Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.

Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.

The C-BIG Repository: an Institution-Level Open Science Platform.

In January 2016, the Montreal Neurological Institute-Hospital (The Neuro) declared itself an Open Science organization. This vision extends beyond efforts by individual scientists seeking to release individual datasets, software tools, or building platforms that provide for the free dissemination of such information. It involves multiple stakeholders and an infrastructure that considers governance, ethics, computational resourcing, physical design, workflows, training, education, and intra-institutional reporting structures. The C-BIG repository was built in response as The Neuro's institutional biospecimen and clinical data repository, and collects biospecimens as well as clinical, imaging, and genetic data from patients with neurological disease and healthy controls. It is aimed at helping scientific investigators, in both academia and industry, advance our understanding of neurological diseases and accelerate the development of treatments. As many neurological diseases are quite rare, they present several challenges to researchers due to their small patient populations. Overcoming these challenges required the aggregation of datasets from various projects and locations. The C-BIG repository achieves this goal and stands as a scalable working model for institutions to collect, track, curate, archive, and disseminate multimodal data from patients. In November 2020, a Registered Access layer was made available to the wider research community at https://cbigr-open.loris.ca , and in May 2021 fully open data will be released to complement the Registered Access data. This article outlines many of the aspects of The Neuro's transition to Open Science by describing the data to be released, C-BIG's full capabilities, and the design aspects that were implemented for effective data sharing.

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy.

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 three-dimensional 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.

A Multistep Workflow to Evaluate Newly Generated iPSCs and Their Ability to Generate Different Cell Types.

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality-control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs. Our workflow tests four benchmarks: cell growth, genomic stability, pluripotency, and the ability to form the three germline layers. We also outline a simple test for assessing cell growth and highlight the need to compare different growth media. Genomic integrity in the human iPSCs is analyzed by G-band karyotyping and a qPCR-based test for the detection of common karyotypic abnormalities. Finally, we confirm that the iPSC lines can differentiate into a given cell type, using a trilineage assay, and later confirm that each iPSC can be differentiated into one cell type of interest, with a focus on the generation of cortical neurons. Taken together, we present a multistep quality-control workflow to evaluate newly generated iPSCs and detail the findings on these lines as they are tested within the workflow.