Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory.

Retinoblastoma (Rb) is a type of malignant tumor due to abnormal retinogenesis with biallelic mutations of the gene. Its pathogenesis has been proposed as a "two-mutation hypothesis" by Knudson since 1971; however, there remain some debates on disease onset sufficiency of the biallelic mutations. To obtain straightforward evidence for this hypothesis, we investigated whether two-hit mutations of the gene drive tumorigenesis in patient-induced pluripotent stem cell (hiPSC)-derived human retinal organoids (hROs) and whether single allelic mutation hiPSC-derived hROs exhibit molecular and cellular defects. We generated hiPSCs with a heterozygous germline mutation ( ) from a Rb patient. A second-allele gene mutation was knocked in to produce compound heterozygous mutations ( ) in the hiPSCs. These two hiPSC lines were independently developed into hROs through a stepwise differentiation. The hiPSC- derived organoids demonstrated tumorigenesis in dishes, consistent with Rb profiles in spatiotemporal transcriptomes, in which developmentally photoreceptor fate-determining markers, CRX and OTX2, were highly expressed in hiPSC- derived hROs. Additionally, ARR3 maturing cone precursors were co-labeled with proliferative markers Ki67 or PCNA, in agreement with the consensus that human Rb is originated from maturing cone precursors. Finally, we demonstrated that retinal cells of hROs with monoallelic mutation were abnormal in molecular aspects due to its haploinsufficiency. In conclusion, this study provides straightforward supporting evidence in a way of reverse genetics for "two-hit hypothesis" in the Rb tumorigenesis and opens new avenues for development of early intervention and treatment of Rb.