Temporal Uncoupling of Radial Glial Progenitor Cell Lineage Progression in Self-organizing Organoid System – MADM-CloneSeq

Radial glial progenitors (RGPs) produce all excitatory neurons in the developing cerebral cortex. Mosaic Analysis with Double Markers (MADM)-based lineage tracing in vivo revealed a quantitative framework of RGP lineage progression. Here we established MADM technology in embryonic stem cells to probe RGP lineage progression in self-organizing cortical organoid system. We found that RGPs exhibit high level of equipotency in organoid rather than temporally stereotyped lineage progression as observed in vivo. RGPs in organoids showed increased lineage restriction, diminishing cell-type diversity in clones of cortical projection neurons, despite uniform RGP scRNAseq transcriptional signatures and unitary lineage trajectory. Hence, critical non-cell-autonomous cues absent in self-organizing systems and/or genuine stem cell niche are essential for faithful RGP lineage progression and the generation of cortical cell-type diversity.