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 4

In vivo functional characterization in unilateral 6-OHDA-induced Parkinsonian rats.

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In vivo functional characterization in unilateral 6-OHDA-induced Parkins...
(A) Amphetamine-induced rotations per minute measured at the indicated time points after transplantation in different animal groups. (B) The percentage of the adhesive removal time by the contralateral paw (left) and deep miss steps in the ladder rung walking test (right). (C–F) Representative IHC images of grafted cells showing expression of FOXA2, EN1, ALDH1A1, DAT, and TH together with human-specific markers (hNA or hNCAM) in Boost and Boost+ grafts. Arrowheads indicate ALDH1A1-expressing TH+ dopaminergic axons (E). (G) Stereological estimation of total hNA+ and TH+ cell numbers in Boost and Boost+ grafts (logarithmic scale). (H–K) The percentage of graft composition showing TH+ cells out of hNA+ cells (H), FOXA2- or EN1-expressing cells out of hNA+ cells (I), EN1+TH+ and ALDH1A1+TH+ cells out of the total human TH+ cells (J), and human-specific hKi67+ cells out of human-specific hKu80+ cells (K) in Boost and Boost+ cell grafts. Animal numbers are 5–7 per group in A and B and 3 per group in G–K. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. In A and B, the color of * represents the significance between the group with the same color of symbol and the vehicle group. In C–E, scale bar: 100 μm (left panels), 50 μm (right panels). In F, scale bar: 20 μm (right panels). The areas outlined in white lines are shown with higher magnification images in the right panels. (L–O) Whole-cell recordings from grafted mDA neurons (Boost+: 6 months after transplantation), including examples of a spontaneously active neuron (L), spontaneous EPSCs (M), action potentials evoked by current injections (N), and EPSCs evoked by local electrical stimulation (O). Recordings were performed in the absence of synaptic blockers.