胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

本文通过定向诱导人胚胎干细胞分化为心肌细胞,对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测,旨在探索分化过程中细胞能量代谢表型的转换机制.用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞;细胞免疫荧光检测人胚胎干细胞标志物,流式细胞术检测人心肌祖细胞和心肌细胞标志物;应用细胞外流量分析(extracellular flux analysis)方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.研究发现,人胚胎干细胞干性保持稳定,均表达Nanog、OCT4、SOX2细胞标志物;在向心肌分化过程中,第7 d心肌祖细胞标志物Isl1表达99%以上,分化第14 d心肌细胞标志物cTnT表达83%以上;人胚胎干细胞糖酵解代谢能力最强,心肌细胞线粒体功能最强,心肌祖细胞处于两种代谢方式的过度阶段.因此推断,在人胚胎干细胞向心肌细胞分化的过程中,细胞糖酵解能力逐渐减弱,线粒体氧化磷酸化能力逐渐增强,细胞的能量代谢类型发生转变.本研究旨在优化人胚胎干细胞定向分化为心肌细胞的方法,揭示...     

CD4+ T cells mediate murine syngeneic graft-versus-host disease-associated colitis

Syngeneic graft-vs-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA). Following cessation of CsA, this inducible disease is characterized by weight loss, diarrhea, and development of inflammation in the colon and liver. Although nonspecific effector cells and Th1 cytokines have been shown to participate in disease induction, the role of T cells has not been fully elucidated. Initial studies demonstrated significant increases in CD4+ T cells, but not other T cell populations in the colons of diseased animals relative to transplant control animals. To demonstrate a functional linkage between increases in colonic CD4+ T cells and disease induction, in vivo T cell depletion studies were performed. Beginning on the day of bone marrow transplantation, groups of control and CsA-treated animals were treated with mAb against either CD4 or CD8 for 21 days. Treatment with anti-CD4, but not anti-CD8, eliminated clinical symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.     

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

Primary culture and phenotyping of murine chondrocytes

The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.     

Analysis of VEGF--a regulated gene expression in endothelial cells to identify genes linked to angiogenesis

Angiogenesis is important for many physiological processes, diseases, and also regenerative medicine. Therapies that inhibit the vascular endothelial growth factor (VEGF) pathway have been used in the clinic for cancer and macular degeneration. In cancer applications, these treatments suffer from a "tumor escape phenomenon" where alternative pathways are upregulated and angiogenesis continues. The redundancy of angiogenesis regulation indicates the need for additional studies and new drug targets. We aimed to (i) identify novel and missing angiogenesis annotations and (ii) verify their significance to angiogenesis. To achieve these goals, we integrated the human interactome with known angiogenesis-annotated proteins to identify a set of 202 angiogenesis-associated proteins. Across endothelial cell lines, we found that a significant fraction of these proteins had highly perturbed gene expression during angiogenesis. After treatment with VEGF-A, we found increasing expression of HIF-1α, APP, HIV-1 tat interactive protein 2, and MEF2C, while endoglin, liprin β1 and HIF-2α had decreasing expression across three endothelial cell lines. The analysis showed differential regulation of HIF-1α and HIF-2α. The data also provided additional evidence for the role of endothelial cells in Alzheimer's disease.     

Metabolic profiling and biochemical phenotyping of plant systems

Over the last two decades incredible progress has been made in the development of technologies to both create and characterise genetic diversity. In plant systems, the development of knockout populations, transposon insertions and chemical mutagenesis programs has facilitated the generation of an enormous base of diversity. During this period, the elucidation of complete genomes and the rapid development of tools to describe the expression of genes and the protein complement have also been achieved. To complement such studies, methods allowing non-biased, simultaneous and rapid determination of metabolites important in both primary and secondary metabolism have been developed. Our intention here is to review current methodologies for metabolic profiling in plants, highlighting the strategies for biochemical phenotyping of plants by determining the steady-state concentrations of a broad spectrum of metabolites. Emphasis will be placed on cells cultured in vitro, and the future prospects of this rapidly expanding research field will be discussed     

Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions

Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Mechanical phenotyping of stem cells

Elasticity and visco-elasticity are mechanical properties of cells which directly reflect cellular composition, internal structure (cytoskeleton), and external interactions (cell-cell and/or cell-surface). A variety of techniques involving probing, pulling, or deforming cells have been used to characterize these mechanical properties. With continuing advances in the technology, it may be possible to establish mechanical phenotypes that can be used to identify cells at specific points of differentiation and dedifferentiation with direct applications to regenerative medicine, therapeutics, and diagnostics.     

Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.     

Morphological alterations in cultured endothelial cells induced by arachidonic acid

The addition of arachidonic acid (20:4), but not other fatty acids, including the structurally similar eicosapentaenoic acid (20:5), induced specific morphological changes in cultured endothelial cells derived from bovine aorta and pulmonary artery. Cells exhibited a time- and dose-dependent change from their normal, epithelioid morphology to become elongated, polygonal, and spindle-shaped. Cells isolated from aorta appeared more sensitive to these changes than those from pulmonary artery. The effect was observed as early as 12 h after exposure to 20:4, required 48 h for maximal expression, and could be reversed in 2-5 h after change to normal media. The morphological alteration was not observed in cells treated with leukotrienes or PGE2. When cells were pretreated with ibuprofen, aspirin, or indomethacin to block prostaglandin synthesis and then exposed to 20:4, the dose-response effect was shifted to the left. This increased sensitivity to 20:4 suggests either a direct effect of 20:4 on cell morphology or an indirect effect due to metabolites of 20:4 which are not dependent on the cyclooxygenase pathway.     

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 microM Hcy alone or in combination with 2.5 microM genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting. Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation; and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect on apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action.     

A novel method to establish primary cultures of bovine corneal endothelial cells

This paper presents a novel, safe and time-saving method to obtain uncontaminated primary cultures of corneal endothelial cells from calf eyes. These primary cultures can be used either for subcultivation or for preparation of extracellular matrix in vitro. The method eliminates the risk of deleterious mechanical or chemical influence on the cells.     

Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells

Interleukin 6 (IL-6) is a cytokine that acts on a wide range of tissues influencing cell growth and differentiation. Here we show that IL-6 plays a role in the early vascular development (vasculogenesis) in the central nervous system (CNS). We report that IL-6 induces the proliferation of brain microvascular endothelial cells in vitro. Furthermore, IL-6 significantly accelerates the formation of tube-like structures by these cells in Matrigel basement matrix. Moreover, IL-6 mRNA is expressed in vivo in two physiological conditions in which vascularization in the CNS is important: (1) during normal brain development, (2) during the healing process of a traumatic brain injury. Expression of IL-6 mRNA coincides with the expression of vascular endothelial growth factor (VEGF) mRNA in the developing brain with decreasing expression following birth. However, IL-6 mRNA can be detected in the healing adult murine brain tissue by in situ hybridization coinciding with the period of intense tissue reorganization. The transient upregulation of IL-6 mRNA during normal brain development and at brain injury site and the effect of IL-6 on in vitro vasculogenesis suggest that IL-6 may play a role in normal physiology of vascularization in the CNS.     

Interferometric studies of endothelial cells in primary culture

Summary A population of endothelial cells growth from Xenopus laevis tadpole hearts was investigated by microinterferometry. The resulting interferograms were evaluated by an automatic image analyzer. The mean values of dry mass were 778±340 pg (10^–15 g) for whole cells, 648±309 pg for cytoplasm, 116±45 pg for nuclei, and 19±10 pg for nucleoli. Two subpopulations of cells were identified, an actively growing one and a less active one. The density (dry mass per m²) of the nuclei and nucleoli of less active cells was greater than that of the nuclei and nucleoli of actively growing cells. In addition, the inactive cells were always large and possessed a considerable amount of cytoplasm. The entrance of cells into S-phase could not be detected by microinterferometry; and no differences were apparent between cells possessing one nucleolus and those containing two nucleoli. The values obtained in these amphibian cells were compared with those derived from mammalian cells.