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Although significant advances have been made over the last decade with respect to our understanding of stem cell biology,progress has been limited in the development of successful techniques for clinically significant ex vivo expansion of hematopoietic stem and progenitor cells. We here describe the effect of Notch ligand density on induction of Notch signaling and subsequent cell fate of human CD34+CD38- cord blood progenitors. Lower densities of Delta1(ext-IgG) enhanced the generation of CD34+ cells as well as CD14+ and CD7+ cells,consistent with early myeloid and lymphoid differentiation,respectively. However,culture with increased amounts of Delta1(ext-IgG) induced apoptosis of CD34+ precursors resulting in decreased cell numbers,without affecting generation of CD7+ cells. RNA interference studies revealed that the promotion of lymphoid differentiation was primarily mediated by Delta1 activation of Notch1. Furthermore,enhanced generation of NOD/SCID repopulating cells was seen following culture with lower but not higher densities of ligand. These studies indicate critical,quantitative aspects of Notch signaling in affecting hematopoietic precursor cell-fate outcomes and suggest that density of Notch ligands in different organ systems may be an important determinant in regulating cell-fate outcomes. Moreover,these findings contribute to the development of methodology for manipulation of hematopoietic precursors for therapeutic purposes. View Publication

OBJECTIVE: To compare the suppressive effect of mesenchymal stem cells (MSC),derived from normal individuals or severe aplastic anemia patients (SAA),on T-cell activation. PATIENTS AND METHODS: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3),following immunosuppressive therapy (IS) (n = 16),or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR),phytohemaglutinin (PHA)-primed cultures,activation surface markers,gamma-IFN production,hematopoietic colony formation (CFC),production of cyclic ADP-ribose (cADPR). RESULTS: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures,resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR,a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. CONCLUSION: The ability of MSC to downregulate T-cell priming,proliferation,and cytokine release is deficient in patients with SAA,persists indefinitely after immunosuppressive therapy,but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined. View Publication

The self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid,massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J. We obtained a similar phenotype by blocking the Notch cascade with a gamma-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus,maintenance of undifferentiated,proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that gamma-secretase inhibitors,developed for Alzheimer's disease,might be of therapeutic benefit in colorectal neoplastic disease. 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

Imatinib,a Bcr-Abl tyrosine kinase inhibitor,is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients,relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus,alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency,selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article,we compared imatinib,AMN107,and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing textgreater90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested,with the exception of T315I. Thus,both inhibitors hold promise for treating imatinib-refractory CML. 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

Oligonucleotides with a CpG" motif trigger a proinflammatory response through activation of Toll-like receptor 9 (TLR9) and are being studied to exploit these properties for use as adjuvants and cancer therapies. However� View Publication

Hereditary hemochromatosis (HH),an iron overload disease associated with mutations in the HFE gene,is characterized by increased intestinal iron absorption and consequent deposition of excess iron,primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin,a peptide hormone produced by the liver in response to iron loading. In this study,we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading,accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely,wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. View Publication

Evidence is presented that bone marrow (BM) in addition to CD45(positive) hematopoietic stem cells contains a rare population of heterogenous CD45(negative) nonhematopoietic tissue committed stem cells (TCSC). These nonhematopoietic TCSC (i) are enriched in population of CXCR4(+) CD34(+) AC133(+) lin(-) CD45(-) and CXCR4(+) Sca-1(+) lin(-) CD45(-) in humans and mice,respectively,(ii) display several markers of pluripotent stem cells (PSC) and (iii) as we envision are deposited in BM early in development. Thus,since BM contains versatile nonhematopoietic stem cells,previous studies on plasticity trans-dedifferentiation of BM-derived hematopoietic stem cells (HSC) that did not include proper controls to exclude this possibility could lead to wrong interpretations. Therefore,in this spotlight review we present this alternative explanation of 'plasticity' of BM-derived stem cells based on the assumption that BM stem cells are heterogenous. We also discuss a potential relationship of TCSC/PSC identified by us with other BM-derived CD45(negative) nonhematopoietic stem cells that were recently identified by other investigators (eg MSC,MAPC,USSC and MIAMI cells). Finally,we discuss perspectives and pitfalls in potential application of these cells in regenerative medicine. 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

Zebularine (2(1H)-pyrimidinone riboside,Zeb),a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase,has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-,di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb,the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT. View Publication

Adult mammalian cardiomyocytes are considered terminally differentiated and incapable of proliferation. Consequently,acutely injured mammalian hearts do not regenerate,they scar. Here,we show that adult mammalian cardiomyocytes can divide. One important mechanism used by mammalian cardiomyocytes to control cell cycle is p38 MAP kinase activity. p38 regulates expression of genes required for mitosis in cardiomyocytes,including cyclin A and cyclin B. p38 activity is inversely correlated with cardiac growth during development,and its overexpression blocks fetal cardiomyocyte proliferation. Activation of p38 in vivo by MKK3bE reduces BrdU incorporation in fetal cardiomyocytes by 17.6%. In contrast,cardiac-specific p38alpha knockout mice show a 92.3% increase in neonatal cardiomyocyte mitoses. Furthermore,inhibition of p38 in adult cardiomyocytes promotes cytokinesis. Finally,mitosis in adult cardiomyocytes is associated with transient dedifferentiation of the contractile apparatus. Our findings establish p38 as a key negative regulator of cardiomyocyte proliferation and indicate that adult cardiomyocytes can divide. View Publication