Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons

Abstract

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i Neurons) or lower motor neurons (i LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i Neurons or i LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc.

cis-Regulatory Chromatin Contacts in Neural Cells Reveal Contributions of Genetic Variants to Complex Neurological Disorders

“…1a, b ). Finally, lower motor neurons were induced from a WTC11 line containing integrated, isogenic, and inducible copies of NGN2, ISL1 , and LHX3 at the AAVS1 safe-harbor locus (i 3 LMN iPSCs) 10 . The cells exhibited homogenous expression of the lower motor neuron markers HB9 and SMI32 in culture ( Supplementary Fig.…”

“…Human lower motor neurons were differentiated from WTC11 iPSCs with a doxycycline inducible transgene expressing NGN2, ISL1, and LHX3 integrated at the AAVS1 safe-harbor locus (i 3 LMN iPSCs) as previously reported 10 . Briefly, i 3 LMN iPSCs were maintained on growth factor reduced Matrigel in StemFit media (Nacalai USA).…”

“…Therefore, charting the landscape of epigenomic regulation in well-characterized, physiologically relevant cell types should offer significant advantages for identifying causal variants, deciphering their functions, and enabling novel therapies for previously intractable diseases. Towards this goal, we used wild type human induced pluripotent stem cells (iPSCs) from the WTC11 line 7 to generate three neuronal cell types: excitatory neurons 8 , hippocampal dentate gyrus (DG)-like neurons 9 , and lower motor neurons 10 . GFAP-positive astrocytes from the gastrulating brains of two individuals were also included for their relevance to human brain development and disease.…”

Clinical and Molecular Aspects of Senataxin Mutations in Amyotrophic Lateral Sclerosis 4

“…Stem cells were differentiated into motor neuron-like cells by stable insertion of an inducible transcription factor cassette expressing neurogenin-2 (NGN2), islet-1 (ISL1), and LIM homeobox 3 (LHX3; hNIL) into the CLYBL safe harbor locus as previously described. 12 Differentiation was initiated using Dulbecco modified Eagles medium (DMEM)/F12 containing N2 supplement, nonessential amino acids, L-glutamine, 10μM ROCK inhibitor, 0.2μM compound E, and 2μg/ml doxycycline. After 2 days of differentiation, the cells were dissociated with accutase and plated on poly-l-ornithine-coated dishes in DMEM/F12 media containing N2 supplement, nonessential amino acids, L-glutamine, B27 supplement, 10ng/ml brain-derived neurotrophic factor, and 1μg/ml laminin (density of 10,000 cells per well of a 96-well plate for TGF-β characterization).…”

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

“…ELISA-based measurements of Aβ42 in i 3 Neurons I 3 Neurons were differentiated from WT and JIP3 KO IPSCs as described previously (Fernandopulle et al, 2018). i 3 Neurons were grown on Poly-Ornithine(PO)-coated 6 well plates for 21 days before Aβ42 ELISA measurements were performed as per a previously validated ELISA assay for the measurement of human Aβ42 (Teich et al, 2013).…”