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 3

In vivo cell type composition by snRNA-seq.

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In vivo cell type composition by snRNA-seq. (A) Representative microscop...
(A) Representative microscopy images of 1-month-old (equivalent of day 45 differentiation in vitro) intrastriatal grafts from Boost versus Boost+ patterned progenitors (day 16) on various markers, FOXA2, SC121, EN1, and ALDH1A1. (B) Schematic illustration of the snRNA-seq from the grafts 1- and 9-months postimplantation of the mDA neuron progenitor (day 16) derived from the Boost and Boost+ method. (C) UMAP plot of 1-month grafted cells from the Boost and Boost+ protocols, colored by cell types. (D) Dotplot of canonical marker genes of midbrain progenitors, mDA neuron subtypes, and off-targets cells (E) UMAP plot colored by protocol at 1 month. (F and G) Percentages of each cell type colored by protocol (F) and different neuronal subtypes in each protocol at 1 month (G). (H) UMAP plot of 9 months grafted cells from the Boost and Boost+ protocols, colored by cell types. (I) Dotplot of canonical marker genes of midbrain progenitors, mDA neuron subtypes, and off-target cells at 9 months. (J) UMAP plot, colored by protocol at 9 months. (K and L) Percentages of each cell type, colored by protocol (K) and different neuronal subtypes in each protocol at 9 months (L). (M) UMAP of adult midbrain dopamine (mDA) neurons, divided by SOX6-and CALB1-positive neurons that were selected to determine highly specific markers for A9 and A10 DA neurons, respectively. (N and O) Boxplots showing the distribution of enrichment scores for each cell type, protocol, and time point according to A9 (N) and A10 (O) mDA neurons of adult signatures. Mann-Whitney rank test and Benjamini-Hochberg correction; *P < 0.001.