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.