Retinoblastoma (Rb) is a childhood cancer of the developing retina, accounting for up to 17% of all tumors in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we established 2 disease models via retinal organoid differentiation of a pRB (retinoblastoma protein)-depleted human embryonic stem cell line (RB1-null hESCs) and a pRB patient-specific induced pluripotent (iPSC) line harboring a RB1 biallelic mutation (c.2082delC). Both models were characterized by pRB depletion and accumulation of retinal progenitor cells at the expense of amacrine, horizontal and retinal ganglion cells, which suggests an important role for pRB in differentiation of these cell lineages. Importantly, a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+) was observed in both pRB-depleted organoid models, which were defined as Rb-like clusters by single-cell RNA-Seq analysis. The pRB-depleted retinal organoids displayed similar features to Rb tumors, including mitochondrial cristae aberrations and rosette-like structures, and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. In both models, the Rb cones expressed retinal ganglion and horizontal cell markers, a novel finding, which could help to better characterize these tumors with possible therapeutic implications. Application of Melphalan, Topotecan, and TW-37 led to a significant reduction in the fraction of Rb proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.

Retinoblastoma (Rb) is a rare malignant disorder affecting the developing retina of children under the age of five. Chemotherapeutic agents used for treating Rb have been associated with defects of the retinal pigment epithelium (RPE), such as hyperplasia, gliosis, mottling, and visual acuity. Herein, we have developed two pluripotent stem cell (PSC)-RPE models to assess the cytotoxicity of known Rb chemotherapeutics such as Melphalan, Topotecan, and TW-37. Our findings demonstrate that these drugs alter the RPE cell size, decrease the monolayer barrier's trans-epithelial resistance and affect the cells' phagocytic activity. Our transcriptional analyses demonstrate an altered expression of genes involved in melanin and retinol processing, tight junction and apical-basal polarity pathways in both models. None of the drug treatments applied within the clinical range caused significant cytotoxic effects, changes to the apical-basal polarity, tight junction network or cell cycle. Together, our results demonstrate that although the most commonly used Rb chemotherapeutic drugs do not cause cytotoxicity in RPE, their application in vitro leads to compromised phagocytosis and strength of the barrier function, in addition to changes in gene expression that could alter the visual cycle.

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