scholarly journals Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaoya An ◽  
Guoliang Wang ◽  
Mengyi Jin ◽  
Xiaoping Zhou ◽  
Shubin Gao ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Fate ◽  
Proliferative Activity ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Transplantation Surgery ◽  
Epithelial Sheets

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

scholarly journals The O-GlcNAc modification promotes terminal differentiation of human corneal epithelial cells

Glycobiology ◽  
2020 ◽  
Vol 30 (11) ◽  
pp. 872-880 ◽  
Author(s):  
Nicole M McColgan ◽  
Marissa N Feeley ◽  
Ashley M Woodward ◽  
Damien Guindolet ◽  
Pablo Argüeso
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Ocular Surface ◽  
Barrier Function ◽  
Terminal Differentiation ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Human Corneal Epithelial Cells ◽  
Glcnac Transferase

Abstract Dynamic modification of nuclear and cytoplasmic proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in orchestrating the transcriptional activity of eukaryotic cells. Here, we report that the O-GlcNAc modification contributes to maintaining ocular surface epithelial homeostasis by promoting mucin biosynthesis and barrier function. We found that induction of human corneal epithelial cell differentiation stimulated the global transfer of O-GlcNAc to both nuclear and cytosolic proteins. Inflammatory conditions, on the other hand, were associated with a reduction in the expression of O-GlcNAc transferase at the ocular surface epithelia. Loss- and gain-of-function studies using small interfering RNA targeting O-GlcNAc transferase, or Thiamet G, a selective inhibitor of O-GlcNAc hydrolase, respectively, revealed that the presence of O-GlcNAc was necessary to promote glycocalyx barrier function. Moreover, we found that Thiamet G triggered a correlative increase in both surface expression of MUC16 and apical epithelial cell area while reducing paracellular permeability. Collectively, these results identify intracellular protein O-glycosylation as a novel pathway responsible for promoting the terminal differentiation of human corneal epithelial cells.

Potential role for laminin 5 in hypoxia-mediated apoptosis of human corneal epithelial cells

2001 ◽  
Vol 114 (22) ◽  
pp. 4033-4040
Author(s):  
Miechia A. Esco ◽  
Zhiyu Wang ◽  
Mark L. McDermott ◽  
Michelle Kurpakus-Wheater
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mitochondrial Membrane ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Laminin 5 ◽  
Corneal Epithelial ◽  
Human Corneal Epithelial Cells ◽  
Cells Cultured

Laminin 5 functions to promote cell-matrix adhesion and therefore is hypothesized to abrogate apoptosis initiated through the loss of epithelial cell contact with extracellular matrix. Laminin 5 levels are decreased in epithelial cells cultured in a hypoxic environment. Exposure of epithelial cells to hypoxia may induce apoptotic pathways transmitted through changes in mitochondrial membrane potential. Using an apoptosis assay based on mitochondrial membrane integrity, the effect of hypoxia (2% oxygen) on human corneal epithelial cell viability was determined. Both a virally transformed corneal epithelial cell line and third passage corneal epithelial cells were resistant to hypoxia-mediated apoptosis for up to 5 days in culture. However, at 7 days in culture, a statistically significant increase in apoptosis was noted in hypoxic corneal epithelial cells compared to normoxic (20% oxygen) controls. Increased apoptosis in hypoxic epithelium at 7 days in culture correlated with decreased deposition of laminin 5 into the extracellular matrix, as determined by western blot analysis and immunofluorescence microscopy. Additionally, the extracellular processing of the α3 and γ2 chains of laminin 5 was negatively impacted by corneal epithelial cell exposure to hypoxia for 7 days. Treatment of human corneal epithelial cells cultured in 20% oxygen with function-inhibiting antibodies to laminin 5 for 2 or 3 days resulted in a statistically significant decrease in proliferation, and concomitant increase in apoptosis, compared with untreated normoxic controls. Based on these results, it appears that mechanisms of hypoxia-mediated apoptosis in human corneal epithelial cells may be initiated by the loss of processed laminin 5 in the extracellular matrix or by the loss of laminin 5-epithelial cell communication and transmitted through mitochondria.

scholarly journals The core planar cell polarity gene, Vangl2 , directs adult corneal epithelial cell alignment and migration

2016 ◽  
Vol 3 (10) ◽  
pp. 160658 ◽  
Author(s):  
Amy S. Findlay ◽  
D. Alessio Panzica ◽  
Petr Walczysko ◽  
Amy B. Holt ◽  
Deborah J. Henderson ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Corneal Epithelium ◽  
Planar Cell Polarity ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
The Core

