Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Summary Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4–8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7–10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.     

The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.     

Isolation and characterization of biliary epithelial cells from rainbow trout liver

Summary Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.     

Isolated hepatocyte couplets and bile duct units - Novel preparations for the in vitro study of bile secretory function

Studies of the mechanisms of bile formation have been facilitated by the development of in-vitro models that enable the transport functions of hepatocytes and bile duct epithelial cells to be analyzed in isolation. Isolated hepatocyte couplets represent a primary bile secretory unit that retain secretory polarity. Isolated bile duct units are intact polarized segments of interlobular bile duct segments. Both preparations can be maintained in short term culture, respond appropriately to choleretic agonists and facilitate a variety of functional applications.     

The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine

Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 μg/L, 7.5 μg/L, 15 μg/L, or 30 μg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver.     

Alteration in N-acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N-acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E₄-PHA. The increased GnT-V activity led to the elevation of the β1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.     

Identification and characterization of label-retaining cells in mouse pancreas

Pancreatic stem cells (PSCs) may play an important role in maintaining and repairing pancreatic tissues. However, both the existence and localization of PSCs in adult mammalian pancreas still remain elusive. In order to locate the potential pancreatic progenitor/stem cells, we used the tracing label-retaining cells (LRCs) method and identified slow-cycling cells in mouse pancreas. Characterization of the LRCs revealed that the differentiation marker-negative LRCs were located not only within and around the islets but also around the acini and ducts. About 30% of the LRCs around the acini and ducts expressed c-Met, which is a putative pancreatic progenitor/stem cell marker. Moreover, the LRCs around the acini could be activated to form duct-like structures in response to pancreatic damage, and the involvement of these LRCs in the neogenesis of islets and focal areas could also be observed in acini. Our data suggest that the LRCs located around the acini and ducts may represent potential pancreatic progenitor/stem cells, and characterization of these cells may aid in further identification of the specific markers of pancreatic progenitor/stem cells.     

Telocytes and interstitial cells of Cajal in the biliary system

A novel type of interstitial tissue cells in the biliary tree termed telocytes (TCs), formerly known as interstitial Cajal‐like cells (ICLCs), exhibits very particular features which unequivocally distinguish these cells from interstitial cells of Cajal (ICCs) and other interstitial cell types. Current research substantiates the existence of TCs and ICCs in the biliary system (gallbladder, extrahepatic bile duct, cystic duct, common bile duct and sphincter of Oddi). Here, we review the distribution, morphology and ultrastructure of TCs and ICCs in the biliary tree, with emphasis on their presumptive roles in physiological and pathophysiological processes.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: A culture study

Cellular senescence is reportedly involved in cholangiopathy in primary biliary cirrhosis and oxidative stress is proposed as a pathogenetic factor in biliary epithelial cells (BECs). This study investigated the involvement of proinflammatory cytokines (IFN-β, IFN-γ and TNF-α) and ataxia telangiectasia-mutated (ATM)/p53/ p21^WAF1/Cip1 pathway with respect to oxidative stress in cellular senescence of BECs. H₂O₂ treatment (oxidative stress) induced phosphorylation (activation) of ATM and p53 and also p21^WAF1/Cip1 expression in BECs. Treatment with inflammatory cytokines generated reactive oxygen species (ROS) in cultured BECs followed by activation of the ATM/p53/p21^WAF1/Cip1 pathway and the induction of cellular senescence. Pre-treatment with ATM inhibitor (2-aminopurine) and antioxidant (N-acetylcysteine) significantly blocked the cellular senescence of BECs induced by oxidative stress or inflammatory cytokines. In conclusion, proinflamamtory cytokines induce ROS generation and activate the ATM/p53/p21^WAF1/Cip1 pathway, followed by biliary epithelial senescence. This senescent process may be involved in the development of destructive cholangiopathy in humans.     

Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: effect on epithelial barrier function and leukocyte survival

This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0 x 10(6) and 1.0 x 10(6)cells/ml, respectively, appeared to affect both the survival of leukocytes and epithelial barrier function. Cell viability counts of immune cells showed 95% and 72.5% cell survival after isolation and after 4 days in co-culture with EEC, respectively, but only 11% cell survival when cultured alone for 4 days without EEC. Short-circuit current (I(sc)) results also showed that EEC and PBL co-culture exhibited a four-fold increase in the transepithelial resistance (TER) as compared to EEC culture alone, indicating enhanced protective barrier function. Taken together, the currently established in vitro co-culture model of endometrial epithelial cells and immune cells may provide a means to investigate local cellular immune responses upon uterine infections.     

Wnt/β‐catenin signalling controls bile duct regeneration by regulating differentiation of ductular reaction cells

Recently, the incidence of bile duct‐related diseases continues to increase, and there is no effective drug treatment except liver transplantation. However, due to the limited liver source and expensive donations, clinical application is often limited. Although current studies have shown that ductular reaction cells (DRCs) reside in the vicinity of peribiliary glands can differentiate into cholangiocytes and would be an effective alternative to liver transplantation, the role and mechanism of DRCs in cholangiole physiology and bile duct injury remain unclear. A 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐enriched diet was used to stimulate DRCs proliferation. Our research suggests DRCs are a type of intermediate stem cells with proliferative potential that exist in the bile duct injury. Meanwhile, DRCs have bidirectional differentiation potential, which can differentiate into hepatocytes and cholangiocytes. Furthermore, we found DRCs highly express Lgr5, and Lgr5 is a molecular marker for neonatal DRCs (P < .05). Finally, we confirmed Wnt/β‐catenin signalling achieves bile duct regeneration by regulating the expression of Lgr5 genes in DRCs (P < .05). We described the regenerative potential of DRCs and reveal opportunities and source for the treatment of cholestatic liver diseases.     

Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma

Background

At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA).

Methods

Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium.

Results

The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44^high, CD24^low, and AC133⁺. These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α₂β₁, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α₂β₁, and CD146 surface marker expression than the epithelial type cells.

Conclusion

The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.