Screening Of Peptides Binding Specificially To Surface Molecules Of Mouse Embryonic Stem Cells R1

Embryonic stem cells (ESCs) are cells derived from the inner cell mass of theblastocyst of early-stage embryo. ESCs are distinguished by two distinctive properties:self-renewal and pluripotency. Self-renewal means that, under defined conditions, ESCsare capable of propagating themselves indefinitely. Additionally, ES cells are pluripotent,which means they are able to differentiate into all derivatives of the three primary germlayers, including all the cell types in the adult body. Because of their plasticity andpotentially unlimited capacity for self-renewal, ESCs have many important applicationson both theoretical research and treatment of many human diseases.Previous studies have suggested that specific molecules are expressed on ESCssurfaces, which can serve as markers for the isolation, identification and purification ofESCs. However, only a few markers have been identified for ESCs and these markersare often not entirely specific, impeding their application on ESCs research. Therefore,it is of great significance to screen and identify new ESCs surface markers and studytheir functions.Phage display is a powerful gene engineering tool for selecting peptides, proteins,or antibodies with specific binding properties from libraries containing a large numberof variants. The technology involves insertion of coding sequences of exogenouspeptides or proteins into proper location of the structural genes of phage coat proteins sothat exogenous peptides or proteins can be co-expressed with coat proteins on the phagesurfaces with the reassembly of descendant phages. These expressed exogenouspeptides or proteins then constitute a screen library.Our study aimed to screen and identify peptides that specifically bind to surfacemolecules of mouse ESC R1using the phage display technology to provide foundationfor identifying new R1cell surface markers. We first established the culture systems forR1self-renewal and differentiation induction and managed to obtain sufficient R1cells,differentiated cells derived from R1and feeder cells (MEFs). Subsequently, we set outto screen peptides from a phage library that could bind to R1cell surface molecules.During the screening process, only peptides that could bind to R1surface mmoleculesbut not differentiated cells surface molecules were considered potential target peptides.After three rounds of screening, phages specifically binding to R1surface molecules were obtained and the corresponding peptides’ sequences were determined throughanalyses of the inserted DNA sequences. Then we performed ELISA to validate ourresults using differentiated cells and MEF cells as controls and confirmed the bindingspecificity of these peptides to R1. Finally, we analyzed the homology between ourpotential target peptides with known proteins and discussed possible correspondingprotein identities, which provided important cues for identification of R1surfacemarkers.