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BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2,CD3,CD7,CD14,CD16,CD24,CD36,CD38,CD56,CD66b,or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that,in combination with other markers,allows easy isolation of the stem cell fraction at unprecedented purities. View Publication

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation,such as expression of specific cell surface antigens,inhibition of cell proliferation,and polyploidization. Conversely,knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin,the expression of Fli-1,Ets-1,and Ets-2 transcription factors,but not GATA-1 or MafB,is blocked. Thus,tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation. View Publication

NK cells express different TLRs,such as TLR3,TLR7,and TLR9,but little is known about their role in NK cell stimulation. In this study,we used specific agonists (poly(I:C),loxoribine,and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12,IL-15,or IFN-alpha,and investigated the secretion of IFN-gamma,cytotoxicity,and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine,in conjunction with IL-12,but not IL-15,triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also,secretion of IFN-gamma was dependent on MEK1/ERK,p38 MAPK,p70(S6) kinase,and NF-kappaB,but not on calcineurin. IFN-alpha induced a similar effect,but promoted lesser IFN-gamma secretion. However,cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged,as well as the expression of the NKG2D receptor. Excitingly,IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12,and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12,and this effect can be further enhanced by costimulation through NKG2D. Hence,integration of the signaling cascades that involve TLR3,TLR7,IL-12,and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions,which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer. View Publication

The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study,constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML,expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression. View Publication

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway,which utilizes beta-catenin to relay the signal,have been demonstrated to regulate HSC function. In this study,we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation,such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data,we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway,resulting in a pool of quiescent HSCs. View Publication

OBJECTIVE Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP,PEPCK,and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes,and metformin-and adenovirus SHP (Ad-SHP)-mediated hepatic glucose production was measured in B6-Lep(ob/ob) mice. RESULTS Hepatic SHP gene expression was induced by metformin,5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR),and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK),as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4alpha-or FoxA2-mediated promoter activity of PEPCK and G6Pase,and the inhibition was blocked with siRNA SHP. Additionally,SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore,oral administration of metformin increased SHP mRNA levels in B6-Lep(ob/ob) mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lep(ob/ob) mice. CONCLUSIONS We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP. View Publication

BACKGROUND: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs,as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines,were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT). METHODS AND RESULTS: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay,respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT,the levels of both EPC and VEGF/SDF-1 markedly increased (P textless .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential,but on DSAT the baseline level was incompletely attained. CONCLUSIONS: In response to SAT,patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential. View Publication

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here,we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules,we have identified SB203580,a specific p38 MAP kinase inhibitor,as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations textless10 microM,induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers,so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly,transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly,cardiomyogenesis was strongly inhibited at SB203580 concentrations textgreater or =15 microM. Thus,modulation of the p38MAP kinase pathway,in combination with factors released by END2 cells,plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process. View Publication

Implanted materials,such as medical devices,provoke the body to initiate an inflammatory reaction,known as the foreign body reaction (FBR),which causes several complications for example in hip prostheses,silicone implants,peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood,but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this,acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6h to 7 days. A significant fraction of Sca-1(+) (6h-2 days),c-kit(+),CD34(+) and CD271(+) (2-3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-,osteo-,and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses. View Publication

BACKGROUND Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts,but their actions with regard to bone metabolism are still unclear. In this study,we investigated the effects of adiponectin on the proliferation,differentiation,and mineralization of osteoblastic MC3T3-E1 cells. RESULTS Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator,5-amino-imidazole-4-carboxamide-riboside (AICAR),in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA,and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA,as determined by real-time PCR,and reduced ALP activity and mineralization,as determined by von Kossa and Alizarin red stainings. In contrast,AMP kinase activation by AICAR (0.01-0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation,differentiation,and mineralization. BrdU assay showed that the addition of adiponectin (0.01-1.0 mug/ml) also promoted their proliferation. Osterix,but not Runx-2,appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression,respectively. CONCLUSION Taken together,this study suggests that adiponectin stimulates the proliferation,differentiation,and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. View Publication

OBJECTIVES: Our objective was to develop and assess a novel endogenous progenitor cell (EPC) assay based on aldehyde dehydrogenase (ALDH) activity,and to define the relationship of ALDH-bright (ALDH(br)) cells with previously defined EPCs,patient age,and extent of coronary artery disease. BACKGROUND: Accurate assessment of circulating EPCs is of significant interest,yet current assays have limitations. Progenitor cells display high levels of ALDH activity. An assay based on ALDH activity may offer a simple means for enumerating EPCs. METHODS: We simultaneously determined the numbers of EPCs based on ALDH activity and cell surface expression of CD133,CD34,and vascular endothelial growth factor receptor-2 in 110 patients undergoing cardiac catheterization. We assessed the reproducibility of these estimates,correlation among EPC assays,and the association of ALDH(br) numbers with age and disease severity. RESULTS: Aldehyde dehydrogenase-bright cells were easily identified in nonmobilized peripheral blood with median and mean frequencies of 0.041% and 0.074%,respectively. Aldehyde dehydrogenase-bright cells expressed CD34 or CD133 cell surface markers (57.0% and 27.1%,respectively),correlated closely with CD133+CD34+ cells (r = 0.72; p textless 0.001),and differentiated into endothelial cells with greater efficiency than CD133+CD34+ cells. Aldehyde dehydrogenase-bright cell numbers were inversely associated with patient age and coronary disease severity. CONCLUSIONS: Aldehyde dehydrogenase activity represents a novel simplified method for quantifying EPCs. The correlation of ALDH(br) cells with clinical factors and outcomes warrants further study. View Publication

TRAIL induces apoptosis in a variety of tumor cells. Our laboratory found that human neutrophils contain an intracellular reservoir of prefabricated TRAIL that is released after stimulation with Mycobacterium bovis bacillus Calmette-Guérin. In this study,we examined the subcellular distribution of TRAIL in freshly isolated neutrophils. Neutrophil granules,secretory vesicles (SV),and plasma membrane vesicles were isolated by subcellular fractionation,followed by free-flow electrophoresis,and examined by ELISA and immunoblot. TRAIL was found in all membrane-bound fractions with the highest amounts in the fractions enriched in azurophilic granule (AG) and SV. Immunofluorescence confocal microscopy showed that TRAIL colocalized independently with myeloperoxidase (MPO),lactoferrin (LF),and albumin,respective markers of AG,specific granules,and SV. Furthermore,immunotransmission electron microscopy demonstrated that TRAIL colocalized intracellularly with MPO and albumin. We examined TRAIL expression in PLB-985 cells induced with dimethylformamide and in CD34-positive stem cells treated with G-CSF. Quantitative RT-PCR analysis showed that TRAIL was expressed in each stage of development,whereas MPO and LF were only expressed at distinct times during differentiation. Collectively,these findings suggest that TRAIL is expressed throughout neutrophil development,resulting in a broad distribution among different granule subtypes. View Publication