% human serum albumin (HSA) containing 10mm MgCI2, ImM GDP- fucose and 20mU/ml recombinant human al, 3-fucosyltransferase VI for about 45 minutes at about 37°C. The reaction may be stopped by washing the cells with buffer, for example PEB buffer (1X PBS, O. 1mM EDTA, 0.5% BSA fraction V) at 4°C.

One of ordinary skill in the art can verify that the selectin ligands have been fucosylated. For example, the cells may be incubated with human P-selectin IgM chimeras followed by incubation with FITC-conjugated anti-human IgM antibody recognizing the u chain of the chimeras, followed by flow cytometry. Methods of making such chimeras are known in the art and disclosed for example by Maly et al.

(1996) Cell 86 : 643-653 and Hidalgo et al. (2002) J. Clin. Invest. 110: 559-569, the disclosures of which are incorporated herein by reference. Alternatively, the P- selectin IgG chimeras may be preconjugated with biotinylated protein A, and the cells subsequently incubated with streptavidin-FITC followed by flow cytometry. Binding of the P-selectin chimera to the treated cells is an indication that the selectin ligand has been fucosylated. Accordingly, flow cytometry or a similar assay may be used to compare treated and untreated cells. A statistically significant increase in P-selectin binding to the treated versus the untreated cells indicates that a population of cord blood HPCs having increased fucosylation of selectin ligands relative to native cells has been obtained. In a preferred embodiment, at least 70%, and preferably at least 80%, and more preferably at least 90% of the cells in the population of treated cord blood hematopietic are capable of binding to P-selectin.

Similarly, fucosylation of the selectin ligand may be assessed utilizing an antibody that recognizes a fucosylated moiety, such as for example the HECA-452 (cutaneous lymphocyte antigen, CLA) antibody, which recognizes fucosylated/sialylated selectin ligand and is commercially available It has been discovered in accordance with the present invention that cord blood HPCs that have been fucosylated as described hereinabove have enhanced ability to home to and engraft in bone marrow relative to untreated cord blood HPCs.

Accordingly, the present invention further provides a method of bone marrow transplantation utilizing the fucosylated cord blood HPCs.

Hematopoietic reconstitution by bone marrow transplantation is useful in the treatment of various diseases and disorders including for example certain types of anemia, immune deficiencies, cancer, genetic disorders of hematopoiesis, inherited storage diseases, thalassemia, and sickle cell disease. Hematopoietic reconstitution is also useful as a bone marrow rescue procedure for patients who have received chemotherapy or radiation. Both autologous and heterologous transplantation are contemplated in accordance. with the present invention. The cord blood HPCs of the present invention may be preserved and stored prior to use by methods known in the art and disclosed for example in U. S. Patent No. 6,461, 645, the disclosure of which is incorporated herein by reference.

Compositions comprising the fucosylated cord blood HPCs of the present invention and a pharmaceutically acceptable carrier are used for bone marrow transplantation in accordance with transplantation methods known in the art. Suitable carriers are those that are biologically and physiologically compatible with the recipient, such as buffered saline solution. Other carriers include water, isotonic common salt solutions, alcohols, polyols, glycerine and vegetable oils. The composition for administration must be formulated, produced and stored according to standard methods complying with proper sterility and stability. The compositions are introduced into a subject in need of a bone marrow transplant in an amount sufficient to reconstitute the hematopoietic system. Introduction may be by any means known in the art, including systemically, e. g., intravenously, into various delivery sites or directly into the bone Systemic infusion of cells is a preferred means of administration.

The following non-limiting examples serve to further illustrate the present invention. All references cited herein are incorporated herein in their entirety.

EXAMPLE 1 METHODS The following methods were used in Examples 2-8.

Antibodies and selectir chimeras. For in vivo studies, rat mAbs against mouse VCAM-1 (MK 2.7) and P-selectin (clone RB40.34) were purified from hybridoma supernatants (American Type Culture Collection, Rockville, Maryland, USA), and control rat IgG was obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

Potential endotoxin contamination was removed using a polymyxin B column (Detoxi-Gel ; Pierce Biotechnology Inc., Rockford, Illinois, USA). The rat anti- mouse E-selectin Ab (clone 9A9) was a gift of B. Wolitzky (MitoKor, San Diego, California, USA). Anti-human PSGL-1 (KPL1) and FITC-conjugated anti-human CD38 were purchased from Pharmingen (San Diego, California, USA), and phycoerythrin-conjugated (PE-conjugated) anti-human CD34 (clone AC136) was from Miltenyi Biotec (Bergisch Gladbach, Germany). The mouse anti-human PSGL- 1 Ab (PSL-275) and human P-selectin-IgG chimera were generously provided by R.

Schaub (Genetics Institute, Cambridge, Massachusetts, USA). Murine E-and P- selectin-IgM chimeras were produced by transfection of COS-7 cells with selectin- IgM DNA vector (a gift of John Lowe, University of Michigan, Ann Arbor, Michigan, USA) using the DEAE-dextran method described by Maly (1996) Cell 86 : 643-653.

Hzrman samples and CD34+ cell isolatiotz. Fresh human hematopoietic cell samples were obtained from unused portions of three different clinical sources: steady-state BM, mPB, and CB. BM samples were collected from normal donors at the time of harvest for allogeneic transplantation. mPB cells were collected by leukapheresis from either healthy donors (n = 19) or patients with hematopoietic malignancies in remission (n = 15). Both BM and mPB samples were obtained from the Mount Sinai Bone Marrow Transplantation Program. Umbilicl CB samples were obtained from normal fullterm deliveries ( : 38 weeks). All human samples were obtained in accordance with protocols approved by the Internal Review Board of Mount Sinai.

For the enrichment of CD34+ cells, low-density mononuclear cells (MNCs) were collected after centrifugation at 250 g over Ficoll-Hypaque (d = 1.077 g/ml).

After two washes in PBS containing 2 mM EDTA and 0.5% BSA, contaminating red blood cells (RBCs) were lysed in a 0.8% NH4CI solution. CD34+ cells were purified from the MNC fraction using the CD34-isolation mini-MACS kit (Miltenyi Biotec) following the manufacturer's instructions. The purity of the isolated CD34+ cells ranged from 70% to 99% (average-89% for CB and-95% for mPB). For intravital microscopy experiments, purified human CD34+ cells were fluorescently labeled by incubation with 33 uM carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes Inc. , Eugene, Oregon, USA) for 30 minutes at room temperature and washed three times in RPMI before injection into mice via the carotid artery catheter.

