Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.     

小鼠胚胎干细胞分化扩增期连续低密度传代对原始细胞中Oct-4阳性细胞比例及神经分化能力的影响

目的：探讨在分化扩增期采用连续低密度传代的方法是否能降低小鼠胚胎干细胞向神经细胞分化的前体细胞中Oct-4阳性细胞的比例,以及对神经分化能力的影响。方法：采用“五步法”将小鼠胚胎干细胞向神经元分化,进入扩增期后采用连续低密度传代的方法连续传10代。然后应用细胞免疫组化鉴定Oct-4阳性细胞、神经元与胶质细胞、流式细胞仪检测Oct-4阳性细胞比例、凋亡试剂盒测定细胞凋亡。结果：流式细胞仪检测出扩增期连续低密度传代得到的前体细胞中Oct-4阳性细胞的比例由16.17±4.8％降至4.33±1.63％,扩增期低密度传代细胞和正常高密度传代细胞的细胞凋亡率鉴定分别为15.16±3.64% 和11.88±2.63%,步骤5诱导分化后的细胞GFAP和Tuj-1免疫组化染色呈阴性。结论：低密度传代培养可以减少分化后Oct-4阳性细胞的比例,且该比例下降不是由Oct-4阳性细胞的凋亡引起的。同时可能是因为低密度传代培养和高密度相比引起了细胞的质变、或者改变了前体细胞向神经元分化的某种微环境,导致了前体细胞不能分化为神经细胞。提示高密度培养在前体细胞向神经元分化过程中具有重要作用,高密度和低密度培养的比较,提供了研究ES细胞向神经元分化机制的新平台和研究方向。     

TwinGFP, a marker for cell cycle analysis in transiently transfected cells

Green fluorescent protein (GFP) is widely used as a marker to identify transfected cells either by fluorescence microscopy or flow cytometry. However, cell cycle analysis with propidium iodide typically employs ethanol for cell permeabilization. During this treatment, soluble GFPs generally leak out of cells, probably due to their small size. We have now significantly improved cellular retention by creating an in-frame fusion of two GFP DNA sequences, thereby generating a double-sized GFP (TwinGFP, 57 kDa). Permeabilized HeLa cells transfected with pTwinGFP showed a strong green fluorescent signal localized throughout the cells that could easily be detected by fluorescence microscopy and flow cytometry, in contrast to cells transfected with a standard single GFP construct. The experiment indicates that protein size constitutes the major determinant of the loss of fluorescence in permeabilized cells. As a proof of principle, pTwinGFP was cotransfected with the p53 tumor suppressor gene into HeLa cells, and cells transiently expressing p53 could be identified and phenotypically characterized by flow cytometry.     

Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.     

Cell Growth Suppression of Astrocytoma C6 Cells by Glial Fibrillary Acidic Protein cDNA Transfection

Abstract: The cellular functions of the intermediate filament family including glial fibrillary acidic protein (GFAP) are not well known yet beyond their roles as structural elements of cells. Expression of GFAP, which is specific in astrocytes and regulated developmentally, suggests its involvement in cell growth and differentiation of astrocytes. We transfected murine GFAP cDNA into a rat astrocytoma C6 cell line to assess the specific effect of GFAP on cells. Two stable GFAP-transfected cell lines, GFC6-5 and GFC6-6, exhibited a series of morphological and growth characteristics that distinguish them from their counterparts, i.e., NeoC6 cells transfected only with the neomycin-resistant gene, and native C6 cells. Both GFC6-5 and GFC6-6 cells showed elongated cell shapes with extended processes rich in GFAP, markedly suppressed cell growth, and decreased bromodeoxyuridine uptake. Western blot analysis revealed a remarkable increase of GFAP expression in GFC6-5 and GFC6-6 compared with that in NeoC6 and C6, in contrast to similar vimentin expression in all cell lines. The results indicate that the expression of GFAP has dramatic effects on cell morphology and cell growth suppression in C6 cells, suggesting that GFAP may function as a tumor suppressor in astrocytoma.     

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.     

Gene therapy in a rodent model of Parkinson's disease using differentiated C6 cells expressing a GFAP-tyrosine hydroxylase transgene.

Cells expressing a tyrosine hydroxylase (TH) cDNA under control of the promoter of the human glial fibrillary acidic protein (GFAP) gene were tested for therapeutic efficacy in a rat model of Parkinson's disease. The GFAP gene encodes an intermediate filament protein found almost exclusively in astrocytes. Its promoter is of interest for gene therapy as it is expressed in astrocytes throughout postnatal life and is upregulated in response to almost any damage to the central nervous system, including Parkinson's disease. We previously showed that a line of C6 rat glioma cells that expresses a GFAP-TH transgene, C6-THA, displays increased transgene activity when differentiated by forskolin treatment. Accordingly, the effects were investigated of implantation of both undifferentiated and differentiated C6-THA cells into the striatum of rats that had been lesioned with 6-hydroxydopamine. Implantation of either cell type produced significant behavioral recovery one week after transplantation, as judged by the turning response to apomorphine. At two and three weeks after transplantation, the behavioral effect of the undifferentiated cells was no longer statistically significant, whereas that for the forskolin-differentiated cells remained robust. Transgenic TH mRNA and protein could be detected in implants of both cell types, and in agreement with the behavioral results, levels were higher for the differentiated C6-THA cells than for the undifferentiated cells. These results indicate that the GFAP promoter is sufficiently active to enable production of therapeutic levels of dopamine from a GFAP-TH transgene, and suggest the use of astrocytes for gene therapy for Parkinson's disease. They also show that beneficial modifications of cells produced by treatment while in culture may be maintained following implantation.     

The relationship of glial fibrillary acidic protein to the shape,motility, and differentiation of human astrocytoma cells

Glial fibrillary acidic protein (GFAP), a protein largely limited to astrocytes, was studied in relation to the shape, motility, and differentiation and malignancy of astrocytoma cells in tissue culture by use of time-lapse photography and the immunoperoxidase method.A relationship was observed between the shape of astrocytes and the distribution of GFAP. Spindle-shaped cells showed abundant GFAP in the cell body and processes. In round or polyhedral cells without well developed processes the GFAP was largely perinuclear. As processes developed, GFAP extended out from the nucleus iri dense parallel arrays that radiated into the developing processes. Fully differentiated cells with stellate shape had abundant GFAP throughout.A relationship was also observed between the motility of astrocytes and GFAP. Stellate-shaped cells, showing paucity of locomotion and relatively rigid postures of processes, contained an abundance of GFAP which tended to form dense parallel arrays extending into the processes during their development. Spindle-shaped cells with extending and retracting processes and active migration also contained an abundance of GFAP but not organized into parallel arrays. Bulbous dilatations at the tips of processes (growth cones) contained abundant GFAP. There was also abundant GFAP in the intermittent dilatations along the processes of stellate cells. In contrast to these observations, a retraction of processes, a high degree of plasticity (undulating motion) and multidirectional locomotion were often associated with a paucity of GFAP in less differentiated cells. We hypothesize that GFAP filaments may be inhibitory to great plasticity of motion but not to extension-retraction movements.During mitosis GFAP was sparse at the spindle and in intercellular bridges. Colcemid caused GFAP to disappear from processes and peripheral parts of the cell and to become concentrated near the nucleus.In cultures derived from malignant tumors, undifferentiated and large multinucleated cells usually showed sparsity of GFAP, but occasional well differentiated stellate or spindle-shaped cells containing abundant GFAP were seen. Conversely, although cultures derived from benign tumors may have scattered less well differentiated cells, the differentiated cells with well developed processes were most densely stained and account for the high concentration of GFAP in tissue from these tumors.     

Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway

A single bipotential glial progenitor cell of newborn rat optic nerve (the O-2A progenitor) characterized by its reactivity with antibodies to surface gangliosides (A2B5) and the presence of vimentin, can grow in microcultures in conditions which favor this progenitor's differentiation into oligodendrocytes. We selected at 8 days larger clones derived from such bipolar progenitors which had steadily proliferated on a layer of Type-1 astrocytes during the first week. Clonal growth and ratio of progenitor cells to oligodendrocytes was measured over the next two weeks by phase microscopy and double immunofluorescence labeling for the specific markers A2B5, GC (galactocerebroside, a surface marker for differentiated oligodendrocytes), O4 (a glycolipid marker of oligodendrocytes and some progenitors), GFAP (an astrocyte specific intermediate filament protein) and vimentin. Two types of clones were identified: type A clones (the majority), which were still slowly expanding at three weeks, and type B clones (the minority), which had stopped proliferating during the second week. In type A clones, some cells became GC positive multipolar oligodendrocytes, while other multipolar cells remained GC negative for days and expressed A2B5 and O4, but not GFAP or vimentin. Type B clones contained only GC positive, vimentin negative oligodendrocytes, which were generated during the second week and then decreased in number because of their restricted lifespan. Type B clones only developed in the presence of insulin or neurons. The number of GC negative cells in type A clones increased when insulin or neurons were deleted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of Stably Transformed Bm5 and Sf9 Cells Displaying Green Fluorescence by Using Autographa californica Nuclear Polyhedrosis Virus IE1 Gene

Transformed Bm5 or Sf9 cells displaying green fluorescence were constructed by using Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early gene (ie 1). Green fluorescent protein (gfp) gene was introduced under the control of the AcNPV ie 1 promoter to yield expression plasmid pAcIE1-GFP. It was transfected into Sf9 or Bm5 cells and cell clones expressing GFP were selected by fluorescence microscopy. Genomic DNA from transformed cells was isolated and integration of AcNPV ie 1 gene harboring gfp gene was confirmed by PCR using AcNPV ie 1 gene-specific primers. The GFP was successfully expressed in the cytoplasm of insect cells transformed with pAcIEI-GFP and the expressed GFP was maintained during cell division. Furthermore, GFP expression by AcNPV ie 1 promoter in transformed cells was not interfered with viral replication. This suggests that transformed cells displaying foreign gene product by using AcNPV ie 1 promoter will be useful for the diverse applications of the insect cells.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

Hemato-endothelial differentiation from lentiviral-transduced human embryonic stem cells retains durable reporter gene expression under the control of ubiquitin promoter

Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes controlled under ubiquitin promoter are expressed during hematopoietic-endothelial differentiation in hES cells. Undifferentiated hES cells (H1) were transduced by lentivirus encoding green fluorescent protein (GFP) gene under ubiquitin promoter. GFP-expressing hES cells (GFP-H1) were established after several rounds of mechanical selection under fluorescence microscope. GFP gene was stably expressed in hES cells throughout prolonged (> 50 passages) cultivation, and in differentiated embryo body (EB) and teratoma. Hematopoietic and endothelial markers, including KDR (VEGFR2), CD34, CD31, Tie-2, GATA-1 and GATA-2, were expressed at similar levels during hES cell differentiation in parent hES cells and GFP-H1 hES cells. CD34⁺ cells isolated from GFP-H1 hES cells were capable to generate hematopoietic colony-forming cells and tubular structure-forming cells. Differentiated GFP-EB formed vasculature structures in a semi-solid sprouting EB model. These results indicated that a transgene under ubiquitin promoter in lentiviral transduced hES cells retained its expression in undifferentiated hES cells and in hES-derived hematopoietic and endothelial cells. With the view of embryonic mesodermal developing events in humans, genetic modification of hES cells by lentiviral vectors provides a powerful tool for study of hematopoiesis and vasculogenesis.     

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5?/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP? progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5?/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP? progenitors, A2B5?/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP? progenitors, but their lipid profile was different from that of A2B5?/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.     

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.