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.
Highlights
Progress toward producing more accurate in vitro models of human brain cell types continues to be made (Brennand et al, 2015; Pasxca et al, 2015)
Patterning of NGN2-Induced human PSCs (hPSCs) with Dual SMAD and WNT Inhibition Previously, it has been shown that forced expression of the NGN2 transcription factor in hPSCs can induce rapid differentiation into cells with excitable membranes and capable of synaptic function (Zhang et al, 2013)
To induce patterning toward a forebrain phenotype, cells were neuralized by inhibiting transforming growth factor b (TGF-b) and bone morphogenetic protein (BMP) signaling, and they were dorsalized by inhibiting Wnt signaling for 3 days
Summary
Progress toward producing more accurate in vitro models of human brain cell types continues to be made (Brennand et al, 2015; Pasxca et al, 2015). Directed differentiation approaches aim to mimic embryonic development by stepwise specification of neuronal subtypes (Chambers et al, 2009; Espuny-Camacho et al, 2013; Zhang et al, 2013; Ho et al, 2015) In one such strategy, pluripotent stem cells (PSCs) can be neuralized through the inhibition of bone morphogenetic protein (BMP) and transforming growth factor b (TGF-b) signaling (Chambers et al, 2009; Maroof et al, 2013), regionally specified with morphogens, and allowed to differentiate. Expression of the neuralizing transcription factor NGN2 in human PSCs (hPSCs) was reported to induce an excitatory neuronal identity in a similar time frame (Zhang et al, 2013) While these methods allow more rapid production of human neurons, insight into the heterogeneity of differentiated neurons remains limited. Uncertainty surrounding the identity of iN populations raises concerns that programming methods might not produce cells with strong relevance for disease studies
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