Alice. A NOD/SCID mouse colony was established from breeding pairs obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). Mice with P-and E-selectin double null mutations (P/E-/-) or a single E-selectin null mutation (E4-) (Frenette et al. (1996) Cell 84: 563-574) were backcrossed into the NOD/SCID background. After each generation, blood counts were monitored to select for the SCID mutation. Third generation littennates, which display a lymphopenia similar to that in pure NOD/SCID animals, were used to set up colonies of selectin knockouts and control wild-type NOD/SCID mice. Wild-type and selectin-deficient NOD/SCID mice had numbers of T (CD3) cells, B (B220) cells, and NK (NKl. 1) cells similar to the parental NOD/SCID pure strain as assessed by FACS analysis of the peripheral blood and BM. Transgenic mice expressing the enhanced green fluorescent protein under the control of the ß-actin promoter (EGFP-Tg mice ; a gift from M. Okabe, Osaka University, Japan) were bred in the barrier facility at Mount Sinai School of Medicine. Mice were fed with sterilely irradiated chow and autoclaved water.

Experimental procedures on animals were approved by the Animal Care and Use Committee at the Mount Sinai School of Medicine.

I7ltravital microscop) of the BAI. Mice 6-9 weeks old were used for BM intravital microscopy (BM-IVM). To avoid the retention of injected human cells in the spleen, mice were splenectomized upon weaning as described by Frenette et al.

(1998) Proc. Natl. Acad. Sci. USA 95: 14423-14428 and allowed to recover for at least 2 weeks before BM-IVM experiments. Mice were anesthetized by intraperitoneal injection (6 ml/kg) of 100 mg/ml urethane and 20 mg/ml chloralose (both from Sigma-Aldrich) in PBS. This anesthetic combination was chosen because it does not significantly alter blood flow in the BM microcirculation. The hair in the submandibular area of the neck and on the skullcap was removed using a hair removal lotion (Nair; Carter Products, New York, New York, USA). The trachea was cannulated with PE-160 polyethylene tubing to facilitate spontaneous respiration, and a PE-10 catheter (Becton, Dickinson and Co., Frai} clin Lakes, New Jersey, USA) was inserted into the left common carotid artery for injection of fluorescent cells and Abs.

The scalp was incised in the midline to expose the frontoparietal skull, and the conjunctive tissue covering the skull was carefully removed. A plastic ring was inserted in the incision area to allow application of endotoxin-free PBS. The mouse's cranium was kept in place using a stereotactic holder (David Kopf Instruments, Tujunga, California, USA). A mouse thus prepared was positioned under a fixed- stage, custom-designed intravital microscope (MM-40) equipped with a mercury fluorescent lamp and water immersion objectives (Nikon Corp. , Tokyo, Japan). To block potential sites of retention of human cells, 107 unlabeled human MNCs from each sample were injected immediately before the injection of labeled CD34+ cells.

Human CD34+ cells are rapidly cleared (within minutes) from the circulation of NOD/SCID mice after injection. Approximately 1 x 106 to 2 x 106 CD34+ cells were injected per experiment to visualize 50-500 cells in the left parietal BM using a lOx water-immersion objective (0.3 NA; Nikon Corp.). For determination of the hemodynamic parameters, 107 CFSE-labeled human MNCs or RBCs were injected into the carotid artery at the end of each experiment. In the experiments designed to determine the role of VCAM-1 or P-selectin, mice were preinjected with control rat IgG (or no IgG in two experiments), followed by labeled human CD34+ cells. When cells from the first injection were no longer detected in the circulation (10-15 minutes), the same mouse was injected with anti-VCAM-1 (80 ug) or anti-P-selectin (60 ug) at least 15 minutes before a second injection of labeled human CD34+ cells.

In the experiments aimed at evaluating the role of PSGL-1, fluorescently labeled CD34* cells were incubated with 2 ug KPL1 or control IgG per 106 cells prior to intracarotid injection. The images were captured using an SIT camera with a C2400 camera controller (Hamamatsu, Hamamatsu City, Japan) and were recorded using a VHS video recorder (SVO-9500MD ; Sony Corp. , Tokyo, Japan).

Irnage afralvsis. Vessel diameter and cell velocities were measured using a video caliper and sequential singleframe analysis. The maximal velocity (may), which represents the average velocity of free-flowing CFSElabeled CD34-cells or RBCs, was determined for each BM vessel. The mean blood flow velocity (via) was thus calculated as Vmean = Vmax(2 - #2), where s is the ratio of the CD34+ cell diameter (-7 urn) to the vessel diameter (Dv). The wall shear rate and critical velocity (Vcrit) were obtained from the following formulas: wall shear rate = 8 (Vmean/Dv), and Vent = Vmean x £ (2-£). Any cell traveling below Vcrit was considered to be rolling on the vessel wall. Cells that remained stationary for 5 seconds or more were considered arrested.

Flow cytometry and selectin chimera binding assay. For double staining of CD34 and P-selectin ligands, 106 total CB or mPB MNCs depleted of RBCs were washed and resuspended in staining buffer (RPMI, 5% FCS, and 0.02% NaN3). Fc receptors were blocked with I wu human IgG (Sigma-Aldrich), and cells were incubated with PE-conjugated anti-human CD34. After one wash, cells were incubated with 4 ug of the HUMAN P-selectin-IgG chimera that had been preconjugated with 0.3 llg biotinylated protein A. Preliminary experiments showed that this concentration saturates P-selectin ligands on CD34+ cells. Cells were subsequently incubated with an excess of streptavidin-FITC (1.5 wg ; Jackson ImmunoResearch Laboratories Inc. , West Grove, Pennsylvania, USA) and washed once before analysis using a FACSCalibur flow cytometer (Becton, Dickinson and Co. ). All incubations were performed for 20 minutes at 6°C. For triplecolor labeling with anti-CD34-PE, P-selectin-IgG-Cy5, and anti-CD38-FITC, the procedure was the same except that an anti-CD38-FITC Ab (Pharmingen) was coincubated with an excess of streptavidin-Cy5 (Jackson ImmunoResearch Laboratories Inc. ) before the final wash. In control samples, staining was carried out in the presence of 5 mM EDTA. For double staining of CD34 and PSGL-1, CB or mPB MNCs prepared as described above were incubated with an anti-PSGL-1 Ab (PSL-275 or KPL1), washed once, and incubated with an FITCconjugated anti-mouse Ab (Pharmingen).

