Epithelial Cell Adhesion Molecule (EpCAM) Marks
Hepatocytes Newly Derived from Stem/Progenitor
Cells in Humans
So-Mi Yoon,1* Domniki Gerasimidou,2* Reiichiro Kuwahara,3 Prodromos Hytiroglou,2
Jeong Eun Yoo,1 Young Nyun Park,1,4 and Neil D. Theise5
Epithelial cell adhesion molecule (EpCAM) is a surface marker on human hepatic stem/
progenitor cells that is reported as absent on mature hepatocytes. However, it has also
been noted that in cirrhotic livers of diverse causes, many hepatocytes have EpCAM surface expression; this may represent aberrant EpCAM expression in injured hepatocytes or,
as we now hypothesize, persistence of EpCAM in hepatocytes that have recently derived
from hepatobiliary progenitors. To evaluate this concept, we investigated patterns of
EpCAM expression in hepatobiliary cell compartments of liver biopsy specimens from
patients with all stages of chronic hepatitis B and C, studying proliferation, senescence
and telomere lengths. We found that EpCAM(1) hepatocytes were rare in early stages of
disease, became increasingly prominent in later stages in parallel with the emergence of
ductular reactions, and were consistently arrayed around the periphery of cords of keratin
19(1) hepatobiliary cells of the ductular reaction, with which they shared EpCAM expression. Proliferating cell nuclear antigen (proliferation marker) and p21 (senescence marker)
were both higher in hepatocytes in cirrhosis than in normal livers, but ductular reaction
hepatobiliary cells had the highest proliferation rate, in keeping with being stem/progenitor cell–derived transit amplifying cells. Telomere lengths in EpCAM(1) hepatocytes in
cirrhosis were higher than EpCAM(2) hepatocytes (P < 0.046), and relatively shorter
than those in the corresponding ductular reaction hepatobiliary cells (P 5 0.057). Conclusion: These morphologic, topographic, immunophenotypic, and molecular data support
the concept that EpCAM(1) hepatocytes in chronic viral hepatitis are recent progeny of
the hepatobiliary stem/progenitor cell compartment through intermediates of the transit
amplifying, ductular reaction hepatobiliary cells. (HEPATOLOGY 2011;53:964-973)
T
here is a growing consensus that some contribution to hepatocyte mass derives from intrahepatic, hepatobiliary stem cells and that the
contribution depends on presence of injury, its form,
and its degree.1-8 Support for this concept is found in
animal models, often employing a two-hit experimental method in which there is poisoning of hepatocytes
to inhibit their replication (e.g., 2-acetylaminofluorene)
Abbreviations: CHB, chronic hepatitis B; CHC, chronic hepatitis C; EpCAM, epithelial cell adhesion molecule; K, keratin; PCNA, proliferating cell nuclear antigen.
From the 1Department of Pathology, Brain Korea 21 Project for Medical Science, Center for Chronic Metabolic Disease, Yonsei University Health System, Seoul,
South Korea; the 2Department of Pathology, Aristotle University Medical School, Thessaloniki, Greece; the 3Division of Gastroenterology, Department of Medicine,
Kurume University School of Medicine, Kurume, Japan; the 4Institute of Gastroenterology, Yonsei University Health System, Seoul, South Korea; and the 5Department of
Pathology and Medicine, Beth Israel Medical Center, New York, NY.
Received July 20, 2010; accepted December 2, 2010.
Supported by a Korea Science and Engineering Foundation grant from the Korean Ministry of Science and Technology (R13-2002-054-03004-0 and
20100008075) and by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare, and Family Affairs, Republic of Korea
(0920300) (to Y. N. P.).
*These authors contributed equally to this work.
Address reprint requests to: Young Nyun Park, M.D., Ph.D., Department of Pathology, Yonsei University Health System 250 Seongsanno, Seodaemun-gu, Seoul,
120-752, South Korea. E-mail: young0608@yuhs.ac; fax: (82)-2-362-0860; or Neil Theise, M.D., Division of Digestive Diseases, Beth Israel Medical Center, First Avenue
at 16th Street, New York, NY 10003. E-mail: ntheise@chpnet.org; fax: 212-420-4373.
