Pluripotent stem cell-derived retinal organoids and retinal pigment epithelium as a model system for screening chemotherapeutic agents in retinoblastoma

Abstract

Retinoblastoma (Rb) is a rare childhood malignancy of the developing retina affecting 1:15,000 infants worldwide. The biallelic inactivation of the RB1 gene and the retinoblastoma protein (pRB) loss accounts for up to 98% of Rb cases. Despite the progress in Rb treatments, standard chemotherapeutic agents have been associated with adverse effects on the retina and defects of the retinal pigment epithelium (RPE). Herein, we developed two disease models through retinal organoid differentiation of a pRB-depleted human embryonic stem cell line (H9 RB1-null hESCs) and an Rb patient-specific induced pluripotent (hiPSC) line displaying an RB1 biallelic mutation (c.2082delC). Both models were characterised by pRB depletion and a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+). The pRB-depleted retinal organoids displayed similar features to Rb tumours, including undergoing cell growth in an anchorage-independent manner indicative of cell transformation in vitro. Applying Rb chemotherapeutics such as melphalan, topotecan and TW-37 significantly reduced the fraction of Rb proliferating cone precursors. Additionally, we established two hESC-RPE and hiPSC-RPE models to assess the cytotoxicity of the same three drugs. Our results indicated that these agents applied in the clinical range decreased the monolayer barrier's trans epithelial resistance and affected the cells' phagocytic activity. Moreover, transcriptional analyses demonstrated an altered gene expression in melanin and retinol processing, tight junction and apical-basal polarity pathways. None of the drug treatments within the clinical range caused significant cytotoxicity or changes to the apical-basal polarity, tight junction network or cell cycle. Although standard Rb chemotherapeutics did not induce cytotoxicity in RPE, their application in vitro led to compromised phagocytosis and strength of the barrier. This thesis highlights the applications of retinal organoids and RPE in disease modelling, confirming the suitability of these in vitro models for testing novel therapeutics for Rb.