pRB-depleted pluripotent stem cell retinal organoids mimic retinoblastoma development

Abstract

Purpose : Retinoblastoma (Rb) is a malignant tumour of developing retina with a typical presentation occurring during the first five years of life. Caused in more than 97% of cases by biallelic inactivation of the RB1 tumour suppressor gene, the frequency of Rb is estimated at about 1 in 15,000 live births. Despite the progress in treatments, and high survival rate in developed countries Rb survivors are left with impaired vision having a negative effect on correct motion processing, depth perception and judging distances. Patients can suffer hearing loss, cataracts, cosmetic deformities as well as a neurocognitive deficit. The urgent need to introduce novel treatments warranted our study to develop and characterise a 3D in vitro model of Rb for drug discovery, repurposing and testing.

Methods : We generated a patient-specific iPSC model (c.2082delC) encompassing the heterozygous (RB1^+/-), homozygous (RB1^-/-) and fully corrected isogenic control. We compared the retinal organoids established from the patient-specific iPSC model to those derived from the RB1 knock-out hESC line applying single cell RNA-Seq analysis combined with IHC, TEM, western-blot and colony-forming assay.

Results : Both pRB-depleted models presented significant enrichment in proliferating cone precursors (RXRγ⁺Ki67⁺), defined as Rb clusters by single cell RNA-Seq analysis and displayed tumorigenic features, including mitochondrial cristae aberrations and rosette-like structures. Cells isolated from pRB-deficient organoids were able to undergo growth in an anchorage-independent manner, indicative of cell transformation in vitro. Importantly, the pRB-depleted models, showed an accumulation of retinal progenitor cells at the expense of amacrine, horizontal and retinal ganglion cells, indicating an important role for retinoblastoma protein in the differentiation of these cell lineages. Remarkedly, 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.

Conclusions : Our data provide robust evidence that pRB-depleted organoids mimic the development and malignant transformation that occurs in vivo, providing a powerful tool for novel drug screening and further retinoblastoma analysis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.