Mapping of clonal lineages across developmental stages in human neural differentiation

The cell lineages across developmental stages remains to be elucidated. We developed single-cell split barcoding (SISBAR) to allow clonally tracking single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. We developed “potential-spective” and “origin-spective” analysis to interrogate the cross-stage lineage relationships, and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. We demonstrated that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multi-lineage fates of a progenitor cell type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells, and identified surface markers that can predict its lineage fate after transplantation and improve graft outcomes. Overall design: We developed SISBAR method, which integrated virus-mediated cellular barcoding, clonal splitting, and scRNA-seq, to investigate the lineage relationships among cells across developmental stages in hPSC-based neural differentiation. SISBAR works by genetically labelling each progenitor cell with a unique viral barcode at an earlier differentiation stage, followed by limited cell division, and then randomly splitting the cells into two parts. Half of the cells are immediately subjected to scRNA-seq and viral barcode recovery, and the remaining cells are allowed to continue differentiating to a later stage, followed by scRNA-seq and viral barcode recovery. We designed three successive but independent labelling experiments to investigate the lineage relationships between stage I-II (Exp #1), stage II-III (Exp #2), and stage III-IV (Exp #3), respectively.