Finite cell lines of turkey sperm storage tubule cells: ultrastructure and protein analysis.

Cell lines of turkey sperm storage tubule (SST) epithelial cells were established. Turkey SSTs were dissected from freshly obtained uterovaginal junction (UVJ) tissue and placed in explant culture on various substrates and media. Primary cultures of SST epithelium only survived and grew from SST explants that were cultured on inactivated Sandoz inbred strain, thioguanine- and ouabain-resistance (STO) mouse feeder-cell layers in 12% fetal bovine serum-supplemented Dulbecco's Modified Eagle Medium mixed 1:1 with F12 nutrient mixture. Three independent primary colonies gave rise to 3 finite cell lines, SST-1, -2, and -3, which were continuously cultured for 8 to 16 passages at 1:3 passage ratios over a period of 3 to 4 mo. The cells were passaged by pretreatment with Y27632 and dissociation with Accutase. The SST cells grew as tightly knit monolayers on top of the feeder cells at a slow rate (approximately 96 h doubling time) at a medium pH of approximately 6.9. Lipid vacuoles were visible by light microscopy in the cells particularly at the periphery of growth. Transmission electron microscopy revealed the cells to be a polarized epithelium with apical microvilli and to have lateral tight-junction-like unions and associated desmosomes. Numerous secretory vesicles filled the upper portion of the cells' cytoplasm, and nuclei and other major organelles such as mitochondria, rough endoplasmic reticulum, and Golgi apparatus were distributed somewhat lower in the cytoplasm. The secretory vesicles resembled mucin secretory vesicles. Proteomic analysis by mass spectroscopy of the conditioned medium of the cells, and of the cells themselves, showed the cell lines did not secrete large amounts of any particular protein, and the analysis confirmed their epithelial character. In conclusion, the SST-derived cell lines resembled the mucus-secreting cells found in the epithelium lining the UVJ of the turkey's reproductive tract.

Correction: Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation.

[This corrects the article DOI: 10.1371/journal.pone.0123282.].

Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines.

The establishment and initial characterization of bovine fetal liver cell lines are described. Bovine fetal hepatocytes were cultured from the liver of a 34-d bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO (SIMS mouse strain, thioguanine- and ouabain-resistant) feeder layers and were cultured in a medium supplemented with 10% fetal bovine serum. After 2-3 wk, primary colonies of hepatocytes were observed by phase-contrast microscopic observation. Individual hepatocyte colonies were colony-cloned into independent bovine fetal liver (BFL) cell lines. Two cell lines, BFL-6 and BFL-9, grew the best of several isolates, and they were further characterized for growth potential and for hepatocyte morphology and function. The two cell lines were found to grow markedly better in the presence of the transforming growth factor (TGF)-beta inhibitor, SB431542 (1 µM). Their continuous culture also depended on a particular medium height-for T12.5 flasks, 3 ml total medium produced optimum growth. Higher or lower amounts of medium caused less cell growth or cessation of growth. The cell lines were propagated for over a year at split ratios of 1:2 or 1:3 at each passage until reaching senescence at approximately 30 passages. The cells were laterally polarized with well-developed canalicular spaces occurring between adjacent BFL cells. Treatment of the cultures with cyclic adenosine monophosphate (cAMP)-stimulating chemicals or peptides (e.g., forskolin or glucagon) caused physical expansion of the canaliculi between the cells within 15 min. The cells secreted a spectrum of serum proteins, were positive for the expression of several hepatocyte-specific genes, and converted ammonia to urea, although at a relatively low rate. The culture system provides an in vitro model of fetal bovine hepatocytes and is the first demonstration of the continuous culture of normal bovine hepatocytes as cell lines.

Generation of induced pluripotent stem cells from domestic goats.

The creation of genetically modified goats provides a powerful approach for improving animal health, enhancing production traits, animal pharming, and for ensuring food safety all of which are high-priority goals for animal agriculture. The availability of goat embryonic stem cells (ESCs) that are characteristically immortal in culture would be of enormous benefit for developing genetically modified animals. As an alternative to long-sought goat ESCs, we generated induced pluripotent stem cells (iPSC) by forced expression of bovine POU5F1, SOX2, MYC, KLF4, LIN-28, and NANOG reprogramming factors in combination with a MIR302/367 cluster, delivered by lentiviral vectors. In order to minimize integrations, the reprogramming factor coding sequences were assembled with porcine teschovirus-1 2A (P2A) self-cleaving peptides that allowed for tri-cistronic expression from each vector. The lentiviral-transduced cells were cultured on irradiated mouse feeder cells in a semi-defined, serum-free medium containing fibroblast growth factor (FGF) and/or leukemia inhibitory factor (LIF). The resulting goat iPSC exhibit cell and colony morphology typical of human and mouse ESCs-that is, well-defined borders, a high nuclear-to-cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the ability to generate teratomas in vivo. Additionally, these goat iPSC demonstrated the ability to differentiate into directed lineages in vitro. These results constitute the first steps in establishing integration and footprint-free iPSC from ruminants. Mol. Reprod. Dev. 82: 709-721, 2015. © 2015 Wiley Periodicals, Inc.

Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

Gene expression profiling of MYC-driven tumor signatures in porcine liver stem cells by transcriptome sequencing.

AIM: To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells. METHODS: In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice. RESULTS: Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology analysis further showed that the MYC-induced, altered gene expression was primarily associated with various cellular processes, such as metabolism, cell adhesion, growth and proliferation, cell cycle, inflammation and tumorigenesis. Interestingly, six genes expressed by PICM-19 cells (CDO1, C22orf39, DKK2, ENPEP, GPX6, SRPX2) were completely silenced after MYC-induction in PICM-19-CSCs, suggesting that the absence of these genes may be critical for inducing tumorigenesis. CONCLUSION: MYC-driven genes may serve as promising candidates for the development of hepatocellular carcinoma therapeutics that would not have deleterious effects on other cell types in the liver.

Proteome array identification of bioactive soluble proteins/peptides in Matrigel: relevance to stem cell responses.

Matrigel and similar commercial products are extracts of the Engelbreth-Holm-Swarm sarcoma that provide a basement-membrane-like attachment substrate or gel that is used to grow cells on or in, respectively. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constituents, an analysis of the expressed liquid of gelled Matrigel was performed using proteome array technology. Among the growth factors/cytokines assayed, high positive detection was found for IGFBP1, IGFBP3, LIF, platelet factor 4, PlGF-2, and VEGF; moderate reactivity was found for cyr61, IGFBP2, IGFBP6, IL-1ra, and NOV; and low, but detectable, responses occurred for aFGF, IL-13, IL-23, M-CSF, and VEGF-B. Among the chemokines assayed, high positive detection was found for MIG and serpin E1; moderate reactivity was found for IP-10, MCP-1, and MCP-5, and low, but detectable, responses occurred for CXCL16, I-TAC, and MIP-1α. Among the other biologically active proteins assayed, high positive detection was found for adiponectin, C5a, endocan, lipocalin-2, sICAM-1, MMP-3, and TIMP-1; moderate reactivity was found for C-reactive protein, coagulation factor III, endoglin, endostatin/collagen XVIII, endothelin-1, ICAM-1, MMP-9, osteopontin, pentraxin-3, and RANTES; and low, but detectable, responses occurred for fetuin A, MMP-8, pentraxin-2, RBP4, resistin, and TIMP-4. The study found several growth factors, chemokines, and biologically active proteins not previously identified in Matrigel, and this may have significance to the interpretations of observed cellular responses when cells are grown on or in Matrigel.

New models of hepatitis E virus replication in human and porcine hepatocyte cell lines.

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells.

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1ß (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media.

A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 µM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays.

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM- 19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with ß-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6ß-OH-, 2α-OH- and 2ß-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD50 were 1.59 µM and 31 µM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture.

Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells (ESC) impedes the establishment and validation of porcine ESC lines. This study evaluated the expression of known mouse ESC and human ESC (hESC) pluripotency markers in in vivo inner cell mass (ICM) and in vitro-cultured undifferentiated porcine epiblast cells isolated from 8-day porcine blastocysts, primary cultures of epiblast-derived neuroprogenitor cells, and endoderm cells. The expression profile of common pluripotency markers (POU domain 5 transcript factor 1, SRY-box containing gene 2, and Nanog homeobox), species-specific markers, ESC-associated factors, and differentiation markers was evaluated. The mRNA of uncultured ICMs, cultured epiblast cells, epiblast-derived neuroprogenitor cells, and endoderm cells was amplified prior to expression analysis of candidate genes by real-time RT-PCR. ESC factors whose expression correlated best with the undifferentiated epiblast state were identified by comparative mRNA expression analysis between porcine epiblast-derived somatic cell lines, fetal fibroblasts, and adult tissues. Across tissue types Nanog homeobox exhibited ubiquitous expression, whereas POU domain 5 transcript factor 1, teratocarcinoma-derived growth factor 1, and RNA exonuclease homolog 1 transcript expression was restricted primarily to undifferentiated epiblasts. Our results suggested that expression of pluripotency markers in undifferentiated pig epiblast cells more closely resembled that observed in hESC. Expression alterations of ESC-associated factors in epiblast cells were also observed during in vitro culture. Our data demonstrate the potential use of some pluripotency factors as markers of porcine epiblast stem cells and indicate that the in vitro environment may influence the cultured epiblast's developmental state.

Granulocyte-macrophage colony-stimulating-factor increases interferon-tau protein secretion in bovine trophectoderm cells.

PROBLEM: Uterine-derived factors are required for optimal conceptus development and secretion of the maternal recognition of pregnancy factor, interferon-tau (IFN-tau). Identifying these factors may lead to the development of schemes for increasing pregnancy success in cattle. METHOD OF STUDY: The objectives were to examine the effects of granulocyte-macrophage colony-stimulating-factor (GM-CSF) on trophectoderm proliferation rates and IFN-tau production, and verify the appropriateness of using an in vitro model of bovine trophectoderm (CT-1 cell). RESULTS: Rate of [(3)H]-thymidine incorporation into DNA was increased by supplementation of CT-1 medium with 10 or 100 ng/mL porcine (po) GM-CSF. GM-CSF supplementation to CT-1 medium also increased IFN-tau secretion. When results were normalized to account for number of CT-1 cells, 10 and 100 ng/mL poGM-CSF increased antiviral activity and IFN-tau concentrations (using an IFN-tau-specific enzyme-linked immunosorbent assay) in CT-1 conditioned medium compared with controls. CONCLUSIONS: These findings indicate that GM-CSF increases proliferation and IFN-tau production in bovine trophectoderm.

Fibroblast growth factor-2 is expressed by the bovine uterus and stimulates interferon-tau production in bovine trophectoderm.

Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-tau (IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [(3)H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants.

The production and characterization of anti-bovine CD14 monoclonal antibodies.

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.

Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and interleukin-6 and granulocyte colony stimulating factor knockout mice.

In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50-75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.

The PICM-19 cell line as an in vitro model of liver bile ductules: effects of cAMP inducers, biopeptides and pH.

The PICM-19 fetal liver cell line was isolated from the primary culture and spontaneous differentiation of pig epiblast cells, i.e. embryonic stem cells. PICM-19 cells were induced to differentiate into mostly ductular formations by culturing at pH 7.6-7.8. The ductules were functionally assayed by treatment with cAMP inducing agents and bioactive peptides reported to influence the secretory activity of liver bile ductules. The secretory response of the cells was assessed by qualitative or quantitative measurement of the cross-sectional area of the ductal lumens and the appearance of biliary canaliculi in between PICM-19 cells that had formed monolayers instead of ducts. Forskolin (10 microM) and 8-bromoadenosine 3':5'-cyclic monophosphate (bcAMP; 2 mM) stimulated fluid transport and expansion of ductal structures in 15-20 min and stimulated the appearance and expansion of biliary canaliculi in 30-60 min. Cholera toxin (50 ng/ml) stimulates fluid transport in both ductules and canaliculi in 1-2 h, while 8-bromoguanosine 3':5'-cyclic monophosphate (bcGMP; 2 mM) stimulated only biliary canaliculi in 2 h. Glucagon (1.4 nM) produced a similar response in 5-10 min in ductal structures only, but the response was transitory and was almost completely reversed within 30 min. Secretin (100 pM) and vasoactive intestinal peptide (75 pM) produced a sustained response with maximal ductal lumen expansion occurring in 5-10 min and neither had an immediate effect on canaliculi. Somatostatin (0.5 microM) and gastrin (1 microM) caused marked reduction or disappearance of ductal lumens in 30-60 min, but was ineffective in reversing secretin (100 nM)-induced duct distension. Application of the adrenergic agonists, epinephrine, isoproterenol, and phenylephrine (100 microM), resulted in the complete shrinkage of ductal lumens in 20-30 min. A shift to pH 7.0-7.2 resulted in almost complete reduction of ductal lumens, while a shift to pH 7.8-8.0 resulted in expansion, although not full expansion, of the ductal lumens. PICM-19 bile duct cultures were positive for cytokeratin-7, aquaporin-1 and aquaporin-9 by Western blot analysis. The amounts of these proteins increased in the cultures as differentiation proceeded over time. Transmission electron microscopy revealed that the ductal structures were usually sandwiched between SIM mouse, thioguanine- and ouabain-resistant (STO) feeder cells that had produced a collagen matrix. Also, the ductular PICM-19 cells possessed cilia, probably occurring as a single cilium in each cell, that projected into the lumens of the ducts. The results indicated that the in vitro-produced ductal structures of the PICM-19 cell line are a functional model for biliary epithelium.