AUSTRALIA PATENTS ACT 1990 REGULATION 3.2 Name of Applicant ROBERT SACKSTEFIN Actual Inventors: ROBERT SACKSTEIN Address for Service: E, F. WELLINGTON & CO, Patent and Trade Mark Attorneys, 312 St. Kilda Road, Melboume, Southbank, Victoria, 3006, Invention Title "COMPOSITIONS AND METHODS FOR MODIFYING CELL SURFACE GLYCANS" Details of Associated Provisional Applications Nos: The following statement is a full description of this invention including the best method of performing it known to us. Next page is page 1A CROSS EFERENCETO RELATED APPLICATIONS [00011 This application is a divisional application derived from Australian Patent Application No. 2007254777 (PCT/US2007013178 WO 2007143204)claiming priority of US Application No. 60/810469, the entire text of which are hereby incorporated herein by re ference. FIELD OF THE INVENTION [0002] This invenion relates to compositions and methods for modifying cell-surface glycans on live cells using exogenous glycosylhransferases. Partiuly the composition of methods of the invention preserves the viability and one or more native phenotypic characteristics of the treated cell, BACKGROUND OF THE INVENTION [0003] The capacity to direct migration of blood-borne cells to a predetermined location (Ihmngt has profound implications for a variety of physiolog and pathologic processes. Recruitment of circulating cells to a specific anatomic site is initiated by discrete adhesive interactions between cels in flow and Vascular endothelium at the target tissue(s). The molecules ta mediate thnacts are called homing receptors"and a dnd historically, these structures pilot tropism of cels in blood to the respective target tissue At present. only three tissuespecific homing receptors are recognized L-selectin for peripheral lymph nodes (LPAM-I) for intestines and gut-associated lymphoid tissue and Cutaneous Lymphocyte Antigen (CLA) fAr skin (1) Apart from these tissues it has also been recognized for several decades that circulating cells especially hematopoietic stem ces, nigate effectively to bone marrow (2. Howeverextensive investigations on this process over several decades have yielded complex and sometimes coniEcting results, providing no direct evidence of a homing receptor uniquely promoting marrow tropism [0004] Front a biophysial perspectie, a homing receptor functions as molecular brake. effectinag initial tethering then sustained rolling contacts of cells ini blood flow onto the Next page is page 2
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vascular endothelium at velocities below that of the prevailing bloodstream (Step 1) (M. Thereafter, a cascade of events ensue, typically potentiated by chemokines, resulting in activation of integrin adhesiveness (Step 2), firm adherence (Step 3) and endothelial transmigration (Step 4X3). This "multi-step paradigm" holds that tissue-specific migration is regulated by a discrete combination of homing receptor and chemokine receptor expression on a given circulating cell, allowing for recognition of a pertinent 'traffic signal" displayed by the relevant vascular adhesive ligands and chemokines expressed within target endothelium in an organ-specific manner. Following engagement of homing receptor(s) directing trafficking of cells to bone marrow, several lines of evidence indicate that one chemokine in particular, SDF-1, plays an essential role in Step 2-mediated recruitment of cells to this site (2, 4,.5) [000s] The most efficient effectors of Step I rolling interactions are the selectins (E-, P- and L-selectin) and their ligands (1). As the name implies, selectins are lectins that bind to specialized carbohydrate determinants. consisting of sialofucosylations containing an a42.3} linked sialic acid substitution(s) and an a(1,3>-linked fucose modification(s) prototypically displayed as the terrasaccharide sialyl Lewis X (sLe; Neu5Aca O3Gall-4[ucl 3]GcNAc81-)) (1, 6). E- and P-selectin are expressed on vascular endothelium (P-selectin also on platelets), and L-selectin is expressed on circulating leukocytes (1). E- and P-selectin are typically inducible endothelial membrane molecules that are prominently expressed only at sites of tissue injury and inflammation, However the microvasculature of bone marrow constitutively expresses these selectins (3. 7), and in vivo studies have indicated a role for E selectin in recruitment of circulating cells to marrow (5, 8). Importantly, SDF- I is constitutively expressed in high concentration within the marrow and is co-localized uniquely with E-selectin on the specialized sinusoidal endothelial beds that recruit blood-borne cells to as the bone marrow (5). M61 Two principal ligands for E-selectin have been identified on human hematopoietic stem/progenitor cells (HSPC), PSGL- (9) and a specialized sialofucosylated CD44 glycoform known as Hematopoletic Cell E-/L-selectin Ligand (HCELL) (10,1). CD44 is a rather ubiquitous cell membrane protein, but the HCELL phenotype is found so exclusively on human HSPCs. In contrast to UCELLUs restricted distribution, PSGL-l is widely expressed among henatopoietic progenitors and more mature myeloid and lymphoid cells within the marrow (9). HCELL is operationally defined as CD44 that binds to E selectin and L-selectin under shear 'conditions, and is identified by Western blot analysis of 2 ,cell lysates as a CD44 glycoform reactive with E-selectin-Ig chimera (&Ig) and with mAb HECA452, which recognizes a sialyl Lewis X-like epitope. Like all glycoprotein selection ligands, HCELL binding to E- and L-selectin is critically dependent on a(2,3)-sialic acid and a(,3)-fucose modifications(10-13. On human HSPCs, HCELL displays the pertinent sialofucosylated selectin binding determinants on N-glycans (10, 12), In vitro assays of E and L-selectin binding under hernodynamic shear stress indicate that HCELL is the most potent ligand for these molecules expressed on any human cell (10, 13) Importantly, though E-selectin is constitutively expressed on microvascular endothelium of the marrow, this molecule is prominently expressed on endothelial beds at all sites of tissue injury (e.g., sites of ischemia-reperfusion injury or trauma) or inflammation. SUMMARY OF THE INVENTION (00071 The invention features compositions and methods for modifying glycans expressed on the surface of living, e g. viable cells. The composition and methods allow 5 modification of cell-surface glycans while preserving cell viability and one or mom phenotypic characteristics of the celL For example, the methods and compositions can be employed to modify particular phenotypic characteristics of the el (such as glycosylation) while preserving one or more other phenotypic characteristics (e.g., mutipotency) of the cell. (W81 In one aspect. the invention features a composition for modifying (ex vivo or o in vitro) a glycan, e.g., a glycan expressed on the surface of a cell or a particle or cell fragment (e.g., a mammalian cell or a platelet or a cell membrane-derived substancelfragment such as a liposome, The composition includes a purified glycosyltransferase (e,g, a recombinant glycosyltransferase) and a physiologically acceptable solution, wherein the physiologically acceptable solution is free of one or more divalent metal co-factors (eg., the 25 solution is free of manganese, magnesium, calcium, zinc, cobalt or nickel). In various embodiments, the glycosyltransferase is a fucosyltransferase (e.g., an alpha 1.3 fucosyltransferase, e.g., an alpha 13 fucosyltransferase IlI, alpha 1,3 fucosyltransferase IV, an alpha 1,3 fucosyltransferase VI, an alpha 1,3 fucosyltransferase VH or an alpha 1,3 fucosyltransferase IX), a galactosyltransferase, or a sialyltransferase. 30 {00091 The composition can include more than one glycosyltransferase and/or may include one or more additional agents, such as a donor substrate (e.g., a sugar). Donor substrates include fucose, galactose sialic acid, or N-acetyl glucosamine. 3 [000101 The glycosyltransferase has enzymatic activity. Optimally, the glycosyltransferase is capable of transferring 1.0 pmole of sugar per minute at pH &5 at 37 *C, The composition does not affect integrin adhesion of the cell or cell particle [Ooo11 The composition can include any physiologically acceptable solution that lacks divalent metal co-factors. In various embodiments, the physiologically acceptable solution is buffered, The physiologically acceptable solution is, e.g, Hank's Balanced Salt Solution, Dulbecco&s Modified Eagle Medium, a Good's buffer (see N.E.Good, GAY Winget, WWinter, TNConolly, S.lzawa and tLM.MSingh, Biochemistry 5 467 (1966); NE. Good, Slzawa, Methods Enzymol. 24, 62(1972) such as a REPES buffer, a 2 Morpholinoethanesulfonic acid (MEBS) buffer, phosphate buffered saline (PBS). [00012] In various embodiments. the physiologically acceptable solution is free of glycerol. [00013] The compositions can be used for modifying a glycan on the surface of a cell such as a stem cell (e.g., a mesenchymal stem cell, a hematopoietic stem cell), a progenitor cell (e.g., a neural stem/progenitor cell or pulmonary stem/progenitor cell) or a cell of hematopoictic lineage (e.g., a leukocyte, a lymphocyte), or a cell particle (e.g t a platelet) or a liposome (000141 In another aspect, the invention features a kit for modifying a glycan on the surface of a cell or particle. The kit includes a purified glycosyltransferase, and instructions for contacting a cell with the glycosyltransferase in a physiologically acceptable solution which is free of one or more divalent metal co-factors. [000151 In another aspect, the invention features a method for modifying a glycan on the surface of a cell or particle. The method includes contacting a cell or cell particle with a glycosyltransferase in a physiologically acceptable solution free of divalent metal co-factors 25 under conditions in which the glycosyltransferase has enzymatic activity and the viability of the cell or cell particle population is at least 70%, 80%, 90%. 95%, 97%. 98%, 99% or more. Viability is measures a 2, 4, 6, 8 12, 24 hours after contact with the glycosyltransferase. 1000161 In various embodiments, the cell or particle is contacted with more than one glycosyltransferase and its appropriate donor substrate (eg sugar). For example, the cell is 30 contacted with two glycosyltransferases simultaneously, or sequentially, each adding a distinct monosaccharide in appropriate linkage to the (extending) core glycan structure). The method is useful, e.g., for modifying glycans on the surface of cells e.g. stem cells or differentiated cells or cell particles such as platelets. Cells include for example a 4 mesenchynal cell, hematopoictic stem cells, tissue stem/progenitor cells such as a neural stern cell, a myocyte stem cell, or a pulmonary stem cell. an umbilical cord stem cell, an embryonic stem cell or a leukocyte. The cell or cell particle expresses CD44, e-g., a (2.3) sialyated CD44. The cell or cell particle does not express CD34 or PSGLI. After modification the cell or cell particle binds E-selectin and orL-selectin. The modified cell or cell particles do not bind P-selectin 10017] In various aspects the methods are useful to increase the affinity of the cells for a ligand, and/or to increase the in vivo engraftment/homing potential of the cells when administered to a subject, to prevent clearance of administered cells or platelets (extend the circulatory half-life), or to alter the ability of a platelet to aggregate or to bind to substrates (e.g,, endothelium, leukocytes, extracellular matrix etc.). [000181 Also included in the invention are the cells or cell particles produced by the methods of the invention. [000191 The invention also features methods of increasing engraftment potential of a cell, treating or alleviating a symptom of an immune disorder, tissue injure or cancer by administering to a subject e.g. human a composition comprising the cells of the invention. [000201 The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS 1000211 Figure 1. Human mesenchynal stem cells (MSC) express CD44 and react with SACK-I mAb, which recognizes a sialic acid-dependent epitope displayed on an N glycan substitution exclusively carried on a CD44 scaffold. (a) SACK-1 staining of Western Ms blots of untreated (-) or N-Glycosidase-F-treated (+) immunoprecipitated CD44 from KGla cells (a human cell line that natively expresses HCELL) resolved on a reducing 4-20% SDS PAGE gel. (b) (Left panel) Flow cytometry analysis of SACK-I expression on untreated (gray histogram) or sialidase treated (white histogram) KG I a cells. (Right panel) SACK-I staining of Western blots of untreated (-) or sialidase-treated (+) immunoprecipitated CD44 30 from KGla cells resolved on reducing 4-20% SDS-PAGE ge. SACK1 reactivity is markedly diminished following sialidase treatment, as shown by both flow cytometry and Western blot. (c) flow cytornetry analysis of E-selecrin ligand activity (E-selectinIg 5 chimera (Eg) binding) and of PSGL-I, CD44, SACK-i, HECA452. KM93 (sLeZ), CDI la/CD18 (LEA-1) CD49d/CD29 (VLA-4) and CXCR4 expression on MSC. Dotted line is isotype control, black line is specific antibody (or E-Ig chimera): shaded histogram on SACK-I profile denotes reactivity following sialidase treatment of MSC Results shown are representative of multiple histograms from MSC derived from multiple matow donors. Note that human MSC express CD44 and a CD44 glycoforrn displaying SACK-i determinants, but do not express E-selectin ligands (no staining with E-selectin-lg chimera); they also lack CXCR4 and PSGL-I 4 and also lack the sLe* determinants recognized by KM93 and HECA452 mAbs. [000221 Figure 2. FTVI treatment of human MSC elaborates sialofucosylations on N linked glycans of CD44 rendering ICELW expression, (a) Flow cytometry analysis of HECA452, KM93 (sLez) and E-Ig reactivity on untreated and FTVI-treated MSC. Dotted line is untreated MSC. black line is FTVI-treated MSC (b) Western blot analysis of HECA452 (left panel) and of BIg (right panel) reactivity of MSC lysates resolved on a reducing 4-20% SDS-PAGE. Amounts of lysates in each lane are normalized for cell number of untreated and FTVI-treated MSC, Staining with E-g was performed in the presence (+) or absence (-) of Ca't Note that FTVI treatment induces HECA452-reactive sialofucosylations and Eg binding selectively on a -I00kDa glycoprotein. (c) MSC were treated (+) or untreated (-> with FTV. Thereafter, CD44 was immunoprecipitated (using anti-CD44 mAb l-ermes4 from equivalent cell lysates of FrVI-treated and untreated cells, and immunoprecipitates were digested with N-glycosidase F(+) or buffer treated (-). Immunoprecipitates were then resolved by reducing SDS-PAGE (4-20% gradient) and blotted with HECA452, E-Ig or another anti-human CD44 mAb (2C5). As shown, N glycosidase F treatment abrogates HECA452 and Eg staining of CD44 from FTVI-treated MSC Results shown are representative of multiple experiments from MSC derived from several marrow donors. 