Transcriptome And Proteome Alterations Of Early Differentiation Of Mouse Embryonic Stem Cells Into Hepatocytes

Embryonic stem(ES) cells are a cell population capable of both self-renewal and differentiation into all three lineages of cells.Directed differentiation of ES cells into hepatocytes is a valuable step for its potential application in liver regeneration,and also represents a useful model for studying early embryonic liver development and liver-related diseases,drug metabolism,as well as toxicological research.Up to the present,although many studies have demonstrated the differentiation of ES cells into hepatocytes both in vitro and in vivo,none of them have clarified the precise molecular mechanisms of hepatic induction.Moreover,the current differentiation is not optimistic and has resulted mixed cell population and low yields of functional hepatocytes.Elucidating the molecular mechanisms underlying the inductive process may help in both understanding embryonic liver development,and promoting directed differentiation.To explore the molecular basis of inductive differentiation of ES cells into hepatocytes,microarray and proteomic techniques were utilized here to examine the global transcriptome and proteome alterations during hepatic differentiation of mouse ES cells induced with combinations of defined hepatogenic factors.Some molecules were found to be possibly associated with hepatic differentiation,providing candidates for further study.The whole study could be divided into three parts as follows:Part One Hepatic differentiation of mouse embryonic stem cellsAccording to the secretion pattern during in vivo mouse embryonic liver development, the undifferentiated mouse ES D3 cells were treated stepwise with combinations of defined chemicals and growth factors,such as retinoic acid(RA),fibroblast growth factor(FGF),hepatocyte growth factor(FGF),oncostatin M(OSM) and dexamethasone(Dex).The differentiated cells on day 3 expressed endoderm marker genes,such as Sox17 and Foxa2.The differentiated cells on day 8 were identified by hepatocyte-like morphology,liver-related genes and proteins expression,such as albumin(ALB),cytokeratin 18(CK18).After maturation with OSM and Dex,these cells possessed hepatocyte-like ultrastructures,such as bile duct-like structures, expressed later markers,such as glucose-6-phosphatase(G6p),and further displayed evidence of glycogen storage.In addition,alpha fetoprotein(Afp) gene expression exhibited firstly upregulation then downregulation,while A1b gene expression increased gradually during the differentiation process.All these results strongly indicated that early hepatic differentiation of ES D3 cells occurred on day 8,and furthermore,the gene expression profile during this differentiation process generally recapitulated that of in vivo embryonic liver development.In this part an in vitro model system of induced hepatic differentiation of mouse ES cells was efficiently established for further study on precise molecular mechanisms.Part Two Transcriptome alteration of hepatic differentiation of mouse embryonic stem cellsTo explore the molecular basis of the above process,and find candidate key signaling pathways and molecules involved in hepatocyte differentiation,gene expression alteration was firstly examined at the mRNA level using oligo microarray technique.To focus on the regulations of the signaling pathways and molecules required for mouse ES cells growth and maintenance,a focused microarray named mouse Oligo stem cell microarray from SuperArray was adopted to comparatively analyze the gene expression profilings of undifferentiated ES D3 cells(abbreviated to "esc"),the differentiated cells on day 3(before addition of FGF and HGF,abbreviated to "esc3") and the differentiated cells on day 8(after addition of FGF and HGF for 5 days, abbreviated to "esc8").Comparisons were performed in three subdivided groups.117 differentially expressed(ratio≥2) genes were detected in all,including 31 in group esc3/esc(when esc3 was compared with esc),48 in group esc8/esc(when esc8 was compared with esc) and 38 in group esc8/esc3(when esc8 was compared with esc3). Quantitative PCR was performed to verify the microarray data.Multifaceted alteration reflected complex and widespread transcriptional regulation during early hepatic differentiation.Further bioinformatics analysis showed the majority of these genes were classified as the components of FGF,Notch,Wnt,BMP signaling pathways and extracellular matrix,intercellular junction and adhesion,suggesting that these alterations may be closely associated with hepatic differentiation of mouse ES cells at early stage.In addition,the regulations focused on BMP,FGF signals during the first three days,but on FGF,Notch and Wnt signals during the next five days. These variations reflected potential roles of various signals in different developmental stages.Association network analysis of the differential,differentiation-related genes, based on STRING database,indicated once again the importance of FGF,BMP, Notch and Wnt signaling pathways.The interactions may provide some clues for further detailed investigations about precise menchanisms.Part Three Proteome alteration of hepatic differentiation of mouse embryonic stem cellsTo gain more insight into the molecular changes during the above process,and find candidate hepatic differentiation-associated proteins,a proteomic strategy was utilized to observe the global protein expression alterations.Proteomic separation,comparison and identification of the above three cell samples were performed with 2-DE followed by MALDI-TOF-MS/MS analysis.Of the 119 differentially displayed(ratio≥2) spots analyzed,71 proteins representing 64 distinct protein species were finally identified,including 25 in group esc3/esc,25 in group esc8/esc and 21 in group esc8/esc3.Some protein expressions were validated by Western blotting such as albumin and cytokeratin-8.Similar to gene expression, protein expression also exhibited varied regulation pattern in different stages. Majority of the identified proteins were upregulated during the first three days,but downregulated during the next five days.Bioinformatic annotations showed that this set of proteins was enriched with DNA maintenance,transcription,translation regulation and protein processing,energy/metabolism and chaperon functions, indicating their important roles during early hepatic differentiation.About 40 percent of the 64 proteins were previously found to be enriched in mouse ES cells.Moreover, some of them had been reported to be involved in ES cell differentiation,such as heat shock cognate 71 kDa protein,78 kDa glucose-regulated protein precursor, T-complex protein 1.When compared with the results from the selected four studies about mouse ES cell differentiation along different directions,23(36%) proteins were commonly identified,including heat shock protein 27(hsp27),vimentin,tubulin, platelet-activating factor acetylhydrolase IB and so on.The study about EB formation had more overlapping with ours,mainly with the results in group esc3/esc,while the other three studies along neural lineage had less overlapping,especially when the results in group esc8/esc3 were compared with those from the dopaminergic differentiation study.Parallel analysis of these results may contribute to identify common regulators,cell type-specific and developmental stage-specific regulators of ES cell differentiation.Another interesting finding of this study was that approximately 40 percent of the identified proteins had been noted to be dysregulated in hepatocellular carcinoma(HCC),including annexin,nucleophosmin,chloride intracellular channel protein 1,ribosomal protein,vimentin,hsp27 and so on.These commonly identified proteins are more likely to be involved in the hepatic differentiation-associated processes.Further protein-protein interaction analysis based on IntAct database,showed that some proteins were not detected in this study but related to many of the identified ones.They became the common nodal points of the networks,such as 14-3-3 protein beta,insulin receptor substrate 1.Multiple interactions,with interactors involved in various signaling pathways,herein were placed together in the differentiation context,providing the candidates for further investigations about the complex regulatory networks.Conclusions1.Early hepatic differentiation of mouse ES D3 cells was successfully induced on day 8,with stepwise addition of hepatogenic factors FGF,HGF and so on. Furthermore,the gene expression profile during this differentiation process generally recapitulated that of in vivo embryonic liver development.2.Majority of the changed genes were classified as the components of FGF,Notch, Wnt,BMP signaling pathways and extracellular matrix,intercellular junction and adhesion,suggesting that these alterations may be closely associated with early hepatic differentiation of mouse ES cells.3.These changed proteins and the involved biological processes of transcription, translation regulation,protein processing,energy/metabolism and chaperon may play important roles during early hepatic differentiation.4.Different alterations on day 3 and day 8 reflected the complex and elaborate regulation during early embryo development,and also indicated that time-course analysis was necessary for ES cell differentiation study.5.Some regulatory mechanisms may be common to the development of many organs,especially at early stage.Parallel analysis of the results from independent studies may contribute to identify common regulators,stage-and lineage-specific regulators of ES cell differentiation.6.The commonly identified proteins both in this study and in HCC related studies are more likely to be involved in the hepatic differentiation-associated processes.The potential value of this work1.To provide candidates for further detailed investigations about the precise regulatory mechanisms underlying hepatic differentiation of ES cells,and provide some clues for better understanding of early embryonic development,and then help us perform more efficient hepatic differentiation of ES cells for clinical applications.2.To provide some clues for studies about hepatic differentiation-associated pathologies,such as HCC.Novelty of the project1.Previous studies used to compare undifferentiated ES cells directly with the terminally differentiated cells.The intricate regulations in the process may be overlooked.In this study,earlier molecular changes during ES cell differentiation were focused on and dynamically examined to gain a better understanding of embryonic liver development.