LIVER REGENERATION 9

0892.6638/96/0010-1 249/$01 .50.0 FASEB 1249

Hepatic stem cells in liver regeneration
SNORRI S. THORGEIRSSON
Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892-4255, USA
The key to understanding liver development, hepato-
carcinogenesis, and the ability to regenerate lies in es-
tablishing the existence of a liver stem cell population
(or populations). By now we should be readily con-
vinced that hepatocytes themselves have the ability to
exit quiescence and replicate. But the fundamental
concept that adult mammalian livers contain stem cells
that can replace hepatic parenchyma has been the sub-
ject of an ongoing controversy, which continues to be
fueled by debate over the role of stem cells in liver
growth and repair. Early studies of liver cell lineages
assumed the role of stem cells as progenitors for both
normal and transformed hepatocytes, including hepato-
cellular carcinoma. In recent years, significant ad-
vances have furthered our understanding of liver stem
cell biology: we are beginning to realize the role of
growth factors, transcriptional activators, and cell-cell
communication in the regulation of stem cell activa-
tion. With the development of in vitro assays, in vivo
genetic marking studies, and identification and charac-
terization of the critical trans-activating factors respon-
sible for cell lineage, we are able to address these
issues. We now recognize and distinguish the unipo-
tential stem cell system of the hepatocyte from the
bipotential hepatoblast from the multipotential facul-
tative stem cell system or oval cell. Do you believe
that the adult liver contains stem cells? After all, we
can induce the formation of pancreatic acini and intes-
tinal metaplasia in hepatic tissue. Moreover, pancreatic
ductules can give rise to cells that exhibit all the phe-
notypic traits and biological properties of differentiated
hepatocytes. Most liver injuries are not due to simple
70% partial hepatectomies, but rather to toxic injuries
from viruses, chemicals, and other agents. It is impera-
tive to understand the role of stem cells in order to ul-
timately control the ability of the liver to regenerate. I
believe that adult liver stem cells exist, and I wager
that you will, too, after reading this last review in the
series.
-Clifford J. Steer, Coordinating Editor
ABSTRACT The concept that the liver contains
epithelial celia that share some of the major proper-
ties of stem cells of the well-characterized, stem
cell-fed lineages found in bone marrow, intestinal
epitheium, and epidermis is now well supported.
Nevertheless, the population dynamics of the major
types of liver epithelial cells, hepatocytes, and bile
epithelia display a strildng difference from the popu-
lation dynamics of the classic stem cell systems. The
focus of this review is on recent studies of the
activation and expansion of liver stem cells in vivo
and the role these cells may play in regeneration of
the liver. The requirement for a selective and sus-
tained expression of growth factors during the early
stages of stem cell activation is highlighted. In addi-
tion, results are presented supporting the hypothesis
that after loss of liver mass, both the quiescent stem
cells as well as the residual differentiated hepatocytes
and bile duct epitheial cells are activated to prolif-
erate. However, significant contribution of the stem
cells to the regeneration process only occurs under
circumstances in which the residual differentiated
cells are functionally compromised and/or cannot
proliferate.-Thorgeirsson, S. S. Hepatic stem cells
in liver regeneration. FASEBJ. 10,1249-1256(1996)
Key Words: growth factors . liver stem cell biology stem cell
activation hepatocytes bile duct epithelial cells . liver paren-
chyma oval cells - partial hepatectomy . prolferwion dfferen-
tiation
THE UVER IS MITOTICALLY a quiescent organ in adult hu-
mans and animals. In spite of this slow cellular turnover,
the two parenchyinal cell lineages, hepatocytes and bile
epithelial cells, have a remarkable capacity to meet re-
placement demands of cellular loss. The best example of
the capacity of adult hepatocytes and bile epithelial cells
to proliferate is seen after partial hepatectomy (PH)2 in
rats and mice, in which the compensatory hyperplasia of
these cells in the remaining lobes restores the liver mass.
Moreover, this process of liver regeneration can be re-
1Addresa correspondence to Dr. Thorgeusson, at:National Cancer
Institute, Bldg. 37, Room 3C28, 37 ConventDr.MSC4255, Bethesda, MD
20892-4255 USA.
2Abbreviations: aFGF, acidic fibroblast growth factor, TGFa, trans-
forming growthfactor-alpha; HCF, hepatocyte growthfactorSCF, stem
cellfactor; AFP, a-fetoprotein; AAF, 2-acetylaminofluorene; EGF, epi-
thelial growth factor.
1250 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON
peated several times in experimental animals. The in-
creased use and success of liver transplantation in clini-
cal medicine have shown that these animal models
correctly reflect the capacity of the human liver to regen-
erate (1). Due to this well-established trait of adult hepa-
tocytes and bile epithelial cells to repeatedly regenerate
the liver after either surgically or toxically induced loss
of the tissue, the existence of hepatic stem cells that may
participate in liver regeneration has been somewhat con-
troversial. The major part of the hepatic stem cell contro-
versy may, however, be due to the failure to recognize
that the adult organism contains many kinds of stem
cells, and that these may exist at different stages of dif-
ferentiation and have very different capacities for gener-
ating multilineage progeny.
Self-maintenance is a fundamental and common trait of
all stem cells. A cell population that has an extensive
self-maintaining capacity is perhaps the only definition
that applies to all stem cells (2). In this context, the adult
liver, having the extensive capacity for maintaining
parenchymal cell number throughout the life span of the
organism, can be viewed as a single lineage stem cell
system in which the hepatocyte is the stem cell. Recent
data from hepatic cell transplantation experiments in a
transgenic mouse model (3) have demonstrated the tre-
mendous growth potential of adult hepatocytes (12-16
doublings per donor cell), further supporting the notion of
the liver parenchyma as a single lineage (or unipotential)
stem cell system. On the other hand, if the classic defi-
nition of stem cells proposed by Potten and Loeffler (4) is
used, one would differentiate between actual stem cells,
potential stem cells, and committed stem celLs. Actual stem
cells are defined as undifferentiated cells capable of 1)
proliferation, 2) self-maintenance, 3) production of a
large number of differentiated progeny, 4) regeneration of
the tissue after injury, and 5) flexibility in the use of
these options. Potten and Loeffler (4) consider that po-
tential stem cells are latent or quiescent counterparts of
the actual stem cells that may be reactivated to become
functional stem cells, and that committed stem cells may
correspond to the dividing transit cells (e.g., dividing-
transit enterocytes), sharing with the terminally differenti-
ated cells the ability to execute tissue-specific functions.
Applying the definition of stemness used by Potten and
Loeffler (4), the hepatocytes appear to be committed
stem cells that are normally quiescent, but can be acti-
vated to produce progeny whose only differentiation op-
tion is hepatocytic.
The fact that the early fetal hepatocytes or hepatoblasts
are progenitors for both adult hepatocytes and bile epi-
thelial cells suggests that the hepatoblasts are at least
bipotential precursors (5). The question then arises
whether either or both of the cell lineages derived from
the hepatoblast retain the bipotential capacity of the
precursor cells. There is at present no substantial evi-
dence indicating that adult hepatocytes are more than a
unipotential committed stem cell system. The possible
exception to this generalization is the concept of ductular
metaplasia or transformation of hepatocytes into ductules.
This topic has been extensively discussed by Desmet and
his colleagues (for review, see ref 6). Studies of chronic
cholestatic diseases in humans using both enzyme histo-
chemistry and cytokeratin immunohistochemistry have
provided evidence for gradual transformation of hepato-
cytes into bile duct-type cells (7). However, evidence
showing that these bile duct-type cells also exhibit func-
tional characteristics of normal bile epithelium is still
lacking. It is therefore questionable, but still possible,
that ductular metaplasia of hepatocytes seen in choles-
tatic diseases may reflect a multipotential (or at least
bipotential) capacity of the hepatocytes.
In contrast to the hepatocyte system, there is strong
evidence indicating that the bile epithelium harbors a
compartment of cells that can produce progeny (oval
cells) capable of differentiating into several lineages in-
cluding bile epithelia, hepatocytes, intestinal epithelia,
and possibly exocrine pancreas (8-11). Also, as pointed
out by Sell (10), there may exist a periductal system of
stem cells capable of differentiating into all the hepatic
lineages. These ductular/periductal cells are frequently
referred to as the hepatic stem cell compartment. There
is also strong evidence that oval cells, the early progeny
from the hepatic stem cell compartment (8-11), are more
primitive and more poorly differentiated than are hepato-
cytes or bile epithelial cells, and the multiple differentia-
tion options of oval cells are firmly established.
Therefore, oval cells meet more of the stem cell criteria
of Potten and Loeffler (4) than do hepatocytes: 1) they
are poorly differentiated, 2) they demonstrate extensive
proliferation, and 3) they have multiple differentiation
options, including hepatocytes and bile ductular epi-
thelial cells. Whether oval cells possess the capacity to
proliferate and differentiate throughout the life span of an
animal is still uncertain, as appropriate studies have not
been done. Nevertheless, the immediate precursors of
oval cells appear to approximate Potten and Loefflers
definition of potential stem cell, whose functions of stem-
ness are latent but can be reactivated by appropriate con-
ditions. This definition of a potential stem cell
corresponds to the hypothesis of facultative liver stem
cell proposed earlier by Grisham (12), whose activation
he predicted would lead to the proliferation of oval cells.
In light of these observations, I should like to propose
that the liver be viewed as composed of two stem cell
systems: the unipotential committed stem cells (hepato-
cytes) and the multipotential nonparenchymal epithelial
(ductular) systems (Fig. 1). A comprehensive discussion
of the founding of hepatocytes and bile duct epithelial
cells from hepatoblasts has recently been published (5),
and will not be included in this review.
HEPATIC STEM CELL COMPARTMENT
The existence of cells with stem-like properties that may
differentiate into hepatocytes was first postulated nearly
HEPATIC STEM CELLS 1251
Figure 1. Schematic diagram showing the development of cell lineages in the liver.
60 years ago by Kinosita (13), and later suggested as a
possibility by other investigators (14-16). Wilson and
Leduc (16) postulated, based on experiments involving
liver regeneration in the mouse after chronic injury, that
indifferent cholangiole cells proliferated to form oval
cells that might differentiate to form both hepatocytes and
biliary epithelial cells.
The major support for the existence of hepatic stem
cells has, perhaps not surprisingly in light of the earlier
work by Wilson and Leduc (16), come from extensive
TABLE 1.Markers commonly used to assess differention and totracelineageofliverepithelial cell?
Markers Hepatoblasts Oval cells Hepatocytes Bileductcells References
CK7 - - - + 17,56,57
CK8
+ + + + 17,56
CK18
+ + + + 17,56
CK19
-1- + - + 17,56,58
CK14 [+] [+1 - - 17,59,60
1UJ3 + +1- + - 17,56,58
AFP
+ + - - 17,56,58
GGT
+ + - + 17,56
OV-6
(+) + . + 61
OV-1 (+) + - 4- 61
BDS7 + 4- - + 58,62
BD1 - - - +
63
BPC5
+ - - - 64
HES6 - - +
- 58,62
OC.2
4- + - 4 27,65,66
OC.3
+ + - 4- 27,65,66
H.1
- - 4 - 27,65,66
H.2
+(Transient) - - - 27,65,66
HBD.1 4-
- 4- 4 27,65,66
A6
AFro,,, Crishain
+1-
and Thorgeirsson (ref 5)Wi
+
th permission.