After washing, cells were incubated with 1 llg mouse IgG (Sigma-Aldrich) and anti- CD34-PE Ab, washed, and analyzed by flow cytometry. To evaluate E-selectin ligand expression, 105 CB-or mPB-derived CD34+ cells were incubated with supernatants from COS-7 cells transfected with the murine E-selectin-IgM construct, followed by PE-labeled anti-human IgM goat Ab (Sigma-Aldrich). A similar procedure was followed for the detection of P-selectin ligands using the murine P- selectin-IgM chimera, using supernatants from COS-7 cells transfected with the murine P-selectin-IgM construct.

Engraftment of EGFP progenitors in the cranial BM and homing of human cells into NOD/SCID femoral BM. To map the areas of hematopoiesis in the calvaria, BM nucleated cells (106 cells) from F1 generation EGFP-Tg NOD/SCID mice were injected into sublethally irradiated (300 cGy) NOD/SCID recipients. Fourteen days after transplantation, recipient mice were prepared for fluorescence BM-IVM of the parietal bone as described above. For homing assays, MNCs were isolated from mPB samples as indicated above and resuspended in RPMI. Irradiated (375 cGy) wild-type and P/E-/-NOD/SCID mice were injected intravenously with 2 x 107 MNCs immediately after irradiation. Two hours after injection, mice were sacrificed and femoral BM cells were harvested. After lysis of RBCs and blocking of Fc receptors with mouse and human IgG, 106 total BM nucleated cells were stained with both PE- conjugated anti-CD34 and FITC-conjugated anti-CD45 Abs (Immunotech, Marseilles, France). Flow cytometry was gated on the lymphocyte population since more than 90% of CD34+ cells fall in this region. Analyses were performed on 5 x 105 events per transplanted animal.

Engraftment of human CD34+ cells into NOD/SCID mice. CD34+ cell (105) purified from CB or G-CSF mPB were injected intravenously into sublethally irradiated (350-375 cGy) NOD/SCID or ß2 microglobulin-deficient NOD/SCID mice (The Jackson Laboratory). Mice were sacrificed 4 weeks after injection. After lysis of RBCs and blocking of Fc receptors with mouse and human IgG, 106 BM nucleated cells were stained with an FITC-conjugated anti-human CD45 Ab (Immunotech). Depending on the level of engraftment found by FACS analysis, 0.25 x 105 to 2.5 x 105 BM nucleated cells were plated into methylcellulose media (MethoCult H4433; Stem Cell Technologies, Vancouver, Canada) and incubated at 37°C in a humidified atmosphere containing 5% COz. Human myeloid colonies, erythroid colonies, and colonies containing both myeloid and erythroid cells were counted on day 14.

Statistical analysis. All values are reported as mean SEM. Statistical significance for two paired or unpaired groups was assessed by the Student t test. The Mann-Whitney test was used to determine statistical significance in engraftment studies.

EXAMPLE 2 Human CD34+ cells roll and arrest in NOD/SCID BM microvessels.

The thin cortex of murine cranial bones allows one to observe the behavior of fluorescently labeled cells in the BM of living animals using epifluorescence intravital microscopy. Mazo et al. (1998) J. Exp. Med. 188 : 465-474. Because of the protection afforded by the surrounding bone, the BM microvasculature can be observed without direct injury from the surgical preparation. The bulk of cranial BM is concentrated around the bone sutures of the calvaria. However, individual BM microvessels are not clearly visible in most areas by fluorescence intravital microscopy except for a section in the middle of the parietal bone, which displays a well defined vascular network composed of sinusoids draining into at least two collecting venules that run perpendicular or parallel to the sagittal sinus. The blood flow in the midparietal area of the NOD/SCID BM is centripetal, draining into the sagittal sinus. To evaluate the distribution of active hematopoiesis in the calvaria, BM nucleated cells from EGFP- Tg mice were transplanted into sublethally irradiated (300 cGy) NOD/SCID recipient mice. Fourteen days after transplantation, EGFP-expressing hematopoietic cells had heavily repopulated the parasagittal areas of the parietal bone, including the aforementioned midparietal section, but also areas in the frontal and occipital bones near the coronal and lambdoidal sutures, respectively. These results indicate that the midparietal area carries active hematopoiesis and that the distribution of hematopoietic sites in the cranium of NOD/SCID animals is similar to that described in C57BL/6 animal studies using rhodamine 6G uptake as reported by Mazo et al.

(1998).

Having established that the midparietal vascular network in NOD/SCID mice encompasses BM tissue, the behavior of human HPCs in the BM microcirculation was next evaluated. CD34+ cells from G-CSF-mobilized healthy donors or from patients with hematologic malignancies in remission were isolated. Purified CD34+ cells were fluorescently labeled and injected via the carotid artery of NOD/SCID mice prepared for intravital microscopy. Labeled cells could be individually tracked in BM microvessels. For example, a free flowing CD34+ cell (t = Os) was visualized in the central Cv, tethering to the vessel wall at t = 0.30s, and rolling during the following 0.53 seconds. The percentages of rolling and arrested cells were determined by careful analysis of video recordings. To comparatively evaluate CD34+ cell interactions in sinusoids and collecting venules, the numbers of rolling and arrested HPCs were initially studied in each vessel type separately. Although there was a trend toward a larger CD34+ cell rolling fraction in sinusoids compared with collecting venules, the difference was not significant (Figure 1; P = 0.14). Therefore, the numbers of rolling and arrested cells in both types of microvessels were pooled in subsequent analyses. Most cell arrests in collecting venules (92 of 147, or 63% of arrested cells) occurred within two vessel diameters of vascular junctions, suggesting hotspots for the recruitment of HPCs in the BM. Although the average diameters and centerline velocities (Vm) were lower in sinusoids than in collecting venules (Table 1, P < 0.05), the shear rates were similar between these two groups of microvessels.

The mean percentage of rolling human CD34+ cells (-22%), similar to that described for a murine cell line and fetal liver progenitor cells in the BM of C57BL/6 mice (Mazo et al. , supra. ), indicates that human CD34+ cells can efficiently interact with NOD/SCID BM microvessels.

EXAMPLE 3 P-and E-selectins are required for human CD34+ cell rolling in BM microvessels and homing to the BM compartment.

Previous studies have suggested roles for P-and E-selectins and VCAM-I in mouse progenitor homing to the BM, and that each pathway (VCAM-1/p1 integrin and selectin/mucin ligands) contributes equally to recruitment of mouse HPCs.

Papayannopoulou et al., supra.; Frenette et al. (1998), supra.; Mazo et al., supra.; Vermeulen et al. (1998) Blood 92: 894-900. To evaluate the role of endothelial selectins and VCAM-1 in the initial interactions of human CD34+ cells with BM microvessels, the P-and E-selectin mutations were backcrossed into the NOD/SCID background and an mAb (MK 2. 7) was used to inhibit VCAM-1 function. CD34+ cells were purified from MNCs prepared from mPB samples and fluorescently labeled with CFSE. Mice were preinjected with either an anti-VCAM-1 Ab (MK 2.7) or rat IgG control prior to the injection of CD34+ cells. Anti-VCAM-1 administration reduced CD34+ cell rolling by about 33% (P = 0.05) in the BM microvasculature of wild-type NOD/SCID mice, compared with IgG injection or no injection (Figure 2).

Strikingly, CD34+ cell rolling was drastically reduced (-95% reduction) in BM microvessels of P/E-/-NOD/SCID mice preinjected with either IgG or anti-VCAM-1 (Figure 2). These differences were not due to alterations in hemodynamics of the BM microcirculation since there was no difference in the shear rate between the two groups of mice (Table 1). These results indicate that, in contrast to murine progenitors, the initial interactions of human CD34+ cells are largely dependent on endothelial selectins that are constitutively expressed in the BM tissue and that VCAM-1 plays a partial role in this activity.

Table 1 Hemodynamic characteristics of BM microvessels Mice Source of No. of Vessel diazster (µm) Vmes(µm/s) WSR(s') CD34 cells Cv 5 Mice Cv S Cv'S Cv S Cv+S Cv S Cv'S NOD/SCIO mP 30 30 10 49 ~ 3 28 ~ 2A 38 ~ 2 3,731 ~ 390 1,578 ~ 146A 2, 655 ~ 249 321 ~ 40 245 ~ 21 283 ~ 23 CD 17 30 8 47 ~ 3 32 ~ 1A 10 ~ 3 3,206 ~ 233 1,625 ~ 166A 2,762 ~ 347 283 ~ 22 216 ~ 20A 297 ~ 27 ti. 15 18 7 54 + 4 29 ~ 2A 37 ~ 2 3,681 ~ 640 1,997 ~ 242A 2,197 ~ 174 309 ~ 42 287 ~ 37 240 ~ 16 N/SW) mPB 18 23 S 42 ~ 2 26 1A 33 ~ 2 3,050 ~ 476 1,326 ~ 143A 2,224 ~ 296 295 ~ 49 222 ~ 26 272 ~ 30 N/S PE% mPB 9 23 5 49 ~ 3 30 ~ 2A 35 ~ 2 3,201 ~ 561 1,555 ~ 211A 2,019 ~ 251 259 ~ 41 252 ~ 28 254 ~ 23 N/S E@/ CB 19 14 7 41 3 32 ~ 1A 37 = 2 1,873 ~ 182 1,267 ~ 159A 1,619 ~ 134 234 ~ 28 208 ~ 29 223 ~ 20 The vzlocity of free-flowing cells (Vmuv) was determined in collccring venules (Cv) and sinuspids (S) aftcr the injection of fluntescently iabeled human erythro- cyttrs or CO34- cells. Vessel diameters were measured using a video caliper, and wall shear raees (WSRs) were calculaoed as desonbed in Methods.Danta zre arichsnerie mean ~ SEM. AP < 0.0Scompated wth Cv.N/S WT, badcrossed wild-type NOD/SCID. N/SPEv-, P-and E-selectin-deflcienr NOD/SCID; N/SE-/@,<BR> E-se4ccin-deficlent NOD/SCID.

To detennine whether the defect in the initial interactions of human CD34+ cells in P/E-/-NOD/SCID mice prevents extravasation into the BM compartment (i. e., homing), unsorted MNCs from mPB were injected into wild-type or P/E-/- NOD/SCID mice. Homed human cells were detected by FACS analysis using species-specific Abs against CD34 and CD45 (Figure 3, a and b). Homing of CD34+/CD45-human cells was reduced by approximately 90% in P/E/~ mice compared with wild-type animals (Figure 3, c-e). CD45+CD34-lymphocytes also lodged in the BM of NOD/SCID mice 2 hours after their injection (Figure 3c, lower region), suggesting that donor lymphocytes also rapidly home to the recipient BM after transplantation. However, the numbers of lymphocytes were only modestly reduced (39%, P = 0. 02) in the BM of P/E-/-animals (Figure 3d). Taken together, these intravital observations and the results from the short-term homing assays indicate that endothelial selectins are necessary for human CD34+ cell rolling on BM microvessels and homing into the BM compartment of NOD/SCID mice.

EXAMPLE 4 Defective interactions of CB-derived CD34+ cells with BM microvessels.

Human HPCs can be routinely harvested for clinical transplantation from three different sources, including the BM, mPB, and CB. To evaluate differential homing mechanisms of human HPCs, CD34+ cells were isolated from fresh samples obtained from these three sources. Fluorescently labeled CD34+ cells were injected via a carotid artery catheter, and their interactions in the contralateral midparietal BM were recorded. Detailed analyses of videotapes revealed that the mean percentages of rolling and arrested cells in NOD/SCID BM microvessels were similar between mPB- and BM-derived CD34+ cells (Figure 4). However, the interactions of CB-derived CD34+ cells were significantly altered (~50% and-60% reduction for rolling and arrested cells, respectively) compared with those derived from adult sources (Figure 4). Hemodynamic parameters were similar among the three groups (Table 1). These results suggest a defect in the capacity of neonate-derived CD34+ cells to interact with the endothelium of BM microvessels.

