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J Vis Exp. 2017 Jan 8;(119). doi: 10.3791/54900.

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.

Author information

1
Department of Cognitive Neurosciences, Radboudumc; Donders Institute for Brain, Cognition and Behaviour, Radboud University.
2
Department of Human Genetics, Radboudumc.
3
Donders Institute for Brain, Cognition and Behaviour, Radboud University; Department of Human Genetics, Radboudumc.
4
Donders Institute for Brain, Cognition and Behaviour, Radboud University; Department of Human Genetics, Radboudumc; Department of Molecular Developmental Biology, Radboud University; kees.albers@radboudumc.nl.
5
Department of Cognitive Neurosciences, Radboudumc; Donders Institute for Brain, Cognition and Behaviour, Radboud University; Department of Human Genetics, Radboudumc; n.nadif@donders.ru.nl.

Abstract

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (>95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

PMID:
28117798
PMCID:
PMC5407693
DOI:
10.3791/54900
[Indexed for MEDLINE]
Free PMC Article

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