SUMMARY Recent advances in three dimensional (3D) culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

Objective We utilized human midbrain‐like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α‐synuclein (α‐syn; SNCA) perturbations to investigate genotype‐to‐phenotype relationships in Parkinson disease, with the particular aim of recapitulating α‐syn– and Lewy body–related pathologies and the process of neurodegeneration in the hMLO model. Methods We generated and characterized hMLOs from GBA1−/− and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body–like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol‐b‐epoxide–treated SNCA triplication hMLOs. Results We identified for the first time that the loss of glucocerebrosidase, coupled with wild‐type α‐syn overexpression, results in a substantial accumulation of detergent‐resistant, β‐sheet–rich α‐syn aggregates and Lewy body–like inclusions in hMLOs. These Lewy body–like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α‐syn with ubiquitin, and can also be formed in Parkinson disease patient–derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body–like inclusions in hMLOs derived from patients carrying the SNCA triplication. Interpretation Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild‐type α‐syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490–505