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

Talbot, Neil C.; Pursel, V. G.; Rexroad, Caird E.; Caperna, Thomas J.; Powell, Anne M.; Stone, R. T.

doi:10.1007/bf02639395

Cited by 32 publications

(30 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have also shown that STO feeder cells will support the growth of pig fetal liver cells that differentiated into hepatocytes and ductal structures (Talbot et al, 1994b). We now show that adult pig intrahepatic bile duct cells can 'To whom correspondence should be addressed.…”

Section: Introductionmentioning

confidence: 69%

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

Talbot

Caperna

1998

In Vitro Cell.Dev.Biol.-Animal

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 69%

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

Talbot

Caperna

1998

In Vitro Cell.Dev.Biol.-Animal

Self Cite

View full text Add to dashboard Cite

show abstract

“…Spontaneous differentiation into endoderm-like cells has been previously shown in cultures derived from pig and bovine epiblasts (30,33), and colonies established from the porcine cells expressed ␣-fetoprotein, albumin, and ␤-fibrinogen mRNA (31,32) and may serve as a source for endodermspecific lines. Differentiation of ES cells which express endoderm-specific factors provides an experimental system for studying endoderm differentiation in vitro.…”

Section: Discussionmentioning

confidence: 87%

Involvement of Hepatocyte Nuclear Factor 3 in Endoderm Differentiation of Embryonic Stem Cells

Levinson-Dushnik

Benvenisty

1997

Molecular and Cellular Biology

116

View full text Add to dashboard Cite

The transcription factors of the hepatocyte nuclear factor 3 (HNF3) family, which are active in the liver, are expressed early during endoderm differentiation. To study their involvement in early murine development, we examined their role in embryonic stem (ES) cells. HNF3␣ or HNF3␤ mRNA transcripts were not detected in ES cells before differentiation, and only low levels of HNF3␤ mRNA were detected at a late stage of differentiation of ES cells to embryoid bodies (EB) (20 days after induction of differentiation). To examine the consequences of overexpressing HNF3␣ or -␤ in ES cells, we transfected the two genes into these cells and determined the levels of expression of tissue-specific genes during EB differentiation. Specifically, we examined expression of albumin, cystic fibrosis transmembrane conductance regulator (CFTR), phosphoenolpyruvate carboxykinase (PEPCK), ␣1-antitrypsin, transthyretin, -globin, and neurofilament 68kd as markers for different cell lineages. Overexpression of HNF3␤ (and to a lesser extent of HNF3␣) induced the expression of genes associated with endodermal lineage, namely, the genes for CFTR and albumin, but did not induce the expression of genes involved in late endoderm differentiation, such as the genes for PEPCK and ␣1-antitrypsin. Moreover, expression of HNF1␤ was highly induced in HNF3-overexpressing cells, while expression of HNF1␣ and HNF4 was only mildly induced in these cells. Therefore, HNF3␣ and -␤ seem to be involved in early endoderm differentiation of ES cells and together with other developmental factors are apparently needed for the induction of the endodermal lineage in vivo.The study of cell-specific transcriptional control has led to the identification of transcription factors which are enriched in hepatocytes, among them the hepatocyte nuclear factor 3 (HNF3) family (consisting of HNF3␣, -␤, and -␥) (42). The HNF3 proteins were first identified in rat liver nuclear extracts by virtue of their ability to bind specifically to sequences required for hepatocyte-specific expression of mouse genes (15). HNF3␤, -␣, and -␥ are sequentially activated during the development of the definitive endoderm (42). HNF3␤ mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated (21). Subsequently, HNF3␣ is transcribed in the primitive endoderm in the region of the invaginating foregut, and HNF3␥ appears upon hindgut differentiation (21). HNF3␣ and -␤ are required for mesoderm and neural axis formation, as well as for liver-specific gene expression (21).Embryonic stem (ES) cells are clonal cells derived from the preimplantation mouse embryo (24). ES cells can multiply in culture and remain pluripotent. There are three main mechanisms by which ES cells can be induced to undergo differentiation to all cell types. First, when injected into the inner cell mass of a host blastocyst, they integrate into the inner cell mass and populate all cell lineages including the germ line. Second, when inject...

show abstract

“…It originated from the spontaneous differentiation of pig epiblast tissue and is therefore derived from pig embryonic stem cells [Talbot et al, 1993[Talbot et al, , 1994a. The cell line is also unique in its characteristic differentiation into two phenotypes that resemble fetal hepatocytes or fetal and adult bile duct epithelium [Talbot et al, 1994b]. The second of these phenotypes, bile duct epithelium, is manifested by a unique and striking in vitro morphological change, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…Besides these markers, the SIM mouse, thioguanine-and ouabain-resistant (STO) co-culture of numerous tissues of fetal and adult pigs (e.g., testis, kidney, pancreas, lung, brain, intestine, mammary gland, muscle, skin and blood) have not yielded cells that are similar in appearance or behavior to the PICM-19 cells. In contrast, the culture of fetal pig liver tissue [Talbot et al, 1994b] and adult pig liver tissue ] resulted in cell cultures that closely resembled the PICM-19 cells morphologically and biochemically. Thus, the PICM-19 cells appear to be bipotent liver stem cells with the ability to differentiate into monolayers of hepatocytes or multicellular ductal structures resembling bile duct epithelium.…”

Section: Introductionmentioning

confidence: 99%

The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

Talbot¹,

Caperna²,

Wells³

2002

Cells Tissues Organs

Self Cite

View full text Add to dashboard Cite

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 µM) 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 µM) and gastrin (1 µM) 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 µM), 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.

show abstract

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

Cited by 32 publications

References 47 publications

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

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

Involvement of Hepatocyte Nuclear Factor 3 in Endoderm Differentiation of Embryonic Stem Cells

The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

Contact Info

Product

Resources

About