(CANCER RESEARCH 50. 3811-3815, July l, 1990]
Perspectivesin CancerResearch
Is There a Liver Stem Cell?
Stewart Sell
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, Texas 77030
Abstract
The role of a putative liver stem cell in liver regeneration and carcinogenesis is reviewed. There is increasing evidence that there is a liver
stem cell that has the capacity to differentiate into parenchyma! hepatocytes or into bile ductular cells. These stem cells may be activated to
proliferate after severe liver injury or exposure to hepatocarcinogens.
They are not activated by moderate liver injury, which is repaired by
proliferation of mature hepatocytes. Exposure to most chemical hepato
carcinogens results in proliferation of a small morphologically indistinct
cell population termed "oval cells." These cells have been shown to have
the capacity to differentiate into hepatocytes or into ductular cells. The
origin of these cells appears to be from transition duct cells, but there is
also evidence of an even less mature periportal liver stem cell. Study of
the development of these cells during carcinogenesis indicates that liver
cancer arises from oval cells by aberrant differentiation of stem cells.
From a biological standpoint two major postulates of the
origin of cancer are: Cancer arises from differentiated expres
sive cells by "dedifferentiation" or from pluripotential stem
cells by aberrant differentiation (1). One of the fundamental
postulates of the stem cell theory of cancer is that the incidence
of cancers arising in a given tissue is directly related to the rate
of cell division in that tissue. Thus tumors appear in relative
high incidence in organs with rapid cellular turnover, such as
skin, gastrointestinal mucosa, or bone marrow, but in low
incidence in tissues with low turn over, such as neurological
tissue. Tumors of neurons, which essentially do not proliferate
in the adult, are extremely rare. One exception to this, which
may prove the rule, is the liver. Although primary malignant
tumors of the liver are rare in the western world, the extremely
high incidence of primary hepatocellular carcinomas in other
parts of the world (Africa, Southeast Asia) actually makes
primary hepatocellular carcinoma one of the world's most
common malignant tumors. This brief review will address the
question, "Do hepatocellular carcinomas arise from dediffer
entiation of adult hepatocytes or do these cancers arise by
activation and aberrant differentiation of liver stem cells?"
Stem cells are defined as multipotent cells that divide to
produce one daughter cell that stays as a stem cell while the
other daughter cell expresses a differentiated phenotype. Tissue
stem cells are determined; i.e., they lack the biochemical and
structural markers of differentiation but are determined for
differentiation to a specific cell type (1). Stem cells respond by
proliferation and differentiation to replace senescent cells under
normal circumstances or to restore destroyed tissues in patho
logical conditions. The epithelium of the skin is the classic
example of the former. The basal cells send daughter cells
through the maturational pathways to replace senescent cells.
The basal cells may not be the true stem cells in this case but
are already partially differentiated. Another example is the cell
type responsible for limb regeneration in the amphibian that
can replicate and differentiate into the composite of mature
cells of the regenerated limb. The limb blastema or stem cell
actually is not multipotential but is limited to expression of
Received 12/4/89; revised 2/14/90.
limb cell phenotypes (2). Stem cells may participate in tissue
replacement in organs that normally undergo renewal, such as
hematopoietic cells, but most of the actively dividing cells in
these organs are not the pluripotent stem cells but mitotically
active partially differentiated expressive cells that give rise by
further division and differentiated to terminally differentiated
cells that make up the "mature" cells of the organ. Whether or
not there are stem cells in an organ such as the liver is critical
in understanding the cellular origin and mechanisms in carci
nogenesis.
Study of the cellular changes that precede the development
of liver cancer in animals exposed to chemical hepatocarcino
gens has led most investigators over the last 20 years to the
conclusion that hepatocellular carcinomas arise by dedifferen
tiation of adult liver cells. These studies have concentrated on
a series of lesions called "foci" and "nodules" which have been
designated "premalignant" (3, 4). The development of focal
and nodular lesions may be presented by a description of the
morphological changes reported by Teebor and Becker (5)
following the cyclic feeding of the carcinogen AL2-acetylaminofluorine to rats. The AAF1 containing diet is fed for 2 weeks
followed by 1 week of normal diet, the full carcinogen regimen
consisting of four 2-week-on/l-week-off cycles for a total of 15
weeks. Approximately 36 weeks after the initiation of the AAF
feeding the rats develop primary hepatocellular carcinomas.
