Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids

Recent in vitro studies using RB1^+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1^+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1^+/- retinal organoids, we performed a high throughput RNA sequencing analysis.

iPSCs were generated from RB1^+/+ and RB1^+/- OAMSCs derived from retinoblastoma patients. RB1^+/+ and RB1^+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1^+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1^+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1^+/- retinal organoids of day 120 compared to that of the RB1^+/+. The results thus revealed enhanced ATP production in the RB1^+/- retinal organoids.

The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.