参考文献

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

OBJECTIVES: We tested the hypothesis that asymmetric dimethylarginine (ADMA) may be an endogenous inhibitor of endothelial progenitor cells (EPCs). BACKGROUND: Endothelial progenitor cells play a pivotal role in regeneration of injured endothelium,thereby limiting the formation of atherosclerotic lesions. Reduced numbers of EPCs may affect progression of coronary artery disease. Regulation of EPC mobilization and function is mediated in part by nitric oxide (NO). Endogenous inhibitors of NO synthases,such as ADMA,contribute to endothelial dysfunction and injury. METHODS: We used flow cytometry and in vitro assays to investigate the relationship between EPC number and function with ADMA plasma levels in patients with stable angina. RESULTS: The plasma concentration of ADMA was related to the severity of coronary artery disease and correlated inversely with the number of circulating CD34+/CD133+ progenitor cells (r = -0.69; p textless 0.0001) and endothelial colony forming units (CFUs) (r = -0.75; p textless 0.0001). Adjusting for all patient characteristics,we confirmed these findings in multivariate regression analyses. In vitro differentiation of EPCs was repressed by ADMA in a concentration-dependent manner. Compared with untreated cells,ADMA reduced EPC incorporation into endothelial tube-like structures to 27 +/- 11% (p textless 0.001). Asymmetric dimethylarginine repressed the formation of CFUs from cultured peripheral blood mononuclear cells to 35 +/- 7% (p textless 0.001). Asymmetric dimethylarginine decreased endothelial nitric oxide synthase activity in EPCs to 64 +/- 6% (p textless 0.05) when compared with controls. Co-incubation with the hydroxymethyl glutaryl coenzyme A reductase inhibitor rosuvastatin abolished the detrimental effects of ADMA. CONCLUSIONS: Asymmetric dimethylarginine is an endogenous inhibitor of mobilization,differentiation,and function of EPCs. This contributes to the cardiovascular risk in patients with high ADMA levels and may explain low numbers and function of EPCs in patients with coronary artery disease. View Publication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia,an aggressive CD4(+) malignancy. Although HTLV-2 is highly homologous to HTLV-1,infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34(+) cells with HTLV-1 Tax (Tax1) induced G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (p27),and marked suppression of hematopoiesis in immature (CD34(+)/CD38(-)) hematopoietic progenitor cells in comparison to CD34(+)/CD38(+) cells. HTLV-1 infection of CD34(+) cells also induced p21 and p27 expression. Tax1 also protected CD34(+) cells from serum withdrawal-mediated apoptosis. In contrast,HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression,failed to induce cell cycle arrest,failed to suppress hematopoiesis in CD34(+) cells,and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax(221)) displayed a phenotype in CD34(+) cells similar to that of Tax1,suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34(+) hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2. View Publication

It is often predicted that stem cells divide asymmetrically,creating a daughter cell that maintains the stem-cell capacity,and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg,in Drosophila),it remains illusive whether primitive hematopoietic cells in mammals actually can divide asymmetrically. In our experiments we have challenged this question and analyzed the developmental capacity of separated offspring of primitive human hematopoietic cells at a single-cell level. We show for the first time that the vast majority of the most primitive,in vitro-detectable human hematopoietic cells give rise to daughter cells adopting different cell fates; 1 inheriting the developmental capacity of the mother cell,and 1 becoming more specified. In contrast,approximately half of the committed progenitor cells studied gave rise to daughter cells,both of which adopted the cell fate of their mother. Although our data are compatible with the model of asymmetric cell division,other mechanisms of cell fate specification are discussed. In addition,we describe a novel human hematopoietic progenitor cell that has the capacity to form natural killer (NK) cells as well as macrophages,but not cells of other myeloid lineages. View Publication

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease,we identified GW2580,an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts,rat calvaria,and rat fetal long bone. In contrast,GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells,human endothelial cells,human fibroblasts,or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF,IL-6,and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration,GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice,inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity,and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly,GW2580 inhibited LPS-induced TNF production in mice,in contrast to effects on monocytes and macrophages in vitro. In conclusion,GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes. View Publication

In recent years,mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However,no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells,whereas they only partially affect the proliferation of activated NK cells. In addition,we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30,NKG2D,and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly,MSCs expressed the known ligands for these activating NK receptors-ULBPs,PVR,and Nectin-2. Moreover,NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation. View Publication

TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6,a frequent target of chromosome translocation in human leukemia. Although both proteins are transcriptional inhibitors binding similar DNA recognition sequences,they have opposite biologic effects: TEL inhibits proliferation while TEL2 promotes it. In addition,forced expression of TEL2 but not TEL blocks vitamin D3-induced differentiation of U937 and HL60 myeloid cells. TEL2 is expressed in the hematopoietic system,and its expression is up-regulated in bone marrow samples of some patients with leukemia,suggesting a role in oncogenesis. Recently we also showed that TEL2 cooperates with Myc in B lymphomagenesis in mice. Here we show that forced expression of TEL2 alone in mouse bone marrow causes a myeloproliferative disease with a long latency period but with high penetrance. This suggested that secondary mutations are necessary for disease development. Treating mice receiving transplants with TEL2-expressing bone marrow with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) resulted in significantly accelerated disease onset. Although the mice developed a GFP-positive myeloid disease with 30% of the mice showing elevated white blood counts,they all died of T-cell lymphoma,which was GFP negative. Together our data identify TEL2 as a bona fide oncogene,but leukemic transformation is dependent on secondary mutations. View Publication

The present study examined whether nestin+ neural-like stem cells detected in the scar tissue of rats 1 week after myocardial infarction (MI) were derived from bone marrow and/or were resident cells of the normal myocardium. Irradiated male Wistar rats transplanted with beta-actin promoter-driven,green fluorescent protein (GFP)-labeled,unfractionated bone marrow cells were subjected to coronary artery ligation. Three weeks after MI,GFP-labeled bone marrow cells were detected in the infarct region,and a modest number were associated with nestin immunoreactivity. The paucity of GFP+/nestin+ cells in the scar tissue provided the impetus to explore whether neural-like stem cells were derived from cardiac tissue. Nestin mRNA and immunoreactivity were detected in normal rat myocardium,and transcript levels were increased in the damaged heart after MI. In primary-passage,cardiac tissue-derived neural cells,filamentous nestin staining was associated with a diffuse,cytoplasmic glial fibrillary acidic protein signal. Unexpectedly,in viable myocardium,numerous nestin+/glial fibrillary acidic protein+ fiberlike structures of varying length were detected and observed in close proximity to neurofilament-M+ fibers. The infarct region was likewise innervated,and the preponderance of neurofilament-M+ fibers appeared to be physically associated with nestin+ fiberlike structures. These data highlight the novel observation that the normal rat heart contained resident nestin+/glial fibrillary acidic protein+ neural-like stem cells,fiberlike structures,and nestin mRNA levels that were increased in response to myocardial ischemia. Cardiac tissue-derived neural stem cell migration to the infarct region and concomitant nestin+ fiberlike innervation represent obligatory events of reparative fibrosis in the damaged rat myocardium. View Publication

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However,only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a,a glycogen synthase kinase (GSK) 3 inhibitor,fibroblast growth factor (FGF) 2,and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin,and a GSK3 inhibitor,SB216763,mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2,with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells,and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells. View Publication

In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication