Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders.

Authors:
Sauter EJ, Kutsche LK, Klapper SD, Busskamp V.
In:
Source: Methods Mol Biol
Publication Date: (2019)
Issue: 1942: 101-121
Research Area:
Neurobiology
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector™ X-Unit
Experiment

iPSC cells dissociated with TrypLE (sopped with mTESR+ROCKI) 8x10e5 cells/100µl Solution P3; 10µg piggyBAC+ 2.5µg transposase or 10µg of Cas9 (px458 or px459) + optional 5µg of donor plasmid;  program CB156 or CB-150. more details in the reference

Abstract

Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.