Irene Faravelli (Italy)
University of Milan- IRCCS Fondazione Ca' Granda Ospedale Maggiore Policlinico Department of Pathophysiology and TransplantationAuthor Of 1 Presentation
HUMAN SPINAL CORD-LIKE ORGANOIDS TO MODEL C9ORF72 ALS AND TEST NEW THERAPIES IN VITRO
- Gianluca Costamagna (Italy)
Abstract
Background and Aims:
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. Underlying genetic pathomechanisms include the C9orf72 repeat expansion, the most frequent genetic cause of ALS (C9ALS) in Western countries. Despite recent progress in unraveling C9ALS pathogenesis, reliable disease models and disease-modifying therapies still lack. Here, we aim to model C9ALS in vitro using 3D human spinal cord organoids (SCOs).
Methods:
We differentiated C9ALS induced pluripotent stem cells (iPSCs) and isogenic controls using a free-floating 3D-culture method. We generated SCOs with a modified Lancaster’s protocol promoting neural caudalization and ventralization. Then, we treated C9ALS SCOs with morpholino antisense oligonucleotides (MO) against C9Orf72 repeat expansion. Finally, we assessed the differentiation of organoids at different time points with morphological, immunohistochemical, and qPCR analysis.
Results:
We generated isogenic and C9ALS SCOs exhibiting different co-existing neuronal subpopulations. SCOs expressed neural progenitor, pan-neuronal, astrocyte, motor neuron, and rostrocaudal markers, including markers of cervicobrachial spinal cells. Compared to controls, C9ALS early SCOs diameters measured from day 2 to day 13 were significantly reduced. C9ALS organoids displayed increased dipeptide repeat proteins (DPRs) levels, DNA damage markers associated with C9orf72 expansion, and cytoplasmic inclusions of translocated TDP-43, C9ALS-specific disease hallmarks. Gene expression analysis using qPCR reported differential expression of genes associated with DNA damage and motor neurons in MO-treated C9ALS organoids.
Conclusions:
SCOs represent a valuable system for modeling features of C9ALS pathology, investigating C9ALS pathomechanisms, and testing possible new treatments in vitro.