Abstract
GABAergic interneurons control the neural circuitry and network activity in the brain. The dysfunction of cortical interneurons, especially those derived from the medial ganglionic eminence, contributes to neurological disease states. Pluripotent stem cell-derived interneurons provide a powerful tool for understanding the etiology of neuropsychiatric disorders, as well as having the potential to be used as medicine in cell therapy for neurological conditions such as epilepsy. Although large numbers of interneuron progenitors can be readily induced in vitro, the generation of defined interneuron subtypes remains inefficient. Using CRISPR/Cas9-assisted homologous recombination in hPSCs, we inserted the coding sequence of mEmerald and mCherry fluorescence protein, respectively, downstream that of the LHX6, a gene required for, and a marker of medial ganglionic eminence (MGE)-derived cortical interneurons. Upon differentiation of the LHX6-mEmerald and LHX6-mCherry hPSCs towards the MGE fate, both reporters exhibited restricted expression in LHX6+ MGE derivatives of hPSCs. Moreover, the reporter expression responded to changes of interneuron inductive cues. Thus, the LHX6-reporter lines represent a valuable tool to identify molecules controlling human interneuron development and design better interneuron differentiation protocols as well as for studying risk genes associated with interneuronopathies.
Highlights
Cortical interneurons are gamma-aminobutyric acid-containing (GABAergic) inhibitory neurons that connect locally in the neocortex
The PV and SST interneurons originate from the Nkx2.1+ neural progenitors of the medial ganglionic eminence (MGE), while the 5HT3aR interneurons are derived from the dorsal caudal ganglionic eminence (CGE)
Using CRISPR/Cas9-assisted gene targeting, we successfully generated knock-in lines in the LHX6 locus for tracking cortical interneurons derived from human PSCs (hPSCs)
Summary
Cortical interneurons are gamma-aminobutyric acid-containing (GABAergic) inhibitory neurons that connect locally in the neocortex. They provide inhibitory inputs that shape the responses of pyramidal cells and prevent runaway excitation [1–4]. A recent single cell transcriptomics study revealed that humans and mice share transcriptional programs for interneuron development [22], providing further support for using LHX6 as a lineage marker for human MGE-derived cortical interneurons. Several methods have been reported to generate GABAergic neurons from human PSCs (hPSCs) [24–29] These pioneering works revealed that, while MGE-like progenitors (NKX2.1+ ) can be abundantly produced, the generation of the LHX6 lineage interneurons, i.e., those that express PV or SST, remains inefficient. The current state-of-the-art reflects our limited knowledge about transcription factor regulation and signaling pathways that control MGE development and interneuron fate specifications despite recent significant advances [22,30,31]
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