Maturation of AMPAR Composition and the GABA_AR Reversal Potential in hPSC-Derived Cortical Neurons

Abstract

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl Ϫ levels, which determines the reversal potential of GABA A R-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties.

Cortical expression of AMPA receptors during postnatal development in a genetic model of absence epilepsy

“…stargazin, which is the protein mutated in stargazers (Pickard et al, 2000). This is similar to the increase in GluA3 expression in adults compared to PN17–18 mice observed in the current study, as well as other studies (Livesey et al, 2014; Talos et al, 2006). Thus, put together, these results demonstrate that there is decreased expression of calcium permeable AMPARs (containing GluA1, 3 and 4 subunits) in the stargazer cortex prior to seizure onset, and the loss of these functional AMPARs in the stargazer somatosensory cortex is likely involved in the generation of SWDs in this model.…”

Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons

“…whether stem cell- vs. primary tissue origin causes differential gene responsiveness) we generated cortical-patterned neurons from mouse embryonic stem cells (Mus-ESC CORT -neurons) using established protocols (Gaspard et al, 2008), which exhibit spontaneous firing and synaptic activity (Figure 2a–d), similar to our Hum-ESC CORT -neurons (Bilican et al, 2014; Livesey et al, 2014). The Mus-ESC CORT -neurons were subject to the same 4h KCl/FPL stimulation and RNA-seq analysis (Figure 2—figure supplement 1a).…”

“…We generated dissociated glutamatergic cortical-patterned neurons from human embryonic stem cells (hESC CORT -neurons), whose characterization is described fully elsewhere (Bilican et al, 2014; Livesey et al, 2014). These cells have a combination of homogeneity and electrical maturity that is hard to achieve with classical human stem cell-derived neurosphere-based preparations, which have a poor dynamic range in terms of gene regulation (Paşca et al, 2011) compared to primary mouse neuronal preparations (Spiegel et al, 2014)…”

“…A detailed description of the derivation of hESC CORT -neurons, including immunohistochemical validation of cortical markers as well as electrophysiological characterization can be found in recent studies from the authors (Bilican et al, 2014; Livesey et al, 2014). Briefly, the hESC line H9 was used, and obtained from WiCell (Madison, WI) under full ethical / IRB approval of the University of Edinburgh.…”

“…Embryonic stem cell (ESC)-based technology enables the generation of glutamatergic cortical-patterned neurons from human embryonic stem cells (hESC CORT -neurons) of sufficient homogeneity and electrical maturity (Bilican et al, 2014; Livesey et al, 2014) to enable the study of activity-dependent gene expression. Such responses can then be compared to both primary mouse cortical neurons (of differing developmental stages) as well as those derived from mouse ESCs, to distinguish species-specific differences from those dependent on developmental stage or origin (primary tissue vs. stem cell line).…”