Generation of human iPSC-derived cerebellar organoids and their application in development of gene therapeutics for Friedreich’s Ataxia

Authors

• S Ryu
• J Inman
• Y Gedik
• V Jovanovic
• A Simeonov
• I Singec
• C Tristan

Abstract

The cerebellum is essential for motor coordination and is involved in various neurological conditions. Our understanding of the human cerebellum has largely been derived from postmortem tissues, animal models, or immortalized cell lines grown in 2D culture conditions, which do not faithfully reproduce human cerebellar development and disease pathophysiology. Recently, human induced pluripotent stem cell (hiPSC)-derived organoid models have shown promise as better in vitro models, that more accurately recapitulate in vivo development. However, effective generation of cerebellar organoid models from hiPSCs has been limited. Friedreich's ataxia (FRDA) is a debilitating neurodegenerative disease caused by a mutation in the FXN gene, leading to severe cerebellar degeneration. There is currently no cure for FRDA, highlighting the need for advanced models to explore therapeutic interventions. This study developed a 60-day protocol to generate cerebellar organoid models from hiPSCs. These models are valuable for investigating cerebellar biology, disease progression, particularly in FRDA, and testing gene therapies. Using hiPSC lines from healthy individuals and FRDA patients, we generated cerebellar organoids that develop the main neuronal and glial cell types of the cerebellum. These FRDA cerebellar organoids exhibit disease-specific signatures such as reduced expression of the mitochondrial enzyme ACO2, increased apoptosis, and abnormal mitochondrial morphology. Organoids with longer GAA repeats showed greater mitochondrial abnormalities. Using this model, we tested gene therapeutic approaches, including an AAV-mediated transgene and CRISPR-Cas9 delivery method, to advance our understanding of FRDA and identify potential treatments. Together, this study establishes our model as a valuable tool for studying cerebellum-targeting diseases.

Scientific Focus Area: Biomedical Engineering and Biophysics

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