- + 67,68
1252 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON
Activationof Facultative
jj StemCells
Figure 2. Schematicrepresentation illustrating thehypothesis that oval
cells develop from facultative stem cells located in the canal of Hering
(biliary ductule). The scheme shows thedevelopmentaloptionsthatoval
cellspossess, including differentiation into hepatocytes and biliary epi-
thelium,formation ofintestinal epithelium, and pancreatic acinarepithe-
hum by differentiation and/or metaplasia, and death.
studies in hepatic carcinogenesis (9-11, 17). Several im-
portant experimental models, mostly in the rat, have
emerged from these studies. The most commonly used
models in the rats are produced by 1) treatment with azo-
dyes (18), 2) feeding of a choline-deficient diet, with or
without supplements of ethionine (19) or 2-acety-
laminofluorene (20), 3) treatment with 2-acetylaminofluo-
rene and partial hepatectomy (AAF/PH) (21), and 4)
treatment with D-galactosamine (22). Central to all these
experimen.tal models is the extensive destruction and/or
compromised function of hepatocytes, coupled with the
apparent inability of the residual hepatocytes to prolifer-
ate. A common cellular response in rats subjected to the
experimental systems listed above is the proliferation of
small periportal cells with scant cytoplasm and ovoid nu-
clei, which have been termed oval cells (17, 23, 24). The
existence of similar cells has been reported in the human
liver (25, 26). These oval cells are thought to represent a
progeny of the hepatic stem cell compartment, and at
least in some instances to be a precursor for the hepatic
tumors (10, 11, 27). The precise anatomical location of
the hepatic stem cell compartment in normal liver is still
unclear, but present data suggest that the terminal duc-
tule cells connecting the canals of Hering with the bile
canaliculi and/or a distinct population of periductal cells
constitute the hepatic stem cell compartment (vide infra;
9-11, 16, 28, 29). Recent studies in the rat liver have
shown that oval cells are capable of differentiating, in ad-
dition to bile epithelium, into at least two lineages in
vivo, including hepatocytes (24, 30) and intestinal type
epithelium (31). Furthermore, isolated oval cells in cul-
ture can be induced to differentiate into both hepatocyte-
like and biliary type of cells (32-34). Markers commonly
used to assess differentiation and to trace lineages of oval
cells are listed in Table 1. These data and other results
not reviewed here have supported the notion that oval
cells have lineage options similar to those displayed by
hepatoblasts in early stages of liver development. As
such, the oval cells can be regarded as bipotential pre-
cursors for the two hepatic parenchymal cell lineages.
However, oval cells may, particularly when the hepatic
microenvironment is drastically disrupted, exhibit a ca-
pacity to differentiate into nonhepatic lineages (Fig. 2)
(35).
LIVER REGENERATION VIA HEPATIC STEM
CELLS
The AAF/PH model in the rat will be used to illustrate
both the cellular biology and growth factor/receptor sys-
tems involved in stem cell-energized liver regeneration.
In this experimental system, a rapid and extensive prolif-
eration of oval cells takes place after PH, first in the
periportal area; later, these cells expand into the liver ac-
inus and differentiate into small basophilic hepatocytes
(Fig. 3) (24, 36). The powerful activation of the stem
cell compartment seen in the AAF/PH model is a conse-
quence of a close to complete mitoinhibitory effect of
AAF on the adult rat hepatocytes that prevents the regen-
eration from the remaining liver tissue (21, 24).
LOCALIZATION OF HEPATIC STEM CELLS
It has been established that proliferation of desmin-posi-
tive Ito cells is closely associated with the early stages of
oval cell proliferation in the AAF/PH model (37). Early
population of oval cells can be identified by the use of
the monoclonal antibody OV-6 (37) and distinguished
Figure 3. Time course of the development and expansion of the oval cell
population in the AAF/PH model. Oval cells are identified by y-GT
staining. The time of partial hepatectomy is day 0 (Od); 6d, 9d, and 13d
indicate number of days after the operation. Note the rapid expansion of
the oval cell population after 6 days and the formation by 13 daysofthe
basophihic focus composed of diphoid hepatocytes that are losing the y-GT
staining.
///ThNN
HGF
aFOF
TGFa
TGFI1
SCF
c-kit
Stem Transitional
Cells
HEPATIC STEM CELLS 1253
- 2 9 II 14y#{149}
Differentiated
Hepatocytes
Figure 4. Schematic diagram showing the temporal relationship between
expression of AFP, growth factors, and differentiation stages of the hepatic
stem cell progeny during liverregeneration intheAAF/PH model.
from proliferating desmin-positive Ito cells (38). Results
from a detailed time course study of activation of hepatic
stem cells in the AAF/PH model, utilizing a combination
of immunohistochemistry with OV-6 and desmin antibod-
ies and autoradiography after [3H]thymidine administra-
tion shortly after the PH, indicate that the earliest
population of proliferating OV-6 positive cells is located
in the small bile ductules (38). In addition, these early
populations of OV-6-positive cells express albumin and
a-fetoprotein (AFP) (36). These data clearly show that
the majority of thymidine-labeled, OV-6-positive cells
first observed after PH in the AAF/PH model reside in
the bile ductules. Moreover, at the time when few of the
OV-6-positive cells in the large bile ducts become la-
beled with thymidine, the ductular-derived OV-6-positive
and thymidine labeled oval cells expressing both albu-
min and AFP have already started to infiltrate into the
liver acinus (36, 38). It therefore seems likely that the
major source of oval cells, at least in the AAF/PH model,
is derived from the lining cells of the biliary ductules and
that these cells constitute the dormant/facultative hepatic
stem cell compartment.
