# Dynamic molecular and cellular characteristics of VSX2-positive retinal progenitor cells in human retinal organoids

## TL;DR

This study explores the characteristics of VSX2-positive retinal progenitor cells in human retinal organoids to improve cell therapy for retinal diseases.

## Contribution

The study identifies a new CD biomarker, TNFRSF1B, for sorting early-stage retinal progenitor cells.

## Key findings

- VSX2-eGFP+ cells show RPC characteristics at early stages and bipolar cell traits at late stages.
- RNA-seq analysis reveals transcriptional heterogeneity in VSX2-eGFP+ cells across developmental stages.
- TNFRSF1B is highly expressed in early RPCs and may serve as a novel CD biomarker.

## Abstract

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-enhanced green fluorescent protein (eGFP) 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 recapitulated the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP-negative (-) 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 in VSX2-eGFP+ cells 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.

The online version contains supplementary material available at 10.1186/s13287-025-04700-z.