STRADA‐mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation

Abstract

Genetic diseases involving overactivation of the mechanistic target of rapamycin (mTOR) pathway, so-called "mTORopathies," often manifest with malformations of cortical development (MCDs), epilepsy, and cognitive impairment. How mTOR pathway hyperactivation results in abnormal human cortical development is poorly understood. To study the effect of mTOR hyperactivity on early stages of cortical development, we focused on Pretzel Syndrome (polyhydramnios, megalencephaly, symptomatic epilepsy; PMSE syndrome), a rare mTORopathy caused by homozygous germline mutations in the STRADA gene. We developed a human cortical organoid (hCO) model of PMSE and examined morphology and size for the first 2 weeks of organoid growth, and cell type composition at weeks 2, 8, and 12 of differentiation. In the second week, PMSE hCOs enlarged more rapidly than controls and displayed an abnormal Wnt pathway-dependent increase in neural rosette structures. PMSE hCOs also exhibited delayed neurogenesis, decreased subventricular zone progenitors, increased proliferation and cell death, and an abnormal architecture of primary cilia. At week 8, PMSE hCOs had fewer deep layer neurons. By week 12, neurogenesis recovered in PMSE organoids, but they displayed increased outer radial glia, a cell type thought to contribute to the expansion of the human cerebral cortex. Together, these findings suggest that megalencephaly in PMSE arises from the expansion of neural stem cells in early corticogenesis and potentially also from increased outer radial glial at later gestational stages. The delayed neuronal differentiation in PMSE organoids demonstrates the important role the mTOR pathway plays in the maintenance and expansion of the stem cell pool.

Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

“…( A-C ) Schematics depicting the hypothesized structural outcomes of brain organoids derived from different mixtures (simulating mosaicism of random X-inactivation) of GFP+ and unlabeled isogenic WT and KO cell lines based on previous data from mouse models (Hoshina et al, 2021; Pederick et al, 2018). ( D-F ) Confocal micrographs of multi-rosette brain organoids generated according to previously published methods (Dang et al, 2021; Qian et al, 2016) for each of the three genotype mixtures: WT/KO ( D ), WT/WT ( E ), and KO/KO ( F ). ( G-I ) SOSRS were generated in parallel with the same three mixtures: WT/KO ( G ), WT/WT ( H ), and KO/KO ( I ).…”

“…The first method is the SOSRS protocol described above, with a minor modification of 1 µM CHIR treatment between days 6-10. The second method followed a published protocol known as Spin-Ω (Qian et al, 2016) which we have used previously (Dang et al, 2021). In brief, we generated EBs (day 0) from mixed iPSC cultures in AggreWell800 plates (STEMCELL Technologies).…”

“…The variability of rosette formation is one of the key causes of a structural heterogeneity challenge in the brain organoid field (Quadrato and Arlotta, 2017). For this reason, many groups have used single-cell RNA-sequencing (scRNA-seq) to investigate development and disease as a non-structural read-out (Camp et al, 2015; Quadrato et al, 2017), while a majority of structural disease phenotypes observed to date in brain organoids reflect a size difference such as microcephaly or macrocephaly (Dang et al, 2021; Iefremova et al, 2017; Lancaster et al, 2013; Li et al, 2017; Qian et al, 2016). Although more detailed structural analyses are possible, the variability in size and shape of rosette structures necessitates both a high number of samples and a high level of observer selection (e.g.…”

Antiepileptogenesis and disease modification: Progress, challenges, and the path forward—Report of the Preclinical Working Group of the 2018 NINDS‐sponsored antiepileptogenesis and disease modification workshop

“…patient-derived preparations, such as inducible pluripotent stem cells (iPSCs) or brain organoids that revealed new mechanisms, targets, or treatments, some of which have entered the clinical arena. 86,87,[125][126][127][128][129][130][131][132][133] Even though animal models cannot fully predict safety or tolerability in humans, 122 they have offered insights toward potential mechanisms leading to mechanism-driven trials, that is, precision medicine trials, in small numbers of such patients. They have also revealed pathways that are critical or have changing functions during brain development, 134,135 raising caution for the implementation of treatments that influence those.…”

Modeling Somatic Mutations Associated With Neurodevelopmental Disorders in Human Brain Organoids

“…Recapitulating this complex patient-specific genetic landscape by gene-editing would be difficult and hence hiPSCs provide a useful resource in this regard. hBOs generated from patient-derived hiPSCs have been used to understand the cellular mechanisms underlying the cortical malformations observed in Miller-Dieker and Pretzel syndromes (Bershteyn et al, 2017 ; Iefremova et al, 2017 ; Dang et al, 2021 ). iPSC-derived hBOs have also been used to identify changes in neuronal composition and molecular alterations associated with Rett syndrome (Gomes et al, 2020 ; Samarasinghe et al, 2021 ), idiopathic ASD (Mariani et al, 2015 ; Chiola et al, 2021 ), microcephaly (Lancaster et al, 2013 ), schizophrenia (Stachowiak et al, 2017 ; Khan et al, 2020 ), and other disorders.…”