Analyzing mRNA isoforms in human stem cell-derived retinal neurons using long-read transcriptomics

Abstract

Purpose : The purpose of this study was to perform long-read sequencing (LRS) on differentiating 2D and 3D retinal stem cell cultures to analyze expression of mRNA isoforms in different retinal cell populations.

Methods : Human stem cells were differentiated into 2D RGC cultures and 3D retinal organoids (ROs) using established protocols. Total RNA was extracted from purified RGCs (n= ~400,000-1,000,000 cells) and whole ROs (n=4-6 pooled) using TRIzol reagent. Full-length cDNA libraries were prepared using Oxford Nanopore Technologies (ONT) direct cDNA sequencing protocol and Ligation Sequencing Kit V14. Each ONT library was loaded onto a single R10.4.1 PromethION flow cell for sequencing using standard run parameters. Real-time basecalling was performed using Guppy, then fastq files were merged based on sample. NanoFilt was used to filter out reads (Q score < 7 and read length < 50bp). Reads were mapped to the hg38 cDNA assembly using Salmon, and isoform quantification was performed using Salmon.

Results : Each ONT library generated an average of over 31 million full-length reads with a mean N50 length of approximately 2.5 kb. We could detect over 50,500 known transcripts on average (> 51,500 and 49,200 transcripts in RGCs and ROs, respectively). To validate the data, we investigated the expression of neuron and photoreceptor-specific isoforms. We found expected expression patterns of alternatively spliced transcripts like neurofibromin (NF1), in which a single cassette exon (30alt31) is preferentially skipped in neurons. This neuron-specific isoform was significantly enriched in purified RGCs compared to whole ROs. Moreover, the long transcript for basigin (BSG), which is highly specific to photoreceptors and encodes for the receptor to rod-derived cone viability factor (nucleoredoxin-like 1, NXNL1), was almost exclusively detected in the older RO samples containing terminally differentiated photoreceptors and nearly absent in purified RGC cultures.

Conclusions : Our LRS data can be used to quantify isoform expression in different retinal cell populations in differentiated 2D cultures and 3D ROs. We are currently comparing isoform expression across RO cell types/time points, as well as 2D stem cell-derived RGC and RPE cultures. Ongoing experiments are characterizing alternative splicing patterns in both healthy and disease states through differentiation of mutant stem cell lines.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.