技术资料

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

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. 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

BACKGROUND Humanized mouse models are still under development,and various protocols exist to improve human cell engraftment and function. METHODS Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34(+) hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM),spleen,and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS Most of the NSG mice tolerated the regimen well. The percentage of hCD45(+) and CD19(+) cells rose significantly in a time-dependent manner. The median percentage of hCD45(+)cells in the BM was 55.5% at week 8,and 67.2% at week 12. The median percentage of hCD45(+) cells in the spleen at weeks 8 and 12 was 42% and 51%,respectively. The median percentage of hCD19(+) cells in BM at weeks 8 and 12 was 21.5% and 39%,respectively (P=0.04). Similarly,the median percentage of hCD19(+) cells in the spleen at weeks 8 and 12 was 10% and 24%,respectively (P=0.04). The percentage of hCD19(+) B cells in PB was 23% at week 12. At week 8,hCD3(+) T cells were barely detectable,while hCD7(+) was detected in the BM and spleen. The percentage of hCD3(+) T cells was 2‒3% at week 12 in the BM,spleen,and PB of humanized NSG mice. CONCLUSION We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45(+) cells than earlier studies without any significant toxicity. And human CD45(+) cell engraftment at week 8 was comparable to that of week 12. View Publication

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid,T,and B lineage lymphoid cells in culture,but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term,stromal-free propagation,and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture,it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition,we demonstrate how it may be experimentally manipulated to generate valuable cell lines. View Publication

MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9,the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA,and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils,diffusely distributed throughout lymphocyte cytoplasm,sparsely localized on a diffuse cytoplasmic background in monocytes,and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34(+) cells of patients,but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC-IIA and postulate that cell-specific regulation mechanisms function in MYH9 disorders. View Publication

The Notch1-RBP-Jkappa and the transcription factor Runx1 pathways have been independently shown to be indispensable for the establishment of definitive hematopoiesis. Importantly,expression of Runx1 is down-regulated in the para-aortic splanchnopleural (P-Sp) region of Notch1- and Rbpsuh-null mice. Here we demonstrate that Notch1 up-regulates Runx1 expression and that the defective hematopoietic potential of Notch1-null P-Sp cells is successfully rescued in the OP9 culture system by retroviral transfer of Runx1. We also show that Hes1,a known effector of Notch signaling,potentiates Runx1-mediated transactivation. Together with the recent findings in zebrafish,Runx1 is postulated to be a cardinal down-stream mediator of Notch signaling in hematopoietic development throughout vertebrates. Our findings also suggest that Notch signaling may modulate both expression and transcriptional activity of Runx1. View Publication

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. 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