Poster Presentations - Poster Session II

104 34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143

POSTER PRESENTATIONS - POSTER SESSION II

251Abstract withdrawn.

252THE CYTOKINE ENVIRONMENT INFLUENCES CLONAL DOMINANCE AND RESPONSE TO IMATINIB BY CML PROGENITOR CELLS IN VITROM. Ojo*, M. Y. Gordon, S. B. MarleyImperial College, London, UK

Clinical observations suggest that the Philadelphia positive clone inCML patients has a proliferative advantage over normal progenitor cells.Based on our data showing that different classes of cytokines modulatenormal progenitor proliferation, we hypothesized that the cytokineenvironment might modify the balance between normal and CML cellproliferation, to the advantage of normal cells. To this end, we used acolony replating assay to measure relative levels of proliferation by normaland CML progenitor cells under different conditions of cytokinestimulation. We found that IL-3 and G-CSF in combination synergisticallyenhanced proliferation by normal cells (p=0.005) but not CML cells(p=0.9). This differential response was increased when we added SCF andGM-CSF to the combination (4 cytokines). The same pattern of differentialresponse was seen when we used a delta assay for primitive progenitorcells, showing that it is not restricted to the clonogenic progenitor cellpopulation (p=0.02 for normal cells and 0.4 for CML cells). We used aliquid culture system to model in vitro 'expansion' cultures. Here, thepresence of the 4 cytokines resulted in the preferential expansion of normalover CML progenitor cells (p=0.01), in fulfilment of the Tortoise v Haretheory proposed by Eaves and colleagues. Since the impact of p210bcr-ablon proliferation was modified by the different cytokines, we tested whetherclonogenic responses to Imatinib varied under different conditions ofstimulation. We found that Imatinib reduced proliferation byapproximately 70% in the presence of G-CSF but had no effect in thepresence of IL-3. We conclude that the differential action of selectedcytokines may be used to confer a proliferative advantage on normalprogenitor cells in vitro, and possibly in vivo.

253THYMIC RECENT OUTPUT FUNCTION IN PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIAYangqiu Li1*, Suxia Geng1, Lijian Yang1, Shaohua Chen1, Xueli Zhang1, Grzegorz Przybylski2, Christian A. Schmidt21Institute of Hematology, Medical College, Jinan University, Guangzhou 510632, China2Dept. of Hematology and Oncology, Ernst-Moritz-Arndt University Greifswald, Greifswald 17487, Germany

It is a common feature that leukemia patients suffer from cellularimmunodeficiency which may play a role in initiation of hematologicalmalignancies. Understanding the mechanisms underlying persistentimmunity in leukemia might suggest treatment strategies to enhanceimmune competence in all. Thymic function is characterized its importanceof thymus to T-cell diversity in the periphery during both health anddisease. During early T lymphopoiesis, rearrangement of the V, D and Jsegments of the TCR genes result in deletion of the interveningchromosomal DNA and formation of circular excision circles, the signaljoint T-cell receptor rearrangement excision circles (TRECs) which hasbeen used to evaluate thymic function in T-cell immune reconstructionafter stem cell transplantation. In order to evaluate the thymic recent outputfunction in patients with chronic myelogenous leukemia (CML),quantitative detection of TRECs in DNA of peripheral blood mononuclearcells, purified CD4+ or CD8+ cells from 26 cases with CML werepreformed by real-time PCR. A dramatic reduction of TRECs values inCML was showed. The mean value of TRECs was 0.36±0.16 copies /1000T cells in patients and 6.36±5.28 copies /1000 T cells in normalindividuals (p=0.0005) (TRECs-number was related to the number of T-cells by determination of the number of CD3-positive cells). Lower valueof TRECs could be identified also in sorted CD4+ or CD8+ cells,1.06±1.11 copies /1000 CD4+cells in CML (3.28±1.24 copies/1000 CD4+cells in normal individuals, p=0.0002) and 1.91±1.52 copies /1000 CD8+

cells in CML (4.67±3.63 copies /1000 CD8+ cells in normal individuals,p=0.03). The results indicated that a prominent reduction of thymic recentoutput function in patients with CML and low numbers of T-cells withrecent recombination of their TCR gene. However, whether this reflectsimpairment of immune function associated to the malignancy, remains anopen question.

254CELL GROWTH INHIBITION AND INDUCTION OF APOPTOSIS OF GLIVEC RESISTANT CELLS BY HISTONE DEACETYLASE INHIBITOR (SK-7068) VIA SUPPRESSION OF TYROSINE KINASE CELL SIGNALING PATHWAYB. S. Kim1*, K. S. Ahn2, Y. J. Kim2, E. K. Bae3, I. H. Kim1, J. S. Lee1, S. Y. Park1,2,4, B. K. Kim1,2,4, S. S. Yoon1,2,41Seoul National University Hospital, Department of Internal Medicine, Seoul, Korea2Seoul National University, College of Medicine, Cancer Research Institute, Seoul, Korea3Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea4Seoul National University Hospital, Clinical Research Institute, Seoul, Korea

Imatinib mesylate (Glivec, Gleevec, STI571) is highly effective inpatients with chronic myeloid leukemia (CML). Despite high rates ofhematologic and cytogenetic responses to Glivec therapy, the emergence ofresistance to Glivec has been recognized as a major problem in thetreatment of CML. To investigate the mechanisms of resistance to Glivec,Glivec resistant cell line (K562-GR) was established. Using this cell line,we found that SK-7068, one of HDAC inhibitors, did enhance theapoptosis with increasing levels of cytochrome C, caspase 7 and caspase 3.In addition, the expression levels of anti-apoptotic genes such as Bcl-2,Bcl-xL, and Bid was down-regulated. For further investigation ofmolecular mechanisms, we analyzed gene-expression profiles of K562-GRcells treated with SK-7068 using a cDNA microarray representing 87,000human genes. 87 genes were highly up-regulated in K562-GR cells treatedwith SK-7068. The expression of candidate genes, which we selected inchip analysis, was confirmed using real-time PCR. Several genes (FLT-3,protein phosphatase 2C, ERBB3, JARID2, INSIG1, RNF41, RGSIPIP1,PRSS12, HUMPPA, PDCD4) were down-regulated by SK-7068 treatment.Those genes may be associated with resistance to Glivec. We suggest thattheir down-regulation by SK-7068 treatment was associated mainly withthe induction of apoptosis. Taken together, our data suggested that SK-7068 could overcome the problems of Imatinib mesylate therapy.

255PLASMIN-ALPHA2-ANTIPLASMIN AND TISSUE PLASMINOGEN ACTIVATOR IN ESSENTIAL THROMBOCYTHEMIA AND POLYCYTHEMIA VERA AND THROMBOTIC COMPLICATIONSE. Cacciola*, A. Cipolla, E. Di Francesco, R. Giustolisi, R. R. CacciolaDepartment of Biomedical Science - Section of Hematology, University of Catania, Catania, Italy

Essential thrombocythemia (ET) and polycythemia vera (PV) arecharacterized by thrombophilic tendency linked to platelet-coagulativeactivation responsible for fibrinolytic abnormalities. However, few studieshave concentrated on the correlation between fibrinolysis andthrombogenesis in patients with myeloproliferative disease. Therefore, weinvestigated b-thromboglobulin (bTG) and platelet factor 4 (PF4), markersof platelet activation, prothrombin fragment 1 (F1+2), marker of clottingactivation, plasmin/alpha2-antiplasmin (PAP), fibrinolytic complexupregulating platelet function and tissue plasminogen activator (t-PA),thrombin-derived fibrinolytic component in patients with ET and PV andthrombotic complications. We included 44 patients, 22 ET (5 men and 17women, mean age 55 years) and 22 PV (17 men and 5 women, mean age60 years) who fulfilled PVSG. The mean duration of disease was 4.5 years.Most received cytoreduction, such as hydroxyurea (13 ET and 14 PV),interferon-alpha (2 ET), anagrelide hydrochloride (5ET) or phlebotomy (8PV). None of studied patients had thrombotic risk factors. Of 44 patients,25 (12 ET and 13 PV) developed thrombosis, whereas 19 (10 ET and 9 PV)did not. Measurements were assayed by ELISA. All patients had increased

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Abstracts

bTG and PF4 (352±608 IU/ml and 129±49 IU/ml, respectively, vs 25±9IU/ml and 5±2 IU/ml, respectively) (p=0.0001 and p<0.0001,respectively) F1+2 (2.8±2.8 nmol/L vs 0.7±0.2 nmol/L) (p=0.0001) andPAP (521±714 ng/ml vs 58±54 ng/ml) (p<0.0001). t-PA was higher inthose patients with previous thrombosis than in asymptomatic patients(130±62 ng/ml vs 89±69 ng/ml) (p=0.043). A positive correlation therewas between bTG and PF4 and PAP (p=0.037 and p=0.045, respectively)and F1+2 and t-PA (p<0.0001). These data suggest that the thrombotictendency might be linked to a hyperfibrinolytic state in ET and PV and thatt-PA could be a predictive indicator of thrombogenesis of each patient.

256ENHANCED HDAC (HISTONE DEACETYLASE) ACTIVITY IN BLOOD CD 34 + CELLS IN PATIENTS WITH AGNOGENIC MYELOID METAPLASIAJ. C. Wang*, K. Hemavathy, K. Suppiah, N. Boghossian, T. S. Chang, A. D. NovetskyMaimonides Hospital Medical Center, Brooklyn, USA

We have previously reported elevated Blood TIMP-1, TPO, OPG levels,reduced blood MMP-3 levels and reduced bone marrow SDF-1 levels inAMM patients. Decreased TGFB1RII mRNA has been reported in bloodCD 34 + cells in patients with AMM previously. Therefore, we postulatedthat Likely the TGF-B1 Type II receptor (TGFB1 RII) defects, results inthe loss of negative regulation of megakaryocyte progenitor cells that leadsto abnormal megakaryocytes proliferation. Abnormal megakaryocytesproliferation then leads to over-production of TGF-B1 which then leads tothe increased TPO, TIMP, OPG and decreased MMP, SDF-1. Thus, studiesof the TGF-B1RII gene defects in AMM is important. We have analyzedand found absence of mutations in the gene or the promoter of TGFB1 RIIand the absence of CpG methylation of the promoter. Since repression ofthe TGF-B1RII expression has been found to be associated with HDAC inpancreas cancers (Zhao et al, Cancer Res 63:2624 2003), we studiedHDAC activity in the blood CD34 +cells of patients with AMM. HDACactivity was measured using the HDAC Colorimetric Activity Assay/ Drugdiscovery Kit (BIOMOL Research Lab. Inc. PA). The HDAC activitiesestimated as the amount of the released Deacetylated Product are shown inTable 1.

This study suggests that HDAC activity is enhanced in patients withAMM, and could be responsible for the decreased TGFB1RII expression.HDAC inhibitors could therefore serve as valuable tools in the treatment ofthis disease.

257IM RESISTANCE OF CML STEM CELLS IS BCR-ABL DEPENDENT AND CANNOT BE OVERCOME BY DOSE ESCALATION IN VITROA. Hamilton*, L. J. Elrick, E. Allan, J. W. Baird, H. G. Jorgensen, T. L. HolyoakeGlasgow Royal Infirmary, Glasgow, UK

Despite inducing an excellent rate of complete cytogenetic response(CCR), Imatinib Mesylate (IM) does not eradicate Ph+/BCR-ABL+ stemcells in vivo. We have previously shown that primitive CML cells aresensitive to the anti-proliferative, but not the pro-apoptotic effect of IM,leading to their persistence in vitro.

To determine whether CML stem cell resistance was BCR-ABLdependent or independent and to overcome potentially increasedexpression of BCR-ABL transcripts and/or tyrosine kinase activity, or lowintracellular drug levels, total CD34+ and more primitive CD34+/38- cellsfrom both chronic phase CML patients and normal donors, were cultured inserum free medium, supplemented with 5 growth factors for 72h, in thepresence and absence of increasing concentrations of IM (1-50 microM).Endpoint analyses included D-FISH and RT-PCR for BCR-ABL andphosphorylation status of CrKL, using an antibody specifically designed torecognise only the phosphorylated form of the protein.

In 8 CML samples tested, cells remaining viable at 72h showed onlysingle copy BCR-ABL by FISH and expressed abundant BCR-ABLtranscripts, excluding gene amplification as an explanation for their IMresistance.

After 16h, total CD34+ CML cells showed significant CrKLdephosphorylation in response to IM 1-10 microM (P=0.03), but at 72h nosignificant dephosphorylation was seen, consistent with enrichment of anIM-resistant population. In the CD34+/38- fraction, no significantdephosphorylation was observed at 16h or 72h, suggesting the majority ofthese cells was inherently resistant to IM. Dose escalation to 25 and 50microM IM caused non-specific cell death of both normal and CML cellswithout inducing dephosphorylation of CrKL. These results confirm thatdose-escalation of IM does not achieve inactivation of BCR-ABL/CrKL invitro, suggesting BCR-ABL dependent resistance. Further work is ongoingto quantify BCR-ABL transcripts and to sequence the kinase domain inthese IM resistant cells.

258IS HUMAN NEUTROPHIL ELASTASE ACTIVITY IN CHRONIC MYELOID LEUKAEMIA A POTENTIAL TARGET FOR THERAPY?E. K. Allan*, K. Stewart, T. L. Holyoake, H. G. JorgensenUniversity of Glasgow, Glasgow, UK

Human neutrophil elastase (HNE) is a serine protease that is aberrantlyexpressed in myeloid disorders including chronic myeloid leukaemia(CML). In CML, such protease activity may contribute to leukaemic clonaldominance through antagonism of granulocyte-colony stimulating factor(G-CSF) activity required for normal haemopoiesis. Furthermore,neutropenia is observed in 15 to 40 percent of imatinib (IM)-treatedchronic phase (CP) CML patients. We aimed to quantitate any differencesbetween HNE levels in bone marrow (BM) and peripheral blood (PB)samples taken from CML patients to determine if HNE may be targetedwith an inhibitor modelled on the naturally occurring anti-protease, elafin.Measurement of plasma HNE by ELISA showed that at diagnosis (CPCML n=17), PB HNE was significantly elevated (2023±95 nanog/mL,mean±SEM) with respect to normal controls (64±3 nanog/mL, n=9;p=0.002). HNE was also significantly elevated in CML BM supernatantseparated from samples taken before IM treatment (5251±162 nanog/mL,n=6) with respect to non-CML controls (2154±695 nanog/mL, n=3;p=0.003). After treatment with IM for longer than 4 weeks, PB HNE levelsnormalised (51±3 nanog/mL) in parallel with correction of WBC. At 12weeks post-IM, there were no differences in PB or BM HNE levelsbetween those patients in complete cytogenetic remission and cytogeneticnon-responders (both groups with normal WBC), with BM samples notsignificantly different from non-CML controls. If HNE levels wereelevated in neutropenic IM-treated patients, this would support furtherinvestigation of anti-elastase therapy to abrogate the neutropenia throughremoving the anti-proliferative effect of HNE on normal progenitors.Alternatively, elevated HNE in IM-induced neutropenia would providefurther rationale for supportive therapy with supra-physiological G-CSF.

259A LONG-ACTING G-CSF (FILGRASTIM SD/01) MOBILIZES MOUSE NEUTROPHILS FROM BONE MARROW TO BLOOD, BUT SLOWS DOWN NEUTROPHIL MATURATION RATEE. Knudsen, A. Bøyum, P.O. Iversen, G. Nicolaysen, I. Strøm-Gundersen, H. B. Benestad*Dept. of Physiology, Inst. of Basic Medical Sciences, University of Oslo, Norway

It is generally thought that G-CSF can accelerate the maturation ofgranulocytes (Lord et al. (1991) Blood 77: 2154-59). However, analternative explanation could be a G-CSF mediated mobilization of matureand almost mature granulocytes from marrow to blood. We have evidencefrom an in vivo culture system (diffusion chamber culturing), where suchmobilization cannot take place, that G-CSF does not accelerate maturation.On the contrary, perturbations that increase proliferation rate ofgranulocyte precursors, retarded their maturation rate (Benestad &Rytømaa (1977) Cell Tissue Kinet 10: 461-68; Wang et al. (1999) Stemcells 17: 253-64).

Mouse granulocyte precursors in S-phase were labeled with bromo-deoxyuridine, and the hematopoiesis perturbed by SD/01. With flowcytometry, utilizing antibodies against BrdUrd incorporated in DNA andthe granulocyte specific maturation antigen Gr1, respectively, we have

Table 1AMM MPD Controls1.40 + 0.45 0.39 + 0.11 0.22 + 0.07

n 8 16 10P<0.01 ( AMM Vs MPD, AMM Vs Controls)P=NS (MPD Vs Controls)



investigated cell fluxes from the proliferative to the non-proliferativegranulocyte compartments in bone marrow, and further from marrow toblood. SD/01 mobilized granulocytes from bone marrow to blood,increasing their concentration markedly for several days. Judged by thedensity of Gr1 on bone marrow and blood cells, SD/01 did not seem toaccelerate granulocyte maturation rate. On the contrary, according to thiscriterion cells of the granulocyte lineage were less mature than control cellsafter the SD/01 treatment. Cell density separation of the bone marrow cellsin combination with flow cytometric analysis showed that SD/01 skewedthe (stimulated) granulopoiesis towards lower cell densities (i.e. moreimmature cells), containing less Gr1 antigen per cell, thus supporting theconclusion that G-CSF does not accelerate granulocyte maturation.

260BCR-ABL MEDIATES A DECREASE IN CCN3 EXPRESSION IN CHRONIC MYELOID LEUKEMIAL. McCallum1*, S. Price1, A. Pierce2, A. D. Whetton2, B. Perbal3, A. E. Irvine11Queen's University, Belfast, UK2University of Manchester, Manchester, UK3Universite Paris, Paris, France

Chronic Myeloid leukemia (CML) is characterized by expression of theconstitutively active Bcr-Abl protein tyrosine kinase. We have previouslyshown reduced expression of the growth regulator, CCN3, as a result ofBcr-Abl kinase activity in the murine FDCP-Mix haematopoietic stem cellline, using microarray technology. In this study we have examined CCN3expression in human CML cell lines and in primary human CML cells atdiagnosis and following treatment.

Real-Time PCR was used to monitor mRNA expression of BCR-ABLand CCN3 in the human CML cell lines K562, KU812, LAMA and also innormal bone marrow (NBM) and in 3 human CML patient samples taken atdiagnosis and following response to treatment. Western blot analysis andconfocal microscopy were used to detect Ccn3 protein expression.

BCR-ABL mRNA expression was strong in the human CML cell lineswhilst CCN3 expression was weak or absent. CCN3 mRNA expression inBCR-ABL negative cells (HL60), and in NBM was 30-fold higher thanexpression in the CML cell lines. Treatment of K562 cells with the Bcr-Abltyrosine kinase inhibitor, Imatinib (1 micromolar, 96 h) resulted in a 5-foldreduction in BCR-ABL expression and a concomitant 4-fold increase inCCN3 mRNA. Primary human CML cells at diagnosis showed a four-foldlower expression of CCN3 mRNA in comparison to NBM (p=0.015).Patients responding to treatment showed that as BCR-ABL expressiondeclined (mean decrease 15-fold) CCN3 expression had increased (meanincrease 19-fold) and was comparable with levels found in NBM.Differential expression of Ccn3 protein at diagnosis and in response totreatment, was also confirmed by confocal microscopy and Westernblotting.

Dysregulation of CCN3 expression has not previously been recognizedin CML. CCN3 and BCR-ABL expression appear to be inversely related.Loss of CCN3 expression is consistent with the CML phenotype.

261ANTI-CD40 UPREGULATES SIGNIFICANTLY TUMOR NECROSIS FACTOR-ALPHA PRODUCTION OF MACROPHAGES FROM PATIENTS WITH MYELODYSPLASTIC SYNDROMES COMPARED TO HEALTHY CONTROL SUBJECTSS. Meers1*, V. Raets1, L. Boon2, A. Kasran3, M. Boogaerts1, M. Delforge11Hematology, University Hospital, Leuven, Belgium2Bioceros NV, Utrecht, The Netherlands3Immunology, University Hospital, Leuven, Belgium

Background: Increased levels of intramedullary apoptosis areresponsible for the paradox between bone marrow hypercellularity andperipheral cytopenia frequently encountered in myelodysplastic syndromes(MDS). Several studies have stressed that TNFalpha produced bymacrophages and fibroblasts is one of the principal triggers of apoptosis inMDS, but the factors that upregulate TNFalpha in MDS are largelyunknown. The aim of this study was to examine in vitro TNFalphaproduction by monocyte-derived macrophages of MDS patients inresponse to various stimuli.

Materials & Methods: Peripheral blood mononuclear cells wereobtained from 19 healthy volunteers and 17 MDS patients (all with marrowblast counts <10% according to FAB and WHO). CD14+ cells were

selected using MACS columns and cultured in IMDM + 15% fetal bovineserum to induce macrophage differentiation. After 7 days cells were leftunstimulated or supplemented with LPS (at 1 microg/ml), or 2 clones ofanti-CD40 MoAb (at 10 microg/ml) with IFN-gamma (at 1000 U/ml).TNFalpha concentrations were measured with ELISA at 16h, 24h and 48h.Statistical analysis was performed using student's t-test.

Results: TNFalpha production of unstimulated macrophages was lowand comparable in all 4 groups. LPS induced lower levels of TNFalpha inpatients than in donors, but not significantly. With the stimulatorymonoclonal antibody (clone 64) directed against CD40, we could induce 2-to 10-fold higher TNFalpha production in MDS patients than in controls (at16h: p=0.02; at 24h: p=0.02; at 48h: p=0.01). For antibody-clone 7, levelswere not significantly different at any of the time points.

Conclusion: We conclude from this study that monocyte-derivedmacrophages from MDS patients produce significantly higher TNFalpha inresponse to CD40 compared to healthy controls. The observation thatstimulation with LPS could not reveal the same results, implies anupregulation of the CD40 pathway in MDS monocytes.

262THE CHEMOTACTIC RESPONSE OF CHRONIC PHASE (CP)-CHRONIC MYELOGENOUS LEUKEMIA (CML) PRIMITIVE PROGENITORS TOWARDS STROMAL CELL DERIVED FACTOR-1 (SDF-1) IS MODIFIED AND SUGGESTS ITS INVOLVEMENT IN THE LEUKEMIC PHENOTYPE OBSERVED IN CPT. Prebet1,2*, K. Chabane3, I. Tigaud3, E. Wattel1,2, M. Michallet1, F. E. Nicolini1,21UMR CNRS 5537, Centre Léon Bérard, Lyon, France2Hematology Department, E. Herriot Hospital, Lyon, France3Laboratory for Cytogenetics, Centre Hospitalier Lyon Sud, Pierre Bénite, France

Adhesion of hematopoietic progenitors to the BM stroma is abnormal inCP CML. SDF-1 is a CXC chemokine expressed by normal stromal cells,involved in progenitor homing and proliferation, mechanisms altered in CPCML. Here, we analyzed the influence of SDF-1 and its inhibitor SDF-1(G2), in 4h-TranswellTM migration assays of lin-CD34+ progenitors inserum free medium (SFM) conditions. Eight CP CML patients at diagnosisand 4 unrelated BM donors as controls were explored. Two x 105 lin-

CD34+ purified cells were comparatively analyzed in the presence of SDF-1, or SDF-1(G2) or no additional chemokine in the lower chamber, forcounts, CD34 and CXCR4 expression by FACS, CFC and LTC-ICcontents. After migration, median numbers of total migrant cells wereconsistently lower in CML vs BM (69250±17700 vs 80000±4660) in SFMarm, but equivalent in CML samples in SDF-1 (81450±30000) vs SFM(69250±18000) (p=0.02) indicating a relative insensitivity of CML totalprogenitors to this chemokine. In addition, SDF-1(G2) significantlyinhibited the migration of total CML cells (57400±15000) vs SDF-1(p=0.02). Absolute numbers of total CXCR4+ CML progenitors weresignificantly reduced in the migrant population vs BM, in SFM (19450 vs61000, p=10-5), SDF-1 (22160 vs 74980, p=0.005) and SDF-1(G2)(14850 vs 72000, p=0.01) conditions. No significant difference was foundfor total CD34+ populations. The migration of HPP-CFC (per 100 inputcells) was reduced for CML vs BM, in SFM (29±50 vs 95±20, p=0.02),SDF-1 (28±37 vs 97±19, p=0.03) and SDF-1(G2) (34±90 vs 85±14,p=ns) conditions; but other types of CFC were not affected. In oneexperiment, LTC-IC values (expressed per 1000 input cells) wereequivalent in CML between the different conditions (SFM: 50 vs SDF-1:54, vs SDF-1(G2): 55.2). This suggests that SDF-1-dependent chemotacticproperties of CP CML primitive (CXCR4+ and HPP-CFC) progenitors aremodified and might participate to the leukemic phenotype.



Abstracts

263USE OF SCORE BASED ON MEDIUM FLUORESCENCE RETICULOCYTE AND MYELOPEROXIDASE INDEX OBTAINED BY ADVIA 120 MIGHT EVALUATE MYELOPOIETIC FUNCTION BEFORE CHEMOTHERAPY AND INDIVIDUATE PATIENT WITH HIGH RISK OF NEUTROPENIA POST CHEMOTHERAPYG. Giordano1*, C. Gasbarrino1, S. Papini1, R. Tambaro1, M. P. Petrilli1, V. Fraticelli1, G. Leone2, G. Scambia1, B. Zappacosta1, S. Storti11UCSC, Campobasso, Italy2UCSC, Rome, Italy

Chemotherapy frequently involves, in neoplastic patients, severemyeloid suppression. Myeloid suppression is sometimes the main cause oftherapy recycling delay, deep, severe and prolonged neutropenia, anaemiaand thrombocytopaenia. Neutropenia is associated with an increasedsanitary expense, number of febrile episodes and days of hospitalization.

The aim of our study was to verify if was present a correlation betweenreticulocytary fraction, reticulocytary indices, myeloperoxidase index(MPXI) and postchemotherapy myelopoietic function (MF) and severeneutropenia. Our study is a monocentric, prospectic, nonrandomized, openlabel study.

53 patients (M/F:29/24, median age 58 years), 24 with lymphoma ormyeloma and 29 with solid neoplasms with bone marrow micrometastaseswere treated with chemotherapy (CT). After CT 25 patients hadneutropenia (ANC<500/mcl) for a median of 7 days (range 3-21). BeforeCT, MF was assessed by above mentioned parameters using hematologicautomated analyser ADVIA 120® (BAYER, Diagnostic Division,Tarrytown, NY). Patients with B12 and/or folate and/or iron deficiency orreceiving growth factors and/or transfusions were excluded from our study.

Among tested parameters, MPXI negative value was related toneutropenia with an OR=5.7 (CI95%:1.7-18.9, p0.008) and mediumfluorescence reticulocyte (MFR)>10.7% were inversely related toneutropenia with an OR=0.4 (CI95%:0.13-1.3, p0.212).

Subsequently we assigned to patients with MPXI value positive andMFR>10.7% a score=1, a score=0 was assigned to the remaining patients.Patients with score=1 showed a lower number of neutropenic events (only1 on 14 patients) than those with score=0 (24 on 39 patients), with a Fisherexact test with p<0.001 and an adjusted contingency coefficient of 0.6(CI95%:0.32-0.68).

MPXI and MFR might be used in myelopoiesis assessment before CTadministration, independently from type of tumor, PCT regimen andnumber of CT cycle, with the aim to identify a patient subset with an higherrisk of neutropenia post CT.

264BONE MARROW MOBILIZATION WITH G-CSF AND TRANSPLANTATION WITH CERTAIN STEM CELL SUBPOPULATIONS INFLUENCE LUNG CELL PRODUCTION IN RADIATION-INJURED MICEJ. M. Aliotta*, P. Keaney, M. Passero, M. S. Dooner, J. Pimentel, G. A. Colvin, M. Abedi, D. A. Demers, D. A. Greer, P. J. QuesenberryRoger Williams Medical Center, Providence, RI, USA

Previously we established that escalating doses of total body irradiation(TBI) and the administration of intramuscular (IM) cardiotoxin increasethe conversion of transplanted hematopoietic marrow cells into lung cells.The role of recombinant granulocyte colony-stimulating factor (G-CSF) inmarrow to lung cell conversion was evaluated by exposing mice to 900cGy TBI, transplanting whole bone marrow (WBM) cells then injectingthem with either 1, 2 or 3 rounds of G-CSF (250 microg/kg) or no G-CSF.In a separate experiment, mice exposed to 500 cGy TBI were transplantedwith WBM or various stem cell populations (Lin-, Lin+, C-Kit-, C-Kit+,Sca- or Sca+) then given IM cardiotoxin. Tissue analysis (9 weeks, firstexperiment; 4 weeks, second experiment) included the quantification ofGFP+/CD45- cells (defined as donor marrow-derived, non hematopoieticcells or lung cells) visualized in lung samples by fluorescence microscopy,expressed as a percentage of all nucleated cells. More GFP+/CD45- cellswere seen in mice exposed to 1, 2 or 3 rounds of G-CSF (5.81%, 7.88%and 5.74%, respectively) compared with non-mobilized mice (2.83%,p<0.01). In addition, higher levels of bone marrow engraftment and moreGFP+/CD45- cells were seen in mice transplanted with Lin -, C-Kit + andSca + cells (1.81%, 1.05% and 1.67%, respectively) compared with Lin+,C-Kit – and Sca – cells (0.03%, 0.02% and 0%, respectively; p<0.01). In

both experiments, most of the GFP+/CD45- cells were also cytokeratin+,identifying them as pulmonary epithelial cells. Some possessed themorphologic and molecular phenotype of Type II and I pneumocytes(surfactant protein B+ or Lycopersicon esculentum lectin+). Rare GFP+/CD45- cells were also von Willebrand+ (pulmonary endothelial cells) andalpha-actin+ (smooth muscle cells). These data indicate that in the lungs ofradiation-injured mice, certain stem cell populations offer a conversionadvantage over others and G-CSF augments conversion.

265MESENCHYMAL STEM CELLS ARE RENOPROTECTIVE IN ACUTE RENAL FAILURE AND ENHANCE RECOVERY THROUGH PARACRINE MECHANISMSF. Togel1,2*, Z. Hu1, K. Weiss1, J. Isaac3, C. Lange4, A. R. Zander4, C. Westenfelder1,21University of Utah, Department of Medicine, Division of Nephrology and VA Medical Center, Salt Lake City, UT, USA2University of Utah, Department of Physiology, Salt Lake City, UT, USA3University of Utah, Department of Pathology, Salt Lake City, UT, USA4Bone Marrow Transplantation Center, D-20251 Hamburg

Severe acute renal failure (ARF) currently is a common treatmentresistant clinical problem with disturbingly high mortality rates. We testedthe effects of mesenchymal stem cells (MSC) in rats with ischemia/reperfusion-induced (I/R) ARF (40 min bilateral renal pedicle clamping)on renal function and outcome and investigated amelioration ofinflammatory, vascular and apoptotic/necrotic manifestations. Intracarotidadministration of MSC (~ 106/animal) either immediately or 24 hours afterrenal ischemia, resulted in significantly improved renal function, higherproliferative and lower apoptotic indices, as well as lower renal injury andunchanged leukocyte infiltration scores. Such renoprotection was notobtained with syngeneic fibroblasts. Using in vivo two-photon laserconfocal microscopy, fluorescence labeled MSC were detected early afterinjection in glomeruli and low numbers attached at microvasculature sites.However, within 3 days of administration, none of the administered MSChad differentiated into tubular or endothelial cell phenotype. At 24 hoursafter injury, expression of pro-inflammatory cytokines IL-1b, TNF-a,IFNg, and iNOS was significantly reduced and that of anti-inflammatoryIL-10, and bFGF, TGF-a and Bcl-2 was highly upregulated in treatedkidneys. We conclude that the early and highly significant renoprotectionobtained with MSC is of considerable therapeutic promise for the cellularmanagement of clinical ARF. The beneficial effects of MSC are primarilymediated via complex paracrine actions and not by their differentiation intotarget cells, which, as such, appears to be a more protracted response thatpossibly becomes important in late stage organ repair.

266MODULATION OF NATURAL KILLER (NK) CELL FUNCTIONS BY MESENCHYMAL STEM CELLS (MSC) RESULTS FROM AN INTERPLAY OF SUPPRESSING AND ACTIVATING EFFECTSL. Fang*, C. Lange, M. Engel, A. R. Zander, B. FehseBone Marrow Transplantation, University Hospital Hamburg-Eppendorf, Hamburt, Germany

Bone marrow-derived mesenchymal stem cells (MSC) have been shownto be 'immune-privileged' themselves and exert a strong immune-modulatory function as 'third-party' cells. We investigated these intriguingMSC properties focusing on their regulatory influence on natural killer(NK) cells. Therefore we made use of a novel flow cytometry-approachwhich enabled us to analyze proliferative reactivity of different immunecell subsets at the single cell level. We found strong proliferative responsesof NK cells to allostimulation which were in vitro as efficiently down-regulated by third-party MSC as previously shown for T cells. Analogsuppressing effects of third-party MSC were noted on the immunologicalreactivity of both NK and T cells against CMV antigens. Similar resultswere obtained with MSC culture supernatant, in a dose-dependent manner,suggesting that immune suppression was mainly mediated by solublefactors. This was in agreement with our finding that at low doses MSC losttheir 'immune privilege' and became almost as allo-stimulatory as PBMC.Allo-stimulation of both T and NK by MSC was again almost completelyinhibited if MSC supernatant was added. Based on an alternative FACS-based method to measure immune responses we were in conclusion able todemonstrate that (i) the immune-modulating effects of MSC on NK cells



are at least as strong as previously described for T cells and (ii) the'immune privileged status' of MSC results from an interplay of immuneactivation and suppression.

267BONE MARROW-DERIVED MESENCHYMAL STEM CELLS AUGMENT WOUND HEALING WITHOUT INDUCING IMMUNOSUPPRESSIONX. H. Gao*, Y. B. Liu, K. McFarlin, D. Dulchavsky, D. Deeb, S. A. Dulchavsky, S. C. GautamDepartment of Surgery, Henry Ford Health System, Detroit, Michigan, USA

Bone marrow-derived mesenchymal stem cells (BMSCs) aremultipotential stem cells capable of differentiation into numerous celltypes, including fibroblasts, bone and cartilage, muscle –skeletal, heart andsmooth, and brain cells. BMSCs also secrete a large number of growthfactors (TGF-beta, EGF, FGF, KGF, and VEGF) and cytokines that arecritical to the repair of injured tissues. Because BMSCs are able to trans-differentiate into multiple cell types and secrete tissue repair factors, weinvestigated the efficacy of BMSCs for augmentation of abdominal fascialwound healing in Sprague Dawley rats. Systemic administration ofsyngeneic BMSC (2 million) once daily for four days or a single treatmentwith 4 million BMSCs 24 h after wounding significantly increased thewound bursting strength (WBS) on days 7 and 14 after wounding [WBS(Newtons)±S.D; day 7, control=4.1±1.0, BMSC=11.6±3.3; day 14,control=8.0±0.4, BMSC=14.8±2.9]. Wound healing was alsosignificantly improved following injection of BMSCs locally at the woundsite. Furthermore, allogeneic BMSCs were as efficient as syngeneicBMSCs in promoting wound healing. The increase in the tensile strength ofwound tissue was partially due to the increased production of collagen inthe wound tissue (microg/25 mg wound tissue±S.D.; day 7,control=17.3±0.3, BMSC=21.1±1.4; day 14, control=24.7±0.7,BMSC=27.3±0.1). The augmentation of wound healing by BMSCsoccurred without induction of immunosuppression, since local or systemicadministration of BMSCs had no effect on the development of alloreactivecytotoxic T cells or T cell proliferative response in vivo. These data suggestthat BMSCs may have therapeutic efficacy in the treatment of difficultwounds without induction of immunosuppression.

268VASCULOGENESIS IN PATIENTS WITH MULTIPLE MYELOMA THROUGH HEMATOPOIETIC STEM CELLSR. Ria1*, A. M. Roccaro1, T. Cirulli1, G. Di Pietro1, F. Falzetti2, R. Iacucci Ostini2, T. Zei2, A. Tabilio2, A. Vacca1, F. Dammacco11Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy2Department of Hematology and Clinical Immunology, University of Perugia Medical School, Bari, Italy

Vasculogenesis is a physiological process typical of fetal development.New blood vessels develop from undifferentiated precursors. Severalstudies have shown the presence of circulating and tissue angioblasts alsoin post-natal life that are able to differentiate into endothelial cells andother several histotypes. In tumors, near angiogenesis, vasculogenesiscontributes to the formation of the microvascular plexus that is importantfor local and systemic diffusion.

Here, we show that hematopoietic stem cells of multiple myeloma (MM)patients, purified with an anti-CD133 antibody from peripheral blood aftermobilization with cyclophosphamide and G-CSF, are able to differentiateinto cells with endothelial phenotype after about 20 days culture uponexposure to VEGF and bFGF. Stem cells were seeded in flasks coated withfibronectin in IMDM with 10% FBS, 10% horse serum, 10-6 Mhydrocortisone, 50 ng/mL VEGF and 10 ng/mL bFGF. Stem cellsgradually changed their phenotype, lost the typical antigen ofundifferentiated stem cells, and acquired a mature endothelial cellphenotype. By western blot and RT-PCR we demonstrated that thisphenotype is characterized by a high expression of VEGFR-2/KDR, factor-VIII-related antigen and VE-cadherin. By immunofluorescence, weshowed that this differentiation process went on slowly in step with theculture. Actually, parallel to the endothelial antigen expression, cellsadhered to the fibronectin, spread and acquired the typical endothelial cellshape. Finally, differentiated cells were able to form a capillary network onMatrigel upon exposure to VEGF and bFGF.

In conclusion, our data suggest that angiogenic VEGF and bFGFreleased by MM plasma cells may induce the differentiation ofhematopoietic CD133+ stem cells into endothelial cells that, nearangiogenesis, contribute to the development of a tumoral vascular tree,thus promoting MM progression and diffusion. These data confirm theusefulness of antiangiogenic drugs, particularly the VEGF and bFGFinhibitors, in the therapeutic management of MM.

269THE EFFECT OF GSK3BETA INHIBITOR ON MURINE MULTIPOTENT ADULT PROGENITOR CELLS (MAPC)M. Oki*, Y. Jiang, C. M. VerfaillieDepartment of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA

The Wnt/b-catenin pathway is important for maintenance of theundifferentiated state of human ESCs, HSCs and other stem cells. We heretested if b-catenin activation, by inhibiting GSK3b with BIO, affects thedifferentiation status of MAPCs. Murine MAPCs were cultured±0.1-2microM BIO, LIF, EGF and PDGF-BB, at low density (plating density of100/cm 2 and cells passed every 2d) or at high confluency (like ESCs).qRT-PCR was done weekly for the ESC-specific pluripotency transcripts,Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes,Sox1 and CK19. After 2-4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and cells evaluated by immunohistochemsitry forOct4, b-catenin and E-cadherin protein. BIO caused a dose-dependentclustering of MAPC from 4 days after treatment at both cell densities.FACS phenotype of low-density MAPCs was not affected by BIO: MHC-I/II, CD45, hematopoietic lineage antigen, and CD44 negative; and c-kit,CD9, E-cadherin, EpCAM, CD49f positive. Nanog mRNA was notdetected in any of the cell populations, and BIO did not affect UTF1 and E-Ras expression. However, Oct4 and Rex1 mRNA levels decreased in aBIO dose-dependent manner in low-density MAPCs maintained at lowdensity, while they acquired higher levels of CK19 and Sox1. However,Oct4, b-catenin and E-cadherin proteins were highly expressed in clustersof low-density MAPCs induced by BIO. When MAPCs were maintained atESC densities, only MAPCs treated with 1 or 2microM BIO containedlarge dense MAPC clusters that expressed very high levels of Oct4, b-catenin and E-cadherin. Thus, BIO may allow culture of MAPCs at ESC-like density without loss of Oct4 and E-cadherin, while BIO may inducelineage commitment of low-density MAPCs e are testing whether thedense clusters contain the cells with in vitro multilineage differentiationpotential and capable of contributing to chimeric animals followinginjection in the blastocyst.

270BONE MARROW-DERIVED MULTIPOTENT ADULT PROGENITOR CELLS (MAPC) IN A RAT MODEL OF MYOCARDIAL INFARCTIONM. Mazo1*, C. Bresolle2, O. Agbulut2, M. Gutierrez1, G. Abizanda1, J. J. Gavira1, J. M. García-Verdugo4, A. Hagège2,5, P. Menasché2,3, F. Prósper11Hematology, Cardiology and Cell Therapy, Clínica Universitaria and Division of Cancer, Foundation for Applied Medical Research, Division of Cancer, University of Navarra, Pamplona, Spain2INSERM U 633, Paris, France3Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; University Paris 5, Paris, France4Instituto Cavanilles, University of Valencia, Valencia, Spain5Hôpital Européen Georges Pompidou, Department of Cardiology; University Paris 5, Paris, France

Objective- To compare the functional effects of multipotent adultprogenitor cells (MAPC) and skeletal myoblasts (SM) followingtransplantation in a rat model of chronic myocardial infarction.

Background- In vitro, bone marrow-derived MAPC feature amultilineage differentiation potential. However, their phenotypic fatefollowing in vivo engraftment into infarcted myocardium is not yetestablished.

Methods- Sixty-four rats underwent coronary ligation and, 14 days later,were randomly allocated to receive in-scar injections (150 µL, 5x106 cells)of rMAPC either green fluorescent protein (eGFP)-transduced (n=9) or(eGFP)-transduced and Resovisttm labeled (n=16), SM, either eGFP-transduced (n=11) or non-transduced (n=9) or culture medium (controls,n=19). Left ventricular (LV) function was blindly assessedechocardiographically before transplantation and 1 month thereafter.



Abstracts

Immunofluorescence, immunohistochemistry, electron microscopy andpolymerase chain reaction (PCR) were used to detect grafted cells. All datawere compared by nonparametric tests.

Results- Baseline LV ejection fractions (LVEF) did not differsignificantly among the groups, ranging from 30% to 37%. One monthlater, LVEF decreased from the corresponding baseline values in controls(-1.1% [-42;20]) and SMeGFP (-5.6% [-29;37]) whereas it increased aftertransplantation of either non-transduced SM or rMAPC (+10% [-25;53])and +5.7% [-31;48]), respectively, with decreased end-diastolic volumes.Although rMAPC could be identified early post transplant, no evidence ofengraftment was further observed at 1 month by fluorescence microscopy,anti-eGFP antibodies or PCR. Conversely, grafted cells were detected in80% of SM-treated hearts and, in the non-eGFP labeled subset, thisengraftment was associated with the best functional outcome.Angiogenesis did not differ between the groups.

Conclusions- Although MAPC limited LV remodeling, they did notpersist beyond two weeks nor we could detect differentiation intocardiomyocytes during the initial 2 weeks. MAPC differentiation into cellswith endothelial markers was however detected.

271TRANSPLANTATION OF AUTOLOGOUS SKELETAL MYOBLAST IN A SWINE MODEL OF MYOCARDIAL INFARCTION: SMOOTH MUSCLE DIFFERENTIATION OF GRAFTED CELLSJ. J. Gavira*, G. Abizanda, M. Pérez-Ilzarbe, A. Pérez, C. Moreno, J. Barba, J. Herreros, J. A. García de Jalón, D. Martínez-Caro, F. PrósperHematology, Cardiology and Cell Therapy, Clínica Universitaria and Division of Cancer, Foundation for Applied Medical Research, Division of Cancer, University of Navarra, Pamplona, Spain

Objective: to compare the role of skeletal myoblast transplant in a largeanimal model of acute and chronic myocardial infarction (MI).

Methods: Goettingen pigs received an average of 390±115 x 106 BrdUlabeled autologous myoblast transplanted surgically either 2 months afterMI (chronic model) or 30 minutes after MI (acute model). Control animalsreceived media alone. Functional analysis was performed by 2Dechocardiography for up to 2 months post transplant. Myoblastengraftment and in vivo cell differentiation was analyzed byimmunohistochemistry.

Results: myoblast transplant was associated with an increased in systolicfunction both in the chronic (LVEF in myoblast treated animals was71.3±2.5% versus 60±8.5% in controls) and in the acute model (LVEF inmyoblast treated animals was 72±3.4% versus 60±8.5% in controls).Myoblast engraftment was detected in 8 out of 10 animals. Interestingly,differentiation of transplanted cells to skeletal fibers and smooth musclefibers was detected and was associated to increased angiogenesis inanimals treated with skeletal myoblast. An inflammatory reaction wasassociated to myoblast transplant with the presence of T-cells, leukocytesand macrophages infiltrate both in the acute and chronic model.

Conclusion: Our results demonstrate the benefit of autologous myoblasttransplant in a large animal model of acute and chronic myocardialinfarction and suggest that increase angiogenesis may be a mechanismcontributing to the beneficial effect, while an inflammatory reaction mayunderlie the reduced cell engraftment observed

272BONE MARROW DERIVED CD45 NEGATIVE NONHEMATOPOIETIC STEM CELLS – ARE WE LOOKING AT THE SAME CELLS FROM DIFFERENT ANGLES?M. Kucia, E. Zuba, R. Reca, M. Majka, J. Ratajczak, M. Z. Ratajczak*Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA

Recently we identified in BM a mobile, SDF-1 responsive population ofCXCR4 positive bone marrow mononuclear cells that express markers ofearly tissue committed stem cells (TCSC) for skeletal muscles, heart,neural tissue, liver, pancreas, epidermis and intestinal epithelium(Leukemia 2004:18;29-40, Circulation Res. 2004;95,1191-1199). Ourrecent data show that these cells are very small (~7 microm in diameter),have large nuclei that contain primitive embryonic-type euchromatin,migrate robustly to SDF-1, HGF, LIF and VEGF gradients, areCXCR4+Sca-1+CD45- and CXCR4+CD34+CD133+CD45- in mice andhuman respectively, express pluripotent stem cell (PSC) markers such asOct-4, Nanog, Rex-1 (RQ-PCR, immunhistochemistry), adhere to

fibronectin, undergo emperipolesis in fibroblasts and are enriched in afraction of BM-derived adherent cells. Their number is highest in BM fromyoung (1–2 month-old) mice and decreases in 1-year-old animals, and issignificantly diminished in short living DBA/2J mice as compared to longliving B6 mice. Furthermore, these cells are mobilized into peripheralblood during tissue/organ injuries (e.g., partial irradiation, heart infarct,stroke). Thus, these findings further support the theory of BM as a 'hideout'of a reserve population of PSC/TCSC and suggest that their presence inBM tissue should be considered before experimental evidence isinterpreted simply as trans-dedifferentiation/plasticity of HSC. Wepostulate that these non-hematopoietic CXCR4 positive CD45 negativecells are deposited in the BM early during development. Furthermore, it ispossible that several of recently described BM-derived CD45 negativestem cell populations such as MAPC, USSC and MIAMI cells could in factoverlap with these rare non-hematopoietic PSC/TCSC CD45 negative stemcells, but due to the differences in the experimental approaches employedfor their isolation and identification, were assigned different names. Wewill discuss the concept that perhaps we are looking on the samepopulation of BM-derived non-hematopoietic CD45 nedative stem cellsfrom different angles.

273HEPATOGENIC POTENTIAL OF T CELL PRECURSORSY. Yamada1*, H. Miyake1, E. Nishimoto1, H. Mitsuya1, Y. Yonemura21Department of Hematology, Kumamoto University School of Medicine, Kumamoto, Japan2Blood Transfusion Service, Kumamoto University School of Medicine, Kumamoto, Japan

Several research groups have recently demonstrated in rodents thatcertain bone marrow (BM) cells differentiate into hepatocytes in vitro andin vivo through either of transdifferentiation or cell fusion, or both.Hematopoietic myelomonocytic cells are thought to be a major source ofhepatocyte fusion partners, shown in a transplantation model oftyrosinaemia type I mice. However, it is as yet to be elucidated which cellshave the potential to differentiate into hepatocytes at what frequencies.

We demonstrated that murine fetal liver cells (FLCs) triggered andsupported in vitro transdifferentiation of Sca-1+ cKit- cells into hepatic-likecells. When hematopoietic cells sorted from green fluorescence protein(GFP)-expressing transgenic mice were co-cultured with FLCs fromROSA26 mice (X-gal+ FLCs) in the presence of hepatocyte growth factor,markedly adherent, alpha-fetoprotein (AFP) expressing cells emerged byday 3 in culture and such adherent cells expressed albumin as assessed withimmunocytochemistry and RNA PCR. The AFP-expressing cells werewithin CD5+ or B220+ BM lymphoid lineage cells, although the majorityof AFP-expressing cells were Sca-1+CD5+. Within thymocytes,CD5+CD4-CD8-and CD5+CD4+CD8+cells were seen to express AFP.When CD5+ BM cells and CD5+ thymocytes sorted from GFP mice wereco-cultured with X-gal+FLCs, the GFP+hepatic-like cells were identified atfrequencies of 1 in 1x103 and 3.5x105, respectively.

The present data suggest that certain T-lymphoid progenitorsdifferentiate into hepatic-like cells, and the hepatogenic potential of T-lymphoid progenitors diminishes during the differentiation of T-lymphoidprogenitors to mature T-lymphocytes.

274A SUBPOPULATION OF CD34+STEM CELLS WITH POTENTIAL TO REPOPULATE TISSUEM. Y. Gordon1*, N. Levicar1, F. Al-Allaf1, I. Dimarakis1, T. Thalji1, J. Welsh1, E. Alexandre2, N. A. Habib11Imperial College and OmniCyte Ltd, London, UK2University of Strasbourg, Fondation Transplantation, France

Two main types of stem cell, embryonic and adult, have been proposedas sources for cell therapy of tissue damage and degeneration. Ethicaldifficulties and practical problems, like contamination with non-humanantigenic material, beset the application of embryonic stem cell lines. As aresult, the search for adult human stem cell populations with broad tissuepotential is likely to intensify. A single stem cell population may have thecapacity to fulfil all needs or several may be required to provide celltherapy for all diseases and conditions that are amenable to this approach.We have isolated, from mobilised and leukapheresed blood, amorphologically and phenotypically (CD34+, CD33-, CD38-, HLA-DR-,Thy-1+) homogeneous subpopulation of CD34+ cells (~1%) that exhibits



the necessary properties. Using nested RT-PCR and sequence verificationof the RT-PCR product, we have demonstrated that these cells(OmniCytes) and/or their progeny generated within 2 weeks in cultureexpress genes corresponding to stem cells (Rex-1, Oct-4, Nanog) andhematopoietic (CD34, CD133, CXCR4), hepatic(albumin, alfa-1antitrypsin, vimentin, HGF, HNF3-B, transferrin), pancreatic beta (Pax-6,PDX-1, beta-cellulin, insulin. IAP), cardiac (MEF-2C/D, MHP a/b,Nkx2.5, NPPA, tropomyosin 1/T1), and endothelial (angiopoietin1/2,desmin, E-selectin, ICAM-2 Tie 2, VEGF, VWF) cell differentiation.Moreover, the OmniCytes generate clonogenic hematopoietic cells capableof self-renewal in vitro. Expression of insulin, albumin and vimentin hasbeen further verified by flow cytometry of cultured cells, which identifiedsubpopulations of cells expressing different proteins. Animal studies haveshown that OmniCytes engraft in chronically damaged liver and we haveinitiated a Phase I study in patients with liver insufficiency. Our studydocuments the existence of stem cells with broad potency that can bedirectly and reproducibly isolated from an accessible in vivo source andwhich may hold promise for the clinical application of cell therapy.

275EX VIVO DIFFERENTIATION INTO HEPATOBLAST FROM HUMAN UMBILICAL CORD BLOOD AND BONE MARROWY. J. Jung1*, K. H. Ryu2, E. S. Yoo2, S. Y. Woo1, J. Y. Seoh11Department of Microbiology, College of Medicine, Ewha Womans University, Seoul, Korea2Department of Pediatrics, College of Medicine, Ewha Womans University, Seoul, Korea

Objective: The purpose of this study was to suggest human umbilicalcord blood (UCB) and bone marrow (BM) as the source of liver stem cellsand to generate mature hepatocyte lineage cells.

Methods: Mononuclear cells from UCB and BM were cultured withfibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), andhepatocyte growth factor. (HGF) Morphology of cultured cells wasmonitored in inverted light microscopy. Cultured cells were analyzed byreverse transcription-polymerase chain reaction (RT-PCR),immunofluorescent (IF) staining analysis for detecting hepatocyte lineagemarkers and by flow cytometry for demonstrating hepatocyte lineagedifferentiation rather than hematopoietic one.

Results: Cell growth in given culture condition supplemented with FGF-1, FGF-2, SCF, and HGF yielded large sized and oval shaped cells whichwere adherent to culture dishes. mRNA of ALB, CK18 and alphafetoprotein were detected from day 7 in both UCB and BM derived cells byRT-PCR. In IF analysis, these cells expressed albumin (ALB),cytokeratin(CK)-19 from 7-day of culture and some of the ALB producingcells contained proliferating cell nuclear antigen. Cellular differentiationexpressing CK-18 molecules was observed by flow cytomery analysis.

Conclusions: Liver stem cells may exist in UCB as well as BM and canproliferate into hepatocyte lineage cells in primary culture system in vitro.

276HUMAN MESENCHYMAL STEM CELL HOME SPECIFICALLY TO RADIATION INJURED TISSUES IN A NOD/SCID MOUSE MODELS. Francois*, M. Mouiseddine, B. Allenet, N. Mathieu, A. Semont, J. Frick, D. Thierry, A. ChapelLaboratoire de Thérapie Cellulaire et de Radioprotection Accidentelle LTCRA/IRSN EA 1638, BP 17, Fontenay-aux-Roses 92262, France

The main cause of radiation responses is stem cell depletion, whichleads to a reduced reconstitution of functional cells. Amelioration of theseside effects will reduce the post therapy care needed. We hypothesized thatradiation-induced tissue injuries might play a role in the recruitment ofhuman MSCs (hMSC) for tissue repair. We exploited a well characterizedmurine model of radio-induced lesion to evaluate how injury inducedengraftment of hMSCs in whole body. In untreated mice that were hMSCsadministered, human DNA accounted for hMSC engraftment in lung, inmuscle and into bone marrow. We have attempt to link tissue damage tohMSC implantation. NOD/SCID mice were total body irradiated at asublethal dose, we observed an increased number of hMSC implantedorgans. Total body irradiation induced an increase in engraftment levels ofhMSC in brain, heart, liver, muscle and in bone marrow as compared withmice untreated. The locale abdomen irradiation induced an significantengraftment in organs of exposed area in kidney, stomach, liver, spleen,and gut The locale right leg exposure induced a bone and skin engraftment

related to the geometry of the exposure for bone. In both cases of localeradiation two distinct phenomenon was observed. First a specificimplantation of hMSC into exposed area which might be in relation withthe geometry of radiation. Second an multiple hMSC organs implantationoccurred which might be related to a profound inflammation and tissuedysfunctions. These observations support our hypothesis that hMSC can berecruited to sites of radiation injury. These studies have laid thefoundations for the design of mouse model in which the mechanisms oforgan-specific stemcell mobilization can be studied. For future clinicalpurposes bone marrow hMSC will be an alternative providing sufficientquantity of cell for cell therapy.

277MOBILIZATION OF HEMATOPOIETIC PROGENITORS INCREASES THE PRESENCE OF BONE MARROW DERIVED HEPATOCYTES THROUGH IN VIVO FUSION EVENTS IN A MURINE MODEL OF CHRONIC LIVER DAMAGEO. Quintana-Bustamante1*, A. Alvarez-Barrientos2, A. V. Kofman3, J. A. Bueren1, N. D. Theise3, J. C. Segovia11Hematopoietic Gene Therapy Division, CIEMAT, Madrid, Spain2Cytometry Unit, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain3Liver and Stem Cell Research Laboratory, Beth Israel Medical Center, New York NY, USA

The presence of bone marrow derived hepatocytes (BMDH) in liver hasbeen described, although the mechanism involved in their origin is not yetclear. Direct differentiation of Hematopoietic stem cell (HSC) progenyand/or in vivo cell fusion between HSC-derived cells and endogenoushepatocytes have been proposed as mechanisms accounting for thatobservation. We have demonstrated that G-CSF treatment significantlyincreases BMDHs in mice that had been damaged with carbontetrachloride (CCl4). In the present study we have analysed themechanisms involved in the generation of BMDHs in the CCl4 damagedliver.

With this aim, 107 BM cells from Ly-5.2 female mice -which alsoexpress the green fluorescent protein (EGFP) under the control of the beta-actin ubiquitous promoter- were transplanted into lethally irradiated Ly5.1male mice. After the establishment of a steady-state hematopoiesis, groupsof 4-6 animals were injected with saline or with CCl4 for three months.Half of each group was mobilized with G-CSF for three more weeks. Oneweek later, animals were sacrificed and the presence of BMDHs, identifiedas EGFP+CD45- cells with hepatocyte morphology and expressinghepatocyte specific proteins, was evaluated by fluorescent immuno-histochemistry. G-CSF mobilization significantly augmented thepercentage of BMDHs. To analyze whether in vivo fusion events weretaking place, Y-chromosome analysis by fluorescent in situ hybridizationwas performed. We detected that most hepatocytes expressing BM derivedmarkers (EGFP expression) had at least two nuclei, one of them positivefor the Y-chromosome, indicating that their origin was the result of in vivofusion between male hepatocyte and female blood cell. Altogether, thisdemonstrates that the mobilization of hematopoietic cells from increasesthe presence of BMDHs in the liver by facilitating the in vivo fusionprocesses in response to CCl4-induced injury. These results open thepossibility of using hematopoietic growth factors to treat of non-hematopoietic degenerative diseases.

278CD133 POSITIVE MESENCHYMAL STEM CELLS ISOLATED FROM UMBILICAL CORD BLOOD AND MOBILIZED PERIPHERAL BLOODT. Tondreau*, N. Meuleman, M. Dejeneffe, M. Massy, C. Mortier, R. Leroy, A. Delforge, D. Bron, L. LagneauxExperimental Hematology, Bordet Institute, Belgium

Background: Umbilical cord blood (UCB) and mobilized peripheralblood (MPB) represent two important hematopoietic sources for stem celltransplantation to replace a defective hematopoietic system or to treatmalignant diseases. Their potential use as source of mesenchymal stemcells (MSC) has been a long time debated and remains to be validated.

Methods: CD133 positive cells from UCB (n=8) and MPB (n=6) wereisolated using immunomagnetic microbeads with a purity of 90,4±1,03 %.After the second passage, MSC derived from UCB and MPB were



Abstracts

compared with bone marrow (BM) MSC and identified by their positiveexpression of mesenchymal markers (SH2, SH3) and negative expressionof hematopoietic markers (CD14, CD34, CD45 and HLA-DR).

Results: Two weeks after mesodermal induction, we assessed lipidvacuoles (adipocytes), caclium deposits (osteocytes) and chondrogenicmatrix (chondrocytes) using oil red O, silver nitrate and toluidin bluerespectively. Differentiation into neuronal/glial cells required 10 days ofinduction in presence of NPBM+ cAMP+ IBMX+ NGF+ Insulin. In theseconditions, cells displayed typical neuron-like morphology with typicallong thin neurites and were positive for Nestin, Tuj-1, MAP-2 and GFAP.

After four passages, 6,8.108 and 3,3.107 mesenchymal stem cells wereobtained from the CD133 positive fraction respectively for UCB and MPB.In comparison with BM-MSC derived from two normal donors afterclassical sternal aspiration or from particle fraction removed by nylonfiltration prior to transplant, 3,8.106 to 7,8.109 MSC were obtainedrespectively. We also demonstrated that UCB- and MPB-MSC expressOct4, an octomer binding transcription factor present on undifferentiatedcells, as embryonic stem cells with high proliferative capacity.

Conclusion: These results demonstrate that CD133 positive cell fractioncontains MSC with high proliferative potential. The CD133 positiveselection could thus not be limited to hematopoietic stem celltransplantation as previously described but allows also collection of MSCfrom UCB and MPB.

279OPTIMIZATION OF PROLIFERATION AND DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS SEEDED ON EXTRACELLULAR MATRICESU. Lindner1*, P. Behrens1, B. Behnke2, H. Kirchner2, P. Schlenke11Institute of Immunlogy and Transfusion Medicine, Luebeck, Germany2Clinic of Orthopaedics, Luebeck, Germany

Purpose: Mesenchymal stem cells (MSCs) can differentiate into bone,cartilage, tendon, muscles or adipose tissue. They might be a important toolfor tissue regneration such as cartilage repair in near future. Therefore,there is an urgent need to verify the usefulness of MSCs instead ofembryonic stem cells in order to circumvent disadvantages of the lattersource such as ethical problems, teratocarcinoma formation, and immunerejection. Here, we investigate the proliferation and differentiation capacityof MSCs by using EM proteins during culturing in order to improve thelongevity of MSCs.

Methods: MSCs were isolated from bone marrow of patients underwenthip surgery. Cells were seeded in alphaMEM plus 20% FCS. Afterreaching subconfluence, cells were cultured in corning dishes coated withextracellular basement membrane, laminin, collagen or fibronectin for fivepassages. MSCs were phenotyped by flow cytometry (CD34-, CD45-,CD29+, CD44+, CD49(+), CD73+, CD105+, CD106, CD166+). Inaddition, their differentiation potential into adipo-, osteo- andchondrogenic cells was investigated by different staining procedures(vonKossa-, OilRedO-, Safranin-O).

