留学生生物医学工程论文发表，SCIENCE FRONTIER
Liver Regeneration and Repair: Hepatocytes,Progenitor Cells, and Stem Cells
Nelson Fausto1
Studies of liver regenerative processes have gainednew prominence, generated from analyses of geneticallyengineered animal models, the transplantationof human livers, and the surging interest in stem cells.
These studies gave rise to expectations regarding the practicalapplications of research on the mechanisms of liverregeneration that were unthinkable just a few years ago.As the field expanded to a broader audience, this new
knowledge also brought confusion and often misinterpretationsregarding the cellular mechanisms responsible forliver regeneration in different types of hepatic growthprocesses. This article is a brief review of the role of
hepatocytes, oval cells (see box for nomenclature), andbone marrow cells in liver regeneration and repopulation.
This topic has generated great excitement, a gooddeal of noise, much controversy, and many surprises.
The review of the rapidly expanding literature presentedhere, particularly as it deals with stem cells, wasguided by a few principles: (1) be wary of dogma1 andof overinterpretation of data; (2) a “new discovery”may be a true new finding, but it may simply be therepackaging of an old discovery; and (3) an excitingstory is not necessarily a true story.What is the Source of Hepatocytes in Liver
Growth Processes?
This question has multiple answers, because the sourceof hepatocytes depends on the nature of the growth process.In every case, it is necessary to ascertain whetherhepatocytes responsible for liver regeneration originated
from the replication of existing hepatocytes, were generatedby differentiation of oval cells, or were producedfrom bone marrow cells.2 Replication of mature hepatocytesin liver regeneration has been documented extensively.
3–5 Differentiation of oval cells has also beenestablished as a mechanism that can generate significantnumbers of hepatocytes (Table 1).6–11 However, the contributionof bone marrow cells to the generation of hepatocytesin liver repopulation and regeneration remainsuncertain, both regarding its extent and the mechanisms
involved.
As a general rule, replication of existing hepatocytes isthe quickest and most efficient way to generate hepatocytes
for liver regeneration and repair. Oval cells usuallyreplicate and differentiate into hepatocytes only when thereplication of mature hepatocytes is delayed or entirelyblocked (Fig. 1). Bone marrow cells can generate hepatocytesin transplanted livers but so far, the frequency ofhepatocytes produced by this route is very low, and suchcells are not always detectable. Note however, that in
transplanted livers, bone marrow cells are an importantsource of nonparenchymal cells such as Kupffer cells andendothelial cells (discussed later).
The Proliferative Capacity of Hepatocytes
Because liver regeneration after 70% hepatectomy requires
no more than two rounds of hepatocyte replication,
it was generally assumed that the proliferative
capacity of mature hepatocytes is very limited. This view
has now been drastically changed. First, experiments with
cultured hepatocytes isolated from transgenic mice that
expressed liver growth factors demonstrated that longterm
hepatocyte replication is compatible with a differentiated
phenotype.12 More striking were the results of
hepatocyte transplantation experiments in urokinaseplasminogen
activator transgenic mice, showing that liver
repopulation by transplanted hepatocytes involved at
least 12 rounds of replication.13 Subsequent serial transplantation
experiments performed in fumarylacetoacetate
hydrolase (FAH)-deficient mice (FAH knockout mice)
demonstrated that hepatocytes could replicate 70 or more
times.14 In the serial transplantation experiments, there
was no evidence that the repopulation capacity was dependent
on stem cells. This conclusion also is supported
by data demonstrating that diploid, tetraploid, and octoploid
hepatocytes have roughly the same capacity to repopulate
damaged livers.15 Thus, although hepatocytes
are quiescent in normal livers and replicate in a limited
Abbreviations: FAH, fumarylacetoacetate hydrolase; AFP, -fetoprotein; HCC,
hepatocellular carcinoma; HSC, hematopoietic stem cell; MAPC, multipotent adult
progenitor cell.
From the 1Department of Pathology, University of Washington School of Medicine,
Seattle, WA.
Received December 16, 2003; accepted March 1, 2004
Address reprint requests to: Nelson Fausto, M.D., Department of Pathology,
University of Washington School of Medicine, Box 357470, 1959 NE Pacific
Street, Seattle, WA 98195-7705. E-mail: [email protected].
Copyright © 2004 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.20214
1477
and regulated manner during liver regeneration after partial
hepatectomy, these cells have an enormous proliferative
potential that can be unleashed under certain
conditions. Nevertheless, there is evidence that the replicative
activity of hepatocytes diminishes in advanced cirrhosis
in humans and in chronic liver injury in mice,
reaching a state of “replicative senescence,” perhaps as a
consequence of telomere shortening.16–19
The Origin of Intrahepatic Bile Ducts in
Liver Development and of Oval Cells in the
Adult Liver
Both hepatocytes and intrahepatic bile ducts originate
from endodermal-derived hepatoblasts (Fig. 1) that express
albumin and -fetoprotein (AFP).20,21 At day 14 (mice) or
15 (rats) of embryonic development, hepatoblasts located
near vascular spaces, the site for portal spaces in later development,
express dual markers of the hepatocyte (albumin
and AFP) and biliary (cytokeratins 7 and 19) lineages.22
These hepatoblasts give rise to the primitive intrahepatic bile
ducts, structures that connect parenchymal hepatocytes with
the larger segments of the biliary system. Primitive intrahepatic
bile ducts correspond to the canals of Hering and terminal
bile ductules of adult livers which may constitute the
niche for intrahepatic stem cells.23–25 The embryological origin
of intrahepatic bile ducts explains some important features
of oval cell proliferation in adult livers.
● In adult rat liver, cells of the canals of Hering and
terminal bile ductules may express AFP.26,27 Oval cells
thought to be generated from them may express AFP and
may contain isozymes of aldolase, pyruvate kinase, and
lactic dehydrogenase present in both adult and fetal liver
cells, and glucose-6-phosphatase, a typical hepatocyte
marker.28–34 However, the extent to which these markers
are expressed in a population of proliferating oval cells
depends on the agent that elicited oval cell proliferation.
● Analysis of marker expression suggests that populations
of proliferating oval cells constitute a heterogeneous
cell compartment (or oval cell compartment) containing
cells that may differ in their differentiation capacity and
stage of differentiation. Some of these cells may function
as hepatocyte progenitors, whereas others may be indistinguishable
from cholangiocytes, cells that do not express
AFP, or hepatocyte markers. Oval cells and cholangiocytes
share epitopes that react with, among others cytokeratins
7, 8, 18, and 19, the antibodies OV6 (an anticytokeratin
19 antibody), OC2 (anti-myeloperoxidase),
and some other members of the OC series, glutamyl
transferase, and the antigens A6 and G7.29,30,35–37
Relationships Between Oval Cells and
Hematopoietic Stem Cells
In addition to expressing markers of the hepatocyte
and bile duct lineage, oval cells express markers of hematopoietic
stem cells. Among these are Thy-1, CD34,
CD45, Sca-1, c-Kit, and flt-3,38 all of which can also be
detected in fetal livers. Stemlike cells positive for CD34
and c-Kit have been isolated from normal and cirrhotic
human livers.39,40 The normal adult murine liver contains
hematopoietic cells that are phenotypically similar to cells
present in the bone marrow,41 including cells of the bone
marrow side population.42 The side population cells are
hematopoietic stem cells purified on the basis of their
efflux capacity on staining with the dye Hoechst 33342.
One of the markers for the side population is the adeno-
Table 1. Origin of Hepatocytes in Liver Regeneration and
Repair
Growth processes that depend of the replication of differentiated hepatocytes
Liver regeneration after partial hepatectomy2
Hepatocyte regeneration after carbon tetrachloride and acetaminophen
(centrolobular) injury131
Conditions in which oval cells proliferate and generate hepatocytes
Experimental
Injury caused by galactosamine132
Choline-deficient diet combined with ethionine or AAF133,134
Partial hepatectomy combined with AAF or Dipin135,136
Carbon tetrachloride combined with AAF137
3,5-dietoxycarbonyl-1-1, 4-dihydrocollidine (DCC)138
Allyl alcohol9
Human disease
Atypical ductular reactions in advanced stages of cirrhosis of various
etiologies
Fatty liver disease
Small cell dysplasias
Massive hepatocyte necrosis17,52,68,139
Conditions in which small hepatocyte precursor cells (SHPC) represent a large
fraction of the proliferating cells
Injury caused by retrorsine61,62 and galactosamine63
Abbreviation: AAF, N-2-acetylaminofluorene.
NOTE. Only representative publications are listed.
Fig. 1. Cell lineages in the liver. During embryonic development,
hepatoblasts give rise to the two epithelial lineages of the liver, producing
hepatocytes and cholangiocytes. Oval cells originate in association with
the intrahepatic biliary system, formed by hepatoblasts located near
portal spaces. In adult livers, hepatocytes and cholangiocytes can replicate.
Oval cells form a bipotential reserve compartment capable of
generating hepatocytes whenever hepatocyte replication is blocked (red
lines).
1478 FAUSTO HEPATOLOGY, June 2004
sine triphosphate-binding cassette transporter ABCG2/
BCRP1, which also has been detected during hepatic oval
cell proliferation.43 On the basis of the existing data, the
expression of hematopoietic markers in the normal adult
liver and after hepatic injury leading to oval cell proliferation
may be interpreted in at least two different ways:
● A very small number of hematopoietic stem cells
present in fetal livers may remain in adult livers. These
cells may be distinct from oval cells, but are induced to
proliferate by the same conditions that cause oval cell
proliferation. In this case, hematopoietic stem cells would
be a component of the oval cell compartment but constitute
a distinct population, which does not acquire markers
of the hepatocyte lineage but shares general stem cell
markers with oval cells originating from the canals of Hering.
● Hematopoietic cells located in the adult liver may be
pluripotent stem cells present in the hepatic tissue, functioning
as the equivalent of embryonic stem cells capable
of generating multiple lineages, including hepatocytes. If
this view is correct, hematopoietic stem cells located in the
liver (perhaps in periductular spaces) would differentiate
progressively,9 first into oval cells and ultimately into
hepatocytes, under stimuli known to cause an oval cell
response.
The relationships between oval cells and bone marrow
cells would be understood more easily if it could be demonstrated
that oval cells can be derived from hematopoietic
cells. This has been shown to occur in some
experimental models,44 but in these experiments, the proportion
of hepatocytes generated through this route was
very small, representing approximately 0.15% of hepatocytes
in the liver. More recent data using the FAH model
of liver injury demonstrated that in this model, oval cells
do not originate from bone marrow precursors but are
generated intrahepatically.45 Another study, using three
different models of rat liver injury, showed that bone
marrow cells were not the source of oval cells that repopulated
these livers.46
Oval Cell Differentiation in Massive
Hepatic Necrosis in Humans
An extensive ductular reaction occurs after massive (or
submassive) hepatic necrosis in humans.47–53 In this type
of injury, ductular proliferation involves mature cholangiocytes
and ductular hepatocytes. The latter, located at
the periphery of portal tracts, proliferate and express
cholangiocyte and hepatocyte markers. Ductular hepatocytes
are considered to be an intermediate form between
ductular cells and hepatocytes, resembling ductal plate
cells in the developing human liver. Such cells are also
present in massive hepatic necrosis in rats.54 It is not
known with certainty whether the generation of hepatocytes
from ductular hepatocytes leads to complete repopulation
of injured human livers, but at least one well-
What’s in a Name?
Many different terms are used to refer to cells originating
in the terminal branches of he bile ductular system
and the canals of Hering in rodents and humans,
which can function as progenitors for hepatocytes and
cholangiocytes, the mature forms of the two hepatic epithelial
cell lineages. In rats and mice, in experimental
situations in which large amounts of these cells proliferate,
the term oval cells is used for single cells or for clusters
of cells that form a ductule. Intermediate forms
between ductular cells and hepatocytes are often referred
to as ductular hepatocytes, and the term neocholangiole has
been used to describe the structures that contain these
cells. Small hepatocytes that are not completely differentiated,
which probably originate from oval cells, often are
referred to as small hepatocyte precursor cells. Cell lines
obtained from normal rodent liver, which have progenitor
cell capabilities, are known as liver epithelial cell
lines.
Editor’s note: The reader is referred to page 1739: “Nomenclature of
the finer branches of the biliary tree.”
For human liver, the term hepatic progenitor cells
frequently is used to refer to cells that are equivalent to
oval cells in rodents. The ductular reaction that involves
these cells is known as an atypical ductular reaction
(to distinguish from typical ductular reactions
that do not involve the generation of hepatic progenitor
cells). The term ductular hepatocyte also is used
commonly to describe intermediate forms, particularly
for cells appearing in the repopulation process after massive
hepatic necrosis. In rodents and humans, no special
name has been given to the cells located in the terminal
branches of the ductular system and in the canals of Hering.
