REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [population]

During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development.

在人类大脑发育过程中，会产生多种具有不同区域特性的细胞类型。本研究报道了一套可从人类胚胎干细胞（human embryonic stem cells, hESCs）中诱导生成早期人类大脑前脑、中脑及后脑细胞类型的体系，并基于单细胞RNA测序（single-cell RNA-Seq）分析，推导并通过实验验证了这些细胞类型的生成谱系树。我们构建了SOX2Cit/+与DCXCit/Y hESC细胞系，以靶向标记神经分化过程中的祖细胞与神经元，用于单细胞转录组分析；随后鉴定出兼具嘴侧（皮层）与尾侧（中/后脑）特性的多种独立细胞类型。我们利用基因表达图谱与人类胎儿大脑单细胞基因表达数据，将这些细胞类型与原代组织进行直接比对，证实其与早期人类大脑细胞类型（包括前板细胞）高度相似。基于单细胞转录组数据，贝叶斯算法构建了统一的谱系树，并预测了新型调控转录因子。该谱系树揭示了皮层细胞类型与中/后脑细胞类型之间存在显著的分支点，这一结论经克隆分析实验得到验证。我们证实，通过调控经典Wnt/β-连环蛋白通路，可以选择性诱导生成任一分支的细胞类型。综上，本研究提供了一个经实验验证的、覆盖多个脑区的谱系树，为阐明人类大脑发育过程中区域特异性神经谱系的分子调控机制提供了新的见解。