This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro . Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.

scholarly journals Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells

2019 ◽  
Vol 15 (6) ◽  
pp. e1007825 ◽  
Author(s):  
Kimberly M. Brothers ◽  
Jake D. Callaghan ◽  
Nicholas A. Stella ◽  
Julianna M. Bachinsky ◽  
Mohammed AlHigaylan ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Corneal Epithelial Cells ◽  
Cellular Death ◽  
Corneal Epithelial

scholarly journals Biocompatibility of Nanocellulose-Reinforced PVA Hydrogel with Human Corneal Epithelial Cells for Ophthalmic Applications

2019 ◽  
Vol 10 (3) ◽  
pp. 35 ◽  
Author(s):  
Tummala ◽  
Lopes ◽  
Mihranyan ◽  
Ferraz
Keyword(s):  
Epithelial Cells ◽  
Cell Viability ◽  
Protein Adsorption ◽  
Direct Contact ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Human Corneal Epithelial Cells ◽  
Hydrogel Surface

Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens–cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies.

scholarly journals Hyaluronate Acid-Dependent Protection and Enhanced Corneal Wound Healing against Oxidative Damage in Corneal Epithelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Jing Zhong ◽  
Yuqing Deng ◽  
Bishan Tian ◽  
Bowen Wang ◽  
Yifang Sun ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Epithelial Cells ◽  
Cell Apoptosis ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Wound Healing ◽  
Corneal Epithelial ◽  
Expression Levels ◽  
Corneal Wound

Purpose. To evaluate the effects and mechanism of exogenous hyaluronate (HA) in promoting corneal wound healing.Methods. Human corneal epithelial cells (HCECs) were incubated with different concentrations of HA to evaluate their efficiency in promoting cell migration and their modulation of repair factors. After inducing hyperosmolar conditions, the cell morphologies, cell apoptosis, and expression levels of TNF-αand MMP-9 were detected to assess the protective role of HA. Corneal epithelium-injured rat models were established to test the therapeutic effects of 0.3% HA. Then, the wound healing rates, the RNA expression levels of inflammatory cytokines, and repair factors were examined.Results. HCECs in the 0.03% and 0.3% HA groups showed fewer morphological alterations and lower rates of cell apoptosis following preincubation with HA under hyperosmolar conditions, as well as the expression levels of MMP-9 and TNF-α. In the rat model, the areas of fluorescein staining in the corneas of 0.3% HA group were significantly smaller than the control group. The expression levels of IL-1βand MMP-9 were decreased, while CD44 and FN were increased in the 0.3% HA group.Conclusion. HA enhanced corneal epithelial cell wound healing by promoting cell migration, upregulating repair responses, and suppressing inflammatory responses.

scholarly journals Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye

2006 ◽  
Vol 190 (2) ◽  
pp. 483-493 ◽  
Author(s):  
Claire U Onyimba ◽  
Neelima Vijapurapu ◽  
S John Curnow ◽  
Pamela Khosla ◽  
Paul M Stewart ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Corneal Epithelium ◽  
Ciliary Body ◽  
Primary Cultures ◽  
Anterior Segment ◽  
Body Tissue ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Rabbit Eye

The prereceptor regulation of glucocorticoids (GCs) by 11β-hydroxysteroid dehydrogenase type-1 (11β-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11β-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11β-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11β-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino ‘pigmented’ ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone→cortisol) and dehydrogenase (cortisol→cortisone) activity indicated predominant 11β-HSD1 oxo-reductase activity from both the intact ciliary body tissue (n=12, median 2.1 pmol/mg per h and range 1.25–2.8 pmol/mg per h; P=0.006) and primary cultures of corneal epithelial cells (n=12, median 3.0 pmol/mg per h and range 1.0–7.4 pmol/mg per h, P=0.008) compared with dehydrogenase activity (median 1.0 pmol/mg per h and range 0.5–2.0 pmol/mg per h; median 0.5 pmol/mg per h and range 0.25–1.9 pmol/mg per h respectively). These findings were supported by expression of 11β-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11β-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11β-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11β-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.

scholarly journals Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Divya Arunachalam ◽  
Shruthi Mahalakshmi Ramanathan ◽  
Athul Menon ◽  
Lekshmi Madhav ◽  
Gopalakrishna Ramaswamy ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Differentially Expressed Genes ◽  
Primary Cultures ◽  
Differentially Expressed ◽  
Corneal Epithelial Cell ◽  
Epithelial Cell Line ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Human Corneal Epithelial Cells

Abstract Background Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. Methods Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. Results Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. Conclusions Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

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