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Lookup NU author(s): Dr Seva TelezhkinORCiD,
Dr Polina YarovaORCiD
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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.
Author(s): Telezhkin V, Schnell C, Yarova P, Yung S, Cope E, Hughes A, Thompson BA, Sanders P, Geater C, Hancock JM, Joy S, Badder L, Connor-Robson N, Comella A, Straccia M, Bombau G, Brown JT, Canals JM, Randall AD, Allen ND, Kemp PJ
Publication type: Article
Publication status: Published
Journal: American Journal of Physiology - Cell Physiology
Print publication date: 01/04/2016
Acceptance date: 28/12/2015
ISSN (print): 0363-6143
ISSN (electronic): 1522-1563
Publisher: American Physiological Society
PubMed id: 26718628
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