Small Hepatocyte-like Progenitor Cells Research Articles

Current Perspectives and Progress in Preoperative Portal Vein Embolization with Stem Cell Augmentation (PVESA).

Portal vein embolization with stem cell augmentation (PVESA) is an emerging approach for enhancing the growth of the liver segment that will remain after surgery (i.e., future liver remnant, FLR) in patients with liver cancer. Conventional portal vein embolization (PVE) aims to induce preoperative FLR growth, but it has a risk of failure in patients with underlying liver dysfunction and comorbid illnesses. PVESA combines PVE with stem cell therapy to potentially improve FLR size and function more effectively and efficiently. Various types of stem cells can help improve liver growth by secreting paracrine signals for hepatocyte growth or by transforming into hepatocytes. Mesenchymal stem cells (MSCs), unrestricted somatic stem cells, and small hepatocyte-like progenitor cells have been used to augment liver growth in preclinical animal models, while clinical studies have demonstrated the benefit of CD133 + bone marrow-derived MSCs and hematopoietic stem cells. These investigations have shown that PVESA is generally safe and enhances liver growth after PVE. However, optimizing the selection, collection, and application of stem cells remains crucial to maximize benefits and minimize risks. Additionally, advanced stem cell technologies, such as priming, genetic modification, and extracellular vesicle-based therapy, that could further enhance efficacy outcomes should be evaluated. Despite its potential, PVESA requires more investigations, particularly mechanistic studies that involve orthotopic animal models of liver cancer with concomitant liver injury as well as larger human trials.

Isolation of Small Hepatocyte-Like Progenitor Cells from Retrorsine/Partial Hepatectomy Rat Livers by Laser Microdissection.

Small hepatocyte-like progenitor cells (SHPCs) are known as liver stem/progenitor cells (LSPCs). SHPCs transiently appear and form clusters in rat livers treated with retrorsine (Ret) and a 70% partial hepatectomy (PH). The Ret/PH model has been used widely to analyze the effectiveness of cell transplantation and the mechanisms of LSPC proliferation. Laser microdissection (LMD) is a powerful tool that can excise and collect specific areas of cells from a tissue slice with a laser under a microscope. These cells exhibiting morphological alterations different from the surrounding cells may be analyzed by gene expression profiling. Specific markers of SHPCs have not yet been identified, in part, because it is difficult to isolate SHPCs from the liver using fluorescence or magnetic-activated cell sorting. To examine the underlying mechanism for SHPC growth, we established comprehensive gene expression profiles for SHPCs captured from liver sections using LMD. In this chapter, we introduce a method to isolate SHPCs from liver tissue sections using LMD for gene expression analysis.

Transplantation of Thy1+ Cells Accelerates Liver Regeneration by Enhancing the Growth of Small Hepatocyte-Like Progenitor Cells via IL17RB Signaling.

Small hepatocyte-like progenitor cells (SHPCs) transiently form clusters in rat livers treated with retrorsine (Ret)/70% partial hepatectomy (PH). When Thy1+ cells isolated from d-galactosamine-treated rat livers were transplanted into the livers of Ret/PH-treated rats, the mass of the recipient liver transiently increased during the first 30 days after transplantation, suggesting that liver regeneration was enhanced. Here we addressed how Thy1+ cell transplantation stimulates liver regeneration. We found that the number and size of SHPC clusters increased in the liver at 14 days after transplantation. GeneChip analysis revealed that interleukin 17 receptor b (IL17rb) expression significantly increased in SHPCs from livers transplanted with Thy1+ cells. We subsequently searched for ligand-expressing cells and found that sinusoidal endothelial cells (SECs) and Kupffer cells expressed Il17b and Il25, respectively. Moreover, extracellular vesicles (EVs) separated from the conditioned medium of Thy1+ cell culture induced IL17b and IL25 expression in SECs and Kupffer cells, respectively. Furthermore, EVs enhanced IL17rb expression in small hepatocytes (SHs), which are hepatocytic progenitor cells; in culture, IL17B stimulated the growth of SHs. These results suggest that Thy1-EVs coordinate IL17RB signaling to enhance liver regeneration by targeting SECs, Kupffer cells, and SHPCs. Indeed, the administration of Thy1-EVs increased the number and size of SHPC clusters in Ret/PH-treated rat livers. Sixty days post-transplantation, most expanded SHPCs entered cellular senescence, and the enlarged liver returned to its normal size. In conclusion, Thy1+ cell transplantation enhanced liver regeneration by promoting the proliferation of intrinsic hepatic progenitor cells via IL17RB signaling. Stem Cells 2017;35:920-931.

