Role of stem cells in regenerative treatment of dry eye disease caused by lacrimal gland dysfunction.

An ageing population and increased screen use in younger people have contributed to a rise in incidence of dry eye disease (DED). Quality of life can be significantly affected by DED, with patients experiencing eye dryness, burning, pain and sensitivity to light. If left untreated, DED may progress to cause lasting damage to the delicate cell layers of the ocular surface. The aqueous-deficient form of DED is characterized by decreased tear volume. This can occur through underlying disease or damage to the lacrimal gland (LG), which results in increased inflammation at the ocular surface and decreased tear secretion. Regenerative therapy for treatment of aqueous-deficient DED would ideally restore LG function without causing adverse side effects and be feasible in terms of cost, production and practical application in the clinic. In this review, we evaluate research directed at the development of clinical procedures for regeneration of the LG using various stem cell types and their products. We also discuss work identifying potential therapeutic targets that may alter pathways to effect healing and ameliorate development of DED. Finally, we discuss shortcomings and recommend future avenues for research. These include determination of the best tissue of origin for mesenchymal cells and transference of knowledge gleaned from animal studies to clinical investigations.

Antibiotic treatment for dry eye disease related to meibomian gland dysfunction and blepharitis - A review.

BACKGROUND: Dry eye disease (DED) is among the most prevalent ophthalmic conditions but is often underdiagnosed and mistreated. Antibiotics are regularly used to treat DED caused by meibomian gland dysfunction (MGD) or blepharitis, but their use has been questioned. OBJECTIVE: To critically evaluate the use of oral and topical antibiotics in DED management. METHODS: A literature search was conducted on November 15th, 2021, in the PubMed database. The search terms were: (antibiotics OR azithromycin OR doxycycline OR minocycline) AND (dry eye disease OR meibomian gland OR blepharitis anterior OR blepharitis posterior OR chronic blepharitis). All relevant original articles with English full-text were included. Case reports and review articles were excluded. RESULTS: The search provided 619 articles, of which 22 met the inclusion criteria. Oral and topical antibiotics appeared to have short-term positive effects on signs and symptoms of blepharitis- or MGD-related DED. However, these improvements often reverted upon cessation of treatment. The need for repeated treatments and mild adverse events were common. CONCLUSIONS: Current evidence suggests that patients with blepharitis- or MGD-related DED experience short-term benefits of antibiotics. However, evidence for lasting improvement after completed treatment was lacking. Given the unclear long-term benefits, common side effects, and increasing antibiotic resistance seen globally, the existing literature is not sufficient to conclude that antibiotics are useful in long-term MGD management. A survival-analysis of a single round of antibiotics, in addition to the effects of repeated rounds of treatment, on DED parameters could provide useful insights.

The use of in vivo confocal microscopy in fungal keratitis - Progress and challenges.

Fungal keratitis (FK) is a serious and sight-threatening corneal infection with global reach. The need for prompt diagnosis is paramount, as a delay in initiation of treatment could lead to irreversible vision loss. Current "gold standard" diagnostic methods, namely corneal smear and culture, have limitations due to diagnostic insensitivity and their time-consuming nature. PCR is a newer, complementary method used in the diagnosis of fungal keratitis, whose results are also sample-dependent. In vivo confocal microscopy (IVCM) is a promising complementary diagnostic method of increasing importance as it allows non-invasive real-time direct visualization of potential fungal pathogens and manifesting infection directly in the patient's cornea. In numerous articles and case reports, FK diagnosis by IVCM has been evaluated, and different features, approaches, sensitivity/specificity, and limitations have been noted. Here, we provide an up-to-date, comprehensive review of the current literature and present the authors' combined recommendations for fungal identification in IVCM images, while also looking to the future of FK assessment by IVCM using artificial intelligence methods.

Characterization of Gfat1 (zeppelin) and Gfat2, Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in Drosophila melanogaster.

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.

Artificial intelligence in dry eye disease.

