Scientific Resources

Successful blood and marrow transplant (BMT),both autologous and allogeneic,requires the infusion of a sufficient number of hematopoietic progenitor/stem cells (HPCs) capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion. At present,the most commonly used surrogate marker for HPCs is the cell surface marker CD34,identified in the clinical laboratory by flow cytometry. Clinical studies have shown that infusion of at least 2 x 10(6) CD34(+) cells/kg recipient body weight results in reliable engraftment as measured by recovery of adequate neutrophil and platelet counts approximately 14 days after transplant. Recruitment of HPCs from the marrow into the blood is termed mobilization,or,more commonly,stem cell mobilization. In Section I,Dr. Tsvee Lapidot and colleagues review the wide range of factors influencing stem cell mobilization. Our current understanding focuses on chemokines,proteolytic enzymes,adhesion molecules,cytokines and stromal cell-stem cell interactions. On the basis of this understanding,new approaches to mobilization have been designed and are now starting to undergo clinical testing. In Section II,Dr. Michele Cottler-Fox describes factors predicting the ability to mobilize the older patient with myeloma. In addition,clinical approaches to improving collection by individualizing the timing of apheresis and adjusting the volume of blood processed to achieve a desired product are discussed. Key to this process is the daily enumeration of blood CD34(+) cells. Newer methods of enumerating and mobilizing autologous blood HPCs are discussed. In Section III,Dr. John DiPersio and colleagues provide data on clinical results of mobilizing allogeneic donors with G-CSF,GM-CSF and the combination of both as relates to the number and type of cells collected by apheresis. Newer methods of stem cell mobilization as well as the relationship of graft composition on immune reconstitution and GVHD are discussed. View Publication

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here,we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10,25,or 50 mWcm(-2) and,(1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells,the photobleaching,and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However,in all of the cases,cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT,as well as confirming the likely role of (1)O(2) in porphyrin-based PDT. View Publication

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo,selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study,we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment,at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore,99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR,compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice,demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity,and CFUs were stained intensely for AGT protein,indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge,these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However,comparing the two assays,cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells,p = 0.0001). Overall,60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells� View Publication

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma,an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis,bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2,respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors,and transduced cells were cultured in a semisolid medium permissive for the development of erythroid,myeloid,and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro,in contrast to Tax2-transduced cells,which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected,suggesting that Tax1 inhibited the maturation of relatively early,uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis,lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells,an activity that is not displayed by Tax2,and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo,the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2. View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

The physiologic function of BUBR1,a key component of the spindle checkpoint,was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro,BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes,which was correlated with an increase in megakaryocytic,but a decrease in erythroid,progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However,enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood,which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together,these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

In mammals,the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region,within cells localized to the ventral floor of the major blood vessels,suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells,their precursors,and HCs,suggestive of a role in the divergence and function of both lineages. Indeed,gene targeting approaches have shown that TIE1,TIE2,and ligands for TIE2,the angiopoietins,are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs,we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly,however,these receptors are specifically required during postnatal bone marrow hematopoiesis. 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