1000231 Figure 3. FTVI-treated human MSC display markedly enhanced shear resistant adhesive interactions with endothelial E-selectin under defined shear stress conditions Untreated. FTVI-treated or sialidase digested FTVI-treated MSC were perused over IL-$1 and TNF-a-stimulated human umbilical vein endothelial cells (HUVEC) at 0.5 dyne/cm'. The accumulation of relevant MSC was determined at shear stress of 0.5,142,5, 10 20 and 30 dyne/cm2. In certain instances, EDTA was added to the assay buffer or 6 HUVEC were pretreated with a function blocking mAb to E-selectin prior to use in adhesion assays. Values are means * SEM (n=4 for each group). [00924] Figure 4. FTVI-treated, HCELL-expressing human MSC home efficiently to bone marrow in vivo. Montage images of parasagittal region of calvarium assembled from representative experiments of NOD/SCID mice. (a) All images shown in this set of panels were obtained at I hour after infusion of relevant cells: Left panel, untreated MSC; Middle panel. FrVI-treated cells digested with sialidase; Right panel, FTV-treated cells. (b) Results shown are from representative images of one mouse at I hour (left panel) and 24 hours (middle panel) after infusion of FTVktreated MSC. Right panel shows high power color image of sinusoidal perivascular region 24 hours after injection of FTVI-treated MSC, revealing extravascular (parenchymal) infiltrates of infused FTVk1treated MSC: Red speckles are DiD-labeled MSC. green color highlights the sinusoidal vessels, visualized by injection of fluorescent quantum dots (805 nm). [000251 Figure 5 is a photograph of a Western Blot showing HECA452 reactivity of 5 Neural Stem Cells Treated with Fucosyltransferase 2 VI [000261 - Figure 6 is Flow Cytometric Analysis of HECA452 Expression on Neural Stem Cells Before and After Fucosyltransferase-VI Treatment. [00027 Figure 7 Flow Cytometric Analysis of HECA452 Expression on Pulmonary Stem Cells Before and After Fucosyltransfermse-VI Treatment DETAILED DESCRIPTION [00028] The invention is based in part on the surprising discovery that glycosyltransferases retain enzymatic activity in the absence of divalent metal co-factors (e.g. divalent cautions such as manganese, magnesium, calcium, zinc, cobalt or nickel) and 25 stabilizers such as glycerol. Previously, divalent metal co-factors had been deemed critical for enzymatic activity. The glycosyltrwasferase compositions according to the invention are particularly useful in modification of glycans on live cells. Previous attempts to modify glyca6 structures on live cells resulted in cell death and phenotypic changes to the cell due the toxic effects of the metal co-factors and enzyme stabilizers such as glycerol. For ex vivo 30 custom engineering of live cell surface glycans using glyosyltransferases, it is essential that the target cells remain viable and phenotypically conserved following treatment(s) In 7 applications utilizing stem cells, it is also important to analyze whether differentiation along characteristic lineages is affected by enzymatic treatment. [000291 Beyond their recognized effects on cell viability (20), divalent metal c factors such as Mn* itself triggers signal transduction (21) and activates integrin adhesiveness (e.g., for VLA-4) at levels well below those employed in forced glycosylation (e.g., fucosylation) (22. 23), These Mn effects are confounders to the effect(s) of glycosylation on cellular trafficking, as the resulting integrin-mediated firm adhesion would be manifest rampantly at endothelial beds and within tissue parenchyma expressing relevant ligand [000301 To address these concerns, a new method for high titer fucosyltransferase production in a Pichia Pastoris system was developed. Additionally, the fucosyltmansferase was stabilized in a buffer (e.g- HlBSS) specifically chosen to minimize cell toxicity. Furthermore, enzyme conditions were refined to utilize physiologic buffers in the coupling reaction without input of divalent metal co-factors (e.g., without input of Mn" ions) s [000311 These experimental modifications resulted in high efficiency fucosylation of CD44 on MSCs with 100% cell viability following enzymatic treatment. -Importantly, kinetic analysis following forced fucosylation showed that cell viability in vitro was retained indefinitely after treatment in all MSC, yet HCELL expression was transient: HCELL levels were stable for 24 hours and declined steadily thereafter to baseline (no HCELL) by 96 hours presumably reflecting cell turnover of membrane CD44. importantly, there was no effect on MSC differentiation into various lineages following FTV1 treatments, assayed daily for up to 2 weeks following treatment. Thus, FTVI treatment had no apparent effect on the phenotype of MSC, with exception only of HCELL expression (Figure 2c). In contrast, FTVI treatment of MSC from commercial available FTVI (e.g., compositions containing Mn" and glycerol; 2s Calbiochem) while enhancing HCELL expression cell viability was compromised following these FTVI treatments, with >95% of cells dying within 8 tours of modification. This loss of viability was attributed to exposure to stabilizers (e.g., glycerol) in the commercial enzyme formulations and to exposure to high levels of Mn (10 mM) used in the enzymatic reaction. Accordingly, the compositions of the invention now make it feasible to ex-vivo engineer 30 glycans on a surface of a viable cell to produce a therapeutic product that is suitable for in vivo administration to a human. [00032] Following forced fucosylation, and despite absence of surface CXCR4 expression, intravenously infused MSC homed robustly to bone marrow, a tissue 8 constitutively expressing vascular E-selectin. These findings establish HCELL as a human bone marrow homing receptor, provide direct evidence that CXCR4 engagement is not obligatory for marrow trafficking, and present new perspectives on the multi-step paradigm. 1000331 , The finding that enforced HCELL expression confers marrow tropism, and that its functional inactivation by sialidase treatment specifically reverses this effect, defines this CD44 glycoform as a "bone marrow homing receptor" As such, the ability to custom modify HCELL expression ex vivo may be useful for improving engraftment of HSPCs in clinical transplantation or for use of MSC in cell-based therapy (e.g., for bone diseases). More generally, the data suggest that enforcing cellular HCELL expression may promote systemic delivery to tissues whose endothelial beds express Eselectin. The high specificity and efficiency of this rather subtle fucose modification of a(2,3)-sialylatedglycafOUDs of CD44 thus provides guiding principles and technologies for strategies to selectively upregulate HCELL expression for adoptive cellular therapeutics The facility with which this can be accomplished suggests that rapid translation of this approach to patients should be 5 straightforward, Because E-selectin is displayed prominently at sites of inflammation and ischemia in affected tissues of primates (29, 30), modulation of HCELL expression could lead to directed migration and infiltration of progenitor/stem cells at injured/damaged tissue(s) for regenerative therapeutics. Beyond implications in stem cell based therapies, these findings also testing of how upregulated E-selectin ligand activity on other cells, such 0 as immunologic effector and regulatory cells, may be harnessed to achieve targeted cell migration in a variety of physiologic and pathologic processes, including immune diseases, infectious diseases, and cancer therapeutics. 