The viral barcode plasmid library contained more than 3 million unique barcodes with a relatively even count distribution, sufficient to label more than 30,000 cells with barcode collision rate less than 1% ( Figure S1F). This allows high-level expression of EGFP and viral barcodes without affecting cell differentiation when labeled at a low multiplicity of infection (MOI ∼ 0.1) ( Figures S1B–S1E). We modified the classical retroviral vector by inserting a 32-bp semi-random barcode into the 3′ untranslated region of the EGFP coding sequences, driven by an EF1α promoter ( STAR Methods Figures 1A and S1A). The barcoded sister cell profiled at the earlier stage serves as a surrogate for the ancestor of the clonally related progenies profiled at the later stage, thereby approximating individual cross-stage lineage trajectories based on single cross-stage clones. SISBAR thus simultaneously provides the transcriptome and viral barcode sequences of individual cells to infer their identities and lineage information. 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 ( Figure 1A). SISBAR works by genetically labeling each progenitor with a unique viral barcode at an earlier differentiation stage, followed by limited cell division, and then randomly splitting the cells into two parts. Inspired by these studies, we developed the SISBAR method to investigate the lineage relationships among cells across developmental stages in hPSC-based neural differentiation. 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 a surface marker that can improve graft outcomes. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage 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. We uncovered many previously uncharacterized converging and diverging trajectories. We developed “potential-spective” and “origin-spective” analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. The cell lineages across developmental stages remain to be elucidated.
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