Insight the transcriptomic landscape of embryonic stem cell pluripotency

Embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of the mammalian blastocyst, have the capability of apparent indefinite self-renewing and are pluripotent, so they can differentiate in vivo and in vitro into most cell types. These features therefore make ES cells ideal for their application in regenerative medicine. The aim of my PhD project was to investigate the transcriptome of mouse ES cells to elucidate possible mechanisms that control the cell differentiation program and the maintenance of self-renewal of the undifferentiated pluripotent state. In order to achieve this goal I used the model of ZHBTc4 ES cells, in which the transgene Oct4 expression can be switched off by adding tetracycline in the culture medium. I used two different approaches to analyze this controlled gene specific loss of pluripotency and differentiation model. The first one was the suppressive subtractive hybridization (SSH) method to obtain six cDNA libraries, which contain genes specifically expressed in distinct time points of the Tc-induced differentiation gradient. The other approach aims to investigate the important role that microRNAs (miRNAs) have in development and stem cell maintenance. Expression of microRNAs was examined by microarray technology and function in post-transcriptional regulation of selected target was studied by luciferare reporter assay. Taken together, both approaches provide a strong foundation to elucidate the complex regulatory network coordinated by OCT4 and mechanisms associated with loss of pluripotency, which is essential to understand the establishment of new gene expression programs that accompany lineage specification and cellular pluripotencial reprogramming.