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Am J Physiol Gastrointest Liver Physiol 291: G575-G583, 2006. First published May 18, 2006; doi:10.1152/ajpgi.00102.2006
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LIVER AND BILIARY TRACT

Evidence for epithelial-mesenchymal transitions in adult liver cells

Jason K. Sicklick,1,2 Steve S. Choi,1 Marcia Bustamante,3 Shannon J. McCall,4 Elizabeth Hernández Pérez,3 Jiawen Huang,1 Yin-Xiong Li,1,5,6 Marcos Rojkind,3 and Anna Mae Diehl1

1Division of Gastroenterology and Department of Medicine, Duke University Medical Center, Departments of 4Pathology, 5Cell Biology, and 6Pediatrics, Durham, North Carolina; 2Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 3Departments of Biochemistry, Molecular Biology, and Pathology, George Washington University Medical Center, Washington, District of Columbia

Submitted 3 March 2006 ; accepted in final form 3 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.

cirrhosis; oval cell; stellate cell

EPITHELIAL RENEWAL in the skin and gastrointestinal tract is fueled by resident populations of progenitor cells in both health and disease (7, 8). In contrast, hepatic progenitors are thought to contribute to liver regeneration only when chronic liver disease results in senescence of mature hepatocytes (14, 51, 71). When the latter occurs, small epithelial progenitor cells, or oval cells (OV), accumulate in the liver. These OV typically localize near hepatic stellate cells (HSC), mesenchymal cells that generate extracellular matrix (5). During liver repair, OV are believed to differentiate into both hepatocytes and cholangiocytes (38), whereas HSCs give rise to collagen-producing myofibroblasts that play a critical role in progressive hepatic fibrosis (i.e., cirrhosis) (15). Cirrhosis and hepatocellular carcinoma typically develop in chronically damaged livers where there are large numbers of epithelial progenitors and myofibroblastic HSC (33, 52).

Recent studies of injured kidneys demonstrate that some renal fibroblasts are derived from resident epithelial cells in a process known as epithelial-mesenchymal transition (EMT) (24, 27, 31, 72). EMT occurs when epithelial cells are exposed to members of the transforming growth factor-beta (TGF-beta) superfamily (70). As epithelial cells transform into fibroblasts, epithelial proteins [e.g., E-cadherin (Ecad)] are downregulated and mesenchymal markers [e.g., matrix metalloproteinase-2 (MMP-2) and {alpha}-smooth muscle actin ({alpha}-SMA)] are induced. This yields populations of cells that coexpress epithelial and mesenchymal markers. Although all of the mechanisms for EMT have not been delineated, several key intermediates, including integrin-linked kinase (ILK) (31), Smad3 (53), and Snail (4), have been identified. Whether adult livers harbor cells that may have the capacity for EMT remains unknown.

Indeed, earlier work in fetal livers suggests that EMT may occur during hepatic development. Some stromal cells in embryonic livers express both mesenchymal and epithelial markers (6). Such cells lack markers of terminal hepatocyte differentiation (such as the transcription factor hepatic nuclear factor-4{alpha}, HNF-4{alpha}) but express an OV marker, OV-6 (12), and are resistant to TGF-beta-induced apoptosis (65). Although EMT is thought to occur in fetal livers (6) and cultured neonatal liver cells (42), there have not been any studies demonstrating EMT nor the converse and less-understood process of mesenchymal-to-epithelial transition in adult liver cells. Moreover, frequently the term "EMT" is used to mean transitions in either direction, i.e., epithelial-mesenchymal transitions. Given precedent for this process during hepatogenesis and evidence that repair responses in some adult organs involves EMT (24, 27, 72), we hypothesized that some adult liver cells may be capable of EMTs. Herein, we provide evidence that supports this concept. Namely, hepatic epithelial progenitors coexpress both epithelial and mesenchymal markers, whereas myofibroblastic HSC can be induced to express genes consistent with an epithelial phenotype.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. Adult, male C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Adult, male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Animal experiments fulfilled National Institutes of Health, Duke University, and George Washington University requirements for humane animal care.

Rodent hepatic stellate cell isolation and culture. The HSC fraction was isolated by in situ portal vein perfusion with pronase-collagenase (16, 21, 48). Primary murine HSCs were collected by density gradient centrifugation through OptiPrep (Accurate Chemical, Norway), which separates HSCs from NPCs (36, 40, 49, 57), whereas primary rat HSCs were isolated through Percoll gradient (Amersham Biosciences, Piscataway, NJ). Cell fractions were pooled from six mice or one rat. On isolation, >95% of the isolated cells exhibited autofluorescence typical of quiescent HSC as has been reported by others (57). Freshly isolated murine HSC were used for RNA analysis or cultured on plastic dishes for up to 7 days in 10% serum-supplemented RPMI 1640 medium (GIBCO-BRL, Carlsbad, CA) and 10 mM HEPES. Rat HSC were culture activated for 10 days and passaged twice as previously described (1, 21, 30). These culture conditions were employed to eliminate contaminating cells including macrophages, endothelial cells, and vascular smooth muscle cells (18, 68).

Culture of rat hepatic stellate cell lines. Clonally derived HSC lines (21) were cultured in 10% serum-supplemented RPMI 1640 medium (GIBCO-BRL) and 10 mM HEPES or serum-free HGM medium (3).

Embryonic stem cell culture and endodermal lineage restriction. Murine embryonic stem (ES) cells were kindly provided by Dr. J. H. Fair (Univ. of North Carolina, Chapel Hill, NC). Cells were cultured for 7 days and endodermally lineage-restricted as previously described (13).

Culture of murine hepatic progenitor line. A murine hepatic progenitor cell line (OV) was kindly provided by Dr. B. E. Petersen (Univ. of Florida, Gainesville, FL) and cultured as previously described (46, 47).

Culture of immortalized murine cholangiocyte line. The immortalized, but untransformed, 603B murine cholangiocyte cell line (Chol) (22, 69) was kindly provided by Dr. G. J. Gores (Mayo Clinic, Rochester, MN) and cultured as previously described (26).

Sourcing and isolation of primary human hepatic epithelial progenitor cells. Donated livers, not suitable for orthotopic liver transplantation, were obtained from federally designated organ procurement organizations by Vesta Therapeutics (Durham, NC). Human livers were then processed as previously described by Vesta Therapeutics (35). Immunoselection was then performed using monoclonal anti-EpCAM antibody coupled to magnetic microbeads (Miltenyi Biotec; Auburn, CA). Freshly isolated human hepatic epithelial progenitors expressing epithelial cell adhesion molecule (EpCAM) were then analyzed. Such EpCAM-immunoselected cells include hepatic progenitors in fetal and adult livers (2, 10, 59), as well as some bile duct epithelial cells and bile canalicular epithelial cells (10), which also harbor hepatic progenitors as demonstrated by several groups in the field (28, 43, 44, 62). Together, this immunoselected population is specific for epithelial, but not mesenchymal, cells in the liver.

Two-step RT-PCR. Total RNA was extracted from primary cells and cell lines with the RNeasy kit followed by RNase-free DNase I treatment (Qiagen, Valencia, CA). The primers were designed using Genbank sequences or as previously described (Table 1) (29, 37, 47, 54, 58, 59). For each experiment, total RNA was reverse transcribed to cDNA templates and amplified using Ready-To-Go You-Prime First-Strand Beads (Amersham) with pd(N)6 first-strand cDNA primers (Amersham). For RT-PCR, 1.5–2.0% of the first-strand reaction was amplified using iQ-SYBR Green Supermix (Bio-Rad, Hercules, CA), an iCycler iQ PCR Machine (Bio-Rad), and specific oligonucleotide primers for target sequences, as well as the beta-glucuronidase (Gus) or Gapdh housekeeping genes. The PCR parameters were as follows: denaturing at 95°C for 3 min followed by 40 cycles of denaturing at 95°C for 15 s and annealing-extension at the optimal primer temperatures for 45–60 s. Amplicon products were separated by electrophoresis on a 2.0% agarose gel buffered with 0.5x TBE. For real-time RT-PCR, threshold cycles (Ct) were automatically calculated by the iCycler iQ real-time detection system. Fold changes of target gene levels in the cells are presented as a ratio to levels detected in the corresponding control cells according to the 2–{Delta}{Delta}Ct method (32). For analyses of relative amounts of target genes in primary cells or cell lines, values were determined according to the differences between the average Ct of the target gene and the average Ct of Gus. These {Delta}Ct values are presented on a log2 scale. For all experiments, amplifications were performed in triplicate.


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  		Table 1. Primers for RT-PCR analysis.

 
Immunoblot. For protein extraction, the HSC lines were cultured in serum-containing medium followed by overnight culture in serum-free medium. Cells were then homogenized in lysis buffer containing stabilizing agents and protease inhibitors. Liver proteins (40 µg/lane) in Laemmli buffer were separated by polyacrylamide gel electrophoresis and transferred to nylon membranes. Membranes were blocked, washed, and exposed to secondary antiserum. The primary antibodies employed were against dopamine beta-hydroxylase (DBH; 1:250, Research Diagnostics, Flanders, NJ), MMP-2 (1:1,000, Chemicon International, Temecula, CA), actin {alpha}-SMA (1:300, Sigma-Aldrich, St. Louis, MO), E-cadherin (1:2,500, BD Pharmingen, Franklin Lakes, NJ), muscle pyruvate kinase (MPK; 1:2,000, Cell Signaling Technologies, Beverly, MA), ILK (2 µg/ml, Upstate Biotechnology, Charlottesville, VA) (31), and beta-actin (1:400, Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate secondary antibodies were used, and the antigens were demonstrated by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Immunostaining of human and murine livers. Sections of formalin-fixed, paraffin-embedded murine and human livers, 7 µm thick, were used for immunofluorescent staining for {alpha}-SMA as previously described (58). For CD56 immunohistochemical staining, 7-µm-thick sections were stained with mouse anti-human CD56/NCAM (clone 1B6, dilution 1:50, Vector Laboratories, Burlingame, CA) using the DAKO Envision system kit (Dako, Carpinteria, CA) as previously reported (64). The secondary antibody was then detected using the avidin-biotin-peroxidase method with 3,3'-diaminobenzidine as the substrate (Vector Laboratories). Negative controls were performed by omitting the primary antibody from the protocol and revealed minimal background staining (data not shown). Following staining, screening analyses of tissues were performed in an unblinded fashion by three independent observers to qualitatively identify positively stained cells and structures.

Murine carbon tetrachloride treatment. Wild-type (n = 3) mice were injected intraperitoneally with CCl4 (0.5 mg/kg, Sigma-Aldrich). The CCl4 was initially prepared as 1 mg/ml in corn oil vehicle (Sigma-Aldrich). The mice were killed 8 wk after initiating treatment. Following euthanasia, livers were fixed in formalin, paraffin-embedded, and serially sectioned at 7 µm thick. Sections were then stained as described above and examined by light microscopy. The CCl4-treated livers were compared with livers from untreated litter mates (n = 5). Positively stained bile ductules in random x20 periportal fields were scored in a blinded fashion by two independent observers.

Statistical analysis. Comparisons between groups were made using Intercooled Stata 8.0 (Stata; College Station, TX). Data are presented as the means ± SD unless otherwise indicated. Comparisons between groups were performed using the Student's t-test for real-time RT-PCR analyses. Significance was accepted at the 5% level.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Characterization of clonal rat hepatic stellate cell lines. Two well-established, clonally derived rat HSC lines derived from the liver of a single adult rat that was treated with CCl4 (20, 21, 25, 50) were evaluated for EMT. When grown in serum-containing culture medium, HSC 8B resembles early culture-activated primary rat HSC. In contrast, HSC 5H has features of an established myofibroblast (20, 21). Both RT-PCR and immunoblot analyses demonstrated that both HSC lines have surprising phenotypic heterogenicity (Fig. 1A). At the mRNA (left) and/or protein (right) levels, both cell lines express neural markers including Nestin (39), Gfap, and DBH and produce mesenchymal factors including {alpha}-sma and MMP-2 as well as embryonic MPK, a widely used marker of OV (9, 34, 60, 63, 66). One of the clones, HSC 5H, expresses E-cadherin (Ecad), another epithelial cell marker (23). This HSC line also expresses Hnf-4{alpha}, a transcription factor that is specific for cells fated to become mature hepatocytes (45). The coexpression of mesenchymal and epithelial gene products suggests that these adult liver cells may be capable of EMT. Indeed, both HSC lines express ILK (Fig. 1B), an enzyme that is required for EMT (31). Whereas it might be argued that gene expression in these HSC lines may diverge from that of primary rat HSC, cells from all three sources express classic HSC markers, including Nestin, Mmp-2, and {alpha}-sma (Figs. 1A, 2, and 3A). We also found similar levels of the OV marker Mpk in HSC 5H, HSC 8B, and primary rat HSC (Fig. 1C). In fact, Mpk expression in all of these HSC was significantly higher than in our murine OV line (P < 0.001, P < 0.015, and P < 0.009, respectively).


Figure 1
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  		Fig. 1. Clonally derived hepatic stellate cell (HSC) lines from adult livers express epithelial and mesenchymal markers. A: RNA and protein were obtained from cultured rat HSC lines (5H and 8B) and respectively analyzed by RT-PCR (left) and immunoblot (right), which demonstrate that these lines have multipotent phenotypes, expressing markers of neural [Gfap, Nestin, dopamine beta-hydroxylase (DBH)], mesenchymal [matrix metalloproteinase-2 (MMP-2), {alpha}-smooth muscle actin ({alpha}-sma)], epithelial (E-cadherin), oval [muscle pyruvate kinase (MPK)], and hepatocytic (Hnf-4{alpha}) cells. Gapdh was employed as the housekeeping gene, and beta-actin was used as a protein-loading control. ND indicates not done. B: immunoblot analysis of integrin-linked kinase (ILK) in HSC 5H and HSC 8B. C: real-time RT-PCR analysis of Mpk expression in an oval cell line (OV), HSC 5H, HSC 8B, and culture-activated, primary rat hepatic stellate cells (1° HSC). Results were normalized to Mpk expression in OV using the 2–{Delta}{Delta}Ct method (where Ct is threshold cycle). {dagger}P < 0.05, *P < 0.01, and {ddagger}P < 0.001 compared with OV.

 

Figure 2
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  		Fig. 2. Primary murine HSC from adult livers express epithelial and mesenchymal markers following culture activation. HSC were isolated and pooled from the livers of 6 healthy adult mice. The cells were cultured on plastic in serum-containing medium for up to 7 days. RNA was harvested at 2 time points (day 0 or 7) for quantitative RT-PCR analysis of markers of neural (Gfap, Nestin), mesenchymal ({alpha}-sma, Col1{alpha}2), oval (Mpk, Afp), biliary (Ck-19), and hepatocytic (Hnf-4{alpha}, Albumin) cells. Expression of the target genes was normalized to expression of the housekeeping gene, Gus, in the respective samples using the {Delta}Ct method. With this method, {Delta}Ct equals the log2 of CtGus – CtTarget. The average Ct values for Gus were 25.2 and 22.5, respectively, for days 0 and 7. For reference, each cycle above 0 represents 2{Delta}Ct-fold higher expression of the target gene compared with Gus. {dagger}P < 0.05, *P < 0.01, and {ddagger}P < 0.001 for HSC day 7 compared with HSC day 0.

 

Figure 3
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  		Fig. 3. Hepatic stellate cells can differentiate in vitro. A: 2 rat HSC lines (8B and 5H) were cultured for 2 wk in serum-containing medium (lanes 1 and 3). HSC 8B was also cultured in serum-free media (supplemented with TGF-{alpha}) (lane 2). Cultures were harvested after 2 wk to obtain RNA for semiquantitative RT-PCR analysis of markers of mesenchymal ({alpha}-sma), oval (Mpk, Afp), biliary (Ck-19), and hepatocytic (Hnf-4{alpha}) cells. RT-PCR analysis demonstrates that the more mesenchymal HSC line, 8B, undergoes epithelial-mesenchymal transitions (EMT) in serum-free media and develops an epithelial, oval cell-like phenotype similar to HSC 5H. Removal of serum from the media of HSC 8B resulted in a cellular morphology change from stellate-like (B) to epithelial-like (C; x10 magnification). D: reintroduction of serum into the culture medium converted cellular morphology back to a more myofibroblastic appearance (x10 magnification).

 
Characterization of primary murine stellate cell isolates before and after culture activation. Given the phenotypic heterogeneity of the two rat HSC lines, it may be argued that studies in cell lines may not be good models to determine gene markers. Therefore, we sought to confirm our findings by an alternative technique. We purified the HSC fraction from the livers of six healthy adult mice. RNA was obtained from freshly isolated HSC. The remaining cells were plated on plastic dishes and cultured for 7 days to eliminate contaminating cells and spontaneously differentiate quiescent HSC into activated myofibroblastic HSC. Standard markers of quiescence (e.g., Gfap) and activation [e.g., Nestin, {alpha}-sma, and type I collagen {alpha}2 (Col1{alpha}2)] were evaluated by real-time RT-PCR. The mRNA levels of these genes were normalized to the levels of Gus, a highly expressed housekeeping gene, using the {Delta}Ct method. Each cycle above zero represents 2{Delta}Ct-fold higher expression of the target gene compared with Gus, whereas each cycle below zero represents 2{Delta}Ct-fold lower expression of the target gene compared with Gus.

During culture, cells enlarged and became polygonally shaped with large nuclei, typical of activated HSC (17, 57) (data not shown). Also, as is known to occur during culture activation of primary rodent HSC, in our mouse HSC cultures, Gfap expression decreased by 1,000-fold (P < 0.027), whereas {alpha}-sma and Col1{alpha}2 expression increased by 2,195 (P < 0.0002)- and 1,290-fold (P < 0.0003), respectively (Fig. 2). Concurrently, Nestin expression fell by 36-fold (P < 0.002). Afp, Hnf-4{alpha}, and albumin mRNA were detected in our preparations of freshly isolated HSC. During culture, levels of each of these hepatocytic genes fell to levels that were 79- to 2,024-fold lower than expression of the housekeeping gene, Gus, in the same sample. This finding is consistent with evidence that contaminating hepatocytes were largely eliminated from the cultures over time. In contrast, freshly isolated HSC expressed high levels of Mpk, and expression of this classic OV marker remained high throughout the culture period. Indeed, at day 7, primary HSC expressed about as much Mpk as Col1{alpha}2. Expression of Ck-19, a biliary marker, also remained relatively constant during culture (P > 0.10), suggesting that either our culture conditions promoted the survival of biliary epithelial cells or that some HSC may be capable of differentiating into cholangiocytes via EMT.

Epithelial-mesenchymal transitions in hepatic stellate cells. To further address the issue of EMT, we performed additional experiments with our clonally derived HSC lines. The Ecad-negative line (HSC 8B) was cultured in the presence and absence of serum-containing medium supplemented with TGF-{alpha} (3). Culture in serum-free conditions promoted expression of epithelial genes in HSC 8B (Fig. 3A), inducing Afp, Ck-19, and Hnf-4{alpha}. As a result, HSC 8B began to resemble HSC 5H and OV. Moreover, as HSC 8B cells upregulated these epithelial genes, they lost their stellate cell morphology (Fig. 3B) and acquired epithelial features, becoming distributed in plates and forming occasional ductlike structures (Fig. 3C). When culture medium was resupplemented with 10% serum, the cells regained their mesenchymal-like HSC morphology (Fig. 3D). These findings demonstrate that adult rat HSC exhibit phenotypic plasticity and can be induced to coexpress epithelial and mesenchymal cell markers, typical of cells undergoing EMT (23, 27). Of note, similar to our primary murine HSC (Fig. 2), both clonal rat HSC lines expressed the biliary marker Ck-19 (Fig. 3). Hence, HSC express markers of immature hepatocytes (Afp) as well as biliary epithelial cells (Ck-19). Coexpression of hepatocyte and biliary markers typifies OV (56, 61). On the other hand, despite culture in these conditions for 2 wk, HSC 8B never expressed detectable albumin mRNA. Similarly, albumin transcripts could not be demonstrated in HSC 5H, although this line exhibits high basal expression of Hnf-4{alpha} mRNA (Fig. 3A). Thus our culture conditions did not permit the terminal differentiation of hepatic epithelial progenitor (OV)-like HSC into hepatocytes.

Characterization of hepatic epithelial progenitors. In adult livers, mesenchymal cells, including vascular smooth muscle cells and activated HSC, express {alpha}-sma. However, {alpha}-sma is not an acknowledged marker of hepatic epithelia. Evidence that adult HSC can undergo EMT suggested that some liver epithelial cells might coexpress mesenchymal markers such as {alpha}-sma. To further evaluate this possibility, we screened RNA from murine ES cells treated for 7 days with acidic fibroblast growth factor to differentiate them into endodermally lineage-restricted ES cells (13), primary human EpCAM+-immunoselected hepatic epithelial progenitors (2, 10, 59), and a murine OV line (46, 47). Such hepatic epithelial progenitors are capable of generating functional hepatocytes in vitro and/or in vivo (2, 10, 13, 46, 47, 55, 59, 61). With the use of real-time RT-PCR, neural and mesenchymal marker expression by these hepatic epithelial progenitors was compared with that of quiescent (HSC day 0) and culture-activated (HSC day 7) primary mouse stellate cells (Fig. 4). As previously, data were analyzed using the {Delta}Ct method. Both murine and human liver epithelial progenitors expressed {alpha}-sma and two other well-accepted HSC markers, Gfap and/or Nestin (39), demonstrating an overlapping expression pattern of neural and mesenchymal markers with primary HSC from adult livers. More specifically, ES cells expressed similar levels of Nestin (P = 1.0) and threefold more {alpha}-sma (P < 0.004) than culture-activated HSC. On the other hand, Gfap expression was less than that of quiescent HSC (P < 0.018) but greater than that of activated HSC (P < 0.073). EpCAM+ cells had the highest levels of Gfap with expression similar to the Gus housekeeping gene (P = 0.98). In these cells, {alpha}-sma expression was 100-fold lower than Gus expression (P < 0.0001). OV expression of Gfap was 77-fold lower than quiescent HSC (P < 0.003), whereas expression of {alpha}-sma was 23-fold higher (P < 0.002). Comparison of the OV line to activated HSC demonstrated that OV had 13-fold more Gfap (P < 0.28), 2,048-fold less Nestin (P < 0.0001), and 97-fold less {alpha}-sma (P < 0.0001). Given evidence that primary murine HSC (Fig. 2) and rat HSC lines (Fig. 3A) express the biliary marker Ck-19, we also analyzed a murine cholangiocyte cell line (22, 26, 69) for expression of HSC markers. The cholangiocyte line (Chol) had 27-fold less Gfap than quiescent HSC (P < 0.006) and 1.6-fold more Nestin than activated HSC (P < 0.10). {alpha}-sma Expression was similar in cholangiocytes and freshly isolated HSC (P < 0.57), suggesting that these cholangiocytes, like OV, share an overlapping pattern of gene expression with primary murine HSC.


Figure 4
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  		Fig. 4. Hepatic epithelial progenitors express neural and mesenchymal markers like HSC. Total RNA was obtained from endodermally lineage-restricted embryonic stem (ES) cells (ES day 7), freshly isolated human EpCAM+ hepatic epithelial progenitors (EpCAM+), a hepatic oval cell line (OV), freshly isolated murine HSC (day 0), culture-activated, murine HSC (day 7), and an immortalized murine cholangiocyte cell line (Chol). RNA was analyzed by real-time RT-PCR to evaluate the cells for expression of neural (Gfap, Nestin) and mesenchymal ({alpha}-sma) HSC markers. For all samples, expression of the target genes was normalized to expression of the housekeeping gene Gus in the respective samples using the {Delta}Ct method. Using this method, {Delta}Ct equals the log2 of CtGus – CtTarget. The average Ct values for Gus were 24.5, 23.8, 20.6, 25.2, 22.5, and 20.7 for the respective RNA samples. For reference, each cycle above 0 represents 2{Delta}Ct-fold higher expression of the target gene compared with Gus. {dagger}P < 0.05 for gene expression compared with HSC day 0. *P < 0.05 for gene expression compared with HSC day 7.

 
Overlapping epithelial and mesenchymal expression pattern in the liver. To determine whether the expression of {alpha}-sma mRNA by cultured progenitor cells accurately represents in vivo expression of {alpha}-SMA protein by liver epithelial progenitors, we performed immunofluorescent staining of liver sections from healthy adult mice and mice with CCl4-induced liver damage. In the healthy mouse liver (Fig. 5A), some small bile ductular-like cells stained positive for {alpha}-SMA. Blinded assessment of the number of stained bile ductules in liver sections from five control and three CCl4-treated mice revealed a 3.2-fold increase in {alpha}-SMA-positive bile ductules in the CCl4-treated livers compared with control livers (P < 0.046; Fig. 5B).


Figure 5
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  		Fig. 5. Liver epithelial progenitor cells express a stellate cell marker in vivo. A: liver sections from a healthy, adult wild-type mouse were stained for {alpha}-SMA (x40 magnification). Nuclei were counterstained with DAPI. Open arrowheads indicate negatively stained bile ductules, and white arrowheads indicate positively stained bile ductules. B: healthy adult mice were treated with biweekly intraperitoneal injections of CCl4 and killed after 8 wk to obtain liver tissue. Random periportal fields in sections from each mouse were examined by immunofluorescence microscopy at x20 magnification to quantify {alpha}-SMA-positive bile ductules in control mice (N = 5) and CCl4-treated mice (N = 3). Results following blinded analysis by 2 observers are shown as the means ± SE ({dagger}P < 0.046). C: liver biopsy sections from an infant with biliary atresia were stained for {alpha}-SMA (x20 magnification). Note that epithelial and mesenchymal cells stained with the antibody. D: liver sections of this same child's explanted liver at the time of orthotopic liver transplantation (x40 magnification) demonstrated numerous {alpha}-SMA-stained bile ductules. E: these sections also demonstrated bile stained for neural cell adhesion molecule, or CD56, at x40 magnification. Black arrowheads indicate positively stained bile ductular cells.

 
Demonstration of {alpha}-SMA in bile ductules is noteworthy because the canals of Herring, terminal branches of the biliary tree, harbor hepatic progenitors (43, 44, 62). Recent work from another group (19) also demonstrates that Nestin-positive cells colocalize with other OV markers in these progenitor niches, providing further evidence for EMT in postnatal livers. Proliferation of these multipotent ductular cells is strongly induced in biliary atresia (67). Therefore, we examined liver sections from an infant liver with biliary atresia to determine whether ductular-type cells express {alpha}-SMA in human. Proliferating bile ductules, as well as cells in fibrous septae, strongly stained with the mesenchymal marker {alpha}-SMA (Fig. 5, C and D). This finding is interesting because earlier work by Torbenson and colleagues (64) demonstrated that in most cases of biliary atresia, approximately two-thirds of the bile ductules positively stained for a neural marker, neural cell adhesion molecule (NCAM or CD56). We confirmed their finding. In unblinded analysis or our biliary atresia samples, approximately one-third to one-half of bile ductules were NCAM-positive (Fig. 5E). Neither mature hepatocytes nor mature cholangiocytes expressed these neural and mesenchymal markers. Together, these findings demonstrate that {alpha}-SMA, a well-accepted marker of activated HSC, is also expressed by subpopulations of immature liver epithelial cells. The overlapping pattern of {alpha}-SMA expression in HSC and epithelial progenitors supports the occurrence of EMT and indicates that {alpha}-SMA is not a specific marker of either cell type.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Using several different, but complementary, approaches, we demonstrated coexpression of epithelial and mesenchymal markers in certain adult liver cells, suggesting that EMT occurs in postnatal livers. Expression of these markers overlaps in HSC and hepatic epithelial progenitors, raising the intriguing possibility that cells within these populations may be capable of transitioning between epithelial and mesenchymal phenotypes. This concept is supported by evidence that freshly isolated, and 7-day culture-activated, primary mouse HSC express relatively stable levels of two epithelial markers, Mpk (an oval cell marker) and Ck-19 (a marker of immature and mature biliary epithelial cells). The argument that EMT occurs in adult HSC populations is further supported by evidence that clonally derived adult HSC with neural and smooth muscle markers can be induced to upregulate Ecad, Hnf-4{alpha}, Afp, and Mpk. These genes are expressed by liver epithelial cells, including OV, bipotent progenitors that differentiate into mature hepatocytes or cholangiocytes. Apparently, our culture conditions supported differentiation of HSC 8B toward the biliary lineage, because expression of Ck-19, but not albumin, mRNA was induced. Evidence for EMT in bile ductular cells parallels data generated in renal models of EMT, in which epithelial-mesenchymal transitions occur most often in cells of renal tubules (27, 31). Finally, our findings suggest that, similar to hepatocytes and cholangiocytes, some HSC may arise from endodermal progenitors, because endodermally lineage-restricted ES cells and OV express several typical HSC markers.

