SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids, differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.

ObjectiveTo determine whether there are nuclear depletion and cellular mislocalization of RNA-binding proteins (RBPs) transactivation response DNA-binding protein of 43 kDa (TDP-43), fused in sarcoma (FUS), and polypyrimidine tract–binding protein (PTB) in MS, as is the case in amyotrophic lateral sclerosis (ALS) and oligodendrocytes infected with Theiler murine encephalomyelitis virus (TMEV), we examined MS lesions and in vitro cultured primary human brain–derived oligodendrocytes.MethodsNuclear depletion and mislocalization of TDP-43, FUS, and PTB are thought to contribute to the pathogenesis of ALS and TMEV demyelination. The latter findings prompted us to investigate these RBPs in the demyelinated lesions of MS and in in vitro cultured human brain–derived oligodendrocytes under metabolic stress conditions.ResultsWe found (1) mislocalized TDP-43 in oligodendrocytes in active lesions in some patients with MS; (2) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; (3) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; and (4) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared with optimal culture conditions.ConclusionTDP-43 has been found to have a key role in oligodendrocyte function and viability, whereas PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment.