MODELING KLEEFSTRA SYNDROME USING HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) DERIVED CORTICAL ORGANOIDS

Kleefstra syndrome (KS) is an ultra-rare neurodevelopmental disorder with approximately 1,000 reported cases worldwide, characterized by intellectual disability, hypotonia, autistic features, seizures, and distinct craniofacial morphology. KS most commonly results from haploinsufficiency of EHMT1, which encodes euchromatic histone methyltransferase 1, an epigenetic regulator responsible for H3K9 mono- and dimethylation (H3K9me1/2). However, how EHMT1 mutations lead to KS remains incompletely understood, and mouse models do not fully recapitulate human brain development.
Human induced pluripotent stem cell (hiPSC)-derived models of EHMT1 haploinsufficiency remain limited. Two-dimensional neuronal cultures and three-dimensional cortical spheroids derived from KS patient hiPSCs and CRISPR-engineered isogenic lines exhibit altered neuronal morphology, activity, proliferation, and neural progenitor organization, but lack the complex cytoarchitecture and developmental staging of the human brain.
We generated the first cortical brain organoid model derived from hiPSCs from KS patients carrying EHMT1mutations, their unaffected parent as control, and genome-edited parental lines harboring the same mutation. Prior to organoid generation, hiPSCs were confirmed to be karyotypically normal and to maintain pluripotency. We found that pathogenic EHMT1 variants did not interfere with cortical organoid generation. KS cortical organoids showed reduced EHMT1 levels, consistent with nonsense-mediated decay, increased in size over time, and we were able to maintain them in culture for several months. Compared to controls, KS organoids displayed only minor differences in size at early time points. Finally, multielectrode array recordings revealed sustained neuronal activity in KS organoids over weeks. This model enables the definition of phenotypes associated with EHMT1 dysfunction during early stages of human corticogenesis.