Generation Of Bipotential Hepatic Organoids From Human Pluripotent Stem Cells

Liver disease is of high prevalence in China,in which end stage liver disease(ELSD)is the most severe and often cause serious damage to human body,leading heavy burden to the social,family and nurses.Nurses have a vital role in developing and promoting high-quality care for the increasing numbers of people with end stage liver disease.Liver transplantation(LT)was deem to the only curable treatment for ELSD.However,the scarcity of donor liver severely limits the application of LT.In the past decade years,the development of stem cell technology open a new avenue to the treatment for ELSD,especially the hepatic stem/progenitor cells(HSCs),which have the potential to self-renew and gives rise to abundant hepatocytes and cholangiocytes,sparked immense interest on regenerative medicine applications.Previously,the obtaining of HSCs are mainly derived from human primary liver tissue.However,the source of human liver tissue is very limited.Therefore,the human pluripotent stem cell(hPSCs)including human embryonic stem cells(hESCs)and induced pluripotent stem cells(iPSCs),which can self-renew and have the potential to generate every cell type of human,representing an attractive cell source toward the generation of hepatic stem cells.But the present method to generate HSCs from hPSCs are dependent on serum,feeder cells or gene overexpression,which are not easy to manipulate and inconformable to the clinical application.Therefore,a more defined,easily operating,and scalable method should be established to generate HSCs.Objective: To establish an easy,serum-free,feeder-free,and scalable method to generate HSCs and verify the hepatic stem cell property of h EHOs,test the differentiation potential in vitro and in vivo,and the safety after transplantation.Methods:1 hESCs was induced to hepatic specification.After the hepatic specification stage,cells were dissociated into single cells and embedded in BME2,and seeded in ultra-low attachment surface 24-well plates covered by Advanced DMEM/F12,supplemented with 1% N2 and 1% B27,1.25 mM N-Acetylcysteine,10 nM Gastrin,1% Glutamax,10 mM Nicotinamide,5?M A83-01,10?M Forskolin,250ng/mL R-spondin 1and 50ng/mLEGF.2 The proliferation ability,karyotype,hepatic stem cell specific markers of hEHOs were analyzed.3 Establishing 3D protocols to induce the hEHOs differentiation to hepatocytes and cholangiocytes.4 Analyzing the morphology,bile duct-associated gene expression,in vitro function of hEHO-derived cholangiocytes.5 Analyzing the morphology,hepatocyte specific gene expression,in vitro function and in vivo function in FRG mouse of hEHO-derived hepatocytes.6 Evaluating the lineage restriction and safety of hEHOs transplanted in epididymal fat pads of NOD/SCID mice.Results:1 hEHOs can be generated from HS in our established organoid culture system and can expand more than 20 passages.2 hEHOs maintain exponential growth with cell doubling times essentially unchanged and the karyotype were essentially normal during the culturing period.3 h EHOs express hepatic stem cell-specific markers and transcription factors.4 hEHOs-derived cholangiocytes express bile duct-associated gene,like CK19,SOX9,CFTR,ASBT,HNF6 and HES1.hEHOs-derived cholangiocytes form appropriate polarity and perform secretion function.5 hEHO-derived hepatocytes express liver-specific gene,like ALB,CK18,AAT,TF,CYP3A4,CYP2C9,CPS1,ASS1 and possess the function of albumin secretion,urea production,CYP3A4 activity,the uptake and release of indocyanine green(ICG),and glycogen storage(PAS).6 hEHOs can generate hepatocytes in vivo and were able to engraft into the liver of FRG mice.7 hEHOs have and only have the capacity to differentiate into liver and bile duct and can not form tumors after transplanted into the epididymal fat pads of NOD-SCID mice.Conclusions:1 In this study,we successfully developed an organoid culture systerm to generate bipotential hepatic organoids from easily accessible h PSCs via a simple,chemically defined,feeder-/serum-free methods.2 We demonstrated the hEHOs possess the property of the hepatic stem cells and acquired the ability to self-renew.h EHOs can give rise to functional hepatocytes and cholangiocytes as 3D format in vitro.3 hEHOs are hepatic lineage-restricted and can be large-scale expansion,cryopreserved and are not tumorigenic.Therefore,our results open up new avenues for the use of hEHOs as a novel platform for in vitro studies,disease modeling,cell-based therapy applications and tissue engineering for the liver.