100034] COMPOSmON {000351 The invention provides compositions for ex vivo modification of cellsurface 26 glycans on a viable cell or cell particle. The compositions include a purified glycosyltransferase polypeptide and a physiologically acceptable solution free of divalent metal co-factors. The composition is free of stabilizer compounds such as for example, glycerol. Glycosyltransferase include for example, fucosyltransferase, galactosyltransferase sialytransferase and N, acetylglucosaminotransferase. The fucosyltransferase is an alpha 1,3 so fucosyltransferase such as an alpha 1,3 fucosyltransferase i, alpha 1,3 fucosyltransferase IV, an alpha 1,3 facosyltransferase VI, an alpha 113 fucosyltransferase VII or an alpha 1,3 fucosyltransferase IX) 9 [000361 Optionally, the composition further includes a sugar donor suitable for the specific glycosyltransferase. For example, when the glycoslytransferase is a fucosyltransferase. the donor is GDP-fucose. Whereas, when the glycosyltransferase is a siayltransferase the donor is CMP-sialic acid, One skilled in the art would recognize suitable i sugar donors. 100037] The glycosyltransferases are biologically active, By biologically active is meant that the glycosyltransferases are capable of transferring a sugar molecule from a donor to acceptor. For example. the glycosyltransferase is capable of transferring o, 02, 13,04, 05, 10, 1.5,10, 2.55, 10 or more pmoles of sugar per minute at pH 6.5 at 37*C. [00038] Physiologically acceptable solution is any solution that does not cause cell damage, e.g. death. For example, the viability of the cell or cell particle is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more after treatment with the compositions of the invention. Suitable physiologically acceptable solutions include for example, Hank's Balanced Salt Solution (HBSS). Dulbecco's Modified Eagle Medium (DMEM), a Good's 5 buffer (see NE.Goodt G.D.Winget, W.Winter, TN Conolly, S.lzawa and RM.M.Singh, Biochemistry 5, 467 (1966); .E. Good, S Izawa, Methods Enzymol 24, 62 (1972) such as a HEPES buffer, a 2-Morpholinoethanesulfonic acid (MES) buffer, or phosphate buffered saline (PBS). [000391 THERAETC METHODS The compositions of the invention, due to their low toxicity on viable cells and high enzymatic activity are useful for the ex vivo or in vitro modification of glycan on the surface of cells or cell particles. Moreover, the modified cells and particles produced using the compositions and methods of the invention are useful in therapeutic settings to achieve targeted cell migration in a variety of physiologic and pathologic processes, including bone 25 disease, immune diseases, infectious diseases, and cancer therapeutics. The Federal Drug Administration imposes (FDA) imposes rigid requirements on all final cell products for human administration. Specifically. the FDA requires a minimum cell viability of 70%, and any process should consistently exceed this minimum requirement. Unlike previous described methods of ex vivo or in vitro modification of glycan on the surface of cells which so utilized glycosyltransferases compositions, containing divalent metal co-factors and stabilizers such as glycerol (which resulted in significant cell death), the methods described berein produce a cell based product that meets or exceeds the FDA requirements, 10 (000403 More specifically, the glycan engineering of the cell surface will drive homing of cells to any site where E-selectin is expressed In particular, since CD44 is a ubiquitously expressed cell membrane protein and is displayed on stem/progenitor cell populations of both "adult" and embryonic types the capacity to modify glycosylation of this protein by ex vivo glycan engineering to create the HCELL (CD44 glycofora) phenotype will drive migration of intravascularly injected (adoptively transferred) cells in vivo to marrow or to any tissue/organ site where E-selectin is expressed. (00041 Gilycans are modified on the surface of a cell or cell particle (e.g. platelet or liposome) by contacting a population of cells with one or more glycosyltransferase compositions according to the invention. The cells are contacted with the glycosyltransferase composition under conditions in which the glycosyltransferase bas enzymatic activity. Olycan modification according to the invention results in cells that have at least 80%, 85%, 90%. 95%. 96%, 97%, 98%, 99% or more viability. Viability is determined by methods known in the art such as trypan blue exclusion. Viability is measured I br, 2 hr, 4 hr, 18 hr, 12 hr 24 hr or more after treatment. The phenotype of the cells (other than the glycan modification) is preserved after treatment. By preserved phenotype it is meant the cell maintains its native function and or activity, For example, if the cell is a stem cell it retains its pluripotency. [00042] After modification, the cell or cell particle binds E-selectin and or L-selectin. o In various aspects, the modified cell does not bind P-selectin. Preferably, after modification the cells express the sialofucosylated CD44 glycoform known as Hematopoictic Cell E-/L selectin Ligand (HCELL). After modification, the cell or cell particle is capable of homing in-vivo to the bone marrow and or sites of ischemia or inflammation., (00043] The cell or cell particle is any cell in which cell surface glycan modification is 2s desired. The cell is a stem cell (i,e., multipotent) or a differentiated celL Stem cells include for example a hematopoietic stem cell, a mesechynial stem cell, a tissue stem/progenitor cell (e.g., a neural stem cell. myocyte stem cell or pulmonary stem cell), an umbilical cord stem cell, or an embryonic stem cell. Differentiated cells includes hernatopoietic4ineage cells such as a leukocyte, e.g., a lymphocyte. The lymphocyte can be a Blymphocyte or T 30 lymphocyte, or a subset of T lymphocytes, e.g., a "regulatory" lymphocyte (CD4*/CD25/FOXP3) . (000441 The cell or cell particle expresses CD44. The CD44 is not sialofucosylated. Alternatively the CD44 is alpha (2,3)-sialylated and lacks relevant fucosylations rendering 11 the HCELL phenotype. Enforced glycosylation of CD44 to render HCELL is useful in improving engraftnent of hematopoictic stem/progenitor cells (HSPCs) in clinical hematopoietic stem cel transplantation, or for use of MSC in cell-based therapy (e.g., for bone diseases). More generally, the data suggest that enforcing cellular HCELL expression i may promote systemic delivery of HCELL-bearing cells to tissues whose endothelial beds express E-selectin. (000453 In various aspects cell does not expressPSOL-1, CD34 or both tOo0461 The modified cels of the invention because of their increased homing capabilities are useful for example in for improving engraftment of flSPCs in clinical transplantation, for use of MSC in cell-based therapy (e.g, for bone diseases) or directing migration and infiltration of progenitor/stem cells at injured/damaged tissue(s) for regenerative therapeutics. [00047] For example, the composition are useful for treating a variety of diseases and disorders such as ischemic conditions (e.g, lim ischemia, congestive heart failure, cardiac 5 ischenia, kidney ischemia and ESRD, stroke, and ischemia of the eye) , conditions requiring organ or tissue regeneration (e.g., regeneration of liver, pancreas, lung, salivary gland, blood vessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney, muscle cardiac muscle, nerve, and limb), inflammatory diseases (e g,, heart disease, diabetes, spinal cord injury, rheumatoid arthritis, osteo-arthritis, inflammation due to hip replacement or revision. Crohn's o disease, and graft versus host disease) auto-immune diseases (e.g., type I diabetes, psoriasis, systemic Iupus, and multiple sclerosis), a degenerative disease, a congenital disease hematologic disorders such as anemia, neutropenia, thrombicytosis, myeloproliferative disorders or hematologic neoplasms and cancer such as leukemia, [00048) Diseases and disorders are treated or a symptom is alleviated by administering 2s to a subject in need thereof a cell composition produced by the methods of the invention. The cell compositions are administered allogeneically or autogeneically. (000491 EXAMPLE I: GENERAL METHODS [OSO] Reagents: The following antibodies were from BD Pharmingen: function blocking marine anti-human E-selectin (68-5411 UIgGO rat anti-human CLA (HECA-452; 30 1gM). marine anti-human PSGL-I (KPL-l; IgGI), purified and FITC-conjugated murine anti human L-selectin (DREG-56; IgG), marine anti-human CXCR4 (12G5; IgGz), FITC conjugated marine anti-human CD18 (L130; IgGI). marine anti-human CD-29 (MAR4; IgGO, PE-conjugated marine anti-human CD49d (9FIO; IgGO. mouse IgGlsc isotype, mouse IgGa 12 isotype, mouse IgM isotype. rat IgG isotype and rat JgM isotype. Rat anti-human CD44 (Hermes-i lgG2a) was a gift of Dr. Brenda Sandmaier (Fred Hutchinson Cancer Research Center; Seattle, WA) Recombinant marine E-selectin/human Ig chimera (E-lg) and marine anti-human CD44 (2C5; IgG) were from R&D Systems. Marine anti-human sLe" (KM93; IgM) was from Calbiochem. FC-conjugated marine anti-human CD Ila (25.3; IgGI) PE conjugated mouse IgG ,,tC isotype and FVTC-conjugated mouse IgMisotype were from Coulter-Immunotech, FITC-conjugated goat anti-rat IgM, FIrC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-mouse IgM, PB-conjugated strepavidin, alkaline phosphatase (AP)-conjugated anti-rat IgM, anti-mouse Ig, and anti-human Ig were from Southern Biotechnology Associates. V. Cholerac sialidase was from Roche. [000511 Humancells: Bone marrow (BM) cells were obtained from harvest filers of healthy individuals donating bone marrow for hematopoietic stem cell transplantation at the Brigham & Women's Hospital/Dana Farber Cancer Institute and Massachusetts General HospitaL BM mononuclear cells (BMMNCs) were collected by icoll-Paque density 5 gradient centrifugation. Human cells were used in accordance with the protocols approved by the Human Experimentation and Ethics Conmaittees of Partners Cancer Care Institutions [Massachusetts General Hospital, Brigham & Women's Hospital and Dana Farber Cancer Institute (Boston, MA)] Human umbilical vein endothelial cells (HUVEC) were obtained from the tissue culture core facility at Brigham & Women's Hospitals Pathology Department a and were maintained in M199 supplemented with 15% FBS, 5 units/m heparin, 50 pg/mI endothelial growth factor and 1% penicillin/streptomycirt To stimulate expression of E selectin, confluent monolayers of HUVEC were pre-treated with I ng/ml IL-1 (Research Diagnostics, Inc; Concord, MA) and 10 ng/ml TINFa (Research Diagnostics, Inc.) for 4-6 hrs prior to use in the adhesion studies. as 100052] MSC culture. MSC were maintained in a humidified incubator at 37*C in an atmosphere of 95% air,5% CO (as per (15) ) or in 3% 02.5% CO292% N 2 (as per (16). "MIAMr' cells). For culture of either type of MSC, BMMNCs were plated initially at a density of 2 x10'/cm in DMEM-low glucose medium supplemented with 10% fetal bovine serum (FBS) from selected lots. After several days, non-adherent cells were removed and s0 adherent cells were harvested by treatment with 0.05% trypsin/0.5 mM EDTA/HBSS (Invitrogen Corp.) and replated at a density of 50 cells/cm 2 . Medium was replaced at 48 to 72 hours and every third or fourth day thereafter. Cells were replated when density approached 40% confluence. For all experiments, MSC were used within the first 3 13 passages, and harvested by treatment with 0,05% trypsin/Q.5 mM EDTA/HBSS for less than 3 minutes at 37C. [000531 Generation of SACK-i PAb HCEL was isolated from KGla cells by immunoaffinity chromatography of cell lysates using anti-CD44 mAb. BALIEc mice were injected with pure HCELL in complete Freund's adjuvant (1:1 emulsion), splitting inoculum 50:50 between skin and intraperitoneal sites. Boosting was performed 2 weeks later with pure HCELL, diluted 1:1 in incomplete Freund's adjuvant and injected intraperitoneally. 10 14 days later, mice were boosted by IV injection of 5 ptg HCELL, then spleens were harvested 3 days following IV boost. Splenocytes were fused with NSO myeloma cells. Screening of hybridoma supernatants was initially performed by flow cytometry, against beznatopoietic cell lines KGla (CD44+/HCELL+/HECA4502+o HL60 (CD444HCELL /HECA452+), RPM18402 (CD44+/HCELL-f*BCA452-), JURKAT and K562 (both of which are CD44d4CELL-/1HECA452-) SACK-I mAb was identified as *CD44specific, carbohydrate-specific", by reactivity to KG I a but not to CD44- cell lines, in conjunction with Western blot evidence of mono-specificity for CD44 expressed on KGla cells, sensitive to digestion with N-glycosidase F (New England Biolabs; N-glycoidase F digestion performed as previously described (10, 12)). [000541 Flow cytometry: Aliquots of cells (2 X iW5 cells) were washed with PBS/2% FS and incubated with primary mnAbs or with isotype control mAbs (either unconjugated or a fluorochrone conjugated). The cells were washed in PBS/2% FBS and, for indirect immunofluorescence, incubated with appropriate secondary fluorochrome-conjugated anti isotype antibodies. After washing cells, FITC or PE fluorescence intensity was determined using a Cytomics FC 500 MPL flow cytometer (Beckman Coulter Inc., Fullerton, CA). [000551 Recombinant expression and formulation of human a(13)-fucosyltransferase Vt. Pichia pastors KM 71 (arg4his4aoxl:ARG4) host strain containing the human a(1.3) fucosyltransferase VI (FIN!) gene and the N-erminal signal sequence of S.cerevisiae a factor were used for stable expression and secretion of highly active a(,3)-fucosyltransferase VT into the medium using online methanol sensing (sterilizable methanol sensor by Raven Biotech. Vancouver. Canada) and regulation of methanol addition by Alitea-pumps (Alitea ao A3, Stockholm, Sweden). After the end of fermentation, the broth was cooled down to 10*C and the Pichia cells were separated by a Petlicon-microfiltration system with 0p2 gm membranes and, subsequently, the final formulation was achieved by buffer exchange with 14 HBSS using a Pellicon-ultrafiltration system with 10 kD-UF-membranes (regenerated cellulose). [00056] Recombinant expression and fonnulation of human sialytransferase: Pichia pastoris KM 71 (arg4his4aoxl:ARG4) host strain containing the human sialytransferase gene and the N-tenninal signal sequence of S.cerevisiae c-factor were used for stable expression and secretion of highly active sialytransferase into the medium using online methanol sensing (sterilizable methanol sensor by Raven Biotech, Vancouver, Canada) and regulation of methanol addition by Alitea-pumps (Alitea A., Stockholm, Sweden). After the end of fermentation, the broth was cooled down to l0"C and the Pichia cells were separated by a Pelficon-microfihtration system with 0.2 pam membranes and, subsequently, the final formulation was achieved by buffer exchange with HBSS using a Pellicon-ultrafiltration system with 10 kD-UF-membranes (regenerated cellulose). 