C 2010 by the American Association for the Study of Liver Diseases.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.24122
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
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HEPATOLOGY, Vol. 53, No. 3, 2011
followed by injury to eliminate significant amounts of
hepatocyte mass, either by application of a second
toxin (e.g., retrosine, monocrotaline) or partial hepatectomy.9 In such procedures, there is stem cell activation leading to expansion of a progenitor pool referred
to as oval cells. In these experiments, whether hepatocytes are derived from other, preexisting hepatocytes or
from stem/progenitor cell activation and differentiation
can be partly evaluated through tracking experiments
where populations of cells from an animal with a distinctive marker are transplanted into animals without
the marker (e.g., wild-type, dipeptidyl peptidase-4 positive cells into dipeptidyl peptidase-4–positive knockout recipients, or male, Y chromosome–positive cells
into female recipients).9,10
These models have provided a working hypothesis
for human liver disease. In acute acetaminophen toxicity, the most severe (lethal) injury leads to activation
of a stem/progenitor cell compartment, predominantly
located in the proximal branches of the biliary tree,
including the bile ductules and the canals of Hering.11
This proliferative response, the human equivalent of
the oval cell response in rodents, is referred to as a
ductular reaction.12 In chronic liver diseases, such as
viral or autoimmune hepatitis and alcoholic or nonalcoholic fatty liver disease, ductular reactions are also
prominent, but generally only in the late stages of disease, leading to the hypothesis that it is only after several years, often decades of chronic injury that the hepatocytes lose their replicative potential necessitating
activation of the stem/progenitor cell response.13-15 In
both of these settings, the stem/progenitor cell
response arises because hepatocytes have been largely
eliminated (acute injury) or have lost their replicative
potential (chronic injury), paralleling the animal data.
These human correlates to the animal models have
depended on data gathered predominantly on the basis
of morphology/architecture (e.g., three dimensional
reconstructions of ductular reactions indicating their
link to regenerating hepatocytes)6,7,11 or immunohistochemical markers of proliferation and/or senescence
(Ki-67, p21 respectively, in most studies).13-15 These
data show that in the early stages of chronic liver
disease, hepatocytes can easily accomplish hepatocyte
restitution through cell division; ductular reactions are
largely absent. However, as disease progresses over
many years to decades, hepatocytes show faltering
proliferation (by Ki-67 expression) and increasing senescence (p21 expression). With these changes there
arise parallel, highly proliferative ductular reactions.
More precise cell tracking experiments of the type
performed in animals are, of course, not easily possible
YOON ET AL.
965
in humans, although the recently published data of
Lin et al.16 exploiting mutational analysis in mitochondrial DNA encoded cytochrome c oxidase enzyme
goes a long way to accomplishing this, convincingly
showing the descent of hepatocytes from stem/progenitor cells of associated ductular reactions. Nonetheless,
in humans, the specific distinction between hepatocytederived hepatocytes and stem/progenitor cell-derived
hepatocytes has to date not been accomplished.
Recently, however, epithelial cell adhesion molecule
(EpCAM) has been identified as a surface marker on
human hepatic stem/progenitor cells that is absent on
mature hepatocytes.2,17,18 Yet, it has also been noted
that in cirrhotic livers of diverse causes, many hepatocytes have EpCAM surface expression2; this may
represent aberrant EpCAM expression in injured hepatocytes versus persistence of EpCAM in hepatocytes
that have recently been derived from hepatobiliary
progenitors. We have hypothesized that EpCAM positive [EpCAM(þ)] hepatocytes are indeed newly
derived hepatocytes, originating from differentiation of
EpCAM(þ) stem/progenitor cells in ductular reactions. To evaluate this concept, we investigated the
patterns of EpCAM expression in hepatocytes and
ductular reactions of liver biopsy specimens from
patients with chronic hepatitis B and C in all stages of
disease, performed immunohistochemical studies of
proliferation and senescence, and evaluated telomere
lengths of all hepatobiliary cells in the sections studied.
