iSCORE-PD: an isogenic stem cell collection to research Parkinson Disease

Abstract

Parkinsons disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of 65 human stem cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G/FS, SYNJ1 R258Q/FS, VPS13C A444P/FS, VPS13C W395C/FS, GBA1 IVS2+1/FS). All mutations were introduced into a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using different genome editing techniques. To ensure the genetic integrity of these cell lines, we implemented rigorous quality controls, including whole-genome sequencing of each line. Our analysis of the genetic variation in this cell line collection revealed that while genome editing, particularly using CRISPR/Cas9, can introduce rare off-target mutations, the predominant source of genetic variants arises from routine cell culture and are fixed in cell lines during clonal isolation. The observed genetic variation was minimal compared to that typically found in patient-derived iPSC experiments and predominantly affected non-coding regions of the genome. Importantly, our analysis outlines strategies for effectively managing genetic variation through stringent quality control measures and careful experimental design. This systematic approach ensures the high quality of our stem cell collection, highlights advantages of prime editing over conventional CRISPR/Cas9 methods and provides a roadmap for the generation of gene-edited hPSC collections at scale in an academic setting. Our iSCORE-PD collection represents an easily accessible and valuable platform to study PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.