技术资料

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment,we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy,c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages,whereas erythroid differentiation was impaired,as demonstrated by clonogenic assay,morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets,which can account for c-myb knockdown effects. Indeed,chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently,the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing,whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment,by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed,we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision. View Publication

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types,namely human umbilical cord vein endothelial cells (HUVECs),endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs),to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However,only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. 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

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation,reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization,and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

Human macrophages are specialised hosts for HIV-1,dengue virus,Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens,because available myeloid cell lines are,by definition,not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined,feeder- and serum-free culture conditions and produces very consistent,pure,high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively,up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+),CD16(low),CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic,HIV-1-infectable,and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate,as they recognise foreign nucleic acids; the lentivector system described here overcomes this,as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells,surmounting issues of transgene silencing. Overall,the method we describe here is an efficient,effective,scalable system for the reproducible production and genetic modification of human macrophages,facilitating the interrogation of human macrophage biology. View Publication

MicroRNAs (miRNAs) are small,noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-,maturation-,and disease-specific miRNA expression has been described,miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics,biochemical analysis,and unbiased and hypothesis-driven miRNA target prediction,we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells,miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression,respectively. However,gene-specific RNAi reveals that this reduction,alone and in combination,is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression,DNA-binding,and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression,indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed,we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly,gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T,$\$-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate,the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%,whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular,atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified,functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling,drug discovery,and regenerative medicine. View Publication