A Keratin 12 Expression-Based Analysis of Stem-Precursor Cells and Differentiation in the Limbal-Corneal Epithelium Using Single-Cell RNA-Seq Data.

The corneal epithelium (CE) is spread between two domains, the outer vascularized limbus and the avascular cornea proper. Epithelial cells undergo constant migration from the limbus to the vision-critical central cornea. Coordinated with this migration, the cells undergo differentiation changes where a pool of unique stem/precursor cells at the limbus yields the mature cells that reach the corneal center.

TGFβ1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFβ1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase.

Following wounding, endogenously secreted TGFβs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFβ effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFβ.

The c-Myc Oncogene Maintains Corneal Epithelial Architecture at Homeostasis, Modulates p63 Expression, and Enhances Proliferation During Tissue Repair.

Purpose: The transcription factor c-Myc (Myc) plays central regulatory roles in both self-renewal and differentiation of progenitors of multiple cell lineages. Here, we address its function in corneal epithelium (CE) maintenance and repair.

Methods: Myc ablation in the limbal-corneal epithelium was achieved by crossing a floxed Myc mouse allele (Mycfl/fl) with a mouse line expressing the Cre recombinase gene under the keratin (Krt) 14 promoter.

Multi-parametric evaluation of autologous cultivated Limbal epithelial cell transplantation outcomes of Limbal stem cell deficiency due to chemical burn.

Background: The sparsity of established tools for the grading of limbal stem cell deficiency hinder objective assessments of the clinical outcome of cultivated limbal epithelial cell transplantation. To advance towards the development of standards for the comparison of the outcomes of these bio-surgical protocols we have now applied a battery of recognized objective and patient-declared subjective outcome criteria to the autologous modality of cultivated limbal epithelial cell transplantation.

Methods: The prospective study involved ten patients (M/F = 9/1; mean age = 42.

LOXL1 folding in exfoliation glaucoma.

Exfoliation syndrome (XFS) is an age-related disease defined by the deposition of aggregated fibrous material (XFM) in the peri-cellular space. Principal morbidity occurs in the eye, where XFM accumulates on the anterior ocular tissues. GWAS have found that certain genetic variants of lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme that is required for elastic fiber formation confer risk for the development of XFS, but are not a single causative factor as many genetically affected individuals do not develop XFS or subsequent glaucoma (XFG).

Exfoliation Syndrome: A Disease of Autophagy and LOXL1 Proteopathy.

Exfoliation syndrome (XFS) is an age-related disease involving the deposition of aggregated fibrillar material (exfoliation material) at extracellular matrices in tissues that synthesize elastic fibers. Its main morbidity is in the eye, where exfoliation material accumulations form on the surface of the ciliary body, iris, and lens. Exfoliation glaucoma (XFG) occurs in a high proportion of persons with XFS and can be a rapidly progressing disease.

Author Correction: Divergent effects of Wnt/β-catenin signaling modifiers on the preservation of human limbal epithelial progenitors according to culture condition.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Divergent effects of Wnt/β-catenin signaling modifiers on the preservation of human limbal epithelial progenitors according to culture condition.

Wnt signaling plays an important role in the regulation of self-renewal in stem cells. Here we investigated the effect of CHIR99021, the primary transducer of the Wnt signaling canonical pathway, and IWP2, a wide action Wnt signal blocker, on the growth and differentiation of the limbal epithelial progenitor cells when these cells are cultured in two different, common culture approaches, outgrowth from limbal biopsy explants and isolated cell seeded in low calcium medium. Consistent with their expected effects, irrespective of the culture system, IWP2 decreased total β-catenin while CHIR99021 increased it in nuclear localization.

Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation.

The cornea is the outermost tissue of the eye and it must be transparent for the maintenance of good visual function. The superficial epithelium of the cornea, which is renewed continuously by corneal stem cells, plays a critical role in the permanence of this transparency. These stem cells are localized at the cornea-conjunctival transition zone, referred to as the limbus.

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry.

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells.

Is Autophagy Dysfunction a Key to Exfoliation Glaucoma?

In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.

Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients.

Purpose: To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma.

Methods: Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features.

Corneal recovery in a rabbit limbal stem cell deficiency model by autologous grafts of tertiary outgrowths from cultivated limbal biopsy explants.

Purpose: To determine the corneal regenerative capacity of sequentially generated primary, secondary, and tertiary limbal explant outgrowths in a limbal stem cell deficiency (LSCD) surgical model.

Methods: Two-millimeter-long limbal shallow biopsies were surgically excised from the upper quadrant of the right eye of rabbits and set on preserved amniotic membrane for explant culture. After the generation of primary outgrowth, the biopsies were sequentially transferred to new amniotic membrane to generate secondary and then tertiary outgrowths.

Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions.

Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these cultures.

Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell-free media.

Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface.

Gastrointestinal complications of diabetes.

This review provides an overview of the vast gastrointestinal tract complications of diabetes that can occur from the mouth to the anus. The presentation, diagnosis, and management of gastrointestinal disorders, ranging from gastroparesis, celiac disease, and bacterial overgrowth to nonalcoholic fatty liver disease, are reviewed to heighten awareness. When managing care of patients with diabetes, one should keep in mind the potential gastrointestinal complications, as well as the frequent disorders that are not related to diabetes.

TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop.

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development.

Functional TRPV1 expression in human corneal fibroblasts.

Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line.

[Ocular Surface Reconstruction with Cultivated Limbal Epithelial Cells in Limbal Stem Cell Deficiency: One-year Follow-up Results].

Purpose: To evaluate the 1-year follow-up results of cultivated limbal epithelial cell (CLEC) transplantation in unilateral limbal stem cell deficiency (LSCD).

Material And Method: One-year follow-up results of five unilateral LSCD patients who had undergone CLEC transplantation were evaluated. Parameters for this evaluation were: fluorescein staining of ocular surface, corneal vascularization and status of epithelium with slit lamp, and visual acuity.

NF-κB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells.

Purpose: The corneal wound healing response to an alkali burn results in dysregulated inflammation and opacity. Transient receptor potential vanilloid type1 (TRPV1) ion channel activation by such a stress contributes to this unfavorable outcome. Accordingly, we sought to identify potential drug targets for mitigating this response, in human corneal epithelial cells (HCEC).

ABCG2-dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants.

Purpose: To determine changes in ABCG2-transport-dependent dye exclusion in outgrowths from limbal explants.

Methods: Human or rabbit limbal strips were deposited onto inserts. Over a month, the segments were twice transferred to new inserts.

Gene expression analysis in SV-40 immortalized human corneal epithelial cells cultured with an air-liquid interface.

Purpose: To compare the global gene expression profile of stratified epithelia generated in vitro using simian virus 40 (SV40) immortalized human corneal epithelial cells with the previously reported gene expression of normal human corneal epithelia.

Methods: Immortalized cells expanded in submerged culture were grown in an air-liquid interface of liquid permeable collagen-coated filters to foster stratification and differentiation. Stratified epithelia displaying resistances exceeding 300 Ω·cm2 were dissolved in an RNA purification lysis buffer.

DUSP5 and DUSP6 modulate corneal epithelial cell proliferation.

Purpose: Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells.

Dependence of resolvin-induced increases in corneal epithelial cell migration on EGF receptor transactivation.

Purpose: To determine whether resolvin E1 (RvE1), an endogenous oxygenation product of eicosapentaenoic acid (EPA), induces increases in migration in human corneal epithelial cells (HCECs) and to identify signal pathways mediating this response.

Methods: Migration was measured with the scratch wound assay. Western blot analysis identified changes in the phosphorylation status of prospective intracellular signal transduction mediators.