Results: In almost completed work, human MSCs were cultured for upto five passages (n=3). Flow-cytometric phenotyping indicates thecharacteristic feature of MSCs during culturing. In general, MSCs seededon extracelluar matrix proteins have a significant higher expansion ratethan those cultured in uncoated plastic dishes. Furthermore, MSCsproliferate most strongly by using extracellular basement membranes ascompared with laminin, collagen or fibronectin. Preliminary data obtainedfrom initial differentiation experiments show no unique pattern with regardto the maintenance of the adipo-, chondro- and osteogenic differentiationcapacity.

Conclusion: Coculturing of MSCs with extracellular basementmenbranes leads to higher growth rates when compared with laminin,collagen, fibronectin or uncoated controls. A high level of in-vitroamplifcation of MSCs might be helpful in tissue engineering andcontributes to replace the use of embryonic stem cells.

280LYMPHOCYTES CONTRIBUTE TO NON-HEMATOPOIETIC CELL LINEAGES DURING DEVELOPMENTJ. M. Nygren1*, K. Liuba1, S. Stott2, M. Breitbach3, S. Jovinge1, W. Röll3, D. Kirik2, B. Fleischmann3, A. Björklund2, S. E. W. Jacobsen11Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden

2Wallenberg Neuroscience Center, Section of Neurobiology, and Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden3Institute of Physiology I, University of Bonn, Bonn, Germany

Recent studies have suggested that bone marrow (BM) cells mightcontribute to non-hematopoietic cell lineages in adult tissues through cellfusion rather than transdifferentiation. Cell fusion between identical celltypes is a frequent phenomenon in skeletal muscle and osteoclastformation, whereas the mechanism and requirements for heterotypic fusionbetween BM cells and non-hematopoietic cells is not understood. As BMcell fusion with non-hematopoietic cells has been observed in multipleorgans without prior infliction of organ-specific insults, it has beenproposed that these events might contribute to replacement of non-hematopoietic cell lineages during steady state, and that BMtransplantation might be developed as a therapeutic modality in diseases ofthese organs. Herein, we show that detectable in vivo heterotypic fusionbetween BM-derived cells and non-hematopoietic cells does not occur insteady state in adult mice, but is potently facilitated by lethal whole bodyirradiation typically utilized to condition recipients of BM transplants inprevious fusion studies. However, also tissue specific injuries promotefusion between blood cells of the myeloid and lymphoid lineages andcardiomyocytes, Purkinje neurons, skeletal muscle and hepatocytes.Noteworthy, lymphocytes also contribute to hepatocytes and Purkinjeneurons in unconditioned fetal recipients of BM cells.

281OPTIMISATION OF FREEZING PROTOCOLS OF HUMAN UMBILICAL CORD CELLS - PRELIMINARY RESULTSM. Jastrzewska1, A. Gajkowska1, T. Oldak1,2, E. K. Machaj1,2, M. Kruszewski1, Z. Pojda1,2*1M. Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland2WIHiE Institute of Hygiene and Epidemiology, Warsaw, Poland

Fibroblast-like cells extracted from Wharton's jelly of umbilical cord,expressing selected mesenchymal stem cell markers (SH2, SH3), but nothematopoietic lineage markers (CD34-, CD45-), are a potential stem cellsource for mesenchymal cell-based therapies. Some future applicationsmay utilize the freshly-isolated, in vitro-expanded cells, the others maydemand the collection of frozen cells, similarly to cord blood banks.

We have exploited the possibility of storage of umbilical cords in liquidnitrogen temperature, which enable the recovery of fibroblast-like cellsresiding in Wharton jelly and perivascular regions of umbilical cord. Thecords were stored after cutting into 4-cm long pieces, or mincing intofragments of 2-4 mm diameter. The cryopreservant, consisting of albumin,dimethyl sulfoxide (DMSO), and/or hydroxyethyl starch (HES), was addedby the way of diffusion into intact tissues or microinjections, and the cordfragments were frozen according to modified protocols using the controlfreezer. After thawing, the fragments of umbilical cords werehomogenized, washed, and extracted fibroblast-like cells were cultured inIMDM medium supplemented with 10-20% fetal calf serum (FCS). Thenumbers of surviving cells, depending on the protocol, did not exceedapproximately of 2-15% of their pre-freezing values, allowing, however,for expansion of sufficient numbers of plastic-adherent cells in culture. Themorphology, phenotype, and growth kinetics of cells originating fromfrozen specimens did not differ substantially from cells extracted fromfreshly collected cords. We conclude, that banking of frozen fragments ofumbilical cords may be easier, cheaper, and more practical way ofumbilical cord stem cell preservation than freezing and storage of theextracted and in vitro expanded cells.

282LIVER REPAIR AFTER BONE MARROW CELL TRANSPLANTATION IN MICE TREATED WITH MIXTURE OF ALLYL ALCOHOL AND CARBONE TETRACHLORIDET. V. TodriaHematological Scientific Center, Moscow, Russia

The objective of this study was to evaluate the possibility of bonemarrow (BM) cells to promote the liver repair after injury induced by allylalcohol (AA) and carbone tetrachloride (CCl4) individually or as a binarymixture. (C57Bl/6xCBA)F1 mice injected ip by AA (5, 20, 35 and 50 mg/kg in distilled water) or as a mixture with CCl4 (2 mg/kg in olive oil -standard dose) simultaneously. The liver injury and restitutive responsewas monitored by serum enzyme alanin aminotransferase (ALT) level and



histopathology for 10 days after injury. All mice receiving of AA and CCl4individually as well as with the mixture of the two chemicals (5-35 mg/kgAA+CCl4) survived at least of 10 days. Quantitative analysis showed dose-dependent decrease of hepatocytes per section in the AA+CCl4 groupcompared with the AA group. At the same time, the progressiveregenerative activity of hepatocytes (mitoses, binucleated hepatocytes)significantly increased in the AA+CCl4 groups. Since the highest dose ofAA (50 mg/kg) yielded mortality, in the second set of experiments thehighest dose of AA with/without CCl4 injected into the lethally irradiatedrecipients 3 months after BM transplantation. BMT significantly improvesthe restitutive capability of injured liver after binary mixture compared toAA alone. Peak elevation of ALT levels was observed 2 days afteradministration of mixture regardless of AA dose, while magnitudecorrelates with the dose of AA, reaching 2-fold increase for the highestdose. Two-days peak of ALT represents a rather low level after BMT. Inconclusion, liver repair met induced injury in a dose-dependent manner atin a wide range of the chemical concentration, however the tissue repairexhausts at the highest doses of AA in binary mixture. BM improved theliver capability to restitutive response after massive necrosis. Themechanism of BM effect is under study.

283ENDOTHELIAL PROGENITOR CELLS (EPC) SHARE PHENOTYPICAL MARKERS WITH CELLS OF THE MONOCYTIC-MACROPHAGE CELL LINEAGEN. López-Holgado*, M. Alberca, E. M. Villarón, F. Sáchez-Guijo, A. Martín, I. Sánchez-Abarca, B. Blanco, J. A. Pérez-Simón, J. F. San Miguel, M. C. CañizoUniversity Hospital of Salamanca, Spain

The presence of circulating Endothelial Progenitor Cells in adulthumans has been recently suggested. In the present study we haveattempted to identify the EPC, which have colony forming capacity within7 days of culture, and look for their immunophenotypic profile.

Material and Methods: 42 healthy donors were analysed (7BM, 20 PB, 4apheresis products and 9 buffy-coats products). Median age was 38 yearsand M/F ratio was 19/23. EPC were obtained by culturing MNC in IMDMwith VEGF and beta-FGF. At day 7 the colonies were counted andimmunophenotype and immunohistochemistry studies were carried out onthem. Sequential studies were performed on days +14, +21 and +35. VonWillebrand gene expression was analysed by RT-PCR. Monocytes weregrowth in the same conditions as EPC until day +21.

Results: The mean number of EPC colonies at day 7 was significantlyhigher in BM (813±695) than in steady-state PB (21.2±2.5), whilemobilised PB displayed intermediate values (272±274). C olonies werepositive for CD45, CD31 and lysozyme and negative for vWf.Phenotypical analysis showed that they were positive for CD4, CD13,CD14, CD31, CD33, CD45 and lysozyme, weak positive for CD15 andCD105, and did not express CD16 or CD133. This phenotypic profileremained unchanged in all time-points analysed. By contrast, monocyticcells did not form colonies at day 7, but cord-like structures could be seenin 7 out 9 cases. Flow citometry analysis showed that theimmunophenotype of cultured monocytes at day +21 was identical to thatpre-cultured monocytes and both were similar to those obtained with EPC.The expression of V-cadherin was weak on monocytes, but after 12h ofadhesion monocytes became positive. PCR analysis showed small amountsof vWF transcripts in EPC as well as in monocytes and its expressionincreased after the culture in presence of VEGF.

Comments: Our results in both cell subtypes suggest that the so-calledEPC and monocytic cells share similar markers, including some specificmarker such as lisozyme or VE-cadherin.

284THE INFLUENCE OF CONDITIONED MEDIUM FROM MOUSE EMBRYONIC LIVER CELLS ON THE COLONY-FORMING ACTIVITY OF INTESTINAL CRYPT STEM CELLS: EFFECT OF INTERLEUKIN-1BETAL. A. Vedina1, S. V. Sennikov1, V. A. Trufakin2, V. A. Kozlov1*1Institute of Clinical Immunology, SB RAMS, Novosibirsk, Russia2Institute of Physiology, SB RAMS, Novosibirsk, Russia.

BACKGROUND: The study of stem cell 'plasticity' has largesignificance in cellular therapy development. The intestine is known to hasphylogenetic importance in the hematopoiesis process and high stem cellsproliferative activity. As shown earlier, the population of intestinalepithelial cells includes hematopoietic precursor cells.

THE AIM: We investigated soluble factors influence from conditionedmedium(CM) produced by mouse liver embryonic cells(LES) with andwithout interleukin(IL)-1beta on the colony-forming activity of intestinalcrypt stem cells(ICSC). LES were taken in the active period of embryonichematopoiesis, when they produced essential mediators.

METHODS: The ICSC were cultured for 2 hours with 30% eitherconditioned medium from LES, which were preliminarily cultured for 24hours with IL-1beta or without it for 24,48,72 hours. Then male ICSC wereintroduced intravenously lethally irradiated female-recipients. The numberof splenic colonies were counted on day 9.

RESULTS: The preliminarily cultivation ICSC with 30% CM fromLES, which were cultured for 24,48 hours, led to significant increase of thesplenic colonies number in 2.5 times (p<0.01). But preliminarilycultivation ICSC with 30% CM from LES, which were cultured for 24hours with IL-1beta, abolished the stimulant effect (p<0.05). Weconfirmed the hematopoietic colonies origin by PCR amplification of theY-chromosome-specific sry gene. Moreover, ICSC offsprings weredetected in hematopoietic and others tissues of recipients after 2,4 and 6months.

CONCLUSIONS: Thus CM produced by LES in the activehematopoiesis period contain necessary mediators which stimulate ICSCability to form hematopoietic colonies. However, IL-1beta additioncontribute to suppression stimulant effect CM. The obtained data open newparticipation mechanisms of soluble factors in the stem cells 'plasticity'.

285A VIRAL ONCOGENE WHICH INDUCES B-CELLS TO SURVIVE IN RESPONSE TO IL-2P. Tsimbouri*, J. B. WilsonUniversity of Glasgow, Glasgow, UK

Epstein-Barr Virus (EBV) is a human herpes virus associated withseveral malignancies including endemic Burkitt's lymphoma (eBL),nasopharyngeal carcinoma (NPC) and B-cell lymphomas inimmunosuppressed individuals. Epstein-Barr virus nuclear antigen-1(EBNA-1) plays a key role in the maintenance of the viral genome and isconsistently expressed in all the EBV associated tumour types. WhetherEBNA-1 contributes to tumourigenesis by means other than its function inviral DNA propagation is controversial. In order to address this, we haveexplored the consequences of EBNA-1 expression in the B-cells of twoindependently derived lines of transgenic mice (Wilson et al., 1996,EMBO J., 15, p3117). With the aim of examining the direct effects ofEBNA-1 expression and not secondary mutations occurring throughtumourigenesis, our studies have been conducted using explantedtransgenic lymphocytes prior to the development of any tumour pathology.Transgenic lymphocytes show enhanced proliferation compared to controlsand prolonged survival when cultured in the presence of IL-2 (Tsimbouri etal., Oncogene 21, p5182-7, Tsimbouri et al., 2005 inpreparation).Surviving cells are B-cells and continue to express EBNA-1.As this phenotype is evident in lymphocytes from mice of both lines it isnecessarily independent of any insertion site effects and can only beattributed to EBNA-1 expression. These properties are characteristiconcogenic activities and in this system are context dependant, upon IL-2signalling, a cytokine normally produced by activated T-cells supportingboth T- and B-cell immune responses.

In order to explore if the DNA binding protein EBNA-1 can influencecellular gene expression, microarrays have been used in a comparativeanalysis between transgenic, EBNA-1 expressing B-cells, and controls. Afew key proteins involved in cell survival and differentiation show



Abstracts

deregulated expression in the EBNA-1 transgenic cells, including the anti-apoptotic protein Bcl-XL and the inhibitor of differentiation Id2. Thesedata will be presented.

286GENOMIC AND PROTEOMIC ANALYSIS OF BCR-ABL SILENCING PROFILE REVEALS DIFFERENT DOWNSTREAM PATHWAYS DEPENDENT ON THE LEVEL OF BCR-ABL EXPRESSIONJ. Rangatia1*, K. Zibara1,2, D. Frith3, N. Michael4, N. Totty4, D. Bonnet11Cancer Research UK, LRI, Haematopoietic Stem Cell Laboratory, London, UK2Cancer Research UK, Medical Oncology Unit, St Bartholomew's Hospital, Medical College, London, UK3Cancer Research UK, LRI, 2-D Gel Electrophoresis Laboratory, London, UK4Cancer Research UK, LRI, Protein Analysis laboratory, London, UK

BCR-ABL expression has been implicated in CML disease maintenanceand progression. The aim of this study is to get detailed insight into the roleof BCR-ABL in regulation of transcriptional and translational programduring CML development, and to identify new relevant markers that maybe used as targets for therapeutic intervention in CML. To this purpose, wehave performed a genomic and proteomic analysis of blast-crisis K562 cellline before and after BCR-ABL silencing. Affymetrix HG-U133Plus GeneChip was used for the broad-range transcriptomic analysis for cellsundergoing different levels of stable BCR-ABL silencing. Comparativeanalysis of gene-expression profiles between the control and BCR-ABL-silenced cell population showed about 350 genes there were differentialexpressed. In parallel, two-dimensional difference gel electrophoresis (2-DDIGE) that enables an increased confidence in detection of proteindifferences and protein identification by peptide fragment massfingerprinting was performed for cells undergoing transient BCR-ABLsilencing. About 80 proteins showed statistically significant changes inexpression upon BCR-ABL silencing by proteomic analysis. Comparisonof BCR-ABL downstream targets identified independently by proteomicsand affymetrix analysis revealed some targets to show consistent changesin expression both at mRNA and at protein levels (e.g. Hsp70, ATPsynthase, NADH dehydrogenase). Hsp70 has been recently shown to beclinically relevant for CML therapeutic targeting. Further pathway analysisof these combined sets of targets is being performed to identify noveltargets involved in cell cycle, proliferation, self-renewal, apoptosis,metabolism, DNA repair and differentiation. Changes in the expression ofselected transcripts of interest are being confirmed by real-timequantitative polymerase chain reaction and immunoblot analysis in CMLpatient samples. In summary, mRNA expression data along with proteomicinformation should provided a more detailed picture of pathways regulatedby BCR-ABL and would help identify novel therapeutic targets.

287AN ANALYSIS OF MAJOR BCR/ABL MEDIATED CHANGES IN THE PHOSPHOPROTEOMEC. A. Evans1*, E. M. Rodriguez1,2, R. D. Unwin1, D. W. Sternberg3, S. J. Gaskell2, A. D. Whetton11Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK2Micheal barber Centre for Mass Spectrometry, University of Manchester, Manchester, UK3Mount Sinai Medical School, New York, USA

BCR/ABL is a protein tyrosine kinase associated with Chronic MyeloidLeukaemia. Currently the downstream targets of BCR/ABL transformationhave not been fully described. Whilst protein tyrosine phosphorylationevents have in part been mapped the major changes in threonine and serinephosphorylation (that constitute >95% of phosphorylation events in cells)remain undocumented. To address this IL-3 dependent Ba/F3 cell line wasused as model system for studying the effect of this oncogene. BCR/ABL-expressing and control Ba/F3 cell lysates were taken for phosphoproteinenrichment to enable subsequent mass spectrometric analysis andidentification of changes in phosphorylation associated with BCR/ABLmediated transformation. Enrichment was performed using Qiagenphosphoprotein purification columns followed by assessment of thefractionation using a specific stain for phosphoproteins. The procedure wasshown to enrich specifically for phosphoproteins. The eluted fractions wereloaded into a 2 dimensional SDS gel stained with phosphoprotein specific

stain then analyzed with Progenesis software to identify phosphoproteinsspecifically associated with BCR/ABL-transformed cells. Sypro Rubyprotein stain comparison to phosphoprotein stain confirmed successfulenrichment. The spots identified as potential changes were cut off the gel,digested by trypsin and characterized by liquid chromatography/tandemmass spectrometry. Over 300 phosphoproteins were compared on the gelsfrom Ba/F3 and Ba/F3-BCR/ABL cells respectively and fifty proteins withsignificant differences between control and transformed cells identified.Proteins that increase in phosphorylation level in response to BCR/ABLactivation include L-plastin, and phospholipase A2, heat shock proteins 90and 75 and hsc interacting protein. Those decreased in phosphorylationlevel include NDGR1, acidic ribosomal phosphoprotein P0 and nucleolin.This experimental approach has successfully identified downstream targetsof BCR/ABL for further characterisation.

288IDENTIFICATION OF LEUKEMOGENETIC MECHANISMS BY CHMERIC MLL-AF4H. Yamaguchi1*, K. Inokuchi1, H. Hanawa2,3, K. Sawaguchi1, M. Inami1, T. Shimada2,3, K. Dan11Division of Hematology, Third Internal Medicine, Nippon Medical School, Tokyo, Japan.2Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan3Division of Gene Therapy Research, Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan

Acute lymphoblastic leukemia (ALL) expressing MLL-AF4, the fusionproduct of t(4;11)(q21;q23), respond poorly to chemotherapy and havepoor prognosis. MLL was required in normal hematopoietic proliferationand differentiation through Hox gene regulation. AF4 is a serine/prolinerich nuclear protein with transcriptional activation domain and plays animportant role in B and T lymphopoiesis. The MLL-AF4 fusion proteinpreserves the AT-hook and methyltransferase domains of MLL and theGTP binding, and nuclear localization regions of AF4. Previous in vitroand vivo study predicted that leukemia-specific chimeric proteins in 11q23leukemia including MLL-AF4 would associated with leukemogenesis,although the mechanisms still unknown. In the present study, wesuccessfully established a cell line expressing MLL-AF4 from proB ALLpatients with t(4;11)(q21;q23). This cell line expressed CD10- CD15+CD19+ phenotype and overexpressed c-myc by duplication chromosome 8.We have also succeeded to clone cDNA of MLL-AF4 from this cell line,and used it to confirm leukemogenetic mechanisms. Murine IL3 dependentcell line 32Dc was transduced with lentiviral vector(pCL20c Mp) encodinghuman MLL-AF4 cDNA. After confirming MLL-AF4 expression by RT-PCR, each clone was isolated by limiting dilution. First we examinedgrowth profile under IL3 deprivation in 32Dc cell line. 32Dc withoutMLL-AF4 did not grow and demonstrated apoptotic cell death under IL3deprivation, by contrast MLL-AF4 transduced cell lines temporally grewand tended to show anti-apoptotic effect. Next we are using a 32Dc cellline showing myeloid differentiation in response to granulocyte colonystimulating factor (G-CSF) and we will analyze inhibition ofdifferentiation in MLL-AF4 transfected 32Dc cell line. These results andongoing studies will clarify that MLL-AF4 plays an important role inleukemogenesis.

289THE VALUE OF BONE MARROW ASPIRATES CULTURE FOR THE DETECTION OF BONE MARROW MICROMETASTASIS IN BREAST CANCERL. W. C. Chow*, W. T. Y. LooDepartment of Surgery, University of Hong Kong Medical Centre, Hong Kong, China

Background: Detection of micrometastasis is an important problem ofclinical significance for a better understanding and control of tumorprogression, which will improve patients' survival time. Tumor cells inbone marrow (BM) aspirates are indicative of the general disseminativemetastasis in patients with early breast cancer and characterization ofbreast cancers by various tumour markers which are appropriate for theidentification of high risk groups. In this study bone marrow was aspiratedfor culture and stained with various cancer markers.



Materials and Methods: Bone marrow aspirates were obtained from 44breast cancer patients at the time of surgery. To identify micrometastases inbone marrow, an immunocytochemical assay for cytokeratin (CK-18),vimentin, HER-2 and cyclooxygenase-2 (COX-2) was performed at thesecond passage after selective culture.

Result: The culture of bone marrow aspirates lasted for 2-6 passagesspanning three months. Cytokeratin and vimentin positive bone marrowdisseminated cancer cells were observed in more than 90% of the patients.The HER 2 and COX-2 were stained positive in 50% of the cases.

Conculsion: This high incidence of positively stained rate is attributed tothe migration of cancer cells into the bone marrow. However, these resultsindicate that this technique can be used as an early diagnostic technique ofbone marrow micrometastasis in the patient with breast cancer therebypromoting the development of therapeutic strategy.

290MICE CHIMERIC FOR SMAD3-/- BONE MARROW STROMAL CELLS DEMONSTRATE DECREASED IRRADIATION LUNG FIBROSISJ. S. Greenberger*, X. Zhang, L. Hricisak, M. W. EpperlyUniversity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Lung irradiation results in pulmonary migration of bone marrow originfibroblasts, collagen deposition and fibrosis. Marrow stromal cells havebeen shown to migrate to the C57BL/6J mouse lung from the bone marrowat 120-130 days after pulmonary irradiation. Increased TGFbeta expressionin the irradiated lung may recruit these fibroblast-progenitors, and inducetheir proliferation forming fibrosis. To determine the role of TGFbeta inthis process, we irradiated C57BL/6J mice to 20 Gy to the lungs. At 80days after irradiation, we injected groups of mice intraperitoneally withvarying numbers of labeled marrow stromal cells. In one experiment,5x105 bone marrow stromal cells isolated from either Smad3-/- or wildtype (Smad3+/+) mice were compared. For labeling, a clonal Smad3-/-bone marrow stromal cell line was transfected and selected for subclonesexpressing GFP from a retrovirus containing the GFP transgene. A clonalSmad3+/+ bone marrow stromal cell line was similarly transfected andsubclones selected for expression of DS-red from a retrovirus containingthe DS-red transgene. Reciprocal color labeled sublines of each were alsoselected. At 130 days after 20 Gy lung irradiation, mice were sacrificed andlungs expanded in OCT, removed, frozen in OCT, sectioned and examinedunder a fluorescent microscope for labeled stromal cell involvement inpulmonary irradiation fibrosis. Mice injected with DS-red positiveSmad3+/+ cells demonstrated significant accumulation of DS-red positivecells in areas of organizing alveolitis/fibrosis. In contrast, mice injectedwith a similar or higher number of GFP labeled Smad3-/- bone marrowstromal cells had few labeled cells localized to areas of organizingalveolitis/fibrosis. Thus, Smad3-/- marrow stromal cells have reducedcapacity for pulmonary migration and/or proliferation, and implicateTGFbeta signaling in irradiation pulmonary fibrosis.

291SMAD3 -/- BONE MARROW STROMAL CELLS ARE RADIORESISTANTM. W. Epperly*, X. Zhang, L. Hricisak, S. Cao, J. S. GreenbergerUniversity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

The Smad3 gene product plays an intricate role in the signaltransduction pathway resulting from TGFbeta binding to the TGFbetareceptor. Smad3-/- mice have been shown to have decreased fibrosisfollowing irradiation. Long-term bone marrow cultures (LTBMCs) fromSmad3-/- mice compared to Smad3+/+ littermates demonstrated increasedlongevity of hematopoiesis. Bone marrow stromal cells (BMCs) wereisolated from Smad3-/-or Smad3+/+ LTBMCs. Smad3-/- BMCs weresmaller in size than Smad3+/+ cells. The cell doubling time of Smad3-/-cells was 24 hours while the cell doubling time for Smad3+/+ cells was 48hours. Smad3-/- cells also grew to significantly increased saturationdensity in 25 cm2 tissue culture flasks. Smad3+/+ cells reached saturationdensity at 3.76±SD 0.14 x 105 cells while Smad3/- cells grew denser to15.3±SD 0.96 x 105 cells (p=0.0003). Both cell lines had equivalentplating efficiency of 15.5±SD 1.7% for Smad3+/+ cells compared to18.3±SD 2.7% for Smad3-/- cells. Smad3+/+ or Smad3-/- cells wereirradiated to doses ranging from 0 to 800 cGy. The cells were plated in 4-well tissue culture plates and stained with crystal violet 7 days later.Colonies of greater than 50 cells were counted and analyzed by linear

quadratic and single-hit, multi-target models. Smad3-/- cells wereradioresistant with a D0 of 1.51±SD 0.16 Gy compared to 0.97±SD 0.01Gy for the Smad3+/+ cells (p=0.0374). Irradiation-induced apoptosis wasincreased in the Smad 3+/+ cells 24 hr following 5 or 8 Gy (15 or 43%)compared to less than 1% in the Smad3 -/- cells. The data indicate that theSmad3 gene plays a role in the longevity of hematopoiesis, the biology ofbone marrow stromal cells, and radiation sensitivity.

292INHIBITION OF MACROPHAGE MIGRATION TO THE LUNGS DECREASES IRRADIATION PULMONARY FIBROSISM. W. Epperly*, Y. Niu, L. Hricisak, J. S. GreenbergerUniversity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

A dose limiting late effect of lung radiation therapy is pulmonaryfibrosis. We have demonstrated in a C57BL/6J mouse model, increasedmigration of macrophages into the lung 100-110 days after irradiation,accompanied by increased TGFbeta expression, and followed by increasedmigration of bone marrow origin fibroblasts, deposition of collagen, andfibrosis. The cellular source of elevated pulmonary tissue TGFbeta isunknown. To determine whether infiltration of macrophages in the lungcontributed to irradiation-induced fibrosis, mice were irradiated to 20 Gy tothe lungs, then at 80 days after irradiation and continuing for 21 days, micewere injected intraperitoneally 5 times per week with 1 mg/kg ofhybridoma-derived antibody specific for mouse macrophages or with anon-specific control antibody. At 120 days after irradiation, mice weresacrificed, lungs expanded in OCT, removed, frozen in OCT and sectioned.The sections were then stained with rabbit anti-macrophage specificantibody or with a control antibody followed with a FITC conjugated anti-rabbit antibody. The sections were then examined under a fluorescentmicroscope for number and anatomic sites of macrophage accumulation.Mice injected with the antibody to macrophages showed decreasedmacrophage accumulation in the lungs. The extent of organizing alveolitis/fibrosis was reduced in treated compared to the control antibody treated oruntreated irradiation control groups. Thus, macrophages are involved in thelesions of late irradiation fibrosis.

293HEMATOPOIETIC GROWTH FACTOR INDUCTION BY IONIZING RADIATION AND RADIATION COUNTERMEASURE 5-ANDROSTENEDIOLM. B. Grace, C. M. Chang, V. K. Singh, C. McLeland, V. I. Parekh, R. L. Shafran, C. E. Inal, W. E. Jackson, M. H. Whitnall*Armed Forces Radiobiology Research Institute, Bethesda, MD, USA

5-androstenediol (5-AED) is a systemic radiation countermeasure thatenhances survival in mice exposed to gamma-irradiation, amelioratesradiation-induced neutropenia in mice and non-human primates, andinduces increases in bone marrow GM-CFC in irradiated mice. Closelyrelated 5-androstene steroids and sex steroids do not have these effects. 5-AED mitigates radiation-induced decreases in platelets, NK cells, redblood cells, and monocytes. Administration of 5-AED causes functionalactivation of circulating granulocytes (phagocytic ability), monocytes(oxidative burst), and NK cells (surface CD11b expression). The effects of5-AED on survival and hematological parameters are consistent withinduction of hematopoietic cytokines. To test this hypothesis, weperformed real time quantitative PCR for mRNAs of cytokines in mousespleens, and correlated the results with ELISA for serum cytokines.Spleens and serum were taken at 3 time points: 4 h after 5-AED or vehicle(PEG-400) injection, and 4 or 24 h after irradiation (7.5 Gy whole-bodygamma) or sham-irradiation. Each group contained 5 mice. PCR wasperformed for GM-CSF, IL-6, IL-10, IL-12, and IFNg, and results wereanalyzed by 2-way ANOVA. Radiation alone induced increases in mRNAsfor all cytokines. 5-AED did not induce a significant increase for anycytokine in unirradiated mice, but there was a significant positiveinteraction between radiation and 5-AED for IL-6. ELISA for serumcytokine protein was measured for G-CSF, IL-6, IL-10, and IL-12, andresults were analyzed by the Mann-Whitney test for exact p-values. 5-AEDcaused increases in circulating G-CSF in irradiated and unirradiatedanimals. Since IL-6 and G-CSF are both important hematopoieticcytokines, the results support our hypothesis (Int J Immunopharm 22:1)that the previously observed increases in numbers of circulating innate



Abstracts

immune cells and platelets, and functional activation of granulocytes,monocytes, and NK cells, result from a cytokine cascade induced by 5-AED in the hematopoietic niche.

294DNA DAMAGE AND REPAIR IN FANCONI ANEMIA CELLS EXPOSED TO IONIZING RADIATIONJ. A. Casado1*, M. I. Nuñez2, J. C. Segovia1, J. M. Ruiz de Almodóvar2, J. A. Bueren11Hematopoietic Gene Therapy Division, CIEMAT, Madrid, Spain2Facultad de Medicina, Universidad de Granada, Granada. Spain

Fanconi anemia is a genetically heterogeneous recessive disease mainlycharacterized by bone marrow failure and cancer predisposition. To date,eleven complementation groups have been identified: FA-A, B, C, D1/BRCA2, D2, E, F, G, I, J and L, and nine FA genes have been alreadycloned. Although it is known that FA proteins are involved in DNA repair,the processes in which FA proteins participate are still unclear. In thisstudy we have investigated the response of FA cells to ionizing radiation,both at a molecular and cellular level. By means of pulsed-field gelelectrophoresis we have firstly observed that ionizing radiation generates asimilar number of DNA double strand breaks (DSBs) in normal and FAcells corresponding to three different complementation groups: FA-A, FA-C and FA-F. Thereafter, the rejoining of the DSBs was investigated. Incontrast to previous studies in which these analyses were conducted inplasmid DNA, we followed the kinetics of DSBs rejoining in genomicDNA from normal and FA cells exposed to radiation. Significantly, notonly proliferating, but also quiescent FA cells showed an evident defect torejoin the DSBs produced by radiation, indicating a defective non-homologous end-joining repair. At a cellular level, however, only a modestincrease in the radiosensitivity of Fanca-/- hematopoietic progenitor cellswas observed compared to Fanca+/+ cells. When animals were exposed toa fractionated total body irradiation of 5 Gy, also a similar hematopoieticsyndrome was observed in wild type and Fanca-/- mice. These resultsindicate that FA cells - in particular those having non functional FAproteins upstream FANCD2 - have a defect in the non-homologous endjoining repair of DSBs produced by ionizing radiation. In addition, ourresults suggest that compensatory mechanisms of DNA repair and/or stemcell regeneration should limit the impact of this defect in irradiatedorganisms.

295SINGLE INJECTION OF STEM CELL FACTOR, FLT-3 LIGAND, THROMBOPOIETIN, INTERLEUKIN-3 COMBINED WITH PEGYLATED G-CSF MITIGATES MYELOSUPPRESSION IN HIGHLY IRRADIATED NONHUMAN PRIMATESM. Drouet*, C. Delaunay, N. Grenier, S. Bauge, F. Mourcin, J. F. Mayol, F. HerodinService de Santé des Armées CRSSA, La Tronche, France

We previously demonstrated the capacity of antiapoptotic cytokines incombination (4F=Stem Cell Factor + FLT3-Ligand + MegakaryocyteGrowth and Development Factor + Interleukin- 3; 50 mcg/kg of eachfactor) to counteract neutropenia [ANC<0.5x109/L] andthrombocytopenia [PLT<20x109/L] in cynomolgus monkeys whenadministered as a single intravenous dose 2 hours after a 5 Gy gammaunilateral total body irradiation (TBI) (Blood 2004,103:878-885). Then weevaluated the efficacy of 4F in 7 Gy irradiated monkeys and showed thatPLT went down within 35 and 50 x109/L for only 4 days after treatment. Incontrast no effect was observed upon neutropenia. Here we evaluated thebenefit of adding Pegylated (PEG)-G-CSF to 4F combination: 4F wasadministered 2 hours after myelosuppression and PEG-GCSF was injectedat H2 and on day 7. Cytokine treatment was well tolerated. In accordancewith previous study, 4F + PEG-GCF treated animals (n=3) experienced nothrombocytopenia. Moreover, the period of neutropenia was significantlyreduced (7.3 days±1.5) when compared with historical untreated control(12 days±3) and 4F treated animals (10.3 days±7.3). 4F + PEG-GCF and4F treated animals showed a similar protection of bone marrow CFUs ondays 1, 4 and 40 when compared with controls. Evaluation of long-termhematopoiesis one year after TBI is under way. These results stronglysuggest that 4F, as a single dose treatment, could be safely combined withPEG-G-CSF to reduce both neutropenia and thrombocytopenia in highlyirradiated victims of nuclear accident or terrorism settings.

296NOVEL SMALL MOLECULE RADIOPROTECTIVE AGENTSM. W. Epperly*, M. P. Fink, L. Hricisak, X. Zhang, J. S. GreenbergerUniversity of Pittsburgh, PA, USA

Two new small molecule radioprotectors we are investigating includethe MnSOD mimetic EUK-134 and a novel compound for radioprotection,ethyl pyruvate (EP). 32D cl 3 cells were incubated: 1) in the absence of anyadded compounds, 2) in the presence of EUK-134 or EP for 1 hour beforeirradiation, 3) in the presence of EP in methylcellulose followingirradiation, or 4) in the presence of EP for 1 hour before irradiation and alsoin the presence of EP in methylcellulose following irradiation. In allgroups, cells were irradiated to doses ranging from 0 to 800 cGy. Cellsincubated in EUK-134 had an increased shoulder on the survival curvewith an n of 5.38 compared to an n of 1.43 for 32D cl 3 cells. 32D cl 3 cellsincubated in the presence of EP 1 hour before irradiation or plated inmethylcellulose containing EP had an increased D0 compared to 32D cellsalone (2.20±SD 0.25, 2.21±SD 0.15, and 1.84±SD 0.25 Gy, respectively)but no change in n (2.00±SD 1.01, 1.11±SD 0.12, or 1.70±SD 0.37,respectively). The combination of incubating 32D cl 3 cells in EP bothbefore irradiation and in methylcellulose media after irradiation resulted inoptimal radioprotection as shown by a significantly increased shoulder onthe survival curve compared to untreated-irradiated 32D cl 3 cells(n=4.14±SD 1.59 and 1.70±SD 0.6, respectively, p=0.0485). EP alsodecreased the percent of 32D cl 3 cells undergoing irradiation apoptosiswhen cells were incubated in EP 1 hour before 10 Gy irradiation. Twenty-four hrs after 10 Gy irradiation, cells incubated for 1 hour in EP had2.8±SD 0.7% apoptotic cells (1.2±SD 0.5% for non-irradiated cells)compared to 23.7±SD 1.8% for irradiated cells. These data indicate that EPas well as the SOD mimetic EUK-134 may be effective radioprotectors.

297PROTECTION OF NORMAL HUMAN CD34+ CELLS FROM RADIATION FOUND IN THE DEEP SPACE ENVIRONMENTG. U. Gangenahalli1, J. M. Millholland1, A. Kalota1, P. Bennett2, B. M. Sutherland2, A. M. Gewirtz1*1University of Pennsylvania School of Medicine, Philadlephia, PA, USA2Brookhaven National Laboratory, Upton, NY, USA

High-LET radiation particles (HZE), which exist naturally in deepspace, are particularly dangerous to cells because, by direct ionization, orradiolysis of water, they induce free radical formation that causes doublestrand breaks (DSB), and other complex DNA damages. The HZEexposure risk to astronaut flight crews on long duration missions has notbeen quantitated. We therefore radiated human CD34+ cells, obtained fromconsenting normal donors, to HZE in the form of Fe, Ti, Si, with or withoutcandidate radioprotectants, and analyzed cells for DNA damage, andcolony forming ability. CD34+ cells demonstrated dose dependent toxicityto HZE with damage detectable even at doses ~15 cGy which significantlydecreased BFU-E, CFU-E, CFU-GM and CFU-GEMM colonies comparedto unirradiated controls. Pro-Methyl Hoechst (PMH) treatment prior toirradiation, enhanced CFU formation by all lineages from 2-4 fold in thedose range of 15-50 cGy (137Cs) as compared to untreated cells. At doses>70 cGy, PMH was less effective but still protected BFU-E and CFU-GEMM. The Mg++-Salen Complex, EUK-134, displayed significantradioprotection between 15-30 cGy as well, but at doses >50cGy,protection was lost. Combining these agents increased protectionsuggesting different mechanisms of action. To explore this possibility, wemeasured: 1] phosphorylated histone H2A.X levels, a sensor of DSB; 2]induction of repair proteins unique for DSB, such as Ku70/80, MRE-11and Rad-50; and 3] the number of DNA ends labeled with dUTP-(FITC)terminal transferease enzyme. Based on these measurements, PMH andEUK-134 appeared to protect cells by induction of repair proteins whoseeffective was demonstrated by a decrease in phosphorylated histoneH2A.X levels at doses less than 50 cGy. We conclude that PMH and EUK-134 provide measureable protection to CD34+ cells and that these agentsmight be useful in protecting cells from deep space radiation damage.



298RADIATION DAMAGE TO BONE MARROW PROGENITOR AND STEM CELLS IN MICE PRETREATED WITH CYCLOPHOSPHAMIDE AND TRANSPLANTABILITY OF NORMAL BONE MARROW CELLS INTO DIFFERENTLY PRETREATED RECIPIENTSL. Sefc*, K. Sulc, V. Sykora, T. Pelichovska, O. Psenak, E. NecasInstitute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic

Cyclophosphamide (CY) is often used as antineoplastic andimmunosuppressive drug. In our experiments, it depleted bone marrowprogenitors (BM) with similar effect in different mouse strains (D0=70mg/kg b.w. for CFU-S). High SDF-1 and SCF expression correlated withactive proliferation in early regeneration phase in BM (1 to 3 days afterCY). Downregulation of mRNA expression for both cytokines wasconnected to cessation of stem cell proliferation and their release intoperipheral blood (5 to 7 days after CY). Ten minutes after irradiation, D0for CFU-S in control, CY-3 and CY-7 mice were 0.775 Gy, 1.046 Gy, and0.753 Gy, respectively. While radiosensitivity of control and CY-3 CFU-Sdid not change significantly when measured 24 hrs after CY, CFU-S inmice 7 days after CY became twice as much radiosensitive (D0=0.362Gy). It means that CY-7 BM did not support maintenance of CFU-S thatsurvived irradiation. Thus CY-3 treatment prior to irradiation wasradioprotective while CY-7 treatment caused radiosensitization.

If mice were lethally irradiated (11 Gy), pretreatment with CY 7 daysprior to irradiation resulted in considerably impaired survival compared toonly irradiated mice. CY-2 pretreatment had radioprotective effect.Transplantation of 5x104 bone marrow cells was enough to save CY-2 plus11Gy mice over 30 days period. To obtain the same effect, 5x105 or 5x106

cells were needed in control plus 11Gy mice or CY-7 plus 11Gy mice,respectively. Nevertheless, transplantation of sufficient amount of bonemarrow cells to CY-7 recipients gave significantly higher engraftmentcompared to CY-2 mice.

We conclude that for high and stable posttransplantation chimerism, it ismore efficient to irradiate and transplant later (5 to 7 days) after CY,compared to transplantation to mice one to three days after CY.Nevertheless, the transplanted cell dose must be much higher to ensuresuccessful engraftment.

299SIDE POPULATION (SP) CELLS ISOLATED FROM BONE MARROW OR ESOPHAGUS ARE RADIATION RESISTANTY. Niu*, H. Shen, M. W. Epperly, J. S. GreenbergerUniversity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Mouse hematopoietic and esophageal stem cells are concentrated in sidepopulation (SP) cells. Irradiation of esophagus results in measurablerecruitment of hematopoietic stem cells to the esophagus. To determine theeffect of irradiation on hematopoietic or esophageal SP cells, we irradiatedmice to either: A) 10 Gy whole body and isolated bone marrow 24 or 48hours later or B) in separate experiments, the esophagus was irradiated to30 Gy and the esophagus was removed 24 or 48 hours after irradiation.Single cell suspensions of bone marrow and esophagus were stained withHoechst dye, and examined for apoptosis using Annexin-5 staining. TheSP cells were isolated from each tissue and analyzed for the percent SPpositive cells as well as percent of cells undergoing apoptosis. In non-irradiated bone marrow, the percent of SP cells was 0.081±SD 0.033compared to 0.093±SD 0.027 at 24 hrs after irradiation. There was nochange in the percent of Annexin-5 positive SP cells before or 24 hr afterirradiation (24.5±SD 8.4 or 22.7±SD 5.9, respectively). In contrast,marrow non-SP cells showed a significant increase in percent of apoptoticcells in irradiated compared to non-irradiated samples (26.8±SD 5.4 and38.7±SD 6.2, p=0.021). In the irradiated esophagus there was no change inthe percent of SP cells comparing non-irradiated and irradiated samples(4.5±SD 1.4 and 4.6±SD 1.4). As with bone marrow, a significant increasewas detected in the percent of apoptotic cells in non-SP but not SP cellsfollowing irradiation. The data demonstrate that SP cells are more resistantto irradiation than non-SP cells in both mouse bone marrow and esophagus.

300SIMULTANEOUS DETECTION OF 7 CELLS POPULATIONS FOR STUDYING HEMATOPOIETIC REGULATION, STEM CELL QUALITY CONTROL AND DRUG EFFECTSI. N. Rich*, K. H. HallHemoGenix Inc, Colorado Springs, CO, USA

The HALO (Hematopoietic/Hemotoxicity Assays via LuminesnceOutput) Platform detects and measures the proliferative status of stem andprogenitor cells as a function of intracellular ATP concentration releasedfrom cells to drive a luciferin/luciferase reaction producing luminescence.Validated against the conventional colony-forming assay, HALO is astandardized, non-subjective, multifunctional and multiparameter systemthat uses a 96-well tissue culture plate format. The system has now beenadapted to detected 7 lympho-hematopoietic populations simultaneouslyfrom human, non-human primate, dog, rat and mouse target cells. The 7populations include 2 stem cell populations, 3 hematopoietic progenitor(BFU-E, GM-CFC and Mk-CFC) and 2 lymphopoietic (T-CFC and B-CFC) cell populations. The 2 stem cell populations include the CFC-GEMM and a primitive stem cell which is designated the HighProliferative Potential Stem and Progenitor Cell (HPP-SP) which can bestimulated to produce both lymphopoietic and hematopoietic cells. TheHPP-SP population can be 'primed' with SCF, IL-3, IL-6 and Flt3-L(S,3,6,F) or stimulated with a combination of growth factors (designatedGEMM3mix) to produce both stem and progenitors cells. The addition ofFl3-L produces at least twice as many CD34+ and CD117+ cells as SCF,IL-3 and IL-6 alone. By re-plating 'primed' HPP-SP cells cultured for 7days in the presence of S,3,6,F into secondary culture for a further 7 dayswith the GEMM3 mix, not only can the presence of stem cells besubstantiated, but their ability to differentiate can be measured. Stimulationand detection of all 7 populations simultaneously is used to measure thegrowth potential of different stem cell populations in processed bonemarrow, peripheral blood, and cord blood and their ability to differentiateinto multilineage populations, thereby providing valuable information onengraftment and reconstitution. The 7-population paradigm is also used topredict the 'global' lympho-hematopoietic effects of drugs and theestimation of clinical trial dosing.

301ROLE OF C-KIT IN THE REGULATION OF EMBRYONIC STEM CELL DIFFERENTIATIONA. Bashamboo1*, A. H. Taylor1, J-J. Panthier2, A. Whetton3, L. M. Forrester11John Hughes Bennett Laboratory, University of Edinburgh, UK2URA-INRA de Genetique Moleculaire, Maisons-Alfort, France3LRF Unit, Faculty of Medicine, University of Manchester, Christie Hospital, Manchester, UK

Embryonic stem (ES) cells have the potential to undergo multilineagedifferentiation,including haematopoetic, in vitro but the molecularmechanisms controlling specific differentiation pathways are not fullyunderstood. We are analysing the role of the SCF receptor, c-KIT, amember of the type III Protein Tyrosine Kinase family, in this process. Toaddress the role of c-Kit in the development of specific cell lineages invitro we have analysed the self renewal and differentiation profile of EScells carrying a homozygous targeted deletion of the c-Kit gene incomparison to heterozygous and wild type (WT) ES cells.

We cultured ES cells in reducing concentrations of LIF and assessedtheir survival and self-renewal capacity by staining for alkalinephosphatase after 5 days. We observed that in the absence of LIF, WT cellsproduce a high number of phenotypically differentiated colonies. Incontrast Kit null cells do not survive in the absence of LIF and cellsheterozygous for the targeted mutation showed an intermediate phenotype.These results indicated a dose dependent role of c-Kit in the survival ofdifferentiating ES cells.

To conclusively prove our hypothesis we have developed a strategywhereby we can pharmacologically activate Kit in Kit-null cells. Wegenerated vectors to knock-in an activable Kit receptor (FKBP12-Kit) intothe c-Kit locus. We observed a reversal of the phenotype in the -/FKBP12cells on pharmacological activation of FKBP12-Kit, confirming our earlierobservations.



Abstracts

We also observed that WT ES cells do not survive in the absence of LIF,when cultured in the presence of a neutralising monoclonal anti-c-KITantibody, ACK2. This gives an indication that the observed phenotype isdependent on kinase activity of the receptor. We are currently assessing thesignal transduction mechanism involved in this process.

This system will allow us to use proteomics technology to follow thepotentially instructive downstream signalling events in this importantdevelopmental pathway and could be used to address similar questionsregarding other receptor tyrosine kinases e.g. Flk-1.

302POTENTIATION OF HAEMATOPOIETIC ACTIVITY IN DIFFERENTIATING EMBRYONIC STEM CELLS BY AORTA-GONAD-MESONEPHROS REGIONS. Gordon-Keylock1*, A. Krassowska1, K. Samuel2, E. Dzierzak3, A. Medvinsky4, J. Ansell1, L. Forrester11John Hughes Bennett Laboratories, University of Edinburgh, Western General Hospital, Edinburgh, UK2Cell Therapy Group, Scottish National Blood Transfusion Service, Edinburgh, UK3Erasmus University Medical Centre, Department of Cell Biology and Genetics, Rotterdam, The Netherlands4Institute of Stem Cell Research, University of Edinburgh, Edinburgh, UK

The differentiation of pluripotential embryonic stem (ES) cells intohaematopoietic lineages in vitro has been particularly well characterised,but the successful generation of substantial numbers of haematopoieticstem cells (HSCs) able to engraft lethally irradiated mice in vivo has beenlimited. It is possible that the in vitro differentiation protocols used to datedo not provide a suitable microenvironment for HSC development from EScells. We describe the effect of co-culturing differentiating ES cells in thepresence of embryonic tissue that was predicted to provide a moreappropriate microenvironment for haematopoietic commitment.

The aorta-gonad-mesonephros (AGM) region is the earliest site in theembryo proper that is able to generate definitive, adult-type HSCsautonomously. Clusters of HSCs appear in the AGM region at day 10 ofgestation in the mouse, where they proliferate before colonising the foetalliver to continue haematopoietic development. Long term repopulatingHSC activity has been found to increase within AGM region explantcultures indicating that elements of the supporting microenvironment fordefinitive HSC expansion can be captured in vitro.

In this study an explant culture system was developed to examine theinductive properties of the AGM region on differentiating ES cells. Ahighly significant increase in the number of primitive haematopoieticprogenitors, as measured by in vitro colony assays, was observed after co-culture of ES cells with the AGM region from a 10.5 day embryo.Furthermore, stromal cell lines derived from the AGM region and foetalliver exhibited diverse effects on the differentiation of ES cells.Preliminary data also suggest that the AGM region-derived factor(s)responsible for the increase in haematopoietic differentiation of ES cells isdependent on direct cell-cell contact.

303GENETIC CELL TRACING OF HEMATOPOIESIS ORIGINSI. M. Samokhvalov1*, N. I. Samokhvalova1, A. Medvinsky2, S. Nishikawa11RIKEN Center for Developmental Biology, Kobe, Japan2Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK

Hematopoietic lineage development in mammalian embryogenesisproceeds through multiple steps and requires a number of specific cellularenvironments. The relationship between different nascent blood cellpopulations is poorly defined and the contribution of primaryhematopoietic sources in embryo to mature hematopoietic system isdisputed. Cell tagging of distinct early embryonic progenitors using agenetic switch system offers the possibility for efficient cell fate analysis.To this end, Mer-Cre-Mer recombinase fusion gene was placed bytargeting under the control of the endogenous Runx1proximal promoter,which is active specifically at the sites of earliest embryonichematopoiesis. Mice bearing the Cre-recombinase knock-in construct werecrossed with ROSA26R LacZ-reporter mice. Pregnant females were theninjected with a single dose of tamoxifen at different stages of gestation toactivate Cre-recombinase. The resulting LacZ-positive hematopoietic cellswere followed during embryo development to term and beyond. No Cre-

recombinase activity was detected in absence of tamoxifen, whereas thetamoxifen-dependent activation was strictly stage-specific. Descendants ofearly blood island cells were detected in endothelium of dorsal aorta andumbilical arteries/veins. Their liver contribution commenced at aroundE10.5, and later was observed in multiple definitive hematopoieticlineages. Substantial fraction of E16.5 lymphoid cell population originatedin E7.5-8.0 yolk sac blood islands. A limited contribution to hematopoieticsystem of one-month old mice was also detected. Descendants of AGMregion progenitors were labeled by tamoxifen injection at E9.5 and theircontribution to adult hematopoiesis is now under investigation.

The data suggest that early angioblasts are capable of movement throughblood stream over large distances within the embryo. This furthercorroborates the concept of a hemogenic endothelium as an adaptation ofnascent hematopoietic cells to the luminal microenvironment. The in vivotracing analysis suggests a common anatomical origin for primitiveembryonic hematopoietic lineages and definitive multipotentialprogenitors.

304CHARACTERIZATION OF HEMATOPOIESIS IN THE PROGENY OF SMAD 5 DEFICIENT HEMATOPOIETIC STEM CELLSS. Singbrant, U. Blank, J. Moody, S.KarlssonMolecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University Hospital, Lund, Sweden

Transforming growth factor-beta (TGF-beta) superfamily members,including TGF-beta, Activins, and Bone Morphogenetic Proteins (BMPs),regulate biological functions like cell proliferation, differentiation, andapoptosis. We have recently defined the role of TGF-beta signaling in theregulation of hematopoietic stem cells (HSC) using mice deficient in TGF-beta receptor type I function (EMBO J, 2001, Blood, 2003). Less is knownabout the effects of BMP and the receptor regulated SMAD1, 5 and 8 thattransduce its signals. However, recent findings suggest that BMP4 maystimulate human HSC proliferation in vitro (J. Exp. Med. 1999) and murineSmad5-/- embryonic bodies generate high-proliferative potential colony-forming cells (HPP-CFCs) that display augmented self-renewal capacity(Blood, 2003). The aim of this study is to evaluate the role of Smad5 in theregulation of HSC fate decisions in adult mice using an Mx-Cre mediatedconditional knockout model for Smad5. Analysis of peripheral blood (PB),spleen and bone marrow (BM) 16 weeks after Cre-mediated excisionshowed normal numbers of PB cells, total BM cells, and Lin-, Sca-1+, c-kit+ (LSK) BM cells. Hematopoietic lineage distribution in PB, BM, andspleen was normal as determined by FACS. When transplanted in acompetitive fashion into lethally irradiated recipients, Smad5 deficient BMcells were able to long-term reconstitute the hosts. Additionally Smad5deficient BM cells from mice with mixed background (C57Bl6,129/Ola,CD1) exhibited increased engraftment efficiency at 12 weeks (PB:34.1±13.6%, n=7 vs. 18.3±4.58%, n=5). This repopulative advantage wasalso seen in secondary recipients (PB 12w post transplant: 23.3±17.8% vs.4.5±5.36% for control). However, when repeated in animals backcrossedonto C57Bl6 this repopulative advantage was no longer apparent,suggesting a generic difference. Taken together our data indicate thatSmad5 deficient HSC have normal differentiation and proliferation understeady state conditions, but an increased repopulative capacity dependenton genetic background.

305STROMAL DERIVED FACTOR-1 AND LYSOPHOSPHOLIPIDS MEDIATE ALTERED PROTEIN ACETYLATION TO PROMOTE PROGENITOR CELL MIGRATIONC. A. Evans*, Y. Lu, E. Spooncer, A. D. WhettonUniversity of Manchester, Manchester, UK

Primitive hematopoietic cells undergo chemotaxis in response to thechemokine, stromal derived factor 1 (SDF-1). Their motility is alsopromoted by the lipid mediator; lysophosphatidic acid (LPA) which actscombinatorially with SDF-1. Cytoskeletal rearrangement is required formotility. Proteomic analysis of hematopoietic progenitor cells suggestedthat microtubule function is regulated by post-translational modification.The role of protein acetylation in cell motility mediated by SDF-1 and LPAwas therefore assessed using Histone deacetylase (HDAC) inhibitors,which increase acetylation in a number of proteins, including tubulin, amicrotubule protein. HDAC inhibitors (Scriptaid, Trichostatin A, sodiumbutyrate) reduced the motile response to SDF-1 and LPA in short- and



long-term murine reconstituting cells. The mechanism by which this isachieved was further investigated. It is known that SDF-1 and LPA activatephosphatidylinositol 3-kinase, a signalling protein upstream of RhoGTPases, which play a role in the regulation of microtubule function. Vav1, a guanine nucleotide exchange factor for Rho/Rac GTPase proteinspotentiates the motile responses of hematopoietic cells. Vav-/- progenitorcells are less motile than their wild type counterparts. We demonstrateddifferential sensitivity to HDAC inhibitors with Vav-/- cell motility beinginhibited by Trichostatin A (TSA) but insensitive to sodium butyrate(which cannot inhibit HDAC6, a tubulin deacetylase) whilst Vav +/+ cellswere sensitive to both. SDF-1 and LPA promoted tubulin acetylation inprimitive hematopoietic cells, an event reduced in the absence of Vavexpression. These data demonstrate motile responses of hematopoieticprogenitor cells involve Vav-dependent and independent mechanisms andacetylation events, including regulation of tubulin acetylation at Lys40.The pharmacologic studies performed implicate HDAC6 in theseprocesses. HDAC inhibitors are of potential value in the clinical treatmentof malignant diseases including leukemias but interfere with stem cellmotility.

306DEEP MINING OF THE NUCLEAR PROTEOME OF ENRICHED POPULATIONS GENERATED DURING EMBRYONAL STEM CELL DEVELOPMENT TO MESODERM AND HEMANGIOBLASTS USING RELATIVE QUANTIFICATION MASS SPECTROMETRYD. L. Smith1,2*, A. J. K. Williamson2, C. Lancrin2, G. Lacaud2, V. Kouskoff2, A. D. Whetton11University of Manchester, Manchester, UK2Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK

Embryonic stem (ES) cell differentiation towards hematopoiesis is apoorly understood process. Regulation of transcription undoubtedly plays arole in lineage restriction and development, however there is no directcorrelation between transcriptional expression and protein levels. We haveadopted a flow cytometry-based enrichment of cells at four distinct stagesof development coupled with novel, highly penetrative liquidchromatography (LC) and mass spectrometry (MS) approaches todetermine how nuclear protein expression changes in differentiating EScells.

Brachyury is one of the best markers for early mesoderm. The GFP genehas been targeted to the Brachyury locus (GFP-Bry) to enable detectionand sorting of cells that are committed to mesoderm. Furthermore thehemangioblast cell population has been shown to be positive for VEGFreceptor (Flk-1) expression. Undifferentiated ES cells, epiblast like cells(GFP-, Flk-), mesoderm-committed cells (GFP+, Flk-) and hemangioblasts(GFP+, Flk+) were sorted using high speed flow cytometry. Expression ofspecific genes (Rex1, nanog, oct4, fgf5, brachyury, flk-1, runx1) wasemployed to confirm cell purity. Ten million sorted cells per sample werethen fractionated into nuclear and cytosolic fractions and confirmation ofsuccessful separation achieved using western blotting for organelle specificmarkers.

Tryptic digests were differentially isotopically labelled using isobarictags that enable sample pooling and relative quantification in a single massspectrometry run. To give comprehensive analysis, peptides werefractionated into 64 fractions using an off-line strong cation exchange LCapproach. Each fraction was subjected to nano-flow LC tandem-MS. Thisoffline fractionation addressed complexity and dynamic range issuesenabling high sensitivity protein relative quantification from stem cells andtheir progeny. The isobaric tagging approach facilitated the quantitativeanalysis by integrating relative quantification data for all peptides from asingle protein in several experiments, generating high confidence data onES, epiblast-like, mesoderm and hemangioblast cells on a wide variety ofproteins.

307CXCR4-DEPENDENT INTERNALIZATION AND SECRETION OF FUNCTIONAL SDF-1 BY BONE MARROW ENDOTHELIAL AND STROMAL CELLSA. Dar1*, O. Kollet1, V. Shinder2, P. Goichberg1, A. Kalinkovich1, M. Zsak3, A. Rot3, T. Lapidot11Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.2Unit of Electron Microscopy, Weizmann institute of Science, Rehovot, Israel3Novartis Institutes for BioMedical Research, Vienna, Austria

Interactions between SDF-1 (CXCL12) and its receptor CXCR4 onendothelial cells (EC) mediate chemotaxis, neovascularization andangiogenesis and have been shown to be involved in hematopoieticprogenitor cell homing, retention and mobilization. However, themechanisms by which SDF-1 is presented by CXCR4 expressing bonemarrow (BM) endothelial and stromal cells, to stimulate hematopoietic celladhesion and migration, to retain hematopoietic stem and progenitor cellsin local microenvironments, and to provide survival and differentiationsignals, remain poorly understood. Herein we describe a novel function forCXCR4 expressed on BM endothelial and stromal cells, which efficientlyinternalize circulating SDF-1, resulting in its translocation into the BM. Asingle i.v injection of SDF-1 increased homing of human CD34+ enrichedprogenitors obtained from G-CSF mobilized PBL from healthy donors andfrom cord blood to the murine BM and spleen. Increased progenitorhoming included more primitive CD34+/CD38-/low cells. Additionally,binding and uptake of intra-bone administrated SDF-1 took placepreferentially in the BM stem cell niche, the endosteum, and in venularendothelium but not in arteriolar endothelium. Post internalization, SDF-1was endocytized via the clathrin-coated pits pathway and further releasedmaintaining its chemotactic activity. Remarkably, in situ binding assay andflow cytometry analysis revealed that the chemokine transporter functionof CXCR4 characterized endothelial and stromal cells but nothematopoietic cells, revealing functional differences betweenhematopoietic and stromal cells. We suggest that SDF-1 translocationacross the blood-BM barrier allows effective transfer of SDF-1 encodedsignals from the periphery to the stem cell niche and leukocyte reservoir.

308PZR A NEGATIVE REGULATOR OF SHP-2-DEPENDENT HUMAN MESENCHYMAL STEM CELL MIGRATIONM. Roubelakis1,2*, S. P. Forde1,2, E. Martin-Rendon1, S. M. Watt1,21National Blood Service, Oxford, UK2University of Oxford, Oxford, UK

Human mesenchymal stem cells (hMSCs) are a population ofmultipotent adherent cells identified mostly in the bone marrowmicroenvironment and that give rise to many mesenchymal phenotypes.MSCs participate in processes such as embryogenesis, angiogenesis andtissue regeneration. MSCs are involved in the production of vessels sincethey are able to migrate and form capillary-like structures. Duringembryogenesis, MSCs and haematopoietic stem cells migrate from theAGM region via the fetal liver and spleen to the bone marrow, wheredefinitive haematopoiesis takes place. MSCs remain prominent in theadult, supporting HSC maintanance and playing an important role in tissueregeneration and cancer. Although one of their major characteristics is theirability to migrate, little is known about the molecular regulation of MSCsmigration. In this study, we demonstrate that protein-zero-related (PZR)protein and, its binding partner, protein phosphatase SHP-2 are importantin controlling MSCs migration. SHP-2 is essential in many cellularprocesses such as stem cell fate, proliferation and vascular development inthe haematopoietic system. We have identified PZR, an its novel isoformPZRb, in human MSCs and haematopoietic porgenitor cells. Thesemolecules are derived by differential splicing from a single genetranscription unit on human chromosome 1q24 and have identicalextracellular and transmembrane domains, but differ in their cytoplasmictails. PZR contains two ITIM-motifs that interact with SHP-2, while PZRblacks these motifs. Our results suggest that PZR is a receptor-bindingpartner of SHP-2 that plays a functional role in integrin-dependent cellmigration/motility. Furthermore, over-expression and knock-downexperiments indicate that PZR, but not PZRb, facilitates the migration offibroblasts and MSCs. Immunoprecipitation, Western blotting andconfocal microscopy studies confirm that PZR interact with SHP-2 and theintegrin VLA-5 and may be part of a larger protein complex that modulatesmigration in stem cells.



