技术资料

The Lyn tyrosine kinase plays essential inhibitory signaling roles within hematopoietic cells by recruiting inhibitory phosphatases such as SH2-domain containing phosphatase-1 (SHP-1),SHP-2,and SH2-domain containing 5'-inositol phosphatase (SHIP-1) to the plasma membrane in response to specific stimuli. Lyn-deficient mice display a collection of hematopoietic defects,including autoimmune disease as a result of autoantibody production,and perturbations in myelopoiesis that ultimately lead to splenomegaly and myeloid neoplasia. In this study,we demonstrate that loss of Lyn results in a stem/progenitor cell-intrinsic defect leading to an age-dependent increase in myeloid,erythroid,and primitive hematopoietic progenitor numbers that is independent of autoimmune disease. Despite possessing increased numbers of erythroid progenitors,and a more robust expansion of these cells following phenylhydrazine challenge,Lyn-deficient mice are more severely affected by the chemotherapeutic drug 5-fluorouracil,revealing a greater proportion of cycling progenitors. We also show that mice lacking SHIP-1 have defects in the erythroid and myeloid compartments similar to those in mice lacking Lyn or SHP-1,suggesting an intimate relationship between Lyn,SHP-1,and SHIP-1 in regulating hematopoiesis. View Publication

Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood,but also contributed to mature muscle fibers. However,the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage,they do not include mature CD11b(+) myelomonocytic cells,such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture,bone marrow,peripheral blood after granulocyte colony-stimulating factor mobilization,or injured muscle,none contributed to muscle. In addition,after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG),no GFP myofibers were detected,demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms,these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors. View Publication

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs),whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac,fetal liver,and adult bone marrow. However,HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also,cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus,GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow. View Publication

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However,a small number of regenerated myofibers were detected,suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally,we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore,we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells. View Publication

Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha,a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation,inhibited proliferation in vitro and in mice,and suppressed leukemogenesis. Moreover,activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells,respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis,as indicated by the effects of p42 C/EBPalpha,which were more potent than those of K298E C/EBPalpha,a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC,including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation,also induced granulocyte differentiation. Thus,these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors,and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. View Publication

The pathogenesis of malarial anemia is multifactorial,and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller,K.L.,J.C. Schooley,K.L. Smith,B. Kullgren,L.J. Mahlmann,and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap,G.S.,and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients,MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon,which are known antagonists of hematopoiesis,even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production,and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia,improved erythroid progenitor development,and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications. View Publication

In this study,we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells,in serum-free medium,supplemented only with fibroblast growth factor (FGF)-1,FGF-2,or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays,high levels of stem cell activity were detectable at 1,3,and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However,cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant,showing that HSC activity originated from LSK cells. Subsequently,we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules,stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response,inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly,although HSC activity is typically rapidly lost after short-term culture in vitro,our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells,its physiological function(s) remain unknown,and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations,followed by competitive bone marrow reconstitution assays,shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent,the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore,PrP is a marker for HSCs and supports their self-renewal. View Publication

Flt3 ligand (Flt3L) is a nonredundant cytokine in type I interferon-producing cell (IPC) and dendritic cell (DC) development,and IPC and DC differentiation potential is confined to Flt3+ hematopoietic progenitor cells. Here,we show that overexpression of human Flt3 in Flt3- (Flt3(-)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) and Flt3+ (Flt3(+)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) hematopoietic progenitors rescues and enhances their IPC and DC differentiation potential,respectively. In defined hematopoietic cell populations,such as Flt3- megakaryocyte/erythrocyte-restricted progenitors (MEPs),enforced Flt3 signaling induces transcription of IPC,DC,and granulocyte/macrophage (GM) development-affiliated genes,including STAT3,PU.1,and G-/M-/GM-CSFR,and activates differentiation capacities to these lineages. Moreover,ectopic expression of Flt3 downstream transcription factors STAT3 or PU.1 in Flt3- MEPs evokes Flt3 receptor expression and instructs differentiation into IPCs,DCs,and myelomonocytic cells,whereas GATA-1 expression and consecutive megakaryocyte/erythrocyte development is suppressed. Based on these data,we propose a demand-regulated,cytokine-driven DC and IPC regeneration model,in which high Flt3L levels initiate a self-sustaining,Flt3-STAT3- and Flt3-PU.1-mediated IPC and DC differentiation program in Flt3+ hematopoietic progenitor cells. View Publication

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH),hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon,we investigated the usefulness of ALDH activity for characterizing HSC graft quality,particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time,and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly,in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells,this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism,whereas all other patients showed early complete donor chimerism. In conclusion,we suggest to measure ALDH activity as a surrogate marker for HSC activity,and to transport and store PBSC under controlled cooling conditions. View Publication

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication