(CANCER RESEARCH 50. 3811-3815, July l, 1990]

Perspectivesin CancerResearch

Is There a Liver Stem Cell?

Stewart SellDepartment of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, Texas 77030

Abstract

The role of a putative liver stem cell in liver regeneration and carci-nogenesis is reviewed. There is increasing evidence that there is a liverstem cell that has the capacity to differentiate into parenchyma! hepato-cytes or into bile ductular cells. These stem cells may be activated toproliferate after severe liver injury or exposure to hepatocarcinogens.They are not activated by moderate liver injury, which is repaired byproliferation of mature hepatocytes. Exposure to most chemical hepatocarcinogens results in proliferation of a small morphologically indistinctcell population termed "oval cells." These cells have been shown to have

the capacity to differentiate into hepatocytes or into ductular cells. Theorigin of these cells appears to be from transition duct cells, but there isalso evidence of an even less mature periportal liver stem cell. Study ofthe development of these cells during carcinogenesis indicates that livercancer arises from oval cells by aberrant differentiation of stem cells.

From a biological standpoint two major postulates of theorigin of cancer are: Cancer arises from differentiated expressive cells by "dedifferentiation" or from pluripotential stem

cells by aberrant differentiation (1). One of the fundamentalpostulates of the stem cell theory of cancer is that the incidenceof cancers arising in a given tissue is directly related to the rateof cell division in that tissue. Thus tumors appear in relativehigh incidence in organs with rapid cellular turnover, such asskin, gastrointestinal mucosa, or bone marrow, but in lowincidence in tissues with low turn over, such as neurologicaltissue. Tumors of neurons, which essentially do not proliferatein the adult, are extremely rare. One exception to this, whichmay prove the rule, is the liver. Although primary malignanttumors of the liver are rare in the western world, the extremelyhigh incidence of primary hepatocellular carcinomas in otherparts of the world (Africa, Southeast Asia) actually makesprimary hepatocellular carcinoma one of the world's most

common malignant tumors. This brief review will address thequestion, "Do hepatocellular carcinomas arise from dediffer

entiation of adult hepatocytes or do these cancers arise byactivation and aberrant differentiation of liver stem cells?"

Stem cells are defined as multipotent cells that divide toproduce one daughter cell that stays as a stem cell while theother daughter cell expresses a differentiated phenotype. Tissuestem cells are determined; i.e., they lack the biochemical andstructural markers of differentiation but are determined fordifferentiation to a specific cell type (1). Stem cells respond byproliferation and differentiation to replace senescent cells undernormal circumstances or to restore destroyed tissues in pathological conditions. The epithelium of the skin is the classicexample of the former. The basal cells send daughter cellsthrough the maturational pathways to replace senescent cells.The basal cells may not be the true stem cells in this case butare already partially differentiated. Another example is the celltype responsible for limb regeneration in the amphibian thatcan replicate and differentiate into the composite of maturecells of the regenerated limb. The limb blastema or stem cellactually is not multipotential but is limited to expression of

limb cell phenotypes (2). Stem cells may participate in tissuereplacement in organs that normally undergo renewal, such ashematopoietic cells, but most of the actively dividing cells inthese organs are not the pluripotent stem cells but mitoticallyactive partially differentiated expressive cells that give rise byfurther division and differentiated to terminally differentiatedcells that make up the "mature" cells of the organ. Whether or

not there are stem cells in an organ such as the liver is criticalin understanding the cellular origin and mechanisms in carcinogenesis.

Study of the cellular changes that precede the developmentof liver cancer in animals exposed to chemical hepatocarcinogens has led most investigators over the last 20 years to theconclusion that hepatocellular carcinomas arise by dedifferentiation of adult liver cells. These studies have concentrated ona series of lesions called "foci" and "nodules" which have beendesignated "premalignant" (3, 4). The development of focal

and nodular lesions may be presented by a description of themorphological changes reported by Teebor and Becker (5)following the cyclic feeding of the carcinogen AL2-acetylami-nofluorine to rats. The AAF1 containing diet is fed for 2 weeks

followed by 1 week of normal diet, the full carcinogen regimenconsisting of four 2-week-on/l-week-off cycles for a total of 15weeks. Approximately 36 weeks after the initiation of the AAFfeeding the rats develop primary hepatocellular carcinomas.

After the first feeding cycle, the livers contain small collections of cells that contain higher levels of some enzymes (e.g.,7-glutamyl transpeptidase, glutathione 5-transferase, etc.) thando normal adult hepatocytes (enzyme altered foci) and thesecells stain more intensely basophilic than normal cells (baso-philic foci). After the second cycle collections of enzyme alteredcells now form small masses that push aside the normal livercells. These are designated "neoplastic nodules." After the third

cycle the neoplastic nodules enlarge and can be seen easily withthe naked eye. If the diet is discontinued after three cycles nocarcinomas will develop and the liver will "remold" to a normal

appearance (6). After four cycles much larger nodules are seenand if the AAF diet is then discontinued, carcinomas will appearabout 15 weeks later. Because of the sequence of morphologicaland enzymatic changes from adult liver cell to foci to nodulesto cancer, this system has been used as a model for the dedifferentiation origin of cancer. However, at the same time thatthese designated "preneoplastic" changes are taking place there

is proliferation and evolution of another cellular lineage. Evidence is accumulating that primary hepatocellular carcinomaactually arises from these latter cells and that this cellularlineage arises by aberrant differentiation of stem cells.

Large numbers of small nondescript cells, termed "oval cells"

(7), also appear in the livers of rats exposed to chemical carcinogens (8, 9), but a role for these cells in hepatocarcinogenesishas been largely ignored. The development and fate of oval cellsduring hepatocarcinogenesis are now becoming increasinglyclear (10, 11). These cells proliferate in most, if not all, models

Received 12/4/89; revised 2/14/90.' The abbreviations used are: AAF, Ar-2-acetylaminofluorene; AFP, a-fetopro-

tein.

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IS THERE A LIVER STEM CELL?

of carcinogenesis. This cell lineage develops prior to the neo-plastic nodules. The oval cells arise from periductular cells earlyafter carcinogen exposure and have the capacity to acquireductal or hepatocellular phenotypes (12, 13). More recently,Evarts et al. (14), following oval cells produced relatively earlyafter carcinogen administration and partial hepatectomy byautoradiography, demonstrated that the radiolabeled cells seenearly after [3H]thymidine administration were oval cells but that

the label later appeared in newly formed hepatocytes. Theyconclude that oval cells differentiate into hepatocytes.

Studies of the in vitro proliferation and phenotypic expressionof liver cells taken from carcinogen treated animals is supportive of the stem cell concept. Earlier studies with cells fromcarcinogen treated rats were directed to determining propertiesof nodular cells. Laishes et al. (15, 16) cultured livers with alarge nodular component and found that mixed cell populationsfrom carcinogen treated rats were resistant to the toxic effectsof carcinogen exposure. Subsequently, Ledda et al. (17), usingcultures of cells from carcinogen treated rats using densitygradient centrifugation to obtain cell populations highly enriched in oval cells (18), demonstrated that the oval cells andnot the larger hepatocytes were resistant to the toxic effects ofcarcinogen exposure. Thus it appears that both oval cells andnodular cells are resistant to the toxic effects of carcinogens.

In general, nodular cells survive better in vitro than normalhepatocytes but do not have an increased tendency to proliferateor transform in vitro (19, 20). On the other hand, culturesenriched for oval cells tend to form clusters in vitro and toproliferate spontaneously (21). Recently, more extensive studiesby Germain et al. (22-24) using cultures of oval cell populations(23) and fetal rat liver cells (24) demonstrate that these populations respond similarly to the differentiating stimuli providedby addition of sodium butyrate and dexamethasone. By examining a variety of phenotypic markers they conclude that bothoval cells and fetal liver cells have the capacity to differentiateinto either bile duct cells or into hepatocytes. In contrast, bileduct cells isolated by density gradient from the livers of ratsafter bile duct ligation (25) do not acquire hepatocyte phenotypes in culture (26), suggesting that bile duct cells are not ableto "dedifferentiate" into hepatocytic lineages. Thus bile ductuleand ducts are "determined" for bile ducts and could not produce

hepatocytes under these conditions. Tsao et al. (27, 28) using acell line with "oval cell" properties derived from a normal rat

liver (29) have shown that treatment of the cell line with acarcinogen (/V-methyl-jV'-nitrosoguanidine) results in a pro

gressively increasing phenotypic diversity, an indication of thegrowth and differentiation potential of these cells. Finally,transplantation into nude mice of epithelial cell lines derivedfrom the livers of carcinogen treated rats after transfection withthe H-ras oncogene resulted in growth of moderate to welldifferentiated hepatocellular carcinomas (30). The significanceof this observation to the induction of hepatocellular carcinomas by chemicals is not clear.

Isolation of oval cells from livers of carcinogen treated ratsby density gradient centrifugation is possible because the ovalcells are much smaller than parenchyma! hepatocytes or nodular cells. One of the interesting changes that occur in the liversof rats treated with carcinogens is a change in the ploidy statefrom largely tetraploid to largely diploid (31). Neoplastic nodules are diploid (32). This has been used to suggest that thetransformed hepatocytes that give rise to tumors are derivedfrom nodules. However, Schwarze et al. (33) found that mostof the diploid cells arising early after carcinogen exposure wereabout half the size of the tetraploid hepatocytes, and Scott et

al. (34) noted that the diploid cells corresponded to «-fetopro-tein positive oval cells. Thus it is postulated that diploid ovalcells give rise to diploid nodular cells that, in turn, give rise tonondiploid tumors that are transplantable (35).

Transplantation of putative premalignant cells into nudemice has been used to determine if a given cell population cangive rise to tumors. Transplantation of nodular cells has generally not given rise to tumors, with a few possible exceptions(36, 37). Tatematsu et al. (37) were able to produce nodules bytransplantation of late nodular liver cells and some histológica!evidence of hepatocellular carcinoma within the transplantednodules. In contrast Yoshimura et al. (38) obtained highlyanaplastic adenocarcinomas upon transplantation of culturesobtained from cells highly enriched for oval cells. Oval cellsinjected into the fat pads of syngeneic rats maintain the morphology of duct-like clusters but may express hepatocyte properties, such as albumin and tyrosine aminotransferase activity(23). Fetal liver cells exposed to carcinogen in vitro will producetumors expressing hepatocyte phenotypes upon transplantationto nude mice (39). Each of these studies illustrates the problemsof working with transplantation of a population of cells. It isnot possible to determine which cell in the transplanted population may eventually grow into a transplantable tumor. Thus,cells used for transplantation may be highly enriched in nodularcells or in oval cells, but upon transplantation, only one cell in10s or IO6 may divide and this cell may not represent the

majority population. However, both in vivo and in vitro Findingsare consistent with the conclusion that oval cells can evolveinto either primary hepatocellular carcinomas or cholangiocar-cinomas.

A critical question is from what cells do the "oval cells" or

stem cells originate. This question has been addressed by sequentially labeling the proliferating cells in the liver very earlyafter exposure to chemical hepatocarcinogens (40, 41). In orderto appreciate the changes seen it is necessary to describe themorphology of the terminal bile ductules and their relationshipto the bile canaliculi of the hepatocytes. The terminal bileductules are composed of two or three ductule cells surroundedby a basement membrane. These ductules are called the canalsof Hering. They connect to the larger bile ducts on one sideand to transitional duct cells on the other. The transitional ductcell connects the bile canaliculi with the canals of Hering. Thetransitional duct cell may be identified by the following characteristics. It does not abut a basement membrane; it bordersone side of a canaliculus with an hepatocyte and, most critically,it forms tight junctions with this hepatocyte. For a clear pictureof the transition ductule cell see the photograph on p. 596 ofBailey's Textbook of Microscopic Anatomy (42). There is an

increasing tendency to refer to the transition ductule cell as a"Hering cell," but this extension of the terminology historically

used for the terminal ductule has led to some confusion. Popperet al. (43) as well as Grisham and Porta (13) first identifiedproliferation of the ducts of Hering after exposure of rats tochemical carcinogens for 3 to 4 weeks and concluded that thesecells were the major proliferating cells. In addition they proposed that so-called terminal duct cells could differentiate intoeither duct cells or hepatocytes. However, when the livers ofcarcinogen exposed rats were examined earlier (1 to 7 daysafter exposure to a rapidly acting carcinogenic diet), extensiveproliferation of periductular cells was seen to precede labelingof the ductules (Fig. 1, a and b). By electron microscopicautoradiography many of these cells had the characteristics ofthe transitional ductule cell (Fig. le). In a poster presented atthe November, 1989 meeting of the American Society of Cell

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IS THERE A LIVER STEM CELL?

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Fig. 1. a, light microscopic autoradiography, 1 day after a choline-devoid diet containing 0.05% w/w A'-2-fluorenyIacetamide (CD-AAF) feeding; b, lightmicroscopic autoradiography 7 days after CD-AAF feeding; c, electron microscopic autoradiography, day 7; d, electron microscopic autoradiography, day 1. Ratswere fed CD-AAF and given injections of 50 >iCiof [3H]thymidine i.v. three times on the day preceding sacrifice. Vertical arrows in a and b, labeled periductular cells;horizontal arrows, labeled ductular cells. Some of the periductular cells in b could be transitional ductule cells, c. L, liver cell; T, transitional ductule cells; P. periportalcell. Arrow, tight junction between one of the transitional ductule cells and the liver cells adjacent to a shared bile canaliculus (markers of transitional duct cells).Transitional duct cells are seen in large numbers after induction of oval cell proliferation. The periportal cell may also be a transitional duct cell. None of there cellsabuts a basement membrane. The hepatocyte shows toxic changes caused by the CD-AAF diet. The labeled cell in d (arrow) is a nondescript periportal cell that doesnot have transitional duct cell or Ito cell characteristics. This may be a putative liver stem cell or a poorly differentiated connective tissue cell. D, ductal cell. For moredetails see Ref. 41.

Biology, Phyllis Novikoff (44) demonstrated a marked increasein the number of transition duct cells within a few weeks ofexposure to a similar carcinogenic regimen. Although it isdifficult to find labeled cells earlier than 2 days after carcinogenexposure, we were able to observe some proliferating cells asearly as the first day (Fig. 1, a and d). Some of these cells weresmaller than the transition duct cells, did not form tight junctions with hepatocytes, and did not form a canaliculus withadjacent liver cells (41). We have tentatively concluded that thisis a true liver stem cell (11). However, the "oval" cells seen

later may arise either from this cell or from the transitionalduct cells.

The finding that most of the cells that proliferate in the liverearly during the early stages of carcinogenesis have a duct ortransition duct cell phenotype does not rule against the presenceof an even less differentiated stem cell. In normal tissues thatundergo rapid renewal, such as bone marrow, skin, or gastrointestinal epithelium, most of the proliferating cells demonstratesome differentiated characteristics and it is very difficult toidentify the stem cell of the tissue. Similarly, when liver cellsare stimulated to proliferate most cells with the capacity toproliferate would be expected to have an identifiable differentiated morphology, i.e., transition duct cells or cells of the canalof Hering, whereas the true stem cells, which are very few in

number, would be difficult to identify, particularly at later timesafter initiation of proliferation when many proliferating differentiated cells are present.

Historically, the presence of liver stem cells has been doubted.In the liver there are very few dividing cells [estimated as lessthan 1/20,000 cells (45)]. In addition, most studies of liver cellregeneration after partial hepatectomy, chemical injury, or viraltoxicity have shown that adult liver cells can divide to restoredestroyed liver parenchyma (45, 46). Localization of AFP producing cells following liver injury and restitution identified largedividing hepatocytes in both rats (47) and mice (48). Thus thereis no apparent need for a stem cell for liver regeneration.

However, Zajick et al. (49, 51) have studied the normal"turnover" of liver cells and find that there is continued pro

duction of hepatocytes (49) and bile duct cells (50). These arisein the portal zones and "stream" toward the central zones

accompanied by littoral cells (51). They conclude that the liverrenews its cells continuously exactly like the gastrointestinaltract and epidermis (51).

In addition, more recent studies indicate that, followingsevere liver injury or exposure of experimental animals tochemical hepatocarcinogenesis, there is proliferation of cells inthe liver with stem cell properties. Engelhardt et al. (52) foundthat whereas most of the AFP containing cells seen after CC14

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IS THERE A LIVER STEM CELL?

injury of mouse liver were differentiated hepatocytes, a fewsmaller cells that resembled oval cells were also positive. In astudy in rats it was found that most, if not all of the cellscontaining mRNA for AFP after CC14 injury were small non-

parynchmal cells (53). More recently, Tournier et al. (54) foundthat although there was a generalized increase in the mRNAfor AFP over most nonnecrotic hepatocytes after CC14 injuryin rats, large amounts of AFP protein and mRNA for AFPwere concentrated over proliferated oval cells and bile duct-likestructures after liver injury induced by D-galactosamine. Long-Evans's cinnamon rats, which spontaneously develop acute

hepatitis, have a proliferation of oval cells which appear todifferentiate into hepatocytes without malignant transformation (55). Thus, even though there is some disagreement amongthese studies in the extent of small cell proliferation and AFPproduction after liver cell injury, they each indicate the participation of a putative liver stem cell.

If there is a liver stem cell, it should play a important roleduring the morphogenesis of the liver during development. Atthis time our understanding of the development of the normalliver is not clear. The conventional view is that the liver arisesfrom a hepatic diverticulum formed from endothelium betweenthe foregut and the yolk sac. This diverticulum extends into themesoderm of the septum transversum where it gives rise to theparenchymal cells of the liver, with the mesodermal cells forming the capsule and fibrous tissue of the liver (56-58). However,

it is also possible that hepatocytes form from mesodermal cellsinto which the ductal endodermal cells extend (59). The conventional view is supported by the findings of Germain et al.(24) that at 12 days of embryological development the emerginghepatic cells in the rat embryo contain both ductal and hepa-tocyte markers. They conclude that there are at this stagebipotential precursor epithelial cells that are capable of differentiation into hepatocytes or into biliary epithelial cells. Theyfurther find that such differentiation in vitro may vary depending on culture medium supplements. Similar results have beenfound in our laboratory (60), but we have also found thatmesenchymal cells can express a liver cell phenotype at restricted times during embryogenesis.2 Nevertheless, there are

sufficient data to support the conclusion that the liver developsfrom a hepatocytic stem cell that has the potential to developinto a duct cell or a hepatocyte.

Studies on experimental injury to the pancreas also supporta liver/pancreas stem cell (61). Hepatocytes appear in the liversof rats with aging (62) or after experimental injury (63, 64), inhumans (65) or hamsters spontaneously (66), or in hamsterstreated with carcinogens (67). In rats these cells arise fromperiductular or ductular cells in a manner very similar, if notidentical, to that seen in the livers of rats exposed to carcinogens(68). Thus it may be concluded that the pancreas also containsa liver stem cell that has the potential of differentiating intoduct cells or hepatocytes.

In conclusion, for over 30 years the study of chemical hepa-tocarcinogenesis in the liver has concentrated on analysis of thenodules, based on the assumption that these were, in fact, truepremalignant lesions. However, in view of the more recentobservations an alternate hypothesis must be considered, i.e.,that liver cancer does not arise by dedifferentiation of hepatocytes through formation of foci and nodules but by aberrantdifferentiation of hepatic stem cells. The transitional duct cellor cells of the canals of Hering may serve as hepatic stem cells,

but the presence of a more primitive precursor must also begiven serious consideration.

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