IPSC-Derived Astrocytes Contribute to In Vitro Modeling of Parkinson's Disease Caused by the GBA1 N370S Mutation

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder that ranks second in prevalence after Alzheimer's disease. The number of PD diagnoses increases annually. Nevertheless, modern PD treatments merely mitigate symptoms rather than preventing neurodegeneration progression. The creation of an appropriate model to thoroughly study the mechanisms of PD pathogenesis remains a current challenge in biomedicine. Recently, there has been an increase in data regarding the involvement of not only dopaminergic neurons of the substantia nigra but also astrocytes in the pathogenesis of PD. Cell models based on induced pluripotent stem cells (iPSCs) and their differentiated derivatives are a useful tool for studying the contribution and interaction of these two cell types in PD. Here, we generated two iPSC lines, ICGi034-B and ICGi034-C, by reprogramming peripheral blood mononuclear cells of a patient with a heterozygous mutation c.1226A>G (p.N370S) in the GBA1 gene by non-integrating episomal vectors encoding OCT4, KLF4, L-MYC, SOX2, LIN28, and mp53DD. The iPSC lines demonstrate the expression of pluripotency markers and are capable of differentiating into three germ layers. We differentiated the ICGi034-B and ICGi034-C iPSC lines into astrocytes. This resulting cell model can be used to study the involvement of astrocytes in the pathogenesis of GBA-associated PD.

Keywords: GBA1; Parkinson’s disease; astrocytes; induced pluripotent stem cells.

Figure 1

Characterization of the induced pluripotent stem cells (iPSC) cell lines ICGi034-B and ICGi034-C: (A) Morphology of iPSC colonies. (B) Quantitative analysis of the expression of pluripotency markers (NANOG, OCT4, SOX2) using RT-qPCR. Error bars show standard deviation. (C) Histochemical detection of alkaline phosphatase. (D) Karyotype analysis (G-banding) (46,XX). (E) Immunofluorescent staining for pluripotency markers OCT4, SOX2, SSEA-4, TRA-1-60. (F) Immunofluorescent staining for differentiation markers: αSMA (red signal) and NKX2.5 (green signal) (mesoderm); TUBB3 (red signal) and MAP2 (green signal) (ectoderm); FOXA2 (red signal) and AFP (green signal) (endoderm). Nuclei are stained with DAPI (blue signal). (G) Sequence images of the GBA1 gene regions vof the patient’s peripheral blood mononuclear cells (PBMCs), iPSC lines ICGi034-B and ICGi034-C, and a healthy donor (GBA-WT). Identified polymorphisms are marked with arrows. All scale bars—100 μm.

Figure 2

Differentiation of iPSC cell lines ICGi034-B and ICGi034-C: (A) Scheme of iPSC differentiation protocols into astrocytes. (B,C) Immunofluorescent staining for markers of astrocyte terminal differentiation GFAP and S100β.