Publications

Glaucoma is an irreversible blinding disease that affects millions of individuals worldwide. Elevated intraocular pressure (IOP), regulated by the trabecular meshwork (TM) in the anterior eye, is the only modifiable risk factor. Human TM cells can be cultured from donor eyes, providing a precious resource for studying factors that induce or prevent glaucoma. The goal of this study was to produce datasets that define the molecular profile of human non-glaucomatous TM cells. Using 18 human TM cell strains cultured from non-glaucomatous individuals deposited from seven laboratories in the USA and UK, this study used transcriptomic, proteomic, lipidomic, and metabolomic analyses to characterize the molecular content of TM cells. The data herein provides the most comprehensive multiomic analyses of human TM cells to date and will be a useful resource for researchers and clinicians in the TM and glaucoma fields.

The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemm's canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type-specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-β signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1 , suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.

Glaucoma is a leading cause of irreversible blindness, with elevated intraocular pressure (IOP) as the most important risk factor. The trabecular meshwork (TM) and Schlemm's canal are the main components of the proximal aqueous humor outflow pathway. Their dysfunction is a major contributor to IOP elevation. This study aims to identify and validate key biomarkers for TM and Schlemm's canal endothelial (SCE) cells. A Microarray was performed on characterized human TM and SCE cells to analyze their transcriptome profiling. Differentially expressed genes (DEGs) were identified and cross-referenced with published single-cell RNA sequencing (scRNA-Seq) datasets to ensure cell-specific relevance. Further validation was performed using qPCR and re-confirmed on the scRNA-seq datasets. One-way ANOVA was used for statistical analysis, and p < 0.05 was considered significant. The Microarray revealed 341 DEGs, with TM cells enriched in metabolic and signaling pathways and SCE cells enriched in adhesion, immune, and morphogenesis-related processes. Cross-referencing with scRNA-Seq data refined the list of candidate biomarkers, and qPCR confirmed the significant gene expression differences between TM and SCE cells. CTTNBP2 and MGARP were identified as TM cell markers. JAM2, PODXL, and IFI27 are new SCE cell biomarkers. The validated biomarkers offer insights into glaucoma pathophysiology and lay the groundwork for targeted therapies.

Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.

Elevated intraocular pressure (IOP) is the most important risk factor for primary open-angle glaucoma (POAG) and currently is the only effective treatment target for glaucoma to prevent vision loss. In POAG patients, the trabecular meshwork (TM) cellularity is reduced which might be the main pathologic reason for the conventional outflow pathway dysfunction leading to elevated IOP. Stem cell-based therapy has been shown promising to reduce IOP and preserve retinal ganglion cells and their function in animal models. In this chapter, we describe the method details on TM stem cell cultivation and identification; induction for differentiation into different cell types, including differentiation to TM cell responsiveness to dexamethasone treatment with phagocytic function; and transplantation into mouse anterior chamber for therapeutic purposes.

BACKGROUND: Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.

METHODS: A total of 24 donor corneal rims with informed consent were used-18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC's conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.

RESULTS: By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters-stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)-were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.

CONCLUSIONS: We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC-in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Intraocular pressure (IOP) elevation is the primary risk factor and currently the main treatable factor for progression of glaucomatous optic neuropathy. In addition to direct clinical and living animal in vivo studies, ex vivo perfusion of anterior segments and whole eyes is a key technique for studying conventional outflow function as it is responsible for IOP regulation. We present well-tested experimental details, protocols, considerations, advantages, and limitations of several ex vivo model systems for studying IOP regulation. These include: (1) perfused whole globes, (2) stationary anterior segment organ culture, (3) perfused human anterior segment organ culture, (4) perfused animal anterior segment organ culture, (5) perfused human corneal rims, and (6) perfused human anterior segment wedges. These methods, with due consideration paid to their strengths and limitations, comprise a set of very strong tools for extending our understanding of IOP regulation.