Study And Screening Of Embryonic Stem Cell Specific Molecular Through Prodction Of Mouse Monoclonal Antibodies

ES cells and their differentiation model in vitro can simulate the development process of embryos that has a wide application prospect in developmental biology, transgenic animal production, cloned animals, disease model, drug screening, gene therapy and transplantation therapy. Now commonly ES cell surface markers are SSEA-1、SSEA-3、 SSEA-4、TRA-1-60、TRA-1-81 and GCTM2 etc. These markers are used for identification and quality control of ES cells. They are initially produced by monoclonal antibody screening with teratoma or embryonic stem cells as immunogen. Other antibodies of transcription factors of ES cell markers such as OCT4 and NANOG are obtained through immunization with protein antigen. However these markers have their limitations. They are expressed in some adult stem cells, also in normal tissues or cells. So discovering new ES cell functional molecules and specific markers is very important.In this study we screened proteomics candidate markers using mouse monoclonal antibody technology. We selected 30 molecules from 100 hES new membrane surface proteins which have no functional annotation and expressed recombinant proteins. We immunized BALB/c mice with these recombinant proteins and got a series of positive clones through WB and ELISA screening. Through ICC staining,9 positive clones against hES are selected. By further ICC staining and IHC tissuemicroarray detection method, we found GRAMD1A protein expresses in a variety of epithelial cells specifically, which can be used as a new generalized epithelial cell marker. This marker is valuable in regenerative medicine and cancer diagnosis.Then, we use monoclonal antibody technology screening mES markers. We immunized BALB/c mice with whole mouse embryonic stem cells. Through mES staining, we screened a large number of positive clones. Six monoclonal antibodies are relatively specific for mES cells. By flow cytometry analysis, we further found that their expression decreased after spontaneous differentiation of mESC. And then we purified antigen through co-immunoprecipitation and identified one protein is nucleophosmin by mass spectrum detection. Recent studies have found that NPM/B23 is essential for embryonic development and maintenance of genomic stability. NPM1 gene inactivation can lead to unlimited centrosome replication and genomic instability. Our experimental results showed that NPM1 is widely expressed in the adult tissues and is highly expressed in most tumor cells. Its expression decreased with spontaneous differentiation of mES cells. The antigen of antibody B3-C1 expressed in undifferentiated mES cells specifically. Immunohistochemical staining results showed that it mainly expressed in primary spermatocytes and uterine endometrial cells. This protein is likely to be a new specific marker of mESC. Although it is unable to identify the protein now, the antibody can be as a meaningful tool for stem cell identification.Recently, it has been shown that Zscan4 (zinc finger and scan domain-containing protein 4), which is expressed specifically in 2-cell stage embryos and mouse ES cells, is required for the maintenance of genome stability and a normal karyotype in mouse ES cells. Although only a small fraction (1-5%) of undifferentiated ES cells express Zscan4 at a given time, essentially all of the mES cells in culture undergo the transient Zscan4+ state with in nine passages. Undifferentiated mES cells thus oscillate between the Zscan4+ state and the Zscan- state, during which dramatic events including telomere eextension, occur. According to this clue, we studied Zscan4 expression pattern in human embryonic stem cells and adult tissue through preparation of high specific Zscan4 mouse monoclonal antibody against human. Although human Zscan4 and mouse Zscan4 gene sequence have a big difference, the experimental result showed that Zscan4 also played an important role in maintaining hESC genome stability. Tissue microarray results showed that Zscan4 expressed mainly in vascular endothelial cells and in different tumor cells, which suggested Zscan4 is positively related with tumorigenesis.