Single-cell transcriptomic approaches have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single-cell behavior.

单细胞转录组学方法已发现多能干细胞 (PSC) 群体内的分子异质性。纵向追踪单细胞谱系及其表型的工具将揭示异质性是否超出分子同一性。因此，我们生成了稳定的 Cre 诱导彩虹报告基因人类 PSC 系，可提供多达 18 个独特的膜靶向荧光条形码。这些条形码能够在单细胞克隆扩增、改变形态和迁移时对其进行重复评估。由于该报告基因的细胞分辨率，我们鉴定出具有增强克隆扩增、同步细胞分裂和持续定位于集落边缘的 PSC 子集。在定向分化为心肌细胞、皮质神经元和成肝细胞的整个过程中，报告基因表达稳定维持。对神经分化的重复检查揭示了来自克隆显性祖细胞的自组装皮质组织。总的来说，这些发现证明了该报告基因线在跟踪单细胞行为方面的广泛实用性和易于实施。