Intrahepatic cholangiocarcinoma: review and update

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies that could develop at any level from the biliary tree. CCA is currently classified into intrahepatic (iCCA), perihilar and distal on the basis of its anatomical location. Of note, these three CCA subtypes have common features but also important inter-tumor and intra-tumor differences that can affect the pathogenesis and outcome. A unique feature of iCCA is that it recognizes as origin tissues, the hepatic parenchyma or large intrahepatic and extrahepatic bile ducts, which are furnished by two distinct stem cell niches, the canals of Hering and the peribiliary glands, respectively. The complexity of iCCA pathogenesis highlights the need of a multidisciplinary, translational and systemic approach to this malignancy. This review will focus on the advances of iCCA epidemiology, histo-morphology, risk factors, molecular pathogenesis, revealing the existence of multiple subsets of iCCA

Peribiliary glands as a niche of extra-pancreatic precursors yielding insulin-producing cells in experimental and human diabetes

Peribiliary glands (PBGs) are niches in the biliary tree and containing heterogeneous endodermal stem/progenitors cells that can differentiate, in vitro and in vivo, towards pancreatic islets. The aim of this study was to evaluate, in experimental and human diabetes, proliferation of cells in PBGs and differentiation of the biliary tree stem/progenitor cells (BTSCs) towards insulin-producing cells. Diabetes was generated in mice by intraperitoneal injection of a single dose of 200 mg/kg (N=12) or 120 mg/kg (N=12) of streptozotocin. Liver, pancreas and extrahepatic biliary trees were en bloc dissected and examined. Cells in PBGs proliferated in experimental diabetes, and their proliferation was greatest in the PBGs of the hepato-pancreatic ampulla, and inversely correlated with the pancreatic islet area. In rodents, the cell proliferation in PBGs was characterized by the expansion of Sox9-positive stem/progenitor cells that gave rise to insulin-producing cells. Insulin-producing cells were located mostly in PBGs in the portion of the biliary tree closest to the duodenum, and their appearance was associated with up-regulation of MafA and Gli1 gene expression. In patients with type 2 diabetes, PBGs at the level of the hepato-pancreatic ampulla contained cells showing signs of proliferation and pancreatic fate commitment. In vitro, high glucose concentrations induced the differentiation of human BTSCs cultures towards pancreatic beta cell fates. The cells in PBGs respond to diabetes with proliferation and differentiation towards insulin-producing cells indicating that PBG niches may rescue pancreatic islet impairment in diabetes. These findings offer important implications for the patho-physiology and complications of this disease. This article is protected by copyright. All rights reserved

Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota's Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs

Adult human biliary tree stem cells differentiate to β-pancreatic islet cells by treatment with a recombinant human Pdx1 peptide

Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells

Cryopreservation protocol for human biliary tree stem/progenitors, hepatic and pancreatic precursors

Human biliary tree stem/progenitor cells (hBTSCs) are being used for cell therapies of patients with liver cirrhosis. A cryopreservation method was established to optimize sourcing of hBTSCs for these clinical programs and that comprises serum-free Kubota's Medium (KM) supplemented with 10% dimethyl sulfoxide (DMSO), 15% human serum albumin (HSA) and 0.1% hyaluronans. Cryopreserved versus freshly isolated hBTSCs were similar in vitro with respect to self-replication, stemness traits, and multipotency. They were able to differentiate to functional hepatocytes,cholangiocytes or pancreatic islets, yielding similar levels of secretion of albumin or of glucose-inducible levels of insulin. Cryopreserved versus freshly isolated hBTSCs were equally able to engraft into immunocompromised mice yielding cells with human-specific gene expression and human albumin levels in murine serum that were higher for cryopreserved than for freshly isolated hBTSCs. The successful cryopreservation of hBTSCs facilitates establishment of hBTSCs cell banking offering logistical advantages for clinical programs for treatment of liver diseases

Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem/progenitor cells

BACKGROUND: Cell therapy of liver diseases with human biliary tree stem cells (hBTSCs) is biased by low engraftment efficiency. Coating the hBTSCs with hyaluronans (HAs), the primary constituents of all stem cell niches, could facilitate cell survival, proliferation, and, specifically, liver engraftment given that HAs are cleared selectively by the liver. METHODS: We developed a fast and easy method to coat hBTSCs with HA and assessed the effects of HA-coating on cell properties in vitro and in vivo. RESULTS: The HA coating markedly improved the viability, colony formation, and population doubling of hBTSCs in primary cultures, and resulted in a higher expression of integrins that mediate cell attachment to matrix components. When HA-coated hBTSCs were transplanted via the spleen into the liver of immunocompromised mice, the engraftment efficiency increased to 11% with respect to 3% of uncoated cells. Notably, HA-coated hBTSC transplantation in mice resulted in a 10-fold increase of human albumin gene expression in the liver and in a 2-fold increase of human albumin serum levels with respect to uncoated cells. Studies in distant organs showed minimal ectopic cell distribution without differences between HA-coated and uncoated hBTSCs and, specifically, cell seeding in the kidney was excluded. CONCLUSIONS: A ready and economical procedure of HA cell coating greatly enhanced the liver engraftment of transplanted hBTSCs and improved their differentiation toward mature hepatocytes. HA coating could improve outcomes of stem cell therapies of liver diseases and could be immediately translated into the clinic given that GMP-grade HAs are already available for clinical use

Peribiliary gland niche participates in biliary tree regeneration in mouse and in human primary sclerosing cholangitis

Background and Aims: Mechanisms underlying the repair of extrahepatic biliary tree (EHBT) after injury have been scarcely explored. The aims of this study were to evaluate, by using a lineage tracing approach, the contribution of peribiliary gland (PBG) niche in the regeneration of EHBT after damage and to evaluate, in vivo and in vitro, the signaling pathways involved. Approach and Results: Bile duct injury was induced by the administration of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 14 days to Krt19CreTdTomatoLSL mice. Human biliary tree stem/progenitor cells (BTSC) within PBGs were isolated from EHBT obtained from liver donors. Hepatic duct samples (n&nbsp;=&nbsp;10) were obtained from patients affected by primary sclerosing cholangitis (PSC). Samples were analyzed by histology, immunohistochemistry, western blotting, and polymerase chain reaction. DDC administration causes hyperplasia of PBGs and periductal fibrosis in EHBT. A PBG cell population (Cytokeratin19-/SOX9+) is involved in the renewal of surface epithelium in injured EHBT. The Wnt signaling pathway triggers human BTSC proliferation in vitro and influences PBG hyperplasia in vivo in the DDC-mediated mouse biliary injury model. The Notch signaling pathway activation induces BTSC differentiation in vitro toward mature cholangiocytes and is associated with PBG activation in the DDC model. In human PSC, inflammatory and stromal cells trigger PBG activation through the up-regulation of the Wnt and Notch signaling pathways. Conclusions: We demonstrated the involvement of PBG cells in regenerating the injured biliary epithelium and identified the signaling pathways driving BTSC activation. These results could have relevant implications on the pathophysiology and treatment of cholangiopathies

Transplantation of human fetal biliary tree stem/progenitor cells into two patients with advanced liver cirrhosis.

Efforts to identify cell sources and approaches for cell therapy of liver diseases are ongoing, taking into consideration the limits recognized for adult liver tissue and for other forms of stem cells. In the present study, we described the first procedure of via hepatic artery transplantation of human fetal biliary tree stem cells in patients with advanced cirrhosis.MethodsThe cells were immune-sorted from human fetal biliary tree by protocols in accordance with current good manufacturing practice (cGMP) and extensively characterized. Two patients with advanced cirrhosis (Child-Pugh C) have been submitted to the procedure and observed through a 12 months follow-up.ResultsThe resulting procedure was found absolutely safe. Immuno-suppressants were not required, and the patients did not display any adverse effects correlated with cell transplantation or suggestive of immunological complications. From a clinical point of view, both patients showed biochemical and clinical improvement during the 6 month follow-up (Table1), and the second patient maintained a stable improvement for 12 months.ConclusionThis report represents proof of the concept that the human fetal biliary tree stem cells are a suitable and large source for cell therapy of liver cirrhosis. The isolation procedure can be carried out under cGMP conditions and, finally, the infusion procedure is easy and safe for the patients. This represents the basis for forthcoming controlled clinical trials

Activation of anti-apoptotic machinery downstream to Fas/FasL pathway in primary mixed and pure mucin producing cholangiocarcinoma cells: key role of c-FLIP

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignancies lacking of effective strategies for prevention and cure. Recently, we have established a protocol for the isolation of primary cells from mixed and mucin specimens of human CCA. To this regard, the aim of this study was to analyze the influence on proliferation and apoptosis as well as the related modifications of apoptotic machinery downstream to Fas/FasL pathway in primary cultures of human mixed and mucin-producing CCA after direct co-culture with peripheral blood mononuclear cells (PBMCs). Our findings show that both IH-CCA subtypes constitutively express high levels of Fas and FasL. Following direct co-culture with PBMCs, the expression of Fas and FasL significantly increased after 24, 48 and 72 hours of exposure (p< 0.05). At the same time, a significant increase of percentage of apoptotic CD4+ and CD8+ T-cells or Natural Killer CD56+ cells was observed along the co-cultures compared to PBMCs cultured alone (

Cholest-4,6-Dien-3-One Promote Epithelial-To-Mesenchymal Transition (EMT) in Biliary Tree Stem/Progenitor Cell Cultures In Vitro

Human biliary tree stem/progenitor cells (hBTSCs), reside in peribiliary glands, are mainly stimulated by primary sclerosing cholangitis (PSC) and cholangiocarcinoma. In these pathologies, hBTSCs displayed epithelial-to-mesenchymal transition (EMT), senescence characteristics, and impaired differentiation. Here, we investigated the effects of cholest-4,6-dien-3-one, an oxysterol involved in cholangiopathies, on hBTSCs biology. hBTSCs were isolated from donor organs, cultured in self-renewal control conditions, differentiated in mature cholangiocytes by specifically tailored medium, or exposed for 10 days to concentration of cholest-4,6-dien-3-one (0.14 mM). Viability, proliferation, senescence, EMT genes expression, telomerase activity, interleukin 6 (IL6) secretion, differentiation capacity, and HDAC6 gene expression were analyzed. Although the effect of cholest-4,6-dien-3-one was not detected on hBTSCs viability, we found a significant increase in cell proliferation, senescence, and IL6 secretion. Interestingly, cholest-4.6-dien-3-one impaired differentiation in mature cholangiocytes and, simultaneously, induced the EMT markers, significantly reduced the telomerase activity, and induced HDAC6 gene expression. Moreover, cholest-4,6-dien-3-one enhanced bone morphogenic protein 4 (Bmp-4) and sonic hedgehog (Shh) pathways in hBTSCs. The same pathways activated by human recombinant proteins induced the expression of EMT markers in hBTSCs. In conclusion, we demonstrated that chronic exposition of cholest-4,6-dien-3-one induced cell proliferation, EMT markers, and senescence in hBTSC, and also impaired the differentiation in mature cholangiocytes