Abstracts

309ERYTHROPOIETIC DIFFERENTIATION OF MURINE ES CELLSS. Kaushal, A. Srivastava, E. Carrier*University of California, San Diego, CA, USA

We hypothesized that dexamethasone induces upregulation oferythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cellstowards erythropoietic differentiation. Murine ES cells (129 CCE)obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem CellBiol., 2001; 115:49-58) were subjected to the in vitro primaryhematopoietic differentiation media containing methylcellulose, IMDM, IL-3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitoryfactor) to promote embryoid body (EB) formation. Total RNA wascollected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR.On day 9 EBs were subjected to secondary differentiation using threedifferent cytokines and growth factors combination 1) SCF, EPO,dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO,EPO. Total RNA from day12 of secondary differentiated ES cells wascollected to study cytokines and growth factors dependent erythrocyticdifferentiation and gene regulation, using RT-PCR. Our resultsdemonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globingenes (a-globin, BH-1 globin, b-major globin, e-globin and z-globin) in the9 days old EBs, whereas, RNA collected from 5 days old EBs showedexpression of HoxB-4, e-globin, v-globin, BH1-globin and FLK-1. Threedays old EBs showed only HoxB-4 and FLK-1 gene expression and lack ofexpression of globin genes, indicating that erythropoiesis-specific genesactivate later. Gene expression studies of RNA collected from secondarydifferentiated ES cells and media containing dexamethasone showeddownregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors mediacombination. These results confirm our hypothesis that dexamethasomeinduces erythropoiesis by down regulating GATA –3 and upregulatingerythropoietic genes such as GATA-1, Flk-1 and Epo-R. Themorphological characteristics of cells after secondary differentiationshowed enhanced production of erythrocytic precursors in dexamethasonecontaining media, which corresponded with molecular studies. Furtherstudies will address the role of wnt/b-catenin and E-cadherin in thisprocess.

310A ROAD MAP TOWARDS DEFINING THE ROLE OF SMAD SIGNALING IN HEMATOPOIETIC STEM CELLST. Utsugisawa1, J. L. Moody1*, M. Aspling1, E. Nilsson1, L. Carlsson2, S. Karlsson11Division of Molecular Medicine and Gene Therapy and the Lund Stem Cell Center, Lund University Hospital, Lund, Sweden2Department of Microbiology, University of Umeå, Umeå, Sweden

The transforming growth factor-beta (TGF-beta) superfamilyencompasses the ligands and receptors for TGF-beta, bone morphogenicproteins (BMP) and Activins. Ligand binding to specific receptorcombinations on the cell surface elicits the activation of receptor Smads(R-Smads 1,2,3,5). These then associate with the common Smad4,translocate into the nucleus, and evoke specific transcriptional responses.Here were have characterized, by quantitative PCR, the expression of theligands, receptors and Smads of the TGF-beta superfamily in sorted, mousehematopoietic stem cells (HSC, LSKCD34-) and in the Lhx-2 cell line. Weshow that primary HSC express the majority of receptors and Smadsnecessary for the transduction of signals from TGF-beta, BMP and Activin.Likewise, the Lhx-2 line has maintained expression of all the moleculesexamined in similar patterns to those seen in primary HSC. Notably absentis expression of Alk-1, a reported alternative receptor for TGF-beta inendothelial cells, and the BMP type I receptors Alk-3 and Alk-6. We haveevaluated the response of primary HSC to various ligands in single cell andbulk proliferation assays performed with SCF, IL-6 and IL-3 under serum-free conditions. We find no effect of BMP-4 on proliferation under theseconditions, and a suppressive effect of Activin A that is similar to that ofTGF-beta in bulk cultures. Furthermore, the response of Lhx-2 cells totested ligands in bulk culture emulates what is seen in primary cells.Signaling studies that verify the phosphorylation of Smad2 (Activin andTGF-beta) and Smad1/5 (BMP) in Lhx-2 cells and lineage negative (Lin-)BM cells confirm cytosolic responses to these ligands. Our studies providea thorough characterization of TGF-beta superfamily expression in HSC

and demonstrate that the Lhx-2 cell line is an appropriate model to definehow regulatory circuits involving Smad signaling control proliferation andquiescence of HSC.

311STIMULATION OF CORD BLOOD CELLS BY STEM CELL FACTOR AND ONCOSTATIN M MAINTAINS NOD/SCID-REPOPULATING CELLSS. Gilfillan1*, S. Kraner1, S. Marz1, K. Götze1, M. Schiemann2, V. Jacobs3, C. Peschel1, R. Oostendorp11III. Med.Klinik, Technical University Munich, Germany2Inst. Microbiology and Immunology, Technical University Munich, Germany3Dept. Obstretrics and Gyneacology, Technical University Munich, Germany

We here investigated whether receptor tyrosine kinase signaling (ckit,flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture,or whether, like in embryonic stem cells, signaling through gp130 is alsorequired. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from humanumbilical cord blood (CB) were cultured in the presence of combinationsof ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and differentstimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of thesecytokines, SCF was the only one that stimulated cell division by itself.When SCF was included in the cytokine cocktail, progenitor levels (CFC,CAFCw2, CAFCw6) after this initital 6-day culture was very similar for allcombinations of cytokines tested. More primitive progenitors were alsoharvested after 6 days of serum-free culture and cultured for another 6weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showedthat in initial 6 day cultures with FL or gp130-stimulators alone, very littlehematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However,combinations of SCF with OSM consistently increased the number ofLTC-CAFCw2 and w6 about two-fold. This finding suggests that veryprimitive hematopoietic progenitors were at least maintained in serum-freecultures. What about HSC? When CB cells were cultured with SCF alone,NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped toabout 20% in 6 days (1 in 5500). Similarly, cultures without growth factorsor with FL or OSM showed a decrease to less than 10% of the originalCRU number. In contrast, the combination of SCF and OSM maintainedCRU at about half the original level (1 in 1900). This study shows thatsignaling through ckit and gp130 maintains CRU level. However,expansion was not observed. To achieve expansion stem cells will requireadditional stimulation, most probably through BMP-signaling pathway.

312HEMATOPOIETIC AND NEURAL STEM CELL ACTIVITIES ARE INCREASED BY TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENTC. Schmittwolf1, D. Vallabhapurapu1, N. Kirchhof1, A. Jauch2, M. Dürr1, H. Harder1, M. Zenke3, A. M. Müller1*1Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), University of Würzburg, D-97078 Würzburg, Germany2Institut für Humanmedizin, Universität Heidelberg, 69102 Heidelberg, Germany3Institute for Biomedical Technology, Cell Biology, RWTH Aachen, D-52057 Aachen, Germany

DNA and chromatin modifications are important for the establishmentand maintenance of cell type-specific gene expression patterns thatconstitute cellular identities. The epigenotype of cells is defined by theexpression of specific genetic programs which is reflected in theirdevelopmental stage- and cell type-specific chromatin organization. Toanalyse whether the potentials of stem cells in bone marrow and in fetalbrain-derived neurospheres can be modified by destabilization theirepigenotypes, murine bone marrow cells and murine neurosphere cellswere transiently treated with chemical compounds that alter acetylationand methylation patterns of chromatin and DNA.

We observe that bone marrow cells are sensitive to Trichostatin A(TSA) treatment as assessed by 7AAD and CFSE labelling studies,respectively. Despite these adverse effects, TSA- and TSA/5-Aza-2'-deoxycytidine (AzaC)-treated cultures yielded greater numbers of lin-/ckit+ cells as compared to untreated cultures along with higher colonyforming cell (CFC) numbers. In competitive repopulation studies we



observe increased hematopoietic engraftment potential in treated comparedto untreated bone marrow cultures. Thus, TSA and AzaC treatmentcounteracts the culture-induced loss of HSC activity in bone marrowcultures.

Inhibitors of DNA methylation and histone deacetylation also act oncultured neural stem cells. After transplantation of neurosphere cells pre-treated with a combination of TSA plus AzaC (TSA/AzaC neurospherecells), recipient mice yielded multi-lineage and transplantable neurosphere-derived hematopoietic engraftment. Untreated neurosphere cells did notengraft. Neurosphere-derived hematopoietic cells showed a diploidkaryotype, indicating that they are unlikely to be products of cell fusionevents. This conclusion is strengthened by multicolour fluorescence in situhybridization (M-FISH).

We conclude that transient treatment of neurosphere and bone marrowcells with TSA or TSA/AzaC results in an increase of neural andhematopoietic progenitor/stem cell activities.

313GENERATION OF MATURE TRYPTASE/CHYMASE DOUBLE POSITIVE MAST CELLS FROM A CYNOMOLGUS MONKEY EMBRYONIC STEM CELLSF. Ma1*, N. Kambe2, D. Wang3, G. Shinoda1, K. Umeda1, H. Suemori4, N. Nakatsuji4, K. Tsuji4, T. Hekei1, T. Nakahata11Department of Pediatrics, Kyoto University School of Medicine, Japan2Department of Dermatology, Kyoto University Graduate School of Medicine, Japan3Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Japan4Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan

Human and non-human primate embryonic stem (ES) cells provideunique tools to study early events occurring in the development. Werecently established an in vitro system to induce hematopoiesis by co-culturing cyonomolgus monkey ES cells with a murine aota-gonada-mesonephros-derived stromal cell line (AGMS). In this system under acondition of 10% to 15% fetal bovine serum but without any other factors,ES cells can differentiate into cobble stone-like cells around day 12, whichare mostly CD34+ and can give rise to various hematopoietic coloniesincluding BFU-E, GM, and mixed lineages in semisolid cultures. Whenreplating onto AGMS-coated plates with SCF, IL-2, IL-3, IL-6, and Flt2/Flk3 ligand, they showed a robust growth. After 3-week culture almost allcultured cells became mast cells (ES-MCs). These ES-MCs hold granules,which were positive for both mast cell-specific tryptase and chymase, andcontained beta-hexosaminidase enzyme activity. A flow cytometricanalysis revealed these ES-MCs were CD45+/Kit highly+ and about 50%cells express a high affinity IgE receptor on their surfaces, which werestrikingly up-regulated after the exposure to IgE. MCs are mainly dividedinto two types, tryptase only+ mucosal-type MC-T cells and tryptase/chymase double + connective tissue-type MC-TC cells, and ES-MCs havea characteristic phenotype of MC-TC cells. Since the developmentalrelation of these two type MCs is still unknown, our model may provide agood tool to uncover the mechanism of mast cell development.

314A NEW MOLECULAR COMPLEX THAT MODULATES CXCR4-DRIVEN MIGRATIONS. P. Forde1,2*, B. Jorgensen Tye1,2, M. Roubelakis1,2, S. M. Watt1,21National Blood Service, Oxford, UK2University of Oxford, Oxford, UK

Haemopoietic stem/progenitor cell homing, migration and adhesion arecomplex, multifactoral processes, involving interactions between multiplereceptors and their ligands. The chemokine axis SDF-1 (Stromal derivedfactor-1, CXCL12)/CXCR4 is a well characterised and importantchemotactic stimulus/receptor unit controlling these events. Othermolecules involved in migration and adhesion of these cells includeintegrins, VLA-4 (Very Late Antigen-4) and VLA-5, and members of thesialomucin (e.g. PSGL-1) and Ig superfamilies. Here we demonstrate thatthe sialomucin, CD164 or endolyn, clusters with the CXCL12 receptor,CXCR4, on CD133+ cord blood cells and modulates their migrationtowards CXCL12. We further show that blockade of the functional class IIglycosylation-dependent epitope on CD164 by the 103B2/9E10 antibodysignificantly reduces CD133+ cell migration on a fibronectin substrate

towards CXCL12. Engagement of this epitope on CD34+CD38-haemopoietic precursors has previously been demonstrated by us to reducethe binding of these cells to bone marrow stromal cells and to preventrcruitment of these cells into cycle. This selective, but transient, clusteringof CD164 with CXCR4 also recruits VLA-4 and VLA-5 to the leadingedge of CD133+ cells, as demonstrated by confocal microscopy andimmunoprecipitation studies. Our findings support a novel associationbetween three distinct families of cell surface receptors that regulate bothcell migratory and proliferative responses and identify a new role forCD164 in modulating CXCL12/CXCR4 mediated cell migration.

315SHORT TERM CULTURE OF CD34+ HUMAN CORD BLOOD CELLS YIELDS LARGE NUMBERS OF MEGAKARYOCYTE PRECURSORS WITHOUT PERTURBING THE ORIGINAL DIFFERENTIATION PROFILE OF SIMULTANEOUSLY MAINTAINED SHORT-TERM REPOPULATING CELLSM. Fischer1*, M. Schmidt1, S. Klingenberg1, C. Eaves2, C. von Kalle1, 3, 4, H. Glimm1, 41Medical Department 1, University Hospital, Freiburg, Germany2Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada3Exp. Hematol. Res. Foundation, CCH, Cincinnati, USA4IMMZ, University, Freiburg, Germany

Human cord blood (CB) contains 2 types of CD34+ short-termrepopulating cells (STRCs) that both engraft NOD/SCID-ß2microglobulin-/- mice more efficiently than NOD/SCID mice. Delayed platelet recoveriesoften obtained in adult recipients of CB transplants may be due to therelatively low number of STRCs in CB and their lower output ofmegakaryocytic and erythroid progeny in mice as compared to STRCsfrom adult sources. To determine whether the numbers and in vivodifferentiation activity of CB STRCs could be modified in vitro, wemeasured their numbers in the initial CD34+ population and then againafter up to 21 days in serum-free medium supplemented with EPO, flt-3ligand, TPO, SCF, IL-3, IL-6 and IL-11. The highest increases in colony-forming cells were reached on day 8, whereas CD41+ and Glycophorin A+cells were maximally increased 6 days later. Serial bone marrow aspiratesperformed on 220 NOD/SCID-ß2microglobulin-/- mice transplanted withthe cultured cells showed an initial 13-fold decrease in STRC-M (myelo-restricted) activity (day 4), followed by a recovery to 15-20% of inputlevels (by days 8 to 21). Assays of STRC-ML (lympho-myeloid) activityshowed these continuously decreased to 70% and 30% of input (days 14and 21). The size and lineage content of the clones produced from theinitial and culture-derived STRCs showed no differences. Thus exposure tothese growth factors for up to 3 weeks in vitro had no effect on thesubsequent proliferative potential or differentiation profile of the cellsregenerated in mice. These findings provide new support for the conceptthat the hematopoietic growth factors tested play little role in influencingthe the process of lineage restriction. Striking differences in the kinetics ofmaturation of STRC-MLs in vivo and most progenitors detectable in vitrosupports the concept that the STRC assays identify unique subsets offunctionally relevant progenitors.

316TRACKING HEMATOPOIESIS AT THE SINGLE CELL LEVELT. SchroederGSF, Munich, Germany

Despite intensive research, many long-standing questions ofexperimental hematology remain unsolved. One major reason is the factthat hematopoiesis is usually followed by analyzing the fate of populationsof cells - rather than individual cells - at very few time points of anexperiment and without knowing (or quickly loosing) their individualidentities. The static picture yielded by this approach makes it impossibleto appreciate the dynamic developmental processes leading to the(re)generation of the hematopoietic system from individual hematopoieticstem cells (HSC). Real-time tracking of individual cells in culture, tissuesor whole organisms would be an extremely powerful approach to fullyunderstand the developmental complexity of hematopoiesis. However,many of the needed tools are still under development and their applicationin hematology remains difficult. Here, a computer aided culture andimaging system was developed to follow the fate of individual cells overlong periods of time with highest temporal resolution. A new softwaremodule was written, which helps to record and display the divisional



Abstracts

history, position, properties etc. of individual cells. This system was usedto analyze the development of multi-lineage cobblestone colonies fromhighly purified individual adult HSC in stroma co-cultures at single celllevel over many generations, yielding new insights in the behavior of HSCunder conditions closely resembling their physiological environment in thebone marrow. The presented data will improve the understanding of thecellular dynamics of hematopoietic development.

317INTRACELLULAR AND SURFACE MARKER LABELING OF HEMATOPOIETIC CELLS USING QUANTUM DOT BIOCONJUGATESC. Wang1*, J. Zheng1, S. Mardyani2, W. C. W. Chan21Dept. Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada2Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada

Luminescent quantum dots (qdots) have the potential to become a newclass of fluorescent labels for biomedical studies. In contrast to traditionalorganic fluorophores, qdots are extremely stable against photobleachingand highly resistant to chemical and biological degradation. Moreover,qdots have narrow and tunable emission spectra and all colors of qdots canbe excited by a single light source. These optical properties make qdotsideal for cellular imaging and fluorescence analysis of multiplex cellularsystems such as hematopoiesis. In this study, we explored the feasibility ofusing qdot-based probes for labeling hematopoietic cells. The ZnS-cappedCdSe qdots used in this study had a size range of 3-6 nanometers. Thestudy used qdot-transferrin conjugates for intracellular delivery of qdotsfor cellular imaging and qdot-antibodies for detecting specific cell markers.For live cell labeling, human cord blood or mouse marrow cells wereincubated in suspension cultures in the continuous presence of qdot-transferrin conjugates. Positive qdot-labeling was detected in the cells andqdot signal was shown to be in cytoplasmic locations as determined byscanning confocal microscope. The intensity and frequency of qdot-labeledcells increased with incubation time. Continuous exposure of cells to qdotconjugates exhibited no cytoxicity and no changes in cell viability up to 7days in cultures. In the controls with unconjugated qdots, onlymacrophages and neutrophils were shown to contain qdot clusters andaggregates. The uptake of qdots by these cells is consistent with non-specific phagocytosis. For surface marker analysis, qdot-antibodyconjugates were prepared for detection of CD19 and CD45 markers onlymphocytes. Qdot-labeling was shown to be bright, photostable, andspecific, and readily detectable by using fluorescence microscopy or flowcytometry. The results of this study demonstrated feasibility and potentialof using qdots as fluorescence probes for hematopoietic analysis.

318ISOLATION AND CARACTERIZATION OF MULTIPOTENT ADULT PROGENITOR CELLS FROM HUMAN AND CYNOMOLOGUS MONKEYC. Clavel1*, X. L. Aranguren1, M. Barajas2, C. Moreno1, E. Rahrmann2, T. O'Briend2, C. M. Verfaillie2, F. Prosper11Cell Therapy, University Clinic, University of Navarra, Spain2Stem Cell Institute, University of Minnesota, MN, USA

MAPCs (multipotent adult progenitor cells) have been recentlydescribed as a population of adult pluripotent stem cells. We havesuccessfully isolated and characterized a cell population from Bone-Marrow of adult human (hMAPC) and adult cynomologus monkey(pMAPC), that co-purify with mesenchymal stem cells. pMAPCproliferate over 150 PD and hMAPC reached 60 PD, both without apparentsenescence. h/pMAPC express stem cells markers (Oct4, Rex1, SSEA4and Nanog) and lack expression of tissue stem cells specific antigens(CD44, Class I and II, CD31, CD34, CD45, CD75, CD105), demonstratinga phenotype consistent with human MAPC described previously by Dr.Verfaillie group. Within h/pMAPC populations, we can positively identifya 'small' population of cells carrying high levels of self-renewal stem cellmarkers. In vitro, h/pMAPC differentiates into cells expressing markers ofmesodermal derived cell tissues (bone, cartilage, muscle, andendothelium), neuroectoderm (neurons) and endoderm (hepatocytes). Also,no transformation or cytogenetic abnormalities has been observed.Interestingly, these populations of h/pMAPC can be isolated and identified

without prolonged culture (as early as after 10-20 PD) allowing earlyexpansion of cells. These cell populations may have a great clinical andpre-clinical potential in cell therapy.

319EFFECT OF PARATHYROID HORMONE ON HEMATOPOIETIC AND STROMAL PRECURSOR CELLSD. A. Svinareva*, I. N. Nifontova, J. L. Chertkov, N. J. DrizeNational Hematology Research Centre, Moscow, Russian Federation

Osteoblasts are suggested to participate in formation of hematopoietic'niche' needed for regulation of hematopoietic stem cells' fate. An influenceof parathyroid hormone (PTH) on hematopoiesis was investigated, as PTHis known as regulator of osteogenesis. Mice were injected i.p. with PTH 80mkg/kg 5 days/week for 4 weeks. Two weeks later concentration ofdifferent hematopoietic precursors was analyzed. PTH-induced alterationsvaried for hematopoietic precursors of different maturity. The number ofCAFC 28-35 and LTC-IC in femoral bone marrow doubled in PTH-treatedmice. On the contrary the CFU-S 9 and CFU-S 13 number decreased about2-fold while their self-maintenance ability (measured by the number ofdaughter CFU-S per 13 day-old spleen colony) was 1,5-2 fold higher.Concentration of CFU-GM and BFU-E did not change. These resultssuggest the ability of PTH to govern the function of 'niches'. Thissuggestion was supported by revealed changes in bone marrow seedingefficiency (f-factor-24 hours) of normal hematopoietic precursors in PTH-treated mice. CFU-S seeding decreased approximately 5-fold from 5% to0,6% while CAFC-28 f-24 increased 1.2 -fold. The f-24 changes mayprove the osteogenic activity of PTH, as spleen f-24 for CFU-S wasunchanged in treated and control mice (5-6%). PTH did not affect thenumber of mesenchymal stem cells measured by their ability to transferhematopoietic microenvironment while it doubled the weight of de novoformed bone shell in the ectopic foci. In normal mice circulatinghematopoietic precursors with lower self-maintenance ability as comparedto bone marrow precursors repopulate the ectopic hematopoietic foci. Theself-renewal capacity of CFU-S in the foci formed in PTH-treated miceincreased 2-3 fold. It is uncertain whether PTH treatment inducesmobilization of more immature precursors from bone marrow or activatesthe precursors during foci formation. The mechanism of hematopoieticeffects of PTH is the object for future studies.

320EXPOSURE TO NICOTINE DURING GESTATION INTERFERES WITH THE COLONIZATION OF FETAL BONE MARROW BY HEMATOPOIETIC STEM/PROGENITOR CELLSN. Serobyan*, S. K. KhaldoyanidiLa Jolla Institute for Molecular Medicine, San Diego, CA, USA

Environmental factors, including cigarette smoke components, can crossthe placental barrier and accumulate in amniotic fluid and fetal tissue, andtherefore, interfere with the normal course of ontogenesis. While cigarettesmoke contains numerous compounds, the most adverse effects onmammalian tissues have been associated with nicotine. The aim of thisstudy, therefore, was to investigate the effect of intrauterine exposure tonicotine on hematopoiesis during fetal development and postpartum.Intrauterine exposure of mice to nicotine resulted in a more than 2-foldreduction of the delayed-type hypersensitivity (DTH) response and a 2.5-fold decrease in the number of plaque forming cell (PFC) in offspring afterone month of postnatal life, and correlated with low counts of maturelymphocytes and lymphoid progenitors in hematopoietic tissues. Neonatesexposed to nicotine during gestation showed a significant decrease in thenumber of bone marrow hematopoietic progenitors, as measured by colonyforming unit (CFU) and long-term culture initiating cell (LTC-IC) assays,and decreased concentration of IL-6 in their serum. Analysis of the fetalbone marrow (E15) obtained from nicotine-exposed fetuses demonstrated alower number of hematopoietic progenitors, while their number in the fetalliver was not significantly changed. Our data provide evidence that bytargeting nicotinic acetylcholine receptor (nAchR), nicotine interferes withthe fetal development of the hematopoietic system. Inferior colonization ofthe fetal bone marrow by hematopoietic stem/progenitor cells (HSPC)subsequently results in an imbalance of mature blood and immune cellproduction after birth.



321CHARACTERIZATION OF DIVERSE ORIGIN AND FUNCTION OF HUMAN UMBILICAL CORD BLOOD-DERIVED ENDOTHELIAL PROGENITOR CELLSE. S. Yoo1*, Y. C. Mun2, K. E. Lee2, Y. K. Bae2, S. E. Lee2, J. Y. Ahn2, C. M. Seong21Departments of Pediatrics, Ewha Womans University College of Medicine, Seoul, Korea2Departments of Internal Medicine, Ewha Womans University College of Medicine, Seoul, Korea

Ex vivo expanded endothelial progenitor cells (EPCs) represent a newpotential approach for the neovascularization of ischemic sites. But apossible limitation to the use of vascular precursors for therapeuticangiogenesis is the relatively low number of these cells and localaccumulation of infused EPCs in these sites is poor. It should be modulatedeffective recruitment into ischemic site. In this study, we investigated theability of umbilical cord blood(UCB) EPCs on in vitro differentiation intomature endothelium in relation with enhancement of EPCs number andmodulation of homing. We cultured total mononuclear cells from humanUCB in M199 medium supplemented with 20% FCS, 15 mM HEPES,antibiotic and antimycotic solution with VEGF, IGF1 and FGF withfibronection. After 21 days, differentiated endothelial colonies wereassayed by morphology, flow cytometry, endothelial progenitor colony,RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCswere also measured by a modified in vitro transmigration assay in presenceof VEGF and SDF-1 and above results were compared with EPCs fromadult peripheral blood(PB). After 21days of culture, two types of colonieswere grown. Early spindle-shaped cells showed peak growth at 2 to 3weeks and positive for VEGFR2, vWF, CD31, VE-Cadherin, L-selectin,CXCR-4 and CD14. Late cobblestone shaped cells were also positiveabove antibodies except CD14. These cells more produced nitric oxide andformed capillary tube better than early spindle-shaped cells and higherexpressed L-selectin, CXCR4 and more readily migrated by VEGF andSDF-1 compared with adult PB. We provide evidence that progenitors forendothelial cell circulate in human cord blood and differentiate into 2 typesof EPCs which might have different role in neovascularization. The higherexpression of adhesion receptor and in vitro migration ability involved inthe vascular homing of EPC better in UCB. This may provide knowledgeabout the substrate to design strategies to improve EPC localization indamaged tissues after infusion of EPC.

322STEM CELL – STROMAL CELL INTERACTIONS: OBSERVATIONS FROM TIME LAPSE PHOTOGRAPHY EXPERIMENTSA. L. Peterson*, M. S. Dooner, P. J. QuesenberryRoger Williams Medical Center, Providence, RI, USA

Stromal cell cultures expressing green fluorescent protein from maleC57BL/6-Tg(ACTB-EGFP)1Osb/J mice were maintained in standardDexter conditions and followed for 3 weeks before use. Lineage-negative,Sca-1 positive cells were obtained from female C57BL/6J mice and wereeither used fresh or cultured in media containing mSCF for up to 72 hours.After culture (if any), stem cells were labeled with PKH26 fluorescent reddye and either added immediately to slide chamber wells containing Dexterstroma or first stimulated for 1 hour at 37 deg C in media containing SDF-1 and mSCF. Stem cells were allowed 20 minutes to settle after beingadded to stromal cultures. Cell interactions were captured by time-lapsephotography at 10-minute, 2-minute, 1-minute, and 30-second intervals fortotal time periods of up to 12 hours.

We observed a variety of interactions. Stem cells moved quickly in theearly stages of interaction (searching), coming to rest in contact withstromal cells (parking). Stem cells would often visit several stromal cellsbefore parking against one, sometimes dodging around other cells to reachtheir goal. We captured stem cells partially entering stromal cells and thenwithdrawing. We also observed entry of marrow cells through stromalproteopods with stromal residence after several hours of co-culture. On oneoccasion we observed exit of a marrow cell from a stromal cell. Stromalcell membranes were quite active and appeared to be used for limited cellmotility in some cases. Proteopods were common, with 10 to 40 percent ofstromal cells expressing proteopods which often extended and retractedover time. Proteopod expression on stem cells was dependent on cultureconditions. We also observed stem cells rolling and sliding along stromalproteopods.

These dynamics suggest a variety of stem cell – stromal cell interactionswhich may provide a model for epigenetic change in the hematopoieticmicroenvironment.

323THE OSTEO-HEMATON: ONTOGENIC DEVELOPMENT AND BIOLOGY OF STEM CELL NICHESI. J. Blazsek1,2*, E. Oberlin1, M. Souyri1, B. Péault1,31Inserm U506, Villejuif, France2I.C.I.G., Paul Brousse Hospital, France3University of Pittsburgh, PA, USA

The biological function of most vertebrate organs is carried out by manytissue-specific functional units like the nephron, Lieberkühn crypt, islet ofLangerhans or hepato-biliary unit. Hemogenic stem cells (HSCs) irrigatefreely the whole body by the blood stream, but adult HSCs proliferate anddifferentiate exclusively in the BM. We have postulated and documentedpreviously that the spongy BM was also organized into discrete, tissue-specific structural-proliferative units, named hematon.

Here we show the spatial-temporal organization of stem cells and nest-forming cells, and basic mechanisms by which they sculpt together thehematon in mouse and human liver and spongy BM. Video microscopyuncovered the constitutive role of cardiomyogenic mesoderm andsubsequent association of mesodermal (desmin/GFAP+), hepato-cholangiogenic (CK19+, c-met+/CD49f+, connexin-43+) andhematopoietic stem/progenitor cells into compact node-like niches, in earlyliver organogenesis.

Single niches developed autonomously into complex hepatic units, andat low seeding density they evolved into an interactive, contractil network.The organization and integrity of self-bounded units was maintained bydistinct cell lineages emitting stimulatory and/or veto signaling factors(FGF, HGF/SF, SHH, BMP4, SDF-1 and ephrinB2/EphB4). Targetedinactivation of motif links disintegrated the network into isolated colonies,resulting in either abortive or extensive growth.

Colonization and amplification of HSC in BM numerically correlatedwith the node-like niches/hematons. Quantitative colony assays, FACS andimmunocytochemistry analyses allowed to identify mesenchymal stemcells, preadipocytes/adipocytes, contractile myogenic cells, a vascularmeshwork, guardian macrophages and hemopoietic stem/progenitor cells(Sca-1+/c-kit+/CD34+/Thy-1+) in the hematon. A hematon unit plays therole of an organizing centre that emits growth, morphogen and community-effect mediating factors. The physical attachment of many hematons tospongy bone suggests that they, together, may form the basic structural-functional unit in the ultimate, fully competent BM. We suggestdesignating these units and refer to as the osteo-hematon.

