Enhanced yield and subtype identity of hPSC-derived midbrain dopamine neuron by modulation of WNT and FGF18 signaling

While clinical trials are ongoing using human pluripotent stem cell-derived midbrain dopamine (mDA) neuron precursor grafts in Parkinsons disease (PD), current protocols to derive mDA neurons remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of some mDA neuron and subtype-specific markers can be further improved. For example, characterization of mDA grafts by single cell transcriptomics has yielded only a small proportion of mDA neurons and a considerable fraction of contaminating cell populations. Here 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. We demonstrate that Boost+ mDA neurons show higher expression of EN1, PITX3 and ALDH1A1. Improvements in both mDA neurons yield and transcriptional similarity to primary mDA neurons is observed both in vitro and in grafts. Furthermore, grafts are enriched in authentic A9 mDA neurons by single nucSeq. 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 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.