Cerebral organoids display dynamic clonal growth with lineage replenishment

During development tissue stem cells expand by symmetrical divisions while asymmetric divisions self-renew a tissue stem cell and produce differentiating cell types that assemble into functional organs. In brain development this process is variable for individual neural stem cells, resulting in differentially sized stem cell clones, but it is unclear how overall brain size is reproducibly generated during neurogenesis. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyze clonal relationships in an entire tissue are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids and show that individual stem cell clones produce progeny on a vastly variable scale. We find that symmetrically dividing cells in a subpopulation of lineages continuously resides within the developing human tissue and drive the variable lineage size distribution. We show that stem cell output is tunable to tissue demands by chemical ablation or genetic fate restriction in chimeric organoids in which perturbed organoid development is completely compensated for by unaffected lineages. This data reveals adaptive plasticity of stem cell populations in developing human brain tissue dependent on tissue needs to ensure normal development. Overall design: Barcode-based Lineagetracing to measure the lineage size distribution in brain organoids, chimeric WT/KO brain organoids, chimeric puro-resistent/puro-susceptible brain organoids, 2D cell cultures and mouse. Chimeric knockout conditions are WT/WT, WT/KO-ASPM, WT/KO-PAX6 and WT/KO-TP53 with each 50/50 mixing conditions. Puromycin was performed in 90/10 and 80/20 (susceptible/resistent) mixing conditions, with and without puromycin administration. Two 2D cell cultures were analyzed: 2D stem cell culture and 2D cell culture with neural induction. Mouse samples were seperated by left, right and mid brain and sequenced seperately. All samples are available as timecourse. Organoid samples and 2D cell culture with neural induction were measured after 0, 1, 3, 6, 9, 11, 13, 16, 21, 25, 32, 42 days. 2D stem cell culture was measured after 7, 14, 21, 28, 35, 42 days. All mouse samples were measured on the same day, but the three injection timepoints E9.5, E10.5 and E13.5 were measured. All day 0 samples are generally available in 6 replicates and all other samples in 3 replicates.