Expansion,Differentiation And Disease Modeling Of Human Progenitor-like Cells Derived From Primary Hepatocytes

Background & Aims:Hepatocellular Carcinoma(HCC)is one of the most common malignant tumors in the world.The annual number of HCC deaths in China accounts for nearly half of that in the world.The occurrence and development of HCC is complicated,and the specific mechanism needs further study.The number of Hepatitis B patients in China is huge,and most patients with HCC carry the Hepatitis B virus(HBV)infection marker.At present,liver transplantation is an effective treatment for End Stage Liver Disease(ESLD)caused by HCC.Although the number of organ donations is increasing year by year,it is still far from meeting the needs of patients.With the great progress in the field of life medical science,the study of liver regeneration has been deepening.On the one hand,it helps people to understand the mechanism of the occurrence and development of HCC,on the other hand,it helps people to further improve the treatment of ESLD.Although the liver has a strong regenerative ability in human body,the primary hepatocytes(PHCs)fail to maintain their functions and multiplication capacity for a long time in vitro,which leads to the failure of cell therapy and disease modeling based on PHCs.Finding the source of hepatocytes has become an urgent problem.Scientists have tried to obtain hepatocyte-like cells in vitro through various ways.By heterotopically expressing hepatic specific transcription factors,Chinese scientists directly differentiate fibroblasts into induced hepatocyte-like cells(iHeps)or hepatic stem-like cells(iHepSCs),which provides a convenient way to obtain hepatocytes in vitro.However,this process involves the insertion of exogenous genes,and its application is limited by genetic variation and tumorigenicity.In order to avoid the risk caused by gene insertion,chemical reprogramming technology based on small molecule has attracted widespread attention in recent years due to its efficiency and safety in controlling cell fate.Scientists of home and abroad have achieved reprogramming of fibroblasts into pluripotent stem cells or neuron-like cells under purely small molecular conditions,and some articles have explained the dynamic molecular mechanism of chemical reprogramming.In terms of digestive system,chemical reprogramming has also achieved certain breakthroughs.Chinese scientists have reported a method of reversion gastric epithelial cells into induced endodermal progenitor cells(iEndoPCs)in the presence of stromal cells.The progenitor cells can be further differentiated into hepatocyte-like cells,pancreatic endocrine-like cells and intestinal epithelium-like cells.Recently,Japanese scientists and our research team reported a method of reversing mouse PHCs as liver progenitor-like cells almost simultaneously,which can achieve massive expansion of hepatocytes in vitro,and this hepatocyte-derived liver progenitor-like cells(HepLPCs)are highly prone to hepatic differentiation,providing a new way for hepatocyte acquisition in vitro.Despite this,there is no systematic study of expansion and differentiation of human PHCs in vitro currently.Based on the above problems,this study will further explore the culture system of human PHCs under the premise of successful reversion and expansion of mouse PHCs.This study will broaden the source of hepatocytes and provide a new strategy for the modeling and treatment of liver diseases in vitro.It has important scientific significance for the development of liver regenerative medicine and the exploration of liver cancer treatment.Methods:1.Isolation of human PHCs from normal tissues adjacent to surgically removed hemangiomas,and exclusion of liver progenitor cell contamination by Percoll density gradient centrifugation and flow cytometry;Human PHCs were cultured in TEM(Transition and Expansion Medium)in vitro.Real-time PCR,immunofluorescence and flow cytometry were used to study the transformation of PHCs;Time-lapse imaging was used to photograph the transformation process of human PHCs into liver progenitor-like cells.Recorded the number of cell division and calculated the transformation efficiency.2.The effect of each small molecule on the transformation of human PHCs into liver progenitor-like cells in TEM was observed by clonogenic assay;Cell counting at different time points,drawing the growth curve of HepLPCs,calculating the doubling time,and detecting the proliferation ability by EdU experiment;Karyotype analysis to detect the karyotype stability of HepLPCs from different donors.3.TBG-EGFP-T2A-puro(TBG is a hepatocyte-specific promoter)lentiviral system was constructed,and lineage tracing experiment was performed to observe the transformation of green fluorescently labeled hepatocytes in TEM;The passage number of HepLPCs from different donors was recorded,and the karyotype analysis was performed to detect the stability of different algebraic karyotypes.4.CCK-8,immunofluorescence,real-time PCR and Western Blot were used to study the effects of epigenetic regulators on the transformation of human PHCs into liver progenitor-like cells;SIRT1 selective inhibitors and SIRT1 interference assays validated the role of SIRT1-related signaling pathways in this transformation.5.Phenotypic analysis of HepLPCs by transcriptome sequencing,real-time PCR,immunofluorescence and flow cytometry.6.Real-time PCR,flow cytometry and transcriptome sequencing were detected about hepatic differentiation of HepLPCs;Glycogen storage,albumin secretion,ammonia clearance and drug metabolism were used to detect the hepatic function of HepLPCs-Hep;Using FAH gene deficient combined with immunodeficient mice to study the ability of differentiated HepLPCs to repair the liver and treat liver failure in vivo.7.After three-dimensional hepatic differentiation of HepLPCs in low-attachment culture,the expression and translation of HBV infection-related host genes were detected by real-time PCR,Western Blot and immunofluorescence;HBV infected 3D-HepLPCs-Hep,and HBV-DNA,HBsAg,HBeAg secretion and cccDNA were tested.8.PHCs from HBV-infected patients were isolated and cultured in TEM to observe the HBV infection.9.Anti-viral study about CRISPR/CAS9 adenovirus targeting cccDNA combine with or without entecavir was explored by means of 3D-HepLPCs-Hep with HBV infection.Results:1.After Percoll density gradient centrifugation,CD24 and EpCAM positive cells were removed by flow cytometry,and ASGPR1 positive PHCs were obtained;In TEM,the hepatic markers decreased and the progenitor-related markers increased gradually;After about 10 days,human PHCs showed clonal growth;Time-lapse imaging results from day 2 to day 8 showed that about 50% of hepatocytes split more than 2 times with efficient transformation.2.After the small molecules were sequentially deleted from the conditioned medium,the clonal growth efficiency decreased,indicating that each factor had a positive effect on the transformation;The doubling time of HepLPCs was about 25 hours,and the cells from some donors maintained strong proliferation ability at passage 10;Karyotype analysis showed that HepLPCs from different donors had high karyotype stability.3.TBG-EGFP labeled hepatocytes were transformed into liver progenitor-like cells and expanded in TEM,and the surface markers of these HepLPCs were similar to those of the sorted cells above;There were individual differences in the passage number of HepLPCs,most of which were concentrated between passage 10 and 20;The karyotypes between different passages within 10 were highly stable.4.Niacinamide promoted cell senescence and apoptosis during transformation,partially affecting transformation efficiency by inhibiting SIRT1-related signaling pathways;Results of SIRT1 selective inhibitors and SIRT1 interference experiments showed that the SIRT1 inhibition affected transformation efficiency;Removal of seven small molecules during passage could affect SIRT1 and induce cell death.5.The global gene expression profiles of HepLPCs was between PHCs and fetal liver cells,expressing both the progenitor-related markers and the hepatic markers.6.HepLPCs could differentiate into hepatocytes rapidly in about one week;Cluster analysis showed that the gene set about hepatocyte function of HepLPCs-Hep was close to that of primary hepatocytes,such as ABC transporter,bile secretion,cytochrome P450,etc.;After hepatic differentiation of HepLPCs,glycogen storage,albumin secretion,ammonia clearance and drug metabolism were close to PHCs;HepLPCs-Hep could reconstitute liver and alleviate the liver failure status of model mice by cell transplantation.7.HepLPCs form spheres in the low-attachment plate,and the HBV receptor gene NTCP was significantly up-regulated after hepatic differentiation,which provided a basis for subsequent HBV infection;3D-HepLPCs-Hep could be infected by HBV with secreting HBV-DNA,HBsAg,HBeAg and forming cccDNA.8.PHCs derived from HBV-infected patients could be transformed and expanded in TEM,and HBV was lost during expansion;HBV could be reactivated after three-dimensional hepatic differentiation of some patient-derived HepLPCs.9.CRISPR/CAS9 could target cccDNA,and could be used alone or in combination with entecavir to treat HBV infection.Conclusions:Based on the previous work,this study used the small molecule conditioned medium to induce the transformation and expansion of human primary hepatocytes to liver progenitor-like cells in vitro.This hepatocyte-derived liver progenitor-like cells proliferated rapidly and their karyotype were stable.Subsequently,the hepatocyte fate transition related to SIRT1 in vitro was identified,and the phenotypic characteristics of HepLPCs were described in detail,and the rapid hepatic differentiation system of HepLPCs was established.In terms of application,the hepatic differentiation of HepLPCs were transplanted into FAH gene-deficient combined immunodeficient mice to reconstitute the diseased liver and significantly relieve liver failure symptoms.HepLPCs could also be used for HBV infection and antiviral research after three-dimensional hepatic differentiation.The role of CRISPR technology targeting cccDNA in the treatment of HBV infection was verified.This study broadens the source of hepatocytes in vitro and provides a new strategy for the modeling and treatment of liver diseases.It has important scientific significance for the development of liver regenerative medicine and the exploration of liver cancer treatment.