Rapid generation of functional and homogeneous excitatory human forebrain neurons using Neurogenin-2 (Ngn2)

Abstract

Induced neuronal (iN) cells are versatile tools for modeling neurological disorders and human synapse development, and provide a novel cell-based platform for drug screening and discovery 1-9. In 2013 Zhang, Y. et al. study, we reported that inducible expression of a single basic helix-loop-helix transcription factor Neurogenin-2 (Ngn2) in human embryonic stem (ES) and induced pluripotent stem (iPS) cells generates a homogenous population of excitatory neurons that resemble those of cortical upper layer 2/3 neurons in the brain 3. Within 3-4 weeks in culture, Ngn2-iN cells display mature molecular, cellular and synaptic properties, which can be readily analyzed using various functional assays. Coupled with genome editing and/or patient iPS cell lines, Ngn2-iN cells provide an excellent experimental system that can harness rapid, reliable, and renewable source of human neurons in a culture dish. Here, we describe a stepwise protocol in generating Ngn2-iN cells from both feeder-free human pluripotent stem cells (previously published) and feeder-dependent human pluripotent stem cells (unpublished).

Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-DerivedNRXN1-Mutant Neurons

“…Guided by our published protocols (Pak et al, 2018; see Methods), we modified the iPS cell dissociation step to efficiently remove mouse feeder cells from human iPS cell colonies. We also improved the initial induction step to provide the optimal medium combination for cell survival and TetO-inducible Ngn2 expression via doxycycline.…”

“…Generation of neurons (human neurons) from iPS cells. Neurons were generated from feederdependent iPS cells as described previously (Pak et al, 2018) Feeder-independent iPS cells were differentiated as described previously (Pak et al, 2015;Pak et al, 2018): (Day -2) iPS cells were plated on Matrigel coated 6-well plate one day before infection. (2) transcript-level TPM values were summed per gene;…”

“…Media change occurred weekly until maturity. Details on lentivirus generation, primary mouse glia isolation, and reagent preparations are published online (Pak et al, 2018).…”

“…Generation of neurons (human neurons) from iPS cells. Neurons were generated from feederdependent iPS cells as described previously (Pak et al, 2018): (Day -1) iPS cells were plated and infected on Matrigel coated 24-well plate. Per 6-well of iPS cells grown on feeders, cells were washed 1x with PBS.…”

“…Cell pellets were resuspended in 50% hES media/50% mTeSR plus Y-27632, and lentiviruses. Typically 200,000 -300,000 cells were plated per well of a 24-well plate with 1 uL of viruses (rtTA and Ngn2- (Pak et al, 2018).…”