(F1103) ENHANCED IN VIVO SURVIVAL AND YIELD OF SUBTYPE-SPECIFIC DOPAMINE NEURONS FROM HUMAN PLURIPOTENT STEM CELLS BY MODULATION OF TNF-NFKB-P53 AXIS AND WNT-FGF18 SIGNALING
Abstract: While clinical trials are ongoing using human pluripotent stem cell (hPSC)-derived midbrain dopamine (mDA) neuron precursor grafts in Parkinson’s disease (PD), unresolved challenges remain, including extensive cell death following transplantation and suboptimal mDA differentiation protocol from hPSC leading to low yield of mDA neuron proportion from the grafted cells. In particular, the yield of TH+ mDA neurons after in vivo grafting and subtype-specific mDA neuron markers can be further improved. For example, characterization of mDA grafts by single cell transcriptomics has yielded a small mDA neuron proportion and a considerable contaminating cell fraction. Through a pooled CRISPR/Cas9 screen, we identified p53-mediated apoptotic and TNFα-NFκB signaling as key contributors to postmitotic dopamine neuron loss. Coupled with novel cell surface marker-based mDA neuron purification strategies, a clinically approved TNFα inhibitor, adalimumab, significantly improved mDA neuron survival and functional recovery in a preclinical PD mouse model, offering a clinically translatable approach to optimize mDA neuron-based therapies in PD. To further improve mDA neuron differentiation, we present an optimized mDA neuron differentiation strategy that builds on our clinical grade (“Boost”) protocol but includes the addition of FGF18 and IWP2 treatment (“Boost+”) at the mDA neurogenesis stage. Boost+ mDA neurons show higher expression of EN1, PITX3, and ALDH1A1. Improvements in both mDA neuron yield and transcriptional similarity to primary mDA neurons are observed both in vitro and in grafts. Furthermore, grafts are enriched in authentic A9 mDA neurons by single-nucleus sequencing. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo, analyses show increased percentages of TH+ mDA neurons resulting in the efficient rescue of amphetamine-induced rotation behavior in the 6-OHDA rat model and rescue of some motor deficits in non-drug induced assays, including the ladder rung assay that is not improved by Boost mDA neurons. The Boost+ conditions present an optimized protocol with advantages for disease modeling and mDA neuron grafting paradigms.
Funding Source: This work was supported by DGIST start-up funds from the Ministry of Science and ICT (2024010330) to T.W.K, and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2024-00351442) to T.W.K.
