Toward Personalized Regenerative Therapies for Parkinson's Disease

Abstract

Parkinson's disease (PD) represents one of the most common neurodegenerative disorders, significantly impacting the quality of life of millions of people worldwide, and posing growing challenges for healthcare systems. Driven by demographic shifts and increasing numbers of patients, PD gains importance and novel treatment options for PD are urgently needed. Cumulative research has shown that stem cell-based therapies offer considerable promise, but still fundamental challenges remain unresolved. In the context of autologous therapy, key challenges include robust and time-efficient generation of iPSCs, their proper characterization, selection of bona-fide iPSC lines for downstream applications and the standardized and reproducible production of both, high-quality iPSCs and pure mDA progenitors. A major barrier lies in the cell line-dependent variability, which impedes consistency and comparability, both essential prerequisites for widespread clinical application of autologous cell therapies. To address these issues, this study presents a comprehensive process for the generation of iPSC-derived mDA cells, aiming to overcome current bottlenecks to support the translation of stem cell therapies. In total we derived 78 iPSC lines from ten unrelated donors, five healthy controls and five PD patients. Using skin punch biopsies from healthy donors, we established a rapid and robust protocol for the generation of iPSCs, yielding high-quality cryopreserved iPSC seed stocks within seven weeks. This process includes rigorous quality control measures to ensure the selection of bona-fide iPSC lines suitable for downstream applications. We optimized existing mDA differentiation protocols, achieving consistent generation of phenotypically defined mDA progenitors from multiple iPSC lines with diverse genetic backgrounds. Functional assessment demonstrated that mDA cells derived from two independent donors normalized amphetamine-induced rotation in a 6-OHDA rat model of PD. For detailed ex vivo graft phenotyping of preclinical animal models, we developed imaging strategies to (i) assess spatial projection and quantification of transplanted cells using 3D light-sheet imaging and (ii) analyze graft composition through cyclic 2D immunofluorescent imaging. To facilitate scalability and standardization, we adapted key elements of the process to semi-automated platforms, paving the way towards future clinical translation. Moreover, we transferred our protocol to relevant patient-derived samples, generating iPSCs from five PD patients. These lines underwent stringent quality assessment and bona-fide iPSCs were successfully differentiated into mDA progenitors. Our data provide evidence that variables such as somatic cell type, reprogramming method, and culture platform strongly favor standardization and comparability of the cells, thereby minimizing batch-to-batch variability. Thus, this study can contribute to the advancement of personalized iPSC-based research and future translational efforts.