A Platform for Studying Molecular and Cellular Mechanisms of Parkinson’s Disease Based on Human Induced Pluripotent Stem Cells

The aim of the study was to obtain induced pluripotent stem cells (iPSCs) from patients with various forms of Parkinson’s disease (PD), and to create on this basis a platform for studying the pathogenesis of the disease at the molecular and cellular level with the development of a protocol of the stem cell differentiation.

Materials and Methods. iPSCs were derived from cultured skin fibroblasts, taken from five patients with various forms of PD (PARK8, PARK2, GBA-associated and sporadic forms), and reprogrammed with the help of lentiviral vectors and on the basis of Sendai virus. The obtained iPSCs clones were cultured to the stage of embryonic bodies and, after spontaneous differentiation, stained immunocytochemically. Gene expression and neural markers in these iPSCs lines were analysed using reverse transcription polymerase chain reaction.

Results. The obtained iPSCs clones had a normal 46 XY karyotype, stained specifically with Oct4, Nanog, TRA-1-81 and SSEA-4 antibodies, and expressed marker genes responsible for maintaining the pluripotent condition. In the cultures of differentiated iPSCs, cells positively stained for the markers of the three primary germ layers (ectoderm, mesoderm, and endoderm) have been revealed. An effective protocol of iPSCs differentiation into dopaminergic neurons has been worked out, and confirmed by the expression of the specific marker — tyrosine hydroxylase enzyme.

Conclusion. On the basis of explicitly characterized iPSCs from patients with various forms of PD and the developed cellular protocol, a platform for studying the pathogenesis of PD at the molecular and cellular level has been created. Obtaining cell population enriched with dopaminergic neurons opens a perspective for their application for personalized cell replacement PD therapy.

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