Although the possibility that HSC might undergo EMT seems somewhat counterintuitive, in fact, the present findings complement and extend recent studies in {alpha}SMA-RFP/COLL-EGFP double transgenic mice (36). That work confirmed earlier evidence that HSC are heterogeneous (20) by demonstrating that the livers of double transgenic mice included subpopulations of {alpha}-SMA-positive cells that were not actively transcribing collagen as well as collagen-expressing cells that lacked {alpha}-SMA and cells that coexpressed both {alpha}-SMA and collagen. These HSC subpopulations were found to express different levels of neural markers and MMPs, but whether or not expression of epithelial markers differed among the HSC subpopulations was not examined. Our rat HSC lines were generated by expanding HSC clones that were isolated from the liver of a cirrhotic rat (21). Similar to the HSC subpopulations in {alpha}SMA-RFP/COLL-EGFP mice, our two rat HSC lines differ somewhat in their expression of mesenchymal and neural markers. These rat HSC clones also exhibit variable expression of several epithelial genes. Given the aggregate evidence for HSC plasticity, the possibility that HSC may undergo EMT seems plausible.

Together, these data suggest that livers harbor populations of cells that are capable of EMT from the early fetal period to late adulthood, similar to other mesodermally and endodermally derived organs such as the kidney and lung, respectively (24, 27, 31, 70, 72). As in these other organs, in adult livers, resident cell populations that play critical roles in tissue repair may be transitioning between epithelial and mesenchymal phenotypes to modulate the final outcomes of injury. This concept has obvious implications for the pathogenesis of cirrhosis, a condition that is characterized by the accumulation of myofibroblastic HSC and nodules of regenerating hepatic epithelia (11). Because EMT is a very dynamic process (31, 73), complete recovery from cirrhosis may be feasible if cells transition back from a mesenchymal phenotype to one that is more typical of hepatic epithelial cells.

Although the mechanisms regulating EMT are not fully understood, EMT may modulate various aspects of tissue remodeling, including epithelial renewal, fibrosis, and neoplasia (65, 72). In summary, our identification of EMT in adult liver cells that mediate liver repair justifies research to clarify how pathways that regulate EMT influence the evolution of hepatocellular carcinoma and cirrhosis. Such work offers the promise of identifying novel pathways that might be targeted to improve the outcome of many types of liver damage.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants R01 AA-010154 (to A. M. Diehl), R01 DK-053792 (to A. M. Diehl), R01 AA-012059 (to A. M. Diehl), R01 AA-01541 (to M. Rojkind), T32 DK-007713 (to J. K. Sicklick), and T32 DK-007568 (to S. S. Choi).


   ACKNOWLEDGMENTS
 
The authors thank Dr. J. H. Fair for the kind gift of endodermally lineage-restricted murine ES cells; Dr. B. E. Petersen for the kind gift of the murine oval cell line; Dr. G. J. Gores for the kind gift of the murine cholangiocyte cell line; Vesta Therapeutics for the kind gift of human EpCAM-expressing hepatic progenitor cells; Dr. R. Jhaveri for criticisms and discussion; and D. F. Sandler for assistance with manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Diehl, Duke Univ. Medical Center, Division of Gastroenterology, Snyderman-GSRB I Suite 1073, 595 LaSalle St., Box 3256, Durham, NC 27710 (e-mail: diehl004{at}mc.duke.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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