Characterization and genetic manipulation of primed stem cells into a functional naïve state withESRRB.
OBJECTIVE
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 toin-vivoincorporation 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 naïve cells relative to the primed cells. Key upregulated genes in naïve cells includeESRRB,ERAS,ATRX,RNF17,KLF-5, andMYC. After over-expressing some of these genes the primed cells were able to incorporate into embryosin-vivo, re-acquiring a feature previously absent in these cells.


CONCLUSIONS
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.
Ricardo Antonio Rossello, Department of Science and Technology, Sistema Universitario Ana G. Méndez, Metropolitan University, San Juan 00928, Puerto Rico.
Ricardo Antonio
RA
Rossello
Ricardo Antonio Rossello, Department of Science and Technology, Sistema Universitario Ana G. Méndez, Metropolitan University, San Juan 00928, Puerto Rico.
Andreas
A
Pfenning
Ricardo Antonio Rossello, Department of Science and Technology, Sistema Universitario Ana G. Méndez, Metropolitan University, San Juan 00928, Puerto Rico.
Jason T
JT
Howard
Ricardo Antonio Rossello, Department of Science and Technology, Sistema Universitario Ana G. Méndez, Metropolitan University, San Juan 00928, Puerto Rico.
Ute
U
Hochgeschwender
C-myc; ERAS; ESRRB; Induced stem cells; Overexpression