GROWTH FACTORS INVOLVED IN HEPATIC
STEM CELL ACTIVATION AND EXPANSION
During normal hepatic regeneration as well as during re-
newal from the stem cell compartment, several growth
factors appear to affect the proliferation and differentia-
tion of hepatic cells (37, 39, 40). The question therefore
arises as to whether the same growth factors known to be
involved in normal hepatic regeneration are also involved
in regeneration from the stem cell compartment.
There are three primary growth factors associated
with normal liver regeneration: transforming growth fac-
tor-alpha (TGF-a), hepatocyte growth factor (HGF), and
acidic fibroblast growth factor (aFGF) (41). Each of these
growth factors is also capable of inducing replication of
primary hepatocytes in vitro (41). In addition, transform-
ing growth factor-beta 1 (TGF-1) is also expressed dur-
ing hepatic regeneration, and it has been proposed that
TGF-1 may provide at least part of the negative growth
signals controlling liver size after the compensatory hy-
perplasia that occurs after loss of liver mass (42). The
first cells entering DNA synthesis after PH in the
AAF/PH model are the OV-6 and desmin-positive bile
duetular and Ito cells, respectively, in the periportal area
(38). Coincident with the appearance of these cells, an
increase in the expression of TGF-a, HGF, and TGF-1
is observed, whereas increased expression of aFGF is
first seen 24 h later (29). All the growth factors are then
expressed at high levels throughout the period of expan-
sion and differentiation of the oval cells and return to lev-
els seen in normal liver at the end of the regeneration
process. The cellular distribution of the growth factor
transcripts differs: TGF-a and aFGF transcripts are
found both in Ito cells and oval cells (37, 40), whereas
the HGF transcripts are only found in Ito cells (39). The
TGF-1 transcripts are located mainly in Ito cells, but
the early population of oval cells also contain the TGF-f31
transcripts (43). The cellular distribution of the tran-
scripts for all the receptors corresponding to the growth
factors has revealed that all are located on oval cells (22,
44, 45). These data suggest that the same primary growth
factors involved in liver regeneration from existing differ-
entiated parenchyma are also involved in regeneration
from the stem cell compartment.
Recently, a novel liganci/receptor system, the stem cell
factor (SCF)/c-kit system, which may be uniquely in-
volved in the earliest stages of hepatic stem cell activa-
tion, was discovered (46). In the AAF/PH model, the
expression of both SCF and c-kit is seen before the ex-
pression of AFP (46), and the levels of both the SCF and
the c-kit transcripts decline before those of TGF-a,
aFGF, HGF, and TGF-31 (46). It has also been shown
that in contrast to TGF-a, HGF, aFGF, and TGF-f31, the
SCF/c-kit system is only slightly and transiently activated
in regeneration after PH in normal liver (46). The SCF/c-
kit signal transduction system is believed to play a funda-
mental role in the survival, proliferation, and migration of
stem cells in hematopoiesis, melanogenesis, and gameto-
genesis (47). It appears that in all cases, SCF and c-kit
are involved in the early stages of stem cell activation. In
the hemopoietic stem cell system, it has also been dem-
onstrated that SCF in combination with selective multipo-
tential colony-stimulating factors can influence the
relative frequency of progenitor cells committed to vari-
ous lineages (48). Whether the SCF/c-kit system in the
early hepatic stem cell population interacts with other he-
patic growth factors so as to influence the frequency of
lineage commitment of progenitor cells is not known at
present. However, the hepatic expression pattern and cel-
lular location of the SCF/c-kit system indicate that this
1254 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON
signal transduction system is required only during the
early activation and transitional phase of the oval cell dif-
ferentiation. Once the oval cells have differentiated into
the small basophilic hepatocytes, the expression of both
SCF and c-kit is abolished (49). This concept is sche-
matically illustrated in Fig. 4.
PARTICIPATION OF LIVER STEM CELLS IN
REGENERATION
After the hepatocyte population is reduced by, for exam-
ple, PH, the residual hepatocytes proliferate promptly,
continue to cycle until the deficit is repaired, and con-
tinue to function while proliferating. Under these condi-
tions, no apparent contribution to the regeneration
process is provided by the stem cell compartment. Acti-
vation of oval cell proliferation and differentiation by in-
jury, which is more severe and/or qualitatively different
from the simple loss that triggers only hepatocyte prolif-
eration, results in transient reestablishment of a hepato-
cytic lineage that has all the characteristics of a potential
or facultative stem cell system (5). Cells in the normally
quiescent stem cell compartment are activated to produce
poorly differentiated oval cell progeny. Oval cells prolif-
erate extensively to yield a large population of cells that
migrate throughout the parenchyma, some of which differ-
entiate as they migrate. Hepatocytic progeny of oval cells
merge into the functional compartment of mature hepato-
cytes and help restore the parenchyma. Similar to the
generation of new hepatocytes after simple loss, the pro-
duction of hepatocytes via the stem cell (oval cell)
mechanism is also episodic and transient. These two dis-
tinct mechanisms of hepatocyte formation are both sub-
jected to several points of stringent control (5). Controls
are required to regulate the reinitiation of hepatocyte for-
mation from the normally quiescent hepatocytes, as well
as to regulate the activation of potential stem cells that
energizes cell flow through the entire lineage. Although
the controls may differ between the two mechanisms of
hepatocyte formation, it is probable that both pathways
are simultaneously activated after loss of liver mass, in-
cluding that after simple PH.
One of the earliest phenotypic indications of liver stem
cell activation is the expression of AFP (5). A transient
expression of AFP is also seen after simple PH and simi-
lar to that seen in stem cell activation in the AAF/PH
model, the AFP transcripts are located in the bile ductu-
les (36, 50). Expression of both SCF and c-kit is also
transiently elevated after standard PH (46). However,
there is no evidence indicating that the stem cell-derived
hepatocytes significantly contribute to regeneration of
liver mass after simple PH (5). These observations sug-
gest that the activation, and in particular, the expansion
of liver stem cells, are stringently controlled during hepa-
tocyte-driven liver regeneration.
Recent studies of the early activation of oval cell pro-
liferation in the AAF/PH model have provided important
clues toward defining the mechanism controlling the early
expansion of oval cells (51, 52). It has been shown that a
low dose of AAF (and its analogs 2-AF and N-OH-2-
AAF) elicited, in the absence of PH, a mitogenic re-
sponse in ductular and periductular cells within 24 h
after administration. The compounds also induced the ex-
pression of HNF1, HNF3y, AFP, and albumin in the
ductular cells, similar to the observations shown earlier
in the complete AAF/PH model (53). In contrast, initia-
tion of bile duct proliferation by ligation of the common
bile duct had no effects on the expression of these genes
in ductal cells. In addition to eliciting a mitogenic re-
sponse, administration of AAF also induced apoptosis of
cells in the portal areas, a process that contributed to the
overall retention of liver morphology. As no significant
increase in the cellularity of the portal areas was ob-
served at this time, it is probable that an equilibrium be-
tween mitosis and apoptosis exists to maintain a constant
number of ductal cells. This observation is in striking
contrast to the condition seen in the complete AAF/PH
Figure 5. Schematic depiction of liver regeneration illustrating the
contribution of hepatic stem cells. Induction of reparative renewal of the
epithelial cell populations in the liver after both simple PH and certain
types of more severe liver injury involves activation of both the normally
quiescent hepatocytes and the potential (facultative) liver stem cells. As
discussed inthe text, the essential requirement for the sustained activa-
tion of the stem cells and their progeny required for generating the
differentiated cell lineages needed for liver regeneration is a prolonged
expression of a set of growth factors, including those known to be involved
in liver regeneration after simple partial hepatectomy.
HEPATIC STEM CELLS
1255
model, in which the oval cells continuously proliferate
and expand into the liver acinus. Furthermore, admini-
stration of AAF alone results in only a modest and tran-
sient increase in expression of hepatotrophic growth
factors, again in contrast to the intense and sustained ex-
pression of growth factors seen in the AAF/PH model
(vide supra).
The importance of some of the growth factors that are
highly expressed during the activation and expansion of
oval cells in the AAF/PH model is beginning to emerge.
It has now been demonstrated that infusion of epidermal
growth factor (EGF) and HGF, either alone or in combi-
nation, can partially substitute for the PH in the AAF/PH
model (52). The results from this study demonstrate that
although both EGF and HGF increase the number of pro-
liferating cells after AAF administration, they preferen-
tially act on different cell populations. Although
administration of AAF alone or in combination with infu-
sion of HGF resulted in proliferation of almost equal
numbers of ductal and Ito cells, infusion of EGF and/or
combination of EGF and HGF resulted in 75-80% of the
proliferating cells having a ductal phenotype. Also, infu-
sion of EGF and HGF resulted in a decreased number of
cells undergoing apoptosis in response to AAF. These re-
sults have important implications for our understanding of
both hepatic stem cell activation and expansion as well
as for the mechanism of liver regeneration. Based on the
data discussed above, the following model of liver regen-
eration can now be proposed (Fig. 5). Induction of re-
parative renewal of the epithelial cell populations in the
liver after simple PH and certain types of more severe
liver injury involves activation of both the normally qui-
escent hepatocytes and the potential (facultative) liver
stem cells. In a healthy liver, the reparative renewal of
the hepatocyte and biliary epithelial cell populations is
accomplished in most instances by proliferation of resid-
ual differentiated cells of each types, resulting in only a
transient activation of the stem cells. However, under
conditions in which the hepatocytes are unable to re-
spond to the regenerative stimuli and/or are functionally
compromised, a sustained activation of the stem cells and
their progeny ensues, generating the differentiated cell
lineages needed for the liver regeneration. An essential
requirement for the stem cell-driven liver regeneration is
a sustained expression of a set of growth factors, includ-
ing those known to be involved in liver regeneration after
simple PH.
Several implications emerge from this integrated model
of liver regeneration. Perhaps the most important predic-
tion arising from the model suggests that in chronic liver
diseases such as viral hepatitis, a condition in which the
hepatocytes are compromised and a continuous regen-
eration exists, the stem cells would be activated and
might contribute to the regenerative process. The obser-
vations that oval-type cells occur in livers harboring
hepatitis B virus-associated liver tumors (26) and that
these cells can be infected by the hepatitis B virus at
early stage of differentiationhave significantimplications
for human hepatocarcinogenesis (54, 55). Also, the possi-
bility of isolating liver stem cells from which hepatic
lineages can be generated in vitro holds promise for fu-
ture approaches to gene therapy of liver-related diseases.
REFERENCES
1. Van Thiel, D. H.,Gavaler, J.S.,Kam, L, Francavilla, A.. Polimeno, L,
Schade, P.R., Smith, J., Diven, W., Penkmt, R. J., and Staral, T. E. (1989)
Hapid growth of an intact human liver transplanted into a recipient larger
than the donor. Castroenterology 93, 1414-1419
2. Lajtha, L. C.(1970)Stemcell concepts. Nouv. Rev. Fr. Hematol. 21,59-65
3. Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., and Brinster, R.
