Dissection Of Liver Stem/Progenitor Subtypes In Mice

Purposes:Liver stem cells present in the liver are responsible for the maintenance of liver homeostasis and repair after injury.Currently,the exact identity of liver stem cells remains a puzzle.In this study,we foucused on two putative liver stem cell subsets,Lin'CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+ CD49f in liver of C57BL/6J mice,and compared them for their differentiation potential,capabilities of glycogen synthesis by differentiated cells derived from them,and gene expression profiles,in order to determine the stem cell properties of these two cell subsets.Methods:(1)Preparation of single-cell suspension of mouse liver:2-3 months old C57BL/6J mice were used in this study.The animals were killed by breaking the neck,and the liver tissue collected.The liver single cell suspension was obtained by mechanical cleavage,collagenase IV digestion and cell filtration.(2)Flow cytometry analysis and sorting of mouse liver stem cells:cells were stained and incubated with 4 different anti-surface marker protein antibodies,and analyzed or sorted by flow cytometry.(3)In vitro differentiation and immune fluorescence analysis of liver stem cells:The target cell populations were cultured in the presence of differentiation medium for 7 days,14 days or 21 days.Recovered cells were stained with fluorescent-conjugated antibodies against specific cell marker proteins,and inspected by inverted fluorescent microscopy or laser confocal microscopy.(4)Glycogen PAS staining:The target cell populations were cultured in the presence of differentiation medium for 7 days,14 days and or 21 days.The glycogen synthesis of differentiated cells was assessed with a commercial PAS staining kit.(5)Detection of apoptosis:After 14 or 21 days of differentiation induction,the cells were harvested and stained with Annexin V and 7-AAD.The apoptotic cells were determined by flow cytometry.(6)Gene expression profiling:FACS-sorted target cells were treated with TRIzol for total RNA extraction.Microarray analysis was conducted by a commercial company.GO analysis and Kegg pathway analysis were performed on the differentially expressed genes.(7)Statistical methods:the experimental data was analyzed with Graphpad Prism software,and the results are expressed as mean±standard deviation.Result:(1)Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells display distinct differentiation potential.After 7d of differentiation induction of the purified cell subpopulations,Lin-CD45-Sca-1-CD49f+ subpopulation were capable of differentiation into hepatocytes(expressing ALB)and bile duct epithelial cells(expressing CK19),while Lin-CD45-Sca-1+CD49f could only generate hepatocytes.The same phenomenon was observed with cells experiencing 14 and 21 d differentiation induction..(2)Lin-CD45-Sca-1-CD49f+and Lin-CD45-Sca-1+CD49f derived hepatocytes exhibit different ability of glycogen synthesis.To determine whether there is any difference in the maturity or functional acitivity between Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f derived hepatocytes,we analyzed their ability of glycogen synthesis.PAS staining indicated that cells derived from the Lin-CD45-Sca-1-CD49f+ population showed significantly strogger production of glycogen than those from the Lin-CD45-Sca-1+CD49f cell subgroup.(3)Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells have characteritical gene expression profiles that undergo specific changes after differentiation.Microarray analysis showed that a total of 1272 genes were differentially expressed between the Lin-CD45-Sca-1-CD49f+and Lin-CD45-Sca-1+CD49f cell population(FDR<0.05,P<0.05,Fold change?2).In comparison,663 genes were up-regulated and 609 genes down-regulated genes in the Lin-CD45-Sca-1-CD49f+ cells.GO analysis further indicated that these differential expression genes were significantly enriched function in various cell processes including differentiation,proliferaton and apoptosis,etc.In addition,we also examined the gene expression profiles of cells derived both of the two populations.There existed 3205 and 4162 differentially expressed genes,respectively,in the Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f differentiated cells when compared to their correspondent progenitors.(4)Both Lin-CD45-Sca-1-CD49f+and Lin-CD45-Sca-1+CD49f cells showed age-related changes in abundance.The content of Lin-CD45-Sca-1-CD49f+ cells in the liver tissue of neonatal mice was significantly higher than that in young,middle and old mice,but no apparent difference was found among young,middle and old mice.In contrast,the content of Lin-CD45-Sca-1+CD49f cell population in the liver tissue of the newborn mice was much lower than that of adult mice,and old mice had the highest levels of cells,with no significant changes between young and middle age mice.Conclusion:Based on analysis of differentiation potential,functionalities of differentiated cells,and gene expression signatures as shown in this study,both Lin-CD45-Sca-1-CD49f+ and Lin"CD45-Sca-1+?CD49f populations are likely bona fide resident liver stem/progenitor cells,which may play different roles in the liver However,whether or not there is a parental relationship in that one originates from the other for the two cell populations remain to be clarified.