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. 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.