Background
It is well known that terminal stage liver diseases can be treated by liver transplantation with relatively good five-year survival. However, limited donor organs and rejection reactions restrict its usage. Thus, it is necessary to establish a novel strategy. Hepatic progenitor cells (HPCs)-based therapy is considered to be a promising replacement for liver transplantation because HPCs are capable of differentiating into both hepatocyte and biliary lineage [
1,
2]. However, this therapeutic strategy cannot be fully exploited until the mechanisms underlying the histogenesis of HPCs are clearly elucidated. It is traditionally assumed that HPCs originate in the smallest ramifications of the bile ducts or in locations outside the liver, such as bone marrow (BM) [
3‐
5]. However, liver stem cells have never been observed in the adult liver. Meanwhile, the restricted potential to differentiate into hepatocytes and cholangiocytes also qualifies HPCs as true stem cells [
6]. Recent studies suggested that EMT is involved in the pathogenesis of liver cirrhosis, a common endpoint of most chronic liver diseases. There is now convincing evidence that TGF-β1 can induce both cultured hepatocytes and cholangiocytes to undergo EMT [
7‐
9]. Notably, some cells undergoing EMT/MET (mesenchymal-epithelial transition) can simultaneously express epithelial/mesenchymal markers and HPCs markers [
10]. Furthermore, they can repair injured liver in rodent models [
11], raising the exciting possibility that HPCs may represent a subpopulation of cells undergoing EMT/MET [
12]. However, most available evidence supporting this hypothesis is indirect and obtains from rodent models or in vitro studies.
The EMT is a dynamic process that cannot be followed serially in humans because of technical limitations. In this study, we examined specimens from patients with benign HBV-related diseases, including hepatitis and cirrhosis. We used double immunofluorescence staining and RT-qPCR (quantitative reverse transcription PCR) to detect characteristic markers that are associated with HPCs, EMT, and epithelial/mesenchymal cells. In this study, EpCAM was used for identification of HPCs, because several studies indicated that EpCAM is capable of relative higher specificity than other HPCs markers such as CD133, CK19, and OV-6 [
13‐
15]. S100A4, the human homolog of murine fibroblast-specific protein 1 (FSP1), has been demonstrated to be a key marker of early fibroblast lineage development and EMT [
16,
17]. Consequently, loss of cytokeratin (CK) and adherens junction components such as E-cadherin promotes the detachment of transitioning cells from primary sites [
18,
19]. At later stages, EMT is accompanied by an increase in motility and matrix invasion, which is consistent with elevated levels of vimentin and matrix metalloproteinases (MMPs) [
19]. EMT-derived fibroblast cells can express α-smooth muscle actin (αSMA) [
20]. Meanwhile, several nuclear transcription factors contribute to the EMT cascade. Twist, a known helix-loop-helix transcription factor, can directly affect its down-stream element Snail to suppress the expression of E-cadherin [
21‐
23].
Discussion
Despite recent advances in pathophysiology, the histogenesis and heterogeneity of HPCs in humans are matters of debate. In this study, we combined data from immunophenotypic and mRNA studies with ultrastructural examination to provide preliminary evidence for the involvement of EMT in the histogenesis of HPCs in HBV-related liver diseases.
S100A4, which modifies cell motility and growth through interactions with the cytoskeleton and the C-terminus of p53, has been proposed as an early marker of EMT [
17]. Co-expression of EpCAM and S100A4 provided clear evidence that although these HPCs have an epithelial phenotype, they are actively engaged in EMT. HPCs also expressed another important EMT marker MMP-2, which possesses the ability to degrade basement membrane and increases cell motility [
27]. The activity of MMPs can alter the expression of E-cadherin and vimentin and promote the EMT process [
28]. High levels of MMP-2 may promote the dispatch of HPCs from DRs.
A hallmark of EMT is the aberrant expression of E-cadherin (encoded by
CDH1), which is always linked to the tumorigenesis of many epithelial cancers [
29]. E-cadherin is a key factor of cell-cell adhesion junctions in the maintenance of cell polarity and structure. Recent studies uncovered its critical roles in proliferation, differentiation, and carcinogenesis. Analogous to cholangitis, the loss of E-cadherin in the liver contributes to the periportal inflammation and later periductal fibrosis [
30]. A study making use of a transgenic mouse model where the expression of cre was directed by the
albumin enhancer/promoter (termed Alb-cre) demonstrated that these lesions arose as a consequence of the loss of E-cadherin in the cholangiocytes [
30]. The reexpression of cre in hepatocytes could not attenuate the effects [
31,
32]. In this study, the expression of E-cadherin and CK7 in HPCs decreased significantly from mild hepatitis to moderate hepatitis, revealing that these transitioning cells might derive from epithelial cells within DRs and were losing cell-cell contacts. However, the number and ratio of E-cadherin- or CK7-positive HPCs increased in sections of severe hepatitis and cirrhosis, suggesting that the transitioning cells might reverse the cascade and reacquire epithelial characteristics.
Twist is a core element during EMT process [
22]. The overexpression or promoter methylation of Twist is always associated, in a statistically significant manner, with the tumor aggressiveness [
33‐
35]. Activated Twist binds to the promoter region of E-cadherin and transcriptionally downregulates E-cadherin expression [
36]. Together with the polycomb protein Bmi1, Twist contributes to the stemness of cells, which is one of the most important features of HPCs [
22].
