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Forced cell-cycle exit and modulation of GABAA, CREB and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons

Telezhkin, Vsevolod, Schnell, Christian, Yarova, Polina L., Yung, Sun, Sanders, Philip, Cope, Emma, Hughes, Alis, Thompson, Belinda A, Geater, Charlene, Hancock, Jane M., Joy, Shona, Badder, Luned, Connor-Robson, Natalie, Comella, Andrea, Straccia, Marco, Bombau, Georgina, Brown, Jon T, Canals, Josep M., Randall, Andrew D., Allen, Nicholas Denby and Kemp, Paul J. 2016. Forced cell-cycle exit and modulation of GABAA, CREB and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons. American Journal of Physiology - Cell Physiology , ajpcell.00166.2015. 10.1152/ajpcell.00166.2015

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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 sub-types and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time-courses of functional maturation which are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to post-synaptic receptor maturation. Here, we describe a simple protocol which employs the sequential addition of just two supplemented media which 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 synchronised 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: i) spontaneous electrical activity; ii) regenerative induced action potential train activity; iii) Na+ current availability, and; iv) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype which was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multi-electro array recordings. Furthermore, since this protocol can exploit expanded and frozen pre-patterned 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
MRC Centre for Neuropsychiatric Genetics and Genomics (CNGG)
Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: American Physiological Society
ISSN: 0363-6143
Date of Acceptance: 28 December 2015
Last Modified: 14 Mar 2019 11:43
URI: http://orca-mwe.cf.ac.uk/id/eprint/85596

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