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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
- Source :
- American Journal of Physiology-Cell Physiology. 310:C520-C541
- Publication Year :
- 2016
- Publisher :
- American Physiological Society, 2016.
-
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 GABAA receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABAA receptor 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.
- Subjects :
- 0301 basic medicine
Patch-Clamp Techniques
Physiology
Neurogenesis
Cellular differentiation
Blotting, Western
Induced Pluripotent Stem Cells
Cell Culture Techniques
Synaptogenesis
Inhibitory postsynaptic potential
Cell Line
Glycogen Synthase Kinase 3
03 medical and health sciences
0302 clinical medicine
Neural Stem Cells
Image Processing, Computer-Assisted
Humans
Cyclic AMP Response Element-Binding Protein
Induced pluripotent stem cell
Glycogen Synthase Kinase 3 beta
GABAA receptor
Chemistry
Cell Cycle
Cell Differentiation
Cell Biology
Receptors, GABA-A
Immunohistochemistry
Embryonic stem cell
Coculture Techniques
Neural stem cell
Culture Media
Cell biology
030104 developmental biology
Microscopy, Electron, Scanning
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 15221563 and 03636143
- Volume :
- 310
- Database :
- OpenAIRE
- Journal :
- American Journal of Physiology-Cell Physiology
- Accession number :
- edsair.doi.dedup.....70d68156237fcb066e6c304b7bd26821