Cell Responses in Injury Models
- Hepatic Stem Cells: In Search of

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 [42]. 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 [43].

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 {alpha}-fetoprotein, as well as mature hepatic markers(e.g., albumin) and biliary markers (e.g., cytokeratin-19) [9].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 [45]. 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 [45].

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 [46]. 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{alpha}-fetoprotein, hepatocytic markers. Again, the use of 2-AAF preventedthe mature hepatocytes from participating in the regenerativeprocess [47]. Paku et al. looked at the effect of increasingdoses of 2-AAF on the oval cell response [48]. 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 [48].

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 [21] and sca-1 [49]. 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 [50]. 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 [51]. 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. [54] 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[54]. 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 [54].

Gordon et al. also explored the therapeutic potential of thesesmall hepatocyte-like progenitor cells through transplantation[56]. Using a model of retrorsine/partial hepatectomy injury,the small hepatocyte compartment was activated. These cellswere harvested, established in short-term culture, and subsequentlytransplanted into livers of syngeneic rats. They found thatthese cells did not proliferate in culture, but they did engraftinto the hepatic plates of the recipient livers. Once engrafted,these cells proliferated and differentiated into mature hepatocytes[56].

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) [57].

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 [58].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 [60]. 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.

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