Self-organizing principles are insufficient for cortical neural stem cell lineage progression

In the developing cerebral cortex, radial glial progenitors (RGPs) produce all excitatory neurons and glia. Using Mosaic Analysis with Double Markers (MADM), we established a quantitative framework of cortical RGP lineage progression at single-cell resolution in vivo. The extent to which these quantitative aspects of RGP lineage progression are conserved in vitro is unclear. Here we established MADM in embryonic stem cells in order to contrast RGP lineage progression in vivo and in cortical organoids. We found that organoid RGP division behavior was less temporally stereotyped, with both persistence of proliferative and early onset of neurogenic divisions. Cell types were mostly generated in a defined order but not in correct proportions, and one third of RGP lineages were restricted to either deep- or superficial-layer neuronal cell types. Altogether, our results demonstrate that self-organizing cortical organoids lack sufficient tissue and/or developmental context for faithful RGP lineage progression at the quantitative level.