Materials and Methods
Liver Samples. Archival, formalin-fixed, paraffinembedded sections of liver specimens were obtained
from the Departments of Pathology at Beth Israel
Medical Center, New York, United States, Kurume
University School of Medicine, Kurume, Japan, Aristotle University Medical School, Thessaloniki, Greece,
and from the Liver Cancer Specimen Bank, part of the
National Research Resource Bank Program, which is
administered by the Korea Science and Engineering
Foundation under the Ministry of Science and Technology. Approvals from the respective institutional
review boards or the equivalent were obtained prior to
beginning all investigations.
The liver biopsy specimens consisted of 33 cases of
chronic hepatitis B (CHB) and 69 cases of chronic
hepatitis C (CHC). Histologically normal (control)
liver specimens were obtained from wedge-biopsied livers of donors for liver transplantation, autopsy, or normal tissue distant from tumor in hepatic resections.
966
YOON ET AL.
HEPATOLOGY, March 2011
Table 1. Immunohistochemistry Systems Used in the Present Study
Antibody Source
Miltenyi Biotec (BIMC)
Novocastra (AUMS)
Calbiochem (KUSM, KOSEF)
Clone
Dilution
Antigen Retrieval
Antigen Detection
HEA-125
VU-1D9
VU-1D9
1:200
1:100
1:3000
Target Retrieval Solution (Dako) at 99 C (30 minutes)
Bond Vision Biosystems
100 mM sodium citrate (pH 6.0) boiled in microwave (15 minutes)
Dako EnVision kit
Bond Vision Biosystems
Dako EnVision kit
AUMS, Aristotle University Medical School; BIMC, Beth Israel Medical Center; KOSEF, Korea Science and Engineering Foundation; KUSM, Kurume University
School of Medicine.
The liver biopsy specimens with chronic hepatitis were
staged for fibrosis according to a modified Ishak staging system19 (1, portal fibrosis; 2, fibrous septa; 3,
transition to cirrhosis; 4, established cirrhosis) and for
grade of necroinflammatory activity (1, mild; 2, moderate; 3, severe [i.e., with confluent necrosis]).
Immunohistochemistry. Four-micron thick tissue
sections were deparaffinized with xylene and rehydrated with graded alcohols. After washing in distilled
water, sections were immersed in 3% hydrogen peroxide to block endogenous peroxidase. Details of
EpCAM staining methods used at the three institutions are given in Table 1. Other antibodies used for
immunohistochemical stains included: keratin (K) 19
(clone RCK108, Dako, Glostrup, Denmark; dilution
1:20), p21WAF1/Cip1 (clone SX118, Dako; dilution
1:50), and proliferating cell nuclear antigen (PCNA)
(clone PC10, Dako; dilution 1:75). These stains were
either performed in sequential cuts of the tissue block
(EpCAM/K19) or in the same slide (double staining
of EpCAM with K19, p21WAF1/Cip1, or PCNA).
We used the DAKO Envision Kit (Dako) for
immunohistochemistry with a single primary antibody,
using 3,3-diaminobenzidine (Dako) as the chromagen.
All slides were counterstained with hematoxylin. For
double immunohistochemical staining, the EnVision
AP system (Dako) and Vector Blue Alkaline Phosphatase Substrate Kit III (SK-5300, Vector Laboratories,
Burlingame, CA) were used to detect the first primary
antibody, and then the EnVision DuoFLEX Doublestain System (SK110) (Dako) and Vector NovaRED
Substrate Kit (SK-4800, Vector Laboratories) were
used to detect the second primary antibody. The
extent of EpCAM(þ) hepatocytes was assessed both in
relationship to K19(þ) cells of ductular reactions, as
well as semiquantitatively by evaluating the entirety of
the tissue on each stained slide (0, no positive hepatocytes; 1þ, <5% of hepatocytes positive for EpCAM;
2þ, 5%-10% of hepatocytes positive for EpCAM; 3þ,
11%-50% of hepatocytes positive for EpCAM; 4þ,
>50% of hepatocytes positive for EpCAM).
