Abstracts

Human Embryonic Stem Cell Derived Cortical Interneuron Models of pcdh19-clustering Epilepsy

Abstract number : 3.07
Submission category : 1. Basic Mechanisms / 1E. Models
Year : 2022
Submission ID : 2204979
Source : www.aesnet.org
Presentation date : 12/5/2022 12:00:00 PM
Published date : Nov 22, 2022, 05:27 AM

Authors :
Miranda Walker, BS – University of Michigan; Wei Niu, PhD – Neurology – University of Michigan; Katherine Lee, student – Neurology – University of Michigan; Jack Parent, MD – Neuroscience Graduate Program, Neurology, MNI – University of Michigan

Rationale: Protocadherin-19 (PCDH19) Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy (DEE) characterized by early childhood intractable clustered seizures, impaired motor and cognitive development, and neuropsychiatric comorbidities. PCE is caused by mutations in the X-linked gene PCDH19, which encodes for a transmembrane cell adhesion molecule important for brain development. PCE is a unique disorder that affects heterozygous girls and mosaic males but spares hemizygous males. Random X-inactivation leads to mosaic populations of neurons expressing only wild-type (WT) or only mutant PCDH19.  Impaired cell-adhesion between these populations is thought to cause abnormal cell segregation and altered cortical development. In many DEEs, seizures are thought to arise from interneuron dysfunction and inhibitory deficits. Here, we use 2D and 3D human embryonic stem cell (hESC) derived cortical interneuron models to investigate the role of PCDH19 in human cortical interneuron development and PCE pathophysiology.

Methods: We used CRISPR gene-edited HA-Flag tagged WT and knock-out (KO) H9 female hESCs to generate interneurons. In our 2D model, we differentiated hESCs into induced GABAergic neurons (iGNs) and induced excitatory neurons (iNeurons) by inducing expression of the human transcription factors ASCL1 and DLX2 or NGN2, respectively. We also generated ventral forebrain ganglionic-eminence (GE)-like organoids using a novel self-organizing single rosette spheroid (SOSRS) model. SOSRS were made from WT, PCDH19 KO, or mixed WT and KO (mosaic) hESCs to determine the effects of PCDH19 loss of function (LoF) and mosaicism on cortical interneuron migration, survival, and cell-type differentiation. We used immunostaining and qRT-PCR to evaluate marker expression.

Results: Our iGNs and iNeurons show expected mature neuronal marker expression and morphology. We are currently mixing PCDH19 WT and KO iGNs and iNeurons to study the effects of PCDH19 mosaicism on synaptogenesis and network excitability in 2D co-culture. We successfully derived SOSRS with medial GE-like gene expression, and applied this protocol to generate PCDH19 mosaic and control ventral organoids. We found a cell segregation phenotype with PCDH19 KO cells localized to the periphery of GE organoids beginning on day 18. This result differed from our previous findings of a striping pattern in dorsal (excitatory) brain organoids. We are fusing dorsal cortical and MGE-like ventral SOSRS to investigate how PCDH19 LoF and mosaic expression affects interneuron migration and brain network connectivity.

Conclusions: We successfully generated PCDH19 KO and mosaic 2D and 3D hESC models and found a cell segregation phenotype in our ventral PCDH19 WT and KO mosaic SOSRS that differed from cell sorting phenotypes in dorsal organoids. Our result suggests that PCDH19 mosaicism effects cortical interneuron development. Further study of these models will provide novel insights into PCDH19 function, PCE mechanisms, and targets for precision therapeutics.

Funding: NIH-NIGMS TPTR fellowship 5T32GM113900-05 (MW), U54 NS117170 (JMP), SFARI (WN and JMP)
Basic Mechanisms