The creation of a platform for investigating the network function of human pluripotent stem cell derived neurons in development and disease

The formation and function of neural networks is a key aspect of normal brain development, while a converging body of evidence from human genetic and clinical/preclinical studies strongly implicates altered synapse and network function in the aetiology of mental health disorders, including autism spectrum disorder (ASD). The advent of induced pluripotent stem cells (iPS cells) and protocols to differentiate them into functional neurons provides exciting opportunities for modelling human development and disease in vitro, although until recently the possibilities for investigating network function in such neurons have been limited. The work in this project describes the development of a platform for the analysis of network behaviour in iPS cell derived neuron cultures, based upon the use of multi electrode arrays (MEAs). The project also looks at the function of neurons derived from ASD patient iPS cells with heterozygous deletions of SHANK3, a post-synaptic density protein, mutations of which are strongly associated with ASD. Chapter 3 describes the formation of a pipeline for the analysis of MEA data. |t focuses on producing key statistics of basal excitably (e.g. Spike rate, number of bursts) and the analysis of synchronised activity states. Chapter 4 describes the adaptation of a neuron differentiation protocol using astrocyte conditioned medium (ACM) and hypoxic (2% O2) incubator environments to increase the functional maturity of iPS cell derived neurons. Cells cultured in ACM/2% conditions had hyperpolarised resting membrane potentials and increased induced and spontaneous action potential activity compared to neurons cultured in standard conditions. Chapter 5 describes the profiling of spontaneous network-driven activity in iPS cell derived neurons. MEA recordings of cultures showed that spontaneous activity changes markedly over development: up to 30 days post plating (DPP) activity is uncoordinated; between 30-40DPP, coordinated activity emerges in the form of synchronised burst firing; activity from 50DPP is characterised by synchronised oscillating periods of high and low activity each lasting > 5 seconds. Coordinated behaviour required both AMPA and NMDA receptor function and the interval between more active periods was attenuated by inhibitors of GABAA receptors and L-type voltage gated calcium channels, possibly via a common mechanism. Chapter 6 studies the function of iPS cell derived neurons from ASD patients with deletions of SHANK3. Patients neurons were less spontaneously active than control neurons as observed with both MEA recordings and calcium imaging, while also showing changes in the shape of calcium transients. Analysis of the shape of extracellular spikes from MEA recordings revealed a small population of spike shapes, characterised by a ‘double-peak’, that were unique to SHANK3 mutant neurons. This project presents a platform for the recording and analysis of network behaviour in iPS cell derived neuron over both development and in disease states. While the focus here has been on modelling ASD, the work provides a framework for the modelling of human neuron network behaviour in a range on neurodevelopmental disorders and, importantly, as method of screening novel therapeutics in a human cell context