The Discovery And Functional Characterization Of Aire As A Novel Specific Marker Of Embryonic Stem Cells

Embryonic stem cells (ESCs) is one of the major pluripotent stem cell types, which possess the ability to self-renewal infinitively in vitro and to differentiate into all cell types of the three germ layers in vitro and in vivo. ESCs have been considered not only an important model for the in vitro study of molecule mechanisms of cellular differentiation but also a critical source cell source for regenerative medicine. Understanding the molecule characteristics and identifying and functional characterizing novel specific markers for ESCs would critically promote the scientific research and application of these cells.Canonicallly pluripotency was thought to be sustained by the expression of ESC specific genes and the supression of tissue specific genes. However, recent researches raised doubts and proposed modifications on this model. It is recently revealed and gradually accepted that ESCs possess open chromatin structure and globally transcribe genes of a large variety of tissue specificity. However, it was still not clear whether the global gene expression at mRNA level was translated to protein level and therefor possess functional significance and if there was any specific markers of ESCs that regulated the global gene expression, which hindered the the understanding of the roles of global gene expression in ESCs.In this study, we first surveyed the surface proteome of mouse ESCs(mESC), human ESCs(hESC) and human sperms(hSperm), a delegate of germ cells which is considered to have similar expression profile as pluripotent stem cells, and established surface proteome datasets of ESCs and Sperms with the largest coverage by the time of the publication of the results. Bioinformatics analysis experimenal examination of the data revealed that ESCs and Sperms globally expressed a large variety of functional and tissue specific surface proteins. Moreover, Immuno-stainign and flow cytometry analysis revealed that ESCs and Sperms expressed functional proteins heterogeneously, and the percentage of cells positive for any given proteins was relatively stable to specific cell types.Tracking down this route, we discovered that Autoimmune regulator(Aire), the core regulator of promiscuous expression of tissue specific genes in medullary thymic epithelium cells(mTECs), was expressed specifically in ESCs. Gene expression and immunostaining analysis showed that Aire promoted the expression of more than 4000tissue specific genes was correlated with prescence of open chromatin state. Genetic modification experiments showed that Aire played critical roles in the self-renewal of ESCs and reprogramming of somatic cells to iPS cells. The research also revealed that Aire promoted the self-renewal of ESCs through the activation of the expression of Lin28and therefor inhibition of the maturation of the differentiation promoting let-7family microRNAs. These results established Aire as a novel specific marker for ESCs and revealed its functional significance in the global gene expression and self-renewal of ESCs.In conclusion, datas presented here revealed for the first time that global expression of tissue specific surface proteins is a critical characteristics of ESCs and germ cells, indicating the functional significance of global gene expression in ESCs. Tracking down this route, Aire was identified as a novel functional marker of ESCs. This was the first reports that discover a regulator of promiscuous gene expression as a marker of ESCs and set up functional relationships between global expression and pluripotency sustenance network, which contributed significantly to the establishment of open chromatin and global gene expression as a major characteristics of pluripotent stem cells. Also, the research for the first time linked Aire with the microRNA world, which expanded the mechanistic study that Aire regulate gene expression significantly.