Extended Data Fig. 5: Confocal microscopy of aggregates at CP 0 and CP I to analyze cell number per aggregate.

a–c, Light microscope pictures of a representative cell sample at CP 0 (scale bar, 500 µm) (a) and confocal images of the central Z-stack of aggregates of the same batch (scale bar, 50 µm) (b and c). d–f, Light microscope pictures of a representative cell sample at CP I (scale bar, 500 µm) (d) and confocal images in a differentiated state as CM aggregates at CP I (scale bar, 50 µm) (e and f). Nuclei were stained with Sytox red after fixation; afterwards, aggregates were dehydrated with ethanol and cleared with methyl salicylate/benzyl benzoate (MSBB). Notable are pronounced cavities at CP 0 as well as at CP I. g, Aggregates treated for confocal imaging do not differ in diameter compared to untreated, equivalent samples measured according to QC3 at CP 0 or CP I. h, Nuclei identified at CP 0 and CP I through automatic counting of confocal z-stacks imaging whole aggregates. This nuclei count should closely resemble the total cell count per aggregate. i and j, Identified nuclei per aggregate in comparison to the individual aggregate diameter for aggregates at CP 0 (i) and CP I (j). Linear regression was added to depict the goodness of fit (CP 0 R²= 0.73; CP I R²= 0.55). For examples, see supplementary file z-stacks for CP 0 and CP I (open with the hyperstack function of FIJI/ImageJ). PFA, paraformaldehyde. The hiPSC line Phoenix was used in all experiments.