Department of Surgery, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
Key Words. Stem cell • Progenitor cell • Oval cell • Liver regeneration
Correspondence: David A. Gerber, M.D., CB#7211, 4026 Burnett-Womack Building, Chapel Hill, North Carolina 27599-7211, USA. Telephone: 919-966-8008; Fax: 919-966-6308; e-mail: [email protected]
Received February 1, 2006; accepted for publication April 28, 2006.
First published online in STEM CELLS EXPRESS, May 4, 2006.
The field of stem cell biology has exploded with the study ofa wide range of cellular populations involving endodermal, mesenchymal,and ectodermal organs. One area of extensive study has includedthe identification of hepatic stem and progenitor cell subpopulations.Liver stem cells provide insights into the potential pathwaysinvolving liver regeneration that are independent of maturehepatocytes. Hepatic progenitor cells are either bipotent ormultipotent and capable of multiple rounds of replication. Theyhave been identified in fetal as well as adult liver. Variousinjury models have been used to expand this cellular compartment.The nomenclature, origin, and function of the hepatic progenitorcell populations are areas of ongoing debate. In this review,we will discuss the different definitions and functions of hepaticprogenitor cells as well as the current research efforts examiningtheir therapeutic potential.
Stem Cells Vol. 24 No. 8 August 2006, pp. 1833 -1840. OPEN ACCESS ARTICLE.
It has been suggested that when the regenerative ability ofmature hepatocytes is insufficient, then the capacity of theliver for innate cellular recovery occurs secondary to a separateand unique population of liver cells. The origin, nomenclature,and function of these cells have been a longstanding area ofstudy and debate. Hepatic progenitor cells have been broadlycharacterized as a wide range of cell populations by multiplescientific teams. These cells have been investigated from thevery early embryonic stages through adulthood, using a diverserange of experimental models.
A stem cell is typically characterized by its capacity for self-renewaland ability to give rise to multiple differentiated cellularpopulations (often termed cellular plasticity) . Despiteprogress in characterizing select stem cell populations fromdistinct organs (e.g., bone marrow, liver, the nervous system,etc.), a pattern of differences has become evident. These distinctionsare associated with the host and/or the injury model involvedwith isolating a specific cellular population. An additionaland oftentimes greater challenge when working with stem cellsis the lack of unique markers to identify these cells. Althoughsome markers are observed in stem cell subsets (e.g., the effluxof certain dyes appears to be a common property of hematopoieticstem cells),  stem cells are routinely characterized by theabsence rather than the presence of specific lineage-relatedmarkers.
The vast majority of liver-related stem cell research has focusedon either fetal-derived hepatic stem cells or oval cells. Theoval cell is typically defined as a unique cellular populationthat is generated from the biliary compartment in response tohepatic injury [3–8]. To complement the ongoing researchefforts involving these two populations, several investigatorshave more recently identified and isolated hepatic progenitorcells from adult tissue. These cells, like their embryonic counterparts,can be bipotent and are capable of multiple rounds of cell division[9, 10]. The nomenclature and function of described liver-relatedstem cell populations, including the progenitor cell populations,remains an area of dispute, as the majority of these cellularpopulations have not been used in a therapeutic approach toprovide organ-associated function. To further complicate thechallenge with nomenclature, adult liver stem cells are oftenreferred to as hepatic progenitor cells, hepatic oval cells,or both.
For the purposes of this review, we will characterize hepaticprogenitor cells as either somatic hepatic progenitor cells(those cells that can be isolated from adult liver without chemicalinsult or partial hepatectomy), fetal hepatic progenitor cells(due to their origin and isolation from within the developingliver bud), or the previously characterized oval cell. The roleof oval cells in liver regeneration and their potential as hepaticprogenitors will be discussed in further detail. With the varyingdescriptions of unique hepatic progenitor cell populations,it is possible that overlap exists among differing populations.
There is currently a dichotomy between our understanding ofthe processes involved in stem cell differentiation and organdevelopment compared with the unanswered questions relatingto the postnatal role of stem or progenitor cell populationsas they persist into adulthood. Are the latter groups undifferentiatedstem cell populations that persist beyond the fetal period orare they de novo stem cells generated by signals from the adultsomatic cellular compartment? Further comprehension of characterizationand the process by which select stem or progenitor cells undergodifferentiation will provide greater insight into tissue developmentand organogenesis . This understanding could also play arole in developing alternative cell therapy strategies. Thisreview focuses on multiple liver-derived stem or progenitorcell populations that have been isolated from liver tissue atvarious stages of development or in response to select injurymodels.
The cellular plasticity associated with various fetal tissuesmakes embryonic development an ideal place to search for stem/progenitorcell populations. During embryogenesis, the liver arises fromthe gut tube as an out-pouching, referred to as the liver bud.The liver bud begins to grow and differentiate, and subsequentcellular contact with the cardiac mesoderm and the productionof fibroblast growth factors (FGFs) in the local environmentinduces the endoderm toward hepatic development . The septumtransversum, another mesodermal derivative, also contributesto this process of hepatic differentiation. The septum transversumis in close proximity to the developing ventral foregut andproduces bone morphogenic proteins that contribute to the differentiationprocess from endoderm to the future liver .
As the liver bud grows, the cellular constituent of the liveris composed of hepatoblasts. Hepatoblasts are defined as theprecursors for hepatocytes as well as for cholangiocytes, thecells that form the biliary ductal system of the liver .The hepatoblasts have been characterized with various markers,including albumin, -fetoprotein, cytokeratin 17 (CK 17), andCK 19 [15, 16]. During the developmental process, the architectureof the mature liver becomes apparent with the differentiationof the hepatoblasts into hepatocytes and sinusoid formation.Examination of the various cell types in the 14-day-old fetalrat liver reveals three distinct cell populations: those solelyexpressing hepatocyte markers, such as -fetoprotein and albumin;a second population expressing biliary cell markers, such ascytokeratin; and a third population of cells expressing bothhepatic and biliary markers . This latter population isbipotent, capable of developing into biliary or hepatic celllines, and is thus thought of as the fetal source of hepaticprogenitor cells . Sandhu et al. transplanted rat fetalliver epithelial cells of varying ages into adult livers. Theydemonstrated that fetal liver epithelial cells from embryonicday (ED) 12–14 engrafted and were capable of forming bothhepatocytes and cholangiocytes . However, fetal liver epithelialcells from ED18 were only capable of producing hepatocytes,suggesting that they had lost their bipotent capacity .
Investigators studying the characterization of the stem cellcompartment in the fetal liver have focused on defining markersassociated with stem cells as well as those associated withhepatic cells and then identifying which cells possess a combinationof the markers. Petersen et al. demonstrated Thy-1, a markerof hematopoietic stem cells,  on specific populations offetal hepatocytes . These authors also established thata Thy-1-positive cell population also expressed CK-18, a hepatocyticmarker, within the fetal liver. Hepatic progenitor cells havealso been reported to express c-kit, a stem cell marker, alongwith CD34 and Thy-1 . Using c-kit as a marker, along with6- and ß1-integrin subunits, Suzuki et al. facilitatedflow-cytometric separation of progenitor type cells from otherhepatocytes in the developing mouse liver [8, 22].
The origin of the fetal hepatic stem cell populations has beena controversial topic. Early in development the fetal liveris the major location of hematopoiesis . It has been shownthat these hematopoietic cells release signals that direct theliver to grow and differentiate . Eventually, the functionof hematopoiesis is shifted out of the liver to the bone marrow.However, there is a question of whether some of the transienthematopoietic stem cells remain behind to form the hepatic stemcell compartment. Those investigators favoring this line ofreasoning point out that hepatic progenitor cells can sharecell surface markers associated with hematopoietic stem cells(such as CD34 [25, 26], Thy-1 , and c-kit ). However,there is a growing body of work that suggests that the hepaticprogenitor cells are an independent stem cell population, distinctfrom the hematopoietic stem cell population. Nierhoff et al.separated a highly enriched population of fetal hepatic progenitorcells using a Sca1+ antibody . These cells expressed bothhepatic and biliary markers (AFP and cytokeratin markers, respectively)but did not express c-kit or CD34 . However, in a conflictingstudy, Minguet et al. showed that neither the c-kit-negativeembryonic cells nor the positive fraction could differentiateinto hepatocytes. Interestingly, it was the c-kit (+low) fractionthat comprised the hepatic stem cells that differentiate intomature liver cells . In a separate study, embryonic hepaticprogenitors cultured in the presence of FGF expressed increasedlevels of c-kit, ck-19, and -fetoprotein . The subject ofmarkers is controversial and remains an area of active study.Current efforts at delineating the origin of the fetal progenitorcell population have also included short-term labeling techniques.Tremblay et al. harvested mouse embryos at various ages andlabeled the cells to observe migration patterns . They foundtwo distinct populations of cells: lateral cells that are constrainedto a specific tissue-fate and position axis, and medial cellsthat migrate along an anterior-posterior axis and contributeto multiple gut tissues . Further work with labeling techniqueswill help us gain understanding into the migration and differentiationof the progenitor cells.
Our group has isolated a hepatic progenitor cell populationfrom adult murine liver without a preceding injury to the liver. (Fig. 1A, 1B show two images of colony formation.) Earlyin culture, these cells express oval cell-like markers [32,33]. During prolonged culture, the expression profile shiftsaway from oval-cell markers toward albumin and cytokeratin,suggestive of differentiation along hepatocytic and biliarylineages . Mitaka et al. have described a similar populationof cells, from adult rat liver, termed "small hepatocytes" .These cells are smaller than their mature counterparts, approximatelyone-third to one-half the size. They are mononuclear and havea less differentiated morphologic appearance . These smallhepatocytes proliferated for more than 2 months in primary culture,whereas the mature cells stopped replicating after one to twocycles. The small hepatocytes formed colonies in culture anddifferentiated into functional mature hepatocytes, as demonstratedby an increasing albumin concentration within the culture media[34, 35]. Overturf et al. demonstrated liver recovery with thesecells by transplanting them into livers of fumarylacetoacetatehydrolase-deficient mice, a model of hereditary tyrosinemia. After transplantation, these adult hepatic cells replicatedand formed colonies, displaying a growth potential similar toembryonic-derived stem cells . Fujikawa et al. isolatedcells from adult murine livers that were -fetoprotein-positivewith immature endodermal characteristics . They found thatduring in vitro culture, these cells were capable of differentiatinginto both hepatic and biliary cell lineages, suggesting cellularbipotency .
Other groups have described novel methods for isolating progenitorcell populations, including isolating them under hypoxic conditionswhile simultaneously inducing cell aggregate formation .Cells isolated from adult murine livers using this method expressalbumin, AFP, and CK-19, markers consistently found on ovalcells and hepatic progenitor cell populations. However, theinvestigators did not find markers for mature hepatocytes, suchas tryptophan-2,3-dioxygenase (TO) or glucose-6-phosphatase(G6P). After the cells proliferated in culture, they began todifferentiate, and at day 40 they expressed both TO and G6P,suggesting that the cells had differentiated to a mature hepatocyte.
Although the potential for many of these adult-derived progenitorcells is promising, there is still a tremendous amount of investigationto be done before their therapeutic potential can be realized.Perhaps most significantly, there is an ongoing challenge withrespect to identifying unique markers that will support theisolation and purification of these cells from the mature hepatocyteand nonparenchymal cell populations within the liver.
The issue of dedifferentiation as a process that generates stemcell populations has been debated in recent years. Tateno etal. demonstrated that hepatocytes in culture expressed biliarymarkers [39, 40]. They also found that a small population ofthe mature hepatocytes began expressing the immature hepaticmarker -fetoprotein [39, 40]. Koenig et al. found that maturehepatocytes placed in culture formed colonies and with the rightmitogen could be stimulated into expressing biliary as wellas extrahepatic progenitor markers .
Since the identification and subsequent isolation of progenitorcells is a challenge in uninjured livers, several groups havedeveloped experimental models of liver injury to activate andaugment specific cell populations. Just as there are severalmodels for inducing liver injury, there are several theoriesas to which cell population is responsible for regeneratingthe lost or damaged liver parenchyma.
The typical response to a cellular vacuum secondary to a chemicalor surgical insult within the liver involves replication ofadult hepatocytes. Investigators have shown that mature hepatocytescan undergo 8 to 12 rounds of cellular division in responseto consecutive partial hepatectomies . However, when thereis massive injury to the liver and the mature hepatocyte isoverwhelmed or unable to replicate to repair the damage, thereis a second level cellular response that is believed to involvea progenitor cell subpopulation. The most well described cellpopulation involves activation of the oval cell compartmentto facilitate liver rebuilding .
The oval cell, located in the terminal bile ducts, is a potentialliver progenitor cell [9, 44]. Its nomenclature is derived fromthe oval-like appearance of the cell. These cells are a uniquepopulation, have a high nuclear to cytoplasmic ratio, and areactivated in the face of liver injury. Oval cells express immaturemarkers such as -fetoprotein, as well as mature hepatic markers(e.g., albumin) and biliary markers (e.g., cytokeratin-19) .Oval cells have been best studied using an injury model with2-acetylaminofluorene (2-AAF) followed by partial hepatectomy.2-AAF is metabolized to an N-hydroxyl derivative by hepatocytes,and this metabolite is cytotoxic, thus preventing the proliferationof the mature hepatocytes. Biliary epithelial cells lack theability to convert 2-AAF to its toxic metabolite. Alison etal. found that the cells in the terminal bile ducts were responsiblefor liver regeneration following 2-AAF treatment and partialhepatectomy . Within 14 days after 2-AAF treatment and partialhepatectomy, the cells of the biliary ductules had not onlyproliferated but also differentiated into hepatocytes. No regenerationof mature hepatocytes occurred following treatment with 2-AAF,further emphasizing the role of the biliary epithelial cells/ovalcells in liver regeneration .
In response to 2-AAF injury, oval cells form new ductular structuresthat are extensions of the canals of Hering and are surroundedby a continuous basement membrane. They attach at their distalend to a hepatocyte . Golding et al. used the 2-AAF/partialhepatectomy model to study proliferation and differentiationof periductal cells. Initially, the oval cells strongly expressedbiliary markers such as cytokeratin-19, but 1 week after partialhepatectomy, the newly formed ductules expressed albumin and-fetoprotein, hepatocytic markers. Again, the use of 2-AAF preventedthe mature hepatocytes from participating in the regenerativeprocess . Paku et al. looked at the effect of increasingdoses of 2-AAF on the oval cell response . They found thatat higher doses of 2-AAF, the differentiation process of theoval cells is delayed, the oval cells penetrate deeper intothe liver lobule, and the differentiating hepatocytes take ona more tortuous conformation. However, they found that at acellular level, the same process of oval cell differentiationinto hepatocyte was occurring, despite the delay and differingorganization at the tissue level .
Much like the controversy surrounding the origin of fetal liverstem cells, there has been some inquiry into the possibilitythat oval cells do not originate from the liver but insteadare activated bone marrow stem cells that migrate to the liverin response to injury. This hypothesis was based in part bythe fact that oval cells can express certain bone marrow stemcell markers, such as c-kit  and sca-1 . A recent studyinvolving a carbon tetrachloride injury model demonstrated thatonly a very small fraction of the oval cells were bone marrow-derived.The investigators demonstrated that this very low percentagewas due to cellular fusion . Another study involving lethallyirradiated mice, which were subsequently transplanted with bonemarrow cells and then subjected to various hepatic injury models,showed that none of the newly formed hepatocyte clusters expressedmarkers of the transplanted bone marrow . These studiesshow only a minor cellular contribution with respect to liverrepopulation.
Another model of liver injury involves retrorsine treatmentfollowed by partial hepatectomy [17, 52, 53]. Retrorsine isa pyrrolizidine alkaloid that inhibits hepatocyte cell division.In a non-retrorsine-treated partial hepatectomy animal model,the mature hepatocytes undergo cell division to compensate forthe loss of parenchyma. However, after retrorsine treatment,the mature hepatocytes are unable to undergo cell division andcannot repair the damage [54, 55]. Gordon et al.  foundthat liver repair was accomplished through a population of cellsthey termed "small hepatocyte like progenitor cells." (Fig. 2A,2B demonstrate expansion of a cluster of "small hepatocyte likeprogenitor cells" from day 6 through day 14 after partial hepatectomy.)The authors reported that these cells share markers with fetalhepatocytes, mature hepatocytes, and oval cells but are a distinctlydifferent population. In their model of retrorsine/partial hepatectomy,clusters of these small hepatocytes emerged and by day 14 occupied50% of the area of the parenchyma. (Fig. 2A, 2B demonstrateproliferation of small-hepatocyte cells.) They went on to demonstratethat the small hepatocyte compartment was not activated in animalsthat only underwent partial hepatectomy or retrorsine treatment. Phenotypic analysis of the small hepatocyte compartmentshowed that the cells expressed markers of hepatocyte differentiation,such as albumin and transferrin, but they did not express biliarymarkers, such as GST and BD.1 .
A majority of the research involving oval cells has been inrat models. However, recent experiments using a retrorsine/partialhepatectomy injury model in mice demonstrated proliferationof a liver progenitor cell compartment. After subjecting miceto retrorsine and partial hepatectomy, the authors found a populationof cells that expressed the hematopoietic stem cell markersc-kit and Thy-1. In vitro, this same population of cells differentiatedinto cells expressing either biliary markers (e.g., CK-19) orhepatic markers (e.g., albumin) .
Investigators have also focused on identifying bipotent cellsin the human liver. Baumann et al. used immunohistochemistryto study human livers in fulminant hepatic failure. They foundupregulation of a population of cells that expressed c-kit .Several investigators have identified subsets of human fetalliver cells that differentiate into hepatocytes and cholangiocytes[59, 60]. As liver development and differentiation progresses,these cells lose their dual marker expression, suggesting thatthey differentiated into a mature cell type . Although thisis a promising beginning, the investigation involving humanliver progenitor cells is in its nascent stages, and much remainsto be learned. Figure 3 is a summation of select cellular populationsalong with their identifying characteristics.
Although the ultimate application using hepatic/stem progenitorcells involves the development of an alternative therapy toliver transplantation for patients with liver failure, the prospectof this clinical reality remains in the future. There are currentlymore than 17,000 people on the waiting list for a liver transplant,with the majority of these patients suffering with cirrhosis,a manifestation of chronic liver injury (http://www.unos.org).In 2005, only approximately one-third of people waiting fora liver actually underwent a transplant . Using hepatocytesor hepatic progenitor cells as cellular therapy to replace damagedlivers could potentially help alleviate some of the challengesin solid organ transplantation. Transplantation of mature hepatocytepopulations has been successfully performed in numerous experimentalmodels, but with less success in the clinical setting. Orenet al. transplanted mature rat hepatocytes into the portal systemof analbuminemic rats and restored serum albumin levels .The limited success with hepatocyte transplantation [63–66]involves the necessity to transplant large numbers of cellsto achieve acceptable function, as well as providing an outletfor biliary excretion.
Several studies have looked at the potential of stem/progenitorcells in transplantation [52, 67–69]. Sandhu et al. isolatedfetal liver epithelial progenitor cells and transplanted themin syngeneic dipeptidyl petidase IV mutant mice subjected tovarious liver injuries . They found that the fetal liverepithelial progenitor cells, as opposed to the control groupof mature hepatocytes, continued to proliferate 6 months aftertransplantation. The fetal cells differentiated into cholangiocytesor hepatocytes depending on where they engrafted within therecipient liver. This is important, as mature hepatocytes donot form biliary structures , and one of the clinical challengesincludes engraftment of functional transplanted cells.
A concern about using hepatic progenitor cells for therapeutictransplantation is the link between oval cells and hepatocellularcarcinoma. An antigenic relationship between oval cells andhepatocellular carcinoma has been previously demonstrated. Inthe 1970s and 1980s, the oval cell was studied for its malignantpotential . Primary hepatocellular carcinoma has been shownto express oval cell markers OV-6, OC-2, and OC-3 [71, 72].In addition, activation of the oval cell compartment occursprior to hepatocellular carcinoma development [73–75].One of the links between hepatocellular carcinoma and hepaticprogenitor cells is the ductular reaction that occurs with chronichepatitis. As the proliferative ability of the mature hepatocytefails, there is activation of a cell population in the intrahepaticbiliary tree that is thought to represent a potential stem cellcompartment . Falkowski et al. also showed that this ductularreaction occurred with various forms of liver injury . Inaddition, many of the phenotypic properties of hepatocellularcarcinomas are shared with hepatic progenitor cells, suggestinga common origin . In addition to the carcinogenic potential,human liver stem cells have been implicated in several diseases,such as alcoholic liver disease and nonalcoholic fatty liverdisease. Roskams et al. present a good review of the role ofliver stem cells in various pathologies . However, no definitivelink between adult or fetal hepatic progenitor cells and carcinomahas been clearly demonstrated. Perhaps this population of cellswill not have the same carcinogenic potential, but this is certainlyan area of research that will require further exploration.
Bioartificial liver (BAL) systems attempt to provide supportivefunction for a patient with liver disease while addressing theissue of malignant potential and immunologic reaction by creatinga barrier between the functioning hepatocytes and the patient.This field has been extensively studied over the past few decades,with several studies reaching preclinical trials as investigatorshave analyzed BAL design and cell source [79–83]. Parket al. present a concise review of BAL including the most recentstage III clinical trial. In the review, the authors point tothe current pitfalls associated with BAL . Many of theseissues, such as cellular viability and xenografts, may be dealtwith by using species-specific stem cells.
In summary, the field of hepatic stem cell study has undergonetremendous growth during the past decade. The initial phaseof this research has focused on isolating and characterizingselect cellular populations, a critical first step. It is anticipatedthat over the next few years we will see an in-depth investigationof the hepatic stem/progenitor cell populations with respectto differentiation signals. A balance must be achieved betweendeveloping a critical mass of functional cells while controllingthe regenerative capacity of the progenitor cells . Furtherexploration into methods of transplantation and engraftmentof these cells will be required. As we move forward in the fieldof hepatic progenitor cell research, these hurdles must be overcomefor cell transplantation involving stem/progenitor cells tobecome a therapeutic possibility.
This work was supported by NIH Grant T32-GM008450 (M.H.W.) andNIH Grant 5K08DK59302 (D.A.G.). We thank Dr. Bill Coleman forsupplying images of small hepatocyte-like progenitor cells.