Fig. 2: Capturing karyotype diversification across consecutive cell generations using 3D Live-Seq.

a, Top: schematic representing confocal imaging of individual PDTO-9 organoids expressing H2B-Dendra (green) to capture high-spatiotemporal-resolution recordings of individual cell divisions, followed by photoconversion of cells of interest to red fluorescence before single-cell isolation. Bottom: representative imaging stills of the procedure. Right: CNA plots from chromosome 7 to chromosome 16 are shown for photoconverted daughter cells 1 and 2 (top two lanes), as well as the remaining single cells of the organoid combined in lane 3 as reference material. The red box indicates the reciprocal gain and loss of chromosome 15, matching the lagging chromatin phenotype. The dots represent measured CNAs per bin (1 Mb). The average deviation from the diploid genome is indicated by color coding (blue = loss; orange = gain). Scale bars, 10 μm. b, Bar graph representing the fraction of PDTO-9 cell divisions that resulted in reciprocal whole-chromosome or sub-chromosomal CNAs among daughter cells per chromatin error class. In total, 37 normal (N) divisions, 18 divisions displaying chromatin bridges (CB) and nine divisions with lagging chromatin (LC) were captured. c, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 13 cells. Top: representative stills of the growing PDTO-9 structure, with nuclei coded in false color by depth. Middle: reconstruction of the true mitotic tree containing representative stills of 3D-rendered anaphases. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of 12 cells (93% recovery) isolated from the imaged PDTO-9 organoid. A consecutive missegregation of chromosome 7 (lineage I) was mapped to the highlighted branch of the mitotic tree using the photoconverted cell (white arrow in top panel) as a reference landmark. Anaphase stills showing lagging chromatin are indicated with red arrows. Chromosome cartoons along the mitotic tree indicate copy-number changes across cell generations. Lineages II and III cannot be accurately mapped onto the mitotic tree.