Small hepatocyte-like progenitor cells may be a Hedgehog signaling pathway-controlled subgroup of liver stem cells.

The present study aimed to investigate the expression levels of components of the Hedgehog signaling pathway (HH) during the proliferation of a liver stem cell subgroup, namely small hepatocyte-like progenitor cells (SHPCs). Retrorsine-treated Fisher 344 rats underwent a partial hepatectomy (PH) to induce the proliferation of SHPCs, after which reverse transcription-polymerase chain reaction (PCR), quantitative PCR, immunohistochemistry and western blot analysis were performed to analyze the expression of various components of the HH in primary SHPCs at different times points post-PH. A number of components of the HH, including Indian hedgehog (IHH), patched (PTCH), smoothened and glioma-associated oncogene (GLI)1, 2 and 3, were continuously expressed and showed dynamic changes in proliferating SHPCs. In addition, the expression levels of IHH, PTCH and GLI1 were significantly different as compared with those of the control group at the same time point, and there were significant differences among the various time points in the experimental group (P<0.01). Furthermore, there was an association between the postoperative day and expression levels of HH components in the retrorsine-treated group. An immunohistochemical analysis demonstrated that PTCH was also expressed at the protein level. In conclusion, the results of the present study suggested that the HH was continuously activated during the proliferation of SHPCs, thus indicating that SHPCs may be a subgroup of stem cells that are regulated by the HH.

Contribution of extrahepatic small cells resembling small hepatocyte-like progenitor cells to liver mass maintenance in transplantation model of retrorsine-pretreated liver

PurposeRetrorsine selectively inhibits hepatocyte proliferation and following liver injury evokes small hepatocyte-like progenitor cells. The aim of this study is to find out whether endogenous extrahepatic cells contribute to small hepatocyte-like progenitor cells after retrorsine treatment.MethodsWild-type Lewis rat liver exposed to retrorsine was transplanted into GFP transgenic Lewis rat. GFP positive, albumin-producing polygonal cells were expected as reciepient-derived hepatocyte-like cells.ResultsFour weeks after transplantation of 50% volume of retrorsine-pretreated liver, the rate of GFP positive hepatocyte-like cells was 0.02365%. Majority of these cells resided as single cells and their cell size was significantly larger than that of normal hepatocytes (mean cell size; 799.4 um2 vs. 451.3 um2, p<0.0001). At eight weeks, clusters of GFP positive small-size albumin-producing cells appeared and occupied 0.00759% of hepatocytes. The morphology of these cells was similar to that of small hepatocyte-like progenitor cells, 12.5% of them were Ki67 positive, majority of them were negative for CYP1A2 staining, and some clusters contained larger cells indicating further maturation.ConclusionEndogenous extrahepatic cells can form a cluster of small cells resembling small hepatocyte-like progenitor cells in a transplanted retrorsine-pretreated liver. The contribution of extrahepatic cells to liver mass maintenance is quite low and its importance is unclear.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-446) contains supplementary material, which is available to authorized users.

Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers

The potential lineage relationship between hepatic oval cells, small hepatocyte-like progenitor cells (SHPCs), and hepatocytes in liver regeneration is debated. To test whether mature hepatocytes can give rise to SHPCs, rats with dipeptidyl peptidase IV (DPPIV) chimeric livers, which harbored endogenous DPPIV-deficient hepatocytes and transplanted DPPIV-positive hepatocytes, were subjected to retrorsine treatment followed by partial hepatectomy (PH). DPPIV-positive hepatocytes comprised about half of the DPPIV chimeric liver mass. Tissues from DPPIV chimeric livers after retrorsine/PH treatment showed large numbers of SHPC clusters. None of the SHPC clusters were stained positive for DPPIV in any analyzed samples. Furthermore, serial sections stained for gamma-glutamyl-transpeptidase (GGT, a marker of fetal hepatoblasts) and glucose-6-phosphatase (G6Pase, a marker of mature hepatocytes) showed inverse expression of the two enzymes and a staining pattern consistent with a lineage that begins with GGT(+)/G6Pase(-) to GGT(-)/G6Pase(+) within a single SHPC cluster. Using double immunofluorescence staining for markers specific for hepatic oval cells and hepatocytes in serial sections, oval cell proliferations with CK-19(+)/laminin(+) and OV-6(+)/C/EBP-α(-) were shown to extend from periportal areas into the SPHC clusters, differentiating into hepatic lineage by progressive loss of CK-19/laminin expression and appearance of C/EBP-α expression towards the cluster side. Cells in the epithelial cell adhesion molecule (EpCAM(+)) SHPC clusters showed membranous EpCAM(+)/HNF-4α(+) (hepatocyte nuclear factor-4α) staining and were contiguous to the surrounding cytoplasmic EpCAM(+)/HNF-4α(-) ductular oval cells. Extensive elimination of oval cell response by repeated administration of 4,4'-methylenedianiline (DAPM) to retrorsine-exposed rats impaired the emergence of SHPC clusters. These findings highly suggest the hepatic oval cells but not mature hepatocytes as the origin of SHPC clusters in retrorsine-exposed rats.

Liver regeneration by small hepatocyte-like progenitor cells after necrotic injury by carbon tetrachloride in retrorsine-exposed rats

Liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the proliferation and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The activation, emergence, and outgrowth of SHPCs in response to the liver deficit generated through surgical PH have been well characterized. However, the participation of these cells in the restoration of hepatocyte numbers and regeneration of liver tissue mass following necrotic injury has not been investigated. To investigate the capacity of SHPCs to respond to necrotizing liver injury, we combined retrorsine treatment with the centrilobular-specific toxin carbon tetrachloride (CCl 4). Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age, followed by CCl 4 treatment (1500 mg/kg ip) 5 weeks later. Liver tissues were harvested at 3, 7, 14, 21, and 30-days post-injection. The dose of CCl 4 employed resulted in the necrotic destruction of 59 ± 2% of liver mass and elicited a regenerative response equivalent to that of surgical PH. Livers from retrorsine-exposed CCl 4-treated rats exhibit SHPC proliferation similar to retrorsine-exposed rats subjected to PH (RP). SHPCs appear at 3-days post-injection, continue to expand at 7-days and 14-days post-injection, and completely regenerate/restore the liver mass and structure in these animals by 30-days post-injection. The magnitude of SHPC response observed in the undamaged periportal zone of the liver in these animals is unaffected (versus RP rats) by the loss of the centrilobular region. The results of this study show that SHPCs are capable of regenerating liver after exposure to necrotizing agents and suggest that the progenitor cell of origin of the SHPCs is not restricted to the centrilobular zone of the liver parenchyma.

Cytokine-dependent activation of small hepatocyte-like progenitor cells in retrorsine-induced rat liver injury

Complete liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The mechanism(s) governing activation of SHPCs after PH in retrorsine-injured rats has not been investigated. We examined the possibility that SHPCs require cytokine priming prior to becoming growth factor responsive in this model of liver injury and regeneration. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age. Retrorsine-exposed and age-matched control rats were randomized into dexamethasone-treated and no DEX groups. DEX-treated animals were either given a single dose of DEX (2 mg/kg ip) at the time of PH or multiple DEX treatments (2 mg/kg ip each) at 24 and 1 h before PH and 1, 2, and 3 days post-PH. A subset of rats received 10 μg of recombinant IL6 protein, administered intravenously 30 min after PH. Liver tissues were harvested at 7, 14, 21, and 30 days post-PH. Treatment of retrorsine-exposed rats with the cytokine inhibitor dexamethasone (DEX) effectively blocked the emergence of SHPCs resulting in an inhibition of liver regeneration and producing significant short-term mortality. The livers of DEX-treated retrorsine-exposed rats displayed decreased numbers and smaller SHPC clusters compared to retrorsine-exposed rats in the absence of DEX treatment. Administration of recombinant IL6 to DEX-treated retrorsine-exposed rats restored the emergence of SHPCs and SHPC-mediated regenerative response. The livers of DEX-treated retrorsine-exposed rats that received IL6 displayed numbers of expanding SHPC clusters comparable to that of retrorsine-exposed rats in the absence of DEX treatment. These results combine to suggest that SHPC activation after PH in retrorsine-exposed rats is cytokine dependent and may specifically require IL6.

Direct In Vivo Cell Lineage Analysis in the Retrorsine and 2AAF Models of Liver Injury after Genetic Labeling in Adult and Newborn Rats

Backgrounds and AimsWhen hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.Methodology/Principal FindingsWe used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.ConclusionsOur results srongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

Bile duct destruction by 4,4′-diaminodiphenylmethane does not block the small hepatocyte-like progenitor cell response in retrorsine-exposed rats

Liver regeneration after surgical partial hepatectomy (PH) in retrorsine-exposed rats is accomplished through the outgrowth and expansion of small hepatocyte-like progenitor cells (SHPCs). The cells of origin for SHPCs and their tissue niche have not been identified. Nevertheless, some investigators have suggested that SHPCs may represent an intermediate or transitional cell type between oval cells and mature hepatocytes, rather than a distinct progenitor cell population. We investigated this possibility through the targeted elimination of oval cell proliferation secondary to bile duct destruction in retrorsine-exposed rats treated with 4,4'-diaminodiphenylmethane (DAPM). Fischer 344 rats were treated with 2 doses (30 mg/kg body weight) retrorsine (at 6 and 8 weeks of age) followed by PH 5 weeks later. Twenty-four hours before PH, select animals were given a single dose of DAPM (50 mg/kg). Treatment of rats with DAPM produced severe bile duct damage but did not block liver regeneration. Oval cells were never seen in the livers of DAPM-treated retrorsine-exposed rats after PH. Rather, liver regeneration in these rats was mediated by the proliferation of SHPCs, and the cellular response was indistinguishable from that observed in retrorsine-exposed rats after PH. SHPC clusters emerge 1 to 3 days post-PH, expand through 21 days post-PH, with normalization of the liver occurring by the end of the experimental interval. These results provide direct evidence that SHPC-mediated liver regeneration does not require oval cell activation or proliferation. In addition, these results provide strong evidence that SHPCs are not the progeny of oval cells but represent a distinct population of liver progenitor cells.

Treatment with 2-AAF blocks the small hepatocyte-like progenitor cell response in retrorsine-exposed rats

Liver regeneration after partial hepatectomy (PH) in retrorsine-exposed rats is accomplished through proliferation and differentiation of small hepatocyte-like progenitor cells (SHPCs). The cells of origin of SHPCs are not known. We investigated the possibility that SHPCs are directly derived from oval cells, a known liver progenitor cell, by combining the retrorsine/PH (RP) model with 2-acetamidofluorene (2-AAF), an anti-mitotic agent that elicits an oval cell reaction in response to liver deficit. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age, with PH 5 weeks after the final treatment. Seven days prior to PH, a 21-day 2-AAF (50mg) time-release pellet was inserted subcutaneously. Livers were harvested at 3, 7, 10, 14, and 21-days post-PH. Liver sections from animals treated with 2-AAF/retrorsine/PH (2-AAF/RP) contain significant numbers of proliferating oval cells, but no SHPCs at 7-days post-PH, while RP animals exhibit significant numbers of SHPCs and minimal oval cell reaction. Between 10 and 14-days post-PH, new hepatocyte clusters appear in 2-AAF/RP treated rats. Labeling of proliferating oval cells with BrdU at 6-days post-PH demonstrated that these new hepatocytes represent the progeny of differentiating oval cells. The observed differences in progenitor cell responses between 2-AAF/RP and RP animals strongly suggest that SHPCs are not the progeny of oval cell precursors, but represent an independent liver progenitor cell population.

The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice

After liver injury, parenchymal regeneration occurs through hepatocyte replication. However, during regenerative stress, oval cells (OCs) and small hepatocyte like progenitor cells (SHPCs) contribute to the process. We systematically studied the intra-hepatic and extra-hepatic sources of liver cell replacement in the hepatitis B surface antigen (HBsAg-tg) mouse model of chronic liver injury. Female HBsAg-tg mice received a bone marrow (BM) transplant from male HBsAg-negative mice, and half of these animals received retrorsine to block indigenous hepatocyte proliferation. Livers were examined 3 and 6 months post-BM transplantation for evidence of BM-derived hepatocytes, OCs, and SHPCs. In animals that did not receive retrorsine, parenchymal regeneration occurred through hepatocyte replication, and the BM very rarely contributed to hepatocyte regeneration. In mice receiving retrorsine, 4.8% of hepatocytes were Y chromosome positive at 3 months, but this was frequently attributable to cell fusion between indigenous hepatocytes and donor BM, and their frequency decreased to 1.6% by 6 months, as florid OC reactions and nodules of SHPCs developed. By analyzing serial sections and reconstructing a 3-dimensional map, continuous streams of OCs could be seen that surrounded and entered deep into the nodules of SHPCs, connecting directly with SHPCs, suggesting a conversion of OCs into SHPCs. In conclusion, during regenerative stress, the contribution to parenchymal regeneration from the BM is minor and frequently attributable to cell fusion. OCs and SHPCs are of intrinsic hepatic origin, and OCs can form SHPC nodules.

Cellular responses in experimental liver injury

Background/Aims Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes. Methods Mature hepatocytes were genetically labeled using retroviral vectors harboring the β-galactosidase gene. After labeling, retrorsine was administered to rats followed by partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later. Results We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed β-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of β-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice. Conclusions The constant proportion of β-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.

Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury

Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes. Mature hepatocytes were genetically labeled using retroviral vectors harboring the beta-galactosidase gene. After labeling, retrorsine was administered to rats followed by a partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later. We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed a high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed beta-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of beta-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice. The constant proportion of beta-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.

Isolation, short-term culture, and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats.

Complete liver regeneration after partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine can be accomplished through the activation, expansion, and differentiation of a novel population of small hepatocyte-like progenitor cells (SHPCs). These cells have not been isolated in pure form, established in primary culture, or transplanted into syngeneic rats to examine their differentiation potential. Primary liver cells enriched for SHPCs were prepared by differential centrifugation of primary liver cell dispersions from retrorsine-exposed rats 6-8 days and 13-15 days after PH. Isolated SHPCs were characterized for cell size, morphology, and expression of cell type-specific markers (including hepatocyte and bile duct-oval cell markers), and established in short-term primary culture. Isolated SHPCs were transplanted into the livers of syngeneic rats to evaluate their ability to engraft and differentiate into mature hepatocytes. SHPCs obtained from retrorsine-exposed rats 6-8 days and 13-15 days after PH were small (10-12 microm in diameter), morphologically resembled hepatocytes, and were predominantly H.4 antigen-positive, alpha-fetoprotein-positive, and OV6-negative. SHPCs did not proliferate in culture and could not be passaged, but short-term cultures were established using protein substrates (collagen or laminin) and defined medium containing epidermal growth factor and nicotinamide. After transplantation into the livers of syngeneic hosts, SHPCs insert into hepatic plates and give rise to differentiated hepatocyte progeny. The SHPC-derived hepatocyte progeny express a differentiated phenotype (albumin-positive, transferrin-positive, alpha-fetoprotein-negative) and are able to proliferate in vivo in response to the growth stimulus provided by PH. The results demonstrate that enriched SHPC populations can be isolated from retrorsine-exposed rats and established in short-term culture, and they can engraft and differentiate after transplantation into the livers of syngeneic rats.

Temporal Analysis of Hepatocyte Differentiation by Small Hepatocyte-Like Progenitor Cells during Liver Regeneration in Retrorsine-Exposed Rats

Liver regeneration after two-thirds surgical partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). We have examined expression of the major liver-enriched transcription factors, cytochrome P450 (CYP) enzymes, and other markers of hepatocytic differentiation in SHPCs during the protracted period of liver regeneration after PH in retrorsine-exposed rats. Early-appearing SHPCs (at 3-7 days after PH) express mRNAs for all of the major liver-enriched transcription factors at varying levels compared to fully differentiated hepatocytes. In addition, SHPCs lack (or have significantly reduced) expression of mRNA for hepatocyte markers tyrosine aminotransferase and alpha-1 antitrypsin, but their expression levels of mRNA and/or protein for WT1 and alpha-fetoprotein (AFP) are increased. With the exception of AFP expression, SHPCs resembled fully differentiated hepatocytes by 14 days after PH. Expression of AFP was maintained by most SHPCs through 14 days after PH, gradually declined through 23 days after PH, and was essentially absent from SHPC progeny by 30 days after PH. Furthermore, early appearing SHPCs lack (or have reduced expression) of hepatic CYP proteins known to be induced in rat livers after retrorsine exposure. The resistance of SHPCs to the mitoinhibitory effects of retrorsine may be directly related to a lack of CYP enzymes required to metabolize retrorsine to its toxic derivatives. These results suggest that SHPCs represent a unique parenchymal (less differentiated) progenitor cell population of adult rodent liver that is phenotypically distinct from fully differentiated hepatocytes, biliary epithelial cells, and (ductular) oval cells.

Liver Regeneration in Rats with Retrorsine-Induced Hepatocellular Injury Proceeds through a Novel Cellular Response

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass.

Temporal Analysis of Hepatocyte Differentiation by Small Hepatocyte-Like Progenitor Cells during Liver Regeneration in Retrorsine-Exposed Rats

Temporal Analysis of Hepatocyte Differentiation by Small Hepatocyte-Like Progenitor Cells during Liver Regeneration in Retrorsine-Exposed Rats