Dry eye disease (DED) has a prevalence of between 5 and 50%, depending on the diagnostic criteria used and population under study. However, it remains one of the most underdiagnosed and undertreated conditions in ophthalmology. Many tests used in the diagnosis of DED rely on an experienced observer for image interpretation, which may be considered subjective and result in variation in diagnosis. Since artificial intelligence (AI) systems are capable of advanced problem solving, use of such techniques could lead to more objective diagnosis. Although the term 'AI' is commonly used, recent success in its applications to medicine is mainly due to advancements in the sub-field of machine learning, which has been used to automatically classify images and predict medical outcomes. Powerful machine learning techniques have been harnessed to understand nuances in patient data and medical images, aiming for consistent diagnosis and stratification of disease severity. This is the first literature review on the use of AI in DED. We provide a brief introduction to AI, report its current use in DED research and its potential for application in the clinic. Our review found that AI has been employed in a wide range of DED clinical tests and research applications, primarily for interpretation of interferometry, slit-lamp and meibography images. While initial results are promising, much work is still needed on model development, clinical testing and standardisation.

Simple limbal epithelial transplantation: Current status and future perspectives.

Damage to limbal stem cells as a result of injury or disease can lead to limbal stem cell deficiency (LSCD). This disease is characterized by decreased vision that is often painful and may progress to blindness. Clinical features include inflammation, neovascularization, and persistent cornea epithelial defects. Successful strategies for treatment involve transplantation of grafts harvested from the limbus of the alternate healthy eye, called conjunctival-limbal autograft (CLAU) and transplantation of limbal cell sheets cultured from limbal biopsies, termed cultured limbal epithelial transplantation (CLET). In 2012, Sangwan and colleagues presented simple limbal epithelial transplantation (SLET), a novel transplantation technique that combines the benefits of CLAU and CLET and avoids the challenges associated with both. In SLET a small biopsy from the limbus of the healthy eye is divided and distributed over human amniotic membrane, which is placed on the affected cornea. Outgrowth occurs from each small explant and a complete corneal epithelium is typically formed within 2 weeks. Advantages of SLET include reduced risk of iatrogenic LSCD occurring in the healthy cornea at harvest; direct transfer circumventing the need for cell culture; and the opportunity to perform biopsy harvest and transplantation in one operation. Success so far using SLET is comparable with CLAU and CLET. Of note, 336 of 404 (83%) operations using SLET resulted in restoration of the corneal epithelium, whereas visual acuity improved in 258 of the 373 (69%) reported cases. This review summarizes the results of 31 studies published on SLET since 2012. Progress, advantages, challenges, and suggestions for future studies are presented.

Coordinated collective migration and asymmetric cell division in confluent human keratinocytes without wounding.

Epithelial sheet spreading is a fundamental cellular process that must be coordinated with cell division and differentiation to restore tissue integrity. Here we use consecutive serum deprivation and re-stimulation to reconstruct biphasic collective migration and proliferation in cultured sheets of human keratinocytes. In this system, a burst of long-range coordinated locomotion is rapidly generated throughout the cell sheet in the absence of wound edges. Migrating cohorts reach correlation lengths of several millimeters and display dependencies on epidermal growth factor receptor-mediated signaling, self-propelled polarized migration, and a G1/G0 cell cycle environment. The migration phase is temporally and spatially aligned with polarized cell divisions characterized by pre-mitotic nuclear migration to the cell front and asymmetric partitioning of nuclear promyelocytic leukemia bodies and lysosomes to opposite daughter cells. This study investigates underlying mechanisms contributing to the stark contrast between cells in a static quiescent state compared to the long-range coordinated collective migration seen in contact with blood serum.

Concise Review: Altered Versus Unaltered Amniotic Membrane as a Substrate for Limbal Epithelial Cells.

Limbal stem cell deficiency (LSCD) can result from a variety of corneal disorders, including chemical and thermal burns, infections, and autoimmune diseases. The symptoms of LSCD may include irritation, epiphora, blepharospasms, photophobia, pain, and decreased vision. There are a number of treatment options, ranging from nonsurgical treatments for mild LSCD to various forms of surgery that involve different cell types cultured on various substrates. Ex vivo expansion of limbal epithelial cells (LEC) involves the culture of LEC harvested either from the patient, a living relative, or a cadaver on a substrate in the laboratory. Following the transfer of the cultured cell sheet onto the cornea of patients suffering from LSCD, a successful outcome can be expected in approximately three out of four patients. The phenotype of the cultured cells has proven to be a key predictor of success. The choice of culture substrate is known to affect the phenotype. Several studies have shown that amniotic membrane (AM) can be used as a substrate for expansion of LEC for subsequent transplantation in the treatment of LSCD. There is currently a debate over whether AM should be denuded (i.e., de-epithelialized) prior to LEC culture, or whether this substrate should remain intact. In addition, crosslinking of the AM has been used to increase the thermal and mechanical stability, optical transparency, and resistance to collagenase digestion of AM. In the present review, we discuss the rationale for using altered versus unaltered AM as a culture substrate for LEC. Stem Cells Translational Medicine 2018;7:415-427.

Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets.

Cultured epidermal cell sheets (CES) containing undifferentiated cells are useful for treating skin burns and have potential for regenerative treatment of other types of epithelial injuries. The undifferentiated phenotype is therefore important for success in both applications. This study aimed to optimize a method for one-week storage of CES for their widespread distribution and use in regenerative medicine. The effect of storage temperatures 4 °C, 8 °C, 12 °C, 16 °C, and 24 °C on CES was evaluated. Analyses included assessment of viability, mitochondrial reactive oxygen species (ROS), membrane damage, mitochondrial DNA (mtDNA) integrity, morphology, phenotype and cytokine secretion into storage buffer. Lowest cell viability was seen at 4 °C. Compared to non-stored cells, ABCG2 expression increased between temperatures 8-16 °C. At 24 °C, reduced ABCG2 expression coincided with increased mitochondrial ROS, as well as increased differentiation, cell death and mtDNA damage. P63, C/EBPδ, CK10 and involucrin fluorescence combined with morphology observations supported retention of undifferentiated cell phenotype at 12 °C, transition to differentiation at 16 °C, and increased differentiation at 24 °C. Several cytokines relevant to healing were upregulated during storage. Importantly, cells stored at 12 °C showed similar viability and undifferentiated phenotype as the non-stored control suggesting that this temperature may be ideal for storage of CES.

Cultured epidermal stem cells in regenerative medicine.

Transplantation of cultured epidermal cell sheets (CES) has long been used to treat patients with burns, chronic wounds, and stable vitiligo. In patients with large area burns this can be a life-saving procedure. The ultimate goal, however, is to restore all normal functions of the skin and prevent scar formation. Increased focus on the incorporation of epidermal stem cells (EpiSCs) within CES transplants may ultimately prove to be key to achieving this. Transplanted EpiSCs contribute to restoring the complete epidermis and provide long-term renewal.Maintenance of the regenerative potential of EpiSCs is anchorage-dependent. The extracellular matrix (ECM) provides physical cues that are interpreted by EpiSCs and reciprocal signaling between cells and ECM are integrated to determine cell fate. Thus, the carrier scaffold chosen for culture and transplant influences maintenance of EpiSC phenotype and may enhance or detract from regenerative healing following transfer.Long-term effectiveness and safety of genetically modified EpiSCs to correct the severe skin blistering disease epidermolysis bullosa has been shown clinically. Furthermore, skin is gaining interest as an easily accessible source of adult epithelial stem cells potentially useful for restoration of other types of epithelia. This review highlights the role of EpiSCs in the current treatment of skin injury and disease, as well as their potential in novel regenerative medicine applications involving other epithelia.

Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia.

Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis.

BACKGROUND: To objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis. METHODS: A spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student's t-test and Pearson's correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution. RESULTS: Caspase-3 expression in staurosporine-incubated cells increased by 471% ± 182% compared to control (P=0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% ± 5%; P<0.001) and nuclear circumference (78 ± 3%; P<0.001), while nuclear form factor was larger (110% ± 1%; P<0.001). Cell nuclei from the staurosporine-group (R=1.12 ± 0.04; P<0.01) and the control (R=1.28 ± 0.03; P<0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P=0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r=-0.475; P<0.001), nuclear area divided by form factor (r=-0.470; P<0.001), nuclear circumference (r=-0.469; P<0.001), nuclear area (r=-0.445; P<0.001), nuclear form factor (r=0.410; P<0.001) and the nuclear area multiplied by form factor) (r=-0.377; P<0.001). CONCLUSIONS: Caspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947.