id_939. LONG-TERM CULTURE OF PATIENT-DERIVED STRIATAL ORGANOIDS AS AN IN VITRO MODEL OF HUNTINGTON’S DISEASE
Wioletta Lech1, Magdalena Czeredys1, Erkan Metin1, Zuzanna Kuczynska1, Pawel Leszczynski1, Aleksandra Duchnowska2, Marzena Zychowicz1, Leonora Buzanska1
1 Department of Stem Cell Bioengineering, Mossakowski Medical Research Institute, Polish Academy of Sciences, 5 Pawinskiego Str, 02-106, Warsaw, Poland
2 Laboratory of Molecular Basis of Neurodegeneration, Mossakowski Medical Research Institute, Polish Academy of Sciences, 5 Pawinskiego Str, 02-106, Warsaw, Poland
INTRODUCTION: Huntington's disease (HD) is a genetic condition caused by an expansion of the CAG trinucleotide repeat in the HTT gene, which encodes the huntingtin protein. An excessive number of CAG repeats leads to cellular alterations, including mitochondrial dysfunction and reduced ATP levels, ultimately resulting in progressive neuronal degeneration and cell death in the striatum and cerebral cortex.
AIM(S): In vitro brain organoid cultures are a valuable platform for modelling the progression and pathogenesis of neurodegenerative diseases. We were analysed control and HD patient-derived striatal organoids to verify differences in their morphology, organisation and viability.
METHOD(S): iPSCs were seeded onto AggreWell plates to generate spheroids. Neural induction was initiated by administering Dual SMAD inhibitors. Striatal differentiation was induced by the addition of Activin A, IWP-2 (a Wnt signalling inhibitor) and SR11237 (a retinoid X receptor agonist). Further maturation was promoted through the following supplementation: BDNF, NT3, ascorbic acid, cAMP and docosahexaenoic acid. Organoid viability was assessed during long-term in vitro culture using an Alamar Blue (AB) assay. 100-day-old organoids were fixed and sectioned into 20 µm-thick slices using a cryostat. To confirm striatal identity, expression of FOXP1, DARPP-32, GABA and GAD65+67 proteins was analysed. Furthermore, the expression levels of HUNTINGTIN, NESTIN and βTUBULINIII were evaluated.
RESULTS: At day 100, the mean organoid diameter ranged from 1.2 to 1.4 mm. The results of the AB assay demonstrated high viability of the organoids. All organoids expressed FOXP1, DARPP-32, GABA and GAD65+67, confirming a striatal phenotype. HUNTINGTIN protein, as well as neural markers NESTIN and βTUBULINIII were detected.
CONCLUSIONS: The utilised protocol enables reproducible and efficient generation of striatal organoids from control and HD samples. These organoids provide an in vitro system for modelling neurodegeneration in Huntington's disease.
FINANCIAL SUPPORT: The work was supported by the Medical Research Agency grant no. KPOD.07.07-IW.07-0105/25.