GSE196355
Generation of an RCVRN-eGFP reporter hiPSC line by CRISPR/Cas9 to monitor photoreceptor cell development and facilitate the cell enrichment for transplantation
Stem cell based-cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors at specific developmental stage suitable for transplantation is highly challenging so far. This study aimed to generate a specific photoreceptor reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker Recoverin  (RCVRN). After confirmation of the successful targeting and gene stability, three-dimensional retinal organoids were induced from this report line. The RCVRN-eGFP reporter faithfully replicated endogenous RCVRN  expression and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP positive photoreceptors could be enriched by fluorescence activated cell sorting and the further RNA sequencing analysis of these purified cells revealed transcriptome signatures and gene networks of photoreceptors. Moreover, potential cluster of differentiation biomarkers  were extracted here for enrichment of photoreceptors for clinical applications. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of RCVRN positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors at specific developmental stage, thus facilitating the photoreceptor cell therapy for the advanced outer retinal disorders. Overall design: At Day 150, ROs derived from the RCVRN-eGFP reporter hiPSCs (5 ROs per experiment, 2 independent experiments), FACS-sorted eGFP+ and eGFP- cells pooled from ROs derived from the reporter hiPSCs (30 ROs per experiment, 2 independent experiments) were collected in Trizol reagent. Total RNA was extracted using Trizol reagent kit and the quality of RNA was assessed by an Agilent 2100 Bioanalyzer and checked using RNase free agarose gel electrophoresis. Oligo (dT) beads were used to isolate the poly mRNA from the total RNA. Then the enriched mRNA was subjected to RNA-seq library construction from each two independent samples of three groups: unsorted (ROs), eGFP+ (FASC-sorted RCVRN-eGFP positive cells) and eGFP- (FASC-sorted RCVRN-eGFP negative cells).Paire-end sequencing was performed using Illumina Novaseq 6000. Reads were further filtered by fastp (Chen et al., 2018) (version 0.18.0). Clean reads were mapped to the reference genome using HISAT2. 2.4 (Kim et al., 2015). For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variations, using RSEM (Li and Dewey, 2011) software.