技术资料

Gene therapy for a wide variety of disorders would be greatly enhanced by the development of vectors that could be targeted for gene delivery to specific populations of cells. We describe here high-efficiency targeted transduction based on a novel targeting strategy that exploits the ability of retroviruses to incorporate host cell proteins into the surface of the viral particle as they bud through the plasma membrane. Ecotropic retroviral particles produced in cells engineered to express the membrane-bound form of stem cell factor (mbSCF) transduce both human cell lines and primary cells with high efficiency in a strictly c-kit (SCF receptor)-dependent fashion. The availability of efficient targeted vectors provides a platform for the development of a new generation of therapies using in vivo gene delivery. View Publication

Stem cells have been identified and characterized in a variety of tissues. In this review we examine possible shared properties of stem cells. We suggest that irrespective of their lineal origin,stem cells have to respond in similar ways to regulate self-renewal and differentiation and it is likely that cell-cycle control,asymmetry/differentiation controls,cellular protective and DNA repair mechanisms,and associated apoptosis/senescence signaling pathways all might be expected to be more highly regulated in stem cells,likely by similar mechanisms. We review the literature to suggest a set of candidate stemness genes that may serve as universal stem cell markers. While we predict many similarities,we also predict that differences will exist between stem cell populations and that when transdifferentiation is considered genes expected to be both similar and different need to be examined. View Publication

Internal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant,implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3,KIT,and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells,we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions,after chemotherapy-induced myelosuppression,and during bone marrow transplantation. In these assays,we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML,at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL-positive acute leukemia,MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols. View Publication

The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell,but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system,we have demonstrated a consistent defect in DBA,regardless of clinical severity,including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1,but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells,and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone,Epo sensitivity was comparable to normal,but erythroid expansion remained subnormal. In clonogenic phase 2 cultures,the number of colonies did not significantly differ between normal cultures and DBA,in the presence or absence of dexamethasone,and at both low and high Epo concentrations. However,colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA,and the defect lies down-stream of the Epo receptor,influencing survival and/or proliferation of erythroid progenitors. View Publication

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses,the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation,with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement,which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore,polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together,our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms. View Publication

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture,purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population,compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse,suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

Mammalian presenilins (PS) consist of two highly homologous proteins,PS1 and PS2. Because of their indispensable activity in the gamma-secretase cleavage of amyloid precursor protein to generate Abeta peptides,inhibition of PS gamma-secretase activity is considered a potential therapy for Abeta blockage and Alzheimer's disease intervention. However,a variety of other substrates are also subject to PS-dependent processing,and it is thus imperative to understand the consequences of PS inactivation in vivo. Here we report a pivotal role of PS in hematopoiesis. Mice heterozygous for PS1 and homozygous for PS2 (PS1(+/)(-)PS2(-)(/)(-)) developed splenomegaly with severe granulocyte infiltration. This was preceded by an overrepresentation of granulocytic cells in the bone marrow and a greatly increased multipotent granulocyte-monocyte progenitor in the spleen. In contrast,hematopoietic stem cells and T- and B-lymphocytes were not affected. Importantly,treatment of wild-type splenocytes with a gamma-secretase inhibitor directly promoted the granulocyte-macrophage colony-forming unit (GM-CFU). These results establish a critical role of PS in myelopoiesis. Our finding that this activity can be directly modulated by its gamma-secretase activity has important safety implications concerning these inhibitors. View Publication

Angiotensin I-converting enzyme (ACE) inhibitors can affect hematopoiesis by several mechanisms including inhibition of angiotensin II formation and increasing plasma concentrations of AcSDKP (acetyl-N-Ser-Asp-Lys-Pro),an ACE substrate and a negative regulator of hematopoiesis. We tested whether ACE inhibition could decrease the hematopoietic toxicity of lethal or sublethal irradiation protocols. In all cases,short treatment with the ACE inhibitor perindopril protected against irradiation-induced death. ACE inhibition accelerated hematopoietic recovery and led to a significant increase in platelet and red cell counts. Pretreatment with perindopril increased bone marrow cellularity and the number of hematopoietic progenitors (granulocyte macrophage colony-forming unit [CFU-GM],erythroid burst-forming unit [BFU-E],and megakaryocyte colony-forming unit [CFU-MK]) from day 7 to 28 after irradiation. Perindopril also increased the number of hematopoietic stem cells with at least a short-term reconstitutive activity in animals that recovered from irradiation. To determine the mechanism of action involved,we evaluated the effects of increasing AcSDKP plasma concentrations and of an angiotensin II type 1 (AT1) receptor antagonist (telmisartan) on radioprotection. We found that the AT1-receptor antagonism mediated similar radioprotection as the ACE inhibitor. These results suggest that ACE inhibitors and AT1-receptor antagonists could be used to decrease the hematopoietic toxicity of irradiation. View Publication

Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies,resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98,and more than one half of these are homeobox transcription factors. By contrast,the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1),a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors,evidenced by a competitive growth advantage in liquid culture,increased replating efficient of colony-forming cells (CFCs),and a marked increase in spleen colony-forming cell output. Moreover,in a murine bone marrow transplantation model,NUP98-TOP1 expression led to a lethal,transplantable leukemia characterized by extremely high white cell counts,splenomegaly,and mild anemia. Strikingly,a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings,together with similar biologic effects reported for NUP98-HOX fusions,suggest unexpected,overlapping functions of NUP98 fusion genes,perhaps related to common DNA binding properties. View Publication

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment,and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells,viral gene expression was undetectable by 10 days after infection. Importantly,viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly,a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency. View Publication