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There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

Monocyte cytokines (ie,monokines) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma),which is critical for monocyte clearance of infectious pathogens and tumor surveillance. Human CD56bright NK cells produce far more IFN-gamma in response to monokines than do CD56dim NK cells. The kinases and phosphatases involved in regulating IFN-gamma production by monokine-activated NK cells are not clearly identified. SHIP1 is a 5' inositol phosphatase that dephosphorylates the phosphatidylinositol-3 kinase (PI-3K) product PI3,4,5P3. Here,we show that constitutive expression of SHIP1 is distinctly lower in CD56bright NK cells compared with CD56dim NK cells,suggesting it could be an important negative regulator of IFN-gamma production in monokine-activated NK cells. Indeed,overexpression of SHIP1 in CD56bright NK cells followed by monokine activation substantially lowered IFN-gamma production. This effect was not seen when NK cells were infected with a SHIP1 mutant containing an inactive catalytic domain. Finally,NK cells in SHIP1-/- mice produced more IFN-gamma in response to monokines in vivo than did NK cells from wild-type mice. Collectively,these results demonstrate that SHIP1 negatively regulates monokine-induced NK cell IFN-gamma production in vitro and in vivo and provide the first molecular explanation for an important functional distinction observed between CD56bright and CD56dim human NK subsets. View Publication

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30,NKp44,and NKp46. Indeed,the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis,and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA,FasL protein synthesis,and release. In turn,FasL interacting with Fas at NK cell surface causes NK cell suicide,as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly,NK cell apoptosis,but not killing of tumor target cells,is inhibited by cyclosporin A,suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity,but also surface receptors involved in NK cell suicide. View Publication

PTPN11 encodes the protein tyrosine phosphatase SHP-2,which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS),a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential,perturbed erythroid growth,and impaired normal differentiation in liquid cultures. In addition,leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments,which supports a primary role of hyperactive Ras in the pathogenesis of JMML. View Publication

In all,78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells,and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%),respectively,which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417,respectively) or platelets (P=0.88 and 0.126,respectively). However,the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001,respectively) and platelets (P=0.023 and 0.010,respectively),whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007,respectively) but not to platelets (P=0.112 and 0.100,respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore,the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage. View Publication

Although there is evidence that endothelial cells are important targets for human pathogenic Bartonella species,the primary niche of infection is unknown. Here we elucidated whether human CD34+ hematopoietic progenitor cells (HPCs) internalize B. henselae and may serve as a potential niche of the pathogen. We showed that B. henselae does not adhere to or invade human erythrocytes. In contrast,B. henselae invades and persists in HPCs as shown by gentamicin protection assays,confocal laser scanning microscopy (CLSM),and electron microscopy (EM). Fluorescence-activated cell sorting (FACS) analysis of glycophorin A expression revealed that erythroid differentiation of HPCs was unaffected following infection with B. henselae. The number of intracellular B. henselae continuously increased over a 13-day period. When HPCs were infected with B. henselae immediately after isolation,intracellular bacteria were subsequently detectable in differentiated erythroid cells on day 9 and day 13 after infection,as shown by CLSM,EM,and FACS analysis. Our data provide,for the first time,evidence that a bacterial pathogen is able to infect and persist in differentiating HPCs,and suggest that HPCs might serve as a potential primary niche in Bartonella infections. View Publication

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function,anemia,and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression,studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils,even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils,and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b,CD35,and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry,whereas a second subset remained negative,consistent with a bimodal expression. Finally,human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst,compared with human control IgG in stem cell-derived neutrophils. Taken together,these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors. View Publication

Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle,fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented,little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here,we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts,physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin,a marker of regenerating skeletal muscle,to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC,human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle. View Publication

Kaposi's sarcoma (KS) is a common neoplasm in HIV-1-infected individuals causing significant morbidity and mortality. Despite recent advances,the pathogenesis of this potentially life-threatening neoplasm remains unclear,and there is currently no cure for KS. Notch proteins are known to play a fundamental role in cell fate decisions including proliferation,differentiation,and apoptosis. It is,therefore,not surprising that Notch proteins have been implicated in tumorigenesis and appear to function as either oncogenes or tumor suppressor proteins depending on cellular context. In this report,we demonstrate elevated levels of activated Notch-1,-2,and -4 in KS tumor cells in vivo and in vitro compared to endothelial cells,the precursor of the KS cell. Notch activation was confirmed through luciferase reporter assays and localization of Hes (Hairy/Enhancer of Split)-1 and Hey (Hairy/Enhancer of Split related with YRPW)1 (primary targets of the Notch pathway) in KS cell nuclei. Studies using gamma-secretase inhibitors (GSI and LY-411,575),which block Notch activation,resulted in apoptosis in primary and immortalized KS cells. Similar studies injecting GSI into established KS cell tumors on mice demonstrated growth inhibition or tumor regression that was characterized by apoptosis in treated,but not control tumors. The results indicate that KS cells overexpress activated Notch and interruption of Notch signaling inhibits KS cell growth. Thus,targeting Notch signaling may be of therapeutic value in KS patients. View Publication

The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population,we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice,we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore,all neurospheres derived from these neurogenic regions express Sox2,suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain,with differing capacities for cell production. In vitro,all neurospheres express Sox2,but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example,both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres,but their expression is mutually exclusive; likewise,these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus,we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain,but that not all NSCs maintain the ability to form all neural cell types in vivo. View Publication

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context,little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional,biochemical,and microarray analyses. When deprived of natural ligands,GM-CSF,interleukin-3,or erythropoietin,AP20187 prevented apoptosis of transduced TF1 cells,induced dose-dependent proliferation,and supported long-term growth. In addition,AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably,mRNAs affiliated with heat stress,including Hsp70 and Hsp105,were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence,in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor,or KDR,signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes. View Publication