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Generation of urine-derived iPSC lines and 3D neural models from Dravet Syndrome patients reveals integrated clinical and proteomic signatures of disease severity

ABSTRACT: Dravet syndrome (DS) is a rare and severe developmental epileptic encephalopathy of childhood, primarily caused by mutations in the SCN1A gene, which encodes a key sodium channel involved in neuronal excitability. Although animal models have provided valuable insights into DS pathophysiology, they fail to fully reproduce the molecular and network complexity of the human brain. Human induced pluripotent stem cell (iPSC)-derived neurons represent a promising alternative, yet conventional 2D cultures lack the structural and functional organization characteristic of neural tissues. Objectives This project aims to establish a human 3D model of Dravet syndrome using patient-derived iPSC neurospheres, in order to: Investigate the molecular correlates of clinical heterogeneity in DS. Identify proteomic signatures linked to disease severity assessed through clinical evaluation. Provide a translational platform to explore DS pathophysiology and test personalized therapeutic strategies. Methodology Urine epithelial cells from three DS patients carrying distinct SCN1A variants were reprogrammed into iPSCs, subsequently differentiated into neural stem cells, and aggregated into 3D neurospheres that recapitulate key features of neural tissue organization. Clinical severity was evaluated using the DANCE checklist, while isobaric quantitative proteomics (TMT-based) was used to characterize the molecular phenotypes and identify dysregulated pathways. Main Findings Comparative analyses revealed a strong correlation between molecular alterations and clinical severity: The cell line derived from the most severely affected patient exhibited major disruptions in cellular homeostasis, including stress and metabolic pathway deregulation. The other lines retained proteomic signatures consistent with preserved neuronal and synaptic function.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Guillaume Nugue

LAB HEAD: Magno Rodrigues Junqueira

PROVIDER: PXD070101 | Pride | 2026-05-19

REPOSITORIES: Pride

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Project description:Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of iPSCs to model DS phenotypes, as a prototypical complex human disease, we generated bona-fide DS and wild-type (WT) non-viral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency, and had remarkably similar lineage potency, differentiation kinetics, proliferation and axon extension at early time points. However, at later time points DS cultures showed a two-fold bias towards glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress induced apoptosis, and this could be prevented by the anti-oxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy-21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Non-viral DS iPSCs can therefore recapitulate features of complex human disease in vitro, and provide a renewable and ethically unencumbered discovery platform. Control (WT) and Down Syndrome iPSC lines were generated via episomal reprogramming of human donor fibroblasts. Two iPSC clones conforming to iPS criteria (determined by immunocytochemistry detection of pluripotency markers) were developed from each fibroblast donor. Two control (WT) and DS lines each were further characterized and underwent neural differentiation. Multiple biological replicates of donor fibroblast and iPSC from both control (WT) and DS lines, including 3 euploid DS samples and 3 MEL1 hESC controls (total 54 samples) were hybridized to Illumina HT-12 v4 microarray for gene expression analysis.

			Action	DRS
	DRAVET_CTRL_SDRF.tsv	Tabular
	DRAVET_E_.msf	Msf
	QE_N_00138_DRAVET_E_F1_1.raw	Raw
	QE_N_00139_DRAVET_E_F1_2.raw	Raw
	QE_N_00140_DRAVET_E_F2_1.raw	Raw

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Generation of urine-derived iPSC lines and 3D neural models from Dravet Syndrome patients reveals integrated clinical and proteomic signatures of disease severity

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