RB1 Depleted Pluripotent Stem Cell Retinal Organoids Recapitulate Cell State Transitions of Retinoblastoma Development

Retinoblastoma (Rb) is a childhood cancer of the developing retina in response to bi-allelic inactivation of RB1 or MYCN amplification, accounting for up to 17% of all tumours in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we developed two models via retinal organoid differentiation of a RB1 depleted human embryonic stem cell line (RB1-null hESCs) and a RB1 patient-specific induced pluripotent (iPSC) line harbouring a RB1 biallelic mutation (c.2082delC). Both models were characterised by RB1 depletion and a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+), which were defined as the cell of origin for Rb by single cell RNA-Seq analysis. The RB1 depleted retinal organoids displayed similar features to Rb tumours, 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. The patient-specific iPSC model displayed an enhanced reduction of amacrine, horizontal and retinal ganglion cells and an accelerated loss of cone photoreceptor markers during transition towards Rb, compared to the RB1-null hESC model. In both models, the Rb cells of origin, intermediate retinoma and/or Rb cells expressed retinal ganglion and horizontal cell markers in addition to cone markers, a novel finding, which could help to better characterise these tumours with possible therapeutic implications. Application of Melphalan, Topotecan and TW-37 led to a significant reduction in the fraction of proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.