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 10

CLSTN2- or PTPRO-enriched progenitors integrate into host circuits and exhibit a higher therapeutic potency.

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CLSTN2- or PTPRO-enriched progenitors integrate into host circuits and e...
(A–D) Typical traces of whole-cell patch-clamp recording of sAPs (A) and sAP frequency (B), hyperpolarizing current injection showing voltage sag (C), and voltage sag measurements (D) from grafted mDA neurons 5 months after transplantation. Recorded cell numbers: n = 24 (unsorted), n = 15 (CLSTN2), n = 22 (PTPRO). **P < 0.01, by 1-way ANOVA followed by Tukey’s multiple-comparison test. (E) Typical traces of sIPSCs (top) and sEPSCs (bottom) in grafted human mDA neurons 5 months after transplantation. (F and G) Frequencies of sIPSCs (F) and sEPSCs (G). Number of mice: n = 4 (unsorted), n = 3 (CLSTN2), n = 4 (PTPRO). Recorded cell numbers for sEPSCs: n = 16 (unsorted), n = 16 (CLSTN2), n = 20 (PTPRO). Recorded cell numbers for sIPSCs: n = 16 (unsorted), n = 18 (CLSTN2), n = 20 (PTPRO). (H and I) Amphetamine-induced rotation behavior changes in PD mice over a 6-month post-transplantation period. The grafting dose per mouse was 100,000 cells (H). n = 5 (aCSF), n = 9 (unsorted), n = 11 (CLSTN2), n = 9 (PTPRO). (I) The grafting dose per mouse was 7500 cells. The H9-CLSTN2-P2A-tdT cell line was used. n = 4 (unsorted), n = 3 (sorted). The tdT ratio for the unsorted group was approximately 29%. **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Dunnett’s multiple-comparison test, compared with the ACSF group (H) or with the unsorted group (I). trpl, transplantation.