1000571 Recombinant expression and formulation of human glycosytransferase: Pichia pastors KM 71 (arg4his4aoxl :ARG4) host strain containing the human glycosytransferase gene and the N-terminal signal sequence of S.cerevisiae a-factor is used for stable expression and secretion of highly active glycosytransferase into the medium using online methanol sensing (sterilizable methanol sensor by Raven Biotech, Vancouver, Canada) and regulation of methanol addition by Alitea-pumps (Alitea At, Stockholm, Sweden), After the end of fermentation, the broth is cooled down to 10*C and the Pichia cells were separated D by a Pelicon-microfiltration system with 0.2 pm membranes and, subsequently, the final formulation is achieved by buffer exchange with HBSS using a Pellicon-ultrafiltration system with 10 dcUFA-membranes (regenerated cellulose). (000581 Recombinant expression and jormulation of human N acet ylgucosaminotransferase Pichia pastoris KM 71 (arg4his4aoxl:ARG4) host strain Ps containing the human N-acetylglucosaminotransferase gene and the N-terminal signal sequence of S.cerevisiae a-factor is used for stable expression and secretion of highly active N.acetylglucosaminotransferase into the medium using online methanol sensing (sterilizable methanol sensor by Raven Biotech, Vancouver, Canada) and regulation of methanol addition by Alitea-pumps (Alitea A.B., Stockholm, Sweden). After the end of fermentation, the broth 3o is cooled down to 10*C and the Pichia cells are separated by a Pellicon-nicrofiltration system with 0.2 p membranes and, subsequently, the final formulation is achieved by buffer 15 exchange with HBSS using a Pellicon-utrafiltration system with 10 kD-UF-membranes (regenerated cellulose). [000591 FTVI and Sialidase treatment: MSC either in confluent monolayer or in suspension were treated with 60 mU/mL FTVM in HESS containing 20mM HEPES. 0.1% human serm albumin and I mM guanosine diphosphate (GDP)-fucose for 40 min. at 37C. After the incubation, MSC were washed with HBSS containing 0,2% BSA and 20mM HEPES. Untreated and FTVI-treated MSC were then used for experiments. In some experiments, MSC were first treated with FTV! and then subjected to sialidase treatment (100 mU/mn V. Cholerae Sialidase, 1 hour, 37"C) ("FTVk Sialidase MSC"). Efficacy of sialidase treatment was confirmed in each case by loss of reactivity to KM93 and HECA452 by flow cytometry. 1000601 Sialyransferase treatment: Cells are treated with 60mU/mnL of N sialyltransferase, ImM CMP-sialic acid or treated with buffer alone (HBSS, 0.1% human serum albumin) for 1 hour at 37 0 C. After the incubation, the cells are washed with HBSS containing 0.2% BSA and 20mM HEES. 000611 Galactosyftransferase treatment: Cells are treated with 6OmUiriL of Galactosyltransferas, ImM UDP-galactose or treated with buffer alone (HBSS, 0.1% human serum albumin) for 1 hour at 37*C, After the incubation, the cells are washed with HBSS containing 0.2% BSA and 20mM HEPES. (00062] N-acetylglucosaminotransferase treatment: Cells are treated with 60mUlmL of N-acetylglucosaminotransferase, I mM UDP-N-acetylglucosamine or treated with buffer alone (HBSS, 0,1% human serum albumin) for 1 hour at 37*C. After the incubation, the cells are washed with HBS5 containing 0.2% BSA and 20mM HEPES. (00063) Western blot analysis: Untreated and FTV! treated MSC were lysed using 2% s NP-40 in Buffer A (150mM NaCl, 50mM Tris-HCI, pH 7.4, 1 mM EDTA, 20 pg/ml PMSF, 0,02% sodium azide; and protease inhibitor cocktail tablet (Roche Molecular Biochemicals)). Western blots of quantified protein lysates or of immunoprecipitated protein were performed under reducing conditions as described previously (10). [000641 Immunoprecipitation studies: Cell lysates of untreated or FTVI treated MSC an were incubated with immunoprecipitating antibodies or with appropriate isotype controls and then incubated with Protein G-agarose. Immunoprecipitates were washed extensively using Buffer A containing 2% NP-40, 1% SDS. In some experiments, immunoprecipitates were treated with N-glycosidase F (New England Biolabs) as previously described (10. 12)). For 16 Western blot analysis, all immunoprecipitates were diluted in reducing sample buffer, boiled, then subjected to SDS-PAGE, transferred toPVDF membrane, and immunostained with HECA-452, E-Lg. SACK-I or 2CS (10) (00065] Parallel plateflow chamber adhesion assay; E-selectin binding capacity of untreated and FTVI-treated MSC was evaluated using a parallel plate flow chamber (Glycotech; Gaithersburg, MD). MSC (0,5 X 106 cells/ml, suspended in HBSS/10mM HEPES/2mM CaCl solution) were drawn over confluent HUVEC monotayers, Initially. the MSC were allowed to contact the HUVEC monolayer at a shear stress of 0.5 dyne/cm 2 ? subsequently the flow rate was adjusted to exert shear stress ranging from 05 to 30 dynes/cm 2 . The number of untreated or FTVI-treated MSC adherent to the HUVEC monolayer was quantified in the final 15 sec interval at shear stress of 0.5, 1, 2, 5, 10,20 and 30 dyne/cm 2 . Each assay was performed at least 3 times and the values averaged. Control assays were performed by adding 5 mM EDTA to the assay buffer to chelate Ca required for selection binding or treating HUVEC with function-blocking anti-human E-selectin mAb ( 68-5411) at 37 "C for 15 min. prior to use in adhesion assays. (000661 In vivo homing: All studies were performed in accordance with NIH guidelines for the care and use of animals and under approval of the Institutional Animal Care and Use Committees of the Massachusetts General Hospital and the Harvard Medical School. For intravital microscopy, NOD/SCID mice were anesthetized and a small incision was made in the scalp to expose the underline dorsal skull surface as previously described (5). aperiments were performed on the same day using littermates to analyze each of four groups of MSC (n=4 for each group); (1) FTVI treated (as above) MSC; (2) Buffer-treated MSC; (3) FTV-treated MSC digested with sialidase (100 mU/mi V. Cholera Sialidase, 37"C, hour); and (4) untreated MSC. Cells were stained with the fluorescent lipophilic tracer dye DiD (10 gM, 37C, 30 min; Molecular Probes) and infused into tail vein of NOD/SCID mice. The interactions of MSC with bone marrow microvascular endothelial cells within the parasagittal region were monitored and imaged at different time points after injection by in vivo confocal