Retinoblastoma protein controls growth, survival and neuronal migration in human cerebral organoids

The tumor suppressor retinoblastoma protein (RB) regulates S-phase cell cycle entry via E2F transcription factors. Knockout (KO) mice have shown that RB plays roles in cell migration, differentiation and apoptosis, in developing and adult brain. In addition, the RB family is required for self-renewal and survival of human embryonic stem cells (hESCs). Since little is known about the role of RB in human brain development, we investigated its function in cerebral organoids differentiated from gene-edited hESCs lacking RB. We show that RB is abundantly expressed in neural stem and progenitor cells in organoids at 15 and 28 days of culture. RB loss promoted S-phase entry in DCX⁺ cells and increased apoptosis in Sox2⁺ neural stem and progenitor cells, and in DCX⁺ and Tuj1⁺ neurons. Associated with these cell cycle and pro-apoptotic effects, we observed increased CCNA2 and BAX gene expression, respectively. Moreover, we observed aberrant Tuj1⁺ neuronal migration in RB-KO organoids and upregulation of the gene encoding VLDLR, a receptor important in reelin signaling. Corroborating the results in RB-KO organoids in vitro, we observed ectopically localized Tuj1⁺ cells in RB-KO teratomas grown in vivo Taken together, these results identify crucial functions for RB in the cerebral organoid model of human brain development.