Novel Live Cell Imaging and Genome Wide Approaches to Elucidate the Dynamic Roles of Reversible mRNA Modifications in Pluripotency

Pluripotency is the ability for embryonic stem cells to differentiate into diverse somatic tissues. The N6-methyladenosine (m6A) mRNA modification affects RNA metabolism. How m6A abundances regulate pluripotency is poorly understood. Some m6A depletion studies have reported pluripotency loss while others show the inability for cells to differentiate. We found that decreasing m6A abundances activates Erk signaling promoting differentiation and the Akt cascade which stimulates pluripotency. Both pathways are activated in m6A depleted cells due to the stabilization of FGF5 mRNA and FGF receptor activation. Using cutting-edge single-cell resolution techniques, we quantified pluripotency transcription factor protein levels in thousands of mouse embryonic stem cells. m6A depletion results in increased disassembly of the pluripotency network with cells more prone to differentiation. Alternatively, abrogating m6A depletion dependent Erk activation promotes robust pluripotency. Hyper pluripotent m6A depleted cells incapable of lineage specification can re-activate Erk in permissive conditions and progressively downregulate pluripotency transcription factor levels. Contrary to current assumptions, our findings reveal that m6A depletion alters the cell signaling architecture. This actively promotes pluripotency transcription factor expression network disassembly.

The project generated a comprehensive manuscript which we believe is of the highest international relevance and standing. We therefore expect that the submitted manuscript will be published in a very high impact factor, peer review journal. The project will also result in the completion and graduation of Mr Kangxuan Jin from the Oslo University PhD Program.

We aim to describe the role of dynamic methylation on mRNA in the control of pluripotency and during its acquisition upon cell reprogramming. Pluripotency is the unique ability for a cell to differentiate into all cell lineages of the body, including mesoderm, ectoderm and endoderm. However, knowledge about the putative role of reversible 6-methyladenine (m6A) modifications on mRNA in stem cell biology is currently in its infancy. The three m6A stem cell studies to date are partially at odds about the precise role of m6A regulation in pluripotency. To clear long-standing debates, we developed double knock-in protein fusion pluripotency reporter stem cell lines and cutting edge live time-lapse imaging approaches. We can monitor heterogeneous and dynamic pluripotent state and/or cell reprogramming choices in response to m6A regulator manipulation. Cell subpopulations of interest, isolated by flow cytometric sorting will undergo innovative, high-throughput genome wide analyses to pinpoint mechanisms behind state transitions. We believe that the project is central to three of the four key objectives; 1) Understanding the impact of m6A regulation in pluripotency and reprogramming. 2) Characterization of novel pluripotency regulatory mechanisms. 3) Discovery of new methods to control stem cell state with profound advantages for long-term cell therapy applications.