The role of L-proline and L-proline activated signalling in the regulation of pluripotency

Abstract

© 2014 Dr. Boon Siang Nicholas TanThe study of pluripotent cell populations in the mouse embryo, and mouse pluripotent cells in culture, has revealed four identifiable pluripotent cell populations, or states, that comprise the pluripotent lineage. The four pluripotent populations are the epiblast precursor cells, epiblast, early primitive ectoderm and late primitive ectoderm in the embryo. They are represented in culture by ground state embryonic stem (ES) cells, primed ES cells, early primitive ectoderm-like (EPL) cells and epiblast stem cells (EpiSCs) respectively. Although these cell populations are relatively well characterised, the mechanisms that control progression between cell states remain poorly understood. Understanding the transition of primed ES cells to EPL cells, representing the progression of epiblast to early primitive ectoderm of the post-implantation embryo, is the focus in this thesis. EPL cells are derived from ES cells in response to factors within the conditioned medium MEDII. The ES to EPL cell transition provides a model to investigate the mechanisms that regulate pluripotent cell lineage progression. Amino acids have been shown to regulate cellular processes through modulation of intracellular cell signalling pathways. The amino acid L-proline has been identified as the bioactive factor of MEDII required for EPL cell formation, and ES cells cultured in L-proline-containing medium form EPL cells. EPL cell formation is accompanied by changes in cell morphology, gene expression and differentiation kinetics. In chapter 3, the main L-proline transporter on ES and EPL cells is determined to be the System A amino acid transporter 2 (SNAT2). Identification of SNAT2 was facilitated by radioactive uptake assays. SNAT2 uptake of L-proline can be inhibited by the addition of excess amounts of other SNAT2 substrates, such as alanine. ES cells cultured in L-proline and excess concentrations of SNAT2 substrates, but not other amino acids, were not able to form EPL cells. This suggests that L-proline uptake through SNAT2 is required for EPL cell formation. A requirement for L-proline uptake by SNAT2 in the regulation of pluripotent cells suggests a role for L-proline in activating intracellular pathways in the formation of EPL cells. In chapter 4, pharmacological inhibitors of cell signalling pathways were used to determine requirements of Src tyrosine kinases, p38 MAPK and Erk in the formation and maintenance of EPL cells. Exposure of ES cells to L-proline increased Src tyrosine kinase and p38 MAPK activity. Chemical inhibition of these pathways prevented the acquisition of many, but not all, features of EPL cells in culture. Although Erk1/2 signalling was not activated in response to L-proline addition, it was required within the cell for EPL cell formation. The involvement of multiple signalling pathways in EPL cell formation and maintenance supports the potential role of L-proline activated pathways in primitive ectoderm formation. Amino acids are critical for the developmental processes of the preimplantation mouse embryo to the blastocyst stage. However, regulatory roles of amino acids beyond the blastocyst stage, specifically in pluripotent lineage progression, are not well understood. In chapter 5, the expression of SNAT1 and SNAT2 transporters was determined in mouse embryos between the 2-cell stage and early post-implantation. Both transporters showed temporal dynamic expression patterns and differences in intracellular localisation within cell types. Changes in transporter expression likely reflect different amino acid needs at different stages of development. Dynamic SNAT2 expression was observed in the pluripotent lineage with up regulation in the epiblast prior to primitive ectoderm formation. Primed ES cells cultured in ground state conditions resulted in reduced Slc38a2 expression suggesting regulation of Slc38a2 in the transition of ground state to primed ES cells. SNAT1 and SNAT2 showed regulated expression in the trophectoderm (TE) lineage. SNAT1 was preferentially expressed on outer cells of the compacted morula that are fated to form TE, while SNAT2 was localised in the nucleus of TE and placenta cells. The unexpected nuclear localisation of SNAT2 suggests a novel role for an amino acid transporter in the nucleus. Localisation of SNAT1, in outer cells may indicate requirements for amino acids in TE formation. Collectively, data presented here support a role for amino acids and amino acid transporters in the progression of pluripotent states in vivo and in vitro. Understanding the role of amino acids in pluripotent cell transitions will significantly contribute to the optimisation of differentiation media and protocols used in regenerative medicine