Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Nat Methods. 2022 Jan;19(1):90-99. doi: 10.1038/s41592-021-01344-8. Epub 2021 Dec 30.
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, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed 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（iPSC）衍生类器官为研究人类器官发育提供了模型。单细胞转录组学能够高度解析这些系统内的细胞状态；然而，需要采用直接测量谱系关系的方法。在这里，我们建立了 iTracer，这是一种谱系记录器，它将报告基因条形码与可诱导的 CRISPR-Cas9 瘢痕结合在一起，与单细胞和空间转录组学兼容。我们应用 iTracer 来探索大脑类器官发育过程中的克隆性和谱系动力学，并确定命运限制的时间窗口以及祖细胞神经元家族之间神经发生动力学的变化。我们还建立了长期的四维光片显微镜，用于大脑类器官中的空间谱系记录，并证实了发育中的神经上皮中的区域性克隆性。我们整合了基因扰动（iTracer-perturb）并评估了镶嵌 TSC2 突变对大脑类器官发育的影响。我们的数据揭示了在大脑类器官形成过程中谱系和命运是如何建立的。更广泛地说，我们的技术可以在任何 iPSC 衍生的培养系统中进行调整，以解析正常或受干扰发育过程中的谱系变化。