Snail is a zinc-finger transcription factor, which can induce EMT by repression of E-cadherin [
23]. TGF-β promotes EMT by up-regulating Snail expression via a Smad-dependent pathway. Snail forms a transcriptional repressor complex with SMAD3/4. The complex then targets the adjacent E-boxes and Smad-binding elements in genes encoding junction proteins such as E-cadherin, CAR and occluding [
37].
In all hepatitis and cirrhotic sections, the majority of HPCs within DRs or bile ducts expressed high levels of Twist or Snail, which are proportional to the severity of HBV-related diseases. RT-qPCR further validated these findings, indicating that Twist and Snail were involved in the EMT process of HPCs via the repression of E-cadherin in HBV-related diseases.
We tried to obtain further evidence for the transdifferentiation from HPCs into mature mesenchymal cells by using antibodies for αSMA and vimentin. However, neither cholangiocytes nor hepatocytes expressed αSMA or vimentin in this study. We considered two possibilities for this situation. First, αSMA- or vimentin-positive HPCs may detach from DRs because of an increase in motility and matrix invasion [
38]. Hence, these transitioning cells at an advanced stage of EMT do not express EpCAM and detach from DRs. Second, as reversibility is an important feature of EMT, it is tempting to speculate that these transitioning cells may re-transdifferentiate into parenchymal cells through MET under certain conditions.
Although we did not obtain substantial results about whether hepatocytes could transdifferentiate into HPCs through EMT, some periportal intermediate hepatocytes in sections from cirrhotic livers showed immunopositivity for MMP-2. We postulated an intriguing possibility that, at least in HBV-related liver diseases, intermediate hepatocytes may represent daughter cells of HPCs rather than a cellular origin of HPCs. Ultrastructural evidence that intercellular junctions existed between HPCs and intermediate hepatocytes further supported this hypothesis. Consistent with our findings, a recent study based on an established rodent model provided convincing evidence for challenging the concept that hepatocytes can acquire a mesenchymal phenotype in vivo via EMT [
39]. In contrast, previous studies demonstrated that hepatocytes may also be capable of undergoing EMT in vitro [
40,
41]. Furthermore, an elaborate study indicated that the Hippo signal pathway can direct hepatocytes to transdifferentiate into HPC-like cells and finally mimic an atypical ductular reaction [
42]. We believe that different microenvironments may result in different outcomes. Most recently, an elaborate study provided supporting evidence [
43]. Foetal HPCs can form hepatic cysts characterized by Albumin-positive/CK19-negative in vitro. However, if foetal HPCs are pre-cultured on gelatin-coated dishes, they are capable of forming cholangiocytic cysts expressing Albumin-negative/CK19-positive, similar to that of HPCs [
43]. This cholangiocytic cysts formation can be hampered by hepatic maturation factors, such as hepatocyte growth factor (HGF) and oncostatin M (OSM). Of note, the study also indicated that TGF-β antagonist A-8301 plays an important role in the cholangiocytic cysts formation. It seems that this conclusion is consistent with our hypothesis and observations in vivo. TGF-β derived from portal parenchymal cells or infiltrating immune cells promotes the entrance of cholangiocytes into the EMT cascade and transdifferentiation into hepatocyte-like cells. However, further experiments will be required to gain more insights into the regulatory mechanisms.
Chronic HBV infection may serve as a powerful driver of EMT. The regeneration capacity of mature hepatocytes is overwhelmed during massive or chronic liver infection. Various cytokines released from injured parenchymal cells establish a stable inflammatory infiltrate through the recruitment of leukocytes from blood [
44]. T-cells mediate both liver injury and viral clearance in animal models of HBV infection and produce regulatory cytokines such as TGF-β, a powerful driver of EMT [
45‐
47]. Furthermore, T-cells can express integrin, allowing the adhesion to E-cadherin of the epithelium [
48]. We speculate that high level of TGF-β induces EMT of DRs at the portal tract. These transitioning cells are capable of transdifferentiation into HPCs and movement towards injured sites, resembling chemotaxis. Since the number of immune cells and parenchymal cells decreases and results in the reduced TGF-β1 level at the lobule boundaries, the transitioning cells/HPCs may reverse EMT cascade and re-transdifferentiate into hepatic lineage to restore parenchyma [
12]. The ultrastructural result that tonofilament bundles reoccurred in the cytoplasm of HPCs favors this opinion.
Non-resolving inflammation is established when the recruitment of inflammatory cells outstrips the mechanisms of resolution, including the apoptosis and the migration through lymphatics [
49]. Non-resolving inflammation is responsible for abnormally elevated expression of TGF-β1 and the receptor for advanced glycation endproducts (RAGE), which plays an important role in the regulation of HPCs activation [
50]. Consequently, most of the HPCs undergo complete EMT and differentiate into mature mesenchymal cells. The repair process is mainly fibrogenic because of the excess production and deposition of extracellular matrix components. The genetic or epigenetic changes may increase the susceptibility of HPCs to the detrimental microenvironment and result in self-renewing cells. Finally, HPCs or their progeny may transform to malignant cells because of the further accumulation of other alterations [
50].