Regeneration of hepatocyte buds in cirrhosis from intrabiliary stem cells

Journal of Hepatology 39 (2003) 357 364 www.elsevier.com/locate/jhep Regeneration of hepatocyte buds in cirrhosis from intrabiliary stem cells Olga Falkowski 1,, Hee Jung An 2,, I. Andreea Ianus 3, Luis Chiriboga 3, Herman Yee 3, A. Brian West 3, Neil D. Theise 4, * 1 Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA 2 Department of Pathology, College of Medicine, Pochon CHA University, Sungnam, South Korea 3 Department of Pathology, New York University, School of Medicine, New York, NY, USA 4 Department of Pathology, Division of Digestive Diseases, Beth Israel Medical Center, First Avenue at 16th Street, New York, NY, USA See Editorial, pages 431 434 Background/Aims: In massive hepatic necrosis, hepatic stem cells constitute a canal of Hering derived, cytokeratin 19 (CK19) positive ductular reaction (DR). Whether DRs in cirrhosis are activated stem cells (so called buds ) or biliary metaplasia of cholestatic, injured hepatocytes is still debated. We investigate derivation of intraseptal hepatocytes (ISHs) from DRs and from the biliary tree in cirrhosis. Methods: Explants of hepatitis B and C, alcohol, primary biliary cirrhosis and primary sclerosing cholangitis-related cirrhosis were examined. ISHs were quantified and their associations with DRs and cholestasis recorded. 3Dreconstruction of ISHs and nearby bile ducts was performed in blocks from hepatitis C and primary sclerosing cholangitis cirrhosis. Results: Seven hundred seventy five/830 (94%) ISHs were associated with CK19 positive DRs. ISHs without ductular reactions were more likely to show cholestatic features (P <0.0001). In 3D, ISHs were seen to bud directly from the biliary tree. In summary: ISHs: (1) are usually associated with stem cell-like DRs; (2) are rarely cholestatic, leaving the associated DRs unexplained; and (3) are linked to the biliary tree in 3D. Dynamic proliferation rates in hepatitis C over time suggest that hepatocyte replication diminishes in late stages, with an associated activation of the biliary stem cell compartment. Conclusions: We therefore suggest that the biliary tree, from at least its smaller branches up to the canals of Hering, are composed of or at least harbor facultative hepatic stem cells, and that ISH largely represent buds of newly formed hepatocytes. q 2003 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. Keywords: Progenitor cells; Liver 1. Introduction In human tissues intrabiliary stem cells contribute to hepatocyte reconstitution following severe acute injury [1,2]. Our work demonstrated that with three dimensional Received 17 March 2003; received in revised form 29 April 2003; accepted 12 May 2003 * Corresponding author. Tel.: þ1-212-420-4246; fax: þ1-212-420-4373. E-mail address: ntheise@chpnet.org (N.D. Theise). Co-first authors. Abbreviations: DR, ductular reaction; ISH, intraseptal hepatocyte; CK19, cytokeratin 19; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis. reconstruction, the ductular reactions (DRs) in massive hepatic necrosis represent complex arborizing networks of highly proliferative cells branching from the biliary tree, largely from the canals of Hering, and differentiating into new hepatocytes [1]. The question of whether the DRs in chronic liver disease also represent activation of a stem cell compartment within the biliary tree has not yet been established. Analysis of staining patterns for biliary-specific and hepatocyte-specific markers in chronically diseased livers of diverse causes shows a typical pattern: gradients of cells which range from cholangiocyte-like to hepatocyte-like through intervening cells of intermediate morphology and immunophenotype 0168-8278/03/$30.00 q 2003 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. doi:10.1016/s0168-8278(03)00309-x

358 O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 [3 10]. However, all these represent two-dimensional analyses that yield uncertain conclusions. While such cellular gradients can be explained as stem cell activation, they can just as readily be explained as biliary metaplasia of chronically damaged, cholestatic hepatocytes [10]. To explore this question we have employed two approaches. The first considers cholestasis of hepatocytes. If the metaplasia hypothesis is correct, then cholestatic hepatocytes will be more likely to be associated with DRs. Also, hepatocellular cholestasis would be an inverse marker of linkage to the biliary tree. Functional hepatocytes without biliary drainage would have to demonstrate features of cholestasis. The absence of cholestasis would imply that the hepatocyte canalicular system had drainage access to the bile ducts. The second approach is similar to that used in our study of massive hepatic necrosis, with three dimensional analysis, attempting to establish whether DRs arise from the biliary tree, regenerating hepatocytes or, conversely, represent severe hepatocellular injury. If the former is correct, we would expect that regenerating hepatocytes in cirrhosis are usually associated with DRs, that DRs are highly proliferative, and that they are invariably linked to the biliary tree. On the other hand, if the latter is correct, then DRs would only variably be associated with hepatocytes, would be a relatively low proliferative cell compartment, and that their linkage to the biliary tree would be at best inconsistent, if not absent. Due to the difficulty of three dimensional reconstructions of large hepatocellular nodules we confined our study to small groupings of intraseptal hepatocytes (ISHs). Wanless has referred to these small nodules as buds, identifying them as one of eight features of an hepatocellular repair complex, the set of histologic changes which can lead to regression of cirrhosis [11]. We examined cases of cirrhosis representing a range of chronic liver injury: toxic injury (alcohol), biliary disease (primary biliary cirrhosis, primary sclerosing cholangitis), and viral hepatitis (hepatitis B and C). 2. Experimental procedures 2.1. Routine histology and immunohistochemistry We selected one formalin-fixed, paraffin embedded tissue specimen from livers of patients undergoing liver transplantation ( explants ) for primary cholestatic liver disease (primary biliary cirrhosis, n ¼ 5; primary sclerosing cholangitis, n ¼ 5) and non-cholestatic liver disease (alcoholic liver disease, n ¼ 5; hepatitis B, n ¼ 5; hepatitis C, n ¼ 5). Two serial slides of each case were examined: one with Haematoxylin and Eosin, one with immunohistochemistry for biliary-type cytokeratins. Immunostaining was performed using monoclonal antibody to cytokeratin 19 (DAKO Corp, Carpinteria CA, 1:100) following standard techniques [1]. Interlobular bile ducts served as an internal positive control. Substitution of bovine serum albumin for the primary antibody served as a negative control. ISHs were defined as hepatocytes completely surrounded by fibrous tissue, but that did not distort the outer contours of the septum. The following histologic features of intraseptal hepatocytes were analyzed: 1. nodules (tight, oval to round collections of hepatocytes with a bulging growth appearance) versus loose clusters (single hepatocytes or small clusters, often with fibrous stroma weaving between them) (Figs. 1a,c); 2. association with cytokeratin 19 positive DRs on adjacent, immunostained sections (Figs. 1b,d); 3. cholestasis ( feathery degeneration or cholate stasis and/or cytoplasmic bile pigment) (Fig. 2). 2.2. Associations of hepatocytes and ductular reactions in three dimensions To address concerns that the two dimensional sampling of nodules might undercount the association of intraseptal hepatocyte nodules with DRs, an additional 23 serial sections were prepared from one case of hepatitis C cirrhosis and from one case of primary sclerosing cholangitis. These serial sections were immunostained for cytokeratin 19 to highlight the DRs, as above. Low power views (2 objective) were then photographed, printed as 8.5 11 hard copies, and numbered to correspond to the slides, level 1 through 23. ISHs were identified on level 1 and then annotated in a table to indicate if that level contained hepatocytes (H), hepatocytes associated with ductular reactions (HD), or hepatocyte/drs associated with bile ducts (HDB). Each nodule was then followed down through subsequent levels, continuing analysis and notation until it, or associated ductular reactions and/or bile ducts also disappeared. This procedure was carried out for all intraseptal hepatocyte nodules contained within the photographic images analyzed. 