Publication Date

5-2018

Advisor(s)

Laura Grabel

Department

Biology

Abstract

The derivation of inhibitory interneurons from a human embryonic stem cell (hESC) source is a step towards designing cell replacement strategies to treat a variety of neurological diseases. The medial ganglionic eminence (MGE) is a transient embryonic structure in the ventral telencephalon that is a major source of cortical GABAergic inhibitory interneuron progenitors. The homeobox domain-containing transcription factor NKX2.1 is highly expressed in the MGE and pre-optic area of the ventral subpallium and is essential for specifying specific subtypes of cortical interneuron fate. To generate NKX2.1-positive MGE-like neural progenitors, hESCs were treated with recombinant human sonic hedgehog (rhSHH) and the smoothened agonist purmorphamine (Pur). We employed high throughput mRNA sequencing and quantitative PCR (qRT-PCR) analysis of the NKX 2 .1-positive and negative progenitor population: 1) to examine the transcriptome profile of in vitro hESC-derived progenitors for comparison with in vivo mouse and human MGE-derived progenitors; 2) to identify gene candidates with expression profiles similar to NKX2.1 that could serve as a biomarker for MGE-derived interneurons and be involved in their specification.

We demonstrate that hESC-derived NKX2.1-positive cells display a transcriptome profile similar to the MGE and ventral telencephalon of both mouse and human MGE. NKX2.1 expression was more than 8-fold higher in the FACS enriched GFP-positive neural progenitors than in the GFP-negative cell population and RNA levels of MGE and subpallial markers MBIP, DLX1, and DBX1 were also elevated relative to the NKX2.1:GFP-negative population. Gene ontology (GO) based hierarchical clustering of the top 100 genes with the largest calculated fold change identified genes specific to the subpallium restricted to the NKX2.1:GFP-positive cell fraction, whereas the NKX2.1:GFP-negative population is associated with a more heterogeneous fate. Using astrocyte co-culture, we showed that the MGE-like cell population differentiates into GABAergic interneurons, including SST and PV subtypes. These findings suggest that the MGE-like cell population can serve as an in vitro model to study cortical interneuron differentiation and pathology, and can be used for testing cell-based therapies to treat a variety of interneuron-related neurological disorders.

We identify SFTA3, which encodes surfactant protein H (SP-H), as a biomarker candidate for MGE. Quantitative real-time PCR analysis of hESC-derived NKX2.1-positive progenitors revealed a similar increase in expression of NKX2.1 and SFTA3 over time during neural progenitor differentiation. To determine if SFTA3 plays a role in MGE-like interneuron progenitor specification, SFTA3-/- and NKX2.1- /- hESC lines were generated using CRISPR-CAS9 mediated genome editing. We show that while knockout of NKX2.1 resulted in the significant depletion of MGE and ventral telencephalon marker expression, SFTA3 mutants displayed a moderate decrease. Expression of GABA was significantly decreased in the NKX2.1 KO, but not in the SFTA3 KO. Overall, our data suggest that the deletion of SFTA3 has minimal effects on NKX2.1 RNA and protein expression, and consequently, does not result in significant changes in the expression profiles of MGE-associated marker.

Available for download on Saturday, June 01, 2019

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