American Journal of Pathology, Vol. 147, No. 2, August 1995

Copyright © American Societ for Investigative Patholokg

Morphometric and ImmunohistochemicalCharacterization of Human Liver Regeneration

Erin M. Rubin,* Andrew A. Martin,*Swan N. Thung,t and Michael A. Gerber*From the Department ofPathology and LaboratoryMedicine,* Tulane University School ofMedicine, NewOrleans, Louisiana; and The Lillian and Henry M. Stratton-Hans Popper Department ofPathology, t The Mount SinaiMedical Center of the City University ofNew York, NewYork, New York

Regeneration in human liver is characterized inpart by theformation ofductular structures, so-caUled ductular bepatocytes in massive hepaticnecrosis and bile ductules in mechanical biliaryobstruction. In an attempt to characterize theliver regenerative process, we performed imageanalysis and immunohistochemical staining ofthe ductular structures in these weU defined hu-man liver disorders, 13 cases ofmassive bepaticnecrosis and 9 cases of mechanical biliary ob-struction. The proliferation index was deter-minedand the expression ofseveral antigens waslocalized by immunohistochemical staining usingantibodies to a-fetoprotein, a-i-antitrypsin, albu-min, and cytokeratin 19. The ductular structuresin adult human liver were compared with the de-veloping ductalplates in 1 fetal livers, ranging inagefrom 9 to 36 weeks ofgestation. Image analy-sis demonstrated that the mean total area, meannuclear area, andmean ceUsize ofductularhepa-tocytes were significantly larger than those ofbileductules (p < 0.05). The proliferation index ofductular hepatocytes and bile ductules was sig-nificantly higher than that ofhepatocytes ofnor-mal livers (p < 0.02). Bile ducts, bile ductules inmechanical biliary obstruction, ductular hepato-cytes in massive hepatic necrosis, and the ductalplate ceUs infetal livershowed strong stainingforcytokeratin 19, which characterizes intermediatefilaments associated with bile duct epithelialcells. Albumin, a liver-specific protein, and a-i-antitrypsin, a protease inhibitor, were stronglyexpressed in ductalplate ceUs offetal liver, hepa-tocytes, and ductular hepatocytes, whereas bile

duct ceUs and bile ductules were negativefor al-bumin. In summary, ductular hepatocytes dem-onstrate morphometric and immunophenotypicfeatures of both hepatocytes and biliary epithe-lial ceUs, whereas bile ductules share character-istics primarily with fetal ductal plates and ma-ture bile ducts. These findings suggest thatductular hepatocytes in massive hepatic necrosismay serve as bipotentialprogenitor cells, and bileductules in mechanical biliary obstruction are re-lated to ductalplates offetal liver. (AmJPathol1995, 147:397-404)

Hepatocytes, bile duct epithelial cells, and hepaticprogenitor or stem cells maintain the potential to mul-tiply during adult life. Depending on the type of theinjurious agent, the nature of liver disease or the num-ber of hepatocytes lost, liver regeneration may occurby at least two mechanisms.1-3 First, adult differen-tiated hepatocytes maintain the capability for severalrounds of replication and respond quickly to liverdamage associated with mild to moderate hepato-cyte loss. Second, several lines of evidence suggestthat liver regeneration in viral or toxin-induced mas-sive hepatic necrosis (MHN) may originate from pro-genitor cells and lead to the generation of ductularhepatocytes (DH). We introduced this term to de-scribe ductular structures composed of relativelysmall cells with oval nuclei and prominent nucleoliwith poorly defined lumen and without basementmembranes45 (Figure 1A). In MHN, DH are locatedaround portal tracts and extend into the necrotic lob-ules. Analogous to oval cells in the rat,6 DH may havethe capacity to differentiate into parenchymal hepa-tocytes or bile duct cells.2 DH, also called neocholan-gioles7 or biliary hepatocytes, are probably derivedfrom hepatic progenitor cells. Sirica et ale observedDH in the furan rat liver model of severe hepatic injury

Supported in part by National Institutes of Health grant CA54576.Accepted for publication May 5, 1995.

Address reprint requests to Michael A. Gerber, M.D., Professorand Chairman, Department of Pathology and Laboratory Medicine,SL79, 1430 Tulane Avenue, New Orleans, LA 70112.

