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The narrow intersection between the cornea and conjunctiva, otherwise known as the limbus, is purported to harbor stem cells (SCs) that replenish the ocular surface epithelium throughout life. Damage to this site or depletion of its SCs can have dire consequences for eye health and vision. To date, various SC and keratin proteins have been used to identify the limbus, however, none could definitively mark its boundaries. Herein, we use the mouse as a model system to investigate whether structural and phenotypic features can be used to define the limbus and its boundaries with adjacent tissues. We demonstrate that differentially aligned blood and lymphatic vessels, intraepithelial nerves, and basal epithelial cellular and nuclei dimensions can be used as structural landmarks of the limbus. Identification of these features enabled approximation of the limbal expanse, which varied across distinct ocular surface quadrants, with the superior nasal and inferior temporal limbus being the widest and narrowest, respectively. Moreover, label-retaining SCs were unevenly distributed across the ocular circumference, with increased numbers in the superior temporal and inferior temporal moieties. These findings will heighten our current understanding of the SC niche, be beneficial for accurately predicting SC distribution to improve their isolation and devising efficacious cell therapies, and importantly, aid the ongoing search for novel SC markers., (© The Author(s) 2024. Published by Oxford University Press.)

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation., (© 2014 AlphaMed Press.)

The cornea contains a reservoir of self-regenerating epithelial cells that are essential for maintaining its transparency and good vision. The study of stem cells in this functionally important organ has grown over the past four decades, partly due to the ease with which this tissue is visualized, its accessibility with minimally invasive instruments, and the fact that its stem cells are segregated within a transitional zone between two functionally diverse epithelia. While human, animal, and ex vivo models have been instrumental in progressing the corneal stem cell field, there is still much to be discovered about this exquisitely sensitive window for sight. This review will provide an overview of the human cornea, where its stem cells reside and how components of the microenvironment including extracellular matrix proteins and their integrin receptors are thought to govern corneal stem cell homeostasis., (Copyright © 2011 AlphaMed Press.)

Introduction: Blindness affects 50 million individuals worldwide; a significant proportion of them require a cell or tissue-based repair or replacement strategy to mend their damaged or diseased cornea. This review will focus on the epithelial stem cell (ESC) population of the cornea, where they reside, how they are identified and what alternative cells can be used as functional substitutes., Source of Data: Data for this review were collated after performing literature searches by inserting key words (cornea, limbal, stem cells (SCs), epithelium, stroma, and endothelium) into the search engine PubMed., Areas of Agreement: The prevailing notion is that corneal ESCs reside in an exclusive niche and their activation is dictated by niche-specific signals., Areas of Controversy: Recent studies refute this hypothesis, as the central cornea of many animal species also possesses similar proliferative and clonogenic activity. The other area of controversy is in relation to the use of autologous and/or allogeneic cell therapies which are mostly contaminated with xenogeneic factors, potentially exposing the recipient to potentially harmful foreign infection., Growing Points: Due to the shortage of donor corneal biomaterial, alternative cellular sources are being sought, discovered and trialed., Areas Timely for Developing Research: With the exception of conjunctival and oral mucosal epithelium, no other cell type has been successfully used to treat patients with severe corneal epithelial defects. Embryonic and foetal SCs may have the greatest potential of all; however there are moral, legal, religious and scientific hurdles to overcome before they become routinely used in the clinic.

The adult cornea harbors stem cells (SCs) in its periphery, in a niche known as the limbus. Over the past 2 decades there has been substantial research into these adult corneal SCs, their limbal niche, and their therapeutic applications. However, few studies have investigated how this niche and its SCs develop in humans. To better characterize this development, human fetal corneas from 8.5- to 22-weeks'-gestation (n = 173), neonatal (n = 2), and adult (n = 10) specimens were obtained. Histological and immunohistochemical assessments were conducted to determine embryological changes and expression of developmental and SC-related genes. Fresh fetal corneas were explanted to propagate corneal progenitors and cells characterized using reverse transcription-polymerase chain reaction, immunohistochemistry, flow cytometry, and colony-forming assays. A novel "ridge-like" structure was identified, circumscribing the fetal cornea, which we hypothesize represents the rudimentary SC niche. Immunohistochemistry disclosed "stem-like" cells across the cornea, becoming confined to this ridge with increasing gestational age. In addition, for the first time, pure long-term cultures of fetal corneal epithelium, which displayed phenotypical and functional properties similar to those of adult limbal SCs, were established. Optimization of culture techniques and purification of this SC population will allow for further investigation of their proliferative ability, with potential research and clinical applications. This study expands our understanding of limbal niche development and opens new avenues for investigation.

UVB irradiation modulates immune responses in the skin and is a major cause of sunburn, during which neutrophils accumulate in the skin. Because of their abundance in skin and ability to produce a variety of proinflammatory mediators, we propose that mast cells may play a key role in ultraviolet B (UVB)-induced skin inflammation. Cord blood-derived human mast cells were treated in vitro with varying doses of UVB and production of multiple cytokines was measured in culture supernatants. UVB exposure significantly increased the release of interleukin (IL)-8 and modestly increased IL-1alpha production, but cytokines such as IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma were unaffected. Cycloheximide reduced the UVB-mediated induction of IL-8 by 30-40%, suggesting that new protein synthesis contributed to IL-8 production. In line with this, UVB treatment of mast cells significantly increased IL-8 mRNA. In contrast to its effect on IL-8 production, optimal doses of UVB did not provoke histamine or tryptase release, indicating little effect on degranulation. Our data suggest that mast cells may play a major role during UVB-induced acute inflammation by selectively inducing cytokines involved in neutrophil recruitment.

Background: The basic region-leucine zipper protein c-Jun has been linked to cell proliferation, transformation, and apoptosis. However, a direct role for c-Jun in angiogenesis has not been shown., Methods: We used human microvascular endothelial cells (HMEC-1) transfected with a DNAzyme targeting the c-Jun mRNA (Dz13), related oligonucleotides, or vehicle in in vitro models of microvascular endothelial cell proliferation, migration, chemoinvasion, and tubule formation, a rat model of corneal neovascularization, and a mouse model of solid tumor growth and vascular endothelial growth factor (VEGF)-induced angiogenesis. All statistical tests were two-sided., Results: Compared with mock-transfected cells, HMEC-1 cells transfected with Dz13 expressed less c-Jun protein and possessed lower DNA-binding activity. Dz13 blocked endothelial cell proliferation, migration, chemoinvasion, and tubule formation. Dz13 inhibited the endothelial cell expression and proteolytic activity of MMP-2, a c-Jun-dependent gene. Dz13 inhibited VEGF-induced neovascularization in the rat cornea compared with vehicle control (Dz13 versus vehicle: 4.0 neovessels versus 30.7 neovessels, difference = 26.7 neovessels; P =.004; area occupied by new blood vessels for Dz13 versus vehicle: 0.35 mm2 versus 1.52 mm2, difference = 1.17 mm2; P =.005) as well as solid melanoma growth in mice (Dz13 versus vehicle at 14 days: 108 mm3 versus 283 mm3, difference = 175 mm3; P =.006) with greatly reduced vascular density (Dz13 versus vehicle: 30% versus 100%, difference = 70%; P<.001)., Conclusion: DNAzymes targeting c-Jun may have therapeutic potential as inhibitors of tumor angiogenesis and growth.

S100 proteins represent a new class of chemoattractants. Here we extend earlier evidence for the proinflammatory properties of human S100A12. A12 induced migration of monocytoid cells, with optimal activity at 10(-10) M and potency of >10(-9) M C5a. Neutrophils were poorly responsive, and lymphocyte migration was not affected. Actin polymerization in monocytoid cells was accompanied by a sustained [Ca(2+)]i flux of a magnitude comparable with C5a. A12 elicited a transient infiltration of neutrophils (4-8 h) and more delayed recruitment of monocytes (8-24 h) in vivo. A12 (approximately 70 nM) was present in synovial fluid (SF) from rheumatoid arthritis patients, and synovium contained A12-positive neutrophils in the sublining and interstitial region, often surrounding the perivasculature but rarely in the synovial lining layer, although some macrophages were positive. The A12 gene was transiently up-regulated in monocytes by tumor necrosis factor alpha (6 h); induction by lipopolysaccharide (LPS) was sustained (12-48 h). A12 may contribute to leukocyte migration in chronic inflammatory responses.

Objective: To investigate the expression and source of chemokines in minor salivary gland biopsies (MSGs) in patients with Sjögren's syndrome (SS)., Methods: Immunohistochemical analysis was used to determine the pattern of chemokine expression in MSGs from patients with (n=6) and without (n=5) SS, as well as to examine the phenotype of both resident and infiltrating cells expressing chemokines., Results: Significant differences in the number of infiltrating mononuclear (MN) cells in patients with and without SS were noted. Ductal epithelial cells of SS biopsies expressed significantly increased levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted). Biopsies from patients with SS showed that MIP-1beta was expressed by 51% of infiltrating cells, while 41% expressed MIP-1alpha, whereas 22 and 7% expressed RANTES and IL-8, respectively., Conclusion: Chemokines expressed by ductal epithelial cells may attract circulating leucocytes, in particular CD4+ T cells, towards the site of inflammation, thereby orchestrating the influx of MN cells characteristically seen in MSGs in SS. Chemokines may be induced directly by a putative triggering agent for SS, or secondary to the release of pro-inflammatory cytokines produced by epithelial cells. These findings further implicate epithelial cells as playing a major role in the pathogenesis of SS and implicate chemokines in the leucocyte recruitment in this setting.

Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory cytokine and mediator of the inflammatory response. It has been implicated in the pathogenesis of many inflammatory disorders, including rheumatoid arthritis (RA), septic shock, and Crohn's disease. Using a specific anti-human TNF-alpha antibody we detected immunoreactivity for this cytokine in the cytoplasm of inflammatory cells in several chronic inflammatory disorders, including RA, scleritis, and polyarteritis nodosa. These cells were identified predominantly as IgG-expressing plasma cells. Lymph nodes from patients with Hodgkin's lymphoma and breast cancer, but not from control subjects, were also found to contain TNF-alpha-positive plasma cells. Cultured EBV-B lymphocytes and a human plasma cell line (ARH-77) when stimulated with phorbol myristate acetate demonstrated cytoplasmic TNF-alpha immunoreactivity. Western blot analysis of cell membranes and conditioned media from both cell types revealed the presence of the 26-kDa membrane-bound from and the 17-kDa soluble from of TNF-alpha, respectively. TNF-alpha was quantitated by enzyme-linked immunosorbent assay and found to be biologically active as determined by the L929 cytotoxicity assay. This is the first demonstration that plasma cells may be capable of modulating immune and inflammatory responses, not only by antibody production, but also by their secretion of a key inflammatory mediator, TNF-alpha.