A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig

In Vitro Cell.Dev.Biol.-Animal

Caperna

1998

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SUMMARYSecondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.

Section: Discussionsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 92%

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

In Vitro Cell.Dev.Biol.-Animal

Caperna

1998

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“…That is, it is possible that the PFL low-density ductal structures resulted because there were a finite number of cells committed to ductal formation in the population. In contrast, PICM-19 appeared to replenish its stem cell compartment over passage, and, therefore, retained its ability to differentiate into ductal structures with relatively high cell density (manuscript submitted, Talbot et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…Unconcentrated, serumfree conditioned media samples or diluted pig serum (1:50) were prepared as previously described (Talbot et al, 1994). Samples were loaded onto a 10% polyacrylamide gel, and electrophoresis was performed under described conditions (Laemmli, 1970) Hybridizations with a human glyceraldehyde-3-phosphate dehydrogenase DNA probe (pSP6-G3PDH, Ambion, Austin, TX) or a rabbit a-actin DNA probe, provided by Dr. Roger T. Stone, were subsequently performed as loading controls.…”

Section: Methodsmentioning

confidence: 99%

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Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

In Vitro Cell Dev Biol - Animal

Pursel

Rexroad

et al. 1994

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SUMMARYThe secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and f-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.

In vitro pluripotency of epiblasts derived from bovine blastocysts

Molecular Reproduction Devel

Powell

Rexroad

1995

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Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state.