Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors.

Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.

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.

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.

Characterization of two subpopulations of the PICM-19 porcine liver stem cell line for use in cell-based extracorporeal liver assistance devices.

Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent PICM-19 pig liver stem cell line and assessed for their potential application in artificial liver devices (ALD). The study included assessments of growth rate and cell density in culture, morphological features, serum protein production, gamma-glutamyltranspeptidase (GGT) activity and hepatocyte detoxification functions, i.e., inducible P450 activity, ammonia clearance, and urea production. The PICM-19H cell line was derived by temperature selection at 33-34 degrees C. After each passage, PICM-19H cells grew to a nearly confluent monolayer of cells of hepatocyte morphology, i.e., cuboidal cells with centrally located nuclei joined by biliary canaliculi. No differentiation and self-organization into multi-cellular bile ductules, as observed in the parental PICM-19 cell line, occurred within the PICM-19H cell monolayers. The PICM-19H cells contained numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles. The cells had a doubling time of 48-72 h and reached a final density of 1.5 x 10(5) cells/cm(2) at approximately10 d post-passage from a 1:6 split ratio. PICM-19H cells displayed inducible P450 activity, cleared ammonia, and produced urea in a glutamine-free medium. The PICM-19B cells were colony-cloned after spontaneous generation from the PICM-19 parental cell line. PICM-19B cells grew as a tightly knit dome-forming monolayer with no visible biliary canaliculi. Their doubling time was 48-72 h with a final cell density of 2.6 x 10(5) cells/cm(2). Ultrastructural analysis of the PICM-19B monolayers showed the roughly cuboidal cells displayed basal-apical polarization and were joined by tight junction-like complexes. Other ultrastructure features were similar to those of PICM-19H cells except that they possessed numerous cell bodies resembling mucus vacuoles. The PICM-19B cells had relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. PICM-19B cells produced markedly less serum proteins than PICM-19H cells. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an ALD.

Comparative proteomic and regulatory network analyses of the elongating pig conceptus.

Embryo loss during peri-implantation can approach 20% in swine following artificial insemination or natural mating and coincides with rapid conceptus elongation. The objective of the present study was to establish a comprehensive profile of the abundant proteins of the pig conceptus at the time prior to implantation and identify stage-specific changes during elongation. The abundant proteins of a homogenous population of gestational day-11 ovoid (0.7-1 cm) and gestational day-12 filamentous (15-20 cm) porcine concepti were compared by extracting proteins from three independent conceptus pools and separating the proteins by 2-DE. Proteins in 305 spots were analyzed by MALDI-TOF or additionally by LC-MS/MS and 275 were positively identified representing 174 distinct proteins. The proteins could be classified into the following functional categories: cell proliferation/differentiation, cytoskeleton, metabolism, and stress response. Based on spot density, 35 proteins associated with cell proliferation, differentiation, apoptosis, and embryo/maternal signaling, were found to be differentially expressed between ovoid and filamentous concepti. A comparison of the protein expression profile with transcriptomic data from pig concepti of the same developmental stages identified similarities and dissimilarities between protein and mRNA expression profiles. This proteomic study helps to elucidate the biological mechanisms underlying the early embryonic development of the pig.

Pancreatic duct-like cell line derived from pig embryonic stem cells: expression of uroplakin genes in pig pancreatic tissue.

The isolation of a cell line, PICM-31D, with phenotypic characteristics like pancreatic duct cells is described. The PICM-31D cell line was derived from the previously described pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. The PICM-31D cell line was morphologically distinct from the parental cells in growing as a monolayer rather than self-assembling into multicellular acinar-like structures. The PICM-31D cells were propagated for over a year at split ratios of 1:3 to 1:10 at each passage without change in phenotype or growth rate. Electron microscopy showed the cells to be a polarized epithelium of cuboidal cells joined by tight junction-like adhesions at their apical/lateral aspect. The cells contained numerous mucus-like secretory vesicles under their apical cell membrane. Proteomic analysis of the PICM-31D's cellular proteins detected MUC1 and MUC4, consistent with mucus vesicle morphology. Gene expression analysis showed the cells expressed pancreatic ductal cell-related transcription factors such as GATA4, GATA6, HES1, HNF1A, HNF1B, ONECUT1 (HNF6), PDX1, and SOX9, but little or no pancreas progenitor cell markers such as PTF1A, NKX6-1, SOX2, or NGN3. Pancreas ductal cell-associated genes including CA2, CFTR, MUC1, MUC5B, MUC13, SHH, TFF1, KRT8, and KRT19 were expressed by the PICM-31D cells, but the exocrine pancreas marker genes, CPA1 and PLA2G1B, were not expressed by the cells. However, the exocrine marker, AMY2A, was still expressed by the cells. Surprisingly, uroplakin proteins were prominent in the PICM-31D cell proteome, particularly UPK1A. Annexin A1 and A2 proteins were also relatively abundant in the cells. The expression of the uroplakin and annexin genes was detected in the cells, although only UPK1B, UPK3B, ANXA2, and ANXA4 were detected in fetal pig pancreatic duct tissue. In conclusion, the PICM-31D cell line models the mucus-secreting ductal cells of the fetal pig pancreas.

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.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection.

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Feeder-cell-independent culture of the pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31.

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the derivative cell line, PICM-31A1. PICM-31A1 cells were adapted to growth on a polymerized collagen matrix using feeder cell-conditioned medium and were designated PICM-31FF. Like the parental cells, the PICM-31FF cells were small and grew relatively slowly in closely knit colonies that eventually coalesced into a continuous monolayer. The PICM-31FF cells were extensively cultured: 40 passages at 1:2, 1:3, and finally 1:5 split ratios over a 1-yr period. Ultrastructure analysis showed the cells' epithelial morphology and revealed that they retained their secretory granules typical of pancreas acinar cells. The cells maintained their expression of digestive enzymes, including carboxypeptidase A1 (CPA1), amylase 2A (AMY2A), and phospholipase A2 (PLA2G1B). Alpha-fetoprotein (AFP), a fetal cell marker, continued to be expressed by the cells as was the pancreas alpha cell-associated gene, transthyretin. Several pancreas-associated developmental genes were also expressed by the cells, including pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor, 1a (PTF1A). Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratin-8 and cytokeratin-18. The PICM-31FF cell line provides an in vitro model of fetal pig pancreatic exocrine cells without the complicating presence of feeder cells.

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.

Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters.

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation.

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.

Isolation and characterization of porcine visceral endoderm cell lines derived from in vivo 11-day blastocysts.

Two porcine cell lines of yolk-sac visceral endoderm, designated as PE-1 and PE-2, were derived from in vivo 11-d porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of the cell lines was done on STO feeder cells. The PE-1 and PE-2 cells morphologically resembled visceral endoderm previously cultured from in vivo-derived ovine and equine blastocysts and from in vitro-derived bovine blastocysts. Analysis of the PE-1- and PE-2-conditioned medium by 2D-gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry demonstrated that they produced serum proteins. Reverse transcriptase polymerase chain reaction analysis showed that the cells expressed several genes typical for yolk-sac endoderm differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, alpha-fetoprotein, and albumin. Unlike a porcine liver cell line, the PE-1 and PE-2 cell lines had relatively low inducible P-450 content and EROD activity, and, while they cleared ammonia from the cell culture medium, they did not produce urea. Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions and by lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated that the PE-1 cells expressed cytokeratin 18 and had robust microtubule networks similar to those observed in in vivo porcine yolk-sac endoderm. Metaphase spreads prepared at passage 26 of the PE-1 cell line indicated a diploid porcine karyotype of 38 chromosomes. The cells have been grown for over 1 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The cell lines will be of interest as an in vitro model of the porcine preimplantation yolk-sac tissue.

