Browsing by Author "Caperna, Thomas J."

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    Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors
    Talbot, Neil C.; Sparks, Wendy O.; Phillips, Caitlin E.; Ealy, Alan D.; Powell, Anne M.; Caperna, Thomas J.; Garrett, Wesley M.; Donovan, David M.; Blomberg, Le Ann (2017-06)
    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.
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    Metabolism of supplemental iron by hepatic parenchymal and sinusoidal cells of the neonatal pig
    Caperna, Thomas J. (Virginia Polytechnic Institute and State University, 1986)
    Methods were developed to isolate and culture the predominant cell types from porcine liver to investigate hepatic accumulation, distribution and intracellular metabolism of supplemental iron. Hepatocytes were prepared from collagenase perfused livers by differential centrifugation, while Kupffer cells and endothelial cells were isolated by centrifugal elutriation. One day old piglets were injected with iron-dextran (Fe-dextran) and the concentration of accumulated iron was determined in all three cell types 1, 5, and 10 days later. The concentration of iron increased markedly in all three cell types when compared to cells isolated from untreated piglets (Kupffer cells > endothelial cells >> hepatocytes). Accumulated iron was subsequently mobilized from all three cell types. The role of ferritin in metabolism and storage of accumulated iron was investigated. An antiserum was prepared against porcine liver ferritin and the quantity of cellular ferritin was measured by immunoelectrophoresis. The amount of cellular iron associated with ferritin was assessed by ion exchange chromatography. All three types of liver cells accumulated ferritin in response to Fe-dextran treatment. Higher concentrations of ferritin-iron and ferritin-protein were present in Kupffer and endothelial cells than in hepatocytes at all times after iron treatment. However, at 1 day after treatment 48% of the total iron within hepatocytes was associated with ferritin; ferritin-iron accounted for only 10% of total cell iron by day 10. In contrast, ferritin-iron represented only approximately 9% of the total iron in sinusoidal cells throughout the study period. The possibility that accumulation of Fe-dextran enhanced peroxidation of membrane lipids was evaluated. Lipids extracted from heart and liver of iron-treated piglets contained increased levels of conjugated dienes. High levels of conjugated dienes were present in endothelial cells and hepatocytes 1 day after treatment and only in endothelial cells by day 5. Although Kupffer cells accumulated substantial quantities of Fe-dextran, conjugated dienes were not detectable. These studies indicate that treatment of piglets with Fe-dextran may selectively impair function of hepatic endothelial cells and perhaps hepatocytes, and define new criteria for evaluating compounds that are used for iron supplementation.