L (1994) Replacement of diseased mouse liver by hepatic cell transplants.
tion. Science 263, 1149-1152
4. Potten, C. S., and Loeffler, M. (1990) Stemcells: attributes, cycles, spirals,
pitfalls and uncertainties. Development 110, 1001-1020
5. Grisham, J. W., and Thorgeirsson, S.S. (1996) Liver stem cells. In The Stem
Cell Handbook (Potten, C. S., ed) Academic Press, New York In press
6. Van Eyken, P., and Desmet, V. J. (1992) Development of intrahepatic bile
ducts, ductular metaplasia of hepatocytes, andcytokeratin patterns in various
types of human hepatic neoplasms. InThe Role of Cell Types in Hepatocar-
cinogenesis (Sirica, A. E., ed) pp. 227-263, CRC Press, Boca Haton, Florida
7. Van Eyken, P., Sciot, R., Callea, F., and Desmet, V. J.(1989) A cytokeratin-
immunohistochemical study of focal nodular hyperplasia ofthe liver further
evidence that ductular metaplasia of hepatocytes contributes to ductular
proliferation. Liver 9,372-377
8. Thorgeirsson, S. S. (1993) Commentary. Hepatic stem cells. Am. I. Pathol.
142, 1331-1333
9. Fausto, N. (1990) Oval cells and liver carcinogenesis: an analysis of cell
lineages in hepatic tumors using oncogene transfection techniques. Prog.
Clin. Biol. Ret. 331,325-334
10. Sell, S. (1990) Is there a liver stem cell? Cancer Ret. 50,3811-3815
11. Sigal, S. H., Brill, S., and Reid, L M. (1992) The liver as a stem cell and
lineage system. Am. J. Physiol. 263, C 139-C 148
12. Crisham,J. W. (1980) Cell types in long-term propagable cultures of rat liver.
Ann. N.Y. Aced. Sci. 349, 128-137
13. Kinosita, R. (1937) Studies on the carcinogenic chemical substances. Trans.
Soc. Pathol. Jpn. 27,665-727
14. Price, J.M.,Harman, J. W., Miller, E. C., and Miller,J. A. (1952) Progressive
microscopic alterations in the livers of rats fed the hepatic carcinogens
3-methyl4-dimethylaminoazobenzene and 4-fluoro-4-dimethylaminoazo-
benzene. Cancer Ret. 12,192-200
15. Farber, E. (1956) Similarities in the sequence of early histological changes
induced in the liver of the rat by ethionine, 2-acetylaminofluorene. and
3-methyl4-dimethylaminoazobenzene. Cancer Re,. 16,142-148
16. Wilson, J.W.,and Leduc, E. H. (1958) Roleofcholangioles in restoration
of the liver of the mouse after dietary injuly. J. Pathol. &icterioL 76,
441-449
17. Marceau, N. (1990) Biology ofdisease. Celllineage and differentiation
programs in epidermal; urothelial and hepatic tissues and their neoplasms.
Lab. Invest. 63,4-20
18. Inaoka, Y. (1967) Significance of the so-called oval cellproliferation during
azo-dye hepatocarcinogenesis. Cairn 58,355-366
19. Shinozuka, H.,Lombardi, B., Sell, S., and lammarino, R. M. (1978)Early
histological and functional alterations of ethionine liver carcinogenesis in
rats feds choline-deficient diet. Cancer Ret. 38, 1092-1098
20. Sell, S., and Salman, J. (1984) Light- and electron-microscopic autoradiog-
raphic analysis of proliferating cells during the early stages of chemical
hepatocaveinogenesis in the rat induced by feeding N-2-fluorenyl-acetamide
in a choline-deficient diet. Am. J. Pathol. 114, 287-300
21. Tatematsu, M., Ho, R. H., Kaku, T., Ekem, J. K., and Farber, E. (1984)
Studies on the proliferation and fate of oval cells in the liver of rats treated
with 2-acetylaminofluorene and partial hepatectomy. Am. I. Pathol. 114,
418-430
22. Lesch, R., Reutter, W., Keppler, D., and Decker, K. (1970) Liver restitution
after acute galactosamine hepatitis: autoradiographic and biochemical stud-
ies in rats. Exp. Mol. Pathol. 1.2,58-69
23. Farber, E. (1984) Cellular biochemistry of the stepwise development of
cancer with chemicals. Cancer Re,. 44, 5463-5474
24. Evarts, H. P., Nagy, P., Marsden, E., and Thorgeirsson, 5. 5. (1987) A
precursor-product relationship exists between oval cells and hepatocytes in
rat liver. Carcinogenesit 8, 1737-1740
25. Gerber, M. A., Thung, S. N., Shen, S., Stmmeyer, F. W., and Ishak, K. C.
(1983) Phenotypic characterization of hepatic proliferation: antigenic ex-
pression of proliferating epithelial cells in fetal liver, massive hepatic ne-
crosis, and nodular transformation of the liver. Am. J. Pathol. 110,70-74
26. Hsia, C. C., Evarts, H. P., Nakatsukasa, N., Maraden, E. H., and Thorgeira-
son, S.S. (1992) Occurrence of oval-type cells in hepatitis B virus-associated
human hepatocarcinogenesis. Hepatology 16,1327-1333
1256 Vol. 10 September 1996 The FASEBjournal THORGEIRSSON
27. Hixson, D.C., Fans, R. A., and Thompson, N. L. (1990) An antigenic portrait
of the liver during carcinogenesis. Pashobiology 58,65-77
28. Factor, V. M., Radaeva, S. A., andThorgeirsson, S.S. (1994) Origin and fate
of oval cells in flipin-induced hepatocarcinogenesis in the mouse. Am. J.
Pathol. 145,409-422
29. Thorgeirsson, S. S., Evarts, R. P., Fujio, K., and Hu, Z. (1994) Cellular
biology oftherat hepatic stem cell compartment. In Liver Carcinogenesis
(Skouteris, C.C.,ed)pp. 129-145, NATO ASI Series, Vol. H88, Springer-
Verlag, Berlin/Heidelberg
30. Lemire, i. M., Shiojin, N., and Fausto, N. (1991) Oval cell proliferation and
the origin of small hepatocytes in liver injury induced by D-galactosamine.
Am. I. Pathol. 139,535-552
31. Tatematsu, M., Kaku, T., Medline, A., and Farber, E. (1985) Intestinal
metaplasia as a common option of oval cells in relation to cholangiofibrosis
in the livers of rats exposed to 2-acetylaminofluorene. Lab. Invest. 52,
354-362
32. Hayner, N. T., Braun, L, Yaswen, P., Brooks, M., and Fausto, N. (1984)
Isozyme profiles of oval cells, parenchymal cells, and biliaiy cells isolated
by centrifugal eluination from normal and preneoplastic livers. Cancer Ret.
44,332-338
33. Germain, L., Coyette, R., and Marceau, N. (1985) Differential cytokeratin
and x-fetoprotein expression in morphologically distinct epithelial cells
emerging at the early stages of rat hepatocarcinogenesis. Cancer Ret. 45,
673-681
34. Cermain, L., Noel, H., Gourdeau, H., and Marceau, N. (1988) Promotion of
growth and differentiation of rat ductular oval cells in primary culture. Cancer
Ret. 48,368-378
35. Thorgeirason, S. S., and Evarts, H. P. (1992) Growth and differentiation of
stem cells in adult liver. In The Role of Cell Types of Hepatocarcinogenesis
(Sirica, A. E., ed) pp. 100-120, CRC Press, Roes Raton, Florida
36. Golding, M..Sarraf, C. E., Lalani, E. N., Anilkumar, T. V., Edwards, R. J.,
Nagy, P., Thorgeirsson, S. S., and Alison, M.R. (1995) Oval cell differentia-
tion into hepatocytes in the acetylamino-fluorene-treated regenerating rat
liver. Hepatology 22, 1243-1253
37. Evarts, R. P., Nakatsukasa, I-I., Maraden, E. H., Hu, Z., and Thorgeirsson, S.
S. (1992) Expression of transforming growth factor-a in regenerating liver
andduring hepatic differentiation. MoE. Carcinogen. 5,25-31
38. Evarts, R. P., Hu,Z., Fujio, K., Marsden, E. R.,andThorgeirsson,S. S. (1993)
Activation of hepatic stem cell compartment in the rat: Role of transforming
growth factor-U, hepatocyte growth factor, and acidic fibroblast growth factor
in early proliferation. Cell Growth & D(ffer. 4,555-561
39. Hu,Z.,Evarts, H.P., Fujio, K., Marsden, E. R., and Thorgeirsson, S. S. (1993)
Expression of hepatocyte growth factor and c-met genes during hepatic
differentiation and liver development in the rat. Am. J. Pathol. 142,
1823-1830
40. Marsden, E. H.. Hu, Z., Fujio, K., Nakatsukasa, H., Thorgeirsson, S. S., and
Evarts, R. P. (1992) Expression of acidic fibroblast growth factor in regen-
erating liver and during hepatic differentiation. Lab. Invest. 67,427-433
41. Michalopoulos, C. (1990) Liver regeneration: molecular mechanisms of
growth control. FASEBJ. 4,176-187
42. Mead, J. E., and Fausto, N. (1989) Transforming growth factor a may be a
physiological regulator of liver regeneration by means of autocrine mecha-
nisms. Proc. Nat!. Acad. Sci. USA 86, 1558-1562
43. Evarts, R. P., Nakatsukasa, H., Maraden, E. R., Hsia, C. C., Dunsford, H.
A., and Thorgeirsson, S. S. (1990) Cellular and molecular changes in the
early stages of chemical hepatocarcinogenesis in the rat. Cancer Ret. 50,
3439-3444
44. Lenzi, R., Liu, M. H., Tarsetti, F., Slott, P. A., Alpini, C., Xhai, W. R.,
Paronetto, F., Lenzen, R., and Tavolim, N. (1992) Histogenesis of bile
duct-like cells proliferating during ethionine hepatocarcinogenesis. Lab.
Invest. 66,390-402
45. Hu, Z., Evarts, R. P., Fujio, K., Omon, N., Omon, M., Marsden, E. H., and
Thorgeirason, S.S. (1996) Expression of transforming growth factor alpha/epi-
dermal growth factor receptor, hepatocyte growth factor/c-met and acidic
fibroblast growth factor/fibroblast growth factor receptors during hepatocar-
cinogenesis. Carcinogenesi.c 17,931-938
46. Fujio, K., Evarts, H. P., Hu,Z., Marsden, E. R.,andThorgeirsson,S. 5. (1994)
Expression of stem cell factor and its receptor, c-kit, during liver regeneration
from putative stem cells in adult rat. Lab. Invest. 70,511-516
47. Morrison-Graham, K., and Takahashi, Y. (1993) Steel factor and c-kit
receptor from mutants to a growth factor system. BioEssays 15,77-84
48. Metcalf, D. (1991) Lineage commitment of hemopoietic progenitor cells in
developing blast cell colonies: influence of colony-stimulating factors. Proc.
