Ruolo delle cellule staminali/progenitrici nella rigenerazione epatica e delle vie biliari: fenotipo e vie di segnalazione coinvolte

Introduzione. Nel fegato e nelle vie biliari sono presenti due distinte popolazioni di cellule staminali/progenitrici: le cellule progenitrici epatiche (HpSC), situate a livello dei duttuli di Hering, e le cellule staminali delle vie biliari (BTSC), nelle ghiandole peribiliari dei dotti biliari intraepatici di grosso calibro e dei dotti biliari extraepatici. Scopo. Lo scopo dello studio è stato quello di studiare l’attivazione delle HpSC e delle BTSC in corso di patologie epatiche e delle vie biliari e le vie di segnalazione coinvolte. Materiali e Metodi. Sono stati analizzati: pazienti affetti da steatosi epatica non alcolica (NAFLD); pazienti affetti da colangite biliare primitiva (PBC) e da colangite sclerosante primitiva (PSC); pazienti affetti da stenosi non-anastomotiche delle vie biliari (NAS) dopo trapianto di fegato; BTSC umane in vitro. I campioni sono stati studiati mediante colorazioni istomorfologiche, immunoistochimiche e di immunofluorescenza. Risultati. Nella NAFLD, l’espansione delle HpSC correla con lo stadio istologico di malattia e con lo stato di attivazione dei miofibroblasti, dei macrofagi e del sistema lipopolisaccaride-TLR4. Nella PBC e nella PSC, l’attivazione delle HpSC correla con lo stadio di fibrosi; il fenotipo e le modalità di attivazione delle HpSC sono differenti, con il pathway di Notch principalmente coinvolto nella PBC e quello di Wnt nella PSC. L’attivazione delle HpSC rappresenta un fattore predittivo della risposta alla terapia di prima linea in pazienti con PBC. Le ghiandole peribiliari sono coinvolte nella patogenesi del danno nelle colangiopatie che colpiscono i dotti biliari extraepatici, risultando attivate e floride nella PSC e, diversamente, fortemente danneggiate nei dotti affetti da NAS. L’attivazione delle BTSC è mediata dai pathway di Wnt e Notch, i cui ligandi sono prodotti dalle cellule stromali ed infiammatorie in corso di PSC. Conclusioni. Le HpSC e le BTSC sono attivate in patologie epatiche e delle vie biliari. Lo stadio del danno istologico è correlato all’attivazione delle cellule staminali/progenitrici e delle cellule di supporto della nicchia. L’attivazione ed il commissionamento verso un destino specifico delle cellule staminali/progenitrici sono mediati dall’attivazione differenziale delle vie di segnalazione, in maniera finalizzata alla riparazione dello specifico danno

Contribution of resident stem cells to liver and biliary tree regeneration in human diseases

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression

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

Overexpression of the vitronectin v10 subunit in patients with nonalcoholic steatohepatitis: Implications for noninvasive diagnosis of NASH

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM prote in vitro nectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosi

Lactoferrin regulate biliary epithelium growth and the activation of hepatic progenitor cell niche

Lactoferin (Lf) is an iron-binding glycoprotein belonging to the transferrin family and it is present at high levels in breast milk and colostrum. This protein has many known functions and it is a potential antibacterial, antiviral, immunostimulatory, antioxidant, and cancer preventive agent.It has been seen that a 105 kDa Lf receptor (LfR) specifically mediates the effects of Lf in several different cell types (1). In human cholangiopathies, cholangiocytes are able to proliferate and replace the cell loss restoring the integrity of damaged biliary epithelium. However, when cholangiocyte proliferation is severely impaired, the activation of facultative hepatic progenitor cells (HPCs) takes place (2). The aims of our study have been i) to investigate the expression of Lf and LfR in cholangiocytes and in HPCs both in rats and in humans; and ii) to evaluate the in vitro effects of bLf on cholangiocyte proliferation and on HPC activation. Liver specimens have been obtained from normal (N=5) and bile duct ligated (BDL) (N=5) rats; from normal patients (N=5) and from patients with primary biliary cirrhosis (PBC, N=5); Specimens were processed for histology, immunohistochemistry and immunofluorescence. Moreover for the in vitro study small and large cholangioytes from mouse, human non malignant cholangiocytes (H69), and HPCs treated or not with lactoferrin were used. Our results showed that: i) cholangiocytes and hepatic progenitor cells express lactoferrin and its receptor, ii) cholangiocytes and HPC proliferation is enhanced by lactoferrin; iii) the treatment with lactoferrin determine the commitment of HPCs towards cholangiocyte fate; this commitment is characterized by HPC morphological and phenotypical changes. Our current findings suggest that modulation of lactoferrin may be an important therapeutic tool for managing the proliferation of cholangiocyte and the activation of progenitor cell compartment in biliary disorders

Recommendations maximising the clinical value of tissue in the management of patients with intrahepatic cholangiocarcinoma

Background & Aims: Patients with intrahepatic cholangiocarcinoma can now be managed with targeted therapies directed against specific molecular alterations. Consequently, tissue submitted to a pathology department must yield molecular information in addition to a diagnosis or, in resection specimens, staging information. The pathologist’s role reporting these specimens has therefore changed to accommodate such personalised approaches. Methods: We have developed recommendations and guidance for pathologists by undertaking a systematic review of existing guidance to generate candidate statements followed by an international Delphi process. 59 pathologists from 28 countries in 6 continents rated statements mapped to all elements of the specimen pathway from receipt in the pathology department to authorisation of the final written report. A separate survey of ‘end-users’ of the report including surgeons, oncologists, and gastroenterologists was undertaken to evaluate what information should be included in the written report to enable appropriate patient management. Results: 48 statements reached consensus for inclusion in the guidance including 10 statements about the content of the written report that also reached consensus by the ‘end-user’ participants. A reporting proforma to allow easy inclusion of the recommended data points is provided. Conclusions: These guiding principles and recommendations provide a framework to allow pathologists reporting intrahepatic cholangiocarcinoma to maximise the informational yield of specimens required for personalised patient management

Recommendations on maximising the clinical value of tissue in the management of patients with intrahepatic cholangiocarcinoma

Background &amp; Aims: Patients with intrahepatic cholangiocarcinoma can now be managed with targeted therapies directed against specific molecular alterations. Consequently, tissue samples submitted to the pathology department must produce molecular information in addition to a diagnosis or, for resection specimens, staging information. The pathologist's role when evaluating these specimens has therefore changed to accommodate such personalised approaches. Methods: We developed recommendations and guidance for pathologists by conducting a systematic review of existing guidance to generate candidate statements followed by an international Delphi process. Fifty-nine pathologists from 28 countries in six continents rated statements mapped to all elements of the specimen pathway from receipt in the pathology department to authorisation of the final written report. A separate survey of ‘end-users’ of the report including surgeons, oncologists, and gastroenterologists was undertaken to evaluate what information should be included in the written report to enable appropriate patient management. Results: Forty-eight statements reached consensus for inclusion in the guidance including 10 statements about the content of the written report that also reached consensus by end-user participants. A reporting proforma to allow easy inclusion of the recommended data points was developed. Conclusions: These guiding principles and recommendations provide a framework to allow pathologists reporting on patients with intrahepatic cholangiocarcinoma to maximise the informational yield of specimens required for personalised patient management. Impact and Implications: Biopsy or resection lesional tissue from intrahepatic cholangiocarcinoma must yield information about the molecular abnormalities within the tumour that define suitability for personalised therapies in addition to a diagnosis and staging information. Here, we have developed international consensus guidance for pathologists that report such cases using a Delphi process that sought the views of both pathologists and ‘end-users of pathology reports. The guide highlights the need to report cases in a way that preserves tissue for molecular testing and emphasises that reporting requires interpretation of histological characteristics within the broader clinical and radiological context. The guide will allow pathologists to report cases of intrahepatic cholangiocarcinoma in a uniform manner that maximises the value of the tissue received to facilitate optimal multidisciplinary patient management.</p

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

Hepatic stem/progenitor cell activation differs between primary sclerosing and primary biliary cholangitis

The activation of hepatic stem/progenitor cells (HPCs) is characterized by the appearance of ductular reaction (DR) in the liver parenchyma [1]. The aims of the present study were to evaluate the activation of HPCs in human cholangiopathies. Human liver tissue was obtained from liver donors (N=5), Primary Sclerosing Cholangitis (PSC; N=20), and Primary Biliary Cholangitis (PBC; N=20) patients. Ductular reaction extension was evaluated by Keratin(K) 7 immunoreactivity. HPC phenotype and signalling pathways were investigated by immunohistochemistry and immunofluorescence [2]. Ductular reaction in PBC is more extensive, appears earlier, and has a higher proliferation index compared to PSC. In PBC the extension of DR strongly correlates with clinical prognostic scores. A higher percentage of Sox9+ and K19+ cells characterized DR in PBC versus PSC. In cirrhotic-PSC, the HPC compartment showed signs of hepatocyte commitment. The study of the HPC niche indicated lower levels of laminin and NOTCH1 but higher expression of WNT pathway components in PSC compared to PBC. In conclusion, PSC and PBC are characterized by different patterns of HPC niche activation, reflecting the involvement of different portions of the biliary tree as primary target of damage. These aspects could have implications in the pathogenesis of cholangiopathies and could add prognostic value

Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study

Abstract Pancreatic duct glands (PDGs) are tubule‐alveolar glands associated with the pancreatic duct system and can be considered the anatomical counterpart of peribiliary glands (PBGs) found within the biliary tree. Recently, we demonstrated that endodermal precursor niches exist fetally and postnatally and are composed functionally of stem cells and progenitors within PBGs and of committed progenitors within PDGs. Here we have characterized more extensively the anatomy of human PDGs as novel niches containing cells with multiple phenotypes of committed progenitors. Human pancreata (n = 15) were obtained from cadaveric adult donors. Specimens were processed for histology, immunohistochemistry and immunofluorescence. PDGs were found in the walls of larger pancreatic ducts (diameters > 300 μm) and constituted nearly 4% of the duct wall area. All of the cells identified were negative for nuclear expression of Oct4, a pluripotency gene, and so are presumably committed progenitors and not stem cells. In the main pancreatic duct and in large interlobular ducts, Sox9+ cells represented 5–30% of the cells within PDGs and were located primarily at the bottom of PDGs, whereas rare and scattered Sox9+ cells were present within the surface epithelium. The expression of PCNA, a marker of cell proliferation, paralleled the distribution of Sox9 expression. Sox9+PDG cells proved to be Pdx1+/Ngn3+/–/Oct4A−. Nearly 10% of PDG cells were positive for insulin or glucagon. Intercalated ducts contained Sox9+/Pdx1+/Ngn3+ cells, a phenotype that is presumptive of committed endocrine progenitors. Some intercalated ducts appeared in continuity with clusters of insulin‐positive cells organized in small pancreatic islet‐like structures. In summary, PDGs represent niches of a population of Sox9+ cells exhibiting a pattern of phenotypic traits implicating a radial axis of maturation from the bottoms of the PDGs to the surface of pancreatic ducts. Our results complete the anatomical background that links biliary and pancreatic tracts and could have important implications for the common patho‐physiology of biliary tract and pancreas