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.

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.

Metabolomic analysis of longissimus from underperforming piglets relative to piglets with normal preweaning growth.

BACKGROUND: Recent increases in intra-litter variability in weaning weight have raised swine production costs. A contributor to this variability is the normal birth weight pig that grows at a slower rate than littermates of similar birth weight. The goal of this study was to interrogate biochemical profiles manifested in skeletal muscle originating from slow growing (SG) and faster growing littermates (control), with the aim of identifying differences in metabolic pathway utilization between skeletal muscle of the SG pig relative to its littermates. Samples of longissimus muscle from littermate pairs of pigs were collected at 21 d of age for metabolomic analysis (Metabolon, Inc., Durham, NC). RESULTS: Birth weights did not differ between littermate pairs of SG and Control pigs (P > 0.05). Weaning weights differed by 1.51 ± 0.19 kg (P < 0.001). Random forest (RF) analysis was effective at segregating the metabolome of muscle samples by growth rate, resulting in a predictive accuracy of 81% versus random segregation (50%). Decreases in sugars in the pentose phosphate pathway (PPP) in the longissimus of SG pigs were detected (P < 0.05). Decreases were also apparent in glycolytic intermediates (glycerol-3-phosphate and lactate) and key glycolysis-derived intermediates (glucose-6-phosphate and fructose-6-phosphate; P < 0.05). SG pigs had increased levels of phospholipids, lysolipids, diacylglycerols, and sphingolipids (P < 0.05). Pathway analysis identified a cluster of molecules associated with muscle and collagen/extracellular matrix breakdown that are increased in the SG pig (glutamate, 3-methylhistidine and hydroxylated proline moieties; P < 0.05). Nicotinate metabolism was altered in SG pigs, resulting in a 78% decrease in the nicotinamide adenine dinucleotide pool (P < 0.05). CONCLUSIONS: These metabolomic data provide the first evidence for biochemical mechanisms that should be investigated to determine if they have a potential role in the slow growth in some normal birth weight piglets that contribute to increased intra-litter variability in weaning weights and provides essential information and potential targets for the development of nutritional intervention strategies.

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.

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.

Identification of alpha-1 acid glycoprotein (AGP) as a potential marker of impaired growth in the newborn piglet.

Two studies were conducted to investigate the relationship between circulating levels of haptoglobin and α-1 acid glycoprotein (AGP) and growth in neonatal pigs. Circulating serum AGP, but not haptoglobin, was higher (P<0.001) in newborn runts than average-sized littermates. At 1 and 3 weeks, AGP and haptoglobin were similar among control and runt piglets. To determine the possible association between AGP and growth rate, blood was collected between the first and second day after birth in piglets from 10 average litters. Birthweight was positively correlated with growth rate through 21 days (linear regression correlation coefficient (CC), 0.43 (P<0.006); 0.299 (P<0.003) in males and females, respectively). Plasma AGP at birth was negatively correlated with growth (CC, -0.429 (P<0.006); -0.351 (P<0.01) in males and females, respectively). When AGP was calculated on a per kg birthweight basis, the CC with growth improved by 25 and 34% in males and females, respectively, compared with birthweight alone. Haptoglobin in blood was not correlated with growth. These data suggest that AGP at birth is reflective of growth conditions in utero or fetal maturation and may serve as an early predictive biomarker for pre-weaning growth rate.

Identification of protein carbonyls in serum of the fetal and neonatal pig.

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITC-avidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be approximately 600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (<1 kg at birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum.

A gel-based reference map of the porcine hepatocyte proteome.

The overall goal of our research is to characterize and identify gene expression profiles of porcine hepatic cells. In this study, we have prepared two-dimensional electrophoresis maps of cytosol and membrane fractions from freshly prepared hepatocytes which were pooled from three crossbred pigs (35-69kg). Following isoelectric focusing with three pH range immobilized pH gradient strips (pH 3-6, 5-8 and 7-10) and staining the second dimension gels with colloidal Coomassie blue, 728 protein spots were picked and digested with trypsin. Extracted tryptic peptides were initially subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis for identification of proteins by peptide mass fingerprinting (PMF). Proteins which were not identified by PMF were analyzed by liquid chromatography-tandem MS. Utilizing publicly available databases [NCBInr, Swiss Prot and expressed sequence tags (EST)], 648 proteins were identified. Of those, 282 were unique proteins and greater than 90% of proteins spots contained single proteins. These data represent the first comprehensive proteomic analysis of porcine hepatocytes and will provide a database for future investigations of endocrine regulation of gene expression and metabolic processes in vitro.

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.