EXAMPLE 5 Neonate-derived CD34+ cell binding to soluble P-selectin is impaired.

Since endothelial selectins are required for rolling of CD34+ cells (Figure 2), the lower rolling numbers of CB CD34+ cells suggested a reduced expression or function of selectin ligands on neonatal progenitor cells. To evaluate this possibility, the ability of CB-and mPB derived CD34+ cells to bind P-selectin or E-selectin was tested in a fluid-phase assay. Mononuclear cells were isolated from fresh mPB and CB samples and stained with anti-CD34 and the human P-selectin-IgG chimera in the presence and absence of divalent cations. Eight individual donor pair samples were thus prepared in parallel and evaluated by FACS analysis. As shown in Figure 5a, the geometric mean of fluorescence of P-selectin-IgG binding among CD34+ cells was 59% lower in CB than in mPB (P = 0.008). Binding was specific since it was abrogated in divalent cation-chelated samples (Figure 5, a and b). Analysis of flow cytometry scattergrams revealed that while the vast majority (90% 1%) of mPB CD34+ cells bound P-selectin-IgG, approximately one-third of CB-derived CD34+ cells (34% 3%) did not bind soluble P-selectin (Figure 5b), suggesting a defect in expression or function of P-selectin ligand on a subpopulation of CB CD34+ cells.

This finding was not exclusive to the human P-selectin chimeric construct since similar results were obtained with murine P-selectin-IgM. To evaluate the function of E-selectin ligands, the binding of a murine E-selectin-IgM chimera to purified adult and neonatal CD34+ cell was analyzed. In contrast to P-selectin binding, only 60-70% of CD34+ cells bound E-selectin-IgM, but there was no significant difference in binding between CB and mPB (68% 2% and 62% 6% for CB and mPB CD34+ cells, respectively; n = 6, P = 0.33). These data indicate that function or expression of P-selectin ligands, but not E-selectin ligands, is significantly altered on neonate- derived CD34+ cells.

Because PSGL-1 has been reported to be the main functional receptor for P- selectin in BM-derived CD34+ cells (Levesque et al. (1999) Immunity 11: 369-378), its expression was analyzed. Using two different mAbs against PSGL-1 (PSL-275 and KPL1), it was found that most adult and neonatal CD34+ cells expressed this antigen (94% 2% in CB and 97% 1% in mPB, n = 5) (Figure 5c). To investigate whether PSGL-1 is the main functional P-selectin ligand on mPB and CB CD34+ cells, MNCs were stained with anti-CD34 Ab and incubated with either function blocking (KPLl) or non-blocking (PSL-275) mAb against PSGL-1 prior to evaluating soluble P-selectin binding. Preincubation of cells with KPL1, but not with PSL-275, almost completely inhibited P-selectin-IgG binding in both CB-and mPB-derived CD34+ cells (Figure 5d). The normal expression of PSGL-1 glycoprotein on CB CD34+ cells, together with the evidence of abnormal function in a subset of CD34+ cells, suggests defective posttranslational modifications of the PSGL-1 protein in a subpopulation of CB CD34+ cells.

EXAMPLE 6 The PSGL-1/P-selectin pathway plays a major role in the initial interactions of CD34+ cells with BM microvessels.

To assess whether PSGL-1 mediates CD34+ cell rolling in vivo, fluorescently labeled mPB CD34+ cells were treated with anti-PSGL-1 or control IgG and their behavior was evaluated in the BM microcirculation after intracarotid injection. As shown in Figure 6a, the number of rolling CD34+ cells was significantly lower (-56% reduction) in the group treated with anti-PSGL-1. To further test whether the reduced rolling capacity of CB-derived CD34+ cells might be due to defective PSGL-1, the individual roles of PSGL-1, P-selectin, and E-selectin were tested using E-/-mice backcrossed in the NOD/SCID background and inhibitory Abs against P-selectin and PSGL-1. It was found that the reduction observed earlier (Figure 2) in CB CD34+ cell rolling compared with cells derived from mPB was similar in wildtype NOD/SCID and E-/-NOD/SCID mice (Figure 6b). However, inhibition of P-selectin function in both wildtype and E-/-NOD/SCID mice greatly reduced (by 74% and 6S%, respectively) the number of rolling CB CD34+ cells in the BM microvasculature (Figure 6b). In addition, blockage of PSGL-1 function on CB CD34+ cells inhibited their interactions in both wild-type and E-selectin-deficient mice (68% and 55% reduction, respectively). Inhibition using an anti-E-selectin Ab (clone 9A9) produced similar results to E-selectin deficiency (n = 2). These data indicate that the PSGL- l/P-selectin pathway plays a major role in initial interactions and suggest that the reduced ability of CB CD34+ cells to interact with the BM microvessels is due to altered P-selectin binding.

EXAMPLE 7 Neonatal CD34+ cells defective in P-selectin binding are enriched in primitive CD34+CD3Sl°/-cells.

CD34+ cells represent a heterogeneous population of progenitors with various degrees of hematopoietic maturation. The lack or dim expression of CD38 in human CD34+ cells is considered to be a surrogate marker of their primitive nature.

Terstappen et al. (1991) Blood 77: 1218-1227. To further characterize the population of CB-derived CD34+ cells that do not bind P-selectin-IgG, P-selectin chimera binding and CD38 expression among CD34+ cells was analyzed. MNCs obtained from CB samples were stained for CD34 (using PE) and CD38 (using FITC) expression, and P-selectin-IgG chimera binding (using Cy5). CD34+ cells that did not bind P-selectin-IgG contained 23% 3% CD3Sl°/-cells, compared with only 9% 1% CD34+ cells that bound soluble P-selectin (n = 8 different donors, P = 0.0006).

Thus, CD34+ progenitors that do not bind P-selectin are enriched in primitive HPCs.

EXAMPLE 8 Engraftment of NOD/SCID mice by CB CD34+ cells is not compromised.

To determine whether the defect of CB CD34+ cell rolling might influence their engraftment into NOD/SCID mice, CD34+ cells were purified from CB and healthy mPB donors and transplanted into sublethally irradiated (350-375 cGy) NOD/SCID animals or NOD/SCID mice deficient in ß2 microglobulin. One month after transplantation, mice were sacrificed to assess the engraftment levels of human derived progenitors. As shown in Figure 7, there was a trend toward higher engraftment levels in the CB group compared with the mPB group, as measured by the frequency of human CD45+ cells and human progenitors in the BM compartment.

