J. Exp. Path. (I990) 71, 4I -50
Development of intrahepatic bile ducts in rat foetal liver explants in vitro J.A.M. Gall and P.S. Bhathal* Department of Pathology, The University of Melbourne, and *Department of Anatomical Pathology, The Royal Melbourne Hospital, Victoria, Australia Received for publication I June I989 Accepted for publication 3 I August I 989
Summary. The origin of intrahepatic bile ducts was investigated in organ cultures of 3, I 6 and I 9-day foetal rat livers embedded for up to IO days in a semi-solid agar gel on a filter-raft assembly. Following IO days in culture, I 3-day foetal explants consisted of liver cell trabeculae lined by endothelial cells. Although maturation of the liver cells and bile canaliculi was observed, duct development was found in less than io% of the explants. Supplementation of the media with putative inducers of bile duct development or culture of explants adjacent to other tissues did not induce regular duct development. By contrast, explants from the porta hepatis of I 9-day foetuses cultured for 4 days, but not days, regularly contained duct-like structures. The formation of the few ducts in cultures of I 3-day foetal liver explants indicates that these cells can arise by transformation of hepatoblasts but that specific inducers of development are required for predictable and continuous differentiation of biliary epithelial cells. 1
io
Keywords: embryology, intrahepatic bile ducts, liver, morphology, ultrastructure
The ontogeny of the intrahepatic biliary tree is still uncertain and there are two main theories. The ductal hypothesis suggests that the intrahepatic bile ducts develop from the main hepatic ducts (Hammar I926; Koga 1971) whilst the hepatocytogenic hypothesis holds that biliary epithelial cells are derived by a transformation of periportal liver cells (Bloom 1926; Horstmann 1939; Elias I955; Du Bois I963; Wilson et al. I963; Wood I965; Shiojiri I984; Van Eyken et al. I988; Gall & Bhathal I989). Bloom (1926) and Horstmann (I939)
organ
culture, cell differentiation,
observed that differentiating biliary epithelial cells were always adjacent to ingrowing portal connective tissue and suggested bile ducts arose from liver cells under its influence. Doljanski and Roulet (I934) observed the in-vitro transformation of foetal liver cells to duct-like structures under the influence of mesenchyme but reasoned that it plays a merely permissive role. The present study was undertaken to observe the morphological changes connected with duct development in cultured foetal liver explants.
Correspondence: Professor P.S. Bhathal, Department of Anatomical Pathology, The Royal Melbourne Hospital, RMH Post Office, Victoria 3050, Australia.
4I
42 Materials and methods
J.A.M. Gall & P.S. Bhathal trolled atmosphere of I0% C02 in air. The medium was changed every 3 days.
Foetal explants Female Sprague-Dawley rats (10-I4 weeks of age) were mated overnight in wire cages. The following morning, day i, waste collection trays under each cage were inspected for evidence of vaginal plugs. At I 3, i 6 or i 9 days' gestation, the pregnant females were placed under ether anaesthesia and a midline abdominal incision made. The uterine horns containing the foetuses were carefully removed and placed in a sterile Petri dish. All subsequent steps were performed in a laminar flow hood. The uteri of rats were opened and the foetuses removed and placed in cold 0.1 5 M phosphate buffered saline (PBS) in an icecold Petri dish. After decapitation, the foetal livers were excised under a dissecting stereomicroscope. The pancreas and stomach, either attached to the liver or separately, and the small intestine, were excised as required. At I 3 days' gestation, the liver consisted of a median and two lateral lobes. It was used either whole or as separately dissected median and lateral lobes. The procedure for obtaining i 6 and 19-day foetal livers was similar but at this age peripheral portions of the left lateral lobe and the porta hepatis region of the median lobe were used.
Culture procedure Culture conditions. Dulbecco's Modified Eagle's medium (DME; Flow Laboratories Australasia Pty Ltd, North Ryde, NSW, Australia) supplemented with I5% heat-inactivated newborn calf serum (NCS; Flow Laboratories) was used for the culture of tissue explants. Foetal tissues, embedded in either agar or collagen gels, as described below, were placed on strips of 0.45 gm filters (Amicon) on gelfoam sponge rafts (size I2, 20 x 6o X 2 mm; Upjohn, Kalamazoo, MI, USA) in I5 ml of culture medium in plastic Petri dishes (go mm outside base diameter; Johns Professional Products, Melbourne, Australia) and incubated at 370C in a con-
Agar gels. Stock solutions of twofold concentrated DME containing sodium bicarbonate and 2% molten Bacto agar (Difco Laboratories, Detroit, MI, USA) were prepared and added to NCS in the ratio of 2: 2: I, respectively, and maintained at 40°C in a waterbath. Molten agar gel (I ml/dish) was pipetted into Petri dishes (36 mm outside base diameter; Kayline Plastics, Thebarton, South Australia) and allowed to set. Explants were placed onto the gel, excess fluid removed and the tissue overlaid with a few drops of gel solution. The gels were trimmed around the explants after gelation.
Collagen gels. Stock collagen was prepared as described by Michalopoulos and Pitot (I975). The gel solution was prepared by mixing 8 volumes of stock collagen solution with 2 volumes of a i: I: I mixture of tenfold concentrated DME, 0.34 M NaOH and NCS, and kept on ice to prevent immediate gelation. Gel solution (i ml/dish) was layered into 36 mm Petri dishes and allowed to set. Explants were then processed as for agar-embedded tissue. 13, i 6 and I 9-day foetal liver explants. Whole I 3-day foetal livers were grown in culture for IO, I5, 20 and 25 days and the median and left lobes were cultured for I0 days. Peripheral blocks of the excised left lateral lobe and the porta hepatis region of the median lobe of i6 and ig-day foetal livers were embedded in agar gels and placed in separate cultures; the I 6-day foetal liver was grown in culture for either 7 or Io days, while the I9-day foetal liver was grown for either 4 or I0 days. For assessment of whole I 3-day livers cultured for I0 days, 9 explants were used, 3 per culture dish. For the remainder, 6 explants were used, 3 per culture dish. Putative inducers of bile duct development. Whole foetal livers, excised on the 13th day of gestation and embedded in agar, were
Development of intrahepatic bile ducts grown in the presence of the following putative inducers of intrahepatic bile duct growth and differentiation at the concentrations indicated: (i) L-proline (io and 20 mM; hydroxy-L-proline free, Sigma Chemical Company, St Louis, MO, USA); (ii) lithocholic acid (LCA, 0.1, 0.25, 0.5 and i mM dissolved in dimethylsulphoxide (DMSO), Sigma; control media contained 0.4% DMSO); (iii) dexamethasone (i X Io-6 and 2 X 1o-6 M; David Bull Laboratories Pty Ltd, Melbourne, Australia); (iv) ascorbic acid (25 and 50 ,ug/ml, Sigma); and (v) ascorbic acid (5o0 4g/ml) + dexamethasone (2 X IO-6 M). All inducers were prepared as stock solutions, I5-75 91 being added to the culture medium. With the exception of LCA, all inducers were added from day o of culture. LCA was added for the last 3 days of culture. Tissues were maintained in culture for I0 days and the medium changed every 3 days. In cultures containing ascorbic acid, the medium was changed every other day. Foetal livers, embedded in agar gels, were
.FX..
43
also cultured with other organs in the following combinations: (i) the pancreas and stomach attached and in the same gel block as the liver; (ii) the pancreas and stomach in a separate gel but positioned on the filter adjacent to the liver; and (iii) adjacent small intestine in the same block as the liver. The three combinations were grown with and without the medium supplemented with dexamethasone at a concentration of 2 X Io6 M. Whole livers, embedded in collagen gels, were similarly cultured both with and without dexamethasone (2 X io-6 M). For all test cultures other than those embedded in collagen gels, three livers were placed in each dish and three separate trials were assessed. For livers embedded in collagen gels, two explants were placed in each dish and two separate trials were assessed. Light and electron microscopy Cultures were fixed on the supportive filter in Bouin's fluid for 20 min and processed by
...
ar Fig. i. Explant of I3-day foetal rat liver grown for io days in an agar gel. a, A single layer of slender cells covers the upper surface ofthe explant and fibroblast-like cells are seen between the lower surface and the ifiter-raft assembly (F). H&E, x 360. b, Electron micrograph showing a bile canaliculus (arrow) formed between four liver cells. Microvilli are absent from the canalicular lining except near tight junctions. x 8300.
J.A.M. Gall & P.S. Bhathal 44 standard methods for histology. Specimens from control and dexamethasone-supplemented (2 X Io-6 M) liver cultures were immersion-fixed in ice-cold 2.5% glutaraldehyde in PBS and processed for electron microscopy by routine methods. Results
13-day foetal liver explants After Io days in culture, whole 1 3-day foetal liver explants consisted ofliver cells arranged in trabeculae separated by endothelial cells lining a sinusoidal network (Fig. ia). Few haemopoietic cells were seen. The explant did not contain any other vascular structures. Mature collagen, as assessed by the Masson trichrome stain, was present in all livers forming a fine anastomosing meshwork. The liver cells were mature in appearance with a finely granular cytoplasm and a large round nucleus with one to three prominent nucleoli, but were smaller than in the adult liver and resembled those observed in the foetal liver from 2i days' gestation to birth. Characteristic apoptotic bodies, appearing as round to ovoid, eosinophilic structures containing a pyknotic nuclear fragment, were observed. Ultrastructurally, liver cells contained a large nucleus with nucleolus, abundant rough endoplasmic reticulum, numerous large mitochrondria of variable shape, Golgi apparatus, and polyribosomes. Bile canaliculi, which were occasionally seen by light microscopy, were present (Fig. ib). Except for sentinel microvilli on either side of the tight junctions, microvilli were generally not found. Development of bile ducts was infrequently observed. Ofthe 39 whole foetal liver explants, three (7.7%) contained duct-like structures lined by cuboidal cells with a large nucleus, prominent nucleolus and granular cytoplasm (Fig. 2). The size of the cells ranged from that of mature biliary epithelial cells to that observed in developing ducts in
Fig. 2. Explant of I3-day foetal rat liver after io days in culture showing four bile ducts (D) surrounded by a few fibroblasts and lined by cuboidal epithelial cells. H&E, x 68o.
foetal livers of 20-2I days' gestation. Fine strands of collagen were found adjacent to these duct structures but did not always circumscribe them. Culture of the separated median and lateral lobes for io days resulted in a microscopic appearance similar to that described above. There was no evidence of duct development. Thirteen-day whole liver cultured for both I 5 and 20 days resulted in smaller explants than in the i o-day cultures. Histologically, the livers were similar to Ioday cultures but there was reduced mitotic activity. Bile ducts were not observed. There was no viable tissue in livers cultured for 2 5 days.
i6-day foetal liver explants The findings in the explants from the porta hepatis region ofthe median lobe and peripheral portion of the left lateral lobe after Io days in culture were essentially identical. The general microscopic appearance was similar to that of the cultures of I3-days foetal livers. The only difference between explants from the different locations within the liver was the appearance in one of the explants, from the porta hepatis region, of a mature single bile duct which was lined by
Development of intrahepatic bile ducts
45
Fig. 3. Explant of 13-day liver grown for IO days in medium supplemented with 2 X Io-6 M dexamethasone. a, Numerous cystically dilated bile canaliculi (BC) are seen. H&E, x 6io. b, Electron micrograph of the cystically dilated bile canaliculi (BC), with microvilli projecting into the lumina. The lining liver cells contain glycogen (G) and lipid vacuoles (L). x 2500.
46
J.A.M. Gall & cuboidal epithelium. Explants from both the porta hepatis region and left lateral lobe cultured for 7 days did not show any evidence of duct development.
P.S. Bhathal by cuboidal cells were regularly seen in all six explants from the porta hepatis region but in none of the six explants from the peripheral part of the left lateral lobe.
i9-day foetal liver explants Explants from both the left lateral lobe and the porta hepatis region of the median lobe cultured for IO days, were similar in appearance. The amount of tissue was minimal and in some cases no viable tissue remained. The liver cells were arranged in poorly defined trabeculae. Bile canaliculi could be seen but bile duct-like structures were not evident. Apoptosis was abundant, vacuolation minimal, and necrosis was present. Explants cultured for 4 days were similar to those cultured for io days but the explant size and the mitotic activity were considerably increased. Bile duct-like structures lined
Development of 13-day foetal liver explants in the presence ofputative bile duct growth inducers Development in thepresence ofputative chemical inducers. Explants grown for IO days in the presence of dexamethasone (i X io-6 and 2 X Io6 M) consisted of liver cells which were more mature in appearance than in control livers (I 3-day foetal livers cultured for io days). The distinguishing feature of these explants was the presence of cystically dilated bile canaliculi (Fig. 3a). However, bile duct development did not occur. Cytoplasmic vacuolation was often present and a small amount of necrosis was occasionally seen.
Fig. 4. I 3-day foetal liver grown in a collagen gel for io days showing several ducts lined by epithelial cells. Surrounding the ducts is a layer of basement membrane collagen. In two ducts (D) the biliary epithelium merges with the surrounding liver cells (arrows). The base of the explant consists of numerous fibroblasts and collagen. L, Cell with liver cell phenotype in duct wall. Masson trichrome, x 475.
Development of intrahepatic bile ducts Deposition of mature collagen in association with the sinusoidal lining cells was reduced. Ultrastructurally, the liver cells contained a large nucleus and prominent nucleolus, numerous large, variably shaped mitochondria, and abundant rough endoplasmic reticulum and glycogen deposits (Fig. 3b). The cystically dilated structures were confirmed as bile canaliculi by the presence of tight junctions between liver cells bordering the lumina. Livers grown in the presence of proline (io and 20 mM) and ascorbic acid (25 and 50 ,ug/ml) did not show any histological variation from livers grown without inducers. LCA, added for the final 3 days of culture only, at a concentration of O.I mm, did not show any difference compared with either the DMSO control or explants without DMSO. However, at 0.25 mM, a decrease in explant size and mitotic activity were noted. Concentrations in excess of 0.25 mM resulted in complete necrosis of the tissue. Growth of liver explants in collagen gels. Histologically, liver cells within these explants were larger and more mature than those in livers grown in agar gels. Bile duct-like structures were found in one of four livers in dexamethasone-free medium (Fig. 4). The lining cells were cuboidal and had a nucleus with prominent nucleoli and a darker-staining, granular cytoplasm. Liver cells formed part of the duct wall in scanty foci. Strands of collagen surrounded the more mature appearing ducts but formed a discontinuous layer around the other, incomplete duct-like structures. Dexamethasone (2 X i1-6 M) produced canalicular dilatation similar to that described above for livers grown in agar gels.
Development of liver explants in the presence of other tissues. The growth of either stomach and pancreas or small intestine explants adjacent to the liver, either in the same gel or adjacent gels, with or without dexamethasone (2 X i1-6 M), did not influence the development of the liver.
47
Discussion The results presented here demonstrate the use of an organ culture system for the investigation of intrahepatic bile duct growth and development. Using this system, explants from I 3-day rat foetuses remained viable for I0 days. Extension of the culture time to I 5 or 20 days resulted in a decrease in explant size and death of the culture by 2 5 days. Similarly, i 6 and i9-day foetal liver explants in culture showed decreased viability with increased time in culture. However, livers grown for the equivalent of the 'gestational' period (i.e. I3-day foetal liver for io days and i 6 and I 9-day foetal liver for 7 and 4 days, respectively) consisted of cells which resembled mature antenatal or neonatal cells seen in vivo. Under the conditions of the experiments, culture of I3-day foetal livers for io days did not result in the regular development of intrahepatic bile ducts, a finding suggesting either the absence of the progenitor cells or the absence of inducer(s) of growth and development. To investigate whether the progenitor cell was absent, I6 and Ig-day foetal liver explants, which contain developing ducts (Bhathal & Gall I984; Van Eyken et al. I988; Gall & Bhathal I989), were placed in culture. As it has been reported that growth of the ducts is from the porta hepatis (Bloom I926; Koga I97I; Van Eyken et al. I988) rather than the reverse (Horstmann 1939; Du Bois I963), tissue was excised and cultured from the porta hepatis region of the median lobe or the peripheral part of the left lateral lobe. Regular duct development was observed only in explants from the porta hepatis region of I 9-day foetuses when cultured for 4 days but not when cultured for io days. This finding and the single observation of duct development in i 6-day explants and absence of ducts in i9-day explants of the peripheral part of the lateral lobe may indicate the arrest of development due to the absence of the necessary growth inducer(s). As bile duct development has been described in fragments of I3.5-day foetal
48
J.A.M. Gall & P.S. Bhathal mouse liver transplanted subcutaneously in ated maturation of the added tissues nor bile syngeneic newborn mice and in fragments of duct development occurred. I2.5, I 3.5 and I8.5-day foetal livers transOne hypothesis for the development of the planted into the testes of adult mice (Shiojiri intrahepatic bile duct system suggests that it I984), the effects of putative inducers of bile occurs by modulation or transformation of duct growth were investigated in 13-day periportal hepatoblasts under the influence foetal liver explants. Dexamethasone, a syn- of connective tissue (Bloom I926; Horstthetic glucocorticoid which has been shown to accelerate morphological and physiological events in vivo (Avery I975) and to maintain the morphology and extend the longevity of liver cells in vitro (Laishes & Williams I 9 76), accelerated the maturation of liver cells in explants. Bile canaliculi with numerous microvilli were present and many were cystically dilated, probably as a result of the induction of liver cell secretion into the sealed-off biliary canaliculi. Duct development, however, did not occur. LCA, when fed to adult animals, produces biliary epithelial hyperplasia (Hunt et al. I964; Palmer & Hruban I966). It has been suggested that this response is due either to LCA itself or a metabolite (Hunt et al. I964; Palmer & Hruban I966). An alternative hypothesis holds that the hyperplasia is the result of a regenerative response to the cytotoxic effects of LCA (Palmer & Hruban I966; Gall & Bhathal I987). Although the growth rate of a cultured rat liver cell line was enhanced with increasing concentrations of LCA to I mM (eannin et al. 19 79), in foetal liver cultures it was toxic to cells above a concentration of 0.25 mM and did not induce duct morphogenesis. Similar toxicity to adult bile duct cultures above a concentration of 0.5 mM has also been reported (Gall & Bhathal I987). Stomach and pancreas or small intestine were grown adjacent to the liver explant to determine whether a diffusible compound from these tissues, which in vivo would be transported via the portal venous system, may induce duct development. In case the pathways for either the actions or production of these compounds had not developed, dexamethasone was added to the medium of some cultures. Other than the effects of dexamethasone on the liver, neither acceler-
mann 1939; Du Bois I963; Wilson et al. i963; Wood i965; Shiojiri i984). The basis for this hypothesis stems from the consistent finding of collagen adjacent to developing ducts and the in-vitro observation of the formation of duct-like structures in combined cultures of foetal liver cells and mesenchyme (Doljanski & Roulet I934). More recently, Shiojiri (i984) demonstrated that bile duct development occurs in fragments of I2.5, I3.5 and i8.s-day foetal mouse liver (intrahepatic bile duct development beginning in vivo at the porta hepatis at I4.5 days' gestation in this strain of mouse) transplanted into testis of syngeneic mice, and in the I 3. s-day foetal liver transplanted subcutaneously in newborn mice. In particular, Shiojiri noted that duct development occurred earlier when the foetal liver was transplanted subcutaneously, especially in regions rich in connective tissue. From these findings, Shiojiri suggested that contact of hepatoblasts with endothelial cells may favour their differentiation into mature liver cells whereas contact with connective tissue would favour their development into bile duct cells. In adult animals, collagen biosynthesis has been shown to be a mandatory cofactor for the maintenance of biliary and mammary duct hyperplasia (Girotra & Isseroff I980; Wicha et al. I980) and proline has been implicated in the induction of enzymes of the biosynthetic pathway leading to collagen deposition in toxic liver injury (Rojkind & Diaz de Leon 1 9 70). Similarly, ascorbic acid, in fibroblast cultures, has growth-promoting effects which have been attributed to its ability to increase the production of a collagenous matrix (Peterkofsky I972; Rowe et al. I977). To assess the possible effects of mesenchyme and collagen deposition on
Development of intrahepatic bile ducts duct development, I 3-day explants were grown in the presence of proline and ascorbic acid, adjacent to gut mesenchyme which is of similar origin to hepatic mesenchyme (Croisille & Le Douarin 9 71), and embedded in a gel of type I collagen. Growth of mesenchyme adjacent to the liver did not induce duct development. Similarly, regular duct morphogenesis was not present in collagenembedded explants, with or without dexamethasone. Overall, regular development of bile ducts did not occur in the explants. The results of the addition of various putative inducers of intrahepatic bile duct development did not point to a particular compound and it is evident that collagen of type I alone does not stimulate duct development but may, as suggested by Doljanski and Roulet (I934), have a permissive role in providing the conditions for morphogenesis and structural maintenance. Considering the effects various hormones have on differentiation and that differentiation may not be due to a single factor, a variety of combinations of the potential inducers could be further investigated using the present experimental model.
References AVERY M.E. (I975) Pharmacological approaches to the acceleration of fetal lung maturation. Br. Med. Bull. 31, I3-I7. BHATHAL P.S. & GALL J.A.M. (I984) Development of the intrahepatic bile ducts in fetal and postnatal rat liver. J. Anat. I39, I83. BLOOM W. (1926) The embryogenesis of human bile capillaries and ducts. Am. J. Anat. 36, 45 1-
465. CROISILLE Y. & LE DOUARIN N.M. (I 97 I) Development and regeneration ofthe liver. In Organogenesis. Eds R.L. De Haan & H. Ursprung. New York: Holt, Reinhart & Winston. pp. 42I-466. DOLJANSKI L. & ROULET FR. (I934) Ueber die gestaltende Wechselwirkung zwischen dem Epithel und dem Mesenchyme, zugleich ein Beitrag zur Histogenese der sogenannten 'Gallengangswucherungen'. Virchows Arch. Path. Anat. 292, 256-267. Du Bois A.M. (I963) The embryonic liver. In The Liver. Volume i. Ed. C.L. Rouiller. New York: Academic Press. pp. 1-39.
49
ELIAs H. (I955) Origin and early development of the liver in various vertebrates. Acta Hepatologica 3, I - 56. GALL J.A.M. & BHATHAL P.S. (198 7) Isolation and culture of intrahepatic bile ducts and its application in assessing putative inducers of biliary epithelial cell hyperplasia. Br. J. Exp. Path. 68,
50I-5IO.
GALL J.A.M. & BHATHAL P.S. (I989) Morphological and immunohistochemical assessment of intrahepatic bile duct development in the rat. J. Gastroenterol. Hepatol. 4, 241-249. GIROTRA K.L. & ISSEROFF H. (I980) Fasciola hepatica: azetidine inhibition ofbile duct hyperplasia in the infected rat. Exp. Parasitol. 49, 4146. HAMMAR J.A. (I926) Ueber die erste Entstehung der nicht kapillaren intrahepatischen Gallengainge beim Menschen. Z. Mikrosk-Anat. Forsch. 5, 59-89. HORSTMANN E. (I939) Entwicklung and Entwicklungsbedingungen des intrahepatischen Gallengangsystems. Willhelm Roux' Arch. Entwicklungsmech. Org. 139, 363-392. HUNT R.D., LEVEILLE G.A. & SAUBERLICH H.E. (I964) Dietary bile acids and lipid metabolism. III Effects of lithocholic acid in mammalian species. Proc. Soc. Exp. Biol. Med. 115, 277280. JEANNIN J.F., CHESSEBEUF M., MARTIN M.S., LANGNEAU A. & MARTIN F. (I 9 79) Proliferative effect of lithocholic acid on rat liver cell in culture. Biomedicine 31I, 207-209. KOGA A. (I97I) Morphogenesis of intrahepatic bile ducts of the human fetus. Light and electron microscopic study. Z. Anat. Entwicklungsgesch. 135, 156-I84. LAISHEs B.A. & WILLIAMs G.M. (I976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. II Dexamethasone enhanced longevity and maintenance of morphology. In vitro 1I2, 82 I-832. MICHALOPOULOs G. & PITOT H.C. (1975) Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Exp. Cell Res. 94, 70-78. PALMER R.H. & HRUBAN Z. (I966) Production of bile duct hyperplasia and gallstones by lithocholic acid. J. Clin. Invest. 45, I25 5I267. PETERKOFSKY B. (I972) The effect of ascorbic acid on collagen polypeptide-synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch. Biochem. Biophys. 1I52, 3 i8328. ROJKIND M. & DIAZ DE LEON L. (I970) Collagen
50
J.A.M. Gall & P.S. Bhathal
biosynthesis in cirrhotic rat liver slices. A regulatory mechanism. Biochim. Biophys. Acta
cytokeratin-immunohistochemical study. Lab. Invest. 59, 52-59.
217, 5I2-522. ROWE D.W., STARMAN B.J., FUJIMOTO W.Y. & WILLIAMS R.H. (I977) Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts. In vitro I3, 824-
WICHA M. LIOTTA L.A., VONDERHAAR B.K. & KIDWELL W.R. (I98o) Effects of inhibition of basement membrane collagen deposition on rat mammary gland development. Dev. Biol. 8o, 253-266. WILSON J.W., GROAT C.S. & LEDUC E.H. (I963) Histogenesis of the liver. Ann. N.Y. Acad. Sci. III, 8-24. WooD R.L. (I965) An electron microscope study of developing bile canaliculi in the rat. Anat. Rec. 151, 507-530.
830. SHIOJIRI N. (I984) The origin of intrahepatic bile duct cells in the mouse. I. Embryol. Exp. Morphol. 79, 25-39. VAN EYKEN P., SCIOT R. & DESMET V.J. (I 9 8 8) Intrahepatic bile duct development in the rat: a
Development of intrahepatic bile ducts in rat foetal liver explants in vitro J.A.M. Gall and P.S. Bhathal* Department of Pathology, The University of Melbourne, and *Department of Anatomical Pathology, The Royal Melbourne Hospital, Victoria, Australia Received for publication I June I989 Accepted for publication 3 I August I 989
Summary. The origin of intrahepatic bile ducts was investigated in organ cultures of 3, I 6 and I 9-day foetal rat livers embedded for up to IO days in a semi-solid agar gel on a filter-raft assembly. Following IO days in culture, I 3-day foetal explants consisted of liver cell trabeculae lined by endothelial cells. Although maturation of the liver cells and bile canaliculi was observed, duct development was found in less than io% of the explants. Supplementation of the media with putative inducers of bile duct development or culture of explants adjacent to other tissues did not induce regular duct development. By contrast, explants from the porta hepatis of I 9-day foetuses cultured for 4 days, but not days, regularly contained duct-like structures. The formation of the few ducts in cultures of I 3-day foetal liver explants indicates that these cells can arise by transformation of hepatoblasts but that specific inducers of development are required for predictable and continuous differentiation of biliary epithelial cells. 1
io
Keywords: embryology, intrahepatic bile ducts, liver, morphology, ultrastructure
The ontogeny of the intrahepatic biliary tree is still uncertain and there are two main theories. The ductal hypothesis suggests that the intrahepatic bile ducts develop from the main hepatic ducts (Hammar I926; Koga 1971) whilst the hepatocytogenic hypothesis holds that biliary epithelial cells are derived by a transformation of periportal liver cells (Bloom 1926; Horstmann 1939; Elias I955; Du Bois I963; Wilson et al. I963; Wood I965; Shiojiri I984; Van Eyken et al. I988; Gall & Bhathal I989). Bloom (1926) and Horstmann (I939)
organ
culture, cell differentiation,
observed that differentiating biliary epithelial cells were always adjacent to ingrowing portal connective tissue and suggested bile ducts arose from liver cells under its influence. Doljanski and Roulet (I934) observed the in-vitro transformation of foetal liver cells to duct-like structures under the influence of mesenchyme but reasoned that it plays a merely permissive role. The present study was undertaken to observe the morphological changes connected with duct development in cultured foetal liver explants.
Correspondence: Professor P.S. Bhathal, Department of Anatomical Pathology, The Royal Melbourne Hospital, RMH Post Office, Victoria 3050, Australia.
4I
42 Materials and methods
J.A.M. Gall & P.S. Bhathal trolled atmosphere of I0% C02 in air. The medium was changed every 3 days.
Foetal explants Female Sprague-Dawley rats (10-I4 weeks of age) were mated overnight in wire cages. The following morning, day i, waste collection trays under each cage were inspected for evidence of vaginal plugs. At I 3, i 6 or i 9 days' gestation, the pregnant females were placed under ether anaesthesia and a midline abdominal incision made. The uterine horns containing the foetuses were carefully removed and placed in a sterile Petri dish. All subsequent steps were performed in a laminar flow hood. The uteri of rats were opened and the foetuses removed and placed in cold 0.1 5 M phosphate buffered saline (PBS) in an icecold Petri dish. After decapitation, the foetal livers were excised under a dissecting stereomicroscope. The pancreas and stomach, either attached to the liver or separately, and the small intestine, were excised as required. At I 3 days' gestation, the liver consisted of a median and two lateral lobes. It was used either whole or as separately dissected median and lateral lobes. The procedure for obtaining i 6 and 19-day foetal livers was similar but at this age peripheral portions of the left lateral lobe and the porta hepatis region of the median lobe were used.
Culture procedure Culture conditions. Dulbecco's Modified Eagle's medium (DME; Flow Laboratories Australasia Pty Ltd, North Ryde, NSW, Australia) supplemented with I5% heat-inactivated newborn calf serum (NCS; Flow Laboratories) was used for the culture of tissue explants. Foetal tissues, embedded in either agar or collagen gels, as described below, were placed on strips of 0.45 gm filters (Amicon) on gelfoam sponge rafts (size I2, 20 x 6o X 2 mm; Upjohn, Kalamazoo, MI, USA) in I5 ml of culture medium in plastic Petri dishes (go mm outside base diameter; Johns Professional Products, Melbourne, Australia) and incubated at 370C in a con-
Agar gels. Stock solutions of twofold concentrated DME containing sodium bicarbonate and 2% molten Bacto agar (Difco Laboratories, Detroit, MI, USA) were prepared and added to NCS in the ratio of 2: 2: I, respectively, and maintained at 40°C in a waterbath. Molten agar gel (I ml/dish) was pipetted into Petri dishes (36 mm outside base diameter; Kayline Plastics, Thebarton, South Australia) and allowed to set. Explants were placed onto the gel, excess fluid removed and the tissue overlaid with a few drops of gel solution. The gels were trimmed around the explants after gelation.
Collagen gels. Stock collagen was prepared as described by Michalopoulos and Pitot (I975). The gel solution was prepared by mixing 8 volumes of stock collagen solution with 2 volumes of a i: I: I mixture of tenfold concentrated DME, 0.34 M NaOH and NCS, and kept on ice to prevent immediate gelation. Gel solution (i ml/dish) was layered into 36 mm Petri dishes and allowed to set. Explants were then processed as for agar-embedded tissue. 13, i 6 and I 9-day foetal liver explants. Whole I 3-day foetal livers were grown in culture for IO, I5, 20 and 25 days and the median and left lobes were cultured for I0 days. Peripheral blocks of the excised left lateral lobe and the porta hepatis region of the median lobe of i6 and ig-day foetal livers were embedded in agar gels and placed in separate cultures; the I 6-day foetal liver was grown in culture for either 7 or Io days, while the I9-day foetal liver was grown for either 4 or I0 days. For assessment of whole I 3-day livers cultured for I0 days, 9 explants were used, 3 per culture dish. For the remainder, 6 explants were used, 3 per culture dish. Putative inducers of bile duct development. Whole foetal livers, excised on the 13th day of gestation and embedded in agar, were
Development of intrahepatic bile ducts grown in the presence of the following putative inducers of intrahepatic bile duct growth and differentiation at the concentrations indicated: (i) L-proline (io and 20 mM; hydroxy-L-proline free, Sigma Chemical Company, St Louis, MO, USA); (ii) lithocholic acid (LCA, 0.1, 0.25, 0.5 and i mM dissolved in dimethylsulphoxide (DMSO), Sigma; control media contained 0.4% DMSO); (iii) dexamethasone (i X Io-6 and 2 X 1o-6 M; David Bull Laboratories Pty Ltd, Melbourne, Australia); (iv) ascorbic acid (25 and 50 ,ug/ml, Sigma); and (v) ascorbic acid (5o0 4g/ml) + dexamethasone (2 X IO-6 M). All inducers were prepared as stock solutions, I5-75 91 being added to the culture medium. With the exception of LCA, all inducers were added from day o of culture. LCA was added for the last 3 days of culture. Tissues were maintained in culture for I0 days and the medium changed every 3 days. In cultures containing ascorbic acid, the medium was changed every other day. Foetal livers, embedded in agar gels, were
.FX..
43
also cultured with other organs in the following combinations: (i) the pancreas and stomach attached and in the same gel block as the liver; (ii) the pancreas and stomach in a separate gel but positioned on the filter adjacent to the liver; and (iii) adjacent small intestine in the same block as the liver. The three combinations were grown with and without the medium supplemented with dexamethasone at a concentration of 2 X Io6 M. Whole livers, embedded in collagen gels, were similarly cultured both with and without dexamethasone (2 X io-6 M). For all test cultures other than those embedded in collagen gels, three livers were placed in each dish and three separate trials were assessed. For livers embedded in collagen gels, two explants were placed in each dish and two separate trials were assessed. Light and electron microscopy Cultures were fixed on the supportive filter in Bouin's fluid for 20 min and processed by
...
ar Fig. i. Explant of I3-day foetal rat liver grown for io days in an agar gel. a, A single layer of slender cells covers the upper surface ofthe explant and fibroblast-like cells are seen between the lower surface and the ifiter-raft assembly (F). H&E, x 360. b, Electron micrograph showing a bile canaliculus (arrow) formed between four liver cells. Microvilli are absent from the canalicular lining except near tight junctions. x 8300.
J.A.M. Gall & P.S. Bhathal 44 standard methods for histology. Specimens from control and dexamethasone-supplemented (2 X Io-6 M) liver cultures were immersion-fixed in ice-cold 2.5% glutaraldehyde in PBS and processed for electron microscopy by routine methods. Results
13-day foetal liver explants After Io days in culture, whole 1 3-day foetal liver explants consisted ofliver cells arranged in trabeculae separated by endothelial cells lining a sinusoidal network (Fig. ia). Few haemopoietic cells were seen. The explant did not contain any other vascular structures. Mature collagen, as assessed by the Masson trichrome stain, was present in all livers forming a fine anastomosing meshwork. The liver cells were mature in appearance with a finely granular cytoplasm and a large round nucleus with one to three prominent nucleoli, but were smaller than in the adult liver and resembled those observed in the foetal liver from 2i days' gestation to birth. Characteristic apoptotic bodies, appearing as round to ovoid, eosinophilic structures containing a pyknotic nuclear fragment, were observed. Ultrastructurally, liver cells contained a large nucleus with nucleolus, abundant rough endoplasmic reticulum, numerous large mitochrondria of variable shape, Golgi apparatus, and polyribosomes. Bile canaliculi, which were occasionally seen by light microscopy, were present (Fig. ib). Except for sentinel microvilli on either side of the tight junctions, microvilli were generally not found. Development of bile ducts was infrequently observed. Ofthe 39 whole foetal liver explants, three (7.7%) contained duct-like structures lined by cuboidal cells with a large nucleus, prominent nucleolus and granular cytoplasm (Fig. 2). The size of the cells ranged from that of mature biliary epithelial cells to that observed in developing ducts in
Fig. 2. Explant of I3-day foetal rat liver after io days in culture showing four bile ducts (D) surrounded by a few fibroblasts and lined by cuboidal epithelial cells. H&E, x 68o.
foetal livers of 20-2I days' gestation. Fine strands of collagen were found adjacent to these duct structures but did not always circumscribe them. Culture of the separated median and lateral lobes for io days resulted in a microscopic appearance similar to that described above. There was no evidence of duct development. Thirteen-day whole liver cultured for both I 5 and 20 days resulted in smaller explants than in the i o-day cultures. Histologically, the livers were similar to Ioday cultures but there was reduced mitotic activity. Bile ducts were not observed. There was no viable tissue in livers cultured for 2 5 days.
i6-day foetal liver explants The findings in the explants from the porta hepatis region ofthe median lobe and peripheral portion of the left lateral lobe after Io days in culture were essentially identical. The general microscopic appearance was similar to that of the cultures of I3-days foetal livers. The only difference between explants from the different locations within the liver was the appearance in one of the explants, from the porta hepatis region, of a mature single bile duct which was lined by
Development of intrahepatic bile ducts
45
Fig. 3. Explant of 13-day liver grown for IO days in medium supplemented with 2 X Io-6 M dexamethasone. a, Numerous cystically dilated bile canaliculi (BC) are seen. H&E, x 6io. b, Electron micrograph of the cystically dilated bile canaliculi (BC), with microvilli projecting into the lumina. The lining liver cells contain glycogen (G) and lipid vacuoles (L). x 2500.
46
J.A.M. Gall & cuboidal epithelium. Explants from both the porta hepatis region and left lateral lobe cultured for 7 days did not show any evidence of duct development.
P.S. Bhathal by cuboidal cells were regularly seen in all six explants from the porta hepatis region but in none of the six explants from the peripheral part of the left lateral lobe.
i9-day foetal liver explants Explants from both the left lateral lobe and the porta hepatis region of the median lobe cultured for IO days, were similar in appearance. The amount of tissue was minimal and in some cases no viable tissue remained. The liver cells were arranged in poorly defined trabeculae. Bile canaliculi could be seen but bile duct-like structures were not evident. Apoptosis was abundant, vacuolation minimal, and necrosis was present. Explants cultured for 4 days were similar to those cultured for io days but the explant size and the mitotic activity were considerably increased. Bile duct-like structures lined
Development of 13-day foetal liver explants in the presence ofputative bile duct growth inducers Development in thepresence ofputative chemical inducers. Explants grown for IO days in the presence of dexamethasone (i X io-6 and 2 X Io6 M) consisted of liver cells which were more mature in appearance than in control livers (I 3-day foetal livers cultured for io days). The distinguishing feature of these explants was the presence of cystically dilated bile canaliculi (Fig. 3a). However, bile duct development did not occur. Cytoplasmic vacuolation was often present and a small amount of necrosis was occasionally seen.
Fig. 4. I 3-day foetal liver grown in a collagen gel for io days showing several ducts lined by epithelial cells. Surrounding the ducts is a layer of basement membrane collagen. In two ducts (D) the biliary epithelium merges with the surrounding liver cells (arrows). The base of the explant consists of numerous fibroblasts and collagen. L, Cell with liver cell phenotype in duct wall. Masson trichrome, x 475.
Development of intrahepatic bile ducts Deposition of mature collagen in association with the sinusoidal lining cells was reduced. Ultrastructurally, the liver cells contained a large nucleus and prominent nucleolus, numerous large, variably shaped mitochondria, and abundant rough endoplasmic reticulum and glycogen deposits (Fig. 3b). The cystically dilated structures were confirmed as bile canaliculi by the presence of tight junctions between liver cells bordering the lumina. Livers grown in the presence of proline (io and 20 mM) and ascorbic acid (25 and 50 ,ug/ml) did not show any histological variation from livers grown without inducers. LCA, added for the final 3 days of culture only, at a concentration of O.I mm, did not show any difference compared with either the DMSO control or explants without DMSO. However, at 0.25 mM, a decrease in explant size and mitotic activity were noted. Concentrations in excess of 0.25 mM resulted in complete necrosis of the tissue. Growth of liver explants in collagen gels. Histologically, liver cells within these explants were larger and more mature than those in livers grown in agar gels. Bile duct-like structures were found in one of four livers in dexamethasone-free medium (Fig. 4). The lining cells were cuboidal and had a nucleus with prominent nucleoli and a darker-staining, granular cytoplasm. Liver cells formed part of the duct wall in scanty foci. Strands of collagen surrounded the more mature appearing ducts but formed a discontinuous layer around the other, incomplete duct-like structures. Dexamethasone (2 X i1-6 M) produced canalicular dilatation similar to that described above for livers grown in agar gels.
Development of liver explants in the presence of other tissues. The growth of either stomach and pancreas or small intestine explants adjacent to the liver, either in the same gel or adjacent gels, with or without dexamethasone (2 X i1-6 M), did not influence the development of the liver.
47
Discussion The results presented here demonstrate the use of an organ culture system for the investigation of intrahepatic bile duct growth and development. Using this system, explants from I 3-day rat foetuses remained viable for I0 days. Extension of the culture time to I 5 or 20 days resulted in a decrease in explant size and death of the culture by 2 5 days. Similarly, i 6 and i9-day foetal liver explants in culture showed decreased viability with increased time in culture. However, livers grown for the equivalent of the 'gestational' period (i.e. I3-day foetal liver for io days and i 6 and I 9-day foetal liver for 7 and 4 days, respectively) consisted of cells which resembled mature antenatal or neonatal cells seen in vivo. Under the conditions of the experiments, culture of I3-day foetal livers for io days did not result in the regular development of intrahepatic bile ducts, a finding suggesting either the absence of the progenitor cells or the absence of inducer(s) of growth and development. To investigate whether the progenitor cell was absent, I6 and Ig-day foetal liver explants, which contain developing ducts (Bhathal & Gall I984; Van Eyken et al. I988; Gall & Bhathal I989), were placed in culture. As it has been reported that growth of the ducts is from the porta hepatis (Bloom I926; Koga I97I; Van Eyken et al. I988) rather than the reverse (Horstmann 1939; Du Bois I963), tissue was excised and cultured from the porta hepatis region of the median lobe or the peripheral part of the left lateral lobe. Regular duct development was observed only in explants from the porta hepatis region of I 9-day foetuses when cultured for 4 days but not when cultured for io days. This finding and the single observation of duct development in i 6-day explants and absence of ducts in i9-day explants of the peripheral part of the lateral lobe may indicate the arrest of development due to the absence of the necessary growth inducer(s). As bile duct development has been described in fragments of I3.5-day foetal
48
J.A.M. Gall & P.S. Bhathal mouse liver transplanted subcutaneously in ated maturation of the added tissues nor bile syngeneic newborn mice and in fragments of duct development occurred. I2.5, I 3.5 and I8.5-day foetal livers transOne hypothesis for the development of the planted into the testes of adult mice (Shiojiri intrahepatic bile duct system suggests that it I984), the effects of putative inducers of bile occurs by modulation or transformation of duct growth were investigated in 13-day periportal hepatoblasts under the influence foetal liver explants. Dexamethasone, a syn- of connective tissue (Bloom I926; Horstthetic glucocorticoid which has been shown to accelerate morphological and physiological events in vivo (Avery I975) and to maintain the morphology and extend the longevity of liver cells in vitro (Laishes & Williams I 9 76), accelerated the maturation of liver cells in explants. Bile canaliculi with numerous microvilli were present and many were cystically dilated, probably as a result of the induction of liver cell secretion into the sealed-off biliary canaliculi. Duct development, however, did not occur. LCA, when fed to adult animals, produces biliary epithelial hyperplasia (Hunt et al. I964; Palmer & Hruban I966). It has been suggested that this response is due either to LCA itself or a metabolite (Hunt et al. I964; Palmer & Hruban I966). An alternative hypothesis holds that the hyperplasia is the result of a regenerative response to the cytotoxic effects of LCA (Palmer & Hruban I966; Gall & Bhathal I987). Although the growth rate of a cultured rat liver cell line was enhanced with increasing concentrations of LCA to I mM (eannin et al. 19 79), in foetal liver cultures it was toxic to cells above a concentration of 0.25 mM and did not induce duct morphogenesis. Similar toxicity to adult bile duct cultures above a concentration of 0.5 mM has also been reported (Gall & Bhathal I987). Stomach and pancreas or small intestine were grown adjacent to the liver explant to determine whether a diffusible compound from these tissues, which in vivo would be transported via the portal venous system, may induce duct development. In case the pathways for either the actions or production of these compounds had not developed, dexamethasone was added to the medium of some cultures. Other than the effects of dexamethasone on the liver, neither acceler-
mann 1939; Du Bois I963; Wilson et al. i963; Wood i965; Shiojiri i984). The basis for this hypothesis stems from the consistent finding of collagen adjacent to developing ducts and the in-vitro observation of the formation of duct-like structures in combined cultures of foetal liver cells and mesenchyme (Doljanski & Roulet I934). More recently, Shiojiri (i984) demonstrated that bile duct development occurs in fragments of I2.5, I3.5 and i8.s-day foetal mouse liver (intrahepatic bile duct development beginning in vivo at the porta hepatis at I4.5 days' gestation in this strain of mouse) transplanted into testis of syngeneic mice, and in the I 3. s-day foetal liver transplanted subcutaneously in newborn mice. In particular, Shiojiri noted that duct development occurred earlier when the foetal liver was transplanted subcutaneously, especially in regions rich in connective tissue. From these findings, Shiojiri suggested that contact of hepatoblasts with endothelial cells may favour their differentiation into mature liver cells whereas contact with connective tissue would favour their development into bile duct cells. In adult animals, collagen biosynthesis has been shown to be a mandatory cofactor for the maintenance of biliary and mammary duct hyperplasia (Girotra & Isseroff I980; Wicha et al. I980) and proline has been implicated in the induction of enzymes of the biosynthetic pathway leading to collagen deposition in toxic liver injury (Rojkind & Diaz de Leon 1 9 70). Similarly, ascorbic acid, in fibroblast cultures, has growth-promoting effects which have been attributed to its ability to increase the production of a collagenous matrix (Peterkofsky I972; Rowe et al. I977). To assess the possible effects of mesenchyme and collagen deposition on
Development of intrahepatic bile ducts duct development, I 3-day explants were grown in the presence of proline and ascorbic acid, adjacent to gut mesenchyme which is of similar origin to hepatic mesenchyme (Croisille & Le Douarin 9 71), and embedded in a gel of type I collagen. Growth of mesenchyme adjacent to the liver did not induce duct development. Similarly, regular duct morphogenesis was not present in collagenembedded explants, with or without dexamethasone. Overall, regular development of bile ducts did not occur in the explants. The results of the addition of various putative inducers of intrahepatic bile duct development did not point to a particular compound and it is evident that collagen of type I alone does not stimulate duct development but may, as suggested by Doljanski and Roulet (I934), have a permissive role in providing the conditions for morphogenesis and structural maintenance. Considering the effects various hormones have on differentiation and that differentiation may not be due to a single factor, a variety of combinations of the potential inducers could be further investigated using the present experimental model.
