Characterisation of GABAergic neurons derived from mouse embryonic stem cells

Mouse embryonic stem (mES) cells possess a virtually unlimited differentiation potential and have been utilised to generate a variety of different cell types. A great deal of stem cell research focuses on the enrichment of differentiated cultures with a desired cell type. However, differentiation protocols rarely result in homogeneous populations of cells. Generally, embryonic stem cells will differentiate into neural progenitors by default, often giving rise to GABAergic neurons; GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the brain. In this thesis, I investigate some of the physiological properties of GABAergic neurons differentiated from mouse embryonic stem cells. Nearly all mES cell derived neurons derived using a forebrain differentiation protocol responded to the addition of the common neurotransmitters ATP, noradrenaline, acetylcholine and glutamate with elevations of intracellular calcium (Chapter 3). Purinergic receptors have been shown to play a role in modulating GABAergic neuron behaviour in vitro via the P2X2, P2X4 and P2X7 receptor subtypes (Amadio et al., 2007; Papp et al., 2004). The exact subtype(s) of purinergic receptor present on these cultured GABAergic neurons was examined using a variety of agonists and antagonists, as well as pH and Zinc (Chapter 4). The majority of these purinergic receptors were shown to be P2X2, P2X4 and P2Y1. Although reverse transcriptase PCR indicated the presence of other receptor subtypes in culture, the location(s) of these receptors were not pharmacologically characterised. Altering the timing of growth factor addition drove cells towards a midbrain, rather than forebrain, phenotype and the resultant GABAergic neurons responded differently to stimulation with neurotransmitters, particularly dopamine. The dopamine receptor subtype(s) involved in this response were investigated further (Chapter 5). In addition, classification of the types of GABAergic neurons present in cultures was attempted by examining the presence of calcium binding proteins and neuropeptides in culture. Forebrain and midbrain phenotypes were immunoreactive for Neuropeptide Y, however only neurons directed towards a midbrain phenotype displayed co-localisation with cholecystokinin. The role of genes, particularly Math1, Ptf1a and Pitx2, previously associated with GABAergic, or neuronal, differentiation was investigated using lentiviral transduction (Chapter 6). Mis-expression of these genes did not alter the GABAergic neuronal yield, however Math1 transduced mES cells generated more neurons. An siRNA-mediated reduction in Pitx2 gene expression during differentiation did not affect the ability of the cultures to generate GABAergic neurons, but the pharmacological properties of resultant neurons was altered. Cells transfected with Pitx2 siRNA had reduced responses to noradrenaline, acetylcholine and dopamine.