Forced cell cycle exit and modulation of GABAA, CREB, and GSK3{beta} signaling promote functional maturation of induced pluripotent stem cell-derived neurons

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 GABAA receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogen...
Source: AJP: Cell Physiology - Category: Cytology Authors: Tags: Stem Cell Biology CALL FOR PAPERS Source Type: research