GSE185475
VSX2-eGFP reporter in hiPSCs dynamically monitors human retinal development in retinal organoids and reveals cell fate transition and gene networks
The lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-eGFP fluorescence reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs, investigate their features, and facilitate their applications. hiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages, followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs. hiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulates the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP- cells, VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover, the dynamic expression of 394 known CD biomarkers at distinct developmental stages was analyzed herein for the first time. One CD biomarker, TNFRSF1B, which has never been reported to be expressed in RPCs, was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs. This study provides valuable insights into the molecular and cellular characteristics of human RPCs, especially their expression profiles of CD biomarkers, laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs. Overall design: Retinal organoids derived from VSX2 repoter (VSX2-eGFP) hiPSCs and parental hiPSCs were collected at D60. Flow-sorted eGFP positive and eGFP negative cells at D28 were separately collected from VSX2 reprter hiPSC-derived cultures. Flow-sorted eGFP positive and eGFP negative cells at D55, D118, and D170 were separately collected from the neuralretinas of retinal organoids derived from VSX2 reporter hiPSCs. Total RNA from two independent experiments of purified cells and three independent experiments of retinal organoids were extracted using Trizol reagent kit (Invitrogen). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) and checked using RNase free agarose gel electrophoresis. The qualified total RNA was subjected to RNA-seq library construction. Paired-end sequencing was performed using Illumina Novaseq6000. 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.