Molecular mechanisms regulating pluripotency in rat and mouse embryonic stem cells

Abstract

Pluripotency is defined as the capacity at the single cell level to initiate the formation of all lineages of the mature organism in response to the signals from the embryo or cell culture environment. Studying pluripotency is the foundation of mammalian development and embryonic stem cell (ESC) biology. However, how pluripotency can be captured and maintained still remains an intriguing question with many practical implications. ESCs can be derived and propagated from both mouse and rat strains. The rat has long been an essential model of human health and disease and is traditionally preferred over mice in many areas of the biomedical research. However, mouse models have gained popularity over rats as a preferred model due to the inability to genetically manipulate the rat genome in the sophisticated manner as in the mouse. Derivation of germline-­‐ competent rat ESCs from a wide variety of rat strains will pave the way to targeted genetic manipulation of the rat genome. Nevertheless, rat ESC culture system is still in its earlier stages and deeper understanding of molecular mechanisms regulating pluripotency in rat ESCs is essential. We devised to establish authentic Brown Norway rat ESCs. Brown Norway rats are the most widely used strain in the biomedical research and have an edge over the other strains due to the decoding of its genome sequence. In the rat project, Brown Norway ESCs were derived using defined culture conditions and characterized for their pluripotency. The established ESCs expressed the characteristic ESC markers and were able to induce teratoma formation. Due to technical difficulties and host incompatibility issues, it was nevertheless not possible to generate germline competent ESCs. Stabilization of pluripotency in ESCs is a key question in order to efficiently derive and cultivate these cells. Recently, a new gene family (Preferentially expressed antigen in melanoma-­‐like, Pramel) was identified, which might play an important role in maintenance of pluripotency in mouse and rat ESCs. In a parallel project on mouse ESCs (mESCs), we aimed at understanding the molecular factors regulated by Pramel7, a member of this family. Pramel7 is a novel factor involved in maintenance of pluripotency in mESCs, which is regulated through LIF/STAT3 pathway. LIF regulates transcription of Pramel7 via STAT3. In vivo, Pramel7 is expressed in the pluripotent cells of the morula and blastocyst. Knockdown of Pramel7 induces differentiation of mESCs whereas the overexpression maintains them pluripotent even in the absence of LIF. Transcription of Pramel7 is reported to be blocked in the presence of GSK3β inhibitor. GSK3β is an important player in three different signalling pathways, PI3K/Akt, Shp2/MAPK and Wnt/ β-­‐catenin pathway, all of which are known to play key roles in the self-­‐renewal and differentiation of pluripotent ESCs. To deeper understand the link between GSK3β /β-­‐catenin and Pramel7 in ESCs, genetic and biochemical examination of ESCs cultured in defined conditions was carried out. Analysis of intracellular signalling pathways along with the genetic mutants of the target genes, β-­‐catenin and GSK3β sheds light on the circuitry involved. Initially, the question whether there is a link between β-­‐catenin and Pramel7 was addressed. We observed