Leading article Hepatic stem cells
For several decades investigators have been addressing the tantalising question,is there a hepatic stem cell?1Despite widespread attempts to identify and characterise such cells in the liver and a plethora of papers,reviews,and monographs on the subject,2–4doubts as to their very exist-ence have remained.It is only relatively recently that these doubts have been removed,at least in the eyes of most investigators,with convincing evidence from rodent studies and novel developments in the cell biology of the pathogenesis of human liver disease.Moreover,new excit-ingfindings indicate that in tissues,including bone marrow and the brain,there reside cells with an innate ability to di V erentiate into divergent cell types.The stem cellfield is currently a“hot”topic.
Although essentially a quiescent organ,the normal adult liver can fully regenerate following surgical resection or injury.Much of what we have learned about liver growth control derives from the classical two thirds partial hepatec-tomy model in the rat.The process begins with growth activation of mature hepatocytes;other cell types,includ-ing biliary epithelial cells(BEC)and sinusoidal cells,pro-liferate with a delayed response.5However,if liver damage is so severe that hepatocytes are largely obliterated or for some reason are prevented from entering the growth cycle by exposure to hepatotoxins or carcinogens,then activation of a liver stem(progenitor)cell population is postulated,23 giving rise to so-cal
led“oval”cells.These cells are thought to have both clonogenic and bipotential capacity—that is, the ability to proliferate and di V erentiate into cells of either hepatocyte or BEC lineage.67There is also evidence that under certain conditions oval cells can be induced to di V erentiate into non-hepatic lineages including intestinal and pancreatic epithelium.3The origin of oval cells and their precise location within the liver has remained enigmatic.However,recent evidence from a variety of ani-mal models and indeed now human studies has begun to provide answers.
Experimental studies
Oval cells are activated following dosing of animals with a variety of toxins and carcinogens,alone or combined with other surgical or dietary regimens.2389In one of the most studied models,acetylaminofluorene treatment followed by partial hepatectomy,an array of cytokines and growth factors have been shown to be up regulated during the response.2Notably,some of the regulatory mechanisms are beginning to be delineated,for example the recent interesting observation that interferon is implicated in orchestrating the process.10Oval cells themselves however probably represent the activated progeny of a dormant stem cell compartment and while oval cells are readily identified in injured liver,one area of great controversy is the question of where these putative stem cells reside in the normal liver.
Because of the lack of specific markers of liver stem cells (even the so-called oval cell specific marker OV6recognises bile duct epithelium in normal rat liver)11they have been notoriously di Y cult to localise other than in the injury models described above.That they are present is supported by the relative ease with which liver epithelial cell lines have been established from normal liver.9Where however,do these putative stem cells reside?One suggestion is that they are present in the canals of Hering,that is the region where cells are transitional between the periportal hepatocytes and the biliary cells lining the smallest terminal bile ducts.8 Others suggest that there are cells which are found located in the portal tracts,in the periductular region,or even that periportal hepatocytes have stem cell or metaplastic properties.3
Further confusing terminology arises with the descrip-tion of a population of cells isolated from rat liver called “small hepatocytes”12and recently extended to include clonal expansion of“small”human hepatocytes.13These cells appear to have the capacity to clonally expand and yet retain hepatocyte phenotype in vitro.Again,the precise origin or location within the liver of these cells has not been defined.Independently,in a series of hepatocyte transplan-tation studies using a mouse model of hereditary tyrosinaemia,a population of adult mouse hepatocytes with apparent stem cell capacity—that is,multiple rounds of proliferation—demonstrated the ability to largely regen-erate and repopulate t
he liver.14In neither approach was there evidence to suggest that the proliferative cells in question gave rise to epithelium of ductal biliary phenotype.While not proved,it seems likely that the cell populations of Mitaka et al,Hino et al,and Overturf et al are the same or similar.Thus these small hepatocytes dif-fer from oval cells and the term unipotential seems more applicable to this population.
Human liver stem cells
Identifying stem cells or their progeny in human liver has of course been even more of a challenge.In the developing human fetal liver,di V erentiation of hepatoblasts into biliary epithelium is regulated by signals from the portal mesenchyme15;hepatoblasts not in contact with portal mesenchyme mature into di V erentiated hepatocytes.Al-though much is known about the bipotential nature of
Abbreviations used in this paper:BEC,biliary epithelial cells.
Leading articles express the views of the author and not those of the editor and editorial board.
Gut2000;46:743–745743
hepatoblasts,present in fetal liver in large numbers,previ-
ous work has shown that while these cells can proliferate in
vitro16they are thought not to demonstrate clonogenic
potential.Thus although hepatoblasts are considered by
some as liver stem cells,to describe them as equivalent to
oval cells or their progenitors is misleading.
The identity of stem cells in the adult human liver and
their role in response to liver damage/injury has also been
contentious.Numerous morphological studies have high-
lighted the presence of small cells present in diseased
human liver that are suggested to be putative progenitor
cell derivatives,17located in or close to bile ducts or in peri-
portal regions adjacent to hepatocyte margins.18More
convincingly,using markers originally shown to be
expressed or up regulated by rat oval cells(including OV-6,
c-kit,and CD34),several groups have identified oval cells
in hepatoblastoma,19hepatocellular carcinoma,20and
cirrhotic liver.21–23Using double immunolabelling tech-
niques,some of these cells co-express hepatocytic or biliary
phenotypic markers implying lineage progression.2123
However,morphological immunocytochemical studies on
tissue sections convey only a limited picture since they
present a static single“snap shot”of what is undoubtedly a
dynamic process and interpretation is di Y cult.Addition-
ally,it is likely that some(or all)of the markers in question
are expressed on cells transiently.
One means of solving this problem is to develop a
defined cell culture based model where the cell fractions in
question can be isolated,the regulatory events carefully
investigated,and agents responsible for induction of
growth and di V erentiation determined.T o this end,using
antibodies against c-kit and CD34which recognise surface
determinants,cells have been specifically immuno isolated
and cultured from fetal,paediatric,and adult human
liver2425and lineage progression followed using well
characterised phenotypic markers of hepatocyte or biliary
specificity.This work is ongoing and it is hoped will help
resolve the questions which remain concerning the identity
and potential use of liver derived stem cells. Haematopoiesis and liver stem cells
While the debate on the source and location of hepatic
stem cells is ongoing,two recent papers add a new dimen-
sion and o V er a challenging alternative hypothesis to
explain the origin of oval cells.As already discussed,we
know that a number of surface determinants are shared
between haematopoietic derived progenitor cells and oval
cells,including c-kit,26CD34,27and Thy-128in rodents,and
c-kit23and CD3424in humans.These observations have
been brought sharply into focus with the demonstration
that a population of haematopoietic stem cells originating
in the bone marrow may give rise to oval cells in the liver
and have the potential to further di V erentiate into hepato-
cytes and/or ductal cells.29By transplanting rat bone mar-
row into lethally irradiated recipients and following the fate
of syngeneic cells using various markers,they clearly
showed striking changes in the livers of animals induced to
regenerate following2-acetylaminofluorene and CCl
4 treatment.Male donor marrow cells were visualised in
female recipients and in a second model,marrow from
dipeptidyl peptidase IV positive animals was transplanted
into dipeptidyl peptidase IV deficient recipients.In both
cases evidence was presented to suggest that the donor cells
migrated into the livers of recipient animals and subse-
quently underwent di V erentiation to become hepatocytes
although it was less clear whether ductular cells of biliary
phenotype developed.29
A second recently published study describes a similar
approach comprising a mouse marrow transplant model
but which interestingly did not include a liver injury step.30This new report provides important confirma
tory evidence that bone marrow derived haematopoietic stem cells can indeed give rise to hepatocytes.In both studies,the number of cells which undergo the transition appears to be relatively small.Therefore,one is left unsure as to whether the response represents a true physiological or pathophysi-ological phenomenon or simply a quirk of nature uncovered by chance experimental observations.Following bone marrow transplantation,the immune cell chimaerism which ensues will result in cells in the liver with the donor genotype.Therefore,to prove the hypothesis unequivo-cally,it is vital that convincing marker co-expression work be used to confirm the phenotype of the cells purported to di V erentiate from haematopoietic derived cells into cells of hepatic epithelial(hepatocyte)lineage.Such studies are presently underway in a number of centres.
Parallels between liver and brain
Fascinating recentfindings in neural cell biology studies add a further dimension to this emerging stem cellfield. Neural cells found in a post-mitotic epithelial cell layer,the ependyma,overlying the ventricular layer in the brain appear to have stem cell properties.31Rather like the puta-tive hepatic stem cells discussed above,they appear to remain dormant in normal adult tissue but when activated, they proliferate,migrate,and then di V erentiate.They divide asymmmetrically,one daughter cell staying as an undi V erentiated stem cell while the other migrates and gives rise to neuronal or glial cells.31It is n
ot known whether hepatic stem cells demonstrate this property.Thus two organs,the liver and brain,where normally little cell turnover occurs,harbour cells with surprising di V erentia-tion potential.In other studies,32putative neural stem cells transplanted into bone marrow have been shown to di V er-entiate into blood cells.As an interesting caveat,this transdi V erentiation response occurred only in vivo,32 raising the problem of designing culture models with which to investigate the regulatory features of the(neural or hepatic)stem cell di V erentiation process.
Clinical implications
The ability to identify and exploit a human hepatic clonal stem cell could have important clinical implications,as generating large numbers of di V erentiated and therefore fully functional human hepatocytes has enormous poten-tial.Primary hepatocytes remain the ultimate choice for use in bioartificial liver support devices.33Reliance on pri-mary hepatocytes from pigs or other species remains prob-lematical due to the sensitive issue,now under the wider public debate,of xenotransplantation and the perceived inherent risks(possible cross over retroviral infection)of approaches which involve the use of cells or tissue from foreign species.Other important areas where progress has been limited due to lack of su Y cient numbers of good quality primary human hepatocytes include hepatocyte transplantation for the treatment of metabolic disorders or fulminant liv
er failure,34and evaluation of drug toxicology and pharmacokinetics so vital today for the development of safe new therapeutic drugs.35Despite widespread e V orts, no one has yet achieved the goal of generating a safe,fully functional yet clonal,immortalised,or genetically engi-neered human cell model that can be substituted for primary hepatocytes in these various applications.This clearly adds further impetus to the search for the definitive human hepatic stem cell.
Conclusions
In summary,the consensus that there are cells in the liver with stem cell potential has achieved acceptance.T ogether with thefindings from otherfields of cell biology,the plas-
744Strain,Crosby
ticity of certain cell types is clearly more extensive than previously realised,at least when dealing with organs or tissues traditionally regarded as cell quiescent.The poten-tial exploitability of these recent developments in hepatic stem cell biology awaits further investigation.
A J STRAIN
H A CROSBY School of Biosciences,
University of Birmingham and Liver and Hepatobiliary Unit, University Hospital,Birmingham,
Edgbaston,Birmingham B152TH,UK
Email:A.J.Strain@bham.ac.uk/H.A.Crosby.bcm@bham.ac.uk
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Hepatic stem cells745
doi: 10.1136/gut.46.6.743
2000 46: 743-745
Gut
A J STRAIN and H A CROSBY
Hepatic stem cells
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