Modulation of WNT and FGF18 enhances yield and subtype identity of hPSC-derived midbrain dopamine neurons
Tae Wan Kim, Jinghua Piao, Vittoria D. Bocchi, So Yeon Koo, Se Joon Choi, Fayzan Chaudhry, Donghe Yang, Hyein S. Cho, Emiliano Hergenreder, Lucia Ruiz Perera, Subhashini Joshi, Zaki Abou Mrad, Nidia Claros, Shkurte Ademi Donohue, Yeong Eun Im, Hyo Jae Jeong, Anika K. Frank, Ryan M. Walsh, Eugene V. Mosharov, Doron Betel, Viviane Tabar, Lorenz Studer
Tae Wan Kim, Jinghua Piao, Vittoria D. Bocchi, So Yeon Koo, Se Joon Choi, Fayzan Chaudhry, Donghe Yang, Hyein S. Cho, Emiliano Hergenreder, Lucia Ruiz Perera, Subhashini Joshi, Zaki Abou Mrad, Nidia Claros, Shkurte Ademi Donohue, Yeong Eun Im, Hyo Jae Jeong, Anika K. Frank, Ryan M. Walsh, Eugene V. Mosharov, Doron Betel, Viviane Tabar, Lorenz Studer
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Research Article Development Neuroscience

Modulation of WNT and FGF18 enhances yield and subtype identity of hPSC-derived midbrain dopamine neurons

Abstract

While clinical trials of human pluripotent stem cell–derived midbrain dopamine (mDA) neuron precursor grafts for Parkinson’s disease (PD) are ongoing, current protocols remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of certain mDA neuron and subtype-specific markers require improvement. Single-cell transcriptomic analyses of grafts have revealed low proportions of mDA neurons and substantial off-target contamination. Here, we present an optimized mDA neuron differentiation strategy that builds on our clinical-grade (“Boost”) protocol by adding FGF18 and IWP2 treatment (“Boost+”) at the neurogenesis stage. Boost+ mDA neurons show higher expression of EN1, PITX3, and ALDH1A1. Improvements in mDA neuron yield and transcriptional similarity to primary mDA neurons are observed in vitro and following transplantation. Single-nucleus RNA sequencing demonstrates enrichment of A9 mDA neurons within Boost+ grafts. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo analyses show higher percentages of TH+ mDA neurons, resulting in efficient rescue of amphetamine-induced rotation behavior in the 6-OHDA rat model and rescue of deficits in some nondrug-induced assays, including the ladder rung assay, which are not improved by Boost mDA neurons. The Boost+ conditions present an optimized differentiation protocol with advantages for disease modeling and mDA neuron grafting paradigms.

Authors

Tae Wan Kim, Jinghua Piao, Vittoria D. Bocchi, So Yeon Koo, Se Joon Choi, Fayzan Chaudhry, Donghe Yang, Hyein S. Cho, Emiliano Hergenreder, Lucia Ruiz Perera, Subhashini Joshi, Zaki Abou Mrad, Nidia Claros, Shkurte Ademi Donohue, Yeong Eun Im, Hyo Jae Jeong, Anika K. Frank, Ryan M. Walsh, Eugene V. Mosharov, Doron Betel, Viviane Tabar, Lorenz Studer

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Figure 1

FGF18 and IWP2 at neurogenic conversion induce improved yield of mDA neurons.

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FGF18 and IWP2 at neurogenic conversion induce improved yield of mDA neu...
(A) Schematic illustration of the Boost and Boost+ mDA differentiation method. (B) Representative flow cytometry dot plots showing intracellular expression of FOXA2 and EN1 in day 16 progenitors differentiated under the indicated conditions. IWP2 and/or FGF18 were treated from day 12 to 16. (C) Quantification of FOXA2+ and FOXA2+EN1+ populations across differentiation conditions (n = 3; Mean ± SEM) from B. (D) qRT-PCR analysis of mDA neurons at day 40 for mDA neuron markers derived from the Boost and Boost+ methods (n = 4; Mean ± SEM). *P < 0.05, **P < 0.01. (E) Representative immunofluorescence images showing expression of mDA neuron markers FOXA2, TH, and EN1 in day 60 neurons. (F) Representative confocal image of RNA FISH of day 45 mDA neurons, colabeled with TH, mature mDA markers (left). Human RNA probes against NURR1 and PITX3 were used. The number of RNA FISH dots for each RNA gene (NURR1 and PITX3) was quantified per TH+ mDA neurons at day 35 and 45 (right). n = 4, 2-tailed Student’s t test, *P < 0.05. Scale bars: 100 μm (E), 7 μm (F left), 5 μm (F right). (G) qRT-PCR analysis of homogeneous postmitotic mDA neurons purified at day 40 using CD49e and CD184 for mDA markers in the Boost and Boost+ conditions (n = 3; Mean ± SEM). *P < 0.05. (H–J) UMAP plots of in vitro scRNA-seq data colored by timepoint (H), protocol (I), and cell type (J). (K) Dotplot of canonical marker genes of midbrain progenitors, DA neuron subtypes, and off target cells. (L) Overall percentage of cell types across protocols. (M) UMAP of fetal midbrain dopamine (DA) neurons (49), selected to determine highly specific markers. (N) Boxplots showing the distribution of enrichment scores for each cell type and protocol according to fetal dopamine neurons. Mann-Whitney rank test and Benjamini-Hochberg correction; ***P < 0.001.