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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

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

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

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication

Human B cells can be cultured ex vivo for a few weeks,following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However,attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells,recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4,the main LPS receptor,prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells,accompanied by an increase in antigen-specific antibody secretion. This result suggested that some,but not all,B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6,a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo,the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria,until all pathogens have been cleared from the organism. View Publication

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

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However,the critical downstream targets of Scl remain undefined. Here,we identified a novel Scl target gene,transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C,in contrast to Scl,is not essential for specification into primitive or definitive hematopoietic lineages. However,adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced,whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore,megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells,but not in erythroid cells. In addition,an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice,characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary,this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. 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

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 development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity,a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells,mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue,suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus,human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. View Publication

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However,transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT),driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs,which can be administered to kill residual untransduced,diseased HSCs,whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells,transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin,leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia. View Publication