Identification and characterization of aquaporin-9 (AQP9) in porcine hepatic tissue and hepatocytes in monolayer culture.

Aquaporins (AQPs) are members of a large family of integral membrane proteins involved in the rapid movement of water and neutral solutes across cell membranes. In this study, we have prepared an affinity-purified porcine-specific polyclonal antiserum to AQP9 and have investigated the distribution and expression of AQP9 in pig liver tissue and in hepatocytes in primary culture. Immunocytochemical analysis showed that AQP9 was primarily localized in the membrane structures of hepatocytes and was not associated with intrahepatic bile ducts or blood vessels. Western blot analysis indicated that AQP9 ranged in apparent molecular mass between 27 and 38 kD in whole liver and hepatocyte membrane fractions; minor components were also observed at approximately 34 kD in the cytosol compartment of hepatocytes, bile duct and gall bladder. A prominent immunoreactive band at 44 kD was shown to be an artifact of Western blot analysis. In primary cultures of porcine hepatocytes, glucagon enhanced absolute levels of AQP9 protein, while gene expression was enhanced by T3 and glucagon. Insulin alone had no discernable influence on AQP9 gene expression or its cellular protein levels. These data suggest that AQP9 is a major AQP in porcine hepatic tissue and appears to be primarily responsive to glucagon induction.

Derivation and Characterization of a Pig Embryonic-Stem-Cell-Derived Exocrine Pancreatic Cell Line.

OBJECTIVES: The aim of this study was to identify an epithelial cell line isolated from the spontaneous differentiation of totipotent pig epiblast cells. METHODS: PICM-31 and its colony-cloned derivative cell line, PICM-31A, were established from the culture and differentiation of an epiblast mass isolated from an 8-day-old pig blastocyst. The cell lines were analyzed by transmission electron microscopy, marker gene expression, and mass spectroscopy-based proteomics. RESULTS: The PICM-31 cell lines were continuously cultured and could be successively colony cloned. They spontaneously self-organized into acinarlike structures. Transmission electron microscopy indicated that the cell lines' cells were epithelial and filled with secretory granules. Candidate gene expression analysis of the cells showed an exocrine pancreatic profile that included digestive enzyme expression, for example, carboxypeptidase A1, and expression of the fetal marker, α-fetoprotein. Pancreatic progenitor marker expression included pancreatic and duodenal homeobox 1, NK6 homeobox 1, and pancreas-specific transcription factor 1a, but not neurogenin 3. Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratins 8 and 18. CONCLUSIONS: The PICM-31 cell lines provide in vitro models of fetal pig pancreatic exocrine cells. They are the first demonstration of continuous cultures, that is, cell lines, of nontransformed pig pancreas cells.

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.].

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.

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.

Isolation and characterization of a bovine visceral endoderm cell line derived from a parthenogenetic blastocyst.

A cell line, BPE-1, was derived from a parthenogenetic 8-d in vitro-produced bovine blastocyst that produced a cell outgrowth on STO feeder cells. The BPE-1 cells resembled visceral endoderm previously cultured from blastocysts produced by in vitro fertilization (IVF). Analysis of the BPE-1 cells demonstrated that they produced serum proteins and were negative for interferon-tau production (a marker of trophectoderm). Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with desmosomes. Rough endoplasmic reticulum was prominent within the cells as were lipid vacuoles. Immunocytochemistry indicated the BPE-1 cells had robust microtubule networks. These cells have been grown for over 2 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The BPE-1 cell line presumably arose from embryonic cells that became diploid soon after parthenogenetic activation and development of the early embryo. However, metaphase spreads prepared at passage 41 indicated that the cell population had a hypodiploid (2n = 60) unimodal chromosome content with a mode of 53 and a median and mean of 52. The cell line will be of interest for functional comparisons with bovine endoderm cell lines derived from IVF and nuclear transfer embryos.

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.