Because they are likely to be the cells that most commonly
give rise to oval cells, they are often referred to as
intrahepatic stem cells or ductular stem cells.
In this article, the term oval cell is used interchangeably
with hepatic progenitor cell. The terms atypical
ductular reaction and ductular hepatocytes are used to
describe, respectively, ductular reactions involving
oval cells and intermediate forms between ductular
cells and hepatocytes.
HEPATOLOGY, Vol. 39, No. 6, 2004 FAUSTO 1479
documented case has been described in a patient who
recovered from massive liver necrosis. Fujita et al.52 performed
sequential biopsies on the natural liver of a patient
with massive necrosis after receiving an auxiliary partial
orthotopic liver transplant. Complete regeneration of the
natural liver was observed 12 to 14 months after transplantation,
through a process that involved an initial
ductular reaction followed by hepatocyte differentiation
from ductular hepatocytes.
Oval Cells in Liver Tumorigenesis and
Advanced Cirrhosis
As oval cells proliferate in response to treatment with
both carcinogenic and noncarcinogenic agents, the detection
of oval cells in a carcinogenic process is not proof of
the role of oval cells as cancer progenitors. Nevertheless, it
has been demonstrated that oval cells can generate hepatocellular
carcinoma (HCC), cholangiocarcinoma, and
hepatoblastoma in rodents.55–57 Although oval cell proliferation
is fairly common in experimental models of hepatocarcinogenesis,
in some models, oval cell and ductular
proliferation is not apparent (for instance, in carcinogenesis
induced by overexpression of growth factors such as
transforming growth factor 58). Indeed, there is no special
reason to support the notion that HCC is generated
exclusively from oval cells.59 Mature hepatocytes also can
function as tumor precursors, as long as they are in a
proliferative state. It is also important to note that in
general, oval cells do not seem to produce tumors directly,
but do so through the generation of hepatocytes, although
these cells “merge” into hepatocytes of neoplastic nodules
in the liver of rats fed the carcinogen 3-methyl-4-dimethylaminoazobenzene.
60 Whether hepatocytes generated
from oval cells in adult liver are abnormal, immature,
or have a high risk for transformation remains to be established.
In retrorsine-induced hepatocellular injury
combined with partial hepatectomy, the great majority of
proliferating cells are incompletely differentiated hepatocytes
called small hepatocyte precursor cells.61,62 Such
cells, which also have been detected in galactosamineinduced
liver injury,63 presumably originate from oval
cells. Their preferential accumulation in some types of
injury probably reflects a variable transit flux between
cellular compartments containing ductular stem cells,
oval cells, small hepatocyte precursor cells, and mature
hepatocytes.64
Oval cells, commonly referred to as hepatic progenitor
cells, have been detected in human livers in small cell
dysplastic foci, hepatocellular adenomas, chronic viral
and alcoholic hepatitis, nonalcoholic fatty liver disease,
hemochromatosis, primary biliary cirrhosis, and cirrhosis
associated with primary sclerosing cholangitis, conditions
associated with an increased risk of neoplastic development.
18,65–70 Oval cell markers such as AFP and cytokeratins
7 and 19 are expressed in approximately 50% of
small cell dysplastic foci and in HCC, suggesting the possible
origin of HCC from cells that express these markers.
65 This conclusion is strengthened by the finding that
such markers are not detected in foci of large cell dysplasia,
lesions that are not considered to be tumor precursors.
Hepatocyte generation from oval cells occurs at late stages
of cirrhosis, apparently at a time at which hepatocyte replication
has diminished.17 However, this process does not
lead to extensive parenchymal regeneration and is essentially
ineffective in restoring the normal parenchyma.
Based on the experimental data discussed above, it may be
suggested that generation of hepatocytes from oval cells in
severely damaged cirrhotic livers produces hepatocytes
that have a high risk for transformation. Answers to this
question may come from studies of populations of cells
isolated from small cell dysplastic foci and HCCs. In both
animals and humans, chromosomal abnormalities present
in tumors have been found in these foci.71 It would be
important to know whether the cells that harbor such
chromosomal defects in dysplastic foci express oval cell
phenotypes.
Factors Associated With Oval Cell
Proliferation and Oval Cell Phenotypes
In both human and rodent livers, oval cells proliferate
and differentiate in close proximity to stellate cells with
myofibroblast morphological features.48,72,73 In rats, oval
cells form ductules, which are extensions of the canals of
Hering and are surrounded by a continuous basement
membrane. Stellate cells penetrate through this basement
membrane and establish direct contact with oval cells in
the ductules.72 Oval cell proliferation is associated with
increased expression of c-KIT, and also of hepatocyte
growth factor, acidic fibroblast growth factor, and transforming
growth factor , which also function as growth
factors for hepatocyte replication.74 In both human and
rodent liver, expression of transcription factors of the hepatocyte
nuclear factor family is detectable shortly after
the start of oval cell proliferation, indicating an early commitment
to hepatocyte differentiation.53,75,76 It is puzzling
that growth factors that stimulate oval cell
proliferation are similar to those that stimulate hepatocyte
replication after partial hepatectomy and that both cell
types require signaling through tumor necrosis factor receptor
type I.77,78 Yet, as discussed, oval cells and hepatocytes
rarely proliferate simultaneously; oval cell
replication generally occurs when hepatocyte proliferation
is blocked. However, the interferon network is
stimulated only in oval cell, but not in hepatocyte, prolif-
1480 FAUSTO HEPATOLOGY, June 2004
eration.79 Studies in progress suggest that interactions between
interferon and cytokines such as tumor necrosis
factor may inhibit hepatocyte proliferation while they
stimulate oval cell replication (Brooling J et al., unpublished
manuscript, 2001).
Other interesting features of oval cell phenotypes are
the expression of proteins of the drug resistance gene families,
of adenosine triphosphate binding cassette transporter
genes, and of neuroendocrine peptides.80–84
Bone Marrow Cells and Hepatocyte
Production: Differentiation,
Transdifferentiation, and Cell Fusion
The tremendous interest generated by stem cells during
the last few years to a great extent is the result of to two
newly discovered properties of these cells.85 Studies of
hematopoietic stem cells (HSC) and bone marrow mesenchymal
stem cells, revealed that they are capable of
generating many different types of tissue cells (a property
known as transdifferentiation) and can choose multiple
differentiation pathways (a property called differentiation
plasticity). Understanding the mechanisms of transdifferentiation
is key to the field of stem cell biology and should
provide important clues for the use of stem cells in organ
repopulation and regeneration. So far, the most impressive
demonstrations of hepatocyte generation from bone
marrow cells are the production of hepatocytes in cultures
of multipotent adult progenitor cells (MAPCs)86,87 and
the repopulation of livers of FAH knockout mice by
transplanted HSCs.88,89 Yet, neither of these conditions
can be considered as examples of transdifferentiation.
In culture, MAPCs can differentiate into cells of mesodermal,
ectodermal, and endodermal lineages. Injected
into a blastocyst, a single MAPC contributes to the formation
of all somatic tissues. Thus, MAPC can be considered
as equivalent to embryonic stem cells, which have
persisted in adult tissues.86,87 Human, mouse, and rat
MAPCs, grown in matrigel in the presence of hepatocyte
growth factor and fibroblast growth factor-4, differentiated
into mature hepatocytes with apparently fully functional
properties.90 If MAPCs are indeed adult embryonic
stem cells, hepatocyte generation from these cells constitutes
a process of differentiation of pluripotent, uncommitted
cells, along a specific differentiation path. This is
similar to the differentiation process of embryonic stem
cells during development and is quite different from
transdifferentiation, which implies a change in differentiation
commitment of an already committed cell. These
exciting results await confirmation from other laboratories.
Much needs to be known about the properties of
MAPCs, and most importantly, whether they can generate
hepatocytes in vivo.
The other dramatic demonstration of hepatocyte generation
from bone marrow cells is the extensive repopulation of
damaged livers of FAH knockout mice transplanted with
HSC.88,89 So far, this is the only example in animals or humans
of extensive repopulation of damaged livers by cells
derived from bone marrow. In this system, the kinetics of
repopulation by HSC is slow and inefficient compared with
that obtained by hepatocyte transplantation, although significant
repopulation eventually occurs. The first hepatocytes
generated from HSC appeared at approximately 7 weeks
after transplantation. By contrast, transplanted hepatocytes
reconstituted more than 50% of the liver in approximately 4
weeks, leading the authors to conclude that “hepatocyte replacement
by bone marrow cells is a slow and rare event.”91
Nevertheless, by 22 weeks after transplantation, repopulation
from transplanted HSC constituted approximately
30% of the entire liver. It has now been shown that hepatocytes
generated from transplanted HSC in FAH knockout
mice are the product of cell fusion rather than a result of
transdifferentiation.92,93 The fusion process created tetraploid
hepatocytes, 6X hepatocytes, and aneuploid hepatocytes.
The liver cell that fuses with HSCs has not been
identified, but the hepatocytes produced by the fusion event
do not express HSC genes. Recent data from studies of muscle
regeneration suggest that HSC are not capable of directly
generating myogenic progenitors.94 Instead, after muscle injury,
circulating inflammatory cells such as macrophages and
neutrophils fuse with myotubes. A similar mechanism may
occur in the liver, that is, fusion may occur between bone
marrow-derived macrophages and hepatic cells, triggering
the proliferation of the fused liver cell.
It is conceivable that fusion between bone marrow and
liver cells occurs in FAH knockouts because of the high
proliferative pressure imposed by this system.However, it
may be argued that high levels of hepatocyte production
from hematopoietic stem cells can be achieved only in a
system in which cell fusion occurs. In any case, these
results make it clear that any attempt to use HSC to
reconstitute livers needs to demonstrate: (1) the mechanism
by which hepatocytes are formed and (2) that hepatocytes
generated from bone marrow cells function
normally, do not have abnormal genomes, and are not
prone to malignancy.95
Injected Bone Marrow Stem Cells Have
Minimal Capacity to Generate Hepatocytes
in Normal Livers of Mice and in Most
Models of Liver Injury
Krause et al.96 injected single, highly purified bone
marrow cells into irradiated mice and obtained engraftment
in several organs, including skin, lung, and liver.
The livers of five mice examined did not contain HSCHEPATOLOGY,
Vol. 39, No. 6, 2004 FAUSTO 1481
derived hepatocytes, and three of these mice also did not
have bone marrow-derived cells in bile ducts. The other
two mice had 0.4% and 2.2% of bile duct cells of bone
marrow origin 11 months after transplantation. Wagers et
al.97 used a different method of bone marrow cell isolation
and marked cells with green fluorescent protein. Singlelabeled
HSC cells injected into irradiated mice failed to
contribute to brain, kidney, gut, muscle, and liver, although
they completely reconstituted the bone marrow of
these animals. The experiments of Krause et al. and Wagers
et al. differ in important technical aspects, including
the type of cells used, but there seems to be complete
agreement regarding the generation of hepatocytes from
injected, purified bone marrow cells: no bone marrowderived
hepatocytes were detected in either of these experiments.
Theise et al.98 transplanted unfractionated bone
marrow cells or purified CD34lin- cells from male mice
into irradiated female mice and searched for cells containing
the Y chromosome and expressing albumin mRNA in
the liver of the recipient mice 1 week to 8 months after
transplantation. Cells positive for both the Y chromosome
and albumin mRNA were detected from 2 to 8
months after transplantation at frequencies of 0.39% to
1.1% of total hepatocytes in the liver. The authors used a
correction factor to compensate for potential error sampling
in detecting the Y chromosome, which raised the
reported frequencies by a factor of 2. In all of these experiments,
the livers of the mice transplanted with bone marrow
cells were morphologically normal and apparently
undamaged.
It is of great interest to determine whether more efficient
production of hepatocytes from injected bone marrow
cells leading to liver repopulation can be achieved in
injured livers. Mallet et al.99 reconstituted the bone marrow
of lethally irradiated mice with bone marrow cells
from Bcl-2 transgenic mice100 and asked if bone marrowderived
Bcl-2 expressing hepatocytes would repopulate
the liver after hepatic injury elicited by repeated injections
of Fas agonist antibody. Even with severe hepatic damage,
the proportion of bone marrow-derived Bcl-2-expressing
hepatocytes found in hepatic tissue was very small, varying
from 0.008% to 0.8% of total hepatocytes, depending
on the extent of damage. Kanazawa and Verma101 studied
the generation of hepatocytes from bone marrow cells in
three different models of liver injury and concluded that
there was little or no contribution of bone marrow cells to
the replacement of hepatocytes in these models. Fujii et
al.102 transplanted green fluorescent protein-positive
bone marrow cells into green fluorescent protein-negative
irradiated recipient mice and identified green fluorescent
protein-positive endothelial cells and Kupffer cells, but no
hepatocytes. A similar result was obtained by Dahlke et
al.103 using a model of acute liver failure. It can be concluded
from these experiments that bone marrow cells
have a minimal capacity to generate hepatocytes in normal
livers and a very low capacity in injured livers. The
exception is the production of hepatocytes from bone
marrow cells in FAH knockout mice, which, as already
discussed, is a consequence of cell fusion.