microscopy using progressive scanning and optical sectioning combined with video-rate imaging as previously described (5). For delineation of bone 30 marrow vasculature. long-circulating fluorescent quantum dots (Qtracker 800, Invitrogen) were injected systemically just prior to imaging. Stock solution of Qtracker 800(2yM) was diluted 1:4 (50 yL mixed in 150 yL PBSIx) and injected into anesthetized mouse via tail vein. In vivo confocal microscopy of the mouse skull bone marrow was performed as 17 previously described (5). DiD-labeled cells were excited with a solid-state 633 urn laser and imaged with a 45 nm bandpass filter centered at 695 nm, while quantum dots were excited with a solid-state Nd:YAG laser at 532 nm and imaged with a 770 rnm longpass filter. [000671 EXAMPLE 2: HUMAN MESENCUYMAL SThm CELs EXPRESS N-.OKED, 3 SIALYLATED GLYCOFORMS OF CD44 AND Do NoT BIND SELECTS [000681 Bone marrow contains two populations of stem cells, hernatopoietic stem cells and mesenchymal stem cells (MSC). MSC represent a small population of cells present within normal marrow, but they can be isolated and expanded in culture. MSC characteristically express CD44 and several other adhesion molecules found on hematopoietic cells (14). However, it is unknown whether these primitive non-hematopoietic cells express any selectin ligands. This paucity of data, and the finding that NCELL is expressed among only the ealiest hematopoietic cells (CD34+/lin- cells) (10 11), prompted us to examine whether MSC display similar carbohydrate modifications on CD44 that could bind selectins. [000691 MSC were cultured from human bone marrow as per two established. published protocols (15, 16). The MSC derived using both methods were capable of multipotential differentiation toward adipocyte, osteocyte and fibroblast differentiation, as previously described (15, 16). Regardless of protocol. MSC displayed no significant differences in any of the measured cell surface markers or in their response to enzymatic wo treatments. By flow cytometry (Figure 1c), MSC lacked expression of PSGL-l or of sialofucosylated determinants that could serve as selectin ligand(s): notably, the cells were devoid of reactivity to mAb KM93 or HECA452 (each of which identify sialyl Lewis X) and to E-Ig by both flow cytometry and Western blot (Figures 1c, 2a and 2b). Additionally, both types of MSC lacked LEA-i (CDI la/CDl 8) but expressed another integrin, VLA-4 25 (CD49d/CD29) (Figure Ic). VLA-4 can mediate rolling interactions and firm adherence on vascular endothelium (17), but both of these adhesive functions require "inside-out" activation usually mediated by the SDF-/CXCR4 pathway (18). Importantly, analysis of immunofluoresence staining on adherent MSC on plates and by flow cytometry showed no expression of CXCR4 (Figure Ic) and, predictably, MSC did not migrate in response to SDF 30 1 in either static or flow-based assays (not shown). 18 [000701 CD44 expression was high among all MSC isolated from numerous donors (Figure Ic). Conspicuously, SACK-i reactivity was also high on all MSC from all donors (Figure Ic), indicating that these cells uniformly expressed N-inked, sialylated glycoforms of CD44. The native absence of selectin ligands but prsence of a sialylated CD44 acceptor made MSC an ideal cell type to examine how HCELL expression affects cellular trafficking to bone marrow. [000711 EXAMPLE 3. Ex vivo FUCOSYLATION OF MESENCHYMAL STEM CELS REsULTs IN CELL EXPRESSION [000721 To enforce HCELL expression. MSC were treated ex vivo with an a(1;3) fucosyltransferase, fucosyltransferase VI (FrV). In all MSC cultured from all donors, forced fucosylation resulted in profound staining with mAb HECA452 and KM93, consistent with expression of sialyl Lewis X epitopes (Figure 2a) Western blot of cell lysates and of immunoprecipitated CD44 from FTVI-treated MSC revealed that the only glycoprotein bearing requisite sialofucosylations recognized by HECA452 was CD44 (Figures 2b and 2c). Moreover, fucosylated MSC bound EIg by flow cytometry and Western blot analysis of cell lysates showed that the only glycoprotein supporting Eig binding was CD44 (Figure 2) The relevant sialofucosylations of HCELL were displayed on N-glycans. as shown by abrogation of E-g binding following digestion with N-glycosidase F (Figure 2c). [000733 To analyze the E-selcctin ligand activity of FTVI-treated MSC under physiologic blood flow conditions, parallel plate flow chamber studies were performed using human umbilical vein endothelial cells (HU VEC) stimulated by cytokines to express 13 selectin, As shown in Figure 3, FTVI-treated MSC showed profound E-selectin ligand activity, which was completely abrogated in the presence of EDTA and by treatment of MSC with sialidase. Consistent with prior studies of cells natively expressing HCELLI robust shear-resistant interactions were observed within usual post-capillary venular shear levels (I 4 dynes/crn) and persisted at upwards of 20 dynecm 2 . well outside the range where PSGL can support E-selectin binding (10). These data indicated that the HCELL created by fucosylation of MSC surfaces was functionally similar to that displayed natively on the surface of KGla cells and human hematopoietic progenitor cells (10, 1) 19 f000741 ExAMsLE 4: CELL EXPRESSION CONFERRED ENIANCED HOMING OF MSC TO BONE MARROW IN VIVO [00075) To determine whether HCELL expression conferred enhanced homing of MSC to bone marrow in vivo. we employed dynamic real-time confocal microscopy to visualize marrow sinusoidal vessels in the calvarium of live inmunodeficient mouse (NOD/SCID) hosts (5). Four groups of cells were injected into tail vein of respective hosts: (1) FrVI-treated MSC, (2) FTVIktreated MSC digested with sialidase CFTV-Sialidase MSC"') (3) buffer-treated MSC, and (4) untreated MSC In vivo microscopy studies showed that FTVI-treated, HCELL-expressing MSC rolled directly on marrow sinusoidal vessels. and infiltrated the marrow parenchyma rapidly, within hours of infusion (Figure 4,). In contrast, untreated MSC and buffer-treated MSC showed minimal binding interactions with sinusoidal endothelium and displayed only modest infiltrates, whereas FTVI-Sialidase MSC typically showed even lower levels of endothelial interactions and marrow infiltrates (Figure 4;). The latter finding highlights the critical role of HCELL in homing. and also indicates that the marrow tropism following FTVI treatment was not a result of fucosylation per se..or of indirect effects on other adhesion molecules, but is a consequence solely of the induced selectin ligand activity, requiring concomitant expression of a(23)-sialic acid and t(1.3) fucose modifications. Images obtained with simultaneous staining of MSC and blood vessels clearly show that HCELL+ MSC infiltrated the marrow parenchyma (Figure 4). The observed marrow infiltrates are striking given that studies herein were performed without injury induction, such as by use of radiation or other preparative manipulations of recipient animals that markedly augment expression of sinusoidal ligands promoting marrow trafficking (27). Collectively, these data provide definitive evidence that HCELL expression directly enhances homing of MSC to bone marrow. as [00076] In the canonical multi-step paradigm, homing receptor-mediated rolling interactions on the