Savaid Medical School, University of Chinese Academy of Sciences, Beijing, 100049, China.
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
Stem Cell Res Ther. 2022 Aug 13;13(1):412. doi: 10.1186/s13287-022-03104-7.
Midbrain dopaminergic (DA) progenitors derived from human pluripotent stem cells are considered to be a promising treatment for Parkinson's disease (PD). However, the differentiation process produces undesired cell types, which influence the in vivo evaluation of DA cells. In this paper, we analyze the cell fate choice during differentiation and provide valuable information on cell preparation.
Human embryonic stem cells were differentiated into DA progenitors. We applied single-cell RNA sequencing (scRNA-seq) of the differentiation cells at different time points and investigated the gene expression profiles. Based on the differentially expressed genes between DA and non-DA cells, we investigated the impact of LGI1 (DA enriched) overexpression on DA differentiation and the enrichment effect of CD99 (non-DA enriched) sorting.
Transcriptome analyses revealed the DA differentiation trajectory as well as non-DA populations and three key lineage branch points. Using genetic gain- and loss-of-function approaches, we found that overexpression of LGI1, which is specific to EN1 early DA progenitors, can promote the generation of TH neurons. We also found that choroid plexus epithelial cells and DA progenitors are major components of the final product (day 25), and CD99 was a specific surface marker of choroid plexus epithelial cells. Sorting of CD99 cells eliminated major contaminant cells and improved the purity of DA progenitors.
Our study provides the single-cell transcriptional landscape of in vitro DA differentiation, which can guide future improvements in DA preparation and quality control for PD cell therapy.
从中胚层多巴胺能（DA）祖细胞衍生而来的人多能干细胞被认为是治疗帕金森病（PD）的有前途的方法。然而，分化过程会产生不理想的细胞类型，这会影响 DA 细胞的体内评估。在本文中，我们分析了分化过程中的细胞命运选择，并提供了有关细胞制备的有价值的信息。
人胚胎干细胞分化为 DA 祖细胞。我们应用单细胞 RNA 测序（scRNA-seq）对不同时间点的分化细胞进行分析，并研究了基因表达谱。基于 DA 和非 DA 细胞之间差异表达的基因，我们研究了 LGI1（DA 丰富）过表达对 DA 分化的影响以及 CD99（非 DA 丰富）分选的富集效应。
转录组分析揭示了 DA 分化轨迹以及非 DA 群体和三个关键的谱系分支点。使用遗传增益和功能丧失方法，我们发现，特异性表达于 EN1 早期 DA 祖细胞的 LGI1 的过表达可以促进 TH 神经元的产生。我们还发现脉络丛上皮细胞和 DA 祖细胞是最终产物（第 25 天）的主要成分，CD99 是脉络丛上皮细胞的特异性表面标记。CD99 细胞的分选消除了主要的污染细胞，提高了 DA 祖细胞的纯度。
我们的研究提供了体外 DA 分化的单细胞转录图谱，这可以为 PD 细胞治疗中 DA 制备和质量控制的未来改进提供指导。
