Chir99021 Promotes Self-renewal Of Mouse Embryonic Stem Cells By Gene Expression And Regulation

In this study, we investigated J1 mouse embryonic stem cells(J1 mESCs) cultured with or without CHIR99021, and then we performed microarry analysis to identify differentially expressed genes that promote the self-renewal of J1 mESCs. Systematic cluster analysis was performed using Cluster3.0 and TreeView software. We performed GO(gene ontology) and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis with the DAVID(Database for Annotation, Visualization and Integrated Discovery) online system with those differentially expressed genes identified. Validating the differnetially expressed genes by dual-luciferase reporter assay system and real time quantitative PCR(RT-qPCR) aimed to reval the fuction of CHIR99021 in J1 mESCs. The main contents and results of this research are as follows:1.In this study, we have ensured the optimum concentration of CHIR99021 to J1 mESCs. Compared with the control and 1 μM CHIR99021-treated cells, 3 μM CHIR99021 enhanced Nanog expression, whereas treatment of J1 mESCs with 5, 10 or 15μM CHIR99021 did not increase Nanog expression further. Compared with control cells,the viability of cells treated with 10 or 15 μM CHIR99021 for 3 d was reduced. In contrast, cells treated with≤5 μM CHIR99021 showed cell viability similar to that of control cells. Therefore, we ensured 3 μM as the optimum concentration of CHIR99021 to J1 mESCs in this study.2. CHIR99021 combined with LIF(leukaemia inhibitory factor) can maintain J1 mESCs colony morphology and elevate the expression levels of core pluripotent ES cell markers. We cultured J1 mESCs with 1,000 U/mL LIF and either 3μM CHIR99021 or an equal volume of DMSO. In the presence of CHIR99021, J1 mESCs showed high alkaline phosphatase(AP) activity, comparing with those cultured without CHIR99021. Immunofluorescence staining result indicated CHIR99021 treatment can elevate the expression level of all four ES cell markers Nanog. In addition, immunofluorescence staining and immunoblotting results demonstrated that β-catenin was incresed sinificantly in J1 mESCs cultured in CHIR99021-containing medium. CHIR99021 treated cells showed incresed Wnt pathway activity comepared with that in control cells.3. We performed microarray analysis to investigate CHIR99021 downstream targets and further understood its effect on ES cells using the total RNA of cells cultured in the presence or absence of CHIR99021. Our microarray data demonstrated that CHIR99021 can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Using those differentially expressed genes identified, we performed GO(gene ontology) and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis with the DAVID(Database for Annotation, Visualization and Integrated Discovery) online system. CHIR99021 was not only shown to influence the Wnt/β-catenin pathway, but also acted on other signaling pathways such as TGF-β and BMP to maintain the stemness of J1 mESCs.4.CHIR99021 enhances BMP signaling via inhibiting Nodal signaling and promoting BMP4 expression to promote self-renewnal and remain pluripotent of J1 mESCs. Real-time PCR results suggested that the expression of Nodal pathway associated genes Nodal,Smad7 and their downstream regulators Lefty1 and Lefty2 were down-regulated following the treatment of CHIR99021. Meanwhile, CHIR99021 caused up-regulation of BMP pathway associated genes BMP and its downstream regulators Ids. The immunoblotting results of pSmad2 and pSmad1/5 were consistent with the results of Real-time PCR.Dorsomorphin(Dorso) is the inhibitor of BMP pathway. We demonstrated that CHIR99021 can maintain ES cell pluripotency through BMP pathway.5. CHIR99021 induced epigenetic modification changes in J1 mESCs. CHIR99021 modulated ES cells epigenetic modification by altering the expression of epigenetic-associated proteins, including Dnmt3 l,Dnmt3a,Dnmt3 b,Hdac9,Smarcal1 and Cbx7. Our study indicated that Dnmt3l、Dnmt3a、Dnmt3b、Hdac9 and Smarcal1 were down-regulated, while Cbx7 was up-regulated in CHIR99021-treated J1 mESCs. Compared with NC-FAM-transfected cells, the expression of Dnmt3 l was significantly up-regulated strikingly upon CHIR99021 treatment. This outcome revealed that Dnmt3 l might be a downstream target of β-catenin. Nevertheless, other epigenetic regulatory genes were regulated by CHIR99021.In general, our study reveals that CHIR99021 promotes self-renewal of J1 mESCs by modulation of protein-encoding gene and long intergenic non-coding RNA expression. These results provided new insights into pluripotency maintenance of stem cells and induced pluripotency stem cells.