技术资料

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

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end,we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells,and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo,while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence,we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase,the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings,two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly,these cocktails were either deprived of thrombopoietin or of stem cell factor,two cytokines known to favor megakaryopoiesis and HPC expansion,respectively. Based on these results,a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established. View Publication

The c-Myb gene encodes a transcription factor required for proliferation and survival of normal myeloid progenitors and leukemic blast cells. Targeting of c-Myb by antisense oligodeoxynucleotides has suggested that myeloid leukemia blasts (including chronic myelogenous leukemia [CML]-blast crisis cells) rely on c-Myb expression more than normal progenitors,but a genetic approach to assess the requirement of c-Myb by p210(BCR/ABL)-transformed hematopoietic progenitors has not been taken. We show here that loss of a c-Myb allele had modest effects (20%-28% decrease) on colony formation of nontransduced progenitors,while the effect on p210(BCR/ABL)-expressing Lin(-) Sca-1(+) and Lin(-) Sca-1(+)Kit(+) cells was more pronounced (50%-80% decrease). Using a model of CML-blast crisis,mice (n = 14) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/w) marrow cells developed leukemia rapidly and had a median survival of 26 days,while only 67% of mice (n = 12) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/d) marrow cells died of leukemia with a median survival of 96 days. p210(BCR/ABL)-transduced c-Myb(w/w) and c-Myb(w/d) marrow progenitors expressed similar levels of the c-Myb-regulated genes c-Myc and cyclin B1,while those of Bcl-2 were reduced. However,ectopic Bcl-2 expression did not enhance colony formation of p210(BCR/ABL)-transduced c-Myb(w/d) Lin(-)Sca-1(+)Kit(+) cells. Together,these studies support the requirement of c-Myb for p210(BCR/ABL)-dependent leukemogenesis. View Publication

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB),cocultured with neonatal mouse cardiomyocytes,acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days,mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions,acquired electrophysiological properties similar to those of cardiomyocytes,and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However,RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion,under our experimental conditions,hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However,the expression of human-specific cardiac genes was undetectable by RT-PCR. View Publication

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy,but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular,the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that,together with other factors,can expand mouse bone marrow HSCs in culture. Here,we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF,TPO,and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers,as assayed by NOD/SCID transplantation. A serum-free culture containing SCF,TPO,FGF-1,angiopoietin-like 5,and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs,a number potentially applicable to several clinical processes including HSC transplantation. View Publication

MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9,the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA,and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils,diffusely distributed throughout lymphocyte cytoplasm,sparsely localized on a diffuse cytoplasmic background in monocytes,and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34(+) cells of patients,but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC-IIA and postulate that cell-specific regulation mechanisms function in MYH9 disorders. View Publication

Platelets are an abundant source of CD40 ligand (CD154),an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases,including systemic lupus erythematosus (SLE),diabetes,and cardiovascular disease. Heretofore considered largely restricted to activated T cells,we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells,megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore,using several established megakaryocyte-like cells lines,we performed promoter analysis of the CD154 gene and found that NFAT,a calcium-dependent transcriptional regulator associated with activated T cells,mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall,these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated. View Publication

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells,we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model,donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly,highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer,accumulated in vivo at advanced proliferative cycles,and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted,apoptosis-resistant phenotype,including: 1) reduced apoptosis after staurosporine addition,serum starvation,or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection,we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4,IL-10,perforin,or Fas ligand; 2) could not be reversed by IL-2,IL-7,or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis,persist in vivo,and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells. View Publication