| Stem cells can proliferate spontaneously and indefinitely under appropriate culture conditions in vitro(self-renewal),while maintaining the potential to differentiate into various types of cells and to form specific tissues and organs(differentiation).Therefore,stem cells have important significance not only for tissue repair and regeneration,but also can provide a powerful tool for modeling disease and understanding biological development.Mouse embryonic stem cells(mESCs)are the first stem cell lines which were established successfully in vitro and have been studied widely nowadays.Since then,the cell lines of human embryonic stem cells(hESCs)had also been constructed successfully.mESCs have some common pluripotency features with hESCs,but there are more differences between them in biological characteristics,such as morphology,culture conditions,internal regulating signals and so on.In 2007,scientists isolated a new type of cells from the mouse post-implanted blastocyst epiblast tissue,this kind of cells can maintain self-renewal state under culture conditions in vitro and can be passaged many times.Further analysis revealed that the new cells can express self-renewal marker gene,such as Nanog and Oct4,which can also differentiated into various types of cells and tissues derived from the three germ layers in the embryoid bodies and teratomas formation assays.All of these results can prove that the new cells have the characteristics of pluripotent stem cells,thus,which was named as mouse epiblast stem cells(mEpiSCs).Interestingly,mEpiSCs and mESCs have the same species properties,but there are also significant differences on the biological characteristics between them,and mEpiSCs have shown many more similar peculiarities with hESCs surprisingly.Due to human origin,hESCs can not be carried out for chimeric or other experiments which involving ethical and moral problems,in this case,mEpiSCs can serve as supplementary material to fill gaps in related aspects,the deep study of mEpiSCs can provide ideas and clues for the research of hESCs,and its own fruits,which will also be benifit for the expansion in the breadth and depth of entire stem cell research fireld,and will be helpful to extend human's understanding and cognition of the signal regulation network and the developmental rules about stem cells.The early isolated mEpiSCs were cultured in the chemically defined medium(CDM)and maintained growth and self-renewal status via the additions containing activin A and fibroblast growth factors-2(FGF2).However,the cells cultured under such conditions were prone to grow slowly,can not tolerance to single cell digestion,easy to apoptosis,and easy to differentiation after passaging.The modified culture conditions were based on 10%FBS content DMEM medium,and added Activin A,bFGF,IWRI(a stabilizer of Axin protein,also an inhibitor of Wnt/?-catenin signaling pathway)and Y27632(an inhibitor of ROCK kinase)these four kinds of cytokines or small molecule compounds,which can improve the viability of mEpiSCs significantly,and made cells maintained in a good state of self-renewal.However,the culture conditions of Activin A + bFGF + IWR1 + Y27632 involved too many cytokines,which is not conducive to further research on intracellular signaling pathway and molecular regulation mechanism of mEpiSCs,and the culture cost was high.Based on this situation,we tried to further refine and optimize the culture conditions of mEpiSCs in the previous work stage.Through the experiments of decreasing cytokines one by one and evaluate the effects of different combinations,we finally abtained the best culture conditions which can maintain the self-renewal state of mEpiSCs by only adding IWR1 and Y27632.In order to explore the specific mechanism of IWR1 and Y27632 to maintain the self-renewal of mEpiSCs,we designed the control experiments,IWR1 and Y27632 were used to culture the cells respectively,and the NT(No Treatment)condition without IWR1 or Y27632 as the control group,then the microarray analysis was performed to detect the differentially expressed genes in mEpiSCs which under the condition of IWR1 or Y27632 compared to control group,and on the basis of this to screened out the target genes that each small molecule may act on.Taking into account the difficulty of completion of the whole workload and the time limit of the subject period,we first select IWR1 as the object of the investigation.According to the result of microarray,Foxd3,as the downstream target gene of IWR1 was picked up,which may play a role in mEpiSCs maintaining self-renewal.After screening out Foxd3 gene,we designed a reverse experiment using antagonistic effect between IWR1 and another small molecule CHIR99021 on the Wnt/?-catenin signal pathway,the results of the experiment showed that the expression of Foxd3 was positively correlated with IWR1.Next,we designed the experiments around Foxd3 to explore the intermedia of IWR1 regulating the self-renewal status of mEpiSCs.Relevant experiments were performed in two directions:Firstly,the overexpression vector containing the target gene was constructed and transferred into mEpiSCs to up-regulate the expression of Foxd3 in the cells,and observe whether it can replace IWR1 to maintain the self-renewal status of the cells.Secondly,the expression level of Foxd3 was down-regulated in mEpiSCs by shRNA interference,and observe whether the cells tend to differentiation.After the expression level of Foxd3 was up-regulated and down-regulated respectively in mEpiSCs,the self-renewal degree and growth status of the cells were detected by morphological observation,cell counting,qRT-PCR,immunofluorescence and other experimental tools.The results showed that,in the absence of IWR1 condition,compared with the control group cells,overexpression of Foxd3 could maintain the normal morphological characteristics of mEpiSCs,and the growth rate of the cells was significantly increased.The results of qRT-PCR showed that the expression of self-renewal marker genes,such as Esrrb,Nanog and Oct4,were at normal levels,and meanwhile the expression of differentiation marker genes,which represent their respective germ layer,such as Gata6,Mixll and Nestin were at low levels.It also could be detected that self-renewal marker proteins OCT4 and NANOG in the Foxd3 overexpression mEpiSCs under the condition without IWR1.All of these can explained that up-regulating the expression of Foxd3 which can replace the role of IWR1 to maintain mEpiSCs in self-renewal state.On the other hand,in the presence of IWR1,down-regulation of Foxd3 in mEpiSCs could lead to abnormal cell morphology,compared with the control group.In addition,the expression of self-renewal marker genes Oct4 and Nanog were down-regulated at both transcriptional and protein levels,in the mean time,the expression levels of markers which represent endodermal,mesendoderm and mesoderm were significantly increased,indicating that down-regulation the expression of Foxd3 could make IWR1 no longer able to maintain the self-renewal of mEpiSCs.Thus,it can be inferred that Foxd3,as a downstream target gene of small molecule IWR1,which played an important role in the regulation of mEpiSCs growth and internal signaling pathways,and was also a key link in the molecular mechanism of maintaining the cells in self-renewal state.The overall results will not only extend the understanding of molecular mechanism of mEpiSCs self-renewal,but also will be beneficial to the basic research and safe application of this unique stem cells in the future,and relevant achievements can provide clues for exploring human embryonic stem cells and other kinds of pluripotent stem cells. |
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