Transcriptome regulation by ALK in cerebral organoids revealed by single-cell RNA sequencing. Transcriptome regulation by ALK in cerebral organoids revealed by single-cell RNA sequencing

Cerebral organoids derived from human pluripotent stem cells has been used as a model to study the development of mammalian brain. In this study, we employed 10x genomic single-cell RNA sequencing analysis to gain mechanical insight into the transcriptional changes of cells in cerebral organoids subject to the regulation by anaplastic lymphoma kinase (ALK) pathway. Based on the marker genes that most strongly contribute to the clusters, we annotated each cluster to cell types including radial glial cells (RGCs, SOX2 dominant), RGCs and intermediate progenitors (IPs, both SOX2 and EOMES positive), proliferating cells (Ki67 positive), early-born neurons (TBR1 and BCL11B dominant) and immature neurons (both SOX2 and TBR1/BCL11B/POU3F2 positive). Then we compared the cell compositions of the ALK inhibitor and vehicle treated groups in annotated clusters. The results show that transient ALK inactivation significantly reduced the number of proliferating NPCs and early-born neurons, with more cells being entrapped at the RGC stage, indicating a delayed development course from NPCs to neurons after ALK inhibition. By characterizing cell-type-specific transcriptomes, we found that transient ALK inactivation in cerebral organoids cause altered transcription of several genes including claudin 5 (CLDN5), transthyretin (TTR), inhibitor of DNA-binding proteins (IDs), Purkinje cell protein 4 (PCP4), transient receptor potential cation channel subfamily M member 3 (TRPM3), 5-HT2C receptor (HTR2C) and pro-melanin concentrating hormone (PMCH). These genes were significantly down-regulated in response to ALK inhibition in RGCs. Interestingly, the same set of genes were significantly up-regulated in proliferating cells in ALK inhibition group. These results revealed transcriptome regulation by ALK pathway in cerebral organoids. Overall design: Single-cell RNA sequencing was performed to dissect the cell compositions and transcriptional landscapes in two sets of human cerebral organoids (35 days in vitro) at 12 days post ALK inhibitor or vehicle treatment. 12,606 cells pooled from 10 ALK inhibitor treated organoids and 10,736 cells pooled from 8 organoids in the vehicle group were analyzed.

由人多能干细胞（human pluripotent stem cells）诱导产生的脑类器官（cerebral organoids）已被用作研究哺乳动物脑发育的经典模型。本研究采用10x Genomics单细胞RNA测序（single-cell RNA sequencing）技术，旨在深入解析间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）通路调控下脑类器官内细胞的转录变化。基于对细胞聚类贡献度最高的标记基因，我们将每个聚类注释为以下细胞类型：以SOX2高表达为特征的放射状胶质细胞（radial glial cells, RGCs）、同时表达SOX2与EOMES的放射状胶质细胞与中间祖细胞（intermediate progenitors, IPs）、Ki67阳性的增殖细胞、以TBR1和BCL11B高表达为特征的早期新生神经元，以及同时表达SOX2与TBR1/BCL11B/POU3F2的未成熟神经元。随后，我们对比了ALK抑制剂处理组与溶剂对照组的注释聚类细胞组成。结果显示，短暂性ALK失活可显著减少增殖性神经前体细胞（neural progenitor cells, NPCs）与早期新生神经元的数量，使更多细胞滞留于RGC阶段，表明ALK抑制会延缓神经前体细胞向神经元的发育进程。通过解析细胞类型特异性转录组，我们发现脑类器官中的短暂性ALK失活会改变多个基因的转录水平，包括紧密连接蛋白5（claudin 5, CLDN5）、转甲状腺素蛋白（transthyretin, TTR）、DNA结合抑制蛋白（inhibitor of DNA-binding proteins, IDs）、浦肯野细胞蛋白4（Purkinje cell protein 4, PCP4）、瞬时受体电位阳离子通道亚家族M成员3（transient receptor potential cation channel subfamily M member 3, TRPM3）、5-羟色胺2C受体（5-HT2C receptor, HTR2C）以及促黑激素浓缩激素（pro-melanin concentrating hormone, PMCH）。上述基因在RGCs中经ALK抑制后显著下调；有趣的是，在ALK抑制组的增殖细胞中，同一组基因却显著上调。这些结果揭示了ALK通路对脑类器官转录组的调控作用。总体实验设计：对两组人源脑类器官（体外培养35天）在ALK抑制剂或溶剂处理后12天开展单细胞RNA测序，以解析其细胞组成与转录图谱。本研究共分析了来自10个ALK抑制剂处理类器官的12606个细胞，以及来自8个溶剂对照组类器官的10736个细胞。