***P<0.0001, Chi2= 28.04 (Map2ab) and 24.59 (Nestin); *P<4.97, Chi2= 4.97 (Tuj1). Since either GABAAreceptor or N-type Ca2+channel blockade diminished the positive effects of high Ca2+on functional maturation, attempts were made to bypass the effects of each by activating alternative Ca2+influx mechanisms. excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca2+channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons. == Introduction == Induced pluripotent stem cells (iPSCs) from human patients with well-defined, and often genetically determined neuronal pathology, have huge potential both for disease modelling and for reliable high-throughput drug screening. Consistent and reproducible generation of functional neurons from iPSC is crucial to the development of cellular models of neurological disease. Although a plethora of protocols are available which might produce such neuronal models, only a few have been driven by robust functional characterisation of neuronal maturation and synaptogenesis [1-3], which limits their utility for both disease modelling and the development of novel therapiesin vitro. Pluripotent stem cells (PSCs) differentiate into neurons through a program of neuralisation, neuronal-subtype fate specification, cell cycle exit, post-mitotic neuronal differentiation and functional maturation of regulated excitability. Despite significant advances in the development of protocols for PSC neuralisation and neural progenitor fate-specification [4], the latter stages of neural differentiation are still difficult to control. In particular, developmental studies are confounded by on-going progenitor proliferation and neurogenesis in long-term cultures, and the protracted time in culture required for differentiated neurons to become functionally mature, often exceeding 100 days [5-7]. Some successful attempts have been made to synchronise the neurogenesis of plated progenitor cells. For example, inhibition of Notch signalling, using the -secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), has been used to promote cell cycle exit [8]. Additionally, recent small molecule screens have identified the receptor tyrosine kinase inhibitor, SU5402, and the GSK3 inhibitor, CHIR99021, which together with DAPT effectively promoted post-mitotic nociceptor neural differentiation [9]. In part, the slow yet progressive nature of neuronal maturation in differentiating PSC cultures may be due to the Rabbit polyclonal to AKAP5 delayed emergence and development of astrocytes [10,11]. Glia/neuron interactions have major positive effects on the functional maturation of neuronsin vitro; both co-culture with astrocytes, and treatment with astrocyte conditioned medium (ACM), have been shown to enhance neuronal synaptogenesis [12-14]. The astrocyte-secreted factors that accelerate Tildipirosin neurogenesis, promote neurite outgrowth and enhance the synaptogenesis of cultured neurons include thrombospondins, transforming growth factor (TGF), WNTs, glial-derived neurotrophic factor (GDNF) and the chemokine C-C motif ligand 5 (CCL5) [12,15,16]. Similarly, the maturation of human PSC-derived neurons is promoted by glial co-culture or ACM [1,17], although the underlying physiological mechanisms have yet to be determined. Here, the effects of ACM on the electrophysiological properties of maturing human iPSC-derived neurons have been investigated. Neuronal maturation was characterized by a hyperpolarised resting membrane potential (Vm) and the transition from a capacity to fire only evoked action potentials to spontaneous activity as a consequence ofin vitrosynaptogenesis. Fundamental to these processes in other systems is the precise regulation of Ca2+homeostasis and the developmental regulation Tildipirosin of voltage-gated ion channels. Indeed, enhanced L-type voltage-gated Ca2+channel activity promotes mouse progenitor cell neurogenesis [18], and changes Tildipirosin in L-type and N-type Ca2+channel functional expression have been implicated in mouse ESC-derived neuronal differentiation [19]. Furthermore, astrocytes increase N-type channel expression in adult hippocampal cultures [20], where Ca2+influx through L-type and N-type Ca2+channels has been implicated in excitation-coupled neurogenesis [21]. Based on these data, it was hypothesized that one mechanism by which ACM might promote neuronal maturation is through the upregulation of voltage-gated Ca2+channel activity. A further neuromodulatory pathway which is active in immature and differentiating neurons is excitatory -amino butyric acid (GABA) signaling [22-24] We have previously reported that human PSC-derived neurons show ubiquitous Ca2+responses to GABA, even at early stages of differentiation [7,25], an observation similar to that reported in mouse neuroepithelial cells [26]. Early GABAA-evoked Ca2+responses, ahead of synaptogenesis, have also been observed in other systems, including retinal neurogenesis, where it was proposed that GABA might act as a trophic factor by activating L-type Ca2+channels [27]. Given the established excitatory role of GABA in early fetal development [24,26,28], the role of GABA in the regulation of the.