Abstract
Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in β-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of β-Catenin and GSK3β proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through β-Catenin stabilization.