Victor H Guaiquil

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers.

In order to identify reliable markers of corneal epithelial stem cells, we employed an inducible transgenic “pulse-chase” murine model (K5Tta × TRE-H2BGFP) to localize, purify, and characterize slow cycling cells in the cornea. The retention of GFP labeling in slowly dividing cells allowed for localization of these cells to the corneal limbus and their subsequent purification by FACS. Transcriptome analysis from slow cycling cells identified differentially expressed genes when comparing to GFP- faster-dividing cells. RNA-Seq data from corneal epithelium were compared to epidermal hair follicle stem cell RNA-Seq to identify genes representing common putative stem cell markers or determinants, which included Sox9, Fzd7, Actn1, Anxa3 and Krt17. Overlapping retention of GFP and immunohistochemical expression of Krt15, ΔNp63, Sox9, Actn1, Fzd7 and Krt17 were observed in our transgenic model. Our analysis presents an array of novel genes as putative corneal stem cell markers.

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation

Purpose The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin–derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model. Methods Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the materials effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies. Results Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls. Conclusions The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDPs potential utility as an ophthalmic therapeutic agent.

Acute corneal epithelial debridement unmasks the corneal stromal nerve responses to ocular stimulation in rats: Implications for abnormal sensations of the eye.

It is widely accepted that the mechanisms for transducing sensory information reside in the nerve terminals. Occasionally, however, studies have appeared demonstrating that similar mechanisms may exist in the axon to which these terminals are connected. We examined this issue in the cornea, where nerve terminals in the epithelial cell layers are easily accessible for debridement, leaving the underlying stromal (axonal) nerves undisturbed. In isoflurane-anesthetized rats, we recorded extracellularly from single trigeminal ganglion neurons innervating the cornea that are excited by ocular dryness and cooling: low-threshold (<2°C cooling) and high-threshold (>2°C) cold-sensitive plus dry-sensitive neurons playing possible roles in tearing and ocular pain. We found that the responses in both types of neurons to dryness, wetness, and menthol stimuli were effectively abolished by the debridement, indicating that their transduction mechanisms lie in the nerve terminals. However, some responses to the cold, heat, and hyperosmolar stimuli in low-threshold cold-sensitive plus dry-sensitive neurons still remained. Surprisingly, the responses to heat in approximately half of the neurons were augmented after the debridement. We were also able to evoke these residual responses and follow the trajectory of the stromal nerves, which we subsequently confirmed histologically. The residual responses always disappeared when the stromal nerves were cut at the limbus, suggesting that the additional transduction mechanisms for these sensory modalities originated most likely in stromal nerves. The functional significance of these residual and enhanced responses from stromal nerves may be related to the abnormal sensations observed in ocular disease.NEW & NOTEWORTHY In addition to the traditional view that the sensory transduction mechanisms exist in the nerve terminals, we report here that the proximal axons (stromal nerves in the cornea from which these nerve terminals originate) may also be capable of transducing sensory information. We arrived at this conclusion by removing the epithelial cell layers of the cornea in which the nerve terminals reside but leaving the underlying stromal nerves undisturbed.

Author Correction: Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Semaphorin3A induces nerve regeneration in the adult cornea-A switch from its repulsive role in development

The peripheral sensory nerves that innervate the cornea can be easily damaged by trauma, surgery, infection or diabetes. Several growth factors and axon guidance molecules, such as Semaphorin3A (Sema3A) are upregulated upon cornea injury. Nerves can regenerate after injury but do not recover their original density and patterning. Sema3A is a well known axon guidance and growth cone repellent protein during development, however its role in adult cornea nerve regeneration remains undetermined. Here we investigated the neuro-regenerative potential of Sema3A on adult peripheral nervous system neurons such as those that innervate the cornea. First, we examined the gene expression profile of the Semaphorin class 3 family members and found that all are expressed in the cornea. However, upon cornea injury there is a fast increase in Sema3A expression. We then corroborated that Sema3A totally abolished the growth promoting effect of nerve growth factor (NGF) on embryonic neurons and observed signs of growth cone collapse and axonal retraction after 30 min of Sema3A addition. However, in adult isolated trigeminal ganglia or dorsal root ganglia neurons, Sema3A did not inhibited the NGF-induced neuronal growth. Furthermore, adult neurons treated with Sema3A alone produced similar neuronal growth to cells treated with NGF and the length of the neurites and branching was comparable between both treatments. These effects were replicated in vivo, where thy1-YFP neurofluorescent mice subjected to cornea epithelium debridement and receiving intrastromal pellet implantation containing Sema3A showed increased corneal nerve regeneration than those receiving pellets with vehicle. In adult PNS neurons, Sema3A is a potent inducer of neuronal growth in vitro and cornea nerve regeneration in vivo. Our data indicates a functional switch for the role of Sema3A in PNS neurons where the well-described repulsive role during development changes to a growth promoting effect during adulthood. The high expression of Sema3A in the normal and injured adult corneas could be related to its role as a growth factor.

Micro- and Nanoscale Topographies on Silk Regulate Gene Expression of Human Corneal Epithelial Cells

Purpose Corneal basement membrane has topographical features that provide biophysical cues to direct cell adherence, migration, and proliferation. In this study, we hypothesize that varying topographic pitch created on silk films can alter epithelial cell morphology, adhesion, and the genetic expression involved in cytoskeletal dynamics-related pathways. Methods Silicon wafers with parallel ridge widths of 2000, 1000, and 800 nm were produced and used to pattern silk films via soft lithography. Human corneal epithelial cells were cultured onto silk. After 72 hours of incubation, images were taken to study cell morphology and alignment. Cytoskeletal structures were studied by immunofluorescent staining. RNA was collected from cultured cells to perform RNA-Seq transcriptome analysis using the Illumina Hiseq 2500 sequencing system. Differentially expressed genes were identified using DNAstar Qseq then verified using quantitative real-time PCR. These genes were used to perform pathway analyses using Ingenuity Pathways Analysis. Results Primary human corneal epithelial cell alignment to the surface pattern was the greatest on 1000-nm features. Fluorescent microscopy of f-actin staining showed cell cytoskeleton alignment either in parallel (2000 nm) or perpendicular (1000 and 800 nm) to the long feature axis. Z-stack projection of vinculin staining indicated increased focal adhesion formation localized on the cellular basal surface. RNA-seq analysis revealed differentially expressed genes involved in actin organization, integrin signaling, and focal adhesion kinase signaling (−log (P)>5). Conclusions Patterned silk film substrates may serve as a scaffold and provide biophysical cues to corneal epithelial cells that change their gene expression, alter cellular adherence, morphology, and may offer a promising customizable material for use in ocular surface repair.