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Updated: May 11, 2026
Characterization of a Novel Human Organotypic Retinal Culture Technique
Published on: June 9, 2021
Liquid Biopsy for Proliferative Diabetic Retinopathy: Single-Cell Transcriptomics of Human Vitreous Reveals
Rachana Haliyur1, David H Parkinson2, Feiyang Ma3
1Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan.
Purpose:
Current therapies for proliferative diabetic retinopathy (PDR) do not specifically target VEGF-independent, cell-type-specific processes that lead to vision loss, such as inflammatory pathways. This study aimed to identify targetable cell types and corresponding signaling pathways by elucidating the single-cell landscape of the vitreous of patients with PDR.
Design:
Case series.
Subjects:
Vitreous and peripheral blood obtained from 5 adult patients (6 eyes) undergoing pars plana vitrectomy for vision-threatening PDR.
Methods:
Single-cell RNA sequencing (scRNA-seq) was performed on vitreous cells obtained from diluted cassette washings during vitrectomy from 6 eyes and peripheral blood mononuclear cells (PBMCs, n = 5). Droplet-based scRNA-seq was performed using the Chromium 10x platform to obtain single-cell transcriptomes. Differences in tissue compartments were analyzed with gene ontology enrichment of differentially expressed genes and an unbiased ligand-receptor interaction analysis.
Main Outcome Measures:
Single-cell transcriptomic profiles of vitreous and peripheral blood.
Results:
Transcriptomes from 13 675 surgically harvested vitreous cells and 22 636 PBMCs were included. Clustering revealed 4 cell states consistently across all eyes with representative transcripts for T cells (CD2, CD3D, CD3E, and GZMA), B cells (CD79A, IGHM, MS4A1 (CD20), and HLA-DRA), myeloid cells (LYZ, CST3, AIF1, and IFI30), and neutrophils (BASP1, CXCR2, S100A8, and S100A9). Most vitreous cells were T cells (91.6%), unlike the peripheral blood (46.2%), whereas neutrophils in the vitreous were essentially absent. The full repertoire of adaptive T cells including CD4+, CD8+ and T regulatory cells (Treg) and innate immune system effectors (i.e., natural killer T cells) was present in the vitreous. Pathway analysis also demonstrated activation of CD4+ and CD8+ memory T cells and ligand-receptor interactions unique to the vitreous.
Conclusions:
In the first single-cell transcriptomic characterization of human vitreous in a disease state, we show PDR vitreous is primarily composed of T cells, a critical component of adaptive immunity, with activity and proportions distinct from T cells within the peripheral blood, and neutrophils are essentially absent. These results demonstrate the feasibility of liquid vitreous biopsies via collection of otherwise discarded, diluted cassette washings during vitrectomy to gain mechanistic and therapeutic insights into human vitreoretinal disease.
Financial Disclosures:
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
Insights
This study reveals that T cells dominate the vitreous of proliferative diabetic retinopathy (PDR) patients, unlike neutrophils. These findings highlight unique immune cell activity in PDR, offering new therapeutic targets for vision loss.
Area of Science:
- Ophthalmology
- Immunology
- Genomics
Background:
- Current proliferative diabetic retinopathy (PDR) treatments lack specificity for VEGF-independent inflammatory pathways driving vision loss.
- Understanding the cellular and molecular mechanisms of PDR is crucial for developing targeted therapies.
Purpose of the Study:
- To identify targetable cell types and signaling pathways in PDR by analyzing the single-cell transcriptomic landscape of the vitreous.
- To characterize the immune cell composition and activation states within the vitreous of PDR patients.
Main Methods:
- Single-cell RNA sequencing (scRNA-seq) was performed on vitreous cells and peripheral blood mononuclear cells (PBMCs) from PDR patients.
- Transcriptomic data were analyzed to identify cell types, differential gene expression, and ligand-receptor interactions.
Main Results:
- Vitreous samples were predominantly composed of T cells (91.6%), with distinct T cell populations (CD4+, CD8+, Tregs) and activated memory T cells observed.
- Neutrophils were virtually absent in the vitreous, contrasting with their presence in peripheral blood.
- Unique ligand-receptor interactions within the vitreous microenvironment were identified.
Conclusions:
- PDR vitreous is characterized by a unique T cell-dominant immune environment, distinct from peripheral blood.
- The study demonstrates the feasibility of using liquid vitreous biopsies for mechanistic and therapeutic insights into PDR.
- These findings pave the way for novel, targeted therapies for PDR-associated vision loss.
