Abstract: Cell type–specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines

Abstract: Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

Abstract: Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR− CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR β-chain variable region (Vβ)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.

Abstract: Isolation of phenotypically-pure cell sub- populations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated micro- fluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm 2 was found to provide most efficient separa- tion.Celladhesionwasshowntofollowpseudo-firstorder kinetics, and an anti-CD19 coated (Raji-depletion) device with � 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally deter- mined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time. 2005 Wiley Periodicals, Inc.

Abstract: Primary fibroblasts represent a heterogeneous population of cells that can be separated into subsets on the basis of cell surface markers such as Thy-1. Deriving fibroblasts initially involves obtaining tissue explants from tissues such as the lung, heart, cornea, skin, and orbit. The tissue is mechanically dissociated and cells are allowed to proliferate from the fragments. Following establishment of a primary culture of fibroblasts, it is necessary to characterize the new strain of cells to ensure their purity and fibroblastic phenotype using immunofluorescence and immunohistochemistry to detect the presence or absence of cell-specific surface markers. Characterizing the cells as expressing or lacking Thy-1 can also be performed by immunofluorescence in concert with microscopy or by flow cytometry using an anti-human Thy-1 antibody. In addition, fibroblasts may be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting and/or magnetic beading; use of these techniques can yield greater than 99% purity. Once separated, the pure Thy-1 expressing or lacking fibroblast subsets can be propagated. These subsets can then be used for experimentation to determine functional differences between fibroblasts derived from normal and pathological tissue such as scarred lung.

Abstract: Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Abstract: We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.

Abstract: MEMS micro-T-switches actuated via electrochemical bubbles for cell sorting applications in a monolithic chip level are proposed and successfully demonstrated. The electrolysis-bubble actuator, which has the features of low operation temperature and high surface-tension force, is developed to actuate the micro-T-switch sorting structure in our device. The double T-structure design, the T-shape microchannel with the movable micro-T-switch structure located at the junction of the T-shape microchannel, with the electrolysis-bubble actuator makes an active-binary switch function available for cell sorting applications. The room temperature operation and the low voltage required for electrolysis actuation minimize the possibility of cell-damage that happens in the conventional high electric separation instruments, such as flow cytometry. The function of our micro-T-switch chip with a low required actuation voltage of 3.0 ∼ 3.5 V is demonstrated by using human hepatoma cells in this paper. The pH-value measurements characterize the pH-value variation and distribution in the actuating chambers and the mainstream microchannels to trace the possible liver-cell injury due to the pH-value variation during electrolysis-actuation operation. The 84.1% cell viability in the sorted human hepatoma cells through our micro-T-switch sorter is observed via the fluorescence assay technique. Furthermore, 70.2% of total injected cells recover in culture after sorting and grow into colonies after micro-T-switch sorting operation. In this paper, we describe the design, microfabrication, and characterization of our micro-T-switch cell-sorting chip. We also report the cell-sorting demonstration and the cell viability results for the mammalian liver cells through our micro-T-switch cell-sorting chip.

Abstract: Background: Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34+ hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation- and activation-linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC. Methods: We analyzed expression of CD antigens on human MC grown from cord blood-derived CD34+ HPC. The HPC were isolated by magnetic cell sorting (MACS) and FACS to >97% purity, and were cultured in stem cell factor (SCF) and interleukin (IL)-6 with or without additional cytokines (IL-4 or IL-10) in serum-free medium. The cell surface phenotype of MC was determined by monoclonal antibodies and flow cytometry. Results: Cultured MC progenitors were found to react with antibodies against various CD antigens including CD58, CD63, CD117, CD147, CD151, CD203c, and CD172a, independent of the growth factors used and time-point investigated (days 14–42). CD116 [granulocyte–macrophage colony-stimulating factor receptor α (GM-CSFRα)] and CD123 (IL-3Rα) were expressed on MC precursors on day 14, but disappeared thereafter. Cultured MC did not express CD2, CD3, CD5, CD10, CD19, or CD25. Addition of IL-10 to MC cultures showed no effect on expression of CD antigens. However, IL-4 was found to promote expression of CD35 and CD88 on cultured MC without changing expression of other CD antigens. Conclusions: Most MC antigens may already be expressed at an early stage of mastopoiesis. Whereas IL-3R and GM-CSFRs are lost during differentiation of MC, these cells may acquire complement receptors (CD35, CD88) under the influence of distinct cytokines.

Abstract: Purpose To isolate and characterize primary retinal astrocytes in culture (RAC) from wild-type and transgenic mice to aid the study of their properties in vitro. Methods Astrocytes were isolated from wild-type and transgenic Immortomice by collagenase digestion of the retina. Affinity purification using magnetic beads coated with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) was used to remove retinal endothelial cells. The remaining cells were cultured and expanded. The majority of these cells were identified as astrocytes. These cells were characterized for expression of astrocytic markers using fluorescence-activated cell sorting (FACS) and immunostaining analysis. The expression of various integrins and other cell adhesion molecules on the surface of retinal astrocytes, their adhesion to various matrix proteins, their migration, and their ability to organize on Matrigel were determined. Results Here we describe a method for the isolation of RAC from wild-type and thrombospondin-1 deficient (TSP1-/-) mice. Our results indicated that nearly 100% of cells isolated expressed the astrocytic markers GFAP, NG2, Pax2, and vimentin. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of astrocytic markers. The RAC expressed alphavbeta3 integrin and other cell adhesion molecules on their surface. The TSP1-/- RAC adhered more strongly to fibronectin and vitronectin compared to the wild-type cells, while neither cell types adhered to collagen and laminin. Wild-type and TSP1-/- RAC exhibited similar migratory characteristics despite alterations in their adhesive properties and production of various matrix proteins. Also, these cells, like endothelial cells, similarly organized into a network in Matrigel. Conclusions The RAC can be readily obtained from wild-type and transgenic mice. This facilitates the comparison and identification of specific gene functions in RAC compared to astrocytes prepared from other sites of central nervous system.

Abstract: Background The host's first line of defense against bloodstream infection with Candida albicans involves the recognition and clearance of the fungus by neutrophils and monocytes/macrophages. The purpose of the present study was to examine changes in the monocytic cell gene-expression profile in response to C. albicans stimulation. Methods RNA was isolated from THP-1 cells 3 h after coculture with live C. albicans SC5314 cells. After hybridization to microarrays, genes differentially expressed by at least 2.0-fold were included in the final data set. Results As expected, TNFA, IL8, CD83, MIP1A, and MIP1B were among the genes up-regulated. This was confirmed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting analysis, and enzyme-linked immunosorbent assay. Furthermore, RGS1, RGS2, RGS16, DSCR1, GROB, EGR3, FLT4, and TNFAIP6 were also up-regulated in response to C. albicans, whereas CCR2 and NCF2 were among the genes down-regulated in response to C. albicans. Differential expression of selected genes was confirmed at several time points by real-time RT-PCR. Conclusions This study defines the gene expression profile of an early response of human mononuclear cells to C. albicans and identifies genes not previously known to be responsive to this pathogen.

Abstract: Intestinal dendritic cells (DC) are likely to regulate immunity to gut microflora, but little is known about their responses to bacterial antigens. Therefore, DC from normal murine colon were characterized and their cytokine responses to components of Gram-negative and/or Gram-positive bacteria assessed. Cells were obtained by digestion of colonic tissue and contained DC that were identified by flow cytometry as CD11c(+) major histocompatibility complex (MHC) class II(+) cells. Purified DC were obtained by immunomagnetic separation plus cell sorting. DC had the morphology of immature myeloid cells, were endocytically active, expressed low levels of co-stimulatory molecules and stimulated a weak allogeneic mixed leucocyte reaction. Analysis of flow cytometry data by a sensitive subtraction method allowed measurement of production of interleukin (IL)-12 and IL-10 by small numbers of gut DC by intracellular staining. Fewer than 5% of unstimulated DC produced either IL-10 or IL-12. IL-10 production was significantly up-regulated following stimulation with Bifidobacteria longum, but not after exposure to lipopolysaccharide (LPS) or Streptococcus faecium. In contrast, colonic DC produced IL-12 in response to both LPS and B.longum. Thus, colonic DC can produce both IL-12 and IL-10 following bacterial stimulation. Cell wall components from different bacteria stimulate distinct responses and may direct immune responses differentially in the gut.

Abstract: Background: A paucity of information exists concerning how estrogen affects cellular function in the gingiva of women. In this study, the behavior of human gingival fibroblasts was examined in the presence of a potent estrogen, estradiol. Methods: Quiescent, premenopausal gingival fibroblasts were incubated in the presence and absence of estradiol (1 nM) and/or raloxifene (100 nM). Cell number was determined and cell cycle analyzed using a flow cytometer. Collagen and non-collagen production in cell cultures grown on various extracellular matrices were determined using a radioactive microassay which measures collagenase-digestible and collagenase-resistant radiolabeled proteins. To ascertain if the gingiva contained specific estrogen-sensitive cell populations, a fluorescence-activated cell sorter was used to detect, sort, and enrich fibroblast populations responsive to estrogen. Results: Cellular proliferation and the number of cells entering the S-phase of the cell cycle were significantly increased in mass cultures of fibroblasts stimulated by estradiol. Raloxifene did not antagonize the action of estradiol on cell proliferation. In regard to protein production, estradiol significantly reduced collagen production on plastic and collagen IV matrices; whereas non-collagen protein production on plastic and collagen I matrices was significantly reduced. Cell sorting of mass fibroblast populations revealed that, on average, 45% of the cells from the resident population selectively accumulated the estrogen probe. These sorted and estrogen-sensitive enriched cell populations proliferated in the presence of 1 nM estradiol, whereas the sorted, estrogen-deficient enriched fibroblast populations did not proliferate when incubated with 1 nM estradiol. Conclusions: These data indicate that estradiol can induce cellular proliferation while depressing protein production in cultures of human, premenopausal gingival fibroblasts. This cellular proliferation appears to be the result of a specific population of cells within the parent culture that responds to physiologic concentrations of estradiol. J Periodontol 2005;76:1391-1397.

Abstract: In Xenopus laevis, patterning of the trunk mesoderm into the dorsal notochord and lateral somites depends on differential regulation of Wnt–β-catenin signaling. To study the cellular requirements for the physical separation of these tissues, we manipulated β-catenin activity in individual cells that were scattered within the trunk mesoderm. We found that high activity led to efficient cell sorting from the notochord to the somites, whereas reduced activity led to sorting in the opposite direction. Analysis of individual cells overexpressing β-catenin revealed that these cells were unable to establish stable contacts with notochord cells but could freely cross the boundary to integrate within the somitic tissue. Interference with cadherin-mediated adhesion disrupted tissue architecture, but it did not affect sorting and boundary formation. Based on these results, we propose that the boundary itself is the result of cell-autonomous changes in contact behavior that do not rely on differences in absolute levels of adhesion.

Abstract: Antibodies reacting to specific cell surface markers can be bound to magnetic beads and used to specifically capture cells exhibiting the marker. This approach allows the selective enrichment of specific cell subpopulations and as the methods are amenable to use in blood, tissue fluids and culture medium living cells can be recovered that can subsequently be subcultured. Negative cell separation can also be used where undesirable cell types are tagged using magnetic beads and removed from the desirable cell population.

Abstract: Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family, which has tropism for glial cells. It was recently reported that HCMV might play important roles in the pathogenesis of malignant glioma. In this study, we investigated the effects of the HCMV IE1 protein on the gene expression profile in the human glioblastoma cell line, U373MG by employing cDNA microarray technology. Using DNA chips containing approximately 1,000 human cDNAs, RNA samples from U373MG cells stably expressing IE1 were compared with those from the control cells lacking IE1 cDNA. Fluorescence intensities of 13 genes were significantly decreased in IE1-expressing cells, while one gene was found to be upregulated. Among these 14 genes, we chose to work further on glial fibrillary acidic protein (GFAP), thrombospondin-1 (TSP-1), and p53, because of their previously known involvement in tumorigenesis. The mRNA levels of all these genes were found to be decreased in IE1-expressing glioblastoma cells by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) as well as Northern blot analysis. The decreased expression of these genes was also observed at protein levels as measured by immunocytochemistry or fluorescence-activated cell sorting (FACS) analysis. Our data strongly suggested that HCMV IE1 could modulate the expression of cellular genes that might play important roles in the pathogenesis of glial tumors.

Abstract: Purpose: To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression. Experimental Design: Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing9s family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging. Results: EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo . In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas). Conclusions: CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

Abstract: To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.

Abstract: We have recently reported a simple technique for the purification of large granular lymphocyte/NK cells with broad anti-tumor cytotoxicity (lymphokine-activated killer (LAK) activity) by the induction of their adherence to plastic surfaces by recombinant IL-2. Using this technique with nylon wool passed human peripheral blood or splenic lymphocytes we generated populations of adherent LAK (A-LAK) cells which were 80 to 95% large granular lymphocyte and up to 97% Leu-19+ cells. Using two-color fluorescence and cell sorting we now demonstrate that these same cells express a high density of surface structures which are immunochemically cross-reactive with affinity purified anti-laminin antibody. Laminin-like structures were expressed on 80 to 90% of Leu-19+/CD3- LAK cells but were not expressed on resting CD3+/CD4+/Leu19- T cells or CD3+/CD8+/Leu19- T cells. Laminin-like molecules were also expressed on resting Leu19+/CD3- large granular lymphocyte/NK cells (purified to more than 98% by cell sorting) and increased in intensity as these cells developed LAK activity in response to rIL-2. The functional involvement of laminin-like molecules in NK and LAK cytotoxicity was demonstrated by the ability of affinity purified anti-laminin F(ab')2 antibody to inhibit cytolytic activity in a dose-dependent manner (50% inhibition with 10 to 20 micrograms F(ab')2). Analysis of the mechanism of inhibition using single cell cytotoxicity assays indicated that the laminin-like structure was involved in activation of cytotoxicity and not in primary target cell adhesion. We believe that this structure may function as a novel recognition/activation structure used by cells mediating non-MHC restricted cytotoxicity.

Abstract: Objectives: Angiogenesis is necessary for sustained neoplastic development. The angiopoietins Ang-1 and Ang-2 have been implicated in the regulation of this process; recent reports have suggested that a net gain in Ang-2 activity may be an initiating factor for tumor angiogenesis. We examined the recruitment of bone marrow-derived endothelial precursor cells into developing tumor neovasculature, and the spatial relationship between these cells and angiopoietin (Ang-1 and Ang-2) expression. Methods: For this study T-cell depleted knockout mice (RAG-2/KO-5.2) were lethally irradiated and their bone marrow was reconstituted by bone marrow cells (BMCs) from transgenic mice (C57BL/Ka-Thy1.1) expressing green fluorescent protein (GFP). Rat glioma cells (RT-2/RAG) were then injected into the transplanted animals to form solid brain tumors. The animals were killed and their brains were analysed using immunohistochemistry and fluorescence-activated cell sorting. Results: We found that BMCs migrated prefer...

Abstract: In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

Abstract: The gastric enterochromaffin-like (ECL) cell plays a major role in the regulation of gastric acid secretion. We have previously described that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is present on myenteric neurons in the rat and colocalizes with its high-affinity receptor, PAC1, expressed on the surface of gastric ECL cells. The study of ECL cell physiology has been hampered by the inability to isolate and purify ECL cells to homogeneity. Density gradient elutriation alone yields only 65–70% purity of ECL cells. In the present study, we used fluorescence-activated cell sorting (FACS) with a novel fluorescent ligand, Fluor-PACAP-38, for isolating pure ECL cells. FACS was used to isolate ECL cells based on their relatively small size, low density, and ability to bind the fluorescent ligand Fluor-PACAP-38. The sorted cells were unambiguously identified as ECL cells by immunohistochemical analysis using anti-PACAP type-I (PAC1), anti-histidine decarboxylase (HDC), and anti-somatostatin antibodies. Further confocal microscopy demonstrated that Fluor-PACAP-38, a ligand with a higher affinity for PAC1, bound to extracellular receptors of these FACS-purified cells. FACS yielded an average of 2 million ECL cells/4 rat stomachs, and >99% of the sorted cells were positive for PAC1 receptor and HDC expression. The absence of immunohistochemical staining for somatostatin indicated lack of contamination by gastric D cells, which are similar in size and shape to the ECL cells. Internalization of PACAP receptors and a rapid Ca2+ response in purified ECL cells were observed upon PACAP activation, suggesting that these cells are viable and biologically active. These ECL cells demonstrated a dose-dependent stimulation of proliferation in response to PACAP, with a maximum of 30% proliferation at a concentration of 10−7 M. Microarray studies were perfor med to confirm the expression of genes specific for ECL cells. These results demonstrate that rat gastric ECL cells can be isolated to homogeneity by using a combination of density gradient centrifugation, followed by cell sorting using Fluor-PACAP. These techniques now allow microarray studies to be performed in ECL cells to characterize their functional gene expression and will facilitate pharmacological, biochemical, and molecular studies on ECL cell function.

Abstract: Background Primary cultured hepatocytes are the closest model to the liver for drug research. However, to overcome its limited availability, the search for hepatic cell lines as an alternative to primary cultures is a matter of current interest. In particular, highly differentiated hepatocellular carcinomas have been proposed as in vitro tools for routine experiments in hepatotoxicity and drug metabolism. Methods Cell populations were selected by fluorescence-activated cell sorting based on low and high relative expressions of P-glycoprotein. These cell lines were characterized after 21 days in culture by multiparametric analysis with flow cytometry providing direct information on key cellular functions (stability in culture, intracellular ionic homeostasis, plasmatic and mitochondrial membrane-related parameters, red-ox status, drug transport, and metabolism). Results Two subpopulations (ADV-1 and ADV-2) from the differentiated and well-characterized human hepatoma BC2 cell line showed increased activity of drug transport and drug biotransformation capability (cytochrome P450 [CYP] 1A2, CYP2B6, CYP3A4, and CYP2Cs). These subpopulations were characterized extensively by multiparametric flow cytometric analysis. Conclusion ADV-1 subpopulation showed greater stability in culture, better efficiency regarding intracellular pH maintenance through the operation of Na+/H+ exchange antiporter, and significantly greater CYP-dependent biotransformation activity than the BC2 parental cells and ADV-2 cells. © 2004 Wiley-Liss, Inc.