Lineage recording in human cerebral organoids

Induced pluripotent stem cell (iPSC) derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly-resolved descriptions of cell states within these systems, however, new approaches are needed to directly measure lineage relationships. Here we establish an inducible lineage recorder, iTracer, that combines reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze state and lineage relationships. We apply iTracer to explore lineage dynamics during cerebral organoid development and identify clonality of brain regions, a time window of fate restriction as well as neurogenic dynamics between progenitor-to-neuron families within a brain organoid region. We establish long-term 4-D lightsheet microscopy in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation into the system (iTracer-perturb), and assess the effect of mosaic TSC2 mutations on early brain organoid development. Our data sheds light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

诱导多能干细胞（Induced Pluripotent Stem Cell，iPSC）衍生类器官可为人类器官发育研究提供模型。单细胞转录组学（Single-cell Transcriptomics）可实现对这些系统中细胞状态的高分辨率解析描述，但目前仍需新方法来直接测定谱系关联。本研究构建了可诱导谱系记录系统iTracer，该系统整合了报告基因条形码、可诱导CRISPR/Cas9疤痕突变技术与单细胞转录组学，用于分析细胞状态与谱系关联。我们利用iTracer探究了大脑类器官发育过程中的谱系动态，鉴定出脑区域的克隆性、细胞命运限定的时间窗口，以及大脑类器官区域内祖细胞向神经元家族转化的神经发生动态。我们在大脑类器官中建立了长期四维光片显微镜成像体系，并证实了发育中神经上皮的区域克隆性。我们将基因扰动技术整合入该系统（命名为iTracer-perturb），并评估了嵌合TSC2突变对早期大脑类器官发育的影响。本研究数据阐明了大脑类器官形成过程中谱系与细胞命运的建立机制。更广泛而言，我们的技术可适配于任何iPSC衍生的培养系统，用于解析正常或扰动发育过程中的谱系改变。