2.3. Proliferation studies of hepatocytes, ductular reactions, and bile ducts Double immunohistochemistry for proliferation marker Ki-67 (monoclonal antibody MIB-1, DAKO) and for cytokeratin 19 was performed on cases of hepatitis C at all stages of disease [12]. Needle biopsy specimens were used for stages 0 (no fibrosis, n ¼ 0), I (portal fibrosis, n ¼ 10), II, (fibrous septum formation, n ¼ 11), and III (transition to cirrhosis with septa and focal nodularity, n ¼ 10). Liver explant specimens were used for stage IV (cirrhosis, n ¼ 14). Three normal liver specimens (post-perfusion biopsies of liver allografts) were also studied. Double immunostaining was performed with standard techniques [1]. Immunostaining for cytokeratin 19 was performed with an alkaline phosphatase method and stained with Fast Red. Immunostaining with Ki-67 primary Ab (Zymed, 1:25) was performed biotinylated anti-mouse IgG and avidin-biotin complex, visualized with DAB/nickel. Cell counts per 500 cells were obtained whenever possible. Hepatocytes were identified by morphology. Cells of the DRs for this part of the study were considered to be intensely cytokeratin 19 positive cells whether morphologically hepatocyte-like, cholangiocyte-like, or intermediate between the two outside of the portal tracts or septa (stages 0) or distinct from interlobular bile ducts with lumens (stage 4). Bile ducts were defined as cholangiocyte-lined structures, positive for cytokeratin 19, with welldefined lumens. 3. Results Numbers of ISH nodules and loose clusters, with and without cholestasis and without and without DRs, are summarized in Tables 1 and 2. A total of 564 ISH nodules and 266 ISH loose clusters were identified in all specimens examined, summarized in Table 1. Only a minority of ISH were cholestatic: 110/564 (19.5%) ISH nodules and 58/266 (22%) ISH loose clusters. The degree of cholestasis between nodules and loose clusters was not statistically significant. The majority of both ISH nodules and loose clusters were associated with ductular reactions: 515/564 (91.3%) of ISH

O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 359 Fig. 2. Cholestatic intraseptal hepatocytes with bile staining, feathery degeneration and Mallory body formation from a patient with primary biliary cirrhosis (H&E, original magnification 203). nodules and 260/266 (97.7%) of ISH loose clusters. Though cholestasis was present in a minority of ISH of either form, cholestasis of ISH was only rarely present if ductular reactions were also associated with them: 81/434 (19%) DR-associated ISH nodules and 53/207 (26%) DR-associated ISH loose clusters. Conversely, if no ductular reaction was present, a majority of ISH were cholestatic: 29/49 (59%) of ISH nodules and 5/6 (83%) of ISH loose clusters without ductular reactions. Thus, to summarize, ISH were much more likely to be cholestatic without DRs, than if they were associated with DRs (P, 0:0001). Analysis by disease category (Table 2), reveals that ISH nodules and clusters without ductular reactions are more common in the chronic cholestatic diseases primary biliary cirrhosis and primary sclerosing cholangitis. 3.1. Three dimensional analysis of ISH nodules, ductular reactions, and interlobular bile ducts Tabulation for three dimensional relationships of ISH nodules, ductular reactions and interlobular bile ducts in the hepatitis C case is summarized in Table 3. In the sections examined, 32 intraseptal nodules were identified. Though on some levels many ISHs appeared to lack associated ductular reactions, in three dimensions all of them were linked to DRs. Moreover, 23/32 (72%) were linked, via the Fig. 1. (A) Intraseptal hepatocyte (ISH) nodule in hepatitis C cirrhosis (H&E, original magnification 103). (B) Adjacent level as in 1A, immunostained for cytokeratin 19, highlighting the association of a ductular reaction with the ISH nodule. (Immunostained with DAB colorizing agent, Mayer s hematoxylin counter stain, original magnification 103). (C) ISH loose cluster in primary sclerosing cholangitis cirrhosis (H&E, original magnification 203). (D) Adjacent level as in 1C, immunostained for cytokeratin 19, highlighting the absence of associated ductular reaction. Note the positive staining bile ducts nearby. (Immunostained with DAB colorizing agent, Mayer s hematoxylin counter stain, original magnification 203). Table 1 Associations of CK19 positive ductular reactions with ISH nodules and ISH loose clusters All nodules/ clusters (%) ISH nodules CK19DR þ 515 (91.3) CK19CR 2 49 (8.7) ISH loose clusters CK19DR þ 260 (97.7) Cholestatic 81 29 53 5 nodules/clusters Non-cholestatic nodules/clusters 434 20 207 1 CK19DR 2 6 (2.3)

360 O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 Table 2 Associations of CK19 positive ductular reactions with ISH nodules and ISH loose clusters All nodules/ clusters (%) ISH nodules CK19DR þ 515 (91.3) ductular reaction, to an intralobular bile duct as illustrated in Figs. 3 and 4 (linkage directly to a bile duct; and linkage via an intermediate canal of Hering-like structure). Without the three dimensional analysis the links would have only rarely have been unrecognized. Similar tabulation in the case of primary sclerosing cholangitis revealed 43 ISH, of which 15 (35%) were associated with ductular reactions in the 23 levels examined. In turn, only three of these 15 connected to an intralobular bile duct within those levels. 3.2. Proliferation rates CK19CR 2 49 (8.7) ISH loose clusters CK19DR þ 260 (97.7) Alcohol 112 1 68 1 Primary biliary 73 12 52 2 cirrhosis Primary sclerosing cholangitis 84 11 54 2 Chronic hepatitis C 124 3 41 0 Chronic hepatitis B 122 2 45 1 CK19DR 2 6 (2.3) Proliferation rates of hepatocytes, cholangiocytes, and cells of the ductular reaction in cases of hepatitis C are displayed in Fig. 5, organized by stage of disease. Proliferation rates for hepatocytes rise until late stage disease, i.e. cirrhosis, when they appear to drop, simultaneously with a rise in proliferation rates of ductular reactions (most pronounced) and, to some extent cholangiocytes of the interlobular bile ducts. 4. Discussion The role of stem or progenitor cells in human liver regeneration has long been a source of controversy [13]. While there is a rich literature on this topic in experimental animals [13 17], studies in human liver tissue were few and largely restricted to immunohistochemical demonstration of structures suggesting developmental lineages in acute and chronic reactive lesions [3 9,18 28]. These were variously termed ductular hepatocytes, ductular proliferations, and ductular reactions. To many investigators, the intermediate, hepatobiliary cells in ductular reactions, recapitulating embyronic/fetal hepatoblast phenotypes [23], were suggestive of activation of an hepatic stem cell compartment. But it was alternatively suggested that they might represent biliarymetaplasia of cholestatic, damaged hepatocytes [10]. However, with recent studies of acutely diseased livers combining immunohistochemistry with three dimensional reconstruction [1] or with serial biopsies [2], consensus has emerged that, indeed, ductular reactions are activation of facultative stem cells within the biliary tree, particularly the Table 3 A truncated portion of the tabulation, in three dimensions, section level by section level, of intraseptal hepatocytes (H), ductular reactions (D), and interlobular bile ducts (B) in one case of chronic hepatitis C cirrhosis over 23 serial four micron sections a 1 HD HD HD HD HD HD HDB HD HD HD HD HDB HD HDB HD 2 HD HD HD HD HD HD HDB HD HD HD HD HD HD HDB HD 3 HD HD HD HD HD HD HD HD HD HD HD HD HD HDB HD 4 D HD HD HD HD HD HD HD HD HD HD HD HD HDB HD 5 D HD HD HD HD HD HD HD HDB HD HD HD D HDB HD 6 D HD HDB HD HD HD HD HD HDB HD HD HD D HD HD 7 D D HDB HD HD HD HD HDB HD HD HD D HD HD HD 8 D D HDB HD HD HD HD HDB HD HD HD D HD HD HD 9 D D HDB HD HD HD HD HDB HD HD HD D HD HD HD 10 D D HDB HD HD HD HD HDB HD HD HDB D HD HD HD 11 D D HD HD HD HD HD B HD HD HDB D HD HD HD 12 D HD HD HD HD HDB B HD HD HD D HD HD HD 13 D HD HD HD HD HDB B HD HD HD D HD HD HD 14 D HD HD HD HD HDB B HD HD HD D HD HD HD 15 D HD HD HDB HDB HD B HD HD HD HD HD HD HD 16 D HD HD HDB HDB HD B D HD HD HD HD HD HD 17 D HD HD HDB HD HD B D HD HD HD HD HD HD 18 D HD HD HDB HD HD HDB D HD HD HD HD HD HD 19 D HD HD D HD HD HDB D HD HD HD HD HD HD 20 D HD HD D HDB HD HDB D HD HD HD HD HD HD 21 D HD HD D HDB HD HDB D HD D HD HD HD HD 22 D HD HD D HDB HD HDB D HD D HD HD HD HD 23 D HD HD D HDB HD HDB D HD D HD HD HD HD a Level tabulation of linked hepatocytes, ductular reactions, and bile ducts.

O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 361 Fig. 3. Serial, 4 micron sections of hepatitis C cirrhosis. A small intraseptal hepatocyte cluster links directly to an interlobular bile duct as seen in three sequential 4 micron slides (Immunostained with DAB colorizing agent, Mayer s hematoxylin counter stain, original magnification 403). Fig. 4. Serial, 4 micron sections of hepatitis C cirrhosis. A small intraseptal hepatocyte nodule links to an interlobular bile duct via a single intermediate, cytokeratin 19 positive, canal of Hering-like structure, as seen in four sequential 4 micron slides. The complete link can only be appreciated with examination of the serial sections (top of each image). (Immunostained with DAB colorizing agent, Mayer s hematoxylin counter stain, original magnification 203).

362 O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 Fig. 5. Proliferation indices for hepatocytes, cholangiocytes, and ductular reactions in cases of chronic hepatitis C, stages 0 (no fibrosis), 1 (portal fibrosis), 2 (fibrous septa), 3 (transition to cirrhosis), and 4 (cirrhosis). Note that hepatocellular proliferation increases dramatically in even the earliest stages of disease, falling off in cirrhosis, while biliary cells of the ductular reaction and bile ducts begin to significantly increase proliferation only in the latest stages of disease. (CoH, Canal of Hering). canals of Hering. Moreover, circulating cells, probably at least partly bone-marrow derived, can also contribute to liver regeneration in acute injury [29 31]. Definitive studies of ductular reactions of chronic liver disease have not been reported. In this study, we juxtaposed the two competing hypotheses for how ductular reactions might arise, investigating whether three dimensional reconstruction and studies of proliferation indices might suggest whether ductular reactions in cirrhosis are derived from activated, intrabiliary hepatic stem cells or simply reactive metaplasia. Our data support the former, stem cell hypothesis for the formation of ductular reactions and ISH in cirrhosis: 1. examination of cirrhotic explants of diverse diseases reveals that most ISH, whether as nodules or as loose clusters, are associated with ductular reactions (Table 1); 2. ductular reactions are more consistently associated with hepatocytes which are not cholestatic, implying functional linkage of ISH to the biliary tree (Table 2); 3. cholestatic hepatocytes are less frequently associated with ductular reactions, implying that biliary metaplasia of cholestatic hepatocytes is not a mechanism of ductular reaction formation (Table 2); 4. three dimensional reconstruction of ISH in hepatitis C- related cirrhosis demonstrates that in that setting virtually all ISH are associated with ductular reactions which form a structural and probably physiological link to the biliary tree (Table 3); 5. proliferation of hepatocytes in the early stages of chronic hepatitis C account for much of hepatocyte regeneration, but in cirrhosis, the biliary tree becomes more proliferative as hepatocyte replication diminishes (Fig. 5). In earlier work we suggested that the canal of Hering is composed of, or at least harbors, facultative hepatic stem cells. This current study is less clear that the canals of Hering are the predominant location of these cells, suggesting that other portions of the biliary tree can also function in this capacity. While in Fig. 4 the nodule of hepatocytes links to the bile duct by a branching, cholangiocyte lined structure without obvious lumen (i.e. a canal of Hering or a terminal ductule), Fig. 3 shows an ISH nodule appearing to arise from an actual intralobular bile duct. Thus, stem/progenitor populations for the liver are not restricted to the canals of Hering. The proliferation data is reminiscent of experimental models of oval cell proliferation in which oval cells proliferate only when hepatocyte replication is blocked following the primary injury [13 17]. Our data may represent a corresponding natural experiment in humans: over the many years of chronic viral hepatitis, the hepatocytes perhaps reach the end of their replicative potential, or perhaps the