技术资料

Platelets are an abundant source of CD40 ligand (CD154),an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases,including systemic lupus erythematosus (SLE),diabetes,and cardiovascular disease. Heretofore considered largely restricted to activated T cells,we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells,megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore,using several established megakaryocyte-like cells lines,we performed promoter analysis of the CD154 gene and found that NFAT,a calcium-dependent transcriptional regulator associated with activated T cells,mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall,these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated. View Publication

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system,receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however,an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects,whereby LPA receptor-expressing cells exposed to,or primed� View Publication

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations,which is lacking in the field,is also crucial. Here,we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine,a small molecule that activates the SHH pathway,could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+,Irx3+,and Pax7-),with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus,the directed neural differentiation system with small molecules,even without further purification,will facilitate basic and translational studies using human motoneurons at a minimal cost. View Publication

We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore,the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival,and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB. View Publication

View Publication

The HER family receptors have an important role controlling cell growth and differentiation. Although the activity of the HER-2 receptor is strictly controlled in normal cells,its overexpression plays a pivotal role in transformation and tumorigenesis. Constitutive phosphorylation of HER-2 protein has been implicated in conferring uncontrolled growth to mammary cancer cells,and to a lesser extent,with adenocarcinoma of uterus,cervix,fallopian tube,and endometrium. This study addresses the role of HER-2 in cervical carcinoma. Firstly,we demonstrate the presence of HER-2 protein expression by flow cytometry in two new cervical carcinoma cell lines CALO and INBL. Secondly,we use the specific tyrosine kinase inhibitors,Tyrphostins to examine HER-2 regulation by the crystal violet assay. Thirdly,we use western blot analysis to assess the state of HER-2 phosphorylation. The most efficient agent,Tyrphostin B42,known as an inhibitor of epithelial growth factor receptor,arrested cervical carcinoma cell lines growth in vitro at micromolar concentrations within 72 h of application. Tyrphostin B42 inhibited the HER2 signal-regulated kinase pathway,as observed by the reduction in the phosphorylated forms of HER2. The loss of phosphorylated forms of HER2 at early time points after Tyrphostin B42 application was associated with suppression of cell growth. Thus,the inhibition of the proliferation of our cervical carcinoma cell lines by Tyrphostin B42 is associated with inhibition of HER2 protein kinase signal. View Publication

BACKGROUND: Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However,the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus,alternative sources of mesenchymal stromal cells need to be explored. In this study,mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product. DESIGN AND METHODS: Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production. RESULTS: The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture,we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo. CONCLUSIONS: Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid,showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres,immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required. View Publication

View Publication

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific,robust,and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of textgreateror=30 AP(+) cells that coexpress OCT4,NANOG,SSEA3,SSEA4,TRA-1-60,and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition,including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly,this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple,reliable,broadly applicable,and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation. View Publication

ROCK kinases,which play central roles in the organization of the actin cytoskeleton,are tantalizing targets for the treatment of human diseases. Deletion of ROCK I in mice revealed a role in the pathophysiological responses to high blood pressure,and validated ROCK inhibition for the treatment of specific types of cardiovascular disease. To date,the only ROCK inhibitor employed clinically in humans is fasudil,which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm. Clinical trials,mostly focusing on the cardiovascular system,have uncovered beneficial effects of fasudil for additional indications. Intriguing recent findings also suggest significant potential for ROCK inhibitors in the production and implantation of stem cells for disease therapies. View Publication

Mef2c is a MADS (MCM1-agamous-deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML,this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis,whereas BM isolated from Mef2c(Delta/-) mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun,but not PU.1,C/EBPalpha,or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover,retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation,coupled with its functional sensitivity to extracellular stimuli,demonstrate an important role in immunity--and,consistent with findings of other myeloid transcription factors,a target of oncogenic lesions in AML. View Publication

Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens,epithelial membrane antigen (EMA) and the common acute lymphoblastic leukaemia antigen (CALLA/CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture,differences which were enhanced by the addition of hydrocortisone,insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positive cells,by contrast,formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle alpha-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However,a significant minority (approximately 15%) of sorted luminal cells consistently expressed the basal-cell marker cytokeratin 14 in culture. A marked difference was noted in the proliferative behaviour of the two types of sorted cells,with myoepithelial cells dividing rapidly in response to the humoural additives,in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepithelial cells were also distinctive; all spread" luminal clones were similar in appearance to each other� View Publication