技术资料

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations,including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis,causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro,osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore,the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy,bone marrow transplantation,or gene therapy resulted in full recovery of the altered bone parameters. Remarkably,untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. View Publication

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

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We have previously shown that a granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-15 (IL-15) 'fusokine' (GIFT15) exerts immune suppression via aberrant signaling through the IL-15 receptor on lymphomyeloid cells. We show here that ex vivo GIFT15 treatment of mouse splenocytes generates suppressive regulatory cells of B cell ontogeny (hereafter called GIFT15 B(reg) cells). Arising from CD19+ B cells,GIFT15 B(reg) cells express major histocompatibility complex class I (MHCI) and MHCII,surface IgM and IgD,and secrete IL-10,akin to previously described B10 and T2-MZP B(reg) cells,but lose expression of the transcription factor PAX5,coupled to upregulation of CD138 and reciprocal suppression of CD19. Mice with experimental autoimmune encephalomyelitis went into complete remission after intravenous infusion of GIFT15 B(reg) cells paralleled by suppressed neuroinflammation. The clinical effect was abolished when GIFT15 B(reg) cells were derived from mmicroMT (lacking B cells),MHCII-knockout,signal transducer and activator of transcription-6 (STAT-6)-knockout,IL-10-knockout or allogeneic splenocytes,consistent with a pivotal role for MHCII and IL-10 by sygeneic B cells for the observed therapeutic effect. We propose that autologous GIFT15 B(reg) cells may serve as a new treatment for autoimmune ailments. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

The multidrug transporter ABCG2 in cell membranes enables various stem cells and cancer cells to efflux chemicals,including the fluorescent dye Hoechst 33342. The Hoechst(-) cells can be sorted out as a side population with stem cell properties. Abcg2 expression in mouse embryonic stem cells (ESCs) reduces accumulation of DNA-damaging metabolites in the cells,which helps prevent cell differentiation. Surprisingly,we found that human ESCs do not express ABCG2 and cannot efflux Hoechst. In contrast,trophoblasts and neural epithelial cells derived from human ESCs are ABCG2(+) and Hoechst(-). Human ESCs ectopically expressing ABCG2 become Hoechst(-),more tolerant of toxicity of mitoxantrone,a substrate of ABCG2,and more capable of self-renewal in basic fibroblast growth factor (bFGF)-free condition than control cells. However,Hoechst(low) cells sorted as a small subpopulation from human ESCs express lower levels of pluripotency markers than the Hoechst(high) cells. Similar results were observed with human induced pluripotent stem cells. Conversely,mouse ESCs are Abcg2(+) and mouse trophoblasts,Abcg2(-). Thus,absence of ABCG2 is a novel feature of human pluripotent stem cells,which distinguishes them from many other stem cells including mouse ESCs,and may be a reason why they are sensitive to suboptimal culture conditions. View Publication

RATIONALE: Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However,the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES: We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS: The study was performed in a cohort of 21 subjects with allergic asthma,21 healthy control subjects,and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS: Dendritic cells cultured with natural regulatory T cells up-regulated IL-10,down-regulated costimulatory molecules,and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore,this defective function of natural regulatory T cells was associated with their decreased IL-10 expression,disease severity,and could be reversed by oral corticosteroid therapy. CONCLUSIONS: These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Because IL-1beta plays an important role in inflammation in human and murine arthritis,we investigated the contribution of the inflammasome components ASC,NALP-3,IPAF,and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA,decreased levels of synovial IL-1beta,and diminished serum amyloid A levels. In contrast,mice deficient in NALP-3,IPAF,or caspase-1 did not show any alteration of joint inflammation,thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes,we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro,as was the production of IFN-gamma,whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice,but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion,these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation. View Publication

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication