Study On The Characteristic Of Hepatic,biliary And Pancreatic Duct Progenitors Using Organoids

The liver and pancreas are important exocrine glands in human,and they provide exocrine secretions necessary for food digestion,which are collected by a network of ducts that acts as a plumbing system,distributing them to the gallbladder for storage.Stem/progenitors for liver,biliary tree,and pancreas exist at early stages of development in the definitive ventral endoderm.Recent studies have shown that multipotent stem cells existed in human biliary glands.They can differentiate into hepatocytes,cholangiocytes and pancreatic endocrine cells.Similar functional progenitors were found in the pancreatic duct glands.The organoids prepared from adult tissue stem/progenitor cells are a three-dimensional epithelial structure culture.Studies have shown that liver organoids have the ability to differentiate into functional hepatocytes,and mouse pancreatic organoids have the ability to differentiate into hepatocytes and pancreatic endocrine cells after transplanting into mice.Combining the above results,we speculated that hepatic,biliary and pancreatic duct epithelial progenitors have similar functions,which can differentiate into hepatocytes,cholangiocytes and pancreatic endocrine cells.Therefore,we used 3D culture technology to establish mouse hepatic duct organoid(mHDO),bile tree organoid(mBTO),and pancreatic duct organoid(mPDO).We verified the similarity of hepatic,biliary and pancreatic duct epithelial progenitors by method of cell differentiation capabilities,cell lineage tracing,and high-throughput transcriptome sequencing.The human bile tree organoids(hBTO)were also prepared,and the methods for differentiation of hBTO into hepatocytes,cholangiocytes and pancreatic ? cells using a 3D culture in vitro was established successfully,which proved that hBTO has the potential to differentiate into multiple lineages.This research opens a new path for disease treatment and drug screening,and explores a wider range of cellular resources for regeneration of tissue and liver/islet transplantation.The main research methods and results of this study:1.The intrahepatic bile duct(hepatic duct),common bile tree duct(biliary duct),and main pancreatic duct(pancreatic duct)were directly separated under the dissection microscope by mechanical method,and the duct epithelial cells were obtained by collagenase digestion.Using Matrigel as a matrix,mPDO,mBTO and mHDO were successfully established.mPDO,mBTO and mHDO can self-renewal in vitro,and still growth normally after frozen storage and resuscitation.All organoids can be passaged continuously for more than 6 months.By primary test with reverse transcription PCR and immunostaining,they showed the potential to differentiate into multiple lineages.2.Three groups of mPDO,mBTO and mHDO were prepared respectively.Using transcriptome sequencing analysis,we compared gene expression difference between organoids and tissue epithelia.The result showed that comparing with ductal epithelium derived from tissues,the numbers and significance of differential gene expression in the three kinds of organoids were reduced.mPDO,mBTO and mHDO have similar gene expression levels.3.Using Matrigel combined with specific cell culture medium,mPDO,mBTO and mHDO were successfully differentiated into hepatocytes-like cells(ALB expressing cells),cholangiocytes,and pancreatic ?-like cells(Insulin secreting cells).In vivo and in vitro differentiation methods were used to prove that the three kinds of organoids have similar differentiation functions.4.Using cell lineage tracing,we observed the serial sections of transgenic mouse tissue,and prepared mPDO,mBTO and mHDO with transgenic mice.The result showed that hepatic,biliary and pancreatic duct epithelial progenitors were all derived from PDX1 expressed cells.Although Lgr5 was expressed in mPDO,mBTO and mHDO,there were no Lgr5+cells in hepatic,biliary and pancreatic duct epithelium of normal mice during embryonic development,the neonatal stage and maturation.5.With the above results,we selected the most readily available human bile duct epithelium to establish the human bile tree duct organoids(hBTO),and successfully established methods for hBTO differentiation into hepatocytes-like cells(ALB expressing cells),cholangiocytes,and pancreatic ?-like cells(Insulin secreting cells).It provides a foundation for studying regeneration and repair after organ injury.Conclusion:mPDO,mBTO and mHDO prepared from mouse hepatic,biliary,and pancreatic duct epithelial cells have high similarities,including similar gene expression levels,the potential to differentiate into hepatic,biliary and pancreatic lineage cells and have similar cell lineage characteristics.Further,methods for the differentiation of human bile duct organoids hBTO into hepatocytes,cholangiocytes and pancreatic endocrine cells were established,which opened a new way for establishing a drug screening model,studying tissue regeneration and repairment and organ transplantation.