Consensus recommendations for trabecular meshwork cell isolation, characterization and culture

Keller, Kate E.; Bhattacharya, Sanjoy K.; Borrás, T; Brunner, Thomas; Chansangpetch, Sunee; Clark, Abbott F.; Dismuke, W. Michael; Du, Yiqin; Elliott, Michael H.; Ethier, C. Ross; Faralli, Jennifer A.; Freddo, Thomas F.; Fuchshofer, Rudolf; Giovingo, Michael; Gong, Haiyan; González, Pedro; Huang, Alex S.; Johnstone, Murray; Kaufman, Paul L.; Kelley, Mary J.; Knepper, Paul A.; Kopczynski, Casey; Kuchtey, John; Kuchtey, Rachel W.; Kuehn, Markus H.; Lieberman, Raquel L.; Lin, Shan; Liton, Paloma B.; Liu, Yutao; Lütjen–Drecoll, Elke; Mao, Weiming; Masis-Solano, Marisse; McDonnell, Fiona; McDowell, Colleen M; Overby, Darryl R.; Pattabiraman, Padmanabhan P; Raghunathan, Vijay Krishna; Rao, P.V.; Rhee, Douglas J.; Chowdhury, Uttio Roy; Russell, Paul; Samples, John R.; Schwartz, Donald R.; Stubbs, Evan B.; Tamm, Ernst R.; Tan, James C.; Toris, Carol B.; Torrejon, Karen Yud; Vranka, Janice A.; Wirtz, Mary K.; Yorio, Thomas; Zhang, Jie; Zode, Gulab; Fautsch, Michael P.; Peters, Donna M.; Acott, Ted S.; Stamer, W. Daniel

doi:10.1016/j.exer.2018.03.001

Cited by 231 publications

(182 citation statements)

References 48 publications

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“…One important characteristic of TM cells is their responsiveness to DEX exposure, 64 including the formation of CLANs 51 . We thus exposed cells to 200 nM DEX for 11 days and visualized the actin component of the cytoskeleton.…”

Section: Resultsmentioning

confidence: 99%

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Adipose‐derived stem cells integrate into trabecular meshwork with glaucoma treatment potential

et al. 2020

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The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma. K E Y W O R D Sadipose stem cells; differentiation, homing, integration, trabecular meshwork | 7161 ZHOU et al.

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Section: Resultsmentioning

confidence: 99%

“…Another characteristic response of TM cells to DEX is upregulation of myocilin ( MYOC) 64,65 . MYOC mRNA levels were significantly elevated in primary TM cells and in ADSC‐TM cells induced by Co‐Culture and ECM + CM after DEX treatment compared to untreated cells (Figure 3C).…”

Section: Resultsmentioning

confidence: 99%

Adipose‐derived stem cells integrate into trabecular meshwork with glaucoma treatment potential

et al. 2020

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“…Within 48 hours post mortem, primary human TM cells were obtained from cadaver eyes without a history of eye disease (26, 27). Tissues were processed in accordance with the Declaration of Helsinki.…”

Section: Methodsmentioning

confidence: 99%

Genome-wide Expression Profiling and Pathway Analysis in Cyclic Stretched Human Trabecular Meshwork Cells

Drewry

Cai

Helwa

et al. 2018

Preprint

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Purpose Regulation of intraocular pressure is dependent upon homeostatic responses of trabecular meshwork (TM) cells to mechanical stretch. Despite the important roles of miRNAs in regulating TM function and aqueous outflow, it remains unclear how miRNA and their target genes interact in response to physiological cyclic mechanical stretch. We aimed to identify differentially expressed miRNAs and their potential targets in human TM cells in response to cyclic mechanical stress.Methods Monolayers of TM cells from non-glaucomatous donors (n=3-6) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/s, for 6 or 24-hours using computer-controlled Flexcell Unit. We profiled the expression of 800 miRNAs using NanoString Human miRNA assays and identified differentially expressed miRNAs using the Bioconductor Limma package. We identified differentially expressed genes using Operon Human Oligo Arrays with GeneSpring software. Pathway analysis with WebGestalt identified stretch-related pathways. We used Integrative miRNA Target Finder from Ingenuity Pathway Analysis to identify potential miRNA-mRNA regulations. ResultsWe identified 540 unique genes and 74 miRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated the significant enrichment of genes involved in Wnt-signaling, receptor protein serine/threonine kinase signaling, TGF-β pathway, and response to unfolded protein. We also identified several miRNA master regulators, including miR-19b-3p and miR-93-5p, which may act as switches to control several mechano-responsive genes. ConclusionsThis study suggests that cyclic mechanical stress of TM cells triggers alterations in the expression of both mRNAs and miRNAs implicated in glaucoma-associated pathways.

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“…Cell culture. TM cells were isolated from juxtacanalicular and corneoscleral regions of the human donors' eyes, as described (15,17), in accordance with consensus characterization recommendations (31).…”

Section: See Si Experimental Procedures For More Detailsmentioning

confidence: 99%

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Yarishkin

Phuong

Baumann

et al. 2020

Preprint

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AbstractMechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]i elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. 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 a novel potential therapeutic target for treating glaucoma.Significance StatementTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor 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. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.

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Consensus recommendations for trabecular meshwork cell isolation, characterization and culture

Cited by 231 publications

References 48 publications

Adipose‐derived stem cells integrate into trabecular meshwork with glaucoma treatment potential

Adipose‐derived stem cells integrate into trabecular meshwork with glaucoma treatment potential

Genome-wide Expression Profiling and Pathway Analysis in Cyclic Stretched Human Trabecular Meshwork Cells

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

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