Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells

GASTROENTEROLOGY 1996;110:1182–1190

Liver Damage in the Rat Induces Hepatocyte Stem Cells From Biliary Epithelial Cells MALCOLM R. ALISON,* MATTHEW GOLDING,* CATHERINE E. SARRAF,* ROBERT J. EDWARDS,‡ and EL–NASIR LALANI* Departments of *Histopathology and ‡Clinical Pharmacology, Royal Postgraduate Medical School, London, England

See editorial on page 1311. Background & Aims: When rat hepatocyte regeneration after partial hepatectomy is blocked by 2-acetylaminofluorene, a proliferation of biliary epithelia sends out ductules into the parenchyma. The ability of these neoductules to act as a significant progenitor compartment for hepatocytes is in dispute. This study aims to resolve this question by varying the amount of 2acetylaminofluorene administered. Methods: Rats were fed 2-acetylaminofluorene for 6 days before and up to 7 days after partial hepatectomy was performed at a dose of either 2.5 (low) or 5 (high) mgrkg01rday01. The response was monitored by the immunohistochemical expression of intermediate filaments and cytochrome P450 enzymes. Results: No regeneration by mature hepatocytes occurred with either dose, and new ductules expressed the biliary cytokeratins 7, 8, 18, and 19 and, in addition, vimentin. At the high dose, hepatocytic differentiation was infrequent, whereas apoptosis and intestinal differentiation were common. At the low dose, almost all ductules differentiated into hepatocytes within 14 days of hepatectomy. Conclusions: Biliary epithelium is an effective and substantiative hepatocyte progenitor compartment under appropriate conditions.

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iver growth after parenchymal damage is usually achieved by the ephemeral entry of normally proliferatively quiescent hepatocytes into the cell cycle.1,2 However, when hepatocyte regeneration is impeded, relatively undifferentiated biliary cells emerge from the portal areas. These cells are often referred to as oval cells3 – 6 and are considered to harbor facultative multipotential stem cells that, in rats, are capable of differentiating into at least hepatocytes7,8 and intestinal-like cells.9 In humans, similar small cells with ovoid nuclei are seen under a variety of pathological conditions.10 – 12 It is generally believed that any component of the biliary tree can give rise to oval cells,13 and it seems that in most instances the newborn cells form ductular structures in continuity with the existing biliary tree.14,15

Ductular cells undergoing hepatocytic differentiation have been seen in mice,16 – 18 rats,19,20 and humans both outside and within the portal tracts.21,22 In rats, a popular model for generating oval cells has been the modified Solt–Farber procedure, in which animals are fed 2-acetylaminofluorene (AAF) before and after a two-thirds partial hepatectomy (PH). Aromatic amines such as AAF are metabolized to their N-hydroxyl derivative by hepatocytes,23 and this cytotoxic metabolite is probably largely responsible for the failure of hepatocytes to respond to PH. However, biliary cells lack this enzymatic capacity and generally escape contact with the metabolite and thus proliferate. In all these cases, the demonstrated ability of the biliary epithelium to differentiate into hepatocytes has been fairly modest, calling into question the real significance of the so-called oval cell population as a reserve stem-cell compartment. Results from studies adopting the modified Solt– Farber model have certainly been equivocal and have provided examples of apoptosis (cytotoxicity?), intestinal metaplasia (inappropriate differentiation?), as well as evidence for and against the conversion of biliary cells into hepatocytes. The present study reinvestigated the modified Solt–Farber model to examine if the dosage level of AAF was important not just for inhibiting hepatocyte proliferation after PH but also crucial for determining the behavior of the newly emerging biliary cells.

Materials and Methods Animals and Experimental Design Thirty-six male Fischer rats weighing 200 g were used; they all received daily oral gavage of AAF dissolved in polyethylene glycol for 6 days before and up to 7 days after PH; half of the animals were dosed at 2.5 mg/kg and the other half at 5 mg/kg. Two-thirds PH was performed under diethyl ether anesthesia at 24 hours after the first six daily gavages; no dosing was performed on the day of surgery. Three rats from Abbreviations used in this paper: AAF, acetylaminofluorene; PH, partial hepatectomy. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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each group were killed at 2, 4, 6, 7, 9, and 14 days after PH with each rat injected intraperitoneally with bromodeoxyuridine (Sigma Biosciences, Poole, Dorset, England) at a dose of 50 mg/kg at 1 hour before death. Liver slices were fixed in methacarn for 24 hours before processing to paraffin wax.

Immunohistochemistry Three-micron-thick sections were dewaxed, taken down graded alcohols, hydrated in distilled water, and blocked for endogenous peroxidase (0.3% H2O2 in phosphate-buffered saline [PBS]). The staining for incorporated bromodeoxyuridine involved the additional step of incubating the sections for 5 minutes with 1 mol/L HCl at 60⬚C and rinsing well before incubating with normal serum. All sections were then incubated for 10 minutes with normal serum (diluted 1:5 in PBS) from the donor species of the secondary antibody, followed by a 1-hour incubation with a mouse monoclonal antibody raised against either cytokeratin 8 (LE41) or cytokeratin 19 (LP2K) (both from Imperial Cancer Research Fund, London, England), vimentin (Clone V9; Sigma Biosciences), or bromodeoxyuridine (Dako, High Wycombe, England). After rinsing with PBS, primary antibodies were detected by incubation for 45 minutes with biotinylated rabbit anti-mouse immunoglobulins (Dako) and, after further rinsing with PBS, by incubation with horseradish peroxidase–conjugated streptavidin/biotin complex (Dako) for 30 minutes. Peroxidase activity was developed for 2 minutes with 0.05% diaminobenzidine and 0.03% H2O2. Cytochrome P450 enzyme immunolocalization was performed using specific rabbit polyclonal antibodies targeted against either CYP1A1, CYP1A2, CYP2E1, or CYP3A1 as described previously.24 This immunostaining was normally performed as a double immunostain in conjunction with the anti–cytokeratin 19 monoclonal antibody. Briefly, sections were first incubated for 10 minutes with normal swine serum followed by a 2-hour incubation with the specific polyclonal antiserum. After rinsing with PBS, the first primary antibodies were detected by incubation for 45 minutes with biotinylated swine anti-rabbit immunoglobulins (Dako) and, after further rinsing with PBS, incubation with horseradish peroxidase– conjugated streptavidin/biotin complex and horseradish peroxidase visualized as before. After rinsing in tap water, the sections were immersed in 0.6% H2O2 in PBS for 30 minutes to quench any remaining horseradish peroxidase activity, rinsed in PBS, and incubated for 5 minutes with normal rabbit serum. This was followed by a 1-hour incubation with the anti– cytokeratin 19 antibody and visualization as before with the exception that the peroxidase was developed by a 2–15-minute incubation with the VIP peroxidase substrate kit (Vector Laboratories, Burlingame, CA), yielding a purple reaction product.

Western Blot Analysis of Antibodies to Intermediate Filaments Normally, the specificity of antibodies is tested by Western blotting of proteins extracted from fresh or frozen tissue. Proteins cannot be extracted for this purpose from tis-

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sues fixed in conventional cross-linking fixatives such as formalin. However, we have been able to extract proteins from methacarn-fixed tissue and perform Western blots as described previously.25 The electrophoresis reagents and buffers for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were as described by Laemmli.26 Extracted protein (25 mg/lane) was electrophoretically separated on a 12% polyacrylamide gel operated at 180 V for 1 hour and blotted to a nitrocellulose membrane for 2 hours at 0.25 mA. Blots were blocked in 0.5% casein in Tris-buffered saline plus 0.5% Tween 20 for 1 hour and incubated with the primary antibody diluted in this casein buffer for 2 hours. After rinsing well with Tris-buffered saline plus 0.5% Tween 20, the blots were incubated for 1 hour with peroxidase-conjugated rabbit anti-mouse immunoglobulins diluted 1:1000 in casein buffer. Blots were rinsed well with Tris-buffered saline and developed using an enhanced chemiluminescence kit (Amersham International, Amersham, Buckinghamshire, England) by exposing the blots to photographic film (Hyperfilm MP; Amersham International) for 5 seconds.

Electron Microscopy Tissue samples, not exceeding 1 mm3 in volume, were fixed in 2% glutaraldehyde for 2 hours. After washing in phosphate buffer (pH 7.2), tissues were osmicated and dehydrated in acidified 2,2-dimethoxypropane before routine embedding in Araldite resin. One-micrometer sections were cut and stained with toluidine blue for observation at the light microscope level and selection of relevant blocks, followed by ultrathin sections of approximately 100 nm, collected on nickel grids and stained with uranyl acetate and lead citrate, for observation on a Philips CM-10 electron microscope (Eindhoven, Holland).

Results Cellular Changes After PH When Treated at 5 mg/kg AAF The cellular changes ensuing in the livers of rats treated with 5 mg/kg AAF were very similar to those reported by us when a daily dose of 10 mg/kg AAF was administered.14,24 Figure 1 shows that by 14 days after PH there were long strings of basophilic cells emanating from each portal tract (Figure 1A); these cells expressed the biliary cytokeratin phenotype, including diffuse cytokeratin 8 immunoreactivity (Figure 1C). However, many ductular profiles exhibited what looked like clear intestinal differentiation with columnar cells and goblet cell formation (Figure 1B). Evidence of hepatocytic differentiation on a limited scale as adjudged by cytochrome P450 expression was evident, but this was accompanied by apoptosis among the ductular cells (Figure 1D). Counts of the density of dead cells per unit area confirmed that rats treated with a daily dose of 5 mg/kg AAF had many more dead cells among the ductular structures than

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Figure 1. Histology of the rat liver at 14 days after PH with a dose of 5 mg/kg AAF. (A ) Long strings of ductular cells fan out from each portal tract (H&E; original magnification 1001), and (B ) many ducts show intestinal-like differentiation with scattered goblet cells (H&E; original magnification 2001). (C ) These ducts exhibit intense cytoplasmic cytokeratin 19 immunoreactivity (purple stain) contrasting with CYP2E1 staining in ‘‘mature’’ hepatocytes (brown staining) (original magnification 1001). (D ) Where mitosis (thin arrow) and apoptosis (thick arrow) are common, these ducts occasionally stain with the CYP2E1 antibody (brown stain) in locations where cytochrome 8 expression (purple stain) has become membranous, as in normal hepatocytes (original magnification 4001).

rats administered a dose at half this level, although even with the lower dose there was more cell death than found in a normal rat liver (Figure 2), and electron microscopy showed that these cells were apoptotic (Figure 3). Cellular Changes After PH When Treated at 2.5 mg/kg AAF As previously shown, the proliferative response began in small bile ducts close to the limiting plate (Figure 4A and B) and with time arborizing ducts spread out into the periportal and midzonal parenchyma. These new ducts expressed all the biliary cytokeratins, including cytokeratin 8 (Figure 4C), and, in addition, expressed vimentin. However, from 1 week and beyond after PH, it was very noticeable that the ductular nature of this new periportal population was disappearing; by 2 weeks after PH, only the tips of the ducts remained and the more proximal cells now resembled hepatocytes and lacked the biliary cytokeratin phenotype (Figure 4D). These new hepatocytes were uniformally small, capable of dividing, and intensely basophilic (Figure 5A–C); they

lacked expression of CYP1A2 and CYP2E1, although they did show some CYP3A1 immunoreactivity (Figure 5D and E). Once again, the biliary cytokeratin phenotype (cytokeratin 19 expression) only persisted at the boundary between the new cells and the mature hepatocytes. Electron microscopy confirmed the hepatocytic nature of these small cells whose basophilia was clearly attributable to the closely packed rough endoplasmic reticulum in the small cytoplasmic volume (Figure 6). This hepatocytic differentiation and restriction of the biliary phenotype to the periphery of the new cell populations (e.g., Figure 5D) was a consistent feature across the right lateral lobe at 2 weeks after PH, but in the caudate lobe the process was much less advanced and long strings of biliary cells were still present. Cell Proliferation After PH DNA synthesis and mitosis were never observed in the ‘‘old’’ hepatocytes. As expected, bromodeoxyuridine incorporation was commonly seen in the new ductular cells and in a few littoral cells (Figure 7). These

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Figure 2. The effect of AAF dose on the incidence of apoptotic cells among the ductular structures at various times after PH was performed. Counts were made in 30 periportal fields (10 fields per animal) using the 401 objective lens and expressed as means / SD. Doses were either 2.5 mg/kg (䊏) or 5 mg/kg ( ). Statistical differences (Student’s t test) between the two groups at each time point are indicated by *P õ 0.05 and **P õ 0.01.

littoral cells are likely to be Ito cells, which accumulate in the periportal regions at this time and express hepatocyte growth factor messenger RNA.27 Some of the newly formed basophilic hepatocytes could also be seen to proliferate (Figure 5B).

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rapidly renewing epithelia (gut and skin), the stem cells represent the ancestors of the migrating pathways,28,29 but in the liver, the hepatic plates do not seem to represent a stem cell–fed hierarchy.30,31 The efficient replacement of damaged liver by mature hepatocytes might argue against the necessity for hepatic stem cells; however, we have designed an efficient in vivo selection strategy that shows that the damaged liver can generate large numbers of new hepatocytes from recently proliferated ducts. The progeny of putative pluripotent liver stem cells are called oval cells,3,4 and similar cells are seen in humans after massive hepatocyte damage.10 – 12,21,22,32 The true stem cells are probably occasional cells close to or in the marginal ducts and cholangioles,4,33 and the proliferating cells we observe radiating centripetally from each portal tract are likely to be the progeny of these activated stem cells. Certainly we cannot make a definitive statement regarding the exact location of the putative liver stem cells from the present study. If they are found immediately adjacent to the cholangioles (transition ducts),4 then they may differentiate either directly into hepatocytes as seen after allyl alcohol poisoning34 or, alternatively, differentiation may occur after passing through a ductular intermediate.14,24 The biliary histogenesis of these cells is not in doubt because they express the two cytokeratin pairs, 8 and 18 as well as 7 and 19, which typify authentic bile ducts.35 The coexpression of cytokeratins and vimentin in the proliferating ductules was a consistent finding, and similar coexpression has been shown in biliary cells both in vitro and in vivo.36,37

Western Blotting The changes in intermediate filament expression observed histologically were confirmed by Western blotting. In rats treated with the high dose of AAF (5 mg/ kg), the antibody to cytokeratin 19 recognized a major band at 40 kilodaltons, consistent with the long strings of ductular cells present at the later times after PH (Figure 8); with the lower dose, staining was much less intense. This was as expected because the ductular cells differentiate into hepatocytes more promptly when the lower dose is used, although a band was still apparent at 14 days after PH because residual new ductules persisted at this time (Figure 5D–F); in normal liver, this technique was not sufficiently sensitive to detect cytokeratin 19.

Discussion Stem cells possess extensive self-renewal capacity existing throughout the life span of the organism. In

Figure 3. Electron micrograph showing apoptosis of what was probably a small juvenile hepatocyte in a rat at 14 days after PH from the 5 mg/kg group. Cell death was much more common in this group (see Figure 2) and combined with the frequent occurrence of cytoplasmic vacuoles (V) containing cellular debris suggests that AAF was highly cytotoxic (bar Å 1.95 mm).

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Figure 4. Intermediate filament phenotype of the proliferating biliary ducts after PH in animals fed AAF at 2.5 mg/kg. (A ) Proliferation indicated by mitoses (arrows) at 2 days after PH in the small bile ducts inside the portal space (LH side of dashed line, see portal vein [PV]) and in the cholangioles just outside. These structures are beginning to bud, and all of the cells are expressing cytokeratin 19, a characteristic of biliary cells (original magnification 2001). (B ) At the same time, these mitotically active (arrows) biliary ducts (B) begin expression of vimentin (original magnification 4001). (C ) At 7 days after PH, long cords of cytokeratin 8–expressing cells are radiating outwards from each portal tract (PT) (original magnification 1001). In contrast, by 2 weeks (D ), the periportal areas are colonized by small hepatocyte-like cells, and the cytokeratin 8–positive ductular structures are restricted to the periphery (arrows) of this new periportal population, no longer being in obvious contact with the ducts in the portal tracts (original magnification 1001).

Evidence that these biliary cells could differentiate into hepatocytes on a massive scale has been lacking, but we have now shown this is entirely possible provided the agent that prevents hepatocyte regeneration does not cause overt cytotoxicity to the newborn ductular cells. Studies adopting the modified Solt–Farber model have varied the daily dose of AAF ranging from 4 mg/kg38 to 6–7 mg/kg6,7 to 10 mg/kg.9,14,24,25,39,40 The present study has highlighted the significance of AAF to the differentiating process. Likewise, when animals are additionally pretreated with diethylnitrosamine, the ability of ductular oval cells to differentiate into hepatocytes is strictly limited.25 Of course, in this original Solt–Farber model, basophilic foci are composed of large dysplastic hepatocytes generated from so-called ‘‘resistant’’ hepatocytes, seemingly located at random in the parenchyma. This is to be distinguished from the present situation, where uniformally small basophilic hepatocytes are exclusively clustered around each portal

tract because they have been derived from the biliary ductules that formerly occupied these areas. The principal cytotoxic metabolite of AAF is likely to be N-OH-AAF,23 although further metabolism through the action of N-acetyltransferase to acetyloxy-AAF may cause additional cytotoxicity.41 These reactive intermediates undoubtedly inhibit hepatocyte regeneration, and so-called preneoplastic nodules often lack CYP1A1 and CYP1A2, explaining their resistance to AAF.42,43 The biliary system is the preferred route of elimination of AAF, and phase II conjugation, primarily mediated by uridine 5ⴕ-diphosphate glucuronyltransferase,44 leads to the conversion of lipophilic reactive intermediates into water-soluble glucuronides, which are excreted in the bile. In the present study, a daily dose of 5 mg/kg AAF may lead to phase II enzyme saturation, making diffusion of lipophilic reactive intermediates of AAF into other

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Figure 5. H&E staining (A–C ) and immunohistochemical localization of cytokeratin 19 (purple staining) and cytochrome P450 (brown staining) expression (D–F ) in the expanding and differentiating ductular cells in the rats treated with AAF at 2.5 mg/kg. (A and B ) At 9 days after PH, some portal tracts (PT) still show clear evidence of the ductular nature (arrows) of the proliferating cells, but in other periportal areas the ductular nature has almost vanished to be replaced by hematoxyphilic hepatocyte-like cells still showing evidence of mitosis (M) (original magnification 2001). (C ) At 2 weeks after PH, the hepatocytic nature of the periportal cells is obvious; small ducts and cords are still apparent at the margins (arrows) (original magnification 2001). At 14 days, the dual localization of cytokeratin 19 and cytochrome P450 enzymes highlights the new cells; (D ) at 2 weeks, the newly differentiated cells lack CYP1A2 expression and remnant ducts are visible at the margins (small arrows) and the parent ducts are likewise cytokeratin 19 positive (thick arrows) (original magnification 1001). (E ) CYP3A1 expression in the new cells was almost comparable with that in the mature hepatocytes close to the hepatic vein (HV); old hepatocyte nuclei are invariably polyploid and often binucleate and can be found trapped among the new cells (small arrows), and remnant cytokeratin 19–positive ducts (thick arrows) can be seen at the interface with the ‘‘old’’ centrilobular cells (original magnification 2001). (F ) CYP2E1 expression was also lacking in the new cells, and the uniform small size of their nuclei strongly suggests these cells do not arise from a metaplasia of existing hepatocytes (original magnification 2001).

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cells a possibility. This overspill could affect the newly formed biliary cells without actually inhibiting their proliferation. AAF-reactive intermediates could thus be responsible for the common occurrence of apoptosis found with daily doses of ¢5 mg/kg AAF; indeed, apoptosis is a common end point of cytotoxicity in the hepatobiliary system.45 – 47 They could also be responsible for the lack

Figure 6. Transmission electron micrographs of cells at 14 days after PH in rats treated with AAF at 2.5 mg/kg. (A ) A small oval cell with few organelles but whose epithelial nature is apparent from the desmosomal contacts made with neighboring hepatocytes (thick arrows) and with an adjoining oval cell (thin arrow) (bar Å 1.37 mm). (B ) A maturing hepatocyte (left-hand side) with concentrated rough endoplasmic reticulum and a high nuclear/cytoplasmic ratio contrasting with a mature hepatocyte with much more expansive cytoplasm (righthand side) (bar Å 3.8 mm).

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of hepatocyte differentiation from the biliary cells as well as inducing inappropriate differentiation in others; in the present study, intestinal-like differentiation was only found with the higher dose, and it is a very common feature when even greater doses of AAF are administered.9 Liver stem cells are now the focus of intense study after years of skepticism regarding their very existence. As noted by Grisham,30 hepatic plates do not represent tracks of age-dependent migration and differentiation, so the relationship of stemlike hepatic progenitor cells to differentiated hepatocytes in the hepatic muralium is unclear. Nevertheless, hepatologists firmly embrace the hepatocyte stem cell hypothesis. In fact, the hepatobiliary tree probably retains determined stem cells with the same

Figure 7. Bromodeoxyuridine immunolocalization after PH. (A ) At 9 days after PH on the low-dose AAF regimen, labeling is confined to the ductular structures (lower half of micrograph) and occasional sinusoidlining cells (arrows) (original magnification 2001). (B ) At 14 days after PH on the high-dose AAF regimen, labeling is once again largely confined to the profusion of ductular structures emanating from each portal tract, although a few sinusoidal-lining cells are also labeled (original magnification 1001).

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Figure 8. Western blot analysis of the antibody to cytokeratin 19. Lanes 1 and 3 are from liver sections prepared from liver removed at 9 and 14 days after PH from rats treated with 2.5 mg/kg AAF. Lanes 2 and 4 are from liver sections prepared from liver removed at 9 and 14 days after PH from rats treated with 5 mg/kg AAF.

capacity as gastrointestinal stem cells, and Paneth cells, goblet cells, endocrine cells, intestinal-type adenocarcinomas, and hepatocellular carcinomas have all been seen in the biliary tree.48 – 51 In summary, this study has shown that after liver cell loss, if hepatocyte regeneration is inhibited, then proliferation begins at the level of the small bile ducts and the cholangioles; new biliary ducts spread out into the liver parenchyma before differentiating into small hepatocytes. We have provided unequivocal proof that oval cells can be a very effective progenitor compartment for hepatocytes when these cells cannot respond to injury.

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