Characterization and genetic manipulation of primed stem cells into a functional na?ve state with ESRRB

AIM To identify differences between primed mouse embryonic stem cells(ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras.Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies.RESULTS Most results show that both primed and naive cells have similar features(morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the na?ve cells relative to the primed cells. Key upregulated genes in na?ve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these na?ve and primed stem cells. These differences can be overcome through overexpression.

AIMTo identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state.METHODSWe have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras. Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies.RESULTSMost results show that both primed and naive cells have similar features (morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the naïve cells relative to the primed cells. Key upregulated genes in naïve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells.CONCLUSIONAlthough there are no notable phenotypic differences, there are key differences in gene expression between these naïve and primed stem cells. These differences can be overcome through overexpression.