Published online Oct 26, 2016. doi: 10.4252/wjsc.v8.i10.355
Peer-review started: January 15, 2016
First decision: March 1, 2016
Revised: July 21, 2016
Accepted: August 6, 2016
Article in press: August 8, 2016
Published online: October 26, 2016
To identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state.
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.
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 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.
Although 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.
Core tip: Derivation and culturing of mouse embryonic stem cells (ESCs) from gene targeting to injection into blastocysts for chimera generation is a lengthy process that is difficult to control. Some stem cells might be in a primed state, having lost some of their characteristics, most importantly their pluripotency. These differences between ESC clones are usually only detected after many months by the failure of chimeric males to transmit the ESC genome through the germline. Here we have determined key expression differences between cells in a primed state and those in a presumed ground state. Detection of these differences will give researchers a powerful tool to quickly distinguish these cells, saving time, money and effort by choosing the best clones to go forward with. Furthermore, we were able to rescue the ground state through overexpression, indicating that the fate of these cells may potentially be controlled.