Telomere Evaluation by Way of Quantitative
Fluorescence In Situ Hybridization. Six of the CHB
cirrhosis cases and nine normal liver cases were eval-
uated for telomere lengths by way of quantitative fluorescence in situ hybridization. The peptide nucleic acid
probes included Cy3 telomere probe (50 -Cy3-OOCCC-TAA-CCC-TAA-CCC-TAA-30 ) and FAM centromere [P2] probe (50 -FAM-OO-ATTCGTTG
GAAACGGGA-30 ), both obtained from Panagene,
Daejon, South Korea. In brief, tissue sections were
deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was performed in citrate buffer
(pH 6.0) in a 700-W microwave oven for 10 minutes,
and the sections were fixed in 10% buffered formalin.
The sections were then treated with protease I solution
(1 mg/mL, Vysis, Downers Grove, IL) at 37 C for 10
minutes, dehydrated in graded alcohols, and air-dried.
The telomere/centromere probe mix (telomere: 2.5 lL
10 lg/mL PNA Cy3-telomere probe, 2.5 lL 25 lg/
mL FAM centromere probe) was then applied, followed by denaturation at 80 C for 3 minutes and
hybridization at 37 C for 2 hours using Vysis
HYBrite. The sections were washed in posthybridization buffer (NP40/20x saline sodium citrate, Vysis) at
room temperature for 30 minutes and then in Trisbuffered saline with Tween 20 for 15 minutes. To
detect EpCAM, incubation with monoclonal antibody
(EpCAM clone VU-1D9; Calbiochem, Darmstadt,
Germany; dilution 1:3000) was performed for 1 hour
at room temperature, after which the secondary antibody (goat anti-rabbit-Alexa flour 633; Invitrogen,
Eugene, OR) was applied. The sections were counterstained with 4’-6-diamidine-2-phenylindole and
mounted with Prolong anti-fade mounting medium
(Molecular Probes, Eugene, OR) for observation. Then
the sections were examined under fluorescent microscope. The telomere fluorescence intensity and the centromere fluorescence intensity were analyzed using
Image Pro Plus 5.0 software (MediaCybernetics, Silver
Spring, MD), and the telomere fluorescence intensity/
centromere fluorescence intensity ratio was calculated
in each telomere dot. To facilitate day-to-day comparison, a fluorescence bead (Molecular Probe) was photographed and analyzed.
Statistical Analysis. Statistical analysis was performed using SPSS software (SPSS, Chicago, IL) and
assessed using a Student t test and Mann-Whitney U
HEPATOLOGY, Vol. 53, No. 3, 2011
YOON ET AL.
Table 2. Patient Demographics and Chronic Hepatitis Stage
Fibrosis Stage
Chronic
hepatitis C
Chronic
hepatitis B
Controls
Median Age (Range)
Male:Female
n
1
2
3
4
54 years (22-73)
39:30
69
21
10
20
18
47 years (15-69)
19:14
33
8
7
5
13
48 years (42-61)
5:7
12
—
—
—
—
test as deemed appropriate. P < 0.05 was considered
statistically significant; P < 0.1 was considered marginally significant.
Results
The number of cases in each disease, including the
successive stages, age, and sex of patients, are summarized in Table 2. All cases showed necroinflammatory
activity graded as mild or moderate without any showing confluent necrosis sufficient to grade it as severe
activity.
