技术资料

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study,we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs),evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect,confirming that VEGF and IL-6,at least in part,mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo,immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs,and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6),DCs only (SI = 9.3),or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

In this study,in an attempt to identify neuroblastoma-associated surface antigens,we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis,unlike anti-disialoganglioside mAb,5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors,thus representing a highly specific marker for identifying neuroblastoma cells. Molecular analysis revealed that the 5B14 mAb-reactive surface glycoprotein corresponded to the recently identified 4Ig-B7-H3 molecule. Remarkably,mAb-mediated masking of the 4Ig-B7-H3 molecule on cell transfectants or on freshly isolated neuroblastoma cells resulted in enhancement of natural killer-mediated lysis of these target cells. These data suggest that 4Ig-B7-H3 molecules expressed at the tumor cell surface can exert a protective role from natural killer-mediated lysis by interacting with a still undefined inhibitory receptor expressed on natural killer cells. View Publication

The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell,but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system,we have demonstrated a consistent defect in DBA,regardless of clinical severity,including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1,but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells,and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone,Epo sensitivity was comparable to normal,but erythroid expansion remained subnormal. In clonogenic phase 2 cultures,the number of colonies did not significantly differ between normal cultures and DBA,in the presence or absence of dexamethasone,and at both low and high Epo concentrations. However,colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA,and the defect lies down-stream of the Epo receptor,influencing survival and/or proliferation of erythroid progenitors. View Publication

Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies,resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98,and more than one half of these are homeobox transcription factors. By contrast,the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1),a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors,evidenced by a competitive growth advantage in liquid culture,increased replating efficient of colony-forming cells (CFCs),and a marked increase in spleen colony-forming cell output. Moreover,in a murine bone marrow transplantation model,NUP98-TOP1 expression led to a lethal,transplantable leukemia characterized by extremely high white cell counts,splenomegaly,and mild anemia. Strikingly,a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings,together with similar biologic effects reported for NUP98-HOX fusions,suggest unexpected,overlapping functions of NUP98 fusion genes,perhaps related to common DNA binding properties. View Publication

BACKGROUND AND OBJECTIVES: Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells,but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts. DESIGN AND METHODS: AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72,SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation,apoptosis and cytokine release. RESULTS: The cross-talk between these two cell populations,achieved via release of soluble mediators,resulted in increased AML blast proliferation,including increased proliferation of clonogenic progenitors,but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk,and normal osteoblasts could also increase the AML blast proliferation. Furthermore,co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8. INTERPRETATION AND CONCLUSIONS: Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however,detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment,samples (n = 49) were labeled with a RosetteSep progenitor antibody cocktail to remove unwanted mature T-cells,B-cells,granulocytes,natural killer cells,neutrophils and myelomonocytic cells. For CD45-negative cell enrichment,samples (n = 14) were labeled with RosetteSep CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences,respectively. Overall,FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false-positive detection,respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using RosetteSep trade mark progenitor enrichment compared with the CD45-negative enrichment method. Sensitivity of the RosetteSep progenitor enrichment approach for detection of fetal cells in this pilot study shows great promise with recovery of cells that are suitable for FISH and automated microscope scanning. This simple and rapid method may also allow expansion in culture and characterization of the fetal cell type(s) that circulate in maternal blood,hence,greatly improving reliability of non-invasive prenatal diagnosis. View Publication

Clinical observations suggest that in chronic myelogenous leukemia (CML),the Philadelphia chromosome (Ph+) clone has a growth advantage over normal hematopoiesis. Patients with CML have high levels of neutrophil elastase,which has recently been shown to antagonize the action of granulocyte-colony-stimulating factor (G-CSF) and other growth factors. We therefore compared the effect of elastase on the growth of normal and CML progenitor cells. In 10-day suspension cultures of normal or CML CD34+ cells supplemented with G-CSF,stem cell factor (SCF),and granulocyte macrophage-colony-stimulating factor (GM-CSF),CML cells had diminished sensitivity to the growth inhibitory effect of elastase. When equal numbers of CML and normal CD34+ cells were cocultured for 10 days,there was no change in the relative proportions of normal and leukemic cells (measured by fluorescence in situ hybridization [FISH] or flow cytometry). However,when elastase was added,CML cells predominated at the end of the culture period (78% vs 22% with 1 microg/mL and 80% vs 20% with 5 microg/mL elastase). CML neutrophils substituted effectively for elastase in suppressing the proliferation of normal CD34+ cells,but this effect was abrogated by serine protease inhibitors. These results suggest that elastase overproduction by the leukemic clone can change the growth environment by digesting growth factors,thereby giving advantage to Ph+ hematopoiesis. View Publication