Like corneal endothelial cells, human trabecular meshwork cells are believed to be of neural crest origin, but demonstrate physiological properties and an antithrombogenic surface similar to vascular endothelial cells. One current method for isolating trabecular meshwork cells utilizes the motile nature of these cells to migrate away from a trabecular meshwork explant in culture to more distal regions of the culture dish. This 'outgrowth' technique is limited in practice by the relatively small number of cells that migrate per explant per unit time, thus hindering the ability to gather sufficient numbers of cells for comprehensive experimentation. For this reason, we have modified an extracellular matrix digestion technique in current use for the isolation of microvascular endothelial cells to isolate human trabecular meshwork cells. This procedure is both efficient and rapid for isolating large numbers of trabecular meshwork cells and results in the availability of trabecular meshwork cells in sufficient quantities for subsequent experimentation.

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion

The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.

https://www.pnas.org/content/pnas/117/23/12856.full.pdf

Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

/pdf/2d-and-3d-cultures-of-human-trabecular-meshwork-cells-a-1eb6k7avof.pdf

2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study.

The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in t...

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2019.1662584

The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress.

Key points Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. Abstract Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Shan C. Lin

Papers

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture