MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs).

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

Aims/hypothesis: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression.

Non-canonical Wnt signaling in the eye.

Wnt signaling comprises a group of complex signal transduction pathways that play critical roles in cell proliferation, differentiation, and apoptosis during development, as well as in stem cell maintenance and adult tissue homeostasis. Wnt pathways are classified into two major groups, canonical (β-catenin-dependent) or non-canonical (β-catenin-independent). Most previous studies in the eye have focused on canonical Wnt signaling, and the role of non-canonical signaling remains poorly understood.

Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea.

Purpose: MiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing.

Systemic diseases and the cornea.

There is a number of systemic diseases affecting the cornea. These include endocrine disorders (diabetes, Graves' disease, Addison's disease, hyperparathyroidism), infections with viruses (SARS-CoV-2, herpes simplex, varicella zoster, HTLV-1, Epstein-Barr virus) and bacteria (tuberculosis, syphilis and Pseudomonas aeruginosa), autoimmune and inflammatory diseases (rheumatoid arthritis, Sjögren's syndrome, lupus erythematosus, gout, atopic and vernal keratoconjunctivitis, multiple sclerosis, granulomatosis with polyangiitis, sarcoidosis, Cogan's syndrome, immunobullous diseases), corneal deposit disorders (Wilson's disease, cystinosis, Fabry disease, Meretoja's syndrome, mucopolysaccharidosis, hyperlipoproteinemia), and genetic disorders (aniridia, Ehlers-Danlos syndromes, Marfan syndrome). Corneal manifestations often provide an insight to underlying systemic diseases and can act as the first indicator of an undiagnosed systemic condition.

Novel nanopolymer RNA therapeutics normalize human diabetic corneal wound healing and epithelial stem cells.

Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met.

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses.

Exosomes from normal and diabetic human corneolimbal keratocytes differentially regulate migration, proliferation and marker expression of limbal epithelial cells.

Limbal epithelial stem cells (LESC) maintenance requires communication between stem cells and neighboring stromal keratocytes. Extracellular vesicles (EVs) are important for intercellular communication in various stem cell niches. We explored the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos), an EV sub-population, in limbal epithelial cells (LEC) in normal and diabetic limbal niche and determined differences in Exo cargos from normal and diabetic LSC.

Concise Review: Stem Cells for Corneal Wound Healing.

Corneal wound healing is a complex process that occurs in response to various injuries and commonly used refractive surgery. It is a significant clinical problem, which may lead to serious complications due to either incomplete (epithelial) or excessive (stromal) healing. Epithelial stem cells clearly play a role in this process, whereas the contribution of stromal and endothelial progenitors is less well studied.

Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus.

Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas.

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

Purpose: Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas.

Persistence of reduced expression of putative stem cell markers and slow wound healing in cultured diabetic limbal epithelial cells.

Purpose: To examine the expression of putative limbal epithelial stem cell (LESC) markers and wound healing rates in primary healthy and diabetic human limbal epithelial cells (LECs) cultured on different substrata.

Methods: Primary limbal epithelial cells were isolated from human autopsy corneas and discarded corneoscleral rims with dispase II treatment. LECs were cultured in EpiLife medium on human amniotic membrane (AM) denuded with mild alkali treatment, on plastic dishes and on glass slides coated with a mixture of human fibronectin, collagen type IV, and laminin (FCL).

Progress in corneal wound healing.

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane.

Targeting miR-146a to treat delayed wound healing in human diabetic organ-cultured corneas.

Limbal epithelial stem cells (LESC) residing at the corneal periphery are largely responsible for maintaining corneal optical transparency by continuously supplying new corneal epithelial cells, which mature during their radial migration to the central cornea. Diabetes mellitus (DM) affects all the structures of the eye including the cornea. Frequent epithelial erosions, delayed wound healing, and microbial infections are common alterations of the diabetic eye that can result in vision loss.

Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells.

Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was previously shown to normalize wound healing, epithelial and stem cell marker patterns in organ-cultured human diabetic corneas. We now examined if gene therapy of limbal cells only would produce similar effects. Eight pairs of organ-cultured autopsy human diabetic corneas were used.

Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.

Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs.

Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays.

Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.

Purpose: Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.

Methods: Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh.

A simple alkaline method for decellularizing human amniotic membrane for cell culture.

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required.

Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy.

Purpose: We have previously identified specific epithelial proteins with altered expression in human diabetic central corneas. Decreased hepatocyte growth factor receptor (c-met) and increased proteinases were functionally implicated in the changes of these proteins in diabetes. The present study examined whether limbal stem cell marker patterns were altered in diabetic corneas and whether c-met gene overexpression could normalize these patterns.

ZBED4, a cone and Müller cell protein in human retina, has a different cellular expression in mouse.

Purpose: ZBED4, a protein in cones and Müller cells of human retina, may play important functions as a transcriptional activator of genes expressed in those cells or as a co-activator/repressor of their nuclear hormone receptors. To begin investigating these potential roles of ZBED4, we studied the developmental expression and localization of both the Zbed4 mRNA and protein of mouse retina.

Methods: northern blots showed the presence of Zbed4 mRNA in retina and other mouse tissues, and western blots showed the nuclear and cytoplasmic expression of Zbed4 at different developmental times.

Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery of c-Met gene.

Purpose. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)-driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities.

Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes.

Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used.

ZBED4, a BED-type zinc-finger protein in the cones of the human retina.

Purpose: To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina.

Methods: The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina.