Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells [RNA-seq 1]

The differentiation of human pluripotent stem cells (hPSCs) into mesencephalic dopaminergic (mesDA) neurons requires a precise combination of extrinsic factors that recapitulates the in vivo environment and timing. Current methods are capable of generating authentic mesDA neurons after long-term culture in vitro; however, when mesDA progenitors are transplanted in vivo, the resulting mesDA neurons are only minor components of the graft. This low yield hampers the broad use of these cells in the clinic. In this study, we genetically modified pluripotent stem cells to generate a novel type of stem cells called lineage-restricted undifferentiated stem cells (LR-USCs), which robustly generate mesDA neurons. LR-USCs are prevented from differentiating into a broad range of nondopaminergic cell types by knocking out genes that are critical for the specification of cells of alternate lineages. Specifically, we target transcription factors involved in the production of spinal cord and posterior hindbrain cell types. When LR-USCs are differentiated under caudalizing condition, which normally give rise to hindbrain cell types, a large proportion adopt a midbrain identity and develop into authentic mesDA neurons. We show that the mesDA neurons are electrophysiologically active, and due to their higher purity, are capable of restoring motor behavior eight weeks after transplantation into 6-hydroxydopamine (6-OHDA)-lesioned rats. This novel strategy improves the reliability and scalability of mesDA neuron generation for clinical use.Competing Interest StatementThe authors have declared no competing interest. Overall design: Gene expression analysis of RNA-seq data for H9, GBX2 knockout and 4X cells in the undifferentiated state and differentiated to day 4 caudal neural progenitors (CNPs).

人类多能干细胞（human pluripotent stem cells, hPSCs）向中脑多巴胺能（mesencephalic dopaminergic, mesDA）神经元分化，需要精准组合的外源因子以重现体内发育环境与时序。现有体外长期培养方案虽可获得纯正的中脑多巴胺能神经元，但将中脑多巴胺能前体细胞移植入体内后，移植物内的中脑多巴胺能神经元占比极低。该低产率极大限制了此类细胞的临床转化应用。本研究通过基因修饰多能干细胞，构建了一类新型干细胞——谱系限制性未分化干细胞（lineage-restricted undifferentiated stem cells, LR-USCs），其可高效定向生成中脑多巴胺能神经元。LR-USCs可通过敲除其他谱系特化的关键基因，阻断其向广泛的非多巴胺能细胞类型分化。具体而言，本研究靶向调控脊髓与后脑前体细胞生成的转录因子。当将LR-USCs置于通常诱导后脑细胞生成的尾侧化培养条件下进行分化时，大部分细胞会获得中脑细胞身份，并发育为纯正的中脑多巴胺能神经元。研究证实，此类中脑多巴胺能神经元具有电生理活性；且因其纯度更高，移植入6-羟基多巴胺（6-hydroxydopamine, 6-OHDA）损伤的大鼠体内8周后，可有效恢复其运动行为。该新型策略提升了临床级中脑多巴胺能神经元制备的可靠性与可扩展性。利益冲突声明：作者声明无竞争利益。实验整体设计：对未分化状态及诱导分化至第4天的尾侧神经前体细胞（caudal neural progenitors, CNPs）的H9细胞、GBX2敲除细胞与4X细胞进行RNA测序基因表达分析。