These data are consistent with those of Wang et al. (1997) Blood 89: 3919-3924, who found a higher level of engraftment of CB MNCs than of either BM or mPB MNCs, and suggest that CB CD34+ cells can compensate for their reduced initial interactions with the BM endothelium.

EXAMPLE 9 Methods The following materials and methods were used in the subsequent examples.

ArTtibodies and selectin chirner°as Anti-human PSGL-1 (KPL1), anti-CLA (HECA 452) and FITC-conjugated anti-human CD38 were purchased from PharMingen (San Diego, CA), and PE-conjugated anti-human CD34 (clone AC136) was from Miltenyi Biotech (Bergisch Gladbach, Germany). The mouse anti-human PSGL-1 Ab (PSL-275) and human P-selectin-IgG chimera were provided by Dr.

Anjali Kumar (Genetics Institute, Cambridge, MA). Murine E-and P-selectin-IgM chimeras were produced by transfection of COS-7 cells with selectin-IgM DNA vector (gift of Dr. John Lowe, University of Michigan) using the DEAE-Dextran method of Maly et al. (1996) Cell 86: 643-653.

Human samples and CD34+ cell isolation and culture. Umbilical CB samples were obtained from normal full-term deliveries ( 38 weeks) in accordance with protocols approved by the Internal Review Board of Mount Sinai. For the enrichment of CD34+ cells, low-densit) t mononuclear cells (MNC) were collected after centrifugation at 1200 rpm over Ficoll-Hypaque (d = 1.077 g/ml) and washed in PBS containing 0.5% BSA and 2mM EDTA (PEB buffer). CD34+ cells were purified from the MNC fraction using the CD34-isolation mini-MACS kit (Miltenyi) or the EasySep kit (StemCell Technologies, Vancouver, Canada) following the manufacturer's instructions. The purity of the isolated CD34+ cells ranged from 70% to 95%. For intravital microscopy, homing and selectin stability experiments, purified human CD34+ cells were fluorescently labeled by incubation with 20 µM carboxyfluorescein succinimidyl ester (CFSE ; Molecular Probes, Eugene, OR) for 30 min at 6°C, and washed thrice in RPMI before injection into mice.

For cell culture, purified CD34+ cells were washed once in IMDM medium and resuspended in culture medium consisting of IMDM, 10% FBS, 1% deionized BSA (dBSA), 100 ng/ml recombinant human (rh)-stem cell factor, 100 ng/ml rh-Flt- 3 ligand and 10 ng/ml rh-Thrombopoietin (all from R&D Systems, Minneapolis, MN). In some experiments, 2% dBSA was used and FBS was substituted for 160 , ug/ml Lecithin/Cholesterol (Sigma), 10 llg/ml rh-insulin (Novo-Nordisk, Princeton, NJ) and 200 ug/ml transferring (Sigma). Fresh medium was added every other day.

These culture conditions were chosen because they can maintain and expand the primitive stem cell fraction (Ueda et al. (2000) J. Clin. Invest. 105: 1013-1021) and both the P-selectin binding and non-binding populations in cord blood CD34+ cells.

Treatment of CD34+ cells with α1,3-fucosyltransferase VI. Half to five million freshly purified CD34+ cells were washed once in Hanks Balanced Salt Solution buffered with 20 mM HEPES, pH 7.4, and then incubated in 250 fil buffered HBSS/0. 1% HSA containing 10 mM Mec12, 1 mM GDP-fucose (Calbiochem, San Diego, CA) and 20 mU/ml recombinant human al, 3-fucosyltransferase VI (FTVI ; Calbiochem), for 45 min at 37°C. The reaction was stopped by washing the cells with PEB buffer at 4°C, and cells were analyzed by flow cytometry for selectin-chimaera binding before use in other assays.

Mice A NOD/SCID mouse colony was established from breeding pairs obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in microisolators at the barrier facility of Mount Sinai. Mice were fed with sterilely irradiated chow and autoclaved water. Experimental procedures on animals were approved by the Animal Care and Use Committee at the Mount Sinai School of Medicine.

Intravital Microscopy of the Bone Marrow Six to nine week-old splenectomized NOD/SCID mice were used for bone marrow intravital microscopy (BM-IVM) as described by Hidalgo et al. (2002) J. Clin. Invest. 110: 559-569. Mice were anesthetized by i. p. injection (6 ml/kg) of 100 mg/ml Urethane and 20 mg/ml Choralose (both from Sigma) in PBS. The trachea was cannulated with PE-160 polyethylene tubing to facilitate spontaneous respiration and a PE-10 catheter (Becton Dickinson, Franklin Lakes, NJ) was inserted into the left common carotid artery for injection of fluorescent cells. The fronto-parietal skull was exposed and maintained in place using a stereotactic holder (David Kopf Instruments, Tujunda, CA). A plastic ring was inserted in the incision area to allow application of endotoxin-free PBS. A mouse thus prepared was positioned under a fixed-stage custom-designed intravital microscope (MM-40, Nikon, Japan), equipped with a mercury fluorescent lamp and water immersion objectives (Nikon). After the injection of 7 million non-labeled human MNC to block non-specific retention sites, a first bolus of untreated CFSE- labeled CD34+ cells was injected. When the cells from the first injection were no longer detected in the circulation (approximately 15 min), the same mouse was injected with a second bolus of FTVI-treated CD34+ cells. The images were captured using a SIT camera (Hamamatsu, Japan), a camera-controller (C2400, Hamamatsu), and recorded using a VHS video recorder (SVO-9500MD, Sony). Hemodynamic parameters were calculated as reported by Hidalgo et al. , supra. Any cell traveling below the Vcrit was considered to be rolling on the vessel wall. Cells that remained stationary for > 5s were considered"arrested"cells.

Flow cytonzehy, selectin clzimera bindizg assay and cell sortizg. For three- color staining, purified CD34 cells were incubated with PE-conjugated anti-CD34 and biotin-conjugated rat anti-CLA (HECA 452) antibodies. After washing once in binding buffer (RPMI 1640 containing 5% FBS and 0.05% sodium azide), cells were incubated in supernatants from transfected COS-7 cells expressing P-or E-selectin chimeras and then with FITC-conjugated anti-human IgM antibody recognizing the w chain of the chimaeras (1 : 50 dilution) and Cy5-conjugated streptavidin (both from Jackson ImmunoResearch, West Grove, PA) were added. For double-staining of CD34+ and P-or E-selectin ligands, an aliquot of purified CD34+ cells were labeled as above except no anti-CLA antibody was added and the anti-CD34+ antibody was added to the chimaera-containing supernatant. Cells were then washed once with binding buffer before analysis. Control samples were performed in the presence of 5 mM EDTA. All incubations were performed for 15 min at 6°C and analysis was performed using a FACScalibur flow cytometer (Becton Dickinson).

For cell sorting, CB-derived CD34+ cultured for 5 to 7 days were triple-labeled for CD34+ and CLA antigen expression and P-selectin binding. Cells were first labeled with PE-conjugated anti-CD34+ and biotinylated-HECA 452 antibodies as indicated above, then washed once in binding buffer and incubated with 3 ug (per million cells) of the human P-selectin-IgG chimera that had been pre-conjugated with 0. 15 ug of biotinylated Protein A. Preliminary experiments showed that this concentration saturates P-selectin ligands on CD34+ cells. Cells were subsequently incubated with an excess of streptavidin-FITC (1. 5 ug ; Jackson Immunoresearch, West Grove, PA) and washed once and sorted on the basis of P-selectin binding and CLA-expression using a FACSVantageTM (Becton Dickinson). All incubations were performed for 15 min at 6°C.

Chemotactic atndstatic adlaesion assays. The protocols followed for transwell migration and static adhesion assays were similar to those described by Hidalgo et al.

(2001) Exp. Hematol. 29 : 345-355. For the chemotatic assays, 105 freshly purified CD34+ cells were resuspended in 100 gel of assay buffer (RPMI containing 0.5% BSA) and added to the upper chamber of Transwells (5 um pore size; Costar). Then, 600 u. l of adhesion medium with or without 100 ng/mL of CXCL12 (R&D Systems) was added to the lower chamber and transwells were incubated at 37°C for 3 hours.

Viable migrated cells were counted in the flow cytometer by analyzing each sample in the same predetermined time and flow conditions. For adhesion to VCAM-1, the soluble seven-extracellular domain recombinant human VCAM-1 (R&D Systems) was used. CFSE-labeled CD34+ cells were resuspended in the same assay medium (6h 104 in 100 gel) and were added in triplicates to 96-well dishes (High-binding; Costar, Cambridge, MA, USA) previously coated with 1 pg/mL of sVCAM-1 alone or with CXCL12 (0.5 gg/nil) in bicarbonate buffer (0. 1M, pH 8.8). Plates were spun for 15 seconds at 400 rpm to place cells in contact with the ligands, incubated for 5 minutes at 37°C, and unbound cells removed by three washes with RPMI. Bound cells were quantified using a fluorescence analyzer (HTS 7000 Plus; Perkin Elmer, Norwalk, CT).

ELISA-based α1,3-Fucosyltransferase assay. For detection of the al, 3- fucosyltransferase activity in sorted CD34+ cells, a modification of the method described by Rabina et al. (1997) Anal Biochem. 246 : 71-78 was used. 6x105 cells were resuspended in 100 ul of 50 uM MOPS-NaOH pH 7.4, 150 mM NaCI buffer, and lysed by sonication on ice by two 6-second burst on a microprobe sonicator. Cell extracts were adjusted to 1% final concentration of Triton X-100 from a 10% stock, and protein concentration of the extracts determined by the BCA Protein Assay kit (Pierce, Rockford, IL). 10 ug/ml of the acceptor 3'sialyl-N-Acetyllactosamine-BSA (14 atom spacer; V-labs, Covington, LA) was immobilized in ELISA plates (Corning Inc. , Coming, NY) in 50 Ill of sodium bicarbonate pH 8. 8 buffer, overnight at 4°C.

Wells were blocked with 150 ul of a 0.5% BSA solution in the bicarbonate buffer for 1 hour at room temperature. After washing the wells twice with H20,50 ttl of cell extracts diluted 1: 2 in assay buffer (50 uM MOPS-NaOH pH 7.4, 50 I1M GDP-fucose, 6 mM MnC12 and 0.5% Triton X-100) were added in triplicates to the wells (50 ug of protein per triplicate). After 2 hours at 37°C, the wells were washed thrice in T-PB (PBS with 0. 5% BSA and 0.05% Tween-20) and incubated with 50 Ill solution of 0.5 pg/ml HECA 452 in PB buffer for 1 hour at room temperature. After washing thrice, the wells were incubated with peroxidase-labeled anti-rat IgM antibody for another hour at room temperature. After washing, 50 ul per well of tetramethyl-benzidine (TMB; Sigma) peroxidase substrate was added to the wells and incubated for 30 min at room temperature. The reaction was stopped by addition of 50 1ll 1M HCI and the plate read at 450 nm in a RQuant ELISA plate reader (Bio-Tek Instruments, Winooski, VT). The specific of this assay was confirmed with recombinant human a1, 3-fucosyltransferase VI or HL60 cell extracts (a cell line expressing selectin ligands, sLex and functional a1, 3-fucosyltransferases), which in the absence of the GDP-fucose donor were unable to yield a product detectable by this assay.

Engraftment and homing of human CD34+ progenitors in the BM of NOD/SCID mice. For the homing assays, CFSE-labeled CD34+ that had been treated or not with the α1,3-fucosyltransferase VI as indicated above were intravenously injected into sublethally irradiated (350 cGy) NOD/SCID mice. 12 to 16 hours after injection, mice were sacrificed and femoral BM cells were harvested for the quantification of human CFSE+ cells by flow cytometry. For the engraftment experiments, serial dilutions of unlabeled CD34+ cells were injected into sublethally irradiated NOD/SCID mice (350 cGy). After 6 weeks, mice were analyzed for the content of human hematopoietic cells by staining total bone marrow nucleated cells with human specific FITC-labeled anti-CD45 antibody (Sigma). 5 x 105 cells per transplanted animal were analyzed.