After the first feeding cycle, the livers contain small collec
tions of cells that contain higher levels of some enzymes (e.g.,
7-glutamyl transpeptidase, glutathione 5-transferase, etc.) than
do normal adult hepatocytes (enzyme altered foci) and these
cells stain more intensely basophilic than normal cells (basophilic foci). After the second cycle collections of enzyme altered
cells now form small masses that push aside the normal liver
cells. These are designated "neoplastic nodules." After the third
cycle the neoplastic nodules enlarge and can be seen easily with
the naked eye. If the diet is discontinued after three cycles no
carcinomas will develop and the liver will "remold" to a normal
appearance (6). After four cycles much larger nodules are seen
and if the AAF diet is then discontinued, carcinomas will appear
about 15 weeks later. Because of the sequence of morphological
and enzymatic changes from adult liver cell to foci to nodules
to cancer, this system has been used as a model for the dedif
ferentiation origin of cancer. However, at the same time that
these designated "preneoplastic" changes are taking place there
is proliferation and evolution of another cellular lineage. Evi
dence is accumulating that primary hepatocellular carcinoma
actually arises from these latter cells and that this cellular
lineage arises by aberrant differentiation of stem cells.
Large numbers of small nondescript cells, termed "oval cells"
(7), also appear in the livers of rats exposed to chemical carcin
ogens (8, 9), but a role for these cells in hepatocarcinogenesis
has been largely ignored. The development and fate of oval cells
during hepatocarcinogenesis are now becoming increasingly
clear (10, 11). These cells proliferate in most, if not all, models
' The abbreviations used are: AAF, Ar-2-acetylaminofluorene; AFP, a-fetoprotein.
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IS THERE A LIVER STEM CELL?
of carcinogenesis. This cell lineage develops prior to the neoplastic nodules. The oval cells arise from periductular cells early
after carcinogen exposure and have the capacity to acquire
ductal or hepatocellular phenotypes (12, 13). More recently,
Evarts et al. (14), following oval cells produced relatively early
after carcinogen administration and partial hepatectomy by
autoradiography, demonstrated that the radiolabeled cells seen
early after [3H]thymidine administration were oval cells but that
the label later appeared in newly formed hepatocytes. They
conclude that oval cells differentiate into hepatocytes.
Studies of the in vitro proliferation and phenotypic expression
of liver cells taken from carcinogen treated animals is support
ive of the stem cell concept. Earlier studies with cells from
carcinogen treated rats were directed to determining properties
of nodular cells. Laishes et al. (15, 16) cultured livers with a
large nodular component and found that mixed cell populations
from carcinogen treated rats were resistant to the toxic effects
of carcinogen exposure. Subsequently, Ledda et al. (17), using
cultures of cells from carcinogen treated rats using density
gradient centrifugation to obtain cell populations highly en
riched in oval cells (18), demonstrated that the oval cells and
not the larger hepatocytes were resistant to the toxic effects of
carcinogen exposure. Thus it appears that both oval cells and
nodular cells are resistant to the toxic effects of carcinogens.