Generation of Hepatocytes by Bone Marrow
Cells and Cell Chimerism in Recipients of
Liver and Bone Marrow Transplants
Theise et al.104 investigated, in patients receiving bone
marrow or liver transplants, whether hepatocytes could be
generated from the bone marrow cells of the transplant
recipient. The frequency of hepatocytes that were considered
to be bone marrow derived varied from 1% to 3.6%
in five patients and was 8% in one patient. The authors
multiplied these values by a factor of approximately 5 to
correct for sampling errors in the detection of the Y chromosome
and reported that hepatocyte engraftment
ranged from 4% to 43%. Alison et al.105 also examined
the livers of patients who received bone marrow or liver
transplants from sex-mismatched donors. The frequency
of bone marrow-derived hepatocytes in the liver of the
recipients was estimated to be 0.5% to 2%. Korbling et
al.106 reported that the frequency of hepatocytes generated
from the bone marrow of recipients of sex-mismatched
liver or bone marrow transplants varied from4%
to 7% and was unrelated to liver injury and to the time
after transplantation. Two other studies of liver transplant
patients did not detect bone marrow-derived hepatocytes
or found only occasional cells.107,108 Several studies have
addressed the question of cellular chimerism in liver transplants.
Hove et al.109 examined the livers of 16 transplanted
patients to identify cells originating from the
recipient and reported chimerism of endothelial cells in
14 patients, bile duct epithelial cell chimerism in five patients,
and hepatocyte chimerism in one patient. Ng et
al.110 recently reported that the vast majority of recipientderived
cells present in transplanted livers were macrophages
or Kupffer cells and that only 1.6% of the total
recipient cells detected in these livers were hepatocytes
(this corresponded to 0.62% of the total number of hepatocytes
in the liver). Finally, Kleeberger et al.111 detected
91% chimerism frequency in liver transplant recipients,
and the presence of hepatocyte chimerism in two of nine
patients in samples obtained 4 weeks after transplantation
and in five of nine patients at 12 months or longer (a
quantitative analysis of the percentage of recipient hepatocytes
in the chimeric livers was not reported).
How can these results be interpreted? First, the lack of
consistency of the results may be a consequence of the use
1482 FAUSTO HEPATOLOGY, June 2004
of different techniques to identify recipient-derived hepatocytes
in transplanted livers. For instance, factors often
are used to correct for the inability to examine the complete
surface of hepatocyte nuclei to detect the Y chromosome.
This type of correction, which involves the
multiplication of observed values by factors that vary from
less than 2 to more than 5 can introduce significant errors
and uncertainties in the reported data. Another difficulty
is that large numbers of mesenchymal cells in transplanted
livers originate from the recipient’s bone marrow. Although
various markers can be used to identify hepatocytes,
the precision of the methods used for this
identification is variable and not always optimal. Superimposition
of images without confocal microscopy may
lead to errors caused by the detection of reaction products
or in situ hybridization signals in endothelial or Kupffer
cells that are in close apposition to a hepatocyte. The
uncertainties about the techniques used in some of these
experiments have been discussed.112,113 Until more definitive
data are obtained, it can be concluded that the generation
of bone marrow-derived hepatocytes occurs in the
livers of some but not all transplant patients, that it is a
highly inefficient process, and that, because of its very low
frequency, its physiological relevance remains unproven.
Perspectives
During the last few years, work with stem cells became
one of the most exciting areas of biomedical research,
generating much enthusiasm from scientists, clinicians,
and the general public. Having as an endpoint the repopulation
and regeneration of tissues and organs, the
promise of this research is far reaching. After the initial
phase of excitement and the publication of findings that
often defied long-accepted knowledge, it is now time to
assess the progress made, to point out pitfalls and misinterpretations,
and, most importantly, to project a realistic
look into the future. It is a sobering thought that definitions
for stem cells are still being debated.114 Does the
expression “Seeing is not being” apply to stem cells?115 Is
a stem cell “an entity or a function,”116 a contextual category,
1 the first, defined, component of a hierarchical system,
or a functionally plastic entity (the “chiaroscuro stem
cell”117)? Can this entity be isolated and studied in culture
in a biologically meaningful way, or are we dealing with
Heisenberg’s uncertainty?1 Carried to their most extreme
implications, and crudely interpreted, some of these ideas
may lead us to an “anything goes” intellectual environment,
in which all results even the most discordant, are
accepted uncritically. Great progress in stem cell research
has been made and will continue to be made by the careful
scrutiny of experimental data, by examining the reliability
and reproducibility of methods to identify stem cells, and
by addressing the issue of the biological relevance of the
findings. Progress in the field is inextricably linked to
the understanding of mechanisms of cell differentiation,
proliferation, and transdifferentiation and knowledge
about the interactions between cells and
extracellular matrix components in normal and injured
tissues. Future work may show that umbilical cord
cells, embryonic stem cells, or fetal liver cells are better
sources of hepatocyte precursors for hepatic repopulation
than bone marrow cells.118 –130
The emphasis on bone marrow stem cells often obscures
the fact that the remarkable regenerative capacity of
the liver primarily is the result of to the replication of
mature hepatocytes. When hepatocyte replication is slow
or inhibited, intrahepatic stem cells give rise to oval cells,
which replicate and differentiate into hepatocytes (Fig. 1).
The proliferative capacity of normally quiescent, highly
differentiated hepatocytes is unique among differentiated
cells in mammalian tissues. As paradoxical as it may sound
to stem cell biologists, the hepatocyte is the most highly
efficient “stem cell” for the liver.
Where Do We Go From Here?
A first observation is that the identification of bone
marrow–derived hepatocytes needs to be carried out according
to rigorous criteria. Methodological accuracy and
reproducibility are critical factors that determine the reliability
of the data reported. From the review of the literature
presented here, I conclude that the generation of
hepatocytes from bone marrow cells is a very rare event in
liver transplantation and repopulation after injury and
that such hepatocytes are produced by cell fusion rather
than by a transdifferentiation mechanism (Fig. 2). This
premise does not exclude a potential functional role for
bone marrow-derived cells in hepatic homeostasis and, in
fact, suggests experimental approaches to study this issue.
Fig. 2. Possible mechanisms for the generation of hepatocytes from
bone marrow cells. Of the mechanisms shown in the figure, only cell
fusion (#4) has been shown to occur in vivo. Generation of hepatocytes
from pluripotent bone marrow stem cells (#2) can occur in culture.90
(Diagram redrawn from Wagers AJ, Weissman IL.140)
HEPATOLOGY, Vol. 39, No. 6, 2004 FAUSTO 1483
● Does cell fusion generate functionally intact hepatocytes
that, despite their abnormal ploidies, may not carry
a high risk for transformation? Can cell fusion techniques
be applied to liver repopulation in a clinical setting? May
this approach be more successful for liver repopulation
than hepatocyte, oval cell, or hepatic embryonic stem cell
transplantation?
● The role of the bone marrow in generating nonparenchymal
cells in liver regeneration and repopulation
seems to be much more significant than the generation of
hepatocytes. Would blockage of the migration of HSC or
bone marrow-derived leucocytes into the liver interfere
with liver regeneration and repopulation? Is the hepatic
seeding of bone marrow-derived endothelial and Kupffer
cells essential for normal liver homeostasis? Do these cells
(or perhaps even the small number of bone-marrow-derived
hepatocytes) produce special cytokines and growth
factors that are required for hepatocyte replication?
● The derivation of oval cells (albeit at very low levels)
from the bone marrow has been reported, but so far not
confirmed in other laboratories. This very important
question must be settled definitively. If oval cells are not
generated by bone marrow cells, could they (or cells in the
canals of Hering) fuse with bone marrow cells to generate
hepatocytes?
● Do MAPCs differentiate in vivo and generate lineages
that populate the liver in adult humans or animals?
Can cultures of MAPCs be used to produce large amounts
of human hepatocytes suitable for transplantation?
● Great progress has been achieved in the purification
of stem cells from embryonic liver and umbilical cord
blood. Would transplantation of these types of cells into
injured livers reconstruct both hepatocytes and bile ducts?
Are these cells more efficient than fetal or adult hepatocytes
for the repair of liver injury?
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