324PHYSIOLOGICAL-LIKE MICROENVIRONMENT FOR CYTOKINE-FREE, EX-VIVO EXPANSION OF TRANSPLANTABLE HEMATOPOIETIC STEM CELLS (HSC)S. Meretzki1, A. Kadouri2, O. Burger1*, U. Barkai11Pluristem Life Systems Inc, Haifa, Israel2Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

HSC are associated with discrete niches within the bone marrow, whichare part of the 'hematopoietic inductive microenvironment' (HIM). TheHIM provides a comprehensive milieu of soluble signals and physicalinteraction sites responsible for HSC self-renewal and downstreamproliferation and differentiation processes.

A preferred approach for ex-vivo expansion of HSC is to mimic theirnatural microenvironment by allowing them to interact with stroma.However, monolayer cultures do not fully reconstitute the natural growthconditions within the marrow HIM. As such, those efforts have met withonly limited success.

We have improved the present cultivation routines by combining 3-Dscaffold methodology with co-culture techniques and packaged all these inthe PluriXTM Bioreactor system. Stroma cells are initially cultivated onporrosive polystyrene carriers under controlled flow condition. Achievedcell densities are thus more than 4*106 cells/ml for primary cultures and14*106 cells/ml for cell lines. Doing so, a close-to-natural mechanical andbiochemical support is simulated.



Abstracts

We have recently proved that stroma cells cultivated on 3-D scaffold aresuperior to 3-D naked scaffolds and to conventional stroma cell cultivatedon conventional 2-D matrixes in supporting the growth of CD34+,CD34+38- and CD34+38-CXCR4+ cells. The 3-D culture system wassignificantly better than the 2-D cultures and than the 3-D naked scaffold.

In addition to cell-cell interactions, the construction of an artificial HIMprovides available bioactive peptides, secreted by stroma cells. Thosepeptides are required for HSC maintenance and expansion and theirintegral secretion prevents the need for the addition of those molecules intogrowth medium. The avoidance of external cytokine addition promotesHSC proliferation while reducing their differentiation rate.

This system offers wide-range potential in clinical applications,including the expansion of cord blood HSC and the production of bloodderivatives.

325SPONTANEOUS MOBILIZATION OF BONE MARROW-DERIVED HEMATOPOIETIC AND ENDOTHELIAL PROGENITOR/STEM CELLS AFTER ORTHOTOPIC LIVER TRANSPLANTATIONL. Catani1*, S. Talarico1, E. Loggi2, A. Gramenzi2, U. Baccarani3, G. L. Grazi4, M. Aluigi1, M. Baccarani1, P. Andreone2, R. M. Lemoli11Institute of Hematology and Medical Oncology L. A. Seràgnoli, University of Bologna, Bologna, Italy2Department of Hepatology, Cardioangiology and Internal Medicine, University of Bologna, Bologna, Italy3Department of Surgery and Organ Transplantation, University of Udine, Udine, Italy4Liver and Multi Organ Transplant Unit, University of Bologna, Bologna, Italy

This study characterized the peripheral blood (PB) stem/progenitor cellscompartment of 24 adult patients before (day –1) and after (days 1, 3, 7 and14) orthotopic liver transplantation (OLT) for liver failure (10) and/orhepatocellularcarcinoma (14). Twelve healthy donors served as normalcontrols. The time-course phenotypic evaluation of circulating cells aftertransplant demonstrated the significant mobilization of CD34+ cells (day+7; p 0.017), together with a significant increase of primitive CD34+/CD90+ cells (day +7; p 0.043). We also observed the significant increaseof circulating mature committed progenitors (CFU-C) on day +14 aftertransplantation (p 0.028 ). The number of circulating endothelial CD133+and CD34+/KDR+ stem/progenitor cells, which contribute toneoangiogenesis after tissue ischemia and organ regeneration in animalmodels, significantly augmented following OLT (day +3; p less than 0.05).When we analyzed the serum level of cytokines involved in stem cellmobilization and/or liver repair after OLT, we observed the significantincrease of Stem Cell Factor (SCF), Granulocyte Colony-StimulatingFactor (G-CSF), Interleukin-6 (IL-6) and Vascular Endothelial GrowthFactor (VEGF). The serum level of stromal cell-derived factor-1 (SDF-1)significantly decreased after transplant at days +1 and +7 (p less than 0.05).Interestingly, the baseline levels of IL-6 and Hepatocyte Growth Factor(HGF) were significantly higher in patients with liver disease than inhealthy controls (p 0.002). Cytogenetic and molecular analyses showed thehost origin of mobilized bone marrow-derived cells and the co-expressionof liver-specific epithelial markers (CK-19 and alpha-fetoprotein) in arepresentative case of sex-mismatch transplant. This is the first studyreporting the mobilization of both hematopoietic and endothelial stem/progenitor cells after OLT. Our data suggest that stem cell recruitment tonon-hematopoietic organs may be a physiological process in case of tissueinjury and underline the potential role of bone marrow-derived cells forcell therapy of liver diseases.

326PROLONGED AND DURABLE CIRCADIAN RHYTHMS OF ISOLATED POPULATIONS OF PER1-LUC AND PER2-LUC TRANSGENIC BONE MARROWM. Dooner1*, T. Yoshikawa2, D. Demers1, J. Pimentel1, G. Dooner1, M. Menaker2, P. J. Quesenberry11Roger Williams Medical Center, Providence, RI, USA2University of Virginia, Charlottesville, VA, USA

In previous studies we have demonstrated a significant circadianvariation of bone marrow long-term engraftability. These variations arehypothesized to be controlled by a circadian clock in the suprachiasmaticnucleus. We have utilized transgenic mouse models Per1-luc and Per2-luc.

The cells in these animals have a luciferase reporter attached to thepromoter on the circadian clock gene. When the gene is turned on and inthe presence of luciferin, a pulse of white light is given off. With thesemodels we have shown circadian rhythm fluctuations in whole marrow,and subsetted marrow granulocytes, B cells, T cells, macrophages and ahighly purified stem cell populations, Hoechst low rhodamine low(LRH)and Lineage negative SCA-1 positive cells for up to 15 days in culture.While organs such as spleen, liver and whole marrow gave clear longlasting rhythms the purified stem cells gave very low level expressionwhen cultured for up to 35 days. To examine this further, we studiedexvivo influences on whole marrow rhythms such as the time cells wereremoved from the animal, time of centrifugation, density of cells, and cellconcentration in culture. We found that when the cells were incubated onice for 3 hours the rhythms were identical to the control (no incubation)with the exception that they were 3 hours delayed. By placing cells on ice,at least for short time periods (3hours) bone marrow cells retain theircircadian rhythms. Centrifugation and cell concentration did not changecircadian rhythms significantly. These studies show circadian rhythms aredurable rhythms for many bone marrow subpopulations including stemcells. These rhythms can be long lasting in culture, up to 35 days and acritical determinant of bone marrow biology.

327CELLS FROM SMALL VOLUME INTEGRAL LINE SEGMENTS OF CRYOPRESERVED CORD BLOOD UNITS CAN BE EXPANDED TO INCREASE CD15+ AND CD33+ CELLSG. Cho1*, D. J. Anstee1,2, A. Blair1,21Bristol Institute for Transfusion Sciences, Bristol, UK2University of Bristol, Bristol, UK

Ex vivo expanded progenitors have the potential to reduce neutropeniaand thrombocytopenia following high dose chemotherapy. Cord blood(CB) cells are increasingly used a source of haemopoietic progenitor cellsfor transplantation. Infusion of ex vivo expanded progenitor cells, derivedfrom the CB stem cell graft, could accelerate neutrophil and plateletrecovery and may reduce the need for platelet transfusions. CB units areoften cryopreserved within one bag requiring the thawing of the whole unitin order to remove an aliquot for ex vivo expansion. Such expanded cellsare not available for infusion until 1-2 weeks after the unmanipulated partof the unit has been given. In this study, the integral line segments attachedto cryopreserved cord blood units were used as a potential source of stemcells for expansion cultures. Integral line segments were detached andthawed following standard protocols. Mononuclear cells were isolatedusing Ficoll density gradient separation and then underwent magnetic cellenrichment for CD34+ cells. The CD34+ cells were cultured in StemSpanH3000 serum free medium. SCF, TPO, FLT3 ligand, and IL-3 were addedat 50 ng/ml. From day 10, cultures were passaged twice weekly by 1:3dilution in fresh medium and cytokines. At days 14 and 21, expression ofCD34, CD15, CD33, CD38 and CD41a was determined by flowcytometry. Over these time points, the CB specimens showed: 1.71-foldand 5.5-fold expansion of TNC (from 1.94x106 to 3.32x106) and (7.09x105

to 3.90x106) respectively, 2.39-fold and 5.12-fold expansion of CD15+

cells (from 1.12x106 to 2.66x106) and (3.80x105 to 1.95x106) and 4.18-fold and 67.78 fold expansions of CD33+ cells (2.85x104 to 1.19x105) and(3.97x103 to 2.69x105). In conclusion, it is possible to isolate CD34+ cellsfrom the small volume of cord blood in an integral line segment of acryopreserved unit and expand them into granulocytic progenitors.

328CHARACTERIZATION, EXPRESSION ANALYSIS AND DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLSE. Montelatici1*, V. Lo Cicero1, G. Cantarella2, R. F. Mazzola3, L. Lazzari11Cell Factory 'Franco Calori', Centro Trasfusionale, Ospedale Maggiore IRCCS, Milan, Italy2Unità Operativa Otorinolaringoiatria, Ospedale Maggiore IRCCS, Milan, Italy3Dipartimento di Scienze Otorinolaringoiatriche, Plastic Surgery Section, Università degli Studi, Milan, Italy

Adult mesenchymal stem cells (MSCs) are believed to be multipotentcells which can develop into other cell lineages such as osteocytes,adipocytes, chondrocytes and neurons. MSCs have become the centre of



attention as the suitable stem cell source, since it would be expected tocontribute to practical use for regenerative medicine. Adipose tissue iscurrently used in vocal fold lipoinjection to treat laryngeal hemiplegia oranatomical defects. We hypothesized that the effect of this procedure couldbe based not only on mechanical support and filling but also on tissueregeneration mediated by adipose-derived mesenchymal stem cells(AMSCs). In this study we analyzed samples (n=6) of adipose tissue fromthe lower abdomen and upper thigh harvested in patients who underwentvocal fold lipoinjection under strictly sterile conditions.

After an initial centrifugation lower density solid phase was collectedand digested at 37 °C for 30 min with 0.075% w/v collagenase. Followingenzyme activity neutralization, the pellet was filtered and incubatedovernight in low glucose DMEM + 10% Fetal Bovine Serum + Penicillin-Streptomycin. Thereafter, non-adherent fraction was removed, while theadherent cells were cultured for two weeks. After the expansion the cellsshowed the MSC morphology. RT-PCR analysis was performed andAMSCs were positive for Desmin, Osteocalcin, PPARgamma2, CK18 andCK19, and negative for MyoD, Myogenin, MRF-4, Myf-5, alpha-FP andalbumin. The cells also expressed Oct-4, Runx-1 and ABCG-2 whichcharacterize the undifferentiated stem cell state. Moreover, adipogenic,osteogenic, endothelial and myogenic differentiations were induced byculturing adherent cells in defined media supplemented with specificgrowth factors. Adipogenic and osteogenic differentiations were obtainedas demonstrated by Nile Red and Alizarin Red staining. Endothelial andmyogenic differentiations are still in progress. These preliminary resultssupport our hypothesis and suggest that adipose MSCs could have a role inrepairing vocal fold damage.

329HUMAN BONE MARROW STROMAL CELLS MAY PROMOTE HEMATOPOIETIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELL LINES. J. Kim1,2*, H. J. Sung1, J. H. Oh2, J. H. Yoo2, C. H. Song2, S. H. Kim2, B. S. Kim1,21Department of Internal Medicine, Division of Oncology and Hematology, Korea University Medical Center, Seoul, Korea2Institute of Stem Cell Research, Korea University College of Medicine, Seoul, Korea

We have tried to promote hematopoietic differentiation from humanembryonic stem cell(hESC) via co-culture of human bone marrow (BM)stromal cells. We developed a human embryonic stem cell line, SNU-3 toEB in EB formation media and cultured for 10 days desiganted as EBgroup. In EB/FBS group, SNU-3 hESCs were cultured in the same mediaadded 20% FBS. In EB/BM co-culture group, EB developed for 5 daysfrom SNU-3 was co-cultured with human BM stromal cells for additional 5days. The percentage of CD34+/CD45- cells was measured in 5 and 10days of culture in these three groups to compare the degree ofhematopoietic differentiation. To assess the effect of co-cuture duration onhematopoietic dofferentiation, EB was co-cultured with human BMstromal cells for 3, 5, 7, and 10 days and the hematopoietic differentiationwas compared in each time points. There was no difference of the meanpercentage of CD34+/CD45- cells between EB and EB/FBS group(0.25%±0.18 vs 0.28%±0.09). However, the percentage of CD34+/CD45-cell (1.36%±0.55) was significantly higher in EB/BM co-culture groupthan EB and EB/FBS group (p<0.01). The mean percentage of CD34+/CD45- cells and the mean number of hematopoietic colony forming unitsat 5 days of co-culture (1.93±0.439 %, 9.9±0.19 × 10) were significantlyhigher than those of 3, 7, and 10 days of co-culture, respectively (p<0.01,p<0.05). Human BM stromal cells could promote hematopoieticdifferentiation of EB developed from SNU-3 hESCs and further study iswarranted to evaluate its underlying mechanism.

330SUCESSFUL EX-VIVO EXPANSION NOD-SCID REPOPULATING HEMATOPOIETIC STEM CELLSS. Loges1*, M. Butzal1, U. Fischer1, U. M. Gehling2, D. K. Hossfeld1, C. Bokemeyer1, W. Fiedler11Department of Internal Medicine, Division of Oncology, Hematology and Bone Marrow Transplantation, University Hospital Hamburg-Eppendorf, Hamburg, Germany2Department of Hepatobiliary Surgery, University Hospital Hamburg-Eppendorf, Hamburg, Germany

Multipotent hematopoietic stem cells capable to reconstitute thehematopoietic system are of great importance in the treatment of patientswith hematological and solid malignancies. This therapeutic option is oftenchallenged by limited numbers of available stem cells. Ex-vivo expansionof multipotent hematopoietic stem cells would therefore be of great clinicalbenefit.

Our group has developed a cell culture system using the recombinantcytokines VEGF, FLT3L and SCGF allowing ex-vivo expansion ofpurified human CD133+ stem cells from mobilized peripheral blood formore than 4 weeks. During this period there was an 2200-fold increase oftotal cell number. FACS analysis revealed that expanded cells contained 6-15% CD133+ cells indicating proliferation of primitive hematopoietic stemcells. During the 35 day culture period the total number of CD133+ cellswas amplified more than 100-fold compared to input cells.

Colony-assays showed an increase of committed progenitor cells duringthe 4 week observation period (CFU-E 60-fold, CFU-GEMM 48-fold,CFU-GM 59-fold, CFU-G 99-fold and CFU-M 1356-fold).

To assess in-vivo functionality of ex-vivo expanded cells we performedtransplantation assays of hematopoietic stem cells expanded for 7, 14, 21and 32 days into sublethally irradiated NOD-SCID mice. After 3 monthsbone marrow of sacrificed mice was analysed by FACS using humanspecific monoclonal antibodies. For each expansion period the meanpercentage of human CD45+ cells was 11, 3, 3 and 1% respectively.Human CD45+ cells contained 19, 37, 44 and 25% CD34+ stem cells, 21,3, 1 and 12% CD13+ granulocytes, 15, 26, 8 and 32% CD3+ T-cells, 9, 0, 7and 21% CD19+ B-cells and 24, 2, 2 and 11% monocytes (analysed miced7 n=5, d14 n=4, d21 n=7, d32 n=6).

In summary our experiments showed multilineage engraftment of ex-vivo expanded human hematopoietic stem cells. Consequently, our cellculture system can be utilized to generate large numbers of transplantablehematopoietic stem cells.

331GENERATION OF HEMATOPOIETIC CELLS FROM HUMAN EMBRYONIC STEM CELLSM. Pick*, E. S. Ng, M. Costa, R. Davis, R. Mayberry, K. Koutsis, L. Azzola, E. Stanley, A. ElefantyMonash University, Clayton, Australia

Hematopoiesis, the dynamic process of blood cell production, isinitiated when pluripotent stem cells are induced to proliferate by growthfactors and cellular interactions. The generation of hematopoietic cellsfrom human embryonic stem cells (HESCs) represents an invaluablesystem for studying the genesis and control of hematopoiesis. Here wedescribe a defined serum and stromal-free culture system for thereproducible generation of hematopoietic cells from HESCs. We haveanalysed these haematopoietic cells for their expression of cell surfacemarkers and transcription factors specific for haematopoiesis. At day 10 ofdifferentiation 27.2±11.4% of cells were CD34+, at which time a highfrequency of colony forming cells was detected, even in the absence ofother hematopoietic markers such as CD45, CD33 or CD38. When cellswere replated in conditions favouring hematopoietic differentiation foranother 6 to 10 days, expression of CD45 reached a peak of 17.9±6.9%with the majority of these cells expressing myeloid (CD33 and CD15),megakaryocyte (CD41a and CD31) and erythroid (EpoR, CD71) markers.The expression of CD34 and CD117, markers of immature hematopoieticcells, was maintained in around 8% of cells from day 10 onwards. Thesefindings provide a framework for a more detailed analysis of the factorsgoverning the generation of hematopoietic cells from HESCs in vitro.



Abstracts

332QUANTITATIVE ANALYSIS OF CD34+ CELL PHENOTYPE ENRICHMENT ON THE EX-VIVO EXPANSION OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLSC. Lobato da Silva1,2*, R. Goncalves1,2, J. M. S. Cabral2, G. Almeida-Porada11Department of Animal Biotechnology, University of Nevada, 89557 Reno, Nevada, USA2Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, 1049-001, Lisboa, Portugal

In the present studies we correlated the CD34+ cell enrichment with theproliferative capacity of human umbilical cord blood (CB) samplescultured in serum-free media with SCF, Flt-3 ligand, LIF and bFGF in amesenchymal stem cell (MSC) - based feeder system. Freshly isolated CBcells were highly enriched for CD34+ phenotype (85-95%) using magneticsorting (High Purity – HP - cells), labelled with the fluorescent dyePKH26, cultured in T-25 flasks and analysed for 14 days. These expansionresults were compared to those obtained with CB samples having a quitelower initial percentage of hematopoietic progenitor CD34+ cells (5-12%)(Low Purity – LP - cells) also stained with PKH26. Maximal fold increasevalues in total cells ranged 50-105 and 7-8 for HP and LP experiments,respectively. Regarding CD34+ phenotype, HP cells presented maxima of48-90 expansion fold values, contrasting to 6-21 fold for LP cells. Inabsolute numbers, maxima of 2-4*107 CD34+ cells for HP versus 1-7*106

for LP cells were achieved. Although the expansion results are trulydependent of the initial CD34 purity, no significant differences were foundin division kinetics: in general, no loss of expression of CD34 antigen wasdetected before cell division; in both cases, intense cell division began byday 2-3 of the culture; and expression of CD34 was basically maintainedafter the 2-week culture period.

Moreover, extended culture of CB cultures would result in furthermaturation of CB cells, with the presence of more mature phenotypes suchas CD14, CD15 and CD33.

Kinetically, expansion rates (ke) obtained using a two-parameter modelfor HP cells were higher than those obtained for LP cultures (0.10-0.15day-1 versus 0.30-0.40 day-1). In addition, a roughly linear correlation(R2>0.90) was found between ke and initial CD34+ cell percentage.

333EARLY STEPS OF HOMING OF HUMAN CD34-POSITIVE CELLS IN IMMUNODEFICIENT MOUSEA. Zobiri, L. Campos, M. Péoc'h, D. Guyotat*UPRES EA3062, Faculté de Médecine, Saint-Etienne, France

The mechanisms of homing of hematopoietic progenitor cells (HPC)into bone marrow after reinfusion in blood are not fully elucidated.Cytokines, such as Stroma-Derived-Factor-1, and adherence to endothelialand microenvironnement cells are implicated in this phenomenon.However the initial steps of circulation and homing of HSP have not beencarefully studied. We report here on the blood and tissue distribution ofleukemic cell lines (KG1a and U937) and normal CD34+ cells during thefirst 24 hours after infusion into SCID mice. Prior to infusion cells wereincubated with the fluorescent dye PKH26. Blood, bone marrow, spleen,liver and lung were harvested at variable timepoints after infusion, andevaluated for content in fluorescent cells by flow cytometry and byfluorescence microscopy.

The percentage of leukemic cells decreased rapidly (15 minutes) in theblood after infusion, but a small proportion of cells (0.5-08%) were stilldetectable at 24 hours. Cells were rapidly detected in bone marrow, with amaximum reached after one hour. On the contrary, homing to the spleenwas slower, with a maximum at 24 hours. Leukemic cells were alsodetectable in the lung with a maximum at one hour, and a persistance at 24hours.

The kinetics of CD34+ cells showed significant differences: cells werealso detectable in the lung and liver one hour after infusion but decreasedrapidly. The homing to spleen and marrow was slower with a maximum at24 hours for both tissus.

This model of early tracking of infused cells shows that homing is aslow process, with recirculation between different tissues. This modelshould allow to study the factors regulating the initial steps of homing.

334ARE BONE MARROW-DERIVED ENDOTHELIAL PRECURSOR CELLS A FUTURE THERAPEUTIC TARGET FOR BURNS PATIENTS?A. Fox1*, J. Smythe1, N. Fisher1, M. P. H. Tyler2, D. A. McGrouther3, A. L. Harris4, S. M. Watt11Stem Cell Immunotherapy Laboratory, National Blood Service, John Radcliffe Hospital, Oxford, UK2Department of Plastic and Reconstructive Surgery, Stoke Mandeville Hospital, Aylesbury, UK3Department of Plastic and Reconstructive Surgery, University of Manchester, UK4Cancer Research Uk, Weatherall Institute of Molecular Medicine, University of Oxford, UK

Bone marrow (BM)-derived endothelial precursor cells (EPCs) havebeen detected in the peripheral blood of patients following thermal injury.They have been shown to mobilise from the bone marrow followingtrauma, and to contribute to wound healing. The purpose of this study is tocharacterise the kinetics of such cells in burns patients and compare withhealthy control subjects.

Male and female adult patients with superficial and full thickness burnshave been recruited to the study, with blood donor volunteers included ascontrols. EPCs identified from surface expression patterns of CD133+,CD144+, and VEGFR-2+ were quantified by four-colour flow cytometry.Burns patients (N=9) showed a rapid increase in circulating EPCs,maximal within the first 24 hours (mean 463±277 EPC/ml), returning tobasal levels by 72 hours. This represented a seven-fold increase (P=0.001)compared to control subjects (N=32, mean 65±70 EPC/ml). In superficialburns (N=6) the rise in EPC levels was related to the burnt surface area.Patients with full thickness burns (N=3) had an overall higher mean(611±275 EPC/ml) compared to those with superficial burns at 24 hours(389±270 EPC/ml). Parallel to the mobilisation of EPCs, there was alsoelevation in plasma VEGF levels as determined by ELISA. MaximalVEGF levels detected within 24 hours (N=8, mean 113±56 pg/ml),represented a four-fold increase compared to controls (N=12, mean 26±4pg/ml), decreasing to basal levels by 72 hours.

Results suggest that there is an acute but transient mobilisation of EPCsfrom the bone marrow following burns. Surface area and depth of injuryappear to affect their mobilisation. Thermal injury may induce the releaseof chemokines such as VEGF which promote the mobilisation of EPCs.Demonstrating a role for BM-derived EPCs in burns patients may providea therapeutic target.

335EFFECT OF WILD TYPE STROMAL CELLS ON HEMATOPOIESIS IN LONG-TERM BONE MARROW CULTURES REPOPULATED WITH BONE MARROW OF TNF KNOCKOUT MICEI. N. Nifontova*, N. J. DrizeNational Hematology Research Centre, Moscow, Russian Federation

Hematopoiesis in long-term bone marrow cultures (LTBMC) derivedfrom TNF knockout (TNFko) mice is maintained for 200 weeks. On wildtype (WT) and TNFko adherent cell layers (ACL) repopulated with TNFkobone marrow cells (2-step cultures) hematopoiesis is also abnormallyprolonged, while repopulation of the same ACLs with WT bone marrowcells shortened hematopoiesis to standard 1-step longevity. This suggeststhat such long-lasting hematopoiesis occurs due to the absence of autocrineexpression of TNF by hematopoietic cells. The hematopoietic and stromalprecursors were studied in 2-step LTBMC. Concentration of CAFC28-35,CAFC7, CFU-S and CFU-GM was much higher in cultures established onthe WT ACL than on the TNFko ones (Table 'Concentration ofhematopoietic precursors after 110 weeks in culture per 105 cells'). Thescores stayed high for more than 110 weeks, whereas PCR analysis showedthat all adherent cells in the mixed types of LTBMC are of TNFko originsince at least 80 weeks in culture. It is supposed that initial expression ofTNF is needed for better survival of hematopoietic progenitors with highproliferative potential. In the suspension fraction of both types of culturesnon-hematopoietic cells with stromal characteristics were revealed after 70weeks of cultivation. In the 1- step TNFko LTBMC the proportion ofCD45+ cells decreased by that time to less then 5%, in suspension fractionof cultures established on WT ACL 63,4% of the cells were CD45+ andmore then 90% - Sca-1+. These stromal cells had high proliferative



potential and were able to generate adherent cell lines. Therefore initialTNF expression by WT stromal cells is principal for better survival of bothTNFko hematopoietic and stromal progenitor cells.

336CIRCULATING ENDOTHELIAL CELLS AS SURROGATE MARKERS FOR ANTI-ANGIOGENIC DRUGSJ. Smythe1*, N. Fisher2, A. Fox1,3, S. Lowndes4, A. L. Harris4, S. M. Watt1,21Stem Cell Immunotherapy Laboratory, National Blood Service, Oxford, UK2Nuffield Department of Clinical Laboratory Science, University of Oxford, UK3Department of Plastic and Reconstructive Surgery, Stoke Mandeville Hospital, Aylesbury, UK4CRUK Clinical Cancer Centre, Oxford, UK

Tumour development is dependent on the formation of new bloodvessels. These may form through angiogenesis, the sprouting of newvessels from adjacent vessels or by vasculogenesis, the mobilisation ofendothelial progenitor cells (EPC) from the bone marrow. Previous reportshave suggested the numbers of circulating endothelial cells (CEC) andEPC may be increased in cancer patients. Drugs are being developed thatblock the formation of new blood vessels and so changes in the numbers ofCEC and EPC during treatment may be an indirect measure of thetreatment. Here we describe an improved assay for the detection of CECand EPC and compare cell numbers in controls with numbers in patientswith solid tumours.

Three phenotypes of endothelial cells have been identified using four-colour flow cytometry as follows: EPC, CD45dim/-ve, CD133+ve,CD144+ve, VEGF-R2+ve; CEC, CD45dim/-ve, CD34+ve, CD144+ve,VEGF-R2+ve; activated CEC, CD45dim/-ve, CD34+ve, CD144+ve,CD62E+ve.

Isotype control antibodies were used to exclude non-specific binding.The assay was tested using blood samples spiked with CD34+ve cells,isolated from cord blood that is known to contain CEC.

Normal controls contained 0-226 EPC per ml (mean 88), 0-280 CEC perml (mean 130) and 0-210 activated CEC per ml (mean 80).

Samples from five cancer patients with solid tumours contained asimilar number of all cell types whereas samples from a further 2 cancerpatients contained a raised number of CEC (575 and 413 cells per ml) andactivated CEC (419 and 543 cells per ml) but levels of EPC within thenormal range.

These results suggest this assay may be used to track the levels of threephenotypes of CEC in cancer patients during treatment and that the raisednumbers of CEC found in some cancer patients may originate fromexisting blood vessels rather than bone marrow.

337CCL18/PARC IS A NOVEL FACTOR THAT REGULATES HEMATOPOIESIS-SUPPORTIVE FUNCTION OF BONE MARROW MICROENVIRONMENTAL NICHES. K. Khaldoyanidi*, I. U. SchraufstatterLa Jolla Institute for Molecular Medicine, San Diego, CA, USA

Chemokines and their receptors are involved in regulation ofhematopoietic stem cell (HSC) homing and retention within themicroenvironmental niche. To investigate whether pulmonary andactivation-regulated chemokine (PARC), a member of the CC chemokinefamily, is involved in regulation of bone marrow hematopoiesis, purifiedhuman recombinant PARC was added in human long-term bone marrowcultures (LTBMC) at every feeding. The number of both non-adherentcells and hematopoietic progenitors produced in PARC-treated LTBMCwas significantly higher in comparison to LTBMC cultured with heat-inactivated PARC or non-treated cultures. Culture of human bone marrowcells in the presence of PARC facilitated formation of the adherent layer inLTBMC. Proliferation of stromal fibroblast-like cells, and the adhesion

and survival of macrophage-like cells in cultures treated with PARC wassignificantly increased as compared to the control cultures. PARCstimulated monocyte/macrophages, but not freshly isolated monocytes asshown in actin polymerization and Ca2+ mobilization assays. Furthermore,conditioned media from PARC-treated macrophages contained colony-promoting activity that increased both the number and the size of colonies,while PARC itself failed to influence colony formation in methylcellulosecultures. Interleukin-15 was identified as one of the cytokines up-regulatedin PARC-treated macrophages. Overall, PARC is involved in regulation ofhematopoiesis-supportive function of the bone marrowmicroenvironmental niche.

338CULTURE OF CORD BLOOD-DERIVED MONONUCLEAR CELLS AND CD34 ENRICHED CELLS IN TWO SERUM-FREE MEDIAG. Cho1*, D. J. Anstee1,2, A. Blair1,21Bristol Institute for Transfusion Sciences, Bristol, UK2University of Bristol, Bristol, UK

Ex vivo expansion may overcome some of the current limitations ofcord blood transplantation such as low cell number, with reduced utility inadult patients, and prolonged periods of neutropenia andthrombocytopenia. Some groups culture mononuclear cells (MNC) toavoid stem cell loss in a CD34-enrichment step. For clinical use, cellsshould be cultured in serum-free conditions. Here, we explore the cultureof cord blood-derived MNC and CD34-selected cells in two serum-freemedia- StemSpan H3000 (StemCell Technologies) and StemPro-34 SFM(Gibco). MNC from 4 units were isolated using Ficoll density gradientseparation. They were placed in suspension cultures for 6-7 days at 5x105

cells per ml in StemSpan or StemPro-34 with cytokines (in ng/ml: SCF 50,GCSF 20, GMCSF 20, IL-3 20, IL-6 20, TPO 50, FLT3 ligand 50, IL-1150). The total nucleated cell (TNC) and CD34+ cell counts were performedusing flow cytometry. The mean (standard deviation, SD) fold expansionsof TNC in StemSpan vs StemPro-34 were 0.07 (0.03) and 0.08 (0.05),respectively. The mean (SD) fold expansions of CD34+ cells were 0.51(0.46) and 0.54 (0.81). Portions of two units underwent CD34-selectionusing magnetic cell enrichment. Mean CD34 purity was 52.2%. These cellswere cultured at CD34+ cell concentrations of 5.8x103 per ml and 9.4x103

per ml. For StemSpan and StemPro media, the mean fold expansions at 7days for TNC were 44.7 (from 1.45x104 to 6.32x105) and 54.1 (from1.45x104 to 7.64x105), respectively. For CD34plus cells, the foldexpansions were 3.8 (from 7.60x103 to 2.71x104) and 3.0 (from 7.60x103

to 2.24x104), respectively. Culture of MNC in the conditions describeddoes not result in expansion. In contrast, CD34-selection prior to cultureand use of lower cell concentration lead to significant expansion of TNCand CD34+ cells. There were no obvious differences between the twomedia examined.

