Group Leader University of Melbourne University of Melbourne, Victoria, Australia
Abstract: The enteric nervous system (ENS) is essential for many key functions of the gastrointestinal tract, including water and nutrient absorption, hormone secretion and motility. Perturbations in the ENS can result in debilitating disease. Understanding the development, function, and communication of this complex neural network in humans will be essential to developing new therapies to treat enteric neuropathies. The development of enteric neurons and glia can now be mimicked in vitro, by the sequential and guided differentiation of human induced pluripotent stem cells (hiPSC) towards an enteric neuronal and glial fate. However greater insight into the developmental and functional properties of hiPSC-derived ENS cells is critically needed to validate their potential as disease models and for cell therapy. In this study, we investigated the functional activity of hiPSC-derived enteric neurons and glia during early time points post-differentiation. Using an established method, hiPSCs were differentiated into enteric neurons and glia. Live calcium imaging was performed to examine neuronal and glial activity at differentiation day (D) 22, D29, D36, D43, D50 and D57. Additionally, we reconstructed a neural network using ‘graph theory’, to investigate crosstalk and network communication dynamics among enteric neurons. Ca2+ transients were observed in response to high K+ application, as well as the neurotransmitter receptor agonists DMPP (10uM) and ATP (10uM). Further, spontaneous Ca2+ transients were observed. Maturation of spontaneous and evoked responses were identified along the cells’ developmental trajectory. Further, network analysis demonstrated that around 15% of neurons play a critical role in network communication, and that modules with stronger connectivity are not organised based on spatial proximity but are dispersed across the neural network. This is the first study to characterise the functional maturation of neuronal and glial hiPSC-derived ENS cells.
