Investigating the impact of TSC2 pathogenic variants on human neurodevelopment using 3-D cortical organoids Open Access

Abstract

Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic condition caused by heterozygous mutations in either the TSC1 or TSC2 gene, manifesting in the growth of benign tumors in multiple organs throughout the body. In addition, TSC also leads to significant neurological impairments, with more than 90% of patients also diagnosed with epilepsy and more than 50% showing symptoms of autism spectrum disorders (ASDs). Previous investigations using rodent and human stem cell models suggest that TSC deficiency caused by TSC1/2 knockout leads to the hyper-activation of mTOR signaling pathway, which might precipitate abnormalities in neurodevelopmental processes. However, how the pathogenic variants of TSC1/2 genes found in TSC patients impact the trajectory of human brain development and how these variants contribute to the neurological manifestations remains largely unexplored. In this study, we generate 3-D cortical organoids from induced pluripotent stem cells (iPSCs) from TSC patients carrying TSC2 mutations as well as age- and sex-matched healthy individuals. Using immunostaining, transcriptomics, and single-cell transcriptomics, we found TSC2 pathogenic variants led to aberrant neurogenesis, synaptogenesis and gliogenesis.

Table of Contents

Introduction............................................................................................................1

Methods..................................................................................................................4

--Subjects and iPSC generation.................................................................................4

--Table 1. iPSC lines used in this study...................................................................... 5

--Human induced pluripotent stem cell-derived cortical organoids cultures.................6

--RNA isolation and bulk RNA sequencing.................................................................7

--Immunocytochemistry.......................................................................................... 8

--Single cell RNA sequencing (scRNA-seq).................................................................9

--Multi-well microelectrode array (MEA) recording....................................................9

Results...................................................................................................................10

--TSC2 pathogenic variants accelerate the development of cortical organoids.............10

--TSC2 pathogenic variants disturb developmental trajectories of specific cell types.....12

--TSC2 pathogenic variants increase synapse formation and enhance neuronal activity.16

Discussion...............................................................................................................19

REFERENCES...........................................................................................................22

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Neuroscience Biology, Genetics
Keyword	Stem cell Tuberous sclerosis complex Cortical organoids
Committee Chair / Thesis Advisor	Zhexing Wen, Emory University
Committee Members	Liang Zhao, Emory University Anita Corbett, Emory University

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