Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson’s disease model
Peibo Xu, Hui He, Qinqin Gao, Yingying Zhou, Ziyan Wu, Xiao Zhang, Linyu Sun, Gang Hu, Qian Guan, Zhiwen You, Xinyue Zhang, Wenping Zheng, Man Xiong, Yuejun Chen
Peibo Xu, Hui He, Qinqin Gao, Yingying Zhou, Ziyan Wu, Xiao Zhang, Linyu Sun, Gang Hu, Qian Guan, Zhiwen You, Xinyue Zhang, Wenping Zheng, Man Xiong, Yuejun Chen
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Research Article Neuroscience

Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson’s disease model

Abstract

Human pluripotent stem cell–based (hPSC-based) replacement therapy holds great promise for the treatment of Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we have characterized the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including the ventral midbrain, the isthmus, and the ventral hindbrain, resulting in a heterogenous donor cell population. We reconstructed the differentiation trajectory of the mDA lineage and identified calsyntenin 2 (CLSTN2) and protein tyrosine phosphatase receptor type O (PTPRO) as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate high enrichment of mDA neurons (up to 80%) following progenitor cell sorting and transplantation. Marker-sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker-sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.

Authors

Peibo Xu, Hui He, Qinqin Gao, Yingying Zhou, Ziyan Wu, Xiao Zhang, Linyu Sun, Gang Hu, Qian Guan, Zhiwen You, Xinyue Zhang, Wenping Zheng, Man Xiong, Yuejun Chen

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Figure 7

CLSTN2 and PTPRO are predictive of mDA neuron differentiation and can give rise to highly enriched mDA neurons after progenitor sorting and transplantation.

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CLSTN2 and PTPRO are predictive of mDA neuron differentiation and can gi...
(A) Diagram of surface marker reporter cell lines and experimental schematics for in vitro and in vivo maturation. (B and C) Neurospheres matured in vitro and (B) immunostained for TH and (C) statistical analysis (n = 3 batches with 5 neurospheres per batch). Scale bars: 25 μm. ***P < 0.001, by multiple unpaired t test with Holm-Šidák correction. (D) Correlation between the surface marker progenitor ratio and the TH+ neuron ratio. See Figure 9A for a diagram of the cell lines used. (E) Unsorted progenitor-, CLSTN2+ progenitor–, and PTPRO+ progenitor–derived grafts immunostained for human nuclei (hN) and TH. Scale bars: 100 μm and 20 μm (for the enlarged insets [i] and [ii], which represent the edge and center area of the graft, respectively). (F) Quantification of the TH+ neurons ratio in grafts. n = 6 (unsorted), n = 5 (CLSTN2), and n = 7 (PTPRO). **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparison test. Sort, sorted; unsort, unsorted.