Biliary tree stem/progenitor cells and perspectives in physiopathology and regenerative medicine

Introduction

We have recently demonstrated the presence of cells expressing a variety of endodermal markers in (peri)-biliary glands (PBGs) of extrahepatic bile ducts (1). The observations in situ in human tissues have been completed by the in vitro demonstration that (PDX1+⁄SOX17+⁄EpCAM+) cells isolated from the biliary epithelium have long-term (in vitro) persistence and self-renewal, and are able to give rise to a more restricted progeny towards different mature lineages such as hepatocytes, cholangiocytes and β-pancreatic cells (1). These cells have been termed human biliary tree stem/progenitor cells (hBTSCs) (1-3). hBTSCs are located deeper inside the glands of the entire biliary epithelium, express typical markers of primitive endoderm and bilio-pancreatic progenitors, which are lost (together with accumulation of PAS positivity) when these cells progress and differentiate from deep inside the gland to the lumen, recapitulating the organization of maturational lineages in the intestine (2).

Perspectives in liver development

Beside the well known niche at the level of the canals of Hering, our results indicate an additional niche formed by a large number of cells with the phenotypes of endodermal stem⁄progenitor cells within PBGs of the large intrahepatic and extrahepatic bile ducts in fetal and adult life, suggesting a newly identified reservoir of stem cells for the renewal of large bile ducts (1-3). In the fetal life, these niches are closely related to the ductal plate, which has been recently demonstrated to generate the liver parenchyma during embryogenesis (4). This relationship is supported by the observation in fetal tissues, where progenitors in large bile ducts were continuous with ductal plates that, in turn, transits into the canals of Hering (5). Moreover, this is in agreement with the hilum to periphery model of bile duct morphogenesis recently proposed by Lemaigre group (6). hBTSCs are endowed with more primitive phenotypes in comparison with human hepatic stem cells (hHpSCs) located in ductal plate (fetal liver) or canals of Hering (adult liver) suggesting the possible existence of a hierarchy (1-3). Based on the theory of the branching tissues and, on the new insights on the biliary tree development from the hilum towards the periphery (6), 
we hypothesized that hBTSCs give rise to hHpSCs in the ductal plate during fetal development. This could explain how extra and intrahepatic bile ducts are connected. Segmental, area and septal intrahepatic bile ducts share several common features with the extrahepatic bile ducts such as the presence of PBGs (7,8) and, interestingly, the presence of endoderm-like⁄progenitor-like cells that are EpCAM±⁄PDX1+⁄SOX17+⁄Lgr5+. These observations are in accordance with the possibility of isolating multipotent stem cells from fetal and adult livers of rodents and other mammals (9,10).

Bile ducts regeneration and hBTSCs homeostasis

The glands of the biliary epithelium represent niches of cells with classic phenotypic features of stem⁄progenitor cells of endodermal origin with respect to transcription factors, surface and cytoplasmic markers, capacity of proliferation, self-renewal and multipotential differentiative capacity (1-3). Recently, Spence et al., by a lineage tracing study, demonstrated that SOX17+⁄PDX1+ cells are the precursors of biliopancreatic cells (11). The recent study of the organization of cells within glands of the biliary epithelium by Carpino et al. has also shown that: (I) the progenitor-like cells and transit-amplifying cells are located at the bottom of the glands; (II) the cells with a phenotype intermediate between progenitor-like cells and mature cells are found in the middle of the glands; and (III) the fully differentiated cells are in continuum with the surface epithelium (2). Cells, secreting low levels of insulin, are found within PBGs, especially in the hepatopancreatic common ducts, whereas cells producing albumin are found in portions of the biliary epithelium near to liver. Endoderm-like/progenitor-like cells are also found in PBGs within intrahepatic large bile ducts, representing a candidate niche for the renewal of intrahepatic ducts. In PBGs, approximately 10% of the cells co-express PDX1, SOX17 and FOXa2 (forkhead box a2), a phenotype indicative of the early stem cell subpopulation (endoderm-like cells). Moreover, we have shown that glands of the biliary tree are also formed by populations of cells with a less primitive phenotype, being characterized by the loss of FOXa2 and PDX1 expression (biliary progenitor-like cells) or, alternatively, by the loss of FOXa2 and SOX17 (pancreatic progenitor-like cells). Both these cell populations maintained the expression of EpCAM, a more widely expressed marker. Several lines of evidence indicate that EpCAM expression is tightly regulated and only occurs when there is a temporary need for proliferation; it is immediately downregulated upon terminal differentiation (12,13). In adults, biliary tree stem cells could represent the remnant of a common stem⁄progenitor (PDX1+ ⁄SOX17+) for liver, bile duct system and pancreas, which exists at earlier stages of development (11), being considered to derive from ventral endoderm (14,15). Within the body of the PBGs, we identified the presence of a committed intermediate (transit-amplifying) compartment in which cells co-expressing both stem cell markers and markers of mature cells were present, i.e., secretin receptor (cholangiocytes), mucins (goblet cells), insulin (endocrine pancreas) and, rarely, albumin (hepatocytes). The relative proportion of cells expressing mature cell markers indicative of liver vs. bile duct vs. pancreas correlates with the location along the length of the biliary epithelium. Accordingly, PBGs are organized along the duct wall such that cells with an undifferentiated phenotype (EpCAM±⁄PDX1+ ⁄SOX17+⁄Lgr5+) are situated mostly in the gland base near the fibromuscular layer. The transit-amplifying progenitor population (EpCAM+⁄PDX1+⁄SOX17+⁄Lgr5-) is concentrated in the body of the glands (corresponding to the middle of the duct wall). Finally, mature cells (e.g., cholangiocytes, goblet cells, hepatocytes, pancreatic cells) are located at the neck of PBGs in close contact with the surface epithelium: the type of mature cell depending on whether the portion of the biliary tree is near the liver (hepatocytes), in the middle of the biliary tree (cholangiocytes) or near the duodenum (pancreatic cells and goblet cells). The distribution of proliferating cells follows a similar gradient. Taken together, these data indicate that PBGs contain progenitor-like cells, which normally proliferate and are responsible for the renewal of the surface epithelium, generating mature cells such as cholangiocytes and goblet cells (in the middle of the biliary tree) or hepatocytes (near the liver) or islet cells (near the pancreas). The endoderm-like⁄progenitor-like cell compartment within PBGs resembles the organization of the stem cell niche within intestine. In the intestine, proliferation takes place at the base of intestinal crypts where Lgr5+ stem cells interspersed by Paneth cells are located (16,17). They give rise to progenitors produced from stem cells in the crypts (transit-amplifying population), migrate upwards and fully differentiate into mature cells. The phenotype of the mature cell (e.g., stomach, duodenum, small intestine or large intestine) depends on the position of the villus in the anterior to posterior axis along the intestine (18).

We have also demonstrated that the biliary extra-cellular bio-matrix is characterized by a defined structural and molecular organization (19). This organization could support and regulate the lineages within the wall of bile ducts. Moreover, bile acids (20,21) and bile flow (22,23) are additional candidate regulators of the hBTSC niche homeostasis and activation. Signaling pathways involved in the homeostasis of intestinal stem cell niche and mature fate specification (24), such as WNT, Notch, BMP (bone morphogenetic protein), Hedgehog have been extensively studied in the biliary epithelium and found to play a pivotal role in many aspects of biliary physiopathology (development, congenital diseases such as Alagille syndrome and biliary atresia, and bile duct regeneration after injury). Unfortunately, in the biliary tree, these pathways have been scarcely investigated as mediators of hBTSCs self-renewal and maintenance in the niche or specification toward mature lineages (cholangiocytes or goblet cells).

Perspectives in biliary diseases and carcinogenesis

Hyperplasia and neuroendocrine differentiation of PBG cells occur in diseases of bile ducts such as Primary Sclerosing Cholangitis (PSC) (8). PSC is a disease primarily affecting large intra and extrahepatic bile ducts (25). This disease involves the entire wall of the bile ducts as evidenced by the involvement of PBGs. From a clinical and pathological point of view, PSC is completely different with respect to Primary Biliary Cirrhosis (PBC), a disease specifically triggering interlobular bile ducts (26). From a clinical point of view, PBC affects mostly women, whereas PSC affects mostly men (27). In PSC, different types of malignancies can develop, whereas this is not the case of PBC (28). Finally, ulcerative colitis is frequently associated with PSC but not PBC. From a pathological point of view, PBC electively targets interlobular bile duct in portal spaces, whereas PSC preferentially triggers large bile ducts (26,28). In biopsy specimens from PSC patients, hairy and enhancer of split 1 (Hes-1) was completely abolished while pancreatic and duodenal homeobox 1 (PDX-1), a transcription factor driving neuroendocrine cell differentiation, was over-expressed. PDX-1 activation was absent in diseases primarily affecting liver parenchyma including viral hepatitis, alcoholic liver disease, non-alcoholic steato-hepatitis and PBC (29). These data suggest that the transcription factors driving the activation and differentiation of the stem cell compartments after injury are different in PBGs of large ducts with respects to canals of Hering. The existence of lineage-dependant diseases could represent the background for hypothesizing also a lineage-dependent carcinogenesis. A large body of recent literature deals with the involvement of stem⁄progenitors in the oncogenesis of the liver (30-35). Human hepatic stem cells are actually considered to be the origin of some subtypes of hepatocellular carcinoma (HCC) (36) or intrahepatic cholangiocarcinomas (32,33,37). It has been shown that stem cell activators such as WNT⁄b-catenin, transforming growth factor (TGF)-b, Notch and Hedgehog signaling pathways also expedite liver tumorigenesis (34,35). Primary liver cancers arise in the context of hepatobiliary diseases of different etiologies (28,38,39). As for cells of the pancreatic duct glands (40), the cells of the PBGs are sites of increased vulnerability to diseases, especially in response to the death of the injured mature cells or to chronic stimuli, which represent the initial step of the oncogenic process. Similarly, cell proliferation and PBG hyperplasia are common features associated with pathologies affecting the large intrahepatic and the extrahepatic bile ducts, which are currently considered risk factors for development of cholangiocarcinoma (CCA) (28). Interestingly, it has been observed that diseases primary targeting large bile ducts, such as PSC and liver flukes, are associated with both intrahepatic (IH) and extrahepatic (EH) cholangiocarcinoma (CCA) (28). On the contrary, parenchymal liver diseases such as cirrhosis of any etiology and chronic HCV- and HBV-related diseases, where only the stem cell niche in Canals of Hering are activated (28), 
are exclusively associated with IH-CCA. A number of recent studies suggest that stem cell niches within the biliary epithelium could be involved in CCA development (41,42). In particular, endoderm-like stem cells within PBGs could represent the cells of origin of pure mucin-producing IH-CCAs (43-45). This histological subtype of CCA is characterized by mucin-producing cells and typically arises at the hepatic hilum or at the level of large intrahepatic bile ducts (46). Our data demonstrated how PBGs contain mucin-producing cells and their location overlaps with the sites in which the mucinous CCA typically occurs. Moreover, CCAs express several markers in common with PBG cells such as EpCAM, OCT4 (POU class 5 homeobox 1), CD133 (prominin 1), Sall-1 (31). Finally, risk factors for hilar CCA, such as PSC and liver fluke infection, are characterized by the proliferation of PBGs (28,39,47). Based on new insights in pathological heterogeneity of CCA (43), one could hypothesize that carcinogenetic events involving the hHpSCs-derived lineage can give rise to combined HCC (hepatocarcinoma)-CCA and mixed-type CCA, whereas the involvement of the hBTSCs-derived lineage can give rise to pure mucin-producing CCA (44,45). The existence of two different stem cell compartments and the associated lineages may result in multiple cells of origin of CCA and could represent the basis of the clinico-pathological and epidemiological heterogeneity of CCA (45). Signaling pathways, such as WNT, Notch, Hedgehog have been extensively studied in the biliary epithelium and found to play a pivotal role in many aspects of biliary pathology and carcinogenesis. Unfortunately, the involvement of these pathways in carcinogenesis involving hBTSC dependent lineages has been so far neglected.

Perspectives in regenerative medicine of the liver

Patients affected by liver diseases need new cell sources to enable a better transition into clinic programs of cell therapy and regenerative medicine. Hepatocyte transplantation is the proof of concept for cell therapy of liver disease (48,49). Unfortunately, the limited supply of donor organs for the isolation of good quality hepatocytes and the low cell engraftment stimulated the search of alternative cell sources (48,49). Stem cells of different origins are currently under study as sources for cell therapy of liver diseases including Embrionic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, HpSCs, and Bone Marrow Stem Cells (CCA) (48-50). Liver parenchymal repopulation with exogenous cells is the prerequisite for successful cell therapy (50). To this regard, hepatic stem/progenitor cells (hHpSCs) discovered both in fetal and adult life are endowed with high proliferative capability with respect to mature hepatocytes (51-53). hHpSCs reside in the ductal plate of fetal liver and in canals of Hering of adult liver, differentiate in parenchymal liver cells and contribute to hepatic tissue homeostasis (52). Immunogenicity of fetal hepatic stem/progenitor cells is limited due to the low or null expression of class I and II major histocompatibility complex (MHC) antigens (54,55). In addition, hHpSCs showed high tolerance to ischemia (52,54). One of the favorable properties of the stem cells derives from the metabolomic pattern. Indeed, high level of unsaturated molecules perpetuates embryonic stem (ES) cells pluripotency, whereas the increase of oxidized metabolites promoted cell differentiation (56). These regulatory mechanisms may modulate the hepatic stem cells proliferation, renewal and differentiation. In recent studies, isolated hepatic progenitor cells (hepatoblasts and hHpSCs) from fetal liver were used to treat lethal congenital liver diseases (57). Moreover second-trimester fetal liver cells, enriched of EpCAM+ cells were infused into the hepatic artery of 25 patients with end stage liver cirrhosis (58). A marked improvement of clinical and biochemical parameters has been observed and the mean Model for End-Stage Liver Disease (MELD) score significantly decreased during six month follow-up without any adverse immune reaction (58). Interestingly, cell tracing showed specific hepatic seeding (58). In general, clinical trials, using autologous bone marrow derived stem cells, provided interesting data in terms of safety and short-term benefits in the treatment of chronic liver diseases (59). However, the mechanisms of action of bone marrow-derived stem cells in the setting of liver disease treatment are unclear (60). We have recently demonstrated the presence of cells endowed with a constellation of markers suggestive of primitive endodermal phenotype in PBGs of extrahepatic bile ducts (1-3). These cells are easily isolated and cultivated and, show a stable phenotype during in vitro expansion (1). They differentiated between hepatocytic and biliary fates and pancreatic islets under defined mediums (1). Transplantation to the liver of SCID (Severe Combined Immunodeficiency) mice resulted in mature hepatic fate lineage restriction (1). hBTSCs are candidate for regenerative medicine of liver and endocrine pancreas. The observation that hBTSCs can engraft and differentiate in mice livers is encouraging for future studies, where BTSCs could be transplanted in much higher numbers via vascular or intrasplenic routes. There is an increasing range of potential applications of stem cells in liver diseases, with many clinical studies already undertaken (61). In general, the cell types so far proposed for regenerative medicine of the liver have encountered a number of obstacles including: (I) limited sourcing for mature liver cells (48,49); (II) side effects such as emboli, ectopic distribution of cells, and transient effects for adult hepatocytes (48,49); and (III) high oncogenic potential for ES cells and induced Pluripotent Stem (iPS) cells (48,49,59,62). the large availability of adult biliary tree tissues and their biological properties guarantee an ideal source of easily isolable and cultivable stem cells and progenitors candidate for regenerative medicine of the liver. Finally, hBTSCs can be easily produced under GMP conditions. The fetal human biliary tree contains progenitor/stem cells of endodermal origin within the surface epithelium and bud of PBGs, which likely represent the fetal precursors of hBTSC populations persisting in adult biliary tree throughout life (63). The hBTSCs from foetal tissue could, theoretically, be considered as a source for cell therapies of liver and pancreas (63). As far as clinical programs are concerned, hHpSCs from foetal livers have been successfully used in clinical trials without requirement of immune-suppression (58). The isolation of both hepatoblasts and hBTSCs from the foetal liver should furnish obvious advantages in terms of cell yield and therapeutic targets (64).

Potential role of hBTSCs in the regenerative medicine of pancreas

Islet transplantation is viewed as an ideal treatment for type I diabetes, but it is constrained by the limited yield of quality donor pancreata that can be used to isolate islets (65). Recently, a new source of islet precursors has been identified in the biliary epithelium of donors of all ages (1-3). It is noteworthy that lineage restriction of ES cells, iPSCs or mesenchymal stem cells (MSCs) to an islet fate requires transfection with key transcription factors (e.g., PDX1) and sequential treatments with a panel of growth factors and matrix components for 4 to 6 weeks (66,67). By contrast, the biliary stem cells are already at the stage 4 of the 5 stages of the differentiation process previously described (68) and are poised to rapidly generate islets in an appropriate microenvironment and without gene manipulation. Given the assumption that pancreatic stem cells are lacking in postnatal tissues, a number of cell sources have been studied including: ES cells (66,69,70), iPS cells (64,65), MSCs (from bone marrow or cord blood) (71), adipose tissue or amniotic fluid-derived stem cells (72,73), transdifferentiation of acinar cells to islet cells by genetic manipulations (74,75). They will not lead to clinical programs until a number of challenges are overcome. In contrast to these different proposals, the biliary tree, in both fetal and adult life is replete with large numbers of multipotent stem cells (1-3,63,64). The ready availability of biliary tree tissues from fetal and adult liver and the extensive expansion potential of the biliary tree stem cells in culture under wholly defined conditions makes biliary tree stem cells a viable option for clinical programs in the treatment of diabetes and other pancreatic diseases.

hBTSCs and pancreas ongoing organogenesis

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells (76). We show that proximal (PBGs) to distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency [NANOG (Nanog homeobox), OCT4, SOX2], proliferation (Ki67), self-replication (SALL4) and early hepato-pancreatic commitment [SOX9, SOX17, PDX1, LGR5 (eucine-rich repeat-containing G protein coupled receptor 5)] and transit to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation [NGN3 (neurogenin 3), MUC6 (mucin 6, oligomeric mucus/gel-forming), insulin] (76). The biliary tree-derived stem cells and their connections to pancreatic-committed progenitors constitute a biological framework for life-long pancreatic organogenesis (76). In a recent paper by Banga et al. (77), SOX9+ cells in the liver have been reprogrammed to pancreatic cells by the overexpression of three transcriptional factors [PDX1, NGN3 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A)]. The Authors (77) observed the appearance within the intrahepatic biliary tree of ectopic duct-like structures where cells express a variety of endocrine pancreatic markers associated with the improvement of glycemic profile. In addition, chromatin configuration of mature liver cells still allows access to pancreatic transcription factors and, so their overexpression can be effective at phenotypic reprogramming (77). Interestingly, in accordance with the local microenvironment, insulin-positive cells were rarely found in intrahepatic PBGs while they are numerous in the hepato-pancreatic ampulla (2). In vitro, the differentiation of BTSCs towards endocrine pancreatic cells is characterized by the loss of pluripotency markers and the sequential acquisition of endocrine committed progenitor markers (NGN3 and MafA), recapitulating the embryologic development of endocrine pancreas from definite endoderm (1-3). In situ, a proximal-to-distal axis lineages were present, characterized by a shift from PBG stem cells with pluripotency gene expression to committed progenitors with NGN3/MafA expression in the pancreatic duct glands (76). In the paper by Banga et al. (77), insulin-positive cells were mostly located in large intrahepatic bile ducts and associated PBGs and, probably, derive from SOX9+ cells showing the phenotype of BTSCs (SOX17+/PDX1+). This suggests that candidate cells responsible for the permanent reprogramming event are the BTSCs of intrahepatic bile ducts, which are easily driven in vivo to differentiate toward pancreatic lineage, recapitulating the embryological development and the possible pancreatic organogenesis throughout life.

Conclusions

The discovery of human biliary tree stem/progenitor cells is opening a new scenario on many aspects of liver and pancreas pathophysiology (Table 1). These cells could represent, in the next future, an ideal cell source for the regenerative medicine of liver and pancreas. They are currently the objects of extensive investigations as cells of origin of bilio-pancreatic cancers. Finally, their involvement in the homeostasis and renewal of biliary and pancreatic ducts as well in the pathogenesis of chronic liver diseases, including PBC and PSC is under evaluation.

Table 1 Areas of interest arising from the discovery of human biliary tree stem/progenitor cells
Full table

Acknowledgements

Funding: E. Gaudio was supported by research project grant from the University “Sapienza” of Rome and FIRB grant # RBAP10Z7FS_001 and by PRIN grant # 2009X84L84_001. D. Alvaro was supported by FIRB grant # RBAP10Z7FS_004 and by PRIN grant # 2009X84L84_002. V. Cardinale was supported by FIRB grant # RBAP10Z7FS_004. The study was also supported by Consorzio Interuniversitario Trapianti d’Organo, Rome, Italy.

Disclosure: Findings from these studies have been included in patent applications belonging to Sapienza University (Rome, Italy) and/or to UNC (Chapel Hill, NC) and licensed to Vesta Therapeutics (Bethesda, MD). The authors do not have equity or a position in Vesta and are not paid consultants to the company.

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