Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission

Abstract

SUMMARYTranscription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

“…More recently, stem cell differentiation strategies relying on the forced expression of neuralizing transcription factors, such as Neurogenin-2 (NGN2), have emerged (Nehme et al, 2018;Zhang et al, 2013). These NGN2-induced neurons are known to form functional synaptic networks in as little as 4 weeks, and have been proven useful in attempts to probe the disease mechanisms underlying several neurodevelopmental disorders (Merkle and Eggan, 2013;Yi et al, 2016).…”

Variant-specific changes in persistent or resurgent Na+current inSCN8A-EIEE13 iPSC-derived neurons

“…As previously described, these cells quickly and efficiently differentiate into neurons after dox addition Busskamp et al, 2014;Lam et al, 2017). Ngn1/2 induction produces neurons expressing upper cortical layer markers, including CUX1, CUX2, and BRN2, but not deeper layer markers like CTIP2 Busskamp et al, 2014;Nehme et al, 2018). Stable insertion was successfully achieved for P1, P3, C2, and C3 lines using TALENs and homology arms targeting the CLYBL gene that is considered a "safe-harbor-like" locus in the human genome (Cerbini et al, 2015).…”

“…We utilized a modification of the Shi et al technique for differentiating iPSCs into excitatory cortical neurons. The neuronal cultures were transduced with lentivirus containing a mouse Camk2a promoter-driven GFP to identify mature neurons (Shcheglovitov et al, 2013;Nehme et al, 2018). See Supplemental material for further details.…”

“…Whole-cell voltage clamp recordings were performed on isolated neurons labeled with GFP driven by the Camk2a promoter, a marker of excitatory cortical neurons that has been shown to identify neurons with more mature electrophysiological characteristics ( Fig. 2F) (Shcheglovitov et al, 2013;Nehme et al, 2018). Representative traces are shown in Fig.…”

Direct readout of neural stem cell transgenesis with an integration-coupled gene expression switch

“…In cultured systems, additive transgenesis is widely used to derive cells homogenously expressing one or more genes of interest, a process instrumental in efforts to direct pluripotent stem cells towards specific neuronal lineages, decipher the mechanisms regulating their differentiation and harness these cells to model neural pathologies (e.g. Kondo et al, 2017;Nehme et al, 2018;Yang et al, 2017). In vivo, somatic transgenesis approaches that directly target neural progenitors have acquired major importance for neurodevelopmental studies by enabling to permanently mark these cells and trace their lineage.…”