NAKED ORGANOIDS: EXPOSING NEURONAL MICROCIRCUITS OF HUMAN MIDBRAIN ORGANOIDS DERIVED FROM PARKINSON’S DISEASE PATIENTS

The study of early human neurodevelopment has remained an elusive topic due to obvious ethical barriers. Nowadays, stem cell technologies have reached a milestone by organoids, which self-assemble into 3D structures that mimic the complexity and functionality of different brain regions. Here we were able to characterize human midbrain organoids (hMOs) during their development at both single cell and network levels using a combination of electrophysiology and fluorescence optical microscopy. Current injections in patched cells (n=181) evoked different firing patterns: from single spike to tonic firing up to 24 Hz frequency. Cell populations were separated into clusters through unsupervised principal components analysis (PCA) based on their electrophysiological parameters (Fig. 1A). The dopaminergic phenotype extracted by PCA was validated by filling some cells with a fluorescent dextran during patch clamp recordings (Fig. 1B). The onset of a synchronized oscillatory activity was observed in hMOs around 60-90 days using multielectrode arrays (MEAs) and Ca²⁺ multiphoton imaging (Fig. 1C). Light-sheet microscopy of TH in the clarified organoids (Fig. 1D), permitted us to reconstruct in 3D the dopaminergic neuronal network and correlate the oscillatory activity frequency to a critical cell density around 10³ neurons/mm³ (Fig. 1E). hMOs derived from patients with Parkinson’s disease presented an impairment in the formation of the oscillatory activity together with reduced number of dopaminergic neurons. These results support the notion of hMOs as a suitable model for investigating neurodegeneration using Parkinson’s disease patient-derived stem cells.