The field of stem cell biology has exploded with the study of a wide range of cellular populations involving endodermal, mesenchymal, and ectodermal organs. One area of extensive study has included the identification of hepatic stem and progenitor cell subpopulations. Liver stem cells provide insights into the potential pathways involving liver regeneration that are independent of mature hepatocytes. Hepatic progenitor cells are either bipotent or multipotent and capable of multiple rounds of replication. They have been identified in fetal as well as adult liver. Various injury models have been used to expand this cellular compartment. The nomenclature, origin, and function of the hepatic progenitor cell populations are areas of ongoing debate. In this review, we will discuss the different definitions and functions of hepatic progenitor cells as well as the current research efforts examining their therapeutic potential.
It has been suggested that when the regenerative ability of mature hepatocytes is insufficient, then the capacity of the liver for innate cellular recovery occurs secondary to a separate and unique population of liver cells. The origin, nomenclature, and function of these cells have been a longstanding area of study and debate. Hepatic progenitor cells have been broadly characterized as a wide range of cell populations by multiple scientific teams. These cells have been investigated from the very early embryonic stages through adulthood, using a diverse range of experimental models.
A stem cell is typically characterized by its capacity for self-renewal and ability to give rise to multiple differentiated cellular populations (often termed cellular plasticity) . Despite progress in characterizing select stem cell populations from distinct organs (e.g., bone marrow, liver, the nervous system, etc.), a pattern of differences has become evident. These distinctions are associated with the host and/or the injury model involved with isolating a specific cellular population. An additional and oftentimes greater challenge when working with stem cells is the lack of unique markers to identify these cells. Although some markers are observed in stem cell subsets (e.g., the efflux of certain dyes appears to be a common property of hematopoietic stem cells),  stem cells are routinely characterized by the absence rather than the presence of specific lineage-related markers.
Liver Stem Cell Populations
The vast majority of liver-related stem cell research has focused on either fetal-derived hepatic stem cells or oval cells. The oval cell is typically defined as a unique cellular population that is generated from the biliary compartment in response to hepatic injury [3, , , , –8]. To complement the ongoing research efforts involving these two populations, several investigators have more recently identified and isolated hepatic progenitor cells 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-related stem cell populations, including the progenitor cell populations, remains an area of dispute, as the majority of these cellular populations have not been used in a therapeutic approach to provide organ-associated function. To further complicate the challenge with nomenclature, adult liver stem cells are often referred to as hepatic progenitor cells, hepatic oval cells, or both.
For the purposes of this review, we will characterize hepatic progenitor cells as either somatic hepatic progenitor cells (those cells that can be isolated from adult liver without chemical insult or partial hepatectomy), fetal hepatic progenitor cells (due to their origin and isolation from within the developing liver bud), or the previously characterized oval cell. The role of oval cells in liver regeneration and their potential as hepatic progenitors will be discussed in further detail. With the varying descriptions of unique hepatic progenitor cell populations, it is possible that overlap exists among differing populations.
There is currently a dichotomy between our understanding of the processes involved in stem cell differentiation and organ development compared with the unanswered questions relating to the postnatal role of stem or progenitor cell populations as they persist into adulthood. Are the latter groups undifferentiated stem cell populations that persist beyond the fetal period or are they de novo stem cells generated by signals from the adult somatic cellular compartment? Further comprehension of characterization and the process by which select stem or progenitor cells undergo differentiation will provide greater insight into tissue development and organogenesis . This understanding could also play a role in developing alternative cell therapy strategies. This review focuses on multiple liver-derived stem or progenitor cell populations that have been isolated from liver tissue at various stages of development or in response to select injury models.
The cellular plasticity associated with various fetal tissues makes embryonic development an ideal place to search for stem/progenitor cell populations. During embryogenesis, the liver arises from the gut tube as an out-pouching, referred to as the liver bud. The liver bud begins to grow and differentiate, and subsequent cellular contact with the cardiac mesoderm and the production of fibroblast growth factors (FGFs) in the local environment induces the endoderm toward hepatic development . The septum transversum, another mesodermal derivative, also contributes to this process of hepatic differentiation. The septum transversum is in close proximity to the developing ventral foregut and produces bone morphogenic proteins that contribute to the differentiation process from endoderm to the future liver .
As the liver bud grows, the cellular constituent of the liver is composed of hepatoblasts. Hepatoblasts are defined as the precursors for hepatocytes as well as for cholangiocytes, the cells that form the biliary ductal system of the liver . The hepatoblasts have been characterized with various markers, including albumin, α-fetoprotein, cytokeratin 17 (CK 17), and CK 19 [15, 16]. During the developmental process, the architecture of the mature liver becomes apparent with the differentiation of the hepatoblasts into hepatocytes and sinusoid formation. Examination of the various cell types in the 14-day-old fetal rat liver reveals three distinct cell populations: those solely expressing hepatocyte markers, such as α-fetoprotein and albumin; a second population expressing biliary cell markers, such as cytokeratin; and a third population of cells expressing both hepatic and biliary markers . This latter population is bipotent, capable of developing into biliary or hepatic cell lines, and is thus thought of as the fetal source of hepatic progenitor cells . Sandhu et al. transplanted rat fetal liver epithelial cells of varying ages into adult livers. They demonstrated that fetal liver epithelial cells from embryonic day (ED) 12–14 engrafted and were capable of forming both hepatocytes and cholangiocytes . However, fetal liver epithelial cells from ED18 were only capable of producing hepatocytes, suggesting that they had lost their bipotent capacity .
Investigators studying the characterization of the stem cell compartment in the fetal liver have focused on defining markers associated with stem cells as well as those associated with hepatic cells and then identifying which cells possess a combination of the markers. Petersen et al. demonstrated Thy-1, a marker of hematopoietic stem cells,  on specific populations of fetal hepatocytes . These authors also established that a Thy-1-positive cell population also expressed CK-18, a hepatocytic marker, within the fetal liver. Hepatic progenitor cells have also been reported to express c-kit, a stem cell marker, along with CD34 and Thy-1 . Using c-kit as a marker, along with α6- and β1-integrin subunits, Suzuki et al. facilitated flow-cytometric separation of progenitor type cells from other hepatocytes in the developing mouse liver [8, 22].
The origin of the fetal hepatic stem cell populations has been a controversial topic. Early in development the fetal liver is the major location of hematopoiesis . It has been shown that these hematopoietic cells release signals that direct the liver to grow and differentiate . Eventually, the function of hematopoiesis is shifted out of the liver to the bone marrow. However, there is a question of whether some of the transient hematopoietic stem cells remain behind to form the hepatic stem cell compartment. Those investigators favoring this line of reasoning point out that hepatic progenitor cells can share cell 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 hepatic progenitor cells are an independent stem cell population, distinct from the hematopoietic stem cell population. Nierhoff et al. separated a highly enriched population of fetal hepatic progenitor cells using a Sca1+ antibody . These cells expressed both hepatic and biliary markers (AFP and cytokeratin markers, respectively) but did not express c-kit or CD34 . However, in a conflicting study, Minguet et al. showed that neither the c-kit-negative embryonic cells nor the positive fraction could differentiate into hepatocytes. Interestingly, it was the c-kit (+low) fraction that comprised the hepatic stem cells that differentiate into mature liver cells . In a separate study, embryonic hepatic progenitors cultured in the presence of FGF expressed increased levels of c-kit, ck-19, and α-fetoprotein . The subject of markers is controversial and remains an area of active study. Current efforts at delineating the origin of the fetal progenitor cell population have also included short-term labeling techniques. Tremblay et al. harvested mouse embryos at various ages and labeled the cells to observe migration patterns . They found two distinct populations of cells: lateral cells that are constrained to a specific tissue-fate and position axis, and medial cells that migrate along an anterior-posterior axis and contribute to multiple gut tissues . Further work with labeling techniques will help us gain understanding into the migration and differentiation of the progenitor cells.
Adult-Derived Hepatic Progenitor Cell Populations
Our group has isolated a hepatic progenitor cell population from adult murine liver without a preceding injury to the liver . (Fig. 1A, 1B show two images of colony formation.) Early in culture, these cells express oval cell-like markers [32, 33]. During prolonged culture, the expression profile shifts away from oval-cell markers toward albumin and cytokeratin, suggestive of differentiation along hepatocytic and biliary lineages . Mitaka et al. have described a similar population of cells, from adult rat liver, termed “small hepatocytes” . These cells are smaller than their mature counterparts, approximately one-third to one-half the size. They are mononuclear and have a less differentiated morphologic appearance . These small hepatocytes proliferated for more than 2 months in primary culture, whereas the mature cells stopped replicating after one to two cycles. The small hepatocytes formed colonies in culture and differentiated into functional mature hepatocytes, as demonstrated by an increasing albumin concentration within the culture media [34, 35]. Overturf et al. demonstrated liver recovery with these cells by transplanting them into livers of fumarylacetoacetate hydrolase-deficient mice, a model of hereditary tyrosinemia . After transplantation, these adult hepatic cells replicated and formed colonies, displaying a growth potential similar to embryonic-derived stem cells . Fujikawa et al. isolated cells from adult murine livers that were α-fetoprotein-positive with immature endodermal characteristics . They found that during in vitro culture, these cells were capable of differentiating into both hepatic and biliary cell lineages, suggesting cellular bipotency .
Other groups have described novel methods for isolating progenitor cell populations, including isolating them under hypoxic conditions while simultaneously inducing cell aggregate formation . Cells isolated from adult murine livers using this method express albumin, AFP, and CK-19, markers consistently found on oval cells and hepatic progenitor cell populations. However, the investigators did not find markers for mature hepatocytes, such as tryptophan-2,3-dioxygenase (TO) or glucose-6-phosphatase (G6P). After the cells proliferated in culture, they began to differentiate, 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 progenitor cells is promising, there is still a tremendous amount of investigation to be done before their therapeutic potential can be realized. Perhaps most significantly, there is an ongoing challenge with respect to identifying unique markers that will support the isolation and purification of these cells from the mature hepatocyte and nonparenchymal cell populations within the liver.
The issue of dedifferentiation as a process that generates stem cell populations has been debated in recent years. Tateno et al. demonstrated that hepatocytes in culture expressed biliary markers [39, 40]. They also found that a small population of the mature hepatocytes began expressing the immature hepatic marker α-fetoprotein [39, 40]. Koenig et al. found that mature hepatocytes placed in culture formed colonies and with the right mitogen could be stimulated into expressing biliary as well as extrahepatic progenitor markers .
Cell Responsesin Injury Models
Since the identification and subsequent isolation of progenitor cells is a challenge in uninjured livers, several groups have developed experimental models of liver injury to activate and augment specific cell populations. Just as there are several models for inducing liver injury, there are several theories as to which cell population is responsible for regenerating the lost or damaged liver parenchyma.
The typical response to a cellular vacuum secondary to a chemical or surgical insult within the liver involves replication of adult hepatocytes. Investigators have shown that mature hepatocytes can undergo 8 to 12 rounds of cellular division in response to consecutive partial hepatectomies . However, when there is massive injury to the liver and the mature hepatocyte is overwhelmed or unable to replicate to repair the damage, there is a second level cellular response that is believed to involve a progenitor cell subpopulation. The most well described cell population involves activation of the oval cell compartment to facilitate liver rebuilding .
The oval cell, located in the terminal bile ducts, is a potential liver progenitor cell [9, 44]. Its nomenclature is derived from the oval-like appearance of the cell. These cells are a unique population, have a high nuclear to cytoplasmic ratio, and are activated in the face of liver injury. Oval cells express immature markers such as α-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 with 2-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 proliferation of the mature hepatocytes. Biliary epithelial cells lack the ability to convert 2-AAF to its toxic metabolite. Alison et al. found that the cells in the terminal bile ducts were responsible for liver regeneration following 2-AAF treatment and partial hepatectomy . Within 14 days after 2-AAF treatment and partial hepatectomy, the cells of the biliary ductules had not only proliferated but also differentiated into hepatocytes. No regeneration of mature hepatocytes occurred following treatment with 2-AAF, further emphasizing the role of the biliary epithelial cells/oval cells in liver regeneration .
In response to 2-AAF injury, oval cells form new ductular structures that are extensions of the canals of Hering and are surrounded by a continuous basement membrane. They attach at their distal end to a hepatocyte . Golding et al. used the 2-AAF/partial hepatectomy model to study proliferation and differentiation of periductal cells. Initially, the oval cells strongly expressed biliary markers such as cytokeratin-19, but 1 week after partial hepatectomy, the newly formed ductules expressed albumin and α-fetoprotein, hepatocytic markers. Again, the use of 2-AAF prevented the mature hepatocytes from participating in the regenerative process . Paku et al. looked at the effect of increasing doses of 2-AAF on the oval cell response . They found that at higher doses of 2-AAF, the differentiation process of the oval cells is delayed, the oval cells penetrate deeper into the liver lobule, and the differentiating hepatocytes take on a more tortuous conformation. However, they found that at a cellular level, the same process of oval cell differentiation into hepatocyte was occurring, despite the delay and differing organization at the tissue level .
Much like the controversy surrounding the origin of fetal liver stem cells, there has been some inquiry into the possibility that oval cells do not originate from the liver but instead are activated bone marrow stem cells that migrate to the liver in response to injury. This hypothesis was based in part by the fact that oval cells can express certain bone marrow stem cell markers, such as c-kit  and sca-1 . A recent study involving a carbon tetrachloride injury model demonstrated that only a very small fraction of the oval cells were bone marrow-derived. The investigators demonstrated that this very low percentage was due to cellular fusion . Another study involving lethally irradiated mice, which were subsequently transplanted with bone marrow cells and then subjected to various hepatic injury models, showed that none of the newly formed hepatocyte clusters expressed markers of the transplanted bone marrow . These studies show only a minor cellular contribution with respect to liver repopulation.
Another model of liver injury involves retrorsine treatment followed by partial hepatectomy [17, 52, 53]. Retrorsine is a pyrrolizidine alkaloid that inhibits hepatocyte cell division. In a non-retrorsine-treated partial hepatectomy animal model, the mature hepatocytes undergo cell division to compensate for the loss of parenchyma. However, after retrorsine treatment, the mature hepatocytes are unable to undergo cell division and cannot repair the damage [54, 55]. Gordon et al.  found that liver repair was accomplished through a population of cells they termed “small hepatocyte like progenitor cells.” (Fig. 2A, 2B demonstrate expansion of a cluster of “small hepatocyte like progenitor cells” from day 6 through day 14 after partial hepatectomy.) The authors reported that these cells share markers with fetal hepatocytes, mature hepatocytes, and oval cells but are a distinctly different population. In their model of retrorsine/partial hepatectomy, clusters of these small hepatocytes emerged and by day 14 occupied 50% of the area of the parenchyma. (Fig. 2A, 2B demonstrate proliferation of small-hepatocyte cells.) They went on to demonstrate that the small hepatocyte compartment was not activated in animals that only underwent partial hepatectomy or retrorsine treatment . Phenotypic analysis of the small hepatocyte compartment showed that the cells expressed markers of hepatocyte differentiation, such as albumin and transferrin, but they did not express biliary markers, such as GST and BD.1 .
Gordon et al. also explored the therapeutic potential of these small hepatocyte-like progenitor cells through transplantation . Using a model of retrorsine/partial hepatectomy injury, the small hepatocyte compartment was activated. These cells were harvested, established in short-term culture, and subsequently transplanted into livers of syngeneic rats. They found that these cells did not proliferate in culture, but they did engraft into the hepatic plates of the recipient livers. Once engrafted, these cells proliferated and differentiated into mature hepatocytes .
A majority of the research involving oval cells has been in rat models. However, recent experiments using a retrorsine/partial hepatectomy injury model in mice demonstrated proliferation of a liver progenitor cell compartment. After subjecting mice to retrorsine and partial hepatectomy, the authors found a population of cells that expressed the hematopoietic stem cell markers c-kit and Thy-1. In vitro, this same population of cells differentiated into cells expressing either biliary markers (e.g., CK-19) or hepatic markers (e.g., albumin) .
Investigators have also focused on identifying bipotent cells in the human liver. Baumann et al. used immunohistochemistry to study human livers in fulminant hepatic failure. They found upregulation of a population of cells that expressed c-kit . Several investigators have identified subsets of human fetal liver cells that differentiate into hepatocytes and cholangiocytes [59, 60]. As liver development and differentiation progresses, these cells lose their dual marker expression, suggesting that they differentiated into a mature cell type . Although this is a promising beginning, the investigation involving human liver progenitor cells is in its nascent stages, and much remains to be learned. Figure 3 is a summation of select cellular populations along with their identifying characteristics.
Liver Stem Cellsand Transplantation
Although the ultimate application using hepatic/stem progenitor cells involves the development of an alternative therapy to liver transplantation for patients with liver failure, the prospect of this clinical reality remains in the future. There are currently more 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 for a liver actually underwent a transplant . Using hepatocytes or hepatic progenitor cells as cellular therapy to replace damaged livers could potentially help alleviate some of the challenges in solid organ transplantation. Transplantation of mature hepatocyte populations has been successfully performed in numerous experimental models, but with less success in the clinical setting. Oren et al. transplanted mature rat hepatocytes into the portal system of analbuminemic rats and restored serum albumin levels . The limited success with hepatocyte transplantation [63, , –66] involves the necessity to transplant large numbers of cells to achieve acceptable function, as well as providing an outlet for biliary excretion.
Several studies have looked at the potential of stem/progenitor cells in transplantation [52, 67, –69]. Sandhu et al. isolated fetal liver epithelial progenitor cells and transplanted them in syngeneic dipeptidyl petidase IV mutant mice subjected to various liver injuries . They found that the fetal liver epithelial progenitor cells, as opposed to the control group of mature hepatocytes, continued to proliferate 6 months after transplantation. The fetal cells differentiated into cholangiocytes or hepatocytes depending on where they engrafted within the recipient liver. This is important, as mature hepatocytes do not form biliary structures , and one of the clinical challenges includes engraftment of functional transplanted cells.
A concern about using hepatic progenitor cells for therapeutic transplantation is the link between oval cells and hepatocellular carcinoma. An antigenic relationship between oval cells and hepatocellular carcinoma has been previously demonstrated. In the 1970s and 1980s, the oval cell was studied for its malignant potential . Primary hepatocellular carcinoma has been shown to express oval cell markers OV-6, OC-2, and OC-3 [71, 72]. In addition, activation of the oval cell compartment occurs prior to hepatocellular carcinoma development [73, –75]. One of the links between hepatocellular carcinoma and hepatic progenitor cells is the ductular reaction that occurs with chronic hepatitis. As the proliferative ability of the mature hepatocyte fails, there is activation of a cell population in the intrahepatic biliary tree that is thought to represent a potential stem cell compartment . Falkowski et al. also showed that this ductular reaction occurred with various forms of liver injury . In addition, many of the phenotypic properties of hepatocellular carcinomas are shared with hepatic progenitor cells, suggesting a 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 liver disease. Roskams et al. present a good review of the role of liver stem cells in various pathologies . However, no definitive link between adult or fetal hepatic progenitor cells and carcinoma has been clearly demonstrated. Perhaps this population of cells will not have the same carcinogenic potential, but this is certainly an area of research that will require further exploration.
Bioartificial liver (BAL) systems attempt to provide supportive function for a patient with liver disease while addressing the issue of malignant potential and immunologic reaction by creating a 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 investigators have analyzed BAL design and cell source [79, , , –83]. Park et al. present a concise review of BAL including the most recent stage III clinical trial. In the review, the authors point to the current pitfalls associated with BAL . Many of these issues, such as cellular viability and xenografts, may be dealt with by using species-specific stem cells.
In summary, the field of hepatic stem cell study has undergone tremendous growth during the past decade. The initial phase of this research has focused on isolating and characterizing select cellular populations, a critical first step. It is anticipated that over the next few years we will see an in-depth investigation of the hepatic stem/progenitor cell populations with respect to differentiation signals. A balance must be achieved between developing a critical mass of functional cells while controlling the regenerative capacity of the progenitor cells . Further exploration into methods of transplantation and engraftment of these cells will be required. As we move forward in the field of hepatic progenitor cell research, these hurdles must be overcome for cell transplantation involving stem/progenitor cells to become a therapeutic possibility.
The authors indicate no potential conflicts of interest.
This work was supported by NIH Grant T32-GM008450 (M.H.W.) and NIH Grant 5K08DK59302 (D.A.G.). We thank Dr. Bill Coleman for supplying images of small hepatocyte-like progenitor cells.