技术资料

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

Trib1,Trib2,and Trib3 are mammalian homologs of Tribbles,an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice,whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members,we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia,whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor,CCAAT/enhancer-binding protein-alpha (C/EBPalpha),is important for leukemogenesis. Similar to Trib2,Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast,Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha,which account for their differential ability to induce leukemia. View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

OBJECTIVES: In this study,we hypothesized that an antihuman-CD34 antibody immobilized on the surface of commercially available sirolimus-eluting stents (SES) could enhance re-endothelialization compared with SES alone. BACKGROUND: Previous experience with antihuman-CD34 antibody surface modified Genous stents (GS) (OrbusNeich Medical,Fort Lauderdale,Florida) has shown enhanced stent endothelialization in vivo. METHODS: In the phase 1 study,stents were deployed in 21 pig coronary arteries for single stenting (9 vessels: 3 GS,3 SES,and 3 bare-metal stents) and overlapping stenting with various combinations (12 vessels: 4 GS+GS,4 SES+SES,and 4 GS+SES) and harvested at 14 days for scanning electron and confocal microscopy. In phase 2,immobilized anti-CD34 antibody coating was applied on commercially available SES (SES-anti-CD34,n = 7) and compared with GS (n = 8) and SES (n = 7) and examined at 3 and 14 days by scanning electron/confocal microscopy analysis. RESULTS: In phase 1,single stent implantation showed greatest endothelialization in GS (99%) and in bare-metal stent (99%) compared with SES (55%,p = 0.048). In overlapping stents,endothelialization at the overlapping zone was significantly greater in GS+GS (95 +/- 6%) and GS+SES (79 +/- 5%) compared with the SES+SES (36 +/- 14%) group (p = 0.007). In phase 2,SES-anti-CD34 resulted in increased endothelialization compared with SES alone at 3 days (SES-anti-CD34 36 +/- 26%; SES 7 +/- 3%; and GS 76 +/- 8%; p = 0.01),and 14 days (SES-anti-CD34 82 +/- 8%; SES 53 +/- 20%; and GS 98 +/- 2%; p = 0.009). CONCLUSIONS: Immobilization of anti-CD34 antibody on SES enhances endothelialization and may potentially be an effective therapeutic alternative to improve currently available drug-eluting stents. View Publication

Recent studies support the notion that there is an intricate relationship between hematopoiesis and bone homeostasis in normal steady states. Using mice undergoing chronic inflammatory arthritis,we investigated the relationship between hematopoiesis and bone homeostasis in pathologic conditions. We demonstrate that mice undergoing chronic inflammatory arthritis displayed osteoporosis resulting from a severe defect in osteoblast function. Despite the defective osteoblast function,however,the hematopoietic stem cells from these mice exhibited normal properties in either long-term repopulation or cell cycling. Therefore,the bone-forming capacity of osteoblasts is distinct from their ability to maintain hematopoietic stem cells in chronic inflammatory conditions. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication

In ischemic congestive heart failure (CHF),anemia is associated with poor prognosis. Whether anemia develops in nonischemic CHF is uncertain. The hematopoietic inhibitors TNF-alpha and nitric oxide (NO) are activated in ischemic CHF. We examined whether mice with ischemic or nonischemic CHF develop anemia and whether TNF-alpha and NO are involved. We studied mice (n = 7-9 per group) with CHF either due to myocardial infarction (MI) or to overexpression of the Ca(2+)-binding protein calsequestrin (CSQ) or to induced cardiac disruption of the sarcoplasmic reticulum Ca(2+)-ATPase 2 gene (SERCA2 KO). Hematopoiesis was analyzed by colony formation of CD34(+) bone marrow cells. Hemoglobin concentration was 14.0 +/- 0.4 g/dl (mean +/- SD) in controls,while it was decreased to 10.1 +/- 0.4,9.7 +/- 0.4,and 9.6 +/- 0.3 g/dl in MI,CSQ,and SERCA2 KO,respectively (P textless 0.05). Colony numbers per 100,000 CD34(+) cells in the three CHF groups were reduced to 33 +/- 3 (MI),34 +/- 3 (CSQ),and 39 +/- 3 (SERCA2 KO) compared with 68 +/- 4 in controls (P textless 0.05). Plasma TNF-alpha nearly doubled in MI,and addition of anti-TNF-alpha antibody normalized colony formation. Inhibition of colony formation was completely abolished with blockade of endothelial NO synthase in CSQ and SERCA2 KO,but not in MI. In conclusion,the mechanism of anemia in CHF depends on the etiology of cardiac disease; whereas TNF-alpha impairs hematopoiesis in CHF following MI,NO inhibits blood cell formation in nonischemic murine CHF. View Publication

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units,called poly-LacNAc chains,characterize the histo-blood group i and I antigens,respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus,which expresses 3 IGnT transcripts,IGnTA,IGnTB,and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation,the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue,with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene,consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model,with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen,granulocytic CD15,and also erythroid CD71 antigens. Taken together,these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism,with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role. 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

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells,and Ras activity enhances the oncogenic ability of Bcr-Abl. However,the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction,resulting in blocking the MAP kinase pathway. In the present study,we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl(+) progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status,resulting in inhibited proliferation of CML cells. Moreover,RKIP up-regulated cell cycle regulator FoxM1 expression,resulting in G(1) arrest via p27(Kip1) and p21(Cip1) accumulation. In colony-forming unit granulocyte,erythroid,macrophage,megakaryocyte,colony-forming unit-granulocyte macrophage,and burst-forming unit erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions,and inhibited colony formation of Bcr-Abl(+) progenitor cells,whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl(+) progenitor cells. Thus,Bcr-Abl represses the expression of RKIP,continuously activates pERK1/2,and suppresses FoxM1 expression,resulting in proliferation of CML cells. View Publication