ABSTRACT
Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.
Subject(s)
Amnion , Epithelium, Corneal , Mesenchymal Stem Cells , Tissue Engineering , Amnion/cytology , Tissue Engineering/methods , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Epithelium, Corneal/cytology , Epithelium, Corneal/metabolism , Animals , Rabbits , Humans , Cells, Cultured , Limbus Corneae/cytology , Limbus Corneae/metabolism , Cell DifferentiationABSTRACT
By overcoming interspecies differences and mimicking the in vivo microenvironment, three-dimensional (3D) in vitro corneal models have become a significant novel tool in contemporary ophthalmic disease research. However, existing 3D corneal models struggle to replicate the actual human corneal environment, especially the dome-shaped physiological structure with adjustable curvature. Addressing these challenges, this study introduces a straightforward method for fabricating collagen/chitosan-alginate eyeball-shaped gel microspheres with a Janus structure via a two-phase aqueous system, used subsequently to construct in vitro 3D corneal epithelial tissue models. By adjusting the diameter ratio of collagen/chitosan to alginate droplets, we can create eyeball-shaped gel microspheres with varying curvatures. Human corneal epithelial cells were seeded on the surfaces of these microspheres, leading to the formation of in vitro 3D corneal epithelial tissues characterized by dome-like multilayers and tight junctions. Additionally, the model demonstrated responsiveness to UVB exposure through the secretion of reactive oxygen species (ROS) and proinflammatory factors. Therefore, we believe that in vitro 3D corneal epithelial tissue models with dome-shaped structures hold significant potential for advancing ophthalmic research.
Subject(s)
Alginates , Chitosan , Epithelium, Corneal , Microspheres , Humans , Epithelium, Corneal/cytology , Alginates/chemistry , Chitosan/chemistry , Collagen/chemistry , Tissue Engineering , Epithelial Cells/metabolism , Epithelial Cells/cytology , Gels/chemistry , Reactive Oxygen Species/metabolismABSTRACT
Purpose: The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Methods: Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). Results: The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. Conclusions: These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. Translational Relevance: The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.
Subject(s)
Amnion , Cell Movement , Cell Proliferation , Humans , Amnion/metabolism , Cells, Cultured , Limbus Corneae/metabolism , Limbus Corneae/cytology , Epithelium, Corneal/metabolism , Epithelium, Corneal/cytology , Wound Healing/physiology , Epithelial Cells/metabolism , Ophthalmologic Surgical Procedures/methods , Laminin/metabolism , Zonula Occludens-1 Protein/metabolismABSTRACT
Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.
Subject(s)
Cell Differentiation , Epithelium, Corneal , Limbus Corneae , Stem Cells , Humans , Stem Cells/cytology , Stem Cells/metabolism , Limbus Corneae/cytology , Epithelium, Corneal/cytology , Animals , Epithelial Cells/cytology , Epithelial Cells/metabolism , Cell ProliferationABSTRACT
PURPOSE: Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent. METHODS: The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively. RESULTS: Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial. CONCLUSIONS: Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.
Subject(s)
Benzalkonium Compounds , Cell Survival , Epithelium, Corneal , Mesenchymal Stem Cells , Preservatives, Pharmaceutical , Benzalkonium Compounds/toxicity , Benzalkonium Compounds/pharmacology , Humans , Culture Media, Conditioned , Mesenchymal Stem Cells/drug effects , Preservatives, Pharmaceutical/toxicity , Epithelium, Corneal/drug effects , Epithelium, Corneal/cytology , Cells, Cultured , Cell Survival/drug effects , Cell Proliferation/drug effectsABSTRACT
A widely used organophosphate flame retardant (OPFR), triphenyl phosphate (TPP), is frequently detected in various environmental media and humans. However, there is little known on the human corneal epithelium of health risk when exposed to TPP. In this study, human normal corneal epithelial cells (HCECs) were used to investigate the cell viability, morphology, apoptosis, and mitochondrial membrane potential after they were exposed to TPP, as well as their underlying molecular mechanisms. We found that TPP decreased cell viability in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 220 µM. Furthermore, TPP significantly induced HCEC apoptosis, decreased mitochondrial membrane potential in a dose-dependent manner, and changed the mRNA levels of the apoptosis biomarker genes (Cyt c, Caspase-9, Caspase-3, Bcl-2, and Bax). The results showed that TPP induced cytotoxicity in HCECs, eventually leading to apoptosis and changes in mitochondrial membrane potential. In addition, the caspase-dependent mitochondrial pathways may be involved in TPP-induced HCEC apoptosis. This study provides a reference for the human corneal toxicity of TPP, indicating that the risks of OPFR to human health cannot be ignored.
Subject(s)
Apoptosis , Cell Survival , Epithelium, Corneal , Flame Retardants , Membrane Potential, Mitochondrial , Mitochondria , Humans , Apoptosis/drug effects , Flame Retardants/toxicity , Flame Retardants/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Epithelium, Corneal/cytology , Membrane Potential, Mitochondrial/drug effects , Cell Survival/drug effects , Caspases/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Organophosphates/pharmacology , Organophosphates/toxicity , Cells, CulturedABSTRACT
PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.
Subject(s)
Cell Culture Techniques , Cell Differentiation , Epithelium, Corneal , Flow Cytometry , Mesenchymal Stem Cells , Humans , Culture Media, Conditioned , Epithelium, Corneal/cytology , Cell Differentiation/physiology , Flow Cytometry/methods , Mesenchymal Stem Cells/cytology , Cell Culture Techniques/methods , Amnion/cytology , Cells, Cultured , Keratin-3/metabolism , Keratin-3/analysis , Keratin-12/metabolism , Reproducibility of ResultsABSTRACT
Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-ß-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-ß-catenin and MAPK signaling pathways. The expression of ß-catenin, active ß-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.
Subject(s)
Epithelium, Corneal , MAP Kinase Signaling System , MicroRNAs , Stem Cells , Wnt Signaling Pathway , beta Catenin , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Epithelium, Corneal/metabolism , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Stem Cells/cytology , Stem Cells/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Purpose: To determine the role of transmembrane mucins in blocking fluorescein ingress to the corneal epithelium and its deficiency in contributing to corneal fluorescein punctate staining. Methods: A dry eye model was established by extirpating lacrimal and Harderian glands in rabbits to correlate the expression of mucins with fluorescein-stained areas on the corneal button using immunofluorescence. Expression of transmembrane mucins was promoted in human corneal epithelial cells (HCECs) by culturing with the mucin-promoting medium (MPM) or diquafosol treatment. Conversely, the expression of mucins was downregulated by knockdown with short hairpin RNA. The role of mucin1 extracellular domain in fluorescein ingress was further verified by overexpression of N-terminally truncated mucin1 in HCECs. Results: In the rabbit dry eye model, the expression level of mucin1 was significantly decreased in superficial corneal epithelial cells where fluorescein punctate staining was observed. Upregulation of mucin1 and mucin16 in HCECs promoted by MPM or by diquafosol treatment impeded intracellular fluorescein ingress. Downregulation of mucin1 and mucin16 enhanced fluorescence ingress in HCECs after fluorescein staining. Overexpression of truncated mucin1 did not alter the fluorescein intensity of fluorescein-stained HCECs, supporting the notion that the ability of mucin1 to block fluorescein ingress was primarily mediated by its extracellular domain. Minimal inherent expression of mucin16 in the rabbit cornea limited the validation of its role in blocking fluorescein ingress in vivo. Conclusion: Transmembrane mucin1 blocks fluorescein ingress in the corneal epithelium, explaining how fluorescein staining is positive when the level of transmembrane mucins is disturbed in dry eyes.
Subject(s)
Dry Eye Syndromes/metabolism , Fluorescein/metabolism , Fluorescent Dyes/metabolism , Mucin-1/physiology , Animals , Biological Transport/physiology , Blotting, Western , Cells, Cultured , Disease Models, Animal , Epithelium, Corneal/cytology , Epithelium, Corneal/metabolism , Flow Cytometry , Gene Expression , Gene Knockdown Techniques , Humans , Immunohistochemistry , RNA, Small Interfering/genetics , Rabbits , Real-Time Polymerase Chain ReactionABSTRACT
Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.
Subject(s)
Epithelial Cells/metabolism , Epithelium, Corneal/metabolism , Extracellular Vesicles/metabolism , MicroRNAs/metabolism , Stem Cells/metabolism , Cell Differentiation , Cell Line , Epithelial Cells/cytology , Epithelium, Corneal/cytology , Humans , Stem Cells/cytologyABSTRACT
Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.
Subject(s)
Acanthamoeba Keratitis/diagnosis , Acanthamoeba/immunology , Antibodies, Protozoan/analysis , Carboxylesterase/immunology , Culture Media, Conditioned/metabolism , Epithelium, Corneal/cytology , Acanthamoeba/classification , Acanthamoeba/growth & development , Acanthamoeba/isolation & purification , Animals , Antibodies, Protozoan/blood , Antibody Specificity , Carboxylesterase/administration & dosage , Carboxylesterase/genetics , Cell Line , Cells, Cultured , Contact Lenses/parasitology , Early Diagnosis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/parasitology , Epithelium, Corneal/metabolism , Epithelium, Corneal/parasitology , Humans , Immunization , Male , Mice , Protozoan Proteins/administration & dosage , Protozoan Proteins/genetics , Protozoan Proteins/immunologyABSTRACT
Human corneal epithelial cells are needed to study corneal pathophysiology in vitro. Due to the limitations of cell lines, the use of primary cells is highly desirable, but the scarcity of human tissues, along with ethical issues, make it difficult to accomplish all required experiments. In advanced surface ablation (ASA), the central corneal epithelium is removed and discarded. We hypothesized that ASA samples could be used to perform in vitro assays. In this study, 29 samples from patients undergoing ASA were recovered in supplemented DMEM/F12 culture medium, RIPA buffer, or RLT lysis buffer. The first aim was to determine whether cells could be maintained in culture. Although with the explant technique, tissue pieces did not attach to the culture surface, after disaggregation, cells showed high viability (90.0 ± 6.0%), attached to plates, and remained viable for up to 14 days. The second aim was to elucidate if ASA samples could be used to study protein or gene expression. Cytokeratin-3, ZO-1, Ki67, and E-cadherin protein expression were confirmed by immunofluorescence. Total protein (485.8 ± 115.8 µg) was isolated from cells in RIPA buffer, and GAPDH was detected by Western blotting, indicating that samples are adequate for protein studies. RNA (9.0 ± 3.6 µg) was isolated from samples in RLT lysis buffer, and GAPDH gene expression was studied by PCR, confirming that samples were also suitable for gene expression studies. These results suggest that samples obtained from corneal surface ablation procedures may constitute a valuable source of human cells to accomplish in vitro studies.
Subject(s)
Corneal Surgery, Laser , Epithelium, Corneal/cytology , Adult , Antigens, CD/metabolism , Biomarkers/metabolism , Blotting, Western , Cadherins/metabolism , Cell Count , Cell Culture Techniques , Cell Survival , Electrophoresis, Polyacrylamide Gel , Epithelium, Corneal/metabolism , Eye Proteins/metabolism , Female , Humans , Keratin-3/metabolism , Ki-67 Antigen/metabolism , Male , Microscopy, Fluorescence , Surgical Flaps , Zonula Occludens-1 Protein/metabolismABSTRACT
PURPOSE: To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK). METHODS: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period. RESULTS: The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients. CONCLUSIONS: Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration.
Subject(s)
Aniridia/complications , Corneal Diseases/surgery , Epithelium, Corneal/cytology , HLA Antigens/immunology , Limbus Corneae/cytology , Stem Cell Transplantation/methods , Stem Cells/immunology , Adult , Aged , Cells, Cultured , Corneal Diseases/diagnosis , Corneal Diseases/etiology , Epithelium, Corneal/immunology , Female , Follow-Up Studies , Graft Survival , Humans , Limbus Corneae/immunology , Male , Middle Aged , Retrospective Studies , Stem