distortions of blood flow in fully established cirrhosis restrict their replicative abilities. Either way, it appears that when hepatocyte replication is interfered with, then the stem cell compartment activates, giving rise to the ductular reaction. The ductular reactions serve at least two distinct physiologic roles: they are facultative progenitor cells and also functional, bile-conducting cholangiocytes. This finding seems unusual when one considers stem/progenitor cells in the lining of the gastrointestinal tract or the hematopoietic system, where stem cells seem to function exclusively as reservoirs for tissue reconstitution [32], but may relate to the greater cell turnover in those organs. On the other hand, if analogies between organs may be drawn, it may indicate that those cells may also have other physiological roles beyond cell replacement, as yet unlooked for. While our data discriminate between the two major hypotheses regarding the emergence of ductular reactions in human liver disease, we cannot exclude the possibility that hepatocytes entrapped within scar tissue might be able to regenerate ductular structures which then find their way, perhaps chemotactically, through the stroma, to link to the biliary tree via a single linear structure (Fig. 3). We also do not address the role of circulating progenitor cells, from the bone marrow or elsewhere, in chronic liver disease. Studies using liver tissue from transplant patients in whom donor and recipient genders are mismatched will clarify the latter question and shed light on the former. For example, one may consider the case of fibrosing cholestatic recurrent hepatitis C in the allograft of a man with a female donor liver which we previously reported [29]. On average, 35 40% of hepatocytes and cholangiocytes, as well as groupings of ductular reactions, were derived from circulating cells, as identified by Y-chromosome in situ hybridization. In the periportal zones, however, where ductular cells were present and proliferating, the rate of marrow-derived cells engrafting in the liver as hepatocytes was approximately 65%. Though not a perfect model for cirrhosis from chronic disease, this case is suggestive of an answer to these

O. Falkowski et al. / Journal of Hepatology 39 (2003) 357 364 363 questions: cells entering the liver from the circulation, through the biliary tree, then becoming hepatocytes. Our use of the term hepatocyte buds in the title of the paper highlights the perhaps prescient interpretation of such structures made by Ian Wanless and colleagues in their studies of regression of cirrhosis [11]. In that work they suggest that if the disease causing agent in cirrhosis can be removed or halted, then the liver is capable of vast remodeling, perhaps back to normal functioning. They describe the hepatic repair complex as a set of histologic features of this architectural restitution, one of which is the hepatocyte bud. We support their interpretation of hepatocyte buds as being a regenerative phenomenon. Thus, as in our previous work, we use simple techniques immunohistochemical staining and careful, non-computer assisted three dimensional reconstruction to suggest answers to important questions. The power of such simple techniques has not yet been fully exploited in the pursuit of answers to long-standing and difficult questions of tissue biology and pathophysiology, and we would recommend their use to academic pathologists interested in diverse organ systems. Extending our observations into three and even four dimensions, using serially obtained tissue samples, we greatly increase the power of our analyses. In this case, from such work, we can confidently suggest that: (1) the biliary tree, from at least its smaller branches up to the canals of Hering, is composed of or at least harbors facultative hepatic stem cells; (2) such intra-organ stem cells have both regenerative and bile physiologic roles to play, dual roles not yet suggested for stem cells of other organ systems; and (3) these hepatocyte buds may play a role in the structural regression of cirrhosis as suggested by Wanless and colleagues. 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