endothelium facilitates exposure to chenokines presumed critical for G protein-coupled upregulation of integrin adhesiveness with resulting firm adhesion followed by transmigration (3). Notably, the MSC used here did not bear CXCR4 or undergo chemotaxis to SDF- 1, the principal chemokine regulating bone marrow homing (4,5), Thus, 30 the capacity of these cells to infiltrate marrow shows that CXCR4 engagement is not compulsory for marrow trafficking. However, Step I interactions are indispensable for cell trafficking to any tissue, and, as shown here, augmentation of E-selectin ligand activity 20 promotes marrow homing, Viewed more broadly, our findings are consistent with a growing body of experi mental evidence indicating that engagement of homing receptors may be sufficient alone (i.e., absent chemokine signaling) to induce integrin adhesiveneness, with accompanying finn adherence and trans-endothelial migration (1). Notably, it has been found that ligation of CD44 itself on lymphocytes results in direct, synergistic upregulation of VLA-4 adhesiveness, leading to transmigration without chemokine involvement (28). Though future studies will be needed to determine whether this axis operates in other cell types, the fact that MSC characteristically express VLA-4 (Figure Ic) raises this possibility. (004771 EXAMPLES: IN Vvo FUCOSYLATiON OF NEURAL STEM CELLS (000781 Neural stem cells were treated with 60mU/mL of FT-VI ImM GDP-fucose or treated with buffer alone (HBSS, 0.1% human serum albumin) for I hour at 37"C. Cells were lysed in a buffer containing 2%NP-40. Proteins were separated on a 4-20% Tris-HCI gradient gel in denaturing conditions and transferred to a PVDF membrane. Membrane was immunoblotted with HECA452 antibody. Resulting blot shows the expression of HECA452 reactive epitopes on a number of proteins after forced fucosylation. FEr-V-treated neural stem cells were also analyzed for HECA4S2 reactivity using flow cytornetry. FT-VI-Cells were incubated with lOug/mL of HECA452 or lOug/mL Rat IgM isotype control for 30min at 4 0 C and subsequently with 20ug/mL of anti-Rat IgM-FITC for 30min at 4"C The flow cytometric results show an increase in HECA452 epitope expression on the cell surface after enforced fucosylation. ExAMPLE 6: IN VIvo FUCOSYLATION OF PULMONARY STEM CELLS Pulmonary stem cells were treated with 6OmU/nL of FT-VI ImM GDP-fucose or treated with buffer alone (HBSS 0.1% human serum albumin) for I hour at 37C, Cells were incubated with lOug/meL of 1ECA452 or lOug/m. Rat IgM isotype control for 30min at 4*C 25 and subsequently with 20ug/mL of anti-Rat IgM-FITC for 30min at 4"C. The flow cytometric results show an increase in H{ECA452 epitope expression on the cell surface after forced fucosylation. References I. R. Sackstein, Curr Opin Hematol 12,444 (2005), 0 2. T Lapidot, A, Dar, 0. Kollet, Blood 106, 1901 (2005). 3. T. A, Springer. Cell 76,301 (1994). 4. A. Peled et at, Science 283, 845 (1999). 5. D. A Sipkins et at, Nature 435,969(2005). 21 6. M I. Polley etal., Proc Nad AcadSci USA 88,6224 (1991). 7. K. MI. Schweitzer er at, Am J Pathol 148, 165 (1996). 8. P. S. Frenette, S, Subbarao, B. Maow U, H. von Andrian, D. D. Wagner, Proc Natd Acad Sci U S A 95,14423 (1998). 9. Z. Laszik et at, Blood 88, 3010 (1996). 10. C. J. Dimitroff, 1. Y. Lee, S. Rafi, R. C. Fuhlbrigge, R, Sackstein, I Cell Biol 153, 1277 (2001). 11. C. J. Dimitroff, 1 Y. Lee, R. C. Fuhlbrigge, R, Sackstein. Proc Nad Acad Sci US A 97,13841 (2000). 12. R. Sackstein, C. I Dimitroff. Blood 96,2765 (2000). 13. C. S. Dimitroff. . Y. Lee, K. S. Schor, B. M. Sandmaier, R. Sackstein, JBiol Chem 276,47623 (2001). 14. M. F. Pittenger, B. 3 Martin, Circ Res 95,9 (2004). 15. M. F. Pittenger et at, Science 284, 143 (1999). 16. G. DIppolito et atJCell Sci 117,2971(2004). 17. R. Alon et at, J Cell Riol 128, 1243 (1995), 18 V. Grabovsky et at, J p Med 192,495 (2000), 19. B. W. Murray, S. Takayama,.IJ Schultz, C H, Wong, Biochemistry 35, 11183 (1996). 20. N. Schrantz et at, Cell Death Difer 6 445 (1999). 21 K. M, de Brayn, S. Rangarajan, K. A. Reedquist. C. G. Figdor, J L. Bos, J Diol Chem 277,29468 (2002). 22. A. Chigaev a at, J Biol Chem 276,48670 (2001). 23. Y. Takamatsu, P. L Sinnons, J. P. Levesque, Cell Adhes Commun 5, 349(1998), 24. M. M. Kobzdej, A. Leppanen, V. Ramachandran, R, D. Cummings, R P. McEver, Blood 100, 4485 (2002). 25. L Xia, 1. M, McDaniel, T Yago, A. Doeden, R, P. McEver. Blood 104, 3091 (2004). 26. A. Hidalgo, P. S. Frenette, Blood 105, 567 (2005) 27. 1. B. Mazo, E. J. Quackenbush, . B. Lowe, U. H. von Andrian, Blood 99,4182 (2002). 30 28. A. Nandi. P. Estess, M. Siegelnan, Immunity 20,455 (2004) 29. L. Yao et at, Blood 94, 3820 (1999). 30. J. Mocco et at, Cire Res 91, 907 (2002). 22 [00079] A number of embodiments of the invention have been described For example. in a further aspect the present invention provides a composition comprising a population of isolated cells aig Eselectin biinng a forced, the cells being stem cells, stm/progenitor cells or differentiated cells the population having an increased level of E.electin binding activity relative to native populations of cells of that type and a viability of at least 70% over a period of 24 hours afler the enforced E-selectin binding activity. In preferred embodiments of this further aspect of the present invention, the composition is characterized in that: (a) the population has increased reactivitywith an Eselectininununoglobulin fusion protein(Fl chimera)relative to native populations of cells of that type; (b) wherein the population has increased reactivity with monoclonal antibody HECA 452 relative to native populations of cells of that type; (c) the population is suspended in a physiologically acceptable solution., carrier or vehicle; (d) the population binds to E-selectin under static conditions; (e) the population binds to F-selectin under fluid shear stess greater than 0S dynes/cn ()the popular ion i!s capable of undergoingi, tetherving or rollinglj cecx-dpndn binding interactions on cytokine-stimulated human unibilial vein endothelial cells at a fluid shear stress of greater than 0.5 dyves/cn (g) the -selectin binding activity is transient (h) the population has a viability of at least 99% over a period ofat least 24 hours from the enforedA E-selectin binding; (i) the enforced Eseiectin binding activity is transient and the populatoi has a viability of at least 70% over the period in which the cells possess E-selectin binding activity; k the population is a population of mesenchynmal stem cells, or a population of hematopoietic sten cells or a population of neural stem cells, or a population of mvnocyte ;Stem cells, or a popuOlation of pulniiary stem cells, o,,r a population of umbilical cord stem cells, or a population of differtiated cells, preferably the population of differentiated cells is a population of leukocytes 23 [00080] Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. [00081] A reference herein to a document or other matter which is given as prior art is not taken as an adiDssion that that document or prior art was part of common general knowledge at the priority date of any of theclaims [00082] With reference to the use of the words) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing the foregoing description and/or the following claims.