Abstract: The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)-derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271(+) population. The recognized CD271(+) populations were fractionated by fluorescence-activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU-F. The results showed that only the CD271(bright) but not the CD271(dim) population contained CFU-F. Two-color flow cytometry analysis revealed that only the CD271(bright) population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271(bright) population but no other BM cells. The new MSC-specific molecules included the platelet-derived growth factor receptor-beta (CD140b), HER-2/erbB2 (CD340), frizzled-9 (CD349), the recently described W8B2 antigen, as well as cell-surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM-MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

Abstract: Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.

Abstract: The initiation, growth, recurrence and metastasis of solid tumours, including squamous cell carcinoma of the head and neck region, have been related to the behaviour of a small subpopulation of 'tumour-initiating' cells. Cells with stem cell characteristics have also been identified in cell lines derived from cancers and the aim of the present work was to extend examination of such cells. Established cell lines were examined for their patterns of colony morphologies and staining, the presence of a Hoechst dye-excluding 'side population', expression of the putative stem cell markers CD44, CD133 and CD29, and their ability to grow as 'cancer spheroids'. Two cell lines, CaLH2 and CaLH3, recently generated from HNSCC tumour biopsies, were similarly examined. All cell lines showed a holoclone/meroclone/paraclone series of colony morphologies and cell sorting indicated that CD44 marker expression was related to clonogenicity. FACS analysis after exposure to Hoechst dye indicated that the CA1, H357 and UK1 cell lines contain a dye-excluding 'side population', a property associated with stem-like subpopulations. When held in suspension, all cell lines formed spheroids that could be re-passaged. These observations indicate that cell lines derived from HNSCC contain cells with stem cell properties and that such cell lines may provide experimental systems relevant to the behaviour of stem cells present in the tumours of origin and to their responses to therapy.

Abstract: Background Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells Therefore, both enrichment and decontamination before transplantation may be necessary This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed Methods Murine testicular cells were contaminated with 5% EL4 cells Fresh and frozen-thawed suspensions were sorted using MACS (CD49f (+)) and FACS (CD49f(+), H-2Kb(-)) and evaluated by FACS, cell culture and transplantation into W/W(v) mice Human testicular cells were contaminated with 5 or 005% CCRF-SB (SB) cells Frozen-thawed suspensions were sorted using FACS (HLA class I(-)) and evaluated by FACS, cell culture and PCR for the B-cell receptor Results In the mouse, the sorted fractions contained 039% H-2K(b)-positive and 7655% CD49f-positive cells After transplantation, 1 in 20 recipient mice developed a malignancy In the human experiments, an average of 058% SB cells was detected after sorting In only 1 of 11 samples, there were no SB cells observed Conclusion MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important

Abstract: Tight junctions (TJs) are an important component of the blood-brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of brain capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 x 10(6) cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the brain capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-brain barrier in cooperation with claudin-5.

Abstract: Recently, our group purified a rare population of primitive Sca1+/Lin−/CD45− cells from murine bone marrow by employing multiparameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). In order to better characterize VSELs, we focused on their morphological parameters (e.g. diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. To examine the morphological features of VSELs, we employed a multi-dimensional approach, including (i) traditional flow cytometry, (ii) a novel approach, which is ImageStream (IS) cytometry and (iii) confocal microscopy. We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than haematopoetic stem cells (HSC) (3.63 ± 0.09 versus 6.54 ±0.17 μm in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

Abstract: Measurement of Aβ toxicity of cells in culture exposes a subpopulation of cells with resistance to Aβ, even at high concentrations and after long periods of treatment The cell-selective toxicity of Aβ resembles the selective damage observed in cells of specific regions of the Alzheimer9s disease (AD) brain and suggests that there must be particular characteristics or stages of these cells that make them exceptionally sensitive or resistant to the effect of Aβ Using flow cytometry and cell sorting, we efficiently separated and analyzed the Aβ-sensitive and the Aβ-resistant subpopulations within a variety of neuronal cell lines (PC12, GT1–7) and primary cultured neurons (hippocampal, cortex) We found that this distinctive sensitivity to Aβ was essentially associated with cell membrane Aβ binding This selective Aβ binding was correlated to distinctive cell characteristics, such as cell membrane exposure of the apoptotic signal molecule phosphatidyl serine, larger cell size, the G 1 cell cycle stage, and a lower than normal cytosolic ATP level The response to Aβ by the cells with high Aβ binding affinity was characterized by a larger calcium response and increased mortality, lactate dehydrogenase release, caspase activation, and DNA fragmentation The distinctive sensitivity or resistance to Aβ of the different subpopulations was maintained even after multiple cell divisions We believe that these distinctive cell characteristics are the determining factors for the selective attack of Aβ on cells in culture and in the AD brain

Abstract: Progenitors that can transdifferentiate into cells with hepatic or pancreatic phenotypes can be isolated from experimentally injured salivary glands of rodents. In this study, we isolated progenitors from "uninjured" adult human salivary glands by fluorescence-activated cell sorting using anti-CD49f and anti-Thy-1 antibodies. The sorted cells that were contained in the CD49f+/Thy-1+ fraction showed good proliferation on type I collagen. Single purified progenitor cells in plate culture expressed intracellular laminin, CD49f, Thy-1, and NGF receptor p75 (p75(NGFR)). Immunohistological analysis revealed the expression of Thy-1 and p75(NGFR) in stromal cells in the periductal area of the salivary gland. Under overconfluent conditions in plate culture, cell clusters containing insulin and glucagon-positive cells were occasionally formed. In order to produce differentiated cell clusters with uniform quality, we used a spherical culture system. Autonomous differentiation of cells in clusters into insulin-positive cells was induced in the spherical culture system. We measured C-peptide to estimate the endogenously produced insulin content. The C-peptide content of the spheroid bodies was low (3.5 ng/mg of protein), and they simultaneously expressed the early islet differentiation factor Nkx6.1, proendocrine gene neurogenin3, and ductal cell marker cytokeratin19. The progenitors existing in the interstitium of the salivary gland were able to transdifferentiate into cells with a pancreatic endocrine phenotype.

Abstract: Research in the field of cell biology and biomedicine relies on technologies that fractionate cell populations and isolate rare cell types to high purity. A brief overview of methods and commercially available products currently used in cell separations is presented. Cell fractionation by size and density and highly selective affinity-based technologies such as affinity chromatography, fluorescence-activated cell sorting (FACS) and magnetic cell sorting are discussed in terms of throughput, yield, and purity.

Abstract: Overexpression of Skp2, the ubiquitin ligase subunit that targets p27 for degradation, is often observed in cancers, and is associated with aggressive tumor proliferation and poor prognosis. As there is no drug at present that specifically targets Skp2, studies were undertaken to examine the effects of commonly used drugs on Skp2 regulation. Doxorubicin is among the most effective antitumor agents used for the management of breast cancer, but its effect on Skp2 expression is unknown. The objective of this study was to examine the effect of doxorubicin on Skp2 expression regulation in breast cancer cell lines. The expression of Skp2 mRNA and the protein levels of Skp2, p27, p21 and cyclin B were examined in doxorubicin-treated MCF-7 and MDA-MB-231 breast cancer cells. The effect of doxorubicin on the cell cycle profile was assessed by fluorescence-activated cell sorting analysis. Doxorubicin decreased Skp2 mRNA and protein levels in MCF-7 cells, but had the opposite effect in MDA-MB-231 cells. p27 levels were slightly decreased, whereas p53 and p21 levels were significantly upregulated in doxorubicin-treated MCF-7 cells. In contrast, p27 levels were unaffected by doxorubicin treatment in MDA-MB-231 cells, but cyclin B levels were markedly increased. Doxorubicin arrested MCF-7 cells at G1/S and G2/M checkpoints, whereas MDA-MB-231 cells were arrested at G2/M only. The differential effects of doxorubicin on Skp2 expression in breast cancer cells depend upon the specific cell cycle checkpoints activated by the drug. These changes induced by doxorubicin, however, do not significantly affect p27 expression in these cell lines, suggesting that the potential of a given drug to alter p27 expression through Skp2 modulation might depend on its specific action on cell cycle arrest.

Abstract: The presence of heterogeneous cell populations in dental pulp may count for the considerable variation in the outcome of in vitro and in vivo experiments. Here, we intended to determine whether a minor cell sub-population of high proliferation and odontogenic potential existed among a larger compartment of perhaps more committed progenitors. In this study, the STRO-1 antigen, defining a mesenchymal stem cell or progenitor subpopulation, was used for separating rat dental pulp cells with fluorescence-activated cell sorting (FACS). Subsequently, the STRO-1 positive cells were tested for their ability to differentiate towards an odontoblast-like phenotype. Three cell populations (STRO-1 positive, STRO-1 negative, and non-sorted cells) were cultured in odontogenic medium containing dexamethasone and beta-glycerophosphate. Cultures were analyzed by light- and scanning electron microscopy (SEM), and assessed for proliferation, ALP activity, and calcium content. Results showed that the STRO-1 positive cell population was able to differentiate into the odontoblast phenotype, similar to the non-sorted population. The negative cells however showed a fibroblast-like phenotype. SEM and real-time PCR confirmed such results. In conclusion, the STRO-1 selection proved applicable for rat-derived material, to obtain a cell population which is more homogeneous. This positive cell fraction was capable of differentiating into the odontogenic pathway, whereas the negative fraction was not. However, the effect was not always advantageous, when compared to non-sorted cells.

Abstract: Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.

Abstract: AIM: To elucidate the role of Rab23 in hepatocellular carcinoma (HCC) by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS: Primary tumors (n = 100) were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of double- stranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fl uorescence- activated cell sorting. RESULTS: High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 (53.5%) and in 49 of 68 HCC patients (72%) respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth.

Abstract: The involvement of matrix metalloproteinases (MMP) has been suggested in cellular mechanisms leading to medulloblastoma, the most common malignant brain tumor in children. A significant association of the expression levels of MMP-9 with survival and M stage suggests that patients with medulloblastoma metastatic disease at diagnosis may benefit from the anti-MMP therapy. Here, we have evaluated the tumorigenicity of medulloblastoma cells after infection with an adenovirus containing a 21-bp short interfering RNA sequence of the human MMP-9 gene (Ad-MMP-9). Infection of Daoy medulloblastoma cells with Ad-MMP-9 reduced MMP-9 activity and protein levels compared with parental and Ad-SV controls. Ad-MMP-9 decreased the number of viable Daoy cells in a concentration-dependent manner. Fluorescence-activated cell sorting analysis indicated that Ad-MMP-9 infection caused a dose-dependent cell cycle arrest in the G(0)-G(1) phase. Ad-MMP-9-induced cell cycle arrest seems to be mediated by the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway and the cell cycle inhibitor p16(INK4a) and is phenotypically indistinguishable from senescence. Ad-MMP-9 treatment inhibited medulloblastoma tumor growth in an intracranial model and was mediated by up-regulation of p16 expression. These studies validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of medulloblastoma.

Abstract: An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC4(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.

Abstract: Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have emerged as potentially useful substrates for neovascularization and tissue repair and bioengineering EPCs are a heterogeneous group of endothelial cell precursors originating in the hematopoietic compartment of the bone marrow MSCs are a rare population of fibroblast-like cells derived from the bone marrow stroma, constituting approximately 0001-001% of the nucleated cells in the marrow Both cells types have been isolated from the bone marrow In addition, EPC can be isolated from peripheral blood as well as the spleen, and MSC has also been isolated from peripheral adipose tissue Several approaches have been used for the isolation of EPC and MSC, including density centrifugation and magnetic bead selection Phenotypic characterization of both cell types is carried out using immunohistochemical detection and fluorescence-activated cell sorting analysis of cell-surface molecule expression However, the lack of specific markers for each cell type renders their characterization difficult and ambiguous In this chapter, we describe the methods that we use routinely for isolation, characterization, and genetic modification of EPC and MSC from human, rabbit, and mouse peripheral blood and bone marrow

Abstract: Purpose: Aberrant c-Met expression has been implicated in most types of human cancer. We are developing Met-directed imaging and therapeutic agents. Experimental Design: To seek peptides that bind specifically to receptor Met, the Met-expressing cell lines S114 and SK-LMS-1 were used for biopanning with a random peptide phage display library. Competition ELISA, fluorescence-activated cell sorting analysis, an internalization assay, and a cell proliferation assay were used to characterize a Met-binding peptide in vitro. To evaluate the utility of the peptide as a diagnostic agent in vivo, 125I-labeled peptide was injected i.v. into nude mice bearing s.c. xenografts of the Met-expressing and hepatocyte growth factor (HGF)/scatter factor–expressing SK-LMS-1/HGF, and total body scintigrams were obtained between 1 and 24 h postinjection. Results: One Met-binding peptide (YLFSVHWPPLKA), designated Met-pep1, reacts with Met on the cell surface and competes with HGF/scatter factor binding to Met in a dose-dependent manner. Met-pep1 is internalized by Met-expressing cells after receptor binding. Met-pep1 inhibits human leiomyosarcoma SK-LMS-1 cell proliferation in vitro. In SK-LMS-1 mouse xenografts, tumor-associated activity was imaged as early as 1 h postinjection and remained visible in some animals as late as 24 h postinjection. Conclusions: Met-pep1 specifically interacts with Met: it is internalized by Met-expressing cells and inhibits tumor cell proliferation in vitro; it is a potential diagnostic agent for tumor imaging.

Abstract: Growth hormone (GH) deficiency is a significant clinical problem, since growth hormone is essential for the regulation of growth, metabolism, and the cardiovascular system. Stem and progenitor cells have been identified in many adult tissues. Recently, our laboratory identified a cell type within the adult pituitary gland with stem cell-like properties, which we have termed pituitary colony-forming cells (PCFCs). Herein we investigate the ability of PCFCs to survive and differentiate in vivo. Enriched populations of PCFCs were transplanted into an in vivo microchamber model. Grafts were harvested at 6 weeks post-transplant and tested for surviving donor cells (LacZ(+)) or for differentiation (GH(+)). The results showed that donor cells survived in chambers (LacZ(+)) and underwent division (phosphohistone-H3-positive). Furthermore, grafted cells showed colocalization of LacZ and GH, suggesting differentiation. To confirm differentiation, donor cells were obtained from a GH-enhanced green fluorescent protein (eGFP) reporter transgenic mouse model that expressed eGFP under control of the GH promoter. Cells that were eGFP(-), that is, GH(-), were selected by fluorescence-activated cell sorting (FACS) and transplanted. After 6 weeks, eGFP(+)GH(+) cells were detected in grafts by immunostaining and by FACS analysis of digested grafts. In conclusion, PCFCs have the capacity to divide and differentiate into GH(+) cells in vivo. The vascularized tissue chamber model is an ideal model to investigate the environmental niche for PCFC expansion and differentiation and has the potential to be developed into a growth hormone-releasing organoid in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract: Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P putida from activated sludge and river water samples The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry

Abstract: Purpose: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines.Materials and methods: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 – 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis.Results: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 μM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 μM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor ad...

Abstract: Hepatic oval cells (OC) are considered to be facultative liver stem cells and, because they may undergo differentiation into a variety of cell lineages, they might have the potential to be used in cellular therapy. Signals delivered by extracellular matrix (ECM) proteins take part in cellular differentiation in cooperation with signals from growth factors; indeed, some ECM proteins, such as laminin (LAM) and fibronectin (FN), have been shown to contribute to beta-cell differentiation and islet development, respectively. Since no previous studies have investigated the effect of ECM proteins on the expression of islet cell markers by cultured OC, the purpose of the present study was to evaluate whether FN and LAM modulate the expression of genes related to the endocrine pancreas in these liver cells. OC proliferation was induced in Wistar rats by prolonged treatment with 2-acetoaminofluorene/allyl alcohol and confirmed by reverse transcription/polymerase chain reaction and hepatic immunocytochemical and histopathological analyses. OC isolation was performed by Ficoll gradient and magnetic-activated cell sorting. OC were cultured for 1 and 2 months under several conditions with specific growth factors, over a FN or LAM substrate or in high glucose, nicotinamide and fetal calf serum. OC cultured on FN substrate expressed Pdx-1, Pax-6, insulin 2 and glucagon. LAM also induced the expression of Pdx-1, insulin 1 and insulin 2, glucagon, somatostatin and GLUT-2. Our results suggest that these ECM proteins can be used in protocols of OC transdifferentiation aimed at reducing the period necessary for complete transdifferentiation.

Abstract: The organization of endocrine cells in pancreatic islets is established through a series of morphogenetic events involving cell sorting, migration, and re-aggregation processes for which intercellular adhesion is thought to play a central role. In animals, these morphogenetic events result in an islet topology in which insulin-secreting cells form the core, while glucagon, somatostatin, and pancreatic polypeptide-secreting cells segregate to the periphery. Isolated pancreatic islet cells self-assemble in vitro into pseudoislets with the same cell type organization as native islets. It is widely held that differential adhesion between cells of the pancreatic islets generates this specific topology. However, this differential adhesion has never been rigorously quantified. In this manuscript, we use tissue surface tensiometry to measure the cohesivity of spherical aggregates from three immortalized mouse pancreatic islet cell lines. We show that, as predicted by the differential adhesion hypothesis, aggregates of the internally segregating INS-1 and MIN6 beta-cell lines are substantially more cohesive than those of the externally segregating alpha-TC line. Furthermore, we show that forced overexpression of P-cadherin by alpha-TC cells significantly perturbs the sorting process. Collectively, the data indicate that differential adhesion can drive the in vitro organization of immortalized rodent pancreatic islet cells.

Abstract: To establish a method for isolating highly purified brain capillary endothelial cells (BCECs) from rat brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs. The cells were prepared from the capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis. From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat brain. The contamination by neurons and astrocytes was markedly less than in the brain capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively. This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-brain barrier.