Fig. 1. EpCAM staining in early and
late stage chronic viral hepatitis. (A)
EpCAM stain in chronic hepatitis C with
portal fibrosis highlights bile ducts and
ductules (cytoplasmic staining) and a
cluster of periportal hepatocytes (membranous staining). (B) On K19 stain of a sequential section, there is no positivity in
hepatocytes. (C) EpCAM stain of hepatitis
B cirrhosis shows cytoplasmic staining of
biliary structures. Ductular reactions, inclusive of intermediate cells show some cytoplasmic staining, but also membranous
staining. The hepatocytes surrounding the
ductular reactions show extensive membranous staining. (D) K19 stain of a sequential section lacks intermediate cell
and hepatocyte staining. (E, F) On double
staining for EpCAM and K19, all cells of
the ductular reaction and surrounding hepatocytes with K19 expression (blue) are
also EpCAM(þ) (brown). Original magnifications: 200 (A, B, E), 40 (C, D, F).
967
Immunohistochemistry. Examples of EpCAM and
K19 stains are shown in Fig. 1. In all livers, normal
and diseased, EpCAM expression was seen in the cytoplasm of cholangiocytes of all branches of the biliary
tree, including canals of Hering, ductules, and small
and large bile ducts (Fig. 1A,C). In normal liver, hepatocytes were rarely if ever positive for EpCAM (2 of
12 samples) and showed only membranous staining
(data not shown). In diseased liver specimens, hepatocellular staining was also membranous, whereas staining of the intermediate cells of the ductular reactions
ranged from cytoplasmic to membranous, depending
on the degree of hepatocellular differentiation.
We compared staining using all three immunohistochemistry methods, by circulating sequential slides
from all three institutions to each institution. Circulated slides included normal controls, early stage
CHC, and CHB cirrhosis. Neither the institutional
staining investigators (R. K., P. H., Y. N. P.) nor the
central coordinating pathologist (N. D. T.) noted any
differences in staining intensity or pattern of
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YOON ET AL.
HEPATOLOGY, March 2011
Table 3. Expression of EpCAM in Hepatocytes of Livers with
Chronic Hepatitis, Semiquantitatively Assessed by Way of
Immunohistochemistry
Extent of EpCAM Hepatocyte Staining
Stage of Disease
Control livers
Portal fibrosis (stage 1)
Fibrous septa (stage 2)
Transition to cirrhosis (stage 3)
Established cirrhosis (stage 4)
0
11
21
31
41
10
12
4
1
0
2
14
6
2
5
0
3
5
10
8
0
0
2
12
8
0
0
0
0
10
localization, emphasizing the ease and reliability of
staining for this antigen with diverse clones, methods
of antigen retrieval, and detection procedures (data not
shown).
When sequential slides were stained for EpCAM
and K19, in all stages of chronic hepatitis, EpCAM(þ)
hepatocytes were always located in contiguity with
K19(þ) ductular cells, with the EpCAM(þ) cells
arrayed around the periphery of ductular reactions
(Fig. 1A-D). In 12 cases in which double staining for
EpCAM and K19 was accomplished (stages 1-4), all
cells of the ductular reaction and surrounding hepatocytes with K19 expression were also EpCAM(þ) (Fig.
1E,F), and EpCAM(þ)/K19() cells, particularly hepatocytes, increased with increasing stage of disease
(data not shown).
Table 3 summarizes the semiquantitative assessment
of the extent of EpCAM(þ) hepatocyte staining in biopsy specimens according to the stage of chronic hepatitis. Normal livers had no EpCAM hepatocyte staining, or only slight staining (<5%). The extent of
EpCAM(þ) hepatocytes, overall, increased in parallel
with the stage of disease. Only cirrhotic livers had 4þ
(>50%) of parenchyma displaying membranous
EpCAM staining. There was no association between
hepatocyte EpCAM expression and grade of necroinflammation, nor were there significant differences
between livers of comparable stage between those with
CHC versus CHB.
As expected, p21WAF1/Cip1 and PCNA were
expressed in cell nuclei. Labeling indices were calculated for various cell types of normal livers (bile duct
lining cells, canal of Hering cells, hepatocytes) and of
CHB cirrhotic livers (bile duct lining cells, hepatobiliary cells of ductular reactions, hepatocytes) (Fig. 2;
Supporting Tables 1 and 2), including EpCAM(þ)
versus EpCAM() hepatocytes (Fig. 3).
For both antigens, there were statistically significant
labeling index differences between CHB cirrhosis and
normal controls, concerning all epithelial cell types. In
particular, hepatocytes in cirrhosis showed significantly
increased p21 expression compared with hepatocytes
in normal livers, whereas reactive ductular cells had
even more marked difference from the normal canal of
Hering cells (Fig. 2A). In addition, the cells of ductular reactions showed markedly elevated proliferation
rates (measured by PCNA expression) compared with
all other cell types of normal or diseased liver (Fig.
2B). Whereas hepatocytes in cirrhosis had a wider rate
of proliferation than those in normal liver, the difference did not reach statistical significance in this study.
Fig. 2. Labeling indices for (A) p21 and (B) PCNA in normal liver
versus hepatitis B cirrhosis. CoH, canals of Hering.
HEPATOLOGY, Vol. 53, No. 3, 2011
YOON ET AL.
969
shortening from ductular reaction to EpCAM(þ) hepatocytes and to EpCAM() hepatocytes (Fig. 5B).
Discussion
Fig. 3. Labeling indices for (A) p21 and (B) PCNA in EpCAM(þ)
versus EpCAM() hepatocytes in hepatitis B cirrhosis. CoH, canals of
Hering.
p21 and PCNA labeling indices showed no significant
difference between EpCAM(þ) hepatocytes and
EpCAM() hepatocytes (Fig. 3).
Telomere Length Evaluation. The telomere lengths
were measured by studying from 50 to 214 telomere
dots (telomere length signal detected by quantitative
fluorescence in situ hybridization) according to cell
type in nine cases of normal liver (Fig. 4G; Supporting
Table 3). In normal liver, there was no significant difference in telomere lengths among normal hepatocytes,
canal of Hering cells and bile duct cells (Fig. 5A), nor
did they differ according to age or sex.
In cirrhotic livers, the telomere lengths were measured by studying from 33 to 189 telomere dots
according to cell type in six cases (Fig. 4G; Supporting
Table 4). When comparing the telomere lengths
between EpCAM(þ) hepatocytes and EpCAM() hepatocytes in cirrhosis, the telomere lengths of
EpCAM() hepatocytes were significantly shorter than
those of EpCAM(þ) hepatocytes (P ¼ 0.046). In
addition, EpCAM(þ) hepatocytes showed relatively
shorter telomere length than ductular reactions (P ¼
0.057), whereas EpCAM() hepatocytes showed significantly shorter telomere length than ductular reactions (P ¼ 0.016) (Figs. 4 and 5A). There was no significant difference in telomere length according to
patient age in both cirrhotic and normal livers.
When the telomere lengths of ductular reactions,
EpCAM(þ) hepatocytes, and EpCAM() hepatocytes
were traced in each patient, there was gradual telomere
A growing body of work in the last few decades has
identified cells of the ductular reactions in human livers in diverse but usually severe acute and chronic liver
diseases as being the equivalent of the oval cells seen
in rodent models of injury.1,2,5,11-16,18 As such, the
ductular reactions are thought to be the transit amplifying cells deriving from activation of the stem cell
compartments of the liver.1,7 Like oval cells, the cells
are thus thought to have at least two possible differentiation pathways, toward hepatocytes and toward cholangiocytes, the dominant direction being determined
by the presence of injury and the nature and severity
of that injury. In diseases with a predominance of hepatocyte injury, the ductular reaction is triggered by
acute destruction of large amounts of parenchyma11 or
by senescence of hepatocytes by chronic low level
injury, and presumably results in hepatocyte replenishment from the stem/progenitor cell compartments.13-
15
In acute liver failure from acetaminophen toxicity,
three-dimensional reconstruction of K19(þ) ductular
reactions suggested that many hepatocytes appeared to
be deriving from the proximal, smallest branches of
the biliary tree (i.e., the canals of Hering and ductules), which comprise a stem cell niche of mammalian
livers.11 In chronic liver disease, similar three-dimensional reconstruction showed that ductular reactions in
chronic viral hepatitis (CHB and CHC), autoimmune
hepatitis, and fatty liver diseases likewise gave rise to
hepatocyte buds indicating hepatocyte repopulation.13-
15
Studies of proliferation in these settings, with
PCNA or Ki-67 as proliferation markers, supported
this hypothesis by confirming that ductular reactions
are highly proliferative, as one would expect in transit
amplifying cells involved in stem cell–derived repopulation.13,20 That this process is likely triggered by
increasing inability of hepatocytes themselves to replicate after years or decades of injury in chronic disease
was then confirmed by immunohistochemical evaluation of p21 as a marker of senescence.14,15 As hepatocytes become increasingly senescent in later stages of
diseases, indicated by increasing p21 expression with
advancing stage, only then does the ductular reaction
emerge, suggesting that stem cells have taken over the
burden of hepatic restitution.
