Objective: The aim of this study is to evaluate in vitro development of human induced pluripotent stem cells (iPSCs) [1] derived from fibroblasts of Parkinson’s disease (PD) patients carrying heterozygous glucocerebrosidase gene (GBA) E326K mutations.

Background: iPSCs are cells that resemble embryonic stem cells and have been used for disease modelling, drug discovery and cell therapy development [1, 2]. In recent years, growing evidence have suggested a relationship between PD and GBA mutations, now recognized as the most common risk factor for PD [3]. Specifically the E326K variant does not cause Gaucher’s disease when presenting in homozygous fashion but it is still associated to PD and Lewy body dementia in the case of heterozygous carriers.

Method: Dermal fibroblast derived from two PD patients carrying GBA mutation (variant p.E326K) [4] and two healthy donors were reprogrammed into iPSCs through virus-free and feeder-free protocol. All iPSCs clones, showing a uniform morphology, were characterized for their pluripotency, both in vitro through embryoid bodies formation and in vivo through teratoma formation assay. Neural stem cells (NSCs) – able to spontaneously differentiate in neural cells such as astrocytes, oligodendrocytes and neurons – were subsequently obtained from iPSCs.

Results: We successfully reprogrammed the fibroblasts into iPSCs. Neural stem cells were obtained from iPSCs lines. We analyzed these cell lines at morphological and molecular level to underline functional and biochemical differences possibly related to the mutation.

Conclusion: We obtained NSCs from two PD patients carrying heterozygous E326K GBA mutation and two healthy donors. PD patients carrying GBA mutations present a clinical phenotype indistinguishable from idiopathic PD, although motor and cognitive decline is generally faster. GBA mutations could provoke substrate accumulation, altering lysosomal function and eventually promoting α-synuclein aggregation [3,5]. However the E326K variant is associated to PD and Lewy pathology through mechanisms yet unknown, that possibly differs by other Gaucher’s related pathway. In this regard, iPSCs can be used as “disease in a dish” model to shed a light on pathophysiology and to discover new therapeutic strategies (e.g. potential use of enzyme-replacement therapy) to counteract the neurodegenerative process.

References: 1. Rosati J, Ferrari D, Altieri F, et al. Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies. Cell Death Dis. 2018 Sep 17;9(10):937. 2. Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017 Feb;16(2):115-130. 3. Riboldi, G. M., & Di Fonzo, A. B. (2019). GBA, Gaucher Disease, and Parkinson’s Disease: From Genetic to Clinic to New Therapeutic Approaches. Cells, 8(4), 364. 4. Berge-Seidl V, Pihlstrøm L, Maple-Grødem J, et al. The GBA variant E326K is associated with Parkinson’s disease and explains a genome-wide association signal. Neurosci Lett. 2017 Sep 29;658:48-52 5. Blauwendraat C, Nalls MA, Singleton AB. The genetic architecture of Parkinson’s disease. Lancet Neurol. 2020 Feb;19(2):170-178.

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