339CO-EXPRESSION OF C-KIT (CD117) AND OSTEOCALCIN IN ACTIVATED STEM CELLS OF HEMATOLOGICAL MALIGNANCYH. Karlic1*, P. Wihlidal1,2, F. Varga2, T. Nösslinger1,3, K. Klaushofer2, E. Pittermann1,3, M. Pfeilstöcker1,31Ludwig Boltzmann Institute for Leukemia Research, Hanusch Hospital, Vienna, Austria2Ludwig Boltzmann Institute for Osteology, Hanusch Hospital, Hanusch Hospital, Vienna, Austria33rd Medical Department, Hanusch Hospital, Vienna, Austria

Bone marrow of hematological diseases such as lymphoma was shownto harbour a stem cell candidate common to MSC (mesenchymal stemcells) and HSC (hematopoietic stem cells) which expressed OCN(osteocalcin) in addition to c-kit. In this study, we analysed cells from bonemarrow samples obtained from 4 patients with AMLs, one patient withCML at blast crisis, one CML patient in chronic phase and 7 patients withmyeloproliferative disease (MPD), as well as stem cells from peripheralblood and bone marrow obtained from healthy donors and cells from theHL60 cell line. In attempt to find out whether the presence of such stemcells is associated with disease status, sequential bone marrow samplesfrom a patient with acute myeloid leukaemia (AML/M2Eo) were analysedat 6 time points including diagnosis, remission (months 4, 6 and 17 afterdiagnosis) and relapse (months 13 and 40 after diagnosis). RT-PCRanalyses were performed in order to characterise spliced and unsplicedmRNA variants of OCN and to quantify mRNA levels of c-kit and the

Progenitors WT ACL TNFko ACLCAFC28-35 >150 <0.1CAFC7 >150 <0.1CFU-S 493±47.5 4±2CFU-GM 8825±6747 31.8±6.3



Abstracts

transcription factors AML1 and AML3, which are known to regulate geneexpression of c-kit and OCN. Results of immunostaining for c-kit andOCN showed that positive signals for OCN were only detected in thosebone marrow smears, which expressed normally spliced OCN-mRNA, i.e.the bone marrow samples from the AML/M2Eo-patient in the relapsephase, the 2 samples of AML/M2, the sample of CML at blast crisis andthe HL60 cells. The same samples and all others, which were positive forthe unspliced OCN-mRNA, were also positive for c-kit (both at the proteinand at the mRNA level) as well as for AML1- and AML3-mRNA,however, at lower quantities. This indicates an association of OCNexpression with hematological malignancies at disease stages whereactivated bone marrow stem cells are present.

340EX VIVO CORD BLOOD HEMATOPOIETIC STEM CELLS EXPANSIONK. Alimoghaddam1*, M. Khalili2, M. Soleimani3, L. Moezi1, P. Ghodsi1, M. Nohyedin1, A. Ghavamzadeh11Hematology, Oncology and BMT Research Center of Tehran University of Medical Sciences, Tehran, Iran2Khatam University, Tehran, Iran3Tarbiat Modaress University, Tehran, Iran

Purpose: to find the best cell culture condition for expansion of cordbloods hematopoietic CD34+/CD38- ex vivo expansion and study the roleof MIP/a in expansion potential.

Material and methods: CD34+/CD38- cells separated from cord bloodsby Mini-MACS and cultured in different culture mediums including 50ng/ml of TPO,IL-6,SCF and flt3-ligand.for some samples we used RPMI+10% FCS or autologous cord blood plasma and for others we used serumfree media(SF). Also we added MIP/a (10-50ng/ml) to some samples. Wecultured the cells for 2 weeks in CO2 incubator and studied expansionpotential by counting of MNCs, CFU-assay, LTC-IC and studied thenumber of CD34+/CD38- cells before expansion 7 days and 14 days afterexpansion.

Results: expansion potential of cord blood hematopoietic cells was goodand maximally expanded to 25 times. Also we found that serum free mediaisbetter than FCS 10% and autologous cord blood plasma. MIP/a did notchanged expansion potential of hematopoietic cells.

Conclusion: serum free media is the best medium for expansion andfrom GMP points of view and MIP/a is useful for expansion to preventmaturation during expansion that may be useful for increasing transductionefficiency without induction of maturation.

341THE SOURCE OF STEM CELLS IN THE PRE-IMPLANTATION EMBRYOD. Brynmor-ThomasUniversity of St Andrews, St Andrews, Scotland, UK

In contrast to the progeny of stem cells, which have understandably beenextensively studied, their precursors have been virtually disregarded. Theyare however clearly worthy of consideration. The cleavage divisions whichcommence with the first division of the zygote establish a small populationof transient primordial cells together with an enormous accumulation ofpositional information. Within a week of fertilization these eventsculminate in the generation of stem cells. Unlike the zygote and itsimmediate progeny, which are transient founder cells, stem cells canpersist by sustaining self-renewal throughout the life of the organism,while exporting an appropriate output of precursors to one or morelineages. The zygote and its immediate progeny are thus stem cellprecursors but as they are transient they are not stem cells. They are thefounder cells of the organism which include pre-stem cells. The first eventin the ontogeny of a stem cell population is thus the transformation of apre-stem cell into a stem cell and clearly the molecular basis of this event isno less important than the molecular changes associated with thetransformation of a stem cell into a progenitor cell. As the transformationof a pre-stem cell into a stem cell can occur in vitro as well as in vivo, it isaccessible to further study. But it is important to recognize that embryonicstem cells induced artificially in vitro (ESC-IA) are not identical withembryonic stem cells generated spontaneously in vivo (ESC-GS). As Evans& Hunter have emphasized ESC-IA 'are off the normal pathway but canreadily revert onto it' (C.R.Biologies, 325, 1003, 2002).

342ALLOGENEIC STEM CELL TRANSPLANTATION IN SEVERE APLASTIC ANEMIA AFTER CONDITIONING WITH FLUDARABINE, ATG, AND REDUCED DOSE OF CYCLOPHOSPHAMIDEJ. W. Lee*, K. S. Eom, Y. J. Kim, H. J. Kim, C. K. Min, S. Lee, D. W. Kim, W. S. Min, C. C. KimThe Catholic University of Korea, Seoul, Korea

Background: High dose (HD) cyclophosphamide (CY, 200mg/kg) plusATG seems to be accepted as standard conditioning regimen in HLA-matched sibling stem cell transplantation (SCT) for severe aplastic anemia(SAA). However, HD CY causes serious cardiac toxicity in some caseswhich may lead to death within few weeks. To avoid HD CY-associatedcardiac toxicity we underwent HLA-matched sibling SCT using ATG,reduced CY to half dose with incorporation of Fludarabine. Methods:Between March 2002 and December 2004, consecutive twenty patientswith SAA (three patients were AA/PNH syndrome) received matchedsibling SCT. The median age of patients was 41 (21-52) and medianinterval between Dx and SCT was 30 months (1-352). The conditioningregimen consisted of Fludarabine (30mg/m2/day, 6 days),cyclophosphamide (50 mg/kg/day, 2 days) and ATG (2.5mg/kg/day, 4days, IMTIX-SangStat). Stem cell sources were bone marrow(BM) plusCD34+ selected PBSC (n=8), BM (n=9), or PBSC (n=3). All patientsreceived CsA and MTx as GVHD prophylaxis. Results: The median doseof CD34+ cells infused was 3.6x106/kg (1.2-11.9). The median time forANC and platelet to reach 0.5x109/L and 20x109/L was 12 (6-16) and 18(10-23) days, respectively. None of patients developed cardiac toxicity orregimen-related toxicities. The incidence of acute GVHD (more than gradeII) was 10% (n=2) and none developed chronic GVHD. The incidence ofCMV infection requiring preemptive treatment was 50 % (n=10) whichwas higher than those of our historical control (14%). Only one patient diedof hepatic failure due to reactivation of chronic hepatitis C with hepaticGVHD posttransplant 3 months. With median follow up of 8.4 months (2-37), the estimated probability of survival at 2 years was 95 %. Conclusions:These data suggest that the conditioning regimen used in this study isfeasible for patients with SAA who received matched sibling SCT. Basedon the observation of high incidence of CMV infection, we are planning toreduce the dose of ATG or Fludarabine.

343COMORBID CONDITIONS AND TRANSPLANT SUCCESS IN PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)D. M. B. Kerbauy*, F. Chyou, T. Gooley, M. L. Sorror, B. Scott, J. M. Pagel, D. Myerson, F. R. Appelbaum, R. Storb, H. J. DeegFred Hutchinson Cancer Research Center, Seattle WA, USA

We analyzed prognostic factors for the outcome of allogeneichematopoietic cell transplantation (HCT) in 43 patients with CMML.Patients were 1-66 (median 48) years of age. They were classifiedaccording to FAB and WHO classifications, the International PrognosticScoring System (IPSS), and the M.D. Anderson prognostic score(MDAPS). Co-morbidity scores were assessed using an HCT-specificcomorbidity index. Several busulfan or total body irradiation (TBI)-basedconditioning regimens were used. Twenty-one patients received transplantsfrom related donors (18 HLA-identical siblings, 3 HLA-non-identicalfamily members), and 22 from unrelated donors (17 HLA matched, and 5HLA-non-identical). Sustained engraftment was achieved in 41 patients.Eighteen patients are alive at 1.9 to 14.1 years, for an estimated relapse-free survival of 41% at 4 years. Ten patients have relapsed, for acumulative incidence of 23% at 4 years, and all have died. Fifteen patients(34%) died from non-relapse causes. Risk categories by IPSS, WHO,MDAPS, proliferative/dysplastic status, or disease duration had nostatistically significant associations with outcome. However, there was asuggestion that the hazard of relapse was higher with a higher riskclassification by MDAPS (HR 3.79, 95%CI, 0.80-17.92; p=0.09). Therewere no statistically significant associations between, conditioningregimen, GVHD and outcome. Patients with comorbidity scores >/=3 hadan increased hazard of mortality compared to those with lower scores (HR2.81, 95%CI, 1.25-6.34; p=0.01). The corresponding probabilities ofsurviving in remission were 15% and 54% for patients with comorbidityscores of 0-2 and >/=3, respectively. In summary, HCT is currently theonly treatment option with curative potential for CMML. While there is



considerable morbidity and mortality, the use of additional clinical orlaboratory data, such as the MDAPS and the HCT-comorbidity index mayhelp in determining the optimum timing of HCT and in selecting the mostappropriate conditioning regimen for a given patient.

344A PILOT STUDY EVALUATING THE SAFETY AND EFFICACY OF AMD3100 FOR THE MOBILIZATION AND TRANSPLANTATION OF HLA-MATCHED SIBLING DONOR HEMATOPOIETIC STEM CELLS IN PATIENTS WITH ADVANCED HEMATOLOGICAL MALIGNANCIESS. M. Devine1*, L. Andritsos1, T. Edwards1, G. Calandra2, G. Bridger2, J. F. DiPersio11Washington University, St Louis, USA2Anormed Inc, Langley, BC, Canada

Cytokine mobilized peripheral blood (MPB) has become the preferredallograft source for patients with advanced hematological malignancies.Recent studies suggest G-CSF induces mobilization by indirectly targetingthe interaction between the chemokine stromal derived factor 1 (SDF-1)and its receptor CXCR-4. Here we report preliminary results using a directantagonist of the SDF-1/CXCR-4 interaction, AMD3100, as a single agentto procure MPB from allogeneic donors. Six HLA-identical siblingsreceived one or two doses of AMD3100 at 240mcg/kg followed 4 hourslater by leukapheresis (LP). After successful collection and a one weekwashout period, the same donors were re-mobilized using G-CSF at10mcg/kg/day followed by LP commencing day 5. The results of themobilizations and allograft composition are presented in the table.Following transplantation, all 4 recipients engrafted neutrophils >500/ul ata median of 10 days (8-11) and platelets >20,000/ul at a median of 18 days(15-24). All are currently being followed as outpatients with trilineageengraftment, no transfusion requirements, and full donor chimerism.Follow-up ranges from 58-277 days. Two patients had no acute GVHD andtwo experienced overall grade I acute GVHD which responded to topicalsteroids. Two recipients did not receive allografts due to progressivedisease. In summary, grafts mobilized following AMD3100 differ from G-CSF mobilized grafts in the content of CD34+ and immune effector cellsyet appear to reconstitute hematopoiesis similarly. This is the first study todemonstrate that a chemokine antagonist alone can safely and rapidlyinduce the mobilization of a functionally competent hematopoieticallograft. Accrual is ongoing.

345IMATINIB FOLLOWED BY REDUCED INTENSITY STEM CELL TRANSPLANTATION AND DONOR LYMPHOCYTE INFUSIONS IS EFFECTIVE IN CHRONIC PHASE CHRONIC MYELOID LEUKAEMIAM. Copland1*, N. Heaney2, K. Stewart1,2, J. Godden1, G. Smith3, C. Crawley4, T. L. Holyoake1,21Section of Experimental Haematology, ATMU and Cancer Division, University of Glasgow, Glasgow, UK2Department of Haematology, North Glasgow University Hospitals NHS Trust, Glasgow, UK3Leeds General Hospital, Leeds, UK4Addenbrookes Hospital, Cambridge, UK

Although Imatinib (IM) induces a complete cytogenetic response (CCR)in the majority of patients with chronic phase chronic myeloid leukaemia(CML), it is unlikely to be curative due to molecular persistence,presumably at the stem cell level. For patients aged <65 years with amatched donor, we developed a strategy of remission induction with IMfollowed by either reduced intensity stem cell transplant (RISCT;fludarabine/melphalan/Campath-1H) for those achieving CCR after 6-12

months or aged >50 years, or allogeneic SCT (alloSCT;cyclophosphamide/TBI) for those who either progressed on IM or did notachieve CCR after 12 months. Patients received IM 400mg/daily,increasing to 600mg/daily if they did not achieve MCR after 3 months orCCR after 6-9 months. GVHD prophylaxis was either cyclosporine alone(RISCT) or cyclosporine/methotrexate (alloSCT). Quantitative RT-PCRfor Bcr-Abl and donor chimerism were performed at 6 week intervalsfollowing engraftment. Donor lymphocyte infusion (DLI) was given at >6months post RISCT to patients with <100% donor chimerism or remainingBcr-Abl+ at >0.02% on 2 repeat analyses 2 weeks apart. To date, 17patients (aged 20-55 years) with Ph+ CML have been recruited and 13have received a transplant (10 sibling RISCT, 2 sibling alloSCT, and 1MUD alloSCT), all with satisfactory engraftment. Pre-transplant, 7 patientsachieved CCR, 2 MCR, 2 had no response to IM and 2 progressed to blastcrisis after 3 months IM therapy. 1/9 RISCT patients developed chronicGVHD and died of sepsis 11 months post transplant (no DLI). 6 patientsare >6 months post RISCT and all have been receiving incremental(>5x105) DLI. As yet, only one patient is consistently PCR negative(median Bcr-Abl level 0.001%; range 0-0.125%). The combination of IMwith RISCT is much less toxic, with fewer septic episodes andreadmissions than alloSCT, but follow-up is required to establish long-termefficacy.

346SHORT-TERM METHOTREXATE PLUS TACROLIMUS FOR PROPHYLAXIS OF GRAFT-VERSUS-HOST DISEASE IN UNRELATED BONE MARROW TRANSPLANTATION FOR NONMALIGNANT DISEASEH. Inoue1*, M. Yabe1, M. Matsumoto1, S. Hamanoue1, T. Koike1, A. Hiroi1, S. Kato2, H. Yabe11Specialized Clinical Science, Pediatrics, Tokai University School of Medicine, Isehara, Kanagawa, Japan2Department of Cell Transplantation and Regenerative Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Graft-versus-host disease (GVHD) is a major problem in allogeneicbone marrow transplantation (BMT) from unrelated donor. To address thisproblem, we examined the efficacy of tacrolimus (FK506) plus short-termmethotrexate (MTX) in 38 patients who underwent BMT from unrelateddonor between January 2000 and November 2004. All patients receivedFK506 beginning the day before transplantation at a dose of 0.02-0.03 mg/kg per day by continuous intravenous (IV) infusion. MTX wasadministered at a dose of 15 mg/m2 IV on day 1, and 10 mg/m2 on days 3,6, and 11. Oral administration of MMF was started at a dose of 15 mg/kgper day from day 14 to 42 in 2 patients with HLA serotype-mismatchedBMT. The preparative regimens used in this study were thoracoabdominalirradiation (TAI), fludarabine, cyclophosphamide (CY) and antithymocyteglobulin (ATG) for severe aplastic anemia and Fanconi anemia, TAI,melphalan and ATG for adrenoleukodystrophy, busulfan, CY and ATG formucopolysaccharidosis and immunodeficiencies. The median age ofpatients was 10 years (range: 2 to 28). Twenty-four donor/recipient pairswere matched for HLA-A, -B, and -DR by serology and molecular typing.Four pairs were minor mismatches at either class I or class II and 10 pairswere major mismatches at either class I or class II. Thirty-six of the 38patients (95%) engrafted. Fifteen of 36 evaluable patients (42%) developedgrades 1-3 acute GVHD; grade 1 in 11, grade 2 in 2, grade 3 in 2. ChronicGVHD developed in 11 of 35 (31%) evaluable patients; limited type in 7,extended type in 4. At a median follow-up of 964 days (range 125-1880days), 36 patients (95%) are alive. These preliminary data indicate thatFK506-based immunosuppression following unrelated donor BMT iseffective in preventing severe acute GVHD and may leads to decreasedincidence of chronic GVHD.

Parameter(mean values) AMD3100 G-CSFDays of Drug Administration 1.7 5.2Peak fold CD34 Increase 7.1 21.1

CD34 Dose (x106/kg) 2.8 3.5

CD3 Dose (x108/kg) 3.9 1.3

CD4 Dose (x108/kg) 2.2 0.8

CD8 Dose (x108/kg) 2.3 0.4

CD56 Dose (x107/kg) 2.7 1.4



Abstracts

347Abstract withdrawn

348NONMYELOABLATIVE VS. MYELOABLATIVE HEMOPOIETIC CELL TRANSPLANTATION (HCT) FOR SECONDARY MYELODYSPLASIA (MDS) AND ACUTE MYELOGENOUS LEUKEMIA WITH MULTILINEAGE DYSPLASIA (TAML)B. L. Scott1,2*, H. J. Deeg1,21Fred Hutchinson Cancer Research Center, Seattle WA, USA2University of Washington Medical Center, Seattle WA, USA

Approximately 20% of patients who develop MDS or tAML have beenexposed to cytotoxic agents or have a pre-existing hematologic disorder.Past experience with HCT has shown that these patients are at a high riskof post-transplant relapse. Furthermore, because of therapy received priorto transplantation, they may also have an increased risk of transplant-related mortality. We performed a retrospective analysis in 40 patients overthe age of 40 years with a diagnosis of secondary MDS or AML. Allpatients had high risk disease (by IPSS classification) and all hadsignificant comorbidities as assessed by a modified Charlson comorbidityindex. Twenty-seven patients were conditioned with a myeloablativeregimen of busulfan, 16mg/kg (targeted to plasma levels of 800-900 ng/ml), and cyclophosphamide 120 mg/kg. Thirteen patients received anonmyeloablative regimen of fludarabine, 90mg/m2, plus total bodyirradiation, 2 Gy. All patients received 10/10 HLA-allele matched relatedor unrelated donor stem cells. Nonmyeloablative patients were, on average,older than myeloablative patients (median age 57 vs. 47 years). There wereno significant differences between the two groups regarding donor type,stem cells used (marrow vs. peripheral blood), gender, or CMV serostatusof donor or recipient. The mean follow up was 929 days and 1192 days inthe myeloablative and nonmyeloablative cohorts, respectively. The overallsurvival was 21% in the nonmyeloablative cohort and 26% in themyeloablative cohort. Relapse was a more common cause of death in thenonmyeloablative cohort (70% vs. 40%), whereas, transplant-relatedmortality was more common in the myeloablative cohort (60% vs. 30%).Thus, while potentially curative, current transplant approaches are not

ideal. Future preparative regimens for patients with secondary MDS orAML will need to reduce the risks of both relapse- and transplant-relatedmortality.

349RADIOIMMUNOTHERAPY WITH BISMUTH-213 AS CONDITIONING FOR NONMYELOABLATIVE MARROW TRANSPLANTATION IN DLA-HAPLOIDENTICAL ALLOGRAFTSF. R. Kerbauy1*, W. Bethge1, D. S. Wilbur2, D. Hamlin2, E. Santos1, R. Storb1,3, B. M. Sandmaier1,31Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA2Department of Radiation Oncology, University of Washington, Seattle, WA, USA3Department of Medicine, University of Washington, Seattle, WA, USA

We previously reported a conditioning regimen with alpha-emitterBismuth-213 (Bi-213) coupled to anti-CD45 monoclonal antibody (MAb)followed by mycophenolate mofetil (MMF) and cyclosporine (CSP) in acanine model of DLA-identical nonmyeloablative hematopoietic celltransplantation (HCT). Here, we study the efficacy of this approach inDLA-haploidentical littermates. Six dogs (Table 1) received 2.3-4.9 mCi/kg Bi-213-anti-CD45 MAb in 6 to 8 injections followed by granulocytecolony-stimulating factor stimulated peripheral blood cells. They receivedas postgrafting immunosuppression, CSP (15 mg/kg; day 1 to +180) andMMF (10 mg/kg; day 0 to +100). All dogs achieved engraftment with 35-58% donor chimerism of PBMC on day +7 after HCT. Dogs receivinghigher doses of Bi-213 achieved higher chimerism on day 30 after HCT.Engraftment was stable in dog# 2 with donor chimerism among PBMCranging from 10% to 50% with follow up of >35 weeks. Two dogs (# 1 and3) rejected their graft 17 and 18 weeks after HCT, respectively. Dogs # 4, 5and 6, with shorter follow-up of 5, weeks have developed 51-58% donorchimerism by day 30 after HCT. No signs of graft-versus-host disease orother toxicities were noted. We demonstrated that low doseradioimmunotherapy with Bi-213-anti-CD45 as conditioning fornonmyeloablative HCT in DLA-haploidentical dog model is safe andallowed prompt T cell donor chimerism and no GVHD. Although thenumber of dogs studied is limited the use of higher doses of Bi-213 mayhelp prevent graft rejection.

350ANTI-LEUKEMIC EFFECT OF GRAFT-VERSUS-HOST DISEASE ON BONE MARROW VERSUS EXTRAMEDULLARY RELAPSE IN ADULT PATIENTS WITH ACUTE LEUKEMIA WHO RECEIVED NON-T-CELL DEPLETED ALLOGRAFTSJ. H. Lee1*, S. J. Choi1, J. H. Lee1, M. Seol1, Y. S. Lee1, S. G. Ryu2, C. J. Park3, H. S. Chi3, S. Yun4, K. H. Lee11Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Ulsan, Korea2Department of Pharmacy, Asan Medical Center, University of Ulsan College of Medicine, Ulsan, Korea3Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Ulsan, Korea4Clinical Research Center, Asan Medical Center, University of Ulsan College of Medicine, Ulsan, Korea

Several reports have suggested that the graft-versus-leukemia (GVL)effect at the extramedullary (EM) sites might be less prominent than thoseat bone marrow (BM). We analyzed the effect of GVHD on BM versus EMrelapse. This study included 194 patients with acute leukemia who received

Dogs given DLA-haploidentical grafts after Bismuth-213

Dog#DoseBi-213/kg(mCi)

CD34+cellsE6/kg

Chimerism in PBMC(%) on days +7, +30 after HCT

Duration of donor chimerism(weeks)

1 2.3 2.4 38, 33 17 (Rejection)2 3.1 12 50, 51 >353 3.3 2.4 35, 38 18 (Rejection)4 3.3 3.0 51, 60 >55 3.9 4.6 58, 69 >46 4.9 2.6 53, 57 >5PBMC: Peripheral blood mononuclear cells; HCT: hematopoietic cell transplantation

abstracts.fm Page 129 Friday, May 13, 2005 3:45 PM


allogeneic HCT at a single institute. Sixty-five patients relapsed after HCT.Forty-one relapses (63%) occurred in BM only, 9 (14%) in both BM andEM sites, and 15 (23%) in EM sites only. Patients who developed acuteGVHD after HCT had significantly higher relapse-free survival (RFS;69.2% vs. 52.4%; P=0.042) and BM RFS (80.7% vs. 59.1%; P=0.030)compared to those who did not. However, EM RFS was similar betweenpatients with and without acute GVHD (76.7% vs. 78.2%; P=0.744).Among 65 patients who relapsed after HCT, 32 patients attained completeremission (CR) with salvage treatments and 22 experienced the 2ndrelapse, which occurred in BM (n=9), BM/EM (n=1), or EM (n=12).Occurrence of GVHD after salvage treatment for the first relapse was anindependent prognostic factor for induction of CR and remission durationin patients with relapsed leukemia after HCT. In conclusion, our studyconfirmed the anti-leukemic effect of GVHD on relapse after allogeneicHCT for acute leukemia, but GVHD seemed not to be effective inpreventing EM relapse. Leukemic cells at the EM sites may escape theGVL effects of allogeneic immune cells.

351CENTRAL AND PERIPHERAL TOLERANCE, TWO POSSIBLE MECHANISMS IN TOLERANCE INDUCTION FOLLOWING IN UTERO STEM CELLS TRANSPLANTATIONA. Torabi1*, J. S. Pixley2, J. Chase1, E. D. Zanjani11Department of Animal Biotechnology, University of Nevada, Reno, NV, USA2Department of Medicine, VA Medical Center, Reno, Nevada, NV, USA

Our laboratory has demonstrated that optimal age for in uterotransplantation in sheep, 'window of opportunity', would be between 55-65days of gestation (term=145 days). In this study we transplanted 1x106human CD34+ progenitor/stem cells into pre-immune fetal sheep at day 55.Five days later, at day 60, we obtained bone marrow (BM), spleen, thymus,and liver of the transplanted sheep and analyzed distribution of humanprogenitor/stem cells by flowcytometry. Five days post-transplant BM andspleen contained the most human cells population, with average of 20%CD34+45+ in the BM, 16% of CD34+45+ and 17% of CD34+45- in thespleen. Liver contained 2% CD34+45- and thymus 6% CD34+45+.

To relate these findings with fetal sheep immune system, we analyzeddistribution of CD4+25+ (phenotypically comparable to T regulatory cells)and CD1+ cells (phenotypically consistence with dendritic cells) from day52 to 65. At day 52, BM and spleen contain CD4+25+ cells (8% and 6%)and CD1+ cells (43% and 15%). At the same time thymus has less than 1%CD4+25+ and 66% CD1+ cells. At day 58, CD4+25+ and CD1+ cellsincrease in the thymus (to 11% and 70%), diminish in spleen (4% and 4%),and remain unchanged or increase in BM (42% and 40%). By day 65, thesetwo populations decrease in all three organs.

These results suggest that donor cells may interact with recipientCD4+25+ and/or CD1+ cells in the fetus BM and spleen. This interactionresult in tolerance induction due to T regulatory cells and peripheralmechanism. Presence of donor cells in the thymus and re-localization ofrecipient CD4+25+ and CD1+ cells into thymus from periphery at day 58,suggest that central deletion also play a role in tolerance inductionfollowing in utero transplantation.

352SELECTIVE DEPLETION OF ALLOREACTIVE DONOR T CELLS BY ANTI-CD25 IMMUNOTOXIN IN ALLOGENEIC TRANSPLANTATIONJ. Michalek1,2*, E. S. Vitetta21Masaryk University, Brno, Czech Republic2University of Texas Southwestern Medical Center, Dallas, TX, USA

Our preliminary data demonstrate that an anti-CD25 immunotoxin (IT),which reacts with a cell surface activation antigen, can selectively depletealloreactive donor T cells activated by non-leukemic recipient white bloodcells while preserving the beneficial third-party (anti-leukemia and anti-microbial) reactivity in vitro. In adult patients with hematologicalmalignancies, we were able to demonstrate that the alloreactive donor-derived T cell clones causing GVHD could be eliminated ex vivo beforehematopoietic stem cell transplantation (HSCT) with the anti-CD25 ITimmunotoxin and patients could receive an infusion of stem cells depletedof alloreactive T cells.

We evaluated 5 HLA-mismatched healthy volunteer pairs in a mixedlymphocyte reaction (MLR) using 108 responder cells. Responder cellswere activated by allogeneic stimulator cells in a cell ratio 1:1 and theirpeak activation (19.6-26.5%, mean 23.1% of CD3+CD25+ responder cells)occurred after 72 hours. If the IT and IT enhancer NH4Cl were added for24 hours after the first 24 hours of the MLR, the remaining populationrepresented 0.15-0.98% (mean 0.44%) of CD3+CD25+ responder cells.The percent recovery of viable responder cells was 62.3-89.7% (mean78.2%). The depletion of alloreactive cells increased following another 24hour exposure to the IT and NH4Cl resulting in 0.00-0.24% (mean 0.10%)of CD3+CD25+ responder cells with 64.7-86.5% (mean 75.4%) viability.Third party reactivity was preserved, reaching 10.3-19.6% (mean 15.5%)on Day 7 when activated on Day 3 with HLA-mismatched PBMCs fromanother healthy donor.

Thus, using the anti-CD25 IT for selective depletion of alloreactivedonor T cells while minimizing GVHD and preserving third partyreactivity should lead to a safer allogeneic HSCT.

