技术资料

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture,they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands,a process which we call vasculogenesis,was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum,fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response,which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis,the development of blood vessels from in situ differentiating endothelial cells,and angiogenesis,the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated. View Publication

A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore,it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus,the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells,as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover,fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells,as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally,fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides,including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. View Publication

A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences,these two clones encode the same 520 amino acid residue protein,which is preceded by a 32-amino acid residue signal peptide. The two clones,whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts,contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay. View Publication

Three new monoclonal anti-cytokeratin antibodies (mabs) potentially useful in cancer research and clinical diagnosis have been evaluated in immuno-histochemistry on cryostat sections of a broad variety of normal human tissues. A45-B/B3 reacts with all cells containing cytokeratins (epithelia and mesothelia). This mab positively identifies epithelial cells of any kind,and it may serve in differentiating carcinomas from tumours of mesenchymal origin. A53-B/A2 recognizes an individual cytokeratin,No. 19,and stains preferably mesothelia,urothelium,and bile duct epithelium. This antibody is suited to discriminate between different epithelial lineages. A51-B/H4 reacts with a subgroup of cytokeratins (probably including Nos. 14,8 and/or 18). It is positive with most epithelia but negative with keratanized stratified epithelium. This antibody shows an interesting,but up to now unexplained,cross-reactivity with nuclei of certain nonepithelial cells. All three mabs also react with epithelial cells from at least three animal species. View Publication

BACKGROUND/AIMS: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success,however,is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. METHODS: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC),and structural derivatives thereof,on lineage-committed progenitor cells by various molecular biological,biochemical,and functional assays. RESULTS: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly,the compound also induced cardiac functional properties: first,cardiac-like sodium currents in skeletal myoblasts,and secondly,spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. CONCLUSION: CgC induces cardiomyogenic function in lineage-committed progenitor cells,and can thus be considered a promising tool to improve cardiac repair by cell therapy. View Publication

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells,suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study,we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC),which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore,anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells,indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs,both as a monotherapy and in combination with lenalidomide,to improve MM patient outcome. View Publication

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology,immunophenotype and multi-lineage potential were analyzed at passages 3,5,10,15,20,and 25 (P3,P5,P10,P15,P20,and P25,respectively). The cell cycle distribution,apoptosis,and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3,P5,P10,P15,P20,and P25. From P3 to P25,the three defining biological properties of hUC-MSCs [adherence to plastic,specific surface antigen expression,multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased,compared with the cells at P3 (P textless 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %),compared to those at P3 (P textless 0.01). Within subculture generations 3-20 (P3-P20),the differences between the cell apoptotic rates were not statistically significant (P textgreater 0.05). There were no detectable chromosome eliminations,displacements,or chromosomal imbalances,as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN,2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations. View Publication

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator,hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces,eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids,however,it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study,neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient,whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly,while pathogen induced neutrophil trans-epithelial migration was substantial,pathogen induced eosinophil trans-epithelial migration was not observed. Further,gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers,whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection,HXA3 does not appear to exhibit chemotactic activity toward eosinophils. ?? 2013 Elsevier Ltd. All rights reserved. View Publication

Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However,existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs,we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs,using size-controlled embryoid bodies (EBs),and addition of triiodothyronine (T3) and taurine to the differentiation medium,with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation,which will help understand retinal diseases and facilitate large-scale preclinical trials. View Publication

Circulating levels of leptin are elevated in type-2 diabetes mellitus (T2DM) and leptin plays a role in immune responses. Elevated circulating IL-18 levels are associated with clinical complications of T2DM. IL-18 regulates cytokine secretion and the function of a number of immune cells including T-cells,neutrophils and macrophages and as such has a key role in immunity and inflammation. Pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM,however,little is known about the role of leptin in modifying monocyte IL-18 secretion. We therefore aimed to investigate the effect of leptin on IL-18 secretion by monocytes. We report herein that leptin increases IL-18 secretion in THP-1 and primary human monocytes but has no effect on IL-18mRNA. Leptin and LPS signalling in monocytes occurs by overlapping but distinct pathways. Thus,in contrast to a strong stimulation by LPS,leptin has no effect on IL-1βmRNA levels or IL-1β secretion. In addition,LPS stimulates the secretion of IL-6 but leptin did not whereas both treatments up regulate IL-8 secretion from the same cells. Although leptin (and LPS) has a synergistic effect with exogenous ATP on IL-18 secretion in both THP-1 and primary monocytes,experiments involving ATP assays and pharmacological inhibition of ATP signalling failed to provide any evidence that endogenous ATP secreted by leptin-stimulated monocytes was responsible for enhancement of monocyte IL-18 secretion by leptin. Analysis of the action of caspase-1 revealed that leptin up regulates caspase-1 activity and the effect of leptin on IL-18 release is prevented by caspase-1 inhibitor (Ac-YVAD-cmk). These data suggest that leptin activates IL-18 processing rather than IL-18 transcription. In conclusion,leptin enhances IL-18 secretion via modulation of the caspase-1 inflammasome function and acts synergistically with ATP in this regard. This process may contribute to aberrant immune responses in T2DM and other conditions of hyperleptinemia. View Publication

An immunohistological study in the human breast and the rodent breast (from inbred Ludwig/Wistar/Olac rats) was conducted with the use of a murine monoclonal antibody,which reacts with the common acute lymphoblastic antigen,a glycosylated polypeptide of a molecular weight of 100,000. The epitope,as recognized by this antibody,is expressed on myoepithelial cells of the normal human and rat breasts and was studied in the developing rodent mammary gland. Ultrastructural studies in the normal human breast clearly demonstrated the presence of the antigen on the lateral membrane of the myoepithelial cells with no staining of luminal cells,blood vessels,or stromal elements. The antigen survived prolonged enzymatic digestion of human breast tissue and could be demonstrated on myoepithelial cells in single-cell suspensions of human breast where it stained approximately 3-14% of the total cell population. The presence of this antigen on myoepithelial cells is discussed in the context of myoepithelial differentiation in the breast and the potential utility of the antibodies for cell separation. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication