技术资料

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However,the critical downstream targets of Scl remain undefined. Here,we identified a novel Scl target gene,transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C,in contrast to Scl,is not essential for specification into primitive or definitive hematopoietic lineages. However,adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced,whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore,megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells,but not in erythroid cells. In addition,an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice,characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary,this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages. View Publication

Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However,the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here,we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells,as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive,suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally,in vivo administration of succinate was found to elevate the levels of hemoglobin,platelets,and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development. View Publication

Scl and Lyl1 encode two related basic-helix-loop-helix transcription factors implicated in T cell acute lymphoblastic leukemia. Previous studies showed that Scl is essential for embryonic and adult erythropoiesis,while Lyl1 is important for B cell development. Single-knockout mice have not revealed an essential function for Scl or Lyl1 in adult hematopoietic stem cells (HSCs). To determine if maintenance of HSCs in single-knockout mice is due to functional redundancy,we generated Lyl1;Scl-conditional double-knockout mice. Here,we report a striking genetic interaction between the two genes,with a clear dose dependence for the presence of Scl or Lyl1 alleles for HSC function. Bone marrow repopulation assays and analyses demonstrated rapid loss of hematopoietic progenitors due to apoptosis. The function of HSCs could be rescued by a single allele of Lyl1 but not Scl. These results show that expression of at least one of these factors is essential for maintenance of adult HSC function. View Publication

The metabolism of oxygen,although central to life,produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer,cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny,and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably,subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing,CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that,similar to normal tissue stem cells,subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny,which may contribute to tumour radioresistance. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here,we show that MLL normally regulates expression of mir-196b,a hematopoietic microRNA located within the HoxA cluster,in a pattern similar to that of the surrounding 5' Hox genes,Hoxa9 and Hoxa10,during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage,mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b,while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore,overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival,as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. View Publication

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. View Publication

BACKGROUND: Multiple measures of endothelial progenitor cells (EPCs) have been described,but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies. METHODS: We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients,EPCs were also identified on the basis of cell surface marker expression (CD133,CD34,and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity. RESULTS: Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however,endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133,CD34,CD133/CD34,and ALDH activity correlated with each other,but not with VEGFR-2(+) cells. CONCLUSIONS: Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells,which give rise to endothelial CFUs,and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research. View Publication

BACKGROUND: Interest in the biology of endogenous progenitor cells (EPCs) continues to grow as evidence of their role in vascular repair mounts. EPC enumeration requires specialized laboratory techniques and is performed immediately after sample acquisition,limiting the clinical contexts in which EPC enumeration can be performed and the ability to increase sample sizes through multi-center participation. METHODS: We compared the numbers of EPCs enumerated in samples processed immediately after acquisition (n = 36) with EPCs enumerated in specimens stored for 24 hours or after cryopreservation of mononuclear cells (MNC) using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH(br) cells). RESULTS: EPCs assessed in fresh samples correlated with EPCs enumerated after whole blood storage (r = 0.699 for CD133(+)CD34(+) cells,r = 0.880 for ALDH(br) cells,P textless 0.005 and P textless 0.0001,respectively) or mononuclear cryopreservation (r = 0.590 for CD133(+)CD34(+) cells,r = 0.894 for ALDH(br) cells,P textless 0.0001 for each); however,correlation based on assessment of ALDH(br) cells was higher (P textless 0.0003 for comparison of correlation coefficients). Initial results from a multi-site clinical trial suggest that EPC enumeration after mononuclear cell cryopreservation is feasible. CONCLUSION: EPC analysis based on ALDH activity is reproducible,even after extended whole blood storage or MNC cryopreservation. View Publication

PTPN11,which encodes the tyrosine phosphatase SHP2,is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis,we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11(D61Y). Expression of Ptpn11(D61Y) in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD),featuring leukocytosis,anemia,hepatosplenomegaly,and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin(-)Sca1(+)cKit(+) (LSK) compartment is expanded and right-shifted� View Publication