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Updated: Jan 13, 2026
Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells
Published on: April 12, 2021
Dynamic molecular and cellular characteristics of VSX2-positive retinal progenitor cells in human retinal organoids
Dandan Zheng1, Yuan Wang1, Yuanyuan Guan1,2
1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, 510060, China.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
This study used retinal organoids to isolate human retinal progenitor cells (RPCs) and analyze their gene expression. A novel CD biomarker, TNFRSF1B, was identified for RPC sorting, advancing cell therapy for retinal diseases.
Area of Science:
- Stem Cell Biology
- Developmental Neuroscience
- Ophthalmology
Background:
- Understanding human retinal progenitor cells (RPCs) is crucial for developing cell therapies for retinal degenerative diseases.
- Current knowledge gaps in RPC molecular and cellular characteristics limit therapeutic applications.
- This study utilizes a novel reporter system for RPC isolation and characterization.
Purpose of the Study:
- To isolate and characterize human retinal progenitor cells (RPCs) using a VSX2-eGFP reporter system in retinal organoids (ROs).
- To investigate the transcriptome profile and identify potential cell surface markers for RPCs.
- To lay the groundwork for clinical translation of hiPSC-derived RPCs in retinal cell therapy.
Main Methods:
- Human induced pluripotent stem cells (hiPSCs) were differentiated into three-dimensional retinal organoids (ROs).
- VSX2-eGFP reporter fidelity was confirmed via immunostaining.
- Fluorescence-activated cell sorting (FACS) isolated VSX2-eGFP-positive (+) cells for bulk RNA sequencing (RNA-seq) and biomarker analysis.
Main Results:
- Successful differentiation of hiPSCs into ROs enriched with RPCs.
- VSX2-eGFP reporter accurately reflected endogenous VSX2 expression dynamics.
- Transcriptome analysis revealed RPC heterogeneity and identified TNFRSF1B as a novel, highly expressed CD biomarker in early-stage RPCs.
Conclusions:
- This research elucidates the molecular and cellular features of human RPCs, including novel CD biomarker expression profiles.
- The identification of TNFRSF1B offers a new avenue for RPC sorting and enrichment.
- Findings provide a foundation for advancing retinal development research and clinical applications of hiPSC-derived RPCs.