Nat!. Aced. Scj. USA 88, 11310-1 1314
49. Fujio, K.,Hu,Z.,Evans, R.P., Marsden, E. H.,Niu,C.-H.,andThorgeirason,
S.S. (1996) Coexpression of stem cell factor and c-kit in embryonic and adult
liver. Exp. Cell Ret. 224,243-250
50. Lemire, J. M., and Fausto, N. (1991) Multiple alpha-fetoprotein RNAs in
adult rat liver cell type-specific expression and differential regulation.
Cancer Ret. 51,4656-4664
51. Bisgaard, H. C., Nsgy P., Santoni-Rugiu E., and Thorgeirason, S. S. (1996)
Proliferation, apoptosis, and induction of hepatic transcription factors are
characteristic of the early response of biliary epithelial (oval) cells to
chemical carcinogens. Hepatology 23,62-70
52. Nagy, P., Bisgaard, H. C., Santoni-Rugiu, E., andThorgeirsson, S. S. (1996)
In vivo infusion of growth factors enhances the mitogenic response of rat
hepatic ductal (oval) cells following administration of 2-acetylaminofluorene.
Hepatology 23,71-79
53. Nagy, P., Bisgaard, H. C., and Thorgeirsson, S. 5. (1994) Expression of
hepatic liven transcription factors during liver development and oval cell
differentiation. J. Cell Biol. 126,223-233
54. Hsia, C.-C., Thorgeirsson, S. S., andlabor, E. (1994) Expression of hepatitis
B surface and core antigens and transforming growth factor-a in oval cells
of the liver in patients with hepatocellular carcinoma. Med. ViroL 43,
216-221
55. Thorgeirsson, 5. 5. (1995) Target cell populations in virally associated
hepatocarcinogenesis. Proceedings of the 25th Intl. Symposium of the Prin-
cess Takamatsu Cancer Research Fund on Hepatitis C Virus and its
Involvement in the Development of Carcinoma, pp. 163-170, Princeton
Scientific Publishing Co., Princeton, New Jersey
56. Shiojiri, N., Lemire, J. M., and Fauston, N. (1991) Cell lineages and oval cell
progenitors in rat liver development. Cancer Re,. 51,2611-2620
57. Van Eyken, P., aiid Desmet, V. J. (1993) Cytokeratins and the liver. Liver
13,113-122
58. Cermain, L., Blouin, M.-J., and Marceau, N. (1988) Biliazy epithelial and
hepatocytic celllineage relationships in embryonic rat liver as determined
by the differential expression of cytokeratins, aipha-fetoprotein, albumin, and
cell surface-exposed components. Cancer Ret. 48,4909-4918
59. Bisgaard, H. C., Nagy, P., Ton, P.1., Hu, Z., and Thorgeirsson, S.S. (1994)
Modulation of keratin 14 and a-fetopmtein expression during hepatic oval
cell proliferation and liver regeneration. J. Cell. Physiol. 159,475-484
60. Bisgaard, H. C., Ton, P. T.,Nagy, P., and Thorgeirsson. S. S. (1994)
Phenotypic modulation ofkeratins, vimentin, and a-fetoprotein in cultured
rat liver epithelial cells after chemical, oncogene, and spontaneous transfor-
mation. J. Cell. Physiol. 159,485-494
61. Dunsford, H. A., and Sell, S. (1989) Production of monoclonal antibodies to
preneoplastic liver cell populations induced by chemical carcinogens in rats
and transplantable Morris hepatomas. Cancer Ret. 49,4887-4893
62. Marceau, N., Germain, L., Coyette, R., Noel, M., and Gourdeau, H. (1986)
Cell of origin of distinct cultured rat liver epithelial cells, as typed by
cytokeratin and surface component selective expression. Biochem. Cell. Rio!.
64, 788-802
63. Yang, L., Fans, H. A., and Hixson, D. C. (1993) Phenotypic heterogeneity
within clonogenic ductal cell populations isolated from normal adult rat liver.
Proc. Soc. Exp. Biol. Med. 204,280-288
64. Marceau, N., Blouin, M.-J., Noel, M., T#{246}rok, N., and Loranger, A. (1992) The
role of bipotential progenitor cells in liver ontogenesis and neoplasia. In The
Role of Cell Types in Repatocarcinogenesis (Sirica, A. E., ed) pp. 121-149,
CRC Press, Boca Raton, Florida
65. Hixson, D. C., and Allison, J. P. (1985) Monoclonal antibodies recognizing
oval cells induced in the liver of rats by N-2-fluorenylacetamide or ethiomne
in a choline-deficient diet. Cancer Ret. 45,3750-3760
66. Fans, H. A., Monfils, B. A., Dunsford, H. A., and Hixson, D. (1991) Antigenic
relationship between oval cells and a subpopulation of hepatic foci, nodules,
and carcinomas induced by the resistant hepatocyte model system. Cancer
Ret. 51, 1308-1317
67. Engelhardt, N. V., Factor, V. M., Yasova, A. K., Poltoranina, V. S., Bazanov,
V. N., and Lasareva, M. N. (1990) Common antigens of mouse oval and biliaiy
epithelial cells. Expression on newly formed hepatocytes. Differentiation 45,
29-37
68. Engelhardt, N. V., Factor, V. M., Medvinsky, A. L, Baranov, V. N., Lazareva,
M. N.,and Poltoranina, V. S. (1993) Common antigen ofovaland biliary
epithelial cells (A6) is a differentiation marker of epithelial and erythroid
cell lineages in early development of the mouse. Differentiation 55, 19-26