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YOON ET AL.
HEPATOLOGY, March 2011
Fig. 4. Quantitative fluorescence in situ hybridization for telomere length assessment combined with immunohistochemical stain for EpCAM.
The red and green signals in the nuclei correspond to telomere and centromere signals, respectively. Red EpCAM(þ) immunostaining can be
seen on the cell membranes of bile ducts, ductular reactions, canals of Hering, and some hepatocytes. (A-D) Representative features of bile
duct (A), ductular reaction (B), EpCAM(þ) hepatocytes (C), and EpCAM() hepatocytes (D) in hepatitis B viral cirrhosis. (E, F) Representative
features of hepatocytes (E), canals of Hering (arrows) (E), and bile ducts (F) in normal liver. (G) Number of telomere dots according to telomere
length in various cell types in cirrhosis (above) and in normal liver (below). Telomere length was measured by the ratio of telomere fluorescence
intensity and centromere fluorescence intensity in each telomere dot.
HEPATOLOGY, Vol. 53, No. 3, 2011
Fig. 5. Telomere lengths in various cell types. (A) Telomere lengths
in epithelial cell types of hepatitis B viral cirrhosis and normal human
liver. (B) Telomere lengths in various cell types of 6 patients with hepatitis B viral cirrhosis.
Only two studies in humans, however, have actually
provided cell tracking data to support the idea that
cells of the ductular reaction become hepatocytes. In
the first, a male patient with hepatitis C cirrhosis
underwent liver transplantation and received an organ
from a female donor, but then developed severe acute
injury in the form of fibrosing cholestatic recurrent
hepatitis C.21 Using colocalization of Y chromosome
(by way of fluorescence in situ hybridization) and K8/
18 (by way of immunohistochemistry), 40% of the hepatocytes in the afflicted liver bore Y chromosomes,
indicating derivation from the recipient (probably of
YOON ET AL.
971
bone marrow origin). However, not only were cells of
the ductular reaction frequently Y chromosome–bearing, but hepatocytes adjacent to the ductular reaction
were more likely to be Y-positive than those in the
perivenular regions (64% versus 16%, respectively).
These data imply that ductular reaction cells become
new hepatocytes, though in this disease setting the full
lineage pathway appeared to be from bone marrow to
ductular reaction to hepatocyte.
The second study, by Lin et al,.16 concerns cirrhosis
that developed in several chronic liver diseases, and
used analysis of mutations in mitochrondrial DNA
encoding cytochrome c oxidase enzyme. This study
unambiguously demonstrated the derivation of hepatocytes containing distinct mutations as deriving from
adjacent ductular reactions with the identical mutation
(a majority of cirrhotic nodules, furthermore, being
clonal, suggesting derivation from a single stem/progenitor cell within a preexisting canal of Hering).
