Derivation And RNA-seq Analysis Of Mouse Endoderm-like Stem Cells

Embryonic stem cells can be divided into two states,na?ve and primed,according to their pluripotency during developmental progression.As the understanding of the mechanism on embryo development become intensive,researchers have derived several novel kinds of pluripotent stem cells with unique pluripotent features.ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor,followed by culturing in ABCL medium containing Activin A,BMP4,CHIR99021 and mouse leukemia inhibitory(m LIF)factor.In this study,we derived the cells that failed to reprogramming during ABCL induction and identified the pluripotent features.Meanwhile,RNA-seq and analysis of cell population sorted by flow cytometry FCS were performed and the sequencing results offer us a better understanding on the regulation mechanisms which involved in induction.According to the molecular and developmental potential identification,we named the novel cells as Mouse endoderm-like stem cell(mEnd-LC),as the features and morphology was similar to endoderm cells.In addition,mEnd-LC is capable of maintain self-renewal in ABCL medium.1.Derivation of mEnd-LC.According to the results of morphology,real-time fluorescence quantitative PCR and immunofluorescence of mEnd-LC induced in ABCL medium,we proved that mEnd-LC was similar to endoderm cells,and can maintain self-renewal in ABCL medium.2.RNA-seq analysis of mEnd-LC.RNA-seq analysis showed that mEnd-LC had different gene expression levels compared with ASC,ESC and Epi SC.Then we compared the RNA-seq between mEnd-LC and ASC by using GSEA,GSVA,differential gene analysis and other analysis methods,respectively.The results showed that there were significant differences on TGF-?,Wnt,Notch and metabolism-related signaling pathways in mEnd-LC.3.Development potential identification of mEnd-LC.The Embryoid body(EB)formation experiments showed that mEnd-LC was more likely to differentiate to endoderm instead of spherical structure in vitro.In summary,we derived mEnd-LC from the ABCL medium in this study.Compared with ASC,mEnd-LC is more similar to endoderm cells in terms of gene expression level and developmental potential,and it is capable of maintain self-renewal like stem cells.However,the induction mechanism,endodermal formation ability and self-renewal ability of mEnd-LC still need to be further studied.