|M.Sc Student||Verbuk Lyudmila|
|Subject||The Involvement of Ras Signaling pathway in Pluripotency|
and Early Differentiation
|Department||Department of Medicine||Supervisor||Professor Ruby Shalom-Feuerste|
|Full Thesis text|
Human pluripotent stem cells (hPSCs) are self-renewing cells that upon the appropriate signals differentiate into virtually any cell type. Unfortunately, these cells which hold great promises in regenerative medicine are difficult to grow and genetically manipulate in single cell culture, and possess reduced karyotype stability as compared to their mouse counterparts (mPSCs). Recently, it was proposed that these differences reflect the stabilization in culture of mPSCs in the blastocyst-like “naïve” pluripotency state, while hPSCs are stabilized in a slightly more differentiated post-implantation epiblast-like pluripotent “primed” state.
A better understanding of the transitions between these states will allow reverting hPSCs into “naïve” state and using them for regenerative medicine. Interestingly, the isolation and self-renewal of mPSCs was shown to be more efficient following the repression of the Ras dependent ERK pathway. Additionally, the conversion of hPSCs into “naïve” state was allowed by using several inhibitors and factors all of which are known to be involved in common Ras-mediated pathways. However, the role of Ras oncoproteins in pluripotency remained unexplored.
Here we investigated the role of the canonical Ras isoforms (H-Ras, K-Ras and N-Ras), their pivotal Ras-downstream signals (e.i. Raf/MEK/ERK and PI3K/AKT), and a non-canonical E-Ras that is known to be specifically expressed by mPSCs. At early exit from pluripotency which was induced through embryoid body differentiation, we observed an enhancement in the activation of all Ras-isoforms and in contrast, the levels of E-Ras were decreased. During differentiation stage, the levels of phosphorylated ERK (P-ERK) were reduced while P-AKT was induced. Interestingly, removal of the self-renewal factor Lif induced significant Ras activation within 4 hours which preceded the reduction in pluripotency markers, suggesting Lif-mediated self-renewal involves repression of Ras-isoform activation. Here too, differentiation was coupled with a decrease in P-ERK but no change in P-AKT. Interestingly, Ras proteins were activated while E-Ras was reduced during the transition of mPSCs from “naïve” to “primed” state, concomitantly with a decrease in P-ERK and induction of P-AKT. Altogether, our data suggests that high E-Ras/low canonical Ras activity is a hallmark of “naïve” pluripotency while the switch to Ras-isoform activation is required for the transition to "primed" state. However, in contrast to the literature, our data raise the possibility that PI3K/AKT but not ERK pathway is involved in early exit from self-renewal of "naïve" PSCs. Finally, this study suggests that inhibition of canonical Ras-isoforms in hPSCs may permit their conversion into “naïve” state for therapeutic applications.