In related, but separate lines of investigation, marker
studies of these hepatobiliary lineages have revealed a
variety of important molecules that indicate varying
shades of hepatocyte and biliary commitment. Of particular importance have been studies showing that
EpCAM is a marker of the hepatobiliary stem cell
niche and that when such cells develop into hepatocytes in culture, the new hepatocytes as well as the
cells with intermediate features between stem/progenitor cells and hepatocytes also display membranous
EpCAM. 2,16 These findings led us to hypothesize that
EpCAM(þ) hepatocytes are derived relatively recently
from the stem cell niche rather than from other, preexisting hepatocytes.
The goal of the present study was to investigate this
possibility within intact tissue specimens from livers of
patients with hepatitis B and C through several means.
The first is by determining whether EpCAM(þ) hepatocytes develop only in the context of ductular reactions, stage by stage, and exploring the topological
relationships of these cells (Fig. 1 and Table 3). Four
important points support our primary hypothesis: (1)
EpCAM(þ) hepatocytes, like ductular reactions,
increase in frequency and extent with increasing stage
of disease; (2) although ductular reactions sometimes
do not have associated EpCAM(þ) hepatocytes,
EpCAM(þ) hepatocytes, when present, are always
associated with ductular reactions; (3) EpCAM(þ) hepatocytes always appear as aggregates surrounding a
core of ductular reaction cells; and (4) cells of intermediate morphology between the smallest progenitor cells
of the ductular reaction and mature appearing,
EpCAM(þ) hepatocytes are always also EpCAM(þ).
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YOON ET AL.
Thus, morphologically, topographically, and immunophenotypically, EpCAM(þ) hepatocytes appear to
derive from cells of the ductular reaction.
Such data, although compelling, are incomplete. We
thus hypothesized that if EpCAM(þ) hepatocytes were
stem cell–derived, they would have telomere lengths
that were longer than those of the EpCAM() hepatocytes. This hypothesis is based on prior data indicating
that ductular reactions have increased telomerase activation22-25 and that senescent hepatocytes, after years
of increased cell turnover, would have shortened telomeres.26-29 We would also expect that EpCAM() hepatocytes in cirrhosis would have telomeres that would
be shorter than those in EpCAM(þ) hepatocytes, and
that telomere length of EpCAM(þ) hepatocytes would
be shorter than that in ductular reactions. These predictions were confirmed in a statistically meaningful
way for hepatocytes in CHB cirrhosis.
We also sought to explore issues of proliferation and
senescence as previous studies had done,13-15,20 but
discriminating between hepatocytes that were
EpCAM(þ) and those that were EpCAM(). However, there was no significant difference of PCNA and
p21 labeling indices between EpCAM(þ) hepatocytes
and EpCAM() hepatocytes. We have no clear explanation for these findings, though we suspect that the
variable nature of staining for both p21 and PCNA in
regard to fixation and staining conditions plays an important role; study of freshly prepared EpCAM(þ)
versus EpCAM() hepatocytes may be required to
more fully evaluate their relative senescence and cell
cycle status.
In summary, we hypothesize that EpCAM(þ) hepatocytes in chronic liver disease represent hepatocytes
that have derived from activation of a stem cell compartment of the liver in the setting of chronic hepatitis, whereas EpCAM() hepatocytes in such livers
have derived from preexisting hepatocytes. In support
of this hypothesis, we present morphologic, topographic, immunophenotypic, and molecular data. Our
most compelling data, which are indicative of current
functional behavior as well as cell behavior over time,
are represented in the finding that EpCAM(þ) hepatocytes have telomere lengths that are longer than those
of EpCAM() hepatocytes and shorter than the ones
of ductular reactions. This finding is in accord with
our hypothesis and with prior published data regarding
telomerase activity in hepatic stem cell and transit
amplifying cell populations.
Because EpCAM is a surface membrane antigen, we
can expect that isolation and immunosorting of hepatocytes from fresh liver specimens may yield quite
HEPATOLOGY, March 2011
interesting data regarding the nature of liver regeneration in both acute and chronic liver diseases, with
stronger statistical significance than found in this
archival tissue study. Moreover, these data may have
practical implications regarding selection of hepatocytes for use in therapeutic cell transplantation or in
populating of artificial liver assist devices.
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