title: Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons.
abstract: Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Vsevolod
authors.initials: V
authors.lastname: Telezhkin
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Christian
authors.initials: C
authors.lastname: Schnell
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Polina
authors.initials: P
authors.lastname: Yarova
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Sun
authors.initials: S
authors.lastname: Yung
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Emma
authors.initials: E
authors.lastname: Cope
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Alis
authors.initials: A
authors.lastname: Hughes
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Belinda A
authors.initials: BA
authors.lastname: Thompson
authors.affiliation: Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;
authors.forename: Philip
authors.initials: P
authors.lastname: Sanders
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Charlene
authors.initials: C
authors.lastname: Geater
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom; and.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Hancock
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Shona
authors.initials: S
authors.lastname: Joy
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Luned
authors.initials: L
authors.lastname: Badder
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Natalie
authors.initials: N
authors.lastname: Connor-Robson
authors.affiliation: Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;
authors.forename: Andrea
authors.initials: A
authors.lastname: Comella
authors.affiliation: Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;
authors.forename: Marco
authors.initials: M
authors.lastname: Straccia
authors.affiliation: Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;
authors.forename: Georgina
authors.initials: G
authors.lastname: Bombau
authors.affiliation: Hatherly Laboratory, Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, United Kingdom.
authors.forename: Jon T
authors.initials: JT
authors.lastname: Brown
authors.affiliation: Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;
authors.forename: Josep M
authors.initials: JM
authors.lastname: Canals
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom; and Hatherly Laboratory, Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, United Kingdom.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Randall
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom; Allennd@cf.ac.uk.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Allen
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom;
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Kemp
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D018920:Coculture Techniques; D003470:Culture Media / Q000737:chemistry*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D055495:Neurogenesis / Q000502:physiology; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism
chemical_list: C115163:CREB1 protein, human; D003470:Culture Media; D017362:Cyclic AMP Response Element-Binding Protein; D011963:Receptors, GABA-A; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3
keywords: EPSCs; GABAA; embryonic stem cells; iPSCs; induced pluripotent stem cells; neural differentiation; neuronal maturation; patch clamp