References AVERY M.E. (I975) Pharmacological approaches to the acceleration of fetal lung maturation. Br. Med. Bull. 31, I3-I7. BHATHAL P.S. & GALL J.A.M. (I984) Development of the intrahepatic bile ducts in fetal and postnatal rat liver. J. Anat. I39, I83. BLOOM W. (1926) The embryogenesis of human bile capillaries and ducts. Am. J. Anat. 36, 45 1-
465. CROISILLE Y. & LE DOUARIN N.M. (I 97 I) Development and regeneration ofthe liver. In Organogenesis. Eds R.L. De Haan & H. Ursprung. New York: Holt, Reinhart & Winston. pp. 42I-466. DOLJANSKI L. & ROULET FR. (I934) Ueber die gestaltende Wechselwirkung zwischen dem Epithel und dem Mesenchyme, zugleich ein Beitrag zur Histogenese der sogenannten 'Gallengangswucherungen'. Virchows Arch. Path. Anat. 292, 256-267. Du Bois A.M. (I963) The embryonic liver. In The Liver. Volume i. Ed. C.L. Rouiller. New York: Academic Press. pp. 1-39.
49
ELIAs H. (I955) Origin and early development of the liver in various vertebrates. Acta Hepatologica 3, I - 56. GALL J.A.M. & BHATHAL P.S. (198 7) Isolation and culture of intrahepatic bile ducts and its application in assessing putative inducers of biliary epithelial cell hyperplasia. Br. J. Exp. Path. 68,
50I-5IO.
GALL J.A.M. & BHATHAL P.S. (I989) Morphological and immunohistochemical assessment of intrahepatic bile duct development in the rat. J. Gastroenterol. Hepatol. 4, 241-249. GIROTRA K.L. & ISSEROFF H. (I980) Fasciola hepatica: azetidine inhibition ofbile duct hyperplasia in the infected rat. Exp. Parasitol. 49, 4146. HAMMAR J.A. (I926) Ueber die erste Entstehung der nicht kapillaren intrahepatischen Gallengainge beim Menschen. Z. Mikrosk-Anat. Forsch. 5, 59-89. HORSTMANN E. (I939) Entwicklung and Entwicklungsbedingungen des intrahepatischen Gallengangsystems. Willhelm Roux' Arch. Entwicklungsmech. Org. 139, 363-392. HUNT R.D., LEVEILLE G.A. & SAUBERLICH H.E. (I964) Dietary bile acids and lipid metabolism. III Effects of lithocholic acid in mammalian species. Proc. Soc. Exp. Biol. Med. 115, 277280. JEANNIN J.F., CHESSEBEUF M., MARTIN M.S., LANGNEAU A. & MARTIN F. (I 9 79) Proliferative effect of lithocholic acid on rat liver cell in culture. Biomedicine 31I, 207-209. KOGA A. (I97I) Morphogenesis of intrahepatic bile ducts of the human fetus. Light and electron microscopic study. Z. Anat. Entwicklungsgesch. 135, 156-I84. LAISHEs B.A. & WILLIAMs G.M. (I976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. II Dexamethasone enhanced longevity and maintenance of morphology. In vitro 1I2, 82 I-832. MICHALOPOULOs G. & PITOT H.C. (1975) Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Exp. Cell Res. 94, 70-78. PALMER R.H. & HRUBAN Z. (I966) Production of bile duct hyperplasia and gallstones by lithocholic acid. J. Clin. Invest. 45, I25 5I267. PETERKOFSKY B. (I972) The effect of ascorbic acid on collagen polypeptide-synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch. Biochem. Biophys. 1I52, 3 i8328. ROJKIND M. & DIAZ DE LEON L. (I970) Collagen
50
J.A.M. Gall & P.S. Bhathal
biosynthesis in cirrhotic rat liver slices. A regulatory mechanism. Biochim. Biophys. Acta
cytokeratin-immunohistochemical study. Lab. Invest. 59, 52-59.
217, 5I2-522. ROWE D.W., STARMAN B.J., FUJIMOTO W.Y. & WILLIAMS R.H. (I977) Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts. In vitro I3, 824-
WICHA M. LIOTTA L.A., VONDERHAAR B.K. & KIDWELL W.R. (I98o) Effects of inhibition of basement membrane collagen deposition on rat mammary gland development. Dev. Biol. 8o, 253-266. WILSON J.W., GROAT C.S. & LEDUC E.H. (I963) Histogenesis of the liver. Ann. N.Y. Acad. Sci. III, 8-24. WooD R.L. (I965) An electron microscope study of developing bile canaliculi in the rat. Anat. Rec. 151, 507-530.
830. SHIOJIRI N. (I984) The origin of intrahepatic bile duct cells in the mouse. I. Embryol. Exp. Morphol. 79, 25-39. VAN EYKEN P., SCIOT R. & DESMET V.J. (I 9 8 8) Intrahepatic bile duct development in the rat: a
