A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels

Abstract

Background Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. Methods Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. Results Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. Conclusions We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

Human neural rosettes secrete bioactive extracellular vesicles enriched in neuronal and glial cellular components

“…hiPSCs cell culture and differentiation into hNRs. 2 hiPSCs clones (F2A112 and F2A121) from a healthy donor were obtained from PLACEMA Foundation where they were reprogrammed and characterized (Casalia et al, 2021). To obtain hNRs we used a protocol previously described (Zhang and Zhang, 2010), with modifications.…”

Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp

“…As observed with hiPSC-CMs, hiPSC-neurons (including several subtypes, such as motor neurons and sensory neurons) do not completely replicate the adult neuron phenotype in terms of morphology, electrical function, and critically, in resembling neurodegenerative disease features (Casalia et al, 2021;Giacomelli et al, 2022). Although iPSC-neurons fail to entirely capture all the characteristics of fully mature brain cells, they express functional ion channels and are capable of displaying primary neuronal functions such as firing action potentials (Franz et al, 2017).…”

“…A further key challenge is to improve the maturity and subtype specificity of hiPSC-derived cell lines. Despite improved protocols for producing specific somatic cell types and subtypes, the vast majority of hiPSC-directed differentiations produce populations that most closely resemble fetal or neonatal cells ( Berry et al, 2019 ), including the extensively studied hiPSC-derived cardiomyocytes (hiPSC-CMs; Kolanowski et al, 2017 ; Di Baldassarre et al, 2018 ; Ahmed et al, 2020 ), and hiPSC-derived neurons (hiPSC-neurons; Casalia et al, 2021 ; de Leeuw et al, 2021 ; Giacomelli et al, 2022 ). In both cases there is a further complexity in creating pure subtype populations, for example atrial or ventricular hiPSC-CMs ( Cyganek et al, 2018 ; Feyen et al, 2020 ) or defining the desired hiPSC-neuron subtype (e.g., sensory, motor, or dopaminergic neurons; Davis-Dusenbery et al, 2014 ; Hartfield et al, 2014 ; Young et al, 2014 ).…”

Perspectives on Mechanisms Supporting Neuronal Polarity From Small Animals to Humans

“…hiPSCs from skin fibroblasts generated using the Yamanaka factors were gently provided by Dr. Fernando Pitossi (Fundación Instituto Leloir, FIL-CONICET, CABA, Argentina). They were differentiated into cortical neurons following established protocols ( Casalia et al, 2021 ). Immunofluorescence and confocal microscopy was performed using established protocols ( Wilson et al, 2020b ).…”