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.

中文翻译：

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人中脑多巴胺能神经元分化标志物预测帕金森病模型中的细胞治疗结果

基于人类多能干细胞（基于 hPSC）的替代疗法对治疗帕金森病 (PD) 具有很大的前景。然而，hPSC 来源的供体细胞的异质性和移植后中脑多巴胺能 (mDA) 神经元的低产量阻碍了其广泛的临床应用。在这里，我们已经描述了 mDA 神经元分化过程中的单细胞分子景观。我们发现这个过程概括了多个但相邻的胎儿大脑区域的发育，包括腹侧中脑、峡部和腹侧后脑，从而产生了异质的供体细胞群。我们重建了 mDA 谱系的分化轨迹，并确定了 Calsyntenin 2 (CLSTN2) 和蛋白酪氨酸磷酸酶 O 型受体 (PTPRO) 作为 mDA 祖细胞的特异性表面标志物，这可以预测 mDA 神经元分化，并且可以促进祖细胞分选和移植后 mDA 神经元的高度富集（高达 80%）。标记分类的祖细胞在纠正 PD 小鼠的运动缺陷方面表现出更高的治疗效力。不同标记分类的移植物具有惊人一致的细胞组成，其中 mDA 神经元富集，而脱靶神经元类型大多耗尽，表明移植物结果稳定。我们的研究提供了对 mDA 神经元分化过程中细胞异质性的更好理解，并建立了一种产生高度纯化的供体细胞以实现稳定和可预测的治疗结果的策略，从而提高了基于 hPSC 的 PD 细胞替代疗法的前景。