技术资料

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB),platelet-derived growth factor (PDGF),hypoxia-inducible factor-1alpha (HIF-1alpha),stromal cell derived factor-1 (SDF-1),matrix metalloproteinase-2 (MMP-2),vascular endothelial growth factor (VEGF),and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells,both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1,MMP-2,and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells. View Publication

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To assess the underlying mechanisms,C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation,expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL,K562,and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha,whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells,alone or upon C/EBPalpha activation. In summary,the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend,in part,on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells,the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets. View Publication

Human interferon (IFN)-alpha is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-alpha and often leads to dose reduction or treatment discontinuation. However,there is little information on how IFN-alpha inhibits human megakaryopoiesis. In this study,we demonstrated that IFN-alpha did not inhibit colony formation of megakaryocytes from human CD34(+) hematopoietic stem cells. IFN-alpha did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-alpha suppressed the expression of transcription factors regulating late-stage megakaryopoiesis,such as GATA-1,p45(NF-E2),MafG. IFN-alpha also significantly reduced the number of human platelets but not megakaryocytes,and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2R gamma(null) (NOG) mice transplanted with human CD34(+) cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic,NIP-004,was effective for treating IFN-alpha-induced thrombocytopenia in hu-NOG mice. From ultrastructural study,IFN-alpha inhibited the maturation of demarcation membranes in megakaryocytes,although NIP-004 prevented the inhibitory effects of IFN-alpha. These results defined the pathogenesis of IFN-alpha-induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics. View Publication

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently,we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e.,39 degrees C). In this study,we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation,and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days,and that early transient culture at 39 degrees C led to even greater Mk yields (ptextless0.03). Surprisingly,cell viability was only found to be significantly reduced in the early stages of culture,suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks,it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely,gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally,two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors. View Publication

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood,as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow,detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al,2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens,with particular attention to the expression of ALDH on erythroid precursors. To this aim,we used three kinds of approach: i) multidimensional analytical flow cytometry,detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells,followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow,we identified three populations of cells,namely ALDH+CD34+,ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52,0.53 and 0.57,respectively). As compared to ALDH-CD34+ cells,ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells,with brighter expression of CD117 and CD133,accompanied by lower display of CD38 and CD45RA. Of interest,ALDH+CD34- population disclosed a straightforward erythroid commitment,on the basis of three orders of evidences. First of all,ALDH+CD34- cells showed a CD71bright,CD105+,CD45- phenotype. Secondly,induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally,ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study,comparing surface antigen expression of ALDH+/CD34+,ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow,clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. View Publication

The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However,the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis,as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver,we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies,we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population,followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis. View Publication

BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53,eventually causing resistance to imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides,resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML,we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore,MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG),which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival,accumulation of p53 but not of p73,and lack of activation of caspase 3 after MNNG treatment. In contrast,parental cells displayed accumulation of p53,p73,and activation of caspase 3,resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C--textgreaterA:T and A:T--textgreaterG:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion,these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype. View Publication

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs),including polycythemia vera,essential thrombocythemia,and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice,which expressed a lower level of JAK2(V617F),showed moderate elevations of blood cell counts,whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid,megakaryocytic,and granulocytic hyperplasia in the bone marrow and spleen,displayed splenomegaly,and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood,spleen,and bone marrow,and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs. View Publication

Mef2c is a MADS (MCM1-agamous-deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML,this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis,whereas BM isolated from Mef2c(Delta/-) mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun,but not PU.1,C/EBPalpha,or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover,retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation,coupled with its functional sensitivity to extracellular stimuli,demonstrate an important role in immunity--and,consistent with findings of other myeloid transcription factors,a target of oncogenic lesions in AML. View Publication

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication