Establishment And Characterization Of Hepatic Progenitor Cell Model Isolated From Normal Adult Mouse Liver

ObjectiveThis study is aimed to isolate and culture candidate cells of liver stem cells or progenitor cells from normal adult mouse liver,by investigating their abilitys to proliferate and potentials to differentiate in vitro,and to establish a stable cell model cultured in vitro,followed by characterizing the fundamental properties of those cells.MethodsMouse hepatocytes,isolated by two-step collagenase perfusion and mechanical centrifugation,were separated into two fractions as fraction of PH(parenchymal hepatocytes,PH) and fraction with enriched AHPC(adult hepatic progenitor cells, AHPC).Two fraction cells were cultured in modified DMEM more than 60 days,and observed by phase contrast microscopy.It was clarified that the morphological and proliferating difference between those hepatocytes from two fractions.The expression of cell markers,such as Albumin,AFP,CK19,c-kit,CD45,CD34,Oct-4,Desmin, CD 16,Thy-1 and nestin in high proliferating cells and their colonies,was illuminated by immunofluorescence at different culture days,to analyze the origin of those cells and their potentials to differentiate.Besides,it was primarily investigated that the impact of NPC(nonparenchymal cells,NPC) contamination and growth on the activation,proliferation and differentiation of AHPC during primary culture,by immunofluorescence and noticing the morphological changes and growing state of NPC and AHPC.In addition,efforts were made to investigate the subculture possilbity of AHPC colonies.ResultsThe total cell quantity and cell vitality of hepatocytes were relatively high in two fractions,with cell vitality more than 90%.The cell size of fraction with enriched AHPC was(22.63±2.04)μm,and PH fraction was(37.03±6.65)μm,with a significant statistical difference(p＜0.05).The percentage of different cell size, however,showed two fractions shared some cells with certain sizes.There was a contamination of HPC in both fractions,and the one of enriched AHPC had more HPC(about 73%of attached cells).In the primary culture,HPC firstly began to divide and proliferate,then attached hepatocytes showed being activated,and all formed colonies expressed Desmin,a relatively specific cell marker of hepatic stellate cell(HSC).The fraction with enriched AttPC manifested a definitely larger colony-forming rate than PH ones,respectively 21.45%±1.25%and 0.28%±0.09%(p＜0.001).It was found that a subpopulation of hepatocytes in the fraction with enriched AHPC(about 13.5%) began to proliferate significantly and to form colonies on being activated after 2～3 days in culture.The colonies could expand continually more than 2 months,with the occupied area of some reaching 0.64mm~2 and cell dividing more than 10 rounds.Some larger cells,morphologically like mature hepatocytes,appeared in colonies around day30.At 24 hours after attachment,all hepatocytes expressed Albumin,but were negative for AFP,CK19,Oct-4,CD34, CD45,Thy-1,nestin and c-kit.After being activated,the proliferating cells expressed AFP and Albumin at day5,no CK19 expression.But at day30,the expanding colonies expressed CK19,and some cells within colonies manifested Albumin negative. Immunofluorescence double staining showed some cells with specific pattern of expression in colonies at day30,such as Albumin~+AFP~-,Albumin~+AFP~+, Albumin~+CK19~+,CK19~+AFP~+,CK19~+AFP~-.All formed colonies didn't express Oct-4, CD34,CD45,Thy-1,nestin and c-kit during being cultured.Besides,AHPC colonies could be continuously subcultured for more than 60 days without losing proliferating ability.Attachment of colony aggregates showed a better proliferating performance than those of detached single cells of colonies.Attached AHPC colonies off-spring cells were small with epithelial cell-like appearance and a relatively larger nucleus plasma ratio.Conclusion1.There exists a kind of liver specific progenitor cells in adult normal mouse, named adult hepatic progenitor cells(AHPC),and mouse AHPC cell model cultured in vitro has been successfully established.2.Mouse AHPC is capable of proliferation,and holds bipotentials to differentiate into hepatocytes and duct cells.In addition,it can be subcultured for more than 60 days without losing proliferating ability.3.The contamination and growth of NPC can promote AHPC to activate and proliferate at early culture days,and however NPC may play a part role in inducing progeny cells in AHPC colonies to differentiate at later days. 4.By comparing with other reported hepatic progenitor cells isolated from normal rats and mouse,the mouse AHPC isolated in this study shows a relatively higher ability to proliferate with differently expressing characters,and holds definite two differentiating potentials.Mouse AHPC may be useful tools as new in vitro cell models to investigate liver diseases,liver development and regeneration,and can facilitate the study of liver stem cells that can be used in cell transplantation.