title: Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons.
abstract: Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca(2+) channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca(2+) imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca(2+) channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca(2+) or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca(2+) channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca(2+) channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.
authors.affiliation: Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rushton
authors.forename: Virginia B
authors.initials: VB
authors.lastname: Mattis
authors.forename: Clive N
authors.initials: CN
authors.lastname: Svendsen
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Allen
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Kemp
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D010641:Phenotype; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D017077:Culture Media, Conditioned; D005680:gamma-Aminobutyric Acid; D002118:Calcium