In general, nodular cells survive better in vitro than normal
hepatocytes but do not have an increased tendency to proliferate
or transform in vitro (19, 20). On the other hand, cultures
enriched for oval cells tend to form clusters in vitro and to
proliferate spontaneously (21). Recently, more extensive studies
by Germain et al. (22-24) using cultures of oval cell populations
(23) and fetal rat liver cells (24) demonstrate that these popu
lations respond similarly to the differentiating stimuli provided
by addition of sodium butyrate and dexamethasone. By exam
ining a variety of phenotypic markers they conclude that both
oval cells and fetal liver cells have the capacity to differentiate
into either bile duct cells or into hepatocytes. In contrast, bile
duct cells isolated by density gradient from the livers of rats
after bile duct ligation (25) do not acquire hepatocyte pheno
types in culture (26), suggesting that bile duct cells are not able
to "dedifferentiate" into hepatocytic lineages. Thus bile ductule
and ducts are "determined" for bile ducts and could not produce
hepatocytes under these conditions. Tsao et al. (27, 28) using a
cell line with "oval cell" properties derived from a normal rat
liver (29) have shown that treatment of the cell line with a
carcinogen (/V-methyl-jV'-nitrosoguanidine)
results in a pro
gressively increasing phenotypic diversity, an indication of the
growth and differentiation potential of these cells. Finally,
transplantation into nude mice of epithelial cell lines derived
from the livers of carcinogen treated rats after transfection with
the H-ras oncogene resulted in growth of moderate to well
differentiated hepatocellular carcinomas (30). The significance
of this observation to the induction of hepatocellular carcino
mas by chemicals is not clear.
Isolation of oval cells from livers of carcinogen treated rats
by density gradient centrifugation is possible because the oval
cells are much smaller than parenchyma! hepatocytes or nodu
lar cells. One of the interesting changes that occur in the livers
of rats treated with carcinogens is a change in the ploidy state
from largely tetraploid to largely diploid (31). Neoplastic nod
ules are diploid (32). This has been used to suggest that the
transformed hepatocytes that give rise to tumors are derived
from nodules. However, Schwarze et al. (33) found that most
of the diploid cells arising early after carcinogen exposure were
about half the size of the tetraploid hepatocytes, and Scott et
al. (34) noted that the diploid cells corresponded to «-fetoprotein positive oval cells. Thus it is postulated that diploid oval
cells give rise to diploid nodular cells that, in turn, give rise to
nondiploid tumors that are transplantable (35).
Transplantation of putative premalignant cells into nude
mice has been used to determine if a given cell population can
give rise to tumors. Transplantation of nodular cells has gen
erally not given rise to tumors, with a few possible exceptions
(36, 37). Tatematsu et al. (37) were able to produce nodules by
transplantation of late nodular liver cells and some histológica!
evidence of hepatocellular carcinoma within the transplanted
nodules. In contrast Yoshimura et al. (38) obtained highly
anaplastic adenocarcinomas upon transplantation of cultures
obtained from cells highly enriched for oval cells. Oval cells
injected into the fat pads of syngeneic rats maintain the mor
phology of duct-like clusters but may express hepatocyte prop
erties, such as albumin and tyrosine aminotransferase activity
(23). Fetal liver cells exposed to carcinogen in vitro will produce
tumors expressing hepatocyte phenotypes upon transplantation
to nude mice (39). Each of these studies illustrates the problems
of working with transplantation of a population of cells. It is
not possible to determine which cell in the transplanted popu
lation may eventually grow into a transplantable tumor. Thus,
cells used for transplantation may be highly enriched in nodular
cells or in oval cells, but upon transplantation, only one cell in
10s or IO6 may divide and this cell may not represent the
majority population. However, both in vivo and in vitro Findings
are consistent with the conclusion that oval cells can evolve
into either primary hepatocellular carcinomas or cholangiocarcinomas.
A critical question is from what cells do the "oval cells" or
stem cells originate. This question has been addressed by se
quentially labeling the proliferating cells in the liver very early
after exposure to chemical hepatocarcinogens (40, 41). In order
to appreciate the changes seen it is necessary to describe the
morphology of the terminal bile ductules and their relationship
to the bile canaliculi of the hepatocytes. The terminal bile
ductules are composed of two or three ductule cells surrounded
by a basement membrane. These ductules are called the canals
of Hering. They connect to the larger bile ducts on one side
and to transitional duct cells on the other. The transitional duct
cell connects the bile canaliculi with the canals of Hering. The
transitional duct cell may be identified by the following char
acteristics. It does not abut a basement membrane; it borders
one side of a canaliculus with an hepatocyte and, most critically,
it forms tight junctions with this hepatocyte. For a clear picture
of the transition ductule cell see the photograph on p. 596 of
Bailey's Textbook of Microscopic Anatomy (42). There is an
increasing tendency to refer to the transition ductule cell as a
"Hering cell," but this extension of the terminology historically
used for the terminal ductule has led to some confusion. Popper
et al. (43) as well as Grisham and Porta (13) first identified
proliferation of the ducts of Hering after exposure of rats to
chemical carcinogens for 3 to 4 weeks and concluded that these
cells were the major proliferating cells. In addition they pro
posed that so-called terminal duct cells could differentiate into
either duct cells or hepatocytes. However, when the livers of
carcinogen exposed rats were examined earlier (1 to 7 days
after exposure to a rapidly acting carcinogenic diet), extensive
proliferation of periductular cells was seen to precede labeling
of the ductules (Fig. 1, a and b). By electron microscopic
autoradiography many of these cells had the characteristics of
the transitional ductule cell (Fig. le). In a poster presented at
the November, 1989 meeting of the American Society of Cell
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IS THERE A LIVER STEM CELL?
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Fig. 1. a, light microscopic autoradiography, 1 day after a choline-devoid diet containing 0.05% w/w A'-2-fluorenyIacetamide (CD-AAF) feeding; b, light
microscopic autoradiography 7 days after CD-AAF feeding; c, electron microscopic autoradiography, day 7; d, electron microscopic autoradiography, day 1. Rats
were fed CD-AAF and given injections of 50 >iCiof [3H]thymidine i.v. three times on the day preceding sacrifice. Vertical arrows in a and b, labeled periductular cells;
horizontal arrows, labeled ductular cells. Some of the periductular cells in b could be transitional ductule cells, c. L, liver cell; T, transitional ductule cells; P. periportal
cell. Arrow, tight junction between one of the transitional ductule cells and the liver cells adjacent to a shared bile canaliculus (markers of transitional duct cells).
Transitional duct cells are seen in large numbers after induction of oval cell proliferation. The periportal cell may also be a transitional duct cell. None of there cells
abuts a basement membrane. The hepatocyte shows toxic changes caused by the CD-AAF diet. The labeled cell in d (arrow) is a nondescript periportal cell that does
not have transitional duct cell or Ito cell characteristics. This may be a putative liver stem cell or a poorly differentiated connective tissue cell. D, ductal cell. For more
details see Ref. 41.
Biology, Phyllis Novikoff (44) demonstrated a marked increase
in the number of transition duct cells within a few weeks of
exposure to a similar carcinogenic regimen. Although it is
difficult to find labeled cells earlier than 2 days after carcinogen
exposure, we were able to observe some proliferating cells as
early as the first day (Fig. 1, a and d). Some of these cells were
smaller than the transition duct cells, did not form tight junc
tions with hepatocytes, and did not form a canaliculus with
adjacent liver cells (41). We have tentatively concluded that this
is a true liver stem cell (11). However, the "oval" cells seen
number, would be difficult to identify, particularly at later times
after initiation of proliferation when many proliferating differ
entiated cells are present.
Historically, the presence of liver stem cells has been doubted.
In the liver there are very few dividing cells [estimated as less
than 1/20,000 cells (45)]. In addition, most studies of liver cell
regeneration after partial hepatectomy, chemical injury, or viral
toxicity have shown that adult liver cells can divide to restore
destroyed liver parenchyma (45, 46). Localization of AFP pro
ducing cells following liver injury and restitution identified large
dividing hepatocytes in both rats (47) and mice (48). Thus there
is no apparent need for a stem cell for liver regeneration.
However, Zajick et al. (49, 51) have studied the normal
"turnover" of liver cells and find that there is continued pro
later may arise either from this cell or from the transitional
duct cells.
The finding that most of the cells that proliferate in the liver
early during the early stages of carcinogenesis have a duct or
transition duct cell phenotype does not rule against the presence
duction of hepatocytes (49) and bile duct cells (50). These arise
in the portal zones and "stream" toward the central zones
of an even less differentiated stem cell. In normal tissues that
undergo rapid renewal, such as bone marrow, skin, or gastroin
accompanied by littoral cells (51). They conclude that the liver
testinal epithelium, most of the proliferating cells demonstrate
renews its cells continuously exactly like the gastrointestinal
some differentiated characteristics and it is very difficult to tract and epidermis (51).
identify the stem cell of the tissue. Similarly, when liver cells
In addition, more recent studies indicate that, following
are stimulated to proliferate most cells with the capacity to severe liver injury or exposure of experimental animals to
proliferate would be expected to have an identifiable differen
chemical hepatocarcinogenesis, there is proliferation of cells in
tiated morphology, i.e., transition duct cells or cells of the canal
the liver with stem cell properties. Engelhardt et al. (52) found
of Hering, whereas the true stem cells, which are very few in that whereas most of the AFP containing cells seen after CC14
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IS THERE A LIVER STEM CELL?
injury of mouse liver were differentiated hepatocytes, a few
smaller cells that resembled oval cells were also positive. In a
study in rats it was found that most, if not all of the cells
containing mRNA for AFP after CC14 injury were small nonparynchmal cells (53). More recently, Tournier et al. (54) found
that although there was a generalized increase in the mRNA
for AFP over most nonnecrotic hepatocytes after CC14 injury
in rats, large amounts of AFP protein and mRNA for AFP
were concentrated over proliferated oval cells and bile duct-like
structures after liver injury induced by D-galactosamine. LongEvans's cinnamon rats, which spontaneously develop acute
hepatitis, have a proliferation of oval cells which appear to
differentiate into hepatocytes without malignant transforma
tion (55). Thus, even though there is some disagreement among
these studies in the extent of small cell proliferation and AFP
production after liver cell injury, they each indicate the partic
ipation of a putative liver stem cell.
If there is a liver stem cell, it should play a important role
during the morphogenesis of the liver during development. At
this time our understanding of the development of the normal
liver is not clear. The conventional view is that the liver arises
from a hepatic diverticulum formed from endothelium between
the foregut and the yolk sac. This diverticulum extends into the
mesoderm of the septum transversum where it gives rise to the
parenchymal cells of the liver, with the mesodermal cells form
ing the capsule and fibrous tissue of the liver (56-58). However,
it is also possible that hepatocytes form from mesodermal cells
into which the ductal endodermal cells extend (59). The con
ventional view is supported by the findings of Germain et al.
(24) that at 12 days of embryological development the emerging
hepatic cells in the rat embryo contain both ductal and hepatocyte markers. They conclude that there are at this stage
bipotential precursor epithelial cells that are capable of differ
entiation into hepatocytes or into biliary epithelial cells. They
further find that such differentiation in vitro may vary depend
ing on culture medium supplements. Similar results have been
found in our laboratory (60), but we have also found that
mesenchymal cells can express a liver cell phenotype at re
stricted times during embryogenesis.2 Nevertheless, there are
sufficient data to support the conclusion that the liver develops
from a hepatocytic stem cell that has the potential to develop
into a duct cell or a hepatocyte.
Studies on experimental injury to the pancreas also support
a liver/pancreas stem cell (61). Hepatocytes appear in the livers
of rats with aging (62) or after experimental injury (63, 64), in
humans (65) or hamsters spontaneously (66), or in hamsters
treated with carcinogens (67). In rats these cells arise from
periductular or ductular cells in a manner very similar, if not
identical, to that seen in the livers of rats exposed to carcinogens
(68). Thus it may be concluded that the pancreas also contains
a liver stem cell that has the potential of differentiating into
duct cells or hepatocytes.
In conclusion, for over 30 years the study of chemical hepatocarcinogenesis in the liver has concentrated on analysis of the
nodules, based on the assumption that these were, in fact, true
premalignant lesions. However, in view of the more recent
observations an alternate hypothesis must be considered, i.e.,
that liver cancer does not arise by dedifferentiation of hepato
cytes through formation of foci and nodules but by aberrant
differentiation of hepatic stem cells. The transitional duct cell
or cells of the canals of Hering may serve as hepatic stem cells,
2 H. A. Dunsford and S. Sell, unpublished observations.
but the presence of a more primitive precursor must also be
given serious consideration.
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Is There a Liver Stem Cell?
Stewart Sell
Cancer Res 1990;50:3811-3815.
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