353HYPERTHERMIA-TREATED PERIPHERAL BLOOD STEM CELL TRANSPLANTATION IN CHILDHOOD HEMATOLOGICAL MALIGNANCYT. Matsushita1*, N. Satou1, H. Uryu1, J. Yamanaka1, T. Takeno1, N. Yasuda1, H. Kanai1, Y. Mitomi1, T. Kuratsuji2, A. Yuo21Dept of Pediatrics, International Medical Center of Japan, Shinjuku, Tokyo, Japan2Research Institute, International Medical Center of Japan, Shinjuku, Tokyo, Japan

The ant-cancer effect of hyperthermia is well recognized and alreadyapplied in clinical setting especially for the treatment of solid tumors.Hyperthermia-treated peripheral blood stem cell transplantation wasperformed to aim for reducing the risk of recurrence of hematologicalmalignancies in children. [Method and Patients] Peripheral blood stemcells (PBSC) were collected with cell separator following high-dose AraCcontaining chemotherapy. Hyperthermia was done for an hour at 42C withalfa-interferon 100 units/ml of medium containing solution before storingin liquid nitrogen. Stored cells were thawed at 37C and infused to patientsat the time of transplantation. All patients were in complete remission (1stCR; 5 ALL, 3 AML, 2 NHL, 2nd CR; 3 ALL children) at the time of thisprocedure. Double transplantation was performed in five (3 ALL and 2AML) children. Single transplantation was done in eight (5 ALL, 1 AML,and 2 NHL) children. Pre-transplantation chemotherapy was McVAC or L-PAM containing regimens for the second transplantation. [Results]Hematological recovery was obtained following these eighteen courses ofconditioning except one course after high-dose chemotherapy, whichresulted death due to sepsis 3 months after this procedure. Of 13 children, 3children (2 ALL, and 1 NHL) were relapsed and died. Remaining thesenine children are living and free from disease (4–12 years aftertransplantation, median; 8 y). [Discussion] Hyperthermia is simple andeasy method for purging possibly contaminated leukemic cells. Ourprevious study revealed hyperthermia treatment reduced to contaminatedcells to 10 or 1 % of processed PBSC. Considering the high risk features ofthese patients, hyperthermia is one of the effective methods for treatinghematological malignancies, if auto-transplantation is thought as one of thetreatment choice.

354ERYTHROBLASTOSIS IS OBSERVED AT THE EARLY RECOVERY PHASE OF THE ENGRAFTMENT ON ALLOGENEIC UMBILICAL CORD BLOOD TRANSPLANTATIONM. Noguchi, H. Tashiro, M. Goto, K. Kawasugi, N. Shirafuji*Teikyo University, Tokyo, Japan

Objective: To make clear the cytological difference of the allogeneichematopoietic cell transplantation between the transplanted grafts, wecounted and evaluated the peripheral blood hemogram at the early phase ofthe engraftment on the transplantation.

Method: The cases were; BMT 10 cases, PBSCT 3 cases, and CBT 10cases, which showed no complications during at the transplantation (day 0)to the engraftment. Hemograms were counted microscopically at each daysduring this period with use of 3 to 5 smear- glass slides which were madeby hand, and stained with May-Giemsa solution.



Abstracts

Result: At CBT cases 2 cases showed autologous recovery. Theremaining 8 cases showed the continuous erythroblastosis (started at Day14 in average), which was followed by the recovery of the neutrophils(WBC count above 1000 was at Day 21.3 in average). This finding was notinfluenced by the difference of blood types between the recipient and thedonor. At BMT cases, and PBSCT cases, no continuous erythroblastosiswas observed. On 2 cases of autologous recovery after umbilical CBT, noerythroblastosis was observed.

Discussion: Erythroblastosis in blood seemed to be a unique finding atthe early engraftment of the umbilical cord blood transplantation. Thisfinding may reflect the different character of the stem cell between in theumbilical cord blood and in bone marrow/ peripheral blood stem cells.

355BLOODSTREAM INFECTION FOLLOWING UMBILICAL CORD BLOOD TRANSPLANTATION USING REDUCED INTENSITY STEM CELL TRANSPLANTATION FOR ADULT PATIENTSH. Narimatsu1*, T. Matsumura1, M. Kami2, S. Miyakoshi1, K. Masuoka1, T. Myojo1, Y. Kishi2, N. Murashige2, Y. Kanda3, S. Taniguchi11Department of Hematology, Toranomon Hospital, Tokyo, Japan2Hematopoietic Stem Cell Transplant Unit, The National Cancer Center Hospital, Tokyo, Japan3The Department of Cell Therapy and Transplantation Medicine, University of Tokyo Hospital, Tokyo, Japan

Bloodstream infection (BSI) represents a significant problem after cordblood transplantation (CBT). However, little information has been reportedon BSI after reduced-intensity CBT (RI-CBT). We retrospectivelyreviewed the medical records of 102 patients. Median age of patients was55 years (range, 17-79 years). Preparative regimens comprised fludarabine125-150 mg/m 2, melphalan 80-140 mg/m 2

or busulfan 8 mg/kg, and total body irradiation 2-8 Gy. Prophylaxisagainst graft-versus-host disease comprised cyclosporin or tacrolimus. BSIdeveloped within 100 days of RI-CBT in 32 patients. Cumulative incidenceof BSI was 25% at day 30 and 32% at day 100. Median onset was day 15(range, day 1-98). Causative organisms included Pseudomonas aeruginosa(n=12), Staphylococcus epidermidis (n=11), Staphylococcus aureus(n=6), Enterococcus faecium (n=4), Enterococcus faecalis (n=4),Stenotrophomonas maltophilia (n=4) and others (n=7). Of the 32 patientswith BSI, 25 (84%) died within 100 days after RI-CBT. BSI was the directcause of death in 8 patients (25%). Univariate analysis failed to identifyany significant risk factors. BSI clearly represents a significant and fatalcomplication after RI-CBT. Further studies are warranted to determineclinical characteristics, identify patients at high risk of BSI and establishtherapeutic strategies.

356NEONATAL HEMATOPOIETIC STEM CELL TRANSPLANTATION CURES OC/OC MICE FROM OSTEOPETROSISM. Johansson1*, M. Ehinger2, A. Fasth3, S. Karlsson1, J. Richter11Dept Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden2Dept of Pathology, Lund University, Lund, Sweden3Pediatric Immunology, The Queen Silvia Childrens Hospital, Gothenburg, Sweden

Infantile malignant osteopeterosis is a rare autosomal recessive disorderaffecting osteoclast function. Fifty % of the affected children have amutation in the Tcirg1 gene coding for one subunit of an osteoclast specificproton pump. The resulting dense, sclerotic bones cause among otherthings pancytopenia and progressive visual loss. The only curativetreatment so far is hematopoietic stem cell transplantation (SCT). The oc/oc mouse has a mutation in the gene homologous to Tcirg1 giving rise tosimilar symptoms as in patients and death occurs around 3 weeks of age.However, previous attempts to cure these mice with SCT have beenunsuccessful (M.F. Seifert and S.C. Marks, Jr, Tissue & Cell 1987). Wewanted to determine if early hematopoietic SCT could cure oc/oc micefrom osteopetrosis and pave way for gene therapy.

One-day-old irradiated (200, 400 or 600 cGy) oc/oc mice transplantedi.p with normal lineage depleted bone marrow cells (1 or 5x106) that reachengraftment levels around 10-15% survive past normal lifespan. X-rayexamination and histology of transplanted animals show normalisation ofbone structure. Although a correction of bone structure occurs, thetransplanted oc/oc mice remain smaller in size than their littermates. In

contrast to untreated animals, teeth develop but with abnormal structureand shape making them unusable. Mice transplanted on day 8 do notsurvive longer than untransplanted oc/oc mice.

For gene therapy experiments we chose d E14.5 fetal liver (FL) cells as acell source since BM cells are limited in oc/oc animals. Oc/oc mice surviveafter transplantation of normal ter119 depleted FL cells. Transduction ofoc/oc ter119 depleted FL cells was performed after 24 hrs of pre-stimulation with a retroviral vector containing the Tcirg1 and GFP genes,with a transduction efficiency of 50-60%. Results from ongoing genetherapy experiments will be reported.

357NOVEL PREPARATORY CONDITIONING CONSISTED OF TOTAL BODY IRRADIATION, ETOPOSIDE AND CYCLOPHOSPHAMIDE FOR CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIAH. Yabe*, A. Kikuchi, K. Koike, K. Tabuchi, H. Kawasaki, A. Ohara, M. Kobayashi, H. Kakuta, T. Mori, A. MakimotoTokyo Children's Cancer Study Group (TCCSG), Tokyo, Japan

Ninety four children with acute lymphoblastic leukemia (ALL) weretreated by allogeneic stem cell transplantation (SCT) with preconditioningregimen consisted of 12 Gy of total body irradiation (TBI), 60 mg/kg (bodyweight less than 30 kg) or 1800 mg/m2 (body weight more than 30 kg) ofetoposide (VP-16) and 120 mg/kg of cyclophosphamide (CY) betweenJuly 1995 and December 2002. GVHD prophylaxis was carried outaccording to the stem cell source; short-term methotrexate (MTX) andcyclosporine (CyA) or CyA alone were used in HLA-identical relatedBMT, both MTX and tacrolimus were given in UBMT. Medianobservation period was 690 days (range 208 to 2989 days). Disease statusat transplant was 1CR in 46 recipients, 2CR in 30, 3CR in 4 and relapse in14. Stem cell sources were HLA-identical sibling in 28, related donor otherthan HLA-identical sibling in 8, unrelated donor BM in 39 and unrelatedcord blood in 19. Event-free survival (EFS) according to the disease statuswas 64.4% in 1CR, 77.3% in 2CR, 75.0% in 3CR and 26.8% in relapse.EFS in 1CR according to stem cell source was 65.6% in HLA-identicalsibling BMT (n=19), 60.9% in UBMT (n=16), 77.8% in UCBT (n=9).EFS in 2CR according to stem cell source was 71.4% in HLA-identicalsibling BMT (n=7), 73.9% in UBMT (n=14), 100% in UCBT (n=5). Earlydeath within 100 days after SCT was observed in 2 (1 patient in 1CR diedof interstitial pneumonitis at +34 day and 1 patient in second relapse diedof SVC syndrome). Cause of deaths in 26 patients were relapse in 13,GVHD in 3, TMA in 2, others in 8. Addition of VP-16 to conventional TBIand CY did not increase transplant-related mortality, and improved resultwas obtained especially in recipients who received graft in 2CR.

358POST TRANSPLANT CHIMERISM ANALYSIS USING THE BECKMAN CEQ 8000J. W. Baird*, A. N. Parker, I. G. McQuaker, P. S. MacLean, F. M. ReidGlasgow Royal Infirmary, Glasgow, UK

Chimerism analysis is a vital part of patient monitoring after allogeneic(Allo) and matched unrelated donor (MUD) stem cell transplant.Differences between patient and donor DNA was, in the past, detectedusing Southern blotting. While this was an adequate method of chimerismanalysis it required a large amount of DNA, was relatively labourintensive, insensitive and slow. The current 'gold standard' method is PCRbased: inherent differences in sizes of microsatellite marker repeat regionsare exploited as a means of distinguishing pre-transplant and donor DNA.These size differences are detected via a capillary electrophoresis systemand quantitative assessment of the contribution each population hastowards the post transplant blood system can be made. Until recently theonly capillary electrophoresis machine routinely available wasmanufactured by Applied Biosystems; here we show the use and functionof the Beckman CEQ 8000 in chimerism analysis for post transplantmonitoring. Standard PCR protocols using primers labelled with Beckmandyes followed by fragment analysis on the CEQ 8000 has allowed us toidentify a number of patients with mixed chimerism. This has enabled theclinicians to make early intervention to bring the patient back to full donorchimerism. In conclusion in our laboratory the Beckman CEQ8000 hasbeen shown to be a sensitive and robust machine in the accuratequantitiation of chimerism with sensitivity around the level of 1% (1transplant cell in 100 donor cells).



We conclude that an alternative to using the ABI capillaryelectrophoresis machine which can achieve the same level of sensitivity ata fraction of the cost.

359CYCLOSPORINE (CSA) THERAPY POST TRANSPLANTATION: A MULTI-CENTER SURVEY IN 174 PATIENTSA. Nagler1*, M. Leiba1, A. Avramoff2, A. Laor2, K. Schott2, S. Melcer2, A. Shimoni11Chaim Sheba Medical Center, Tel Hashomer, Israel2Dexcel Ltd. Or Akiva, Israel

Deximune® (Dexcel Ltd.) and Sandimmun Neoral® (Novartis Inc.) aretwo CSA formulations widely used after stem cells (SCT) and solid organtransplantation (SOT). For assessing toxicity profile and bioavailability,we performed a multi-center survey in 174 pts. receiving CSAformulations after transplantation. Transplants were performed between1987 and 2004. SOT includes: kidney (41%), combined kidney andpancreas (7%) and liver (2%) transplants. Sixty one percent of the patientswere between 40-65 years, 21% between 18-40 years, 13% over 65 yearsand 5% below 18 years. SCT were performed for: AML–29, ALL–10;NHL-11; MM-8; CML-5; HD-2; CLL-1 and others-15. Seventy six percentwere transplanted from matched sibling donors, while 24% from matchedunrelated donors. Ninety six percent were transplanted with MPBSC, while4% with BM grafts. The main indications for immunosuppressive therapywere treatment (51%) or prevention (38%) of rejection, while 11%received CSA for prevention or treatment of GVHD. Most side effectswere mild and transient (hypertension–43%, tremor–16%, headache–16%,hepatotoxicity–16% and hypomagnesemia–11%), while, only 23% of thepatients reported SAE (mainly hypertension–15%). No significantdifferences were observed in creatinine, BUN, K+, Na+, Mg+, Bil, SGPTand cholesterol pre- and during CSA formulation administration, while,Hgb, SGOT and UA increased from 11.5±0.17 to 12.1±0.16 gr%(p=0.01), 27.5±1.8 to 38.4±4.8 IU/L (p=0.02) and 6.0±0.29 to 6.7±0.2mg/dl (p=0.003) and triglycerides decreased from 267.9±17.4 to210.3±14.5 mg/dl (p=0.002) pre- and during CSA therapy, respectively.Twenty percent of the pts. developed infectious complications duringtherapy: UTI–36%, CMV–11%, pneumonia–8%, aspergillosis–7%, herpeszoster-7%, abdominal wall abscess–7% and others. Interestingly, 4 pts.were diagnosed with malignancy while on CSA therapy (SCC-2, BCC-1,Kaposi sarcoma-1). We conclude that Deximune® administration is safe,with similar toxicity profile, side effects and bioavailability as SandimmunNeoral®. Finally, CSA based immunosuppressive therapy posttransplantation may be associated with increase risk of infectiouscomplications (20%) and malignant transformation.

360ANTI-MICROBIAL PROPHYLAXIS AFTER INDUCTION AND MOBILIZATION CHEMOTHERAPY: NO ILL EFFECT UPON SUBSEQUENT AUTO-TRANSPLANTATIONG. J. Elfenbein1,2*, K. Rolen2,3, R. Rathore1,21Boston University, Boston, MA, USA2Roger Williams Medical Center, Providence, RI, USA3Brown University, Providence, RI, USA

Autotransplantation is fraught with life-threatening infections during theperiod of neutropenia, especially from bacteria that were previously treatedduring neutropenia from earlier chemotherapy. We hypothesized that oralfluoroquinolones (trovofloxacin-200mg/day but mostly gatifloxacin-400mg/day) and fluconazole (200mg/day) would not prejudice subsequenttransplant courses.

We performed a prospective trial with historical controls. G-CSF wasgiven to all patients starting day+3. The incidence of febrile neutropenia(FN) after 37 cycles of induction with cyclophosphamide (CY) andetoposide (E) was 13% (95%CI=3-34%) compared to historical controls of>80%; after 37 courses of dexamethasone, cytarabine, and carboplatin (C),5% (CI=0-23%) compared to >40% historically; and after 16 cycles ofifosfamide (I), C, and E, 6% (CI=0-30%) compared to an average of 61%(range 28-94%, depending upon total dosages of mini-ICE). The incidenceof FN after mobilization (n=45) with CY and prednisone (lymphomas) orpaclitaxel (solid tumors) was 40% although 96% of patients had grade 3/4neutropenia.

After induction, FN developed in 8/90 (9%) cycles. Positive cultureswere obtained during 2/8 (25%) episodes of FN. Both were quinoloneresistant, coagulase negative staphylococcus (CNS). After mobilization,neutrophils exceeded 1x109/L day 11 (range 7-43). 18/45 (40%) patientsdeveloped FN. 18 positive cultures of 15 fifteen different strains of 5different species were obtained from 8/18 (44%) FN episodes. Organismsisolated were CNS (n=8), K.pneumoniae (n=3), E.coli (n=2), S.aureus(n=1) and C.jeikeium (n=1). Notably, P.aeruginosa was not detected but11/15 (73%) strains were quinolone resistant. All patients weresuccessfully treated for FN episodes with cefipime or azactam (dependingon penicillin sensitivity) as monotherapy or along with gentamycin orvancomycin depending upon septic appearance, site of tissue infection, orpositive culture. 2/45 (4%) mobilized patients expired from progressivedisease. 43/45 (96%) underwent autotransplantation. Gratifyingly, 0/43transplant patients died of infections with quinolone resistant organismsduring neutropenia.

361A PROSPECTIVE COMPARISON OF CYTOKINES ALONE FOR PERIPHERAL BLOOD PROGENITOR CELL COLLECTION IN PATIENTS WITH ADVANCED GYNAECOLOGICAL MALIGNANCIES: G-CSF VERSUS G-CSF PLUS EPOA. Perillo1,4*, G. Ferrandina1, L. Pierelli2, S. Rutella3,4, G. Bonanno1,4, V. Gallotta1, A. Paglia1, A. Mariotti1,4, S. Mancuso1,4, G. Scambia1,4,51Gynaecologic Oncology Unit, Catholic University of the Sacred Heart, Rome, Italy2Immunohaematology and Transfusion Service, ASL Viterbo, Viterbo, Italy3Haematology and Blood Transfusion, Catholic University of the Sacred Heart, Rome, Italy4Cord Blood Stem Cell Project, 'Fondazione Cassa di Risparmio', Rome, Italy5Oncology, Catholic University of the Sacred Heart, Campobasso, Italy

At present G-CSF alone represents the gold standard for mobilization ofperipheral blood progenitor cell (PBPC) from healthy donors for allogeneictransplantation, but only few studies have used G-CSF alone to mobilizeautologous PBPC in breast cancer patients, while no data are available forgynaecological malignancies. In this context, the PBPC yield collected bygrowth factor alone could be potentially increased by using a combinationof cytokines.

To address this issue twenty-five patients with advanced gynaecologicalmalignancies (13 patients with stage IIIC-IV ovarian carcinoma and 12patients with stage IIB-IVA cervical cancer) were enrolled into aprospective non-randomized study investigating the role of G-CSF plusEPO combination for PBPC mobilization in steady-state conditions. Allpatients had not been previously treated with chemo/radiotherapy. Tenpatients (G-CSF group) received recombinant human G-CSF 10 microg/kg/d subcutaneously from day -4 to day -1, while fifteen patientsunderwent different EPO-based mobilization regimens (G-CSF+EPOgroup).

No statistically significant differences in CD34+ cell count per microLwere observed in our patient series, being the median count of CD34+ cells66.5 per microL in the G-CSF alone group and 49.2 per microL in the G-CSF+EPO group. The median number of leukapheresis procedures toobtain the planned dose of CD34+ cells was comparable in the two groups.Consequently the number of PBPC collected per kg by apheresis wassimilar, being the median count of CD34+ cells 3.2 and 2.4 x106 per kg inthe G-CSF alone group and in the G-CSF+EPO group respectively.

In our series EPO addition to G-CSF in different schedules does notseem to improve PBPC mobilization/collection. Therefore, G-CSF aloneremains the most cost-effective cytokine regimen to promote PBPCrecirculation in cancer patients in steady-state conditions. These findingswill contribute to the future planning of optimal cytokine combinationstrategies for PBPC collection in cancer patients.



Abstracts

362ENGLAFTMENT OF U-BMT WITH JMMOL FOLLOWING CORD BLOOD TRANSPLANTATIONN. Sato*, T. MatsushitaInternational Medical Center of Japan, Tokyo, Japan

JMMoL is a rare myeloproliferative disorder accounting for about 1-2%of all childhood leukemias. Patients present with hepatosplenomegaly,monocytosis, leukocytosis, thrombocytopenia, leukoerythroblastosis andincreased fetal hemoglobin. Allogeneic bone marrow transplantation isconsidered as only one choice of treatments, which resulted in only 25-30% disease free survival at present. Relapse after transplantation is themost common reason for treatment failure. Second allogeneictransplantation for patients who relapse has been associated with a verypoor outcome. [Case] A 3-year-old girl with juvenile myelomonocyticleukemia (JMMoL) underwent allogenic cord blood transplantation (CBT).One month after CBT, her bone marrow revealed autologous recovery. Toenhance the graft-versus-leukemia (GVL) effect, unrelated bone marrowtransplantation (U-BMT) was performed six months after the firstprocedure. First transplantation was done with Bu+CY+L-PAM and non-TBI as conditioning regimen and the second was AraC+CY and TBI. Shehas achieved clinical remission after this second transplantation, whichdeveloped severe side effects: acute and chronic graft-versus-host disease(GVHD), interstitial pneumonia (IP) due to cytomegalovirus, thromoboticmicroangiopthy (TMA) and severe nephrotic syndrome. GVHD was not sosevere and controllable with steroid. But IP was so severe that she neededto use oxygen during night for several months. Treatment with octreotide,intractable diarrhea due to TMA had been gradually improved. The mostsevere side effect was nephrotic syndrome and secondary sepsis. Duringthese two years following the second transplantation, she faced any kindsof complications but overcame all of these unexpected events. This patientremains in remission with disease free 24 months after the second U-BMT.[Discussion] GVHD may be beneficial in preventing relapses, but severeside effects occurred by the unrelated transplantation. Intensive supportivemeasure is essentially important to attain disease free state like this high-risk patient.

363CHRONIC MYELOID LEUKEMIA COMPLICATED WITH SEVERE SUBCUTANEOUS EMPHYSEMA AFTER ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATIONK. Tokuda*, H. Tauchi, Y. IshidaEhime University, Ehime, Japan

We report a 10-year-old girl who developed bronchiolitis obliteransorganizing pneumonia(BOOP) 3 months after allogeneic periperal bloodstem cell transplantation (alloPBSCT) from an HLA-1 locus-mismatchedmother for chronic myeloid leukemia (CML). She was suffering from atrialseptal defect when she diagnosed CML. We performed alloPBSCT withnonmyeloablative conditioning regimen. We successfully confirmedengraftment 16 days without cardiac complications. The patient receivedcyclosporin A and short-term MTX for graft-versus-host disease (GVHD)prophylaxis. A grade II acute GVHD developed on day 31 and was wellcontrolled with oral prednisolone (2mg/kg/day). BOOP developed on day89 and was treated wirh a megadose methyl-prednisolone therapy. Herrespiratory function improved temporally. She had been on a respiratorbecause of progressive respiratory dysfunction.

Generalized subcutaneous emphysema developed more than 5 monthsafter alloPBSCT. She finally died of pneumothorax andpneumomediastinum on day 275.

BOOP is a major complication after allogeneic stem cell transplantation.But generalized subcutaneous emphysema was very rare. We show hersequential changes on CT scan.

364THE EFFECT OF SEROTONIN AND DOPAMINE IN THE REGULATION OF MEGAKARYOCYTOPOIESISM. Yang*, K. Li, W. Z. Huang, N. H. Pong, C. K. LiDepartment of Paediatrics, The Chinese University of Hong Kong, Hong Kong

Thrombopoiesis may be regulated by the nervous system vianeurotransmitters such as serotonin (5-HT) and dopamine. We investigatedthe new functions of these old molecules on megakaryocytopoiesis. 5-HT2A, 2B and 2C receptors were identified on four humanmegakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e, andhuman bone marrow megakaryocytes. Serotonin (100 nM) significantlystimulated murine and human megakaryocyte colony formation.Ketanserin, a 5-HT2 receptor antagonist, was showed in the same study toblock the mitogenic effect on megakaryocytopoiesis. The apoptotic effectof thrombospondin-1 (TSP-1) was abolished by serotonin (100 nM) as wellas TPO (50 ng/ml) in the cultured MK cells. Serotonin also increased thesize of cultured MK cells and had an effect on actin re-organization inMeg-01 cells. Serotonin is the precursor of melatonin. The effect ofmelatonin on hematopoiesis in irradiated mice (4 Gy) was alsoinvestigated. Melatonin had a stimulating effect on thrombopoiesis in thismodel and also stimulated in vitro CFU-MK formation at a dose dependentmanner (10-500 nM). The dopamine D1 and D2 receptors were alsoidentified on human megakaryocytes and human megakaryocytic cell lines.In in-vitro study, Dopamine induced the MK cell apoptosis at high dose(100 uM), but promoted the cell differentiation at low dose (100 nM).These results indicated that serotonin and dopamine might be involved inthe regulation of megakaryocytopoiesis: serotonin acts as a mitogenicfactor, dopamine as maturation factor.

365A LOW MOLECULAR WEIGHT, ORALLY ACTIVE TPOR AGONIST, SB-497115, DOES NOT PRIME PLATELETS FOR ACTIVATION OR AGONIST-INDUCED AGGREGATION IN VITROJ. Erhardt1, P. Tapley2, C. L. Erickson-Miller2*1GlaxoSmithKline, King of Prussia, PA, USA2GlaxoSmithKline, Collegeville, PA, USA

SB-497115 is a small molecular weight, orally active molecule thatrequires Tpo receptor (TpoR) expression for activity and activates JAK/STAT signalling in platelets. When administered to healthy male subjectsat oral doses of 30 mg and above for 10 days a dose dependent increase inthe platelet count was observed. The ability of recombinant Tpo to enhanceagonist-induced platelet aggregation is well-documented, thus this studywas undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01-10 uM) to affect platelet activation. Platelets were obtainedfrom healthy human volunteers, following written informed consent, bystandard venipuncture technique. Platelet aggregation was induced by asubmaximal concentration of adenosine diphosphate (ADP) (1.0 uM) inexperiments examining synergy with rhTpo (150ng/mL) or SB-497115(0.01-10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM),collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115,potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect onnormal ADP, Collagen or TRAP-induced aggregation was seen with eitherSB-497115 or rhTpo. The expression of platelet P-selectin, an early markerof platelet activation, was analyzed by flow cytometry. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62Pexpression above background levels, while rhTpo produced a modest butsignificant and reproducible increase in the percent of CD62P+ platelets.This study demonstrates that the orally active small molecular weightagonist of the Tpo receptor, SB-497115, does not mimic the ability ofrhTpo to enhance ADP-induced aggregation or to induce P-selectionexpression on human platelets. Finally, SB-497115 has no antagonisticeffect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus,SB-497115 is differentiated from rhTpo with respect to platelet activation.



366HYDROXYUREA IN ESSENTIAL THROMBOCYTEMIA: IN VIVO AND IN VITRO STUDIESN. Maugeri1*, G. Giordano2, M. P. Petrilli2, V. Fraticelli2, C. Cerletti1, G. de Gaetano1, S. Storti2, M. B. Donati11Laboratory Cell Biology and Pharmacology of Thrombosis, UCSC Centro di Ricerca e Formazione ad Alta Tecnologia nelle Scienze Biomediche, Campobasso, Italy2Onco Haematology Division, UCSC Centro di Ricerca e Formazione ad Alta Tecnologia nelle Scienze Biomediche, Campobasso, Italy

Hydroxyurea (HU) is a chemotherapeutic agent used to treat essentialthrombocytemia (ET). The role of HU in reducing the risk of thrombosisassociated to HT could go beyond its capacity to reduce the plateletnumber. Indeed, in ET patients increased P-selectin expression on plateletsurface as well as higher proportion of platelet-PMN aggregates wasdemonstrated. P-selectin is a physiologic inducer of tissue factor (TF) andCD11b expression on PMN surface, both markers of leukocyte activationand prothrombotic substances.

We determined here the effect of HU in vivo and in vitro, on markers ofPMN response to P-selectin.

Circulating platelet-PMN aggregates, CD11b up-regulation, totalcontent and TF expression on PMN and platelet markers of function weredetermined using two-colour flow cytometry in 3 ET patients without anychemotherapy treatment and after 15 days of HU treatment. A controlgroup of 15 healthy donors was tested.

For in vitro studies washed platelets and PMN were prepared fromhealthy donors. PMN were incubated with HU (0-1400 micromol/L) for15min at 37C and washed twice. Thrombin-preactivated platelets werecoincubated under dynamic conditions at 37C for 5 min with autologouswashed PMN to induce the formation of P-selectin-dependent platelet-PMN aggregates. TF expression of TF was determined on PMN activatedby purified P-selectin.

An increase in platelet P-selectin expression was detected in all 3patients and was not modified by HU treatment, while all markers of PMNactivation were decreased.

Incubation of PMN with HU prevented the formation of platelet-PMNaggregates and TF expression in a concentration dependent manner.

In conclusion these results are in agreement with previous indicationsthat HU may prevent thrombotic complications in ET. This effect could bedue not only to the reduction of platelet number but also to its effect onleukocyte activation.

367INCREASED BONE MARROW MEGAKARYOCYTES AND SERUM GMP-140 LEVELS IN KAWASAKI DISEASEX-H. Li1, J-S. Li1, X-J. Li1, M. Yang2*1Department of Pediatrics, Chengdu Children's Hospital, Chengdu, PR China2Department of Pediatrics, The Chinese University of Hong Kong, Hong Kong

Kawasaki disease (KD) is an acute multisystemic vasculitis withthrombocytosis affecting predominantly young children. Thrombocytosisis sometimes the cause of complications such as coronary aneurysmalthrombosis and myocardial infarction. However, the mechanism ofthrombocytosis in KD has not been well known. To clarify the pathologyof thrombocytosis, we studied bone marrow (BM) megakaryocytes (MK)in 12 patients (age: 1-10 years) with KD and 12 age-matched controls. Wealso investigated serum GMP-140 levels in 52 patients with KD (age: 7month -7 years) and 26 age-matched controls using ELISA. Our resultsshowed that: (i) Blood platelet and BM total MK cell counts in KD patients(393.2 + 146.1 x109/L, p<0.05; 242.3 + 196.0 MK/field (3.0 x 2.5cm),p<0.05; n=12) were significantly higher than normal control (114.6 + 47.2x109/L; 43.7 + 28.7 MK/ field, n=12). Platelet-producing MKs in KDpatients (149.1 + 114.4 MK/ field, p<0.05; n=12) were also significantlyhigher than normal control (43.8 + 28.5 MK/field; n=12); and (ii) GMP-140 values in the controls were 14.0 + 3.5 ug/ml (n=26). In KD patients,GMP-140 levels were 20.4 + 7.2 (before treatment), 26.0 + 8.9 (treatmentfor one week) and 22.8 + 9.3 ug/ml (treatment for two weeks) (n=52).There are significant differences in both before and after treatmentcompared with normal control (p<0.05). Our study suggests that increasedmegakaryocytes in BM and elevated GMP-140 in serum may have animportant role on the mechanism of thrombocytosis in KD patients.


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