Single-Cell Profiling of an In Vitro Model of Human Interneuron Development Reveals Temporal Dynamics of Cell Type Production and Maturation

Abstract

SummaryGABAergic interneurons are essential for neural circuit function and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and ultimately therapeutic cell replacement. We describe here a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation timecourse of cell types using single-cell RNAseq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access

Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development

“…Together, these results suggest that our modified protocol promotes the formation of MGE-like neural progenitors, which differentiate into GABAergic neurons expressing cIN markers, including PV or SST, which mark the two major subtypes of MGE-derived cINs. This constitutes higher percentages of cells expressing SST or PV than were previously observed without further coculturing (16,17,19).…”

“…At this time, hcINs express both mature subtype markers (SST or PV) and other hallmarks of maturing neurons (e.g., enriched expression of genes involved in GABAergic synaptic neurotransmission). Therefore, while prior work describes stabilization of the transcriptome after d100 (19), some protocol modifications introduced here, such as N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT)-mediated inhibition of Notch signaling and neural rosette selection, enabled mature neurons to be isolated without coculturing by d35. This may enhance the utility of this system for modeling cellintrinsic aspects of normal and altered cIN differentiation and function.…”

SArKS: de novo discovery of gene expression regulatory motif sites and domains by suffix array kernel smoothing

“…We set out to analyze two published RNA-seq datasets (Close et al, 2017;Mo et al, 2015) using SArKS. The first study presented transcriptome data for adult mouse neurons sorted according to cell class (Mo et al, 2015).…”

“…Dysfunction of PV interneurons has been linked to autism and schizophrenia (Lewis et al, 2005), and the ability to access these neurons using a cell type-specific promoter has been a priority for brain scientists. The second study examined transcriptomes of differentiating interneurons at several developmental time points (Close et al, 2017). In the sections below, we describe the parameters and results of SArKS analyses for both datasets.…”

“…We examined RNA-seq data for differentiating human interneurons (Close et al, 2017), applying SArKS to identify promoter motifs associated with elevated gene expression in doublecortin-positive (DCXþ) GABAergic neurons compared with DCXÀ cells. Differential expression was assessed for 6939 genes as detailed in Section S2.7.2 and we analyzed upstream sequences (from Table 1.…”