Semiyuantitatioe RT-PCR. mRNA was isolated from cultured CD34+ cells sorted on the basis of P-selectin binding using the Trizol reagent (Life Technologies) and cDNA was prepared using the ThermoScrip RT-PCR system (Invitrogen Corp., Carlsbad, CA) as indicated by the manufacturer. PCR amplification of the cDNA was carried out following the conditions previously reported to be below the plateau of amplification using the Taq polymerase (Invitrogen). For p-actin the primers used were: forward 5'-TGGGTCAGAAGGACTCCTATG-3' (SEQ ID NO : 1), reverse 5'- CAGGCAGCTCATAGCTCTTCT-3' (SEQ. ID NO : 2) and PCR was performed as follows: 95°C for 5 min followed by 25 cycles of 94°C for 50 s, 56°C for 50 s and , 72°C for 50 s, and a final extension at 72°C for 7 min. (Huang et al. (2000) J. Biol.

Chem. 275: 31353-31360). The same conditions were used for amplification of C2GnT with the following primers: forward 5'-AAAATGCTTCCTCCACTC-3' (SEQ. ID NO : 3) and reverse 5'-TCAGTGTTTTAATGTCTC-3' (SEQ. ID NO : 4) as disclosed by Huang et al (2000) J. Bio. Chem. 275: 31353-31360. For the FTVII gene the primers used were: forward 5'-CTCGGACATCTTTGTGCCCTATG-3' (SEQ. ID NO : 5) and reverse 5'-CGCCAGAATTTCTCCGTAATGTAG-3' (SEQ. ID NO : 6) and the PCR performed as follows: 36 cycles of 94°C for 1 min, 66°C for 1 min and 72°C for 1 min as disclosed by Petretti et al. (1999) Biophys Acta 1428 : 209-218. For the TPST1 gene the primers used were: forward 5'- AAGATGGTTGGAAAGCTGAAGC-3' (SEQ. ID NO : 7) and reverse 5'- TTCTCATCCACCGTTCAGGATG-3' (SEQ. ID NO : 8) and the PCR performed as follows : 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 50 seconds as disclosed by Tu et al. (1999) J. Immunol. 163: 5070-5078. The same PCR conditions as for TPST1 were used for the TPST2 gene, using the following primers: forward 5'-AGCATGCGCCTGTCGGTGCG-3' (SEQ. ID NO : 9 and reverse 5'-CACTTGGAGAGCGCTTCCAG-3' (SEQ. ID NO: 10) disclosed by Tu et al., supra.

Statislical Analysis. All values are reported as mean + SEM. Statistical significance for two paired or unpaired groups was assessed by the Student t test.

Mann Whitney test was used to determine the statistical significance in NOD/SCID engraftment studies.

EXAMPLE 10 Enhancement of bone marrow engraftment by fucosylation of selectin ligands in cord blood derived CD34 P-and E-selectin ligands were detected on the cell surface of cord blood HPCs by flow cytometry using chimeric molecules containing the functional P-or E- selectin extracellular domains and the CH2, CH3 and CH4 domains of human IgM as described in Example 9. As shown in Figs. 8a and b, a large fraction of the cord blood HPCs is unable to bind either selectin. Cells that do not bind selectins lack the expression of the carbohydrate structure recognized by the HECA 452 antibody, which is a fucosylated/sialylated moiety, as shown in Figs. 8c and d. These data demonstrate that selectin ligands require both fucosylation and sialylation for selectin binding.

As shown in Fig. 9, cells that do not bind P-selectin and do not express the HECA 452 epitope are also unable to bind E-selectin. The upper panel of Fig. 9 depicts the strategy for separating CD34+ cells on the basis of selectin binding.

Purified cord blood CD34+ cells were labeled with the HECA 452 antibody and for P- selectin binding. Cells were sorted on the basis of P-selectin binding, using the gates indicated within the boxes. These results show that a common defect in this subpopulation prevents binding to both P-and E-selectins. Reduced sulfotransferase activity is not the common defect, since these enzymes were shown to be essential for only P-selectin binding activity, via tyrosine sulfation.

Expression of enzymes known to play a role in functional posttranslational modifications of selectin ligands was analysed by RT-PCR. As shown in Fig. lova, the expression of fucosyltransferase VII (FTVII) is reduced in the population of cells that do not bind P-selectin (Psel"eg) compared to those cells that bind P-selectin (Psel+).

Fig. lOb demonstrates a drastic reduction in fucosyltransferase enzymatic activity in the Pselneg populaRion. These results show that lack of selectin binding results from reduced fucosylation of selectin ligands.

Cord blood derived purified CD34+ cells were treated with FTVI as described in Example 9. The cells were treated with 20U/ml of FTVI in a buffer containing ImM GDP-fucose as a donor of fucose and lOmM manganese as a cofactor for the enzyme, and P-and E-selectin binding was analyzed by flow cytometry. As shown in Fig. 11, fucosylation of cells with recombinant FTVI restored full selectin ligand activity in all CD34+ cells.

Control experiments were performed to ensure that induced fucosylation does not affect the biological activity of other surface receptors known to be important for HPC homing to the bone marrow. Fig. 12a shows that there is no difference in the migration of FTVI-treated and non-treated CD34+ cells toward the chemokine CXCL12 (SDF-1), indicating that the function of the chemokine receptor CXCR4 is intact. Fig. 12b shows an adhesion assay of CD34+ cells on recombinant VCAM-1.

VCAM-1 is the receptor for the integrin VLA-4 which has been shown to be important for the HPC homing to the bone marrow. As shown, there is no difference in binding to VCAM-1 between FTV1-treated and non-treated CD34+ cells.

Intravital miscroscopy was used to determine whether FTV1 treatment increases the ability of CD34+ cells to interact with bone marrow sinusoids and collecting venules in vivo.

The interactions of human CD34+ cells with the blood vessels of the BM were visualized in real-time in vivo with this technique. Purified CD34+ cells were fluorescently labelled and injected in NOD/SCID mice (which are immunodeficient and do not reject human tissues), and visualized as they flowed through the blood vessels of the BM with a fluorescence microscope, and their interactions with the vessels recorded on video tape for analysis. All fluorescently labelled cells were visualized passing through bone marrow microvessels and the number of rolling and arrested cells were scored by playback video analysis. Results are shown in Fig. 13.

These analyses revealed that interactions with and arrests on the microvasculature of the BM are significantly enhanced when CD34+ cells are treated with the FTVI enzyme.