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398 Rubin et alA/PAAugust 1995, Vol. 147, iVo. 2

with formation of cholangiolar-like structures com-

posed of biliary epithelial cells and typically a singleDH. They proposed differentiation of bile ductularcells to DH.A second type of ductular structure in liver regen-

eration, which must be distinguished from ductularhepatocytes, is the bile ductule (BD) (Figure 1 B). BDare seen in diseases of the biliary tree such as primarybiliary cirrhosis, primary sclerosing cholangitis, andmechanical biliary obstruction. In these diseases,there is a marked increase in the number of bileductules. Characteristically, they are confined to theportal tracts or marginal zone between portal tractsand liver parenchyma and often accompanied bypolymorphonuclear leukocytes.9-11 BD are charac-terized by small epithelial cells with round to oval nu-

Figure 1. M34HN uzib ductuilair bepatocytes. (A) Ductular bepato-cytes (arrowheads) fbrm irregular ductular struclures in necroticlobule extending to the central hepatic venulce ('arrow) (hematoxi-Ylin aCld eosin). afany ductular bepatocytes express albuitz (B),A1AT(C) C7K19 (D). bhut not AMI' ( E) ( immuznoperoxidase stainingcounter;stained wvilh bematoxvlin, X 400). The scoreior immuno-bistochbemical staining of DH in B, C, anzd D wvas 3. 2. and 2. re-.spectivelv.

clei attached to a basement membrane and sur-rounding a central lumen (Figure 2A). Desmet et al.9suggested that BD are probably derived from propa-gation of pre-existing bile ductules since mitoses maybe observed in their lining cells. BD in adult liver sharecharacteristics with ductal plate cells in fetal liver."1

These two types of ductular structures, DH and BD,propagate in a variety of liver diseases. They can beclearly differentiated in two well defined liver disor-ders in man, MHN with regeneration and mechanicalbiliary obstruction (MBO). MHN is defined as acuteloss of virtually all hepatocytes due to viral hepatitis,drugs, or toxins. 12 MBO is characterized by cholesta-sis due to obstruction of the large bile ducts by gall-stones, strictures or neoplasms.13 The purpose of thisstudy was to characterize DH and BD in these two

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Human Liver Regeneration 399AJP August 1995, Vol. 147, No. 2

liver diseases by morphometric, histochemical, andimmunohistochemical methods to elucidate the rela-tionship of these cell types in liver regeneration.

Materials and MethodsFormalin-fixed, paraffin-embedded liver tissues from11 fetuses, 13 cases of MHN with liver regeneration,9 cases of MBO, and 5 normal livers (9 autopsy and29 surgical pathology specimens) were selected fromthe files of the Department of Pathology of Tulane Uni-versity Medical Center and Charity Hospital of NewOrleans, LA, and the Mount Sinai School of Medicineof the City University of New York, NY. The patientswith MHN presented with signs, symptoms, and bio-chemical and serological test results of acute hepa-titis with jaundice, coagulopathy, and hepatic en-

cephalopathy progressing to hepatic failure. Thepatients with MBO had epigastric pain, jaundice, andevidence of large bile duct obstruction by endoscopyor imaging methods. The estimated gestational age ofthe fetuses ranged from 9 to 36 weeks. The selectionof cases was based on the presence of many DH inMHN and BD in MBO. The respective ductular struc-tures in tissues of MHN and MBO as described in theintroduction were identified histologically and ana-

lyzed using the CAS 200 system (Cell Analysis Sys-

Figure 2. Mechanical biliary obstruction with bille ductules (BD).(A) BD (arrows) are confined to the portal tract and surroundedby a basement membrane (bematoxylin and eosin). The BD sbowstrong expression ofCK19 (B) andfocal weak stainingfor albumin(C, arrow). Periportal hepatocytes are negative for CK19 and posi-tive for albumin (arrowheads) ( immunoperoxidase staining coun-terstained witb bematoxylin, X 400). 7he score for immunohisto-chemical staining ofBD in B was 3 and in C was 1.

tems, Elmhurst, IL). In addition, all tissues were stud-ied immunohistochemically with antibodies to a-1-antitrypsin (AlAT), albumin cytokeratin 19 (ALB),(CK19), and a-fetoprotein (AFP), and the proliferationindex (PI) was determined, using the nuclear prolif-eration marker Ki 67.

Morphometry by Image Analysis

The CAS 200 system is an interactive, video imagecytometer complete with hardware, software and amodified optical microscope. The CAS 200 Microme-ter Application Version 1.0 allowed for area and linearmeasurements to be derived from live images. In tis-sue sections, this application uses stereological mea-surements to calculate the appropriate parametersfrom each field. For this analysis, one slide for eachcase was stained with hematoxylin and eosin. Thearea of 200 ductular structures, the x and y coordi-nates, and the area of individual nuclei were deter-mined using the CAS system. The number of nucleicomprising each ductular structure was counted. Ap-proximately 11 ductular structures were measuredper case. Each image of the ductular structure withcorresponding measurements was printed at highresolution.

400 Rubin et alAJP August 1995, Vol. 147, No. 2

Histochemical Staining andImmunohistochemical Analysis

Five cases each of MHN and MBO were stained byperiodic acid-Schiff after diastase digestion (DPAS),and Mayer Mucicarmine, or mucin (Poly Scientific,Bayshore, NY). DPAS is a histochemical reaction forcarbohydrates and other substances with free hy-droxyl groups except for glycogen and is used hereto demonstrate basement membranes.

Three antigens (AlAT, AFP, and ALB) were local-ized by the unlabeled antibody enzyme method withthe use of peroxidase-antiperoxidase complex4 anddiaminobenzidine (DAB) as chromogen using theVentana 320 Immunostainer (Ventana Medical Sys-tems, Inc., Tucson, Arizona.) All reagents, except theantibodies, were produced by Ventana: liquid cover-

slip, APK wash solution, DAB detection kit, hematoxy-lin counterstain, and enzyme protease 1. The 4-pmthick paraffin sections were positioned on electro-charged slides and placed in a 600C oven for 45 min-utes. The sections were hydrated, rinsed in distilledwater, and incubated with antibodies for 32 minutes.The rabbit anti-human AFP (Dako Corp. Carpinteria,CA), at dilution of 1:15,000, did not require an enzy-

matic digestion and had a counterstain step. The rab-bit anti-human AlAT (Dakopatts, Copenhagen, Den-mark), at dilution of 1:15,000, required an enzymaticdigestion with protease 1 and had a counterstainstep. The mouse monoclonal anti-human ALB(Chemicon International, Inc., Temecula, CA) was di-luted at 1:50 and required enzymatic digestion withprotease 1. Non-immune rabbit serum, at dilution of1:10,000 was used as a negative control.CK19 was localized by the following procedure.

Deparaffinized and hydrated sections were rinsed indistilled water and etched for 15 minutes with 0.1%aqueous hydrogen peroxide to remove endogenousperoxidase activity. After rinsing again with distilledwater, the sections were covered for 30 minutes with0.05 gm % saponin (Sigma Chemical Company, St.Louis, MO) to reduce nonspecific background stain-ing. The sections were incubated with 10% normalhorse serum for 30 minutes. For 2 hours, the sectionswere incubated with mouse monoclonal antibody toCK1 9 (Dako Corp.), at dilution of 1 :100. Next, the sec-

tions were covered with biotinylated horse anti-mouseIgG (Vector Laboratories, Inc., Burlingame, CA), as

secondary antiserum, for 30 minutes at room tem-perature. After rinsing with PBS (Incstar Corporation,Stillwater, MN), the sections were incubated withavidin-biotinylated peroxidase complex (VectorLaboratories, Inc.) for 30 minutes. The chromogenicreaction was developed by placing DAB solution (3,3'

diaminobenzidine tetrahydrochloride, PBS, and 3%hydrogen peroxide) on the sections for 1 to 2 minutes.After rinsing in distilled water, the sections were coun-terstained with hematoxylin, dehydrated, and cover-slipped. As controls, normal mouse immunoglobulinwas substituted for the primary antiserum on con-secutive sections, and normal liver was stained dur-ing the procedure for CK19. The intensity and extentof immunohistochemical staining was semiquanti-tated as shown on Table 1 using the given in the leg-end.

Proliferation Index PI

The PI was determined by immunohistochemicalstaining for the nuclear proliferation marker Ki-67using the monoclonal MIB-1 antibody14 (Immuno-tech, Inc., Westbrook, ME) on 1 1 cases of MHN, 8cases of MBO, and 5 normal livers. The procedurerecommended by the manufacturer for fixed paraffin-embedded sections was followed with minor modifi-cations, and ethyl green was used as counterstain.Quantitative assessment of the PI of DH, BD, and nor-mal hepatocytes was performed using the CAS-200Quantitative Proliferation Index program (OPI), appli-cation version 2.0. In tissue sections, this applicationuses stereological measurements to calculate the PIand is updated with each field analyzed. The imagesegmentation function allows ductular structures tobe analyzed by measuring only specific cells within adefined area.

Statistical Analysis

Data from each specimen were recorded on separateflow sheets and entered into a Macintosh Ilsi com-puter for analysis using Microsoft Excel 4.0 (Microsoft

Table 1. Semiquantitative Evaluation qfImmunobistochemical Staining

Markers

Cases (no.) CK19 AlAT AFP ALB

Fetal liver (1 1)Ductal plates 2.86 2.14 1.43 1.00Hepatoblasts 0.30 2.10 1.10 1.90Bile ducts 3.00 1.69 0.13 0.75

Extrahepatic biliaryobstruction (9)

Bile ductules 2.70 0.60 0.00 1.00Hepatocytes 0.00 1.80 0.20 2.80

MHN (13)Bile ductules 2.85 1.62 0.00 0.23Hepatocytes 0.00 2.18 0.00 2.45Ductular hepatocytes 2.23 2.15 0.00 2.08

0 = no staining; 1 focal/weak staining; 2 diffuse/weak orfocal/strong staining; 3 diffuse/strong staining; focal = <10% ofBD or DH; diffuse = >50% of BD or DH.

Human Liver Regeneration 401AJP August 1995, Vol. 147, No. 2

Corp., Redmond, WA), Exstatix 1.5 (Strategic Map-ping, Inc., San Jose, CA), and Cricket Graph 1.3.2(Cricket Software, Malvern, PA) software programs.The data were analyzed for statistical significancewith Yates modification.

Results

Histochemical Analysis

The epithelial cells comprising both the DH and BDwere negative for mucin. BD were surrounded by a

basement membrane as demonstrated by DPASstaining, whereas DH were not attached to a DPAS-positive basement membrane. The macrophages, as

well as the basement membranes and apical portionsof the bile duct epithelial cells, stained positively withDPAS. The last were also mucin-positive.

Image Analysis

Using the CAS 200 Micrometer Application Versionfor cellular measurement, the total area and diameterof the ductular structures was determined. The totalarea of 97 DH in MHN, 956 + 385 pm2 (mean + SD),was compared with the area of 103 BD in MBO, 749+ 383 pMm2. The difference and direction of the re-

quired tvalue were significant at the 99.9% level. Themean area of the individual nuclei within the DH was

46 ± 13 pm2 and was compared with the mean area

of the BD nuclei of 36 ± 10 pm2. The difference anddirection of the required tvalue were significant at the99.9% level. The mean diameter of DH was 33 ± 7 pmand was compared with the mean diameter of the BDof 29 + 7 pm. The difference and direction of the re-

quired t value were significant at the 99% level. Thetotal number of cells in each ductular structure was

counted. This number divided into the total area of theductular structure gave the approximate cellular area.

The mean number of cells per DH was 8 + 3 pm2 andper BD 8 + 4 pm2. Using this calculation, the mean

cellular area of DH was determined to be 129 + 117pm2 and of BD 108 + 44 pm2 (mean ± SD). The dif-ference and direction were statistically significant atthe 95% level for the required tvalue when comparingthe cellular areas of DH versus BD.

Immunohistochemical Analysis

Immunohistochemical staining of histological sec-

tions resulted in a brown reaction product in the cy-

toplasm of antigen-containing cells, whereas thebackground remained unstained. The results are

summarized in Table 1. The controls confirmed thespecificity of the reactions. Nonspecific staining wasvirtually absent. Table 2 summarizes the staining pat-tern in human liver as reported in the literature.

All fetal ductal plate and bile duct cells had diffuse,strong cytoplasmic staining for CK19. Most of thesecells were positive for AlAT and negative for ALB.Anti-AFP stained many ductal plate cells, but not bileduct cells. Varying numbers of fetal hepatoblastswere positive for AlAT, AFP, and ALB, and transiently(gestational age of 9 to 16 weeks) positive for CK19.Adult hepatocytes stained for ALB and AlAT, butwere negative for AFP and CK19. Many DH of MHNreacted strongly with ALB, AlAT, and CK19, but didnot stain for AFP (Figure 1). By contrast, the BD ofMBO had strong staining for CK19 and focal, weakstaining for AlAT and ALB (Figure 2). BD were nega-tive for AFP.

Proliferation Analysis

Immunostaining for Ki-67 was always nuclear withoutsignificant background staining. The PI of DH in MHNwas 4.13 ± 0.9 and of BD in MBO 10.15 ± 2.8, bothsignificantly higher (P< 0.02) than that of hepatocytesin normal liver (0.22 ± 0.1).

DiscussionA brief discussion of liver embryogenesis is neces-sary in order to interpret the morphological and im-munohistochemical results obtained in our study. Inthe third to fourth week of gestation, the liver arises asthe hepatic diverticulum from the foregut, a mass ofimmature endodermal cells, which differentiates cra-nially into hepatic cords and caudally into the gall-bladder and extrahepatic bile ducts. The hepaticcords are composed of immature hepatocytes orhepatoblasts and grow into the mesenchyme of theseptum transversum, which forms the connective tis-sue elements of the hepatic stroma and capsule. Acapillary plexus derived from the vitelline veins in theouter margins of the septum transversum interposesbetween the hepatic cords and forms the primitive

Table 2. Staining Patterns of Liver Cells4"11 15,26,28

Bile ductMarker Hepatocytes epithelial cells

CK19 Negative PositiveAlAT Positive VariableAFP Negative NegativeALB Positive NegativeDPAS No basement Positive basement

membrane membrane

402 Rubin et alAJP August 1995, Vol. 147, No. 2

hepatic sinusoids. The development of the intrahe-patic biliary system begins with the formation of asingle layer of small epithelial cells from periportalhepatoblasts in direct contact with the mesenchymearound the portal veins. Then in the 8 mm embryo (5to 6 weeks of gestation), a second layer of these cellstransforms into the ductal plate, a cylindrical bilami-nar layer of small epithelial cells surrounding portalvein mesenchyme. Finally, the ductal plates undergotransformation into tubular structures in the portaltracts, gradually maturing into intrahepatic bileducts.9 15-17 The process by which the primitive he-patic cords differentiate into the epithelium of the in-trahepatic bile ducts supports the hypothesis that theentire intrahepatic ductal system is derived fromhepatoblasts.The liver stem cell hypothesis proposes that im-

mature precursor cells persist in the human liver andare characterized by phenotypic markers of bothhepatocytes and biliary epithelial cells 1,2,18-21 Fol-lowing massive hepatocyte loss, these progenitorcells may give rise to hepatocyte and biliary epithelialcell lineages. At the present time, however, liver stemcells or progenitor cells have not been clearly definedor localized in human liver. In light of the stem cellhypothesis, we examined the ductular structures inliver regeneration by morphological, histochemical,and immunohistochemical methods.

By standard histochemical methods, DPAS wasuseful in distinguishing BD from ductular hepato-cytes, whereas mucin staining was not. DPAS dem-onstrated basement membranes around BD, but notductular hepatocytes, and therefore could be used todistinguish BD from ductular hepatocytes.

By image analysis, we found that the cell size,nuclear diameter, and total area of the ductular hepa-tocytes were significantly greater than the corre-sponding values for BD. Thus ductular hepatocytes inmassive hepatic necrosis appear to be morphometri-cally different from the bile ductules found in suchprocesses as mechanical biliary obstruction. Bothtypes of ductular structures proliferate during liver re-generation following MHN and MBO as demonstratedby the PI while mature hepatocytes are dormant. Plssimilar to those reported here have been documentedin the literature, ie, < 1 % for normal hepatocytes22 and3 to 26% in fulminant hepatic failure.23'24

Immunohistochemical stains for AFP, ALB, AlAT,carcinoembryonic antigen, and CK19 have beenused to differentiate the hepatocyte and biliary epi-thelial cell lineages. The fetal liver is the main site forAFP synthesis, and the liver is the only organ for ALBsynthesis. AFP reversibly binds fatty acids with highaffinity and may be both a specialized carrier of poly-

unsaturated fatty acids during fetal life and a factor inthe transfer of these fatty acids to cells.25 AFP stainingwas not seen in cases of mechanical biliary obstruc-tion or massive hepatic necrosis. Blankenberg et al17found AFP intensely stained fetal hepatocytes andductal plate cells, but not adult hepatocytes, BD, orbile duct cells. In our study, AFP was expressed infetal ductal plate cells and hepatoblasts.

Strong ALB staining was detected in fetal hepato-blasts, adult hepatocytes, and ductular hepatocyteswhile focal weak staining was observed in ductalplate cells, bile duct cells, and BD. This pattern sug-gests that cell types such as BD and mature bile ductsfollow primary differentiation along the biliary epithe-lial cell lineage rather than hepatocyte lineage. Inagreement with our results, Blankenberg et al17 foundthat both fetal and adult hepatocytes showed strongexpression of ALB, while the number of positive duc-tal cells decreased with increasing duct size. ALBand AFP have been observed in oval cells of the ratliver.19'20 These cells may be analogous to the puta-tive bipotential precursor cells in the human liver.AlAT, synthesized predominantly by hepatocytes, isthe primary protease inhibitor in human sera. In ourstudy, AlAT staining pattern of fetal livers was similarto that of ALB. In the adult liver, we found similar stain-ing patterns of AlAT and ALB, whereas Blankenberget al.17 detected AlAT only in macrophages. Desmetet a19 considers Al AT to be a marker of mature hepa-tocytes. Thus, AlAT and ALB may have shared ex-pression between progenitor cells and adult cells dif-ferentiated along hepatocyte lines.

Cytokeratins are the major structural proteins of in-termediate filaments in epithelial cells; CK19 repre-sents an antigenic marker for bile duct epithelial cellsand BD.26 In the fetal liver, we demonstrated transientexpression of CK19 in hepatoblasts from 9 to 16weeks gestation. In addition, there was intense cy-toplasmic staining of all ductal plate cells and bileduct epithelial cells. Stosiek et a126 found that mono-clonal CK19 antibodies stained hepatoblasts, ductalplates, and the epithelial cells of the bile ducts earlyin gestation. Around 10 weeks gestation, fetal hepa-tocytes lost CK19, while ductal plate and bile ductcells continued to express the cytokeratin. In the stud-ies of Stosiek et al26 and Nomoto et al. 27 and ourstudy, mature hepatocytes failed to express CK19,while the cytoplasm of bile duct epithelial cells, BD,and many ductular hepatocytes contained the anti-gen. The negative staining of adult hepatocytes forCK1 9 most likely reflects loss of this cytokeratin as theprecursor cells differentiate along hepatocyte lines.These findings support the notion that hepatoblasts

Human Liver Regeneration 403AJP August 1995, Vol. 147, No. 2

represent common progenitor cells for both hepato-cytes and bile duct epithelial cells.

Ductular hepatocytes have been proposed as bi-potential progenitor cells for regenerating hepato-cytes and bile duct cells.2 Ductular hepatocytes oroval cells with phenotypic properties of both hepa-tocytes and biliary cells have been described incases of fulminant hepatitis,4'7 cirrhosis,28 furan,8'29D-galactosamine,30 and bile duct-ligated/CCI4-treated rats.31 In our study, similarities were found be-tween ductular hepatocytes and both adult hepato-cytes and biliary epithelial cells. Many ducturalhepatocytes showed intermediate to strong reactivitywith antibodies to CK19, AlAT, and ALB. Thus, manyductular hepatocytes express both AlAT and ALB,markers for adult hepatocytes, as well as CK19, amarker for biliary epithelial cells. Unexpectedly,ductular hepatocytes failed to stain for AFP; this maybe related to transient expression of AFP by thesecells or to limits of the methodology. Taken together,these findings suggest that ductular hepatocytes aresimilar to oval cells in the rat and have the capacityto differentiate into parenchymal hepatocytes andbile duct cells.

In conclusion, ductular hepatocytes expressed im-munohistochemical markers characteristic of biliaryepithelial cells, hepatocytes, and their precursors. Incontrast, BD exhibited immunophenotypic character-istics primarily of bile ducts and ductal plate cells ofthe fetal liver. Although in human material only staticobservations are possible, these findings suggestthat ductular hepatocytes in massive hepatic necro-sis are related to bipotential progenitor cells whichmay persist, in limited numbers, into adulthood.

AcknowledgmentsWe are grateful to Ms. Mary Cheles for expert tech-nical assistance and to Ms. Jamie Threet for excellentsecretarial help.

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