技术资料

Several hematopoietic growth factors,including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1),promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology,released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo,allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3,proliferated poorly,and released high levels of IL-10. Interestingly,blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally,DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively,our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically. View Publication

In the adult,platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage,several aspects of megakaryopoiesis,including progenitors,maturing megakaryocytes,and circulating platelets,were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis,we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors,indicating that primitive hematopoiesis is bilineage in nature. Subsequently,definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bbeta-positive cells in the conceptus were identified in the yolk sac at E9.5,while large,highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time,the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites. View Publication

p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow,including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover,the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases,and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes,including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes. View Publication

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl,the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency,M351T and H396P were less potent,and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity,whereas the kinase activity of E255K,H396P,and T315I did not correlate with transforming capabilities,suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants,a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion,leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors. 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

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use,in either the autologous or allogeneic setting,cEPCs should likely be expanded from CB kept frozen in CB banks. In this study,we compared the expansion,functional features,senescence pattern over culture,and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB,while CB units were matched in terms of initial volume,nucleated and CD34(+) cell number. Moreover,the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established,the proliferation,migration,tube formation,and acetylated-LDL uptake potentials were similar in both groups. In addition,cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo,in a hind limb ischemia murine model,cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus,cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However,the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use. View Publication