Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

The Communication between Ocular Surface and Nasal Epithelia in 3D Cell Culture Technology for Translational Research: A Narrative Review.

There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia.

Human conjunctival goblet cells express the membrane associated mucin MUC16: Localization to mucin granules.

MUC16 is an extraordinarily large 22,152 amino acid membrane spanning mucin that has been shown to be present in the glycocalyx of the apical cells of the human cornea and conjunctiva where it interfaces with the tear film. The ectodomain of the molecule has been demonstrated in tears, where it has been presumed to be from surface epithelial cells. Data presented here from multiple assays, including immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR of RNA isolated from goblet cells isolated by laser capture microdissection, demonstrate that the membrane tethered mucin is also expressed by conjunctival goblet cells both in humans and in mice. The mucin is present in mucin granules and appears to be localized to the mucin granule membrane. Correlation analyses of the amounts of the goblet cell secreted mucin MUC5AC and the amounts of MUC16 and of MUC1 another membrane tethered mucin ectodomain found in human tear samples demonstrated that MUC5AC amounts correlated to the amounts of MUC16 but not to MUC1. These data suggest that goblet cells are a second source of the mucin in tears. The function of the membrane tethered mucin in the mucin granule remains to be determined.

Incidence and Mitigation of Corneal Pseudomicrocysts Induced by Antibody-Drug Conjugates (ADCs).

Purpose of Review: This study is to highlight the incidence of corneal pseudomicrocysts in FDA-approved antibody-drug conjugates (ADCs), and success of preventive therapies for pseudomicrocysts and related ocular surface adverse events (AEs). Recent Findings: ADCs are an emerging class of selective cancer therapies that consist of a potent cytotoxin connected to a monoclonal antibody (mAb) that targets antigens expressed on malignant cells. Currently, there are 11 FDA-approved ADCs with over 164 in clinical trials. Various AEs have been attributed to ADCs, including ocular surface AEs (keratitis/keratopathy, dry eye, conjunctivitis, blurred vision, corneal pseudomicrocysts). While the severity and prevalence of ADC-induced ocular surface AEs are well reported, the reporting of corneal pseudomicrocysts is limited, complicating the development of therapies to prevent or treat ADC-related ocular surface toxicity. Summary: Three of 11 FDA-approved ADCs have been implicated with corneal pseudomicrocysts, with incidence ranging from 41 to 100% of patients. Of the six ADCs that reported ocular surface AEs, only three had ocular substudies to investigate the benefit of preventive therapies including topical steroids, vasoconstrictors, and preservative-free lubricants. Current preventive therapies demonstrate limited efficacy at mitigating pseudomicrocysts and other ocular surface AEs.

Ocular Surface Ion Transport and Dry Eye Disease.

Purpose of Review: To review the role of ocular surface epithelial (corneal and conjunctival) ion transporters in the pathogenesis and treatment of dry eye disease (DED). Recent Findings: Currently, anti-inflammatory agents are the mainstay of DED treatment, though there are several agents in development that target ion transport proteins on the ocular surface, acting by pro-secretory or anti-absorptive mechanisms to increase the tear fluid Film volume. Activation or inhibition of selected ion transporters can alter tear fluid osmolality, driving water transport onto the ocular surface via osmosis. Several ion transporters have been proposed as potential therapeutic targets for DED, including the cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channels (CaCCs), and the epithelial sodium channel (ENaC). Summary: Ocular surface epithelial cell ion transporters are promising targets for pro-secretory and anti-absorptive therapies of DED.

The Effect of Androgens on Proinflammatory Cytokine Secretion from Human Ocular Surface Epithelial Cells.

Purpose: The purpose of this study is to explore the effects of dihydrotestosterone (DHT) on lipopolysaccharide (LPS)-induced proinflammatory cytokine release in human ocular surface epithelial cells exposed to LPS and LPS-binding protein (LBP).Methods: Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were cultured in keratinocyte-free medium. After confluency, they were exposed to a stratification medium Dulbecco's modified Eagle medium (DMEM)/F12 in the presence of fetal bovine serum and were exposed to vehicle, LPS + LBP, or DHT. Culture media were processed for multiplex-bead analysis of specific proinflammatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-8, IL-6, IL-10, IL-1ß, vascular endothelial growth factor (VEGF)-A. Cytokine concentrations were compared by analysis of variance with Tukey post hoc testing. p < 0.05 was considered statistically significant.Results: The results are LPS + LBP-induced the secretion of IFN-γ, IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-2, IL-8, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-8, IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells. When these LPS + LBP-stimulated cells were exposed to DHT for 2 days, it was found that DHT suppressed the secretion of IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells.Conclusion: LPS + LBP is shown to induce the secretion of certain proinflammatory cytokines from ocular surface and adnexal epithelial cells. DHT showed anti-inflammatory activity by suppressing some of those cytokines in these cell lines.

Increased Cell Survival of Human Primary Conjunctival Stem Cells in Dimethyl Sulfoxide-Based Cryopreservation Media.

Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63α- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.