3D Culture,Differentiation And Transplantation Of Hepatic Progenitor Cells

Objective Liver transplantation is the only effective treatment for end-stage liver disease.However,the current shortage of donor organs on a global scale means that many patients will die from waiting for suitable organs.Therefore,there is an urgent need to explore new treatment options for end-stage liver disease.Recently,treatment by hepatic stem cells and progenitor cells for liver diseases has brought new hopes for this late-stage liver disease,and liver progenitor cells(HPCs)in adult livers as an important source of treatment are expected to have the ability to regenerate hepatocytes.It has been confirmed that stem cell marker sex determining gene 9(SOX9)and cholangiocellular marker cytokeratin 7(CK7)can be used to identify bile duct-derived HPCs in adult mice.However,how to isolate HPCs and perform high-efficiency amplification is still a key issue that effect them applying in clinical treatment.In this study,we hope to establish a simple and efficient method for isolating and culturing hepatic progenitor cells from adult mouse liver,and obtain a large number of stable HPCs for studying differentiation of HPCs in vivo and in vitro.In addition,we will initially investigate the integration and differentiation of HPCs after transplantation in injured mouse liver.Previous study has shown that TGF-β plays an important role in liver injury and hepatic fibrosis.Finally,we will initially investigate the effect of TGF-β on the proliferation and differentiation of HPCs.Methods Liver from 6-8 week-old healthy C57BL/6 mice was perfused with red blood cells,and the progenitor cells in the mouse liver tissue were isolated by using the modified method of isolation lung stem cells in the literature.Then a single clone was selected from the cloned progenitor cells for passage,and a large number of stable cell populations were expanded.By immunofluorescence staining,the isolated progenitor cells were identified to be HPCs(CK7+SOX9+HNF4a-Alb-)by the ductual marker CK7,stem cell marker SOX9,mature hepatocyte marker(HNF4a)and albumin(Alb)respectively.The HPCs were further cultured in vitro using 3D culture technology.At 14th day in 3D culture,the formed structures were stained by immunofluorescent with HNF4a and Alb to detect the differentiation of HPCs.We also investigated the effects of fibroblast growth factor 9(FGF9)and hepatocyte growth factor(HGF)on the differentiation of HPCs in 3D environment.Subcutaneous capsule subcutaneous injection was used to observe the differentiation of HPCs in vivo.At 14th day after injection,the formed structure of HPCs under the renal capsule was stained by immunofluorescent with HNF4a and Alb to detect the differentiation of HPCs.Meanwhile,we investigated the effects of FGF9 and HGF on the differentiation of HPCs in vivo.Mouse liver injury model was established by intraperitoneal injection of CCL4.Then HPCs transplantation was performed by injection of spleen,and the differentiation of HPCs after liver transplantation in mice was investigated.In order to study the role of TGF-P in the proliferation and differentiation of HPCs,we used TGF-β and its inhibitor SB431542 to treat HPCs.The cell viability of HPCs was detected by MTT assay,and quantitative PCR was used to detect the effects of TGF-β on CK7,SOX9,Alb,and HNF4a in HPCs at mRNA transcript levels.Results The progenitor cells were successfully isolated from adult mouse liver.We obtained stable and homogeneous passages with uniform morphology and size.Further identification showed that the isolated progenitor cells expressed CK7 and SOX9 with strong expanding ability in vitro,but did not express Alb and HNF4a,suggesting that the progenitor cells isolated in this study were HPCs.The cell population can be massively and stably clonal expanded in vitro,and can undergo a large number of clonal expansions without differentiation,representing the proliferation characteristics of stem cells.This group of HPCs was found to spontaneously aggregate in a 3D culture environment to form a spheroid-like structure with a substructure in the sphere.It was found that some of the HPCs in this type of spheroid-like structure express the mature hepatocyte marker Alb and HNF4a,suggesting that HPCs can differentiate into hepatocyte-like cells in a 3D culture environment,and after treatment with FGF9 and HGF,compared with the untreated group,the number of HPCs expressing Alb and HNF4a increased significantly.In the differentiation enviroment of HPCs induced by the renal capsule,it was found that HPCs can also spontaneously form a spheroid-like structure and expressed Alb and HNF4a,but the formed spheres with different sizes are generally larger than those obtained in a 3D culture environment.In addition,the number of HPCs expressing Alb and HNF4a increased significantly after treatment of FGF9 and HGF.The mouse liver injury model was successfully established by intraperitoneal injection of CC14.In vitro clonally expanded HPCs were transplanted into injured mouse liver by splenic injection.HPCs were detected in mouse liver at 7th day and 14th day after transplantation.partial HPCs Integration and differentiation in the liver.TGF-P was found to pose an inhibitory effect on the clonal growth of isolated HPCs,and after TGF-β treatment,the morphology of HPCs changed from round-like to Spindle-like shape.The related markers CK7,SOX9,Alb,and HNF4a in TGF-β treated HPCs changed at the transcriptional level,suggesting that TGF-β may affect the differentiation of HPCs.In addition,the effect of TGF-β on HPCs can be inhibited after using TGF-β inhibitor SB431542..Conclusion After confirming that HPCs with characteristics of stem cell exist in adult mouse liver,with the modified methods for separation and isolation of lung stem cells reported in the literature,we successfully isolated progenitor cells from mouse liver,and confirmed that these cells were of biliary origin.The cells pose a strong ability to expand in vitro and have the ability to differentiate into hepatocytes in vivo and in vitro.The cell population has the characteristics of spontaneous aggregation into spheres both in vitro and in vivo,and the in vitro and in vivo formation of a sphere-like structure is conducive to the differentiation of HPCs into mature hepatocytes.In addition,this study demonstrated that CK7+SOX9+HNF4α Alb HPCs can colonize in injured mouse liver and pose the ability to differentiate into hepatocyte-like cells.Finally,this study confirmed the inhibitory effect of TGF-β on the proliferation and differentiation of HPCs.This study provided strong support for the study of the relationship between HPCs and liver regeneration,and also provided new ideas for cell therapy research of liver diseases.