技术资料

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

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

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach,we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor,retinoid-inducible nuclear factor (RINF),that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore,short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly,RINF localizes to 5q31.3,a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene. 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

The core-binding factor (CBF) is a master regulator of developmental and differentiation programs,and CBF alterations are frequently associated with acute leukemia. The role of the CBF member RUNX2 in hematopoiesis is poorly understood. Genetic evidence suggests that deregulation of Runx2 may cause myeloid leukemia in mice expressing the fusion oncogene Cbfb-MYH11. In this study,we show that sustained expression of Runx2 modulates Cbfbeta-smooth muscle myosin heavy chain (SMMHC)-mediated myeloid leukemia development. Expression of Runx2 is high in the hematopoietic stem cell compartment and decreases during myeloid differentiation. Sustained Runx2 expression hinders myeloid progenitor differentiation capacity and represses expression of CBF targets Csf1R,Mpo,Cebpd,the cell cycle inhibitor Cdkn1a,and myeloid markers Cebpa and Gfi1. In addition,full-length Runx2 cooperates with Cbfbeta-SMMHC in leukemia development in transplantation assays. Furthermore,we show that the nuclear matrix-targeting signal and DNA-binding runt-homology domain of Runx2 are essential for its leukemogenic activity. Conversely,Runx2 haplo-insufficiency delays the onset and reduces the incidence of acute myeloid leukemia. Together,these results indicate that Runx2 is expressed in the stem cell compartment,interferes with differentiation and represses CBF targets in the myeloid compartment,and modulates the leukemogenic function of Cbfbeta-SMMHC in mouse leukemia. View Publication

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1,where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study,RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies,flow cytometry,and chromatin immunoprecipitation identified two candidate stem cell marker genes,CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1),to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors,we detected a relatively abundant CD133(high)-ASCL1(high)-ALDH1(high) subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133(high) subpopulation depended on continued ASCL1 expression. Whereas CD133(high) cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor,CD133(low) cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity,including variation in key regulatory factors such as ASCL1,can modulate tumorigenicity in SCLC. View Publication

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans,the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD,which extends over many months,is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC,CD1a(-)CD14(+) DC,and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro,each subset has characteristic properties. After transplantation,both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells,but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells,macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages,although unlikely to initiate alloreactivity,may contribute to GVHD by sustaining the responses of previously activated T cells. View Publication