Stages based molecular mechanisms for generating cholangiocytes from liver stem/progenitor cells

Except for the most organized mature hepatocytes,liver stem/progenitor cells(LSPCs)can differentiate into many other types of cells in the liver including cholangiocytes.In addition,LSPCs are demonstrated to be able to give birth to other kinds of extra-hepatic cell types such as insulin-producing cells.Even more,under some bad conditions,these LSPCs could generate liver cancer stem like cells(LCSCs)through malignant transformation.In this review,we mainly concentrate on the molecular mechanisms for controlling cell fates of LSPCs,especially differentiation of cholangiocytes,insulin-producing cells and LCSCs.First of all,to certificate the cell fates of LSPCs,the following three features need to be taken into account to perform accurate phenotyping:(1)morphological properties;(2)specific markers;and(3)functional assessment including in vivo transplantation.Secondly,to promote LSPCs differentiation,systematical attention should be paid to inductive materials(such as growth factors and chemical stimulators),progressive materials including intracellular and extracellular signaling pathways,and implementary materials(such as liver enriched transcriptive factors).Accordingly,some recommendations were proposed to standardize,optimize,and enrich the effective production of cholangiocyte-like cells out of LSPCs.At the end,the potential regulating mechanisms for generation of cholangiocytes by LSPCs were carefully analyzed.The differentiation of LSPCs is a gradually progressing process,which consists of three main steps:initiation,progression and accomplishment.It’s the unbalanced distribution of affecting materials in each step decides the cell fates of LSPCs.

Multiple cells of origin in cholangiocarcinoma underlie biological,epidemiological and clinical heterogeneity

Recent histological and molecular characterization of cholangiocarcinoma(CCA) highlights the heterogeneity of this cancer that may emerge at different sites of the biliary tree and with different macroscopic or morphological features.Furthermore,different stem cell niches have been recently described in the liver and biliarytree,suggesting this as the basis of the heterogeneity of intrahepatic(IH)-and extrahepatic(EH)-CCAs,which are two largely different tumors from both biological and epidemiological points of view.The complexity of the organization of the liver stem cell compartments could underlie the CCA clinical-pathological heterogeneity and the criticisms in classifying primitive liver tumors.These recent advances highlight a possible new classification of CCAs based on cells of origin and this responds to the need of generating homogenous diagnostic,prognostic and,hopefully,therapeutic categories of IH-and EH-CCAs.

Participation of peribiliary glands in biliary tract pathophysiologies

AIM:To investigate the roles of peribiliary glands around the bile ducts in the pathophysiology of the biliary tract.METHODS:The expression of fetal pancreatic markers,pancreatic duodenal homeobox factor 1(PDX1)and hairy and enhancer of split 1(HES1)and endodermal stem/progenitor(S/P)cell markers[CD44s,chemokine receptor type 4(CXCR4),SOX9 and epithelial cell adhesion molecule(EpCAM)]were examined immunohistochemically in 32 normal adult livers(autopsy livers)and 22 hepatolithiatic livers(surgically resected livers).The latter was characterized by the proliferation of the peribiliary glands.Immunohistochemistry was performed using formalin-fixed,paraffin-embedded tissue sections after deparaffinization.Although PDX1and HES1 were expressed in both the nucleus and cytoplasm of epithelial cells,only nuclear staining was evaluated.SOX9 was expressed in the nucleus,while CD44s,CXCR4 and EpCAM were expressed in the cell membranes.The frequency and extent of the expression of these molecules in the lining epithelia and peribiliary glands were evaluated semi-quantitatively based on the percentage of positive cells:0,1+(focal),2+(moderate)and 3+(extensive).RESULTS:In normal livers,PDX1 was infrequently expressed in the lining epithelia,but was frequently expressed in the peribiliary glands.In contrast,HES1was frequently expressed in the lining epithelia,but its expression in the peribiliary glands was focal,suggesting that the peribiliary glands retain the potential of differentiation toward the pancreas and the lining epithelia exhibit properties to inhibit such differentiation.This unique combination was also seen in hepatolithiatic livers.The expression of endodermal S/P cell markers varied in the peribiliary glands in normal livers:SOX9 and EpCAM were frequently expressed,CD44s infrequently,and CXCR4 almost not at all.The expression of these markers,particularly CD44s and CXCR4,increased in the peribiliary glands and lining epithelia in hepatolithiatic livers.This increased expression of endodermal S/P cell markers may be related to the increased production of intestinal and gastric mucin and also to the biliary neoplasia associated with the gastric and intestinal phenotypes reported in hepatolithiasis.CONCLUSION:The unique expression pattern of PDX1and HES1 and increased expression of endodermal S/P cell markers in the peribiliary glands may be involved in biliary pathophysiologies.

Hormonally and chemically defined expansion conditions for organoids of biliary tree Stem Cells

Wholly defined ex vivo expansion conditions for biliary tree stem cell(BTSC)organoids were established,consisting of a defined proliferative medium(DPM)used in combination with soft hyaluronan hydrogels.The DPM consisted of commercially available Kubota's Medium(KM),to which a set of small molecules,particular paracrine signals,and heparan sulfate(HS)were added.The small molecules used were DNA methyltransferase inhibitor(RG108),TGF-βType I receptor inhibitor(A83-01),adenylate cyclase activator(Forskolin),and L-type Ca2+channel agonist(Bay K8644).A key paracrine signal proved to be R-spondin 1(RSPO1),a secreted protein that activates Wnts.Soluble hyaluronans,0.05%sodium hyaluronate,were used with DPM to expand monolayer cultures.Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology[G*]of less than 100 Pa.The combination is called the BTSC-Expansion-Glycogel-System(BEX-gel system)for expanding BTSCs as a monolayer or as organoids.The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors.Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah-/-mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions.The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.

胆管树干细胞在肝脏和胰腺再生中的研究进展

胆管树干细胞(biliary tree stem cells, BTSCs)是一类存在于胆管壁上胆周腺内的干细胞,具有分化为成熟肝细胞、胆管上皮细胞及胰腺内分泌细胞等类型细胞的多向分化潜能,在肝损伤治疗、糖尿病救治等方面具有巨大应用前景。本文结合胆管及其相关组织的胚胎发育过程、解剖学特征、BTSCs谱系分布特点及分子标志、体外扩增特性、基础及临床研究现状等,对BTSCs的生物学特性进行阐述,并对其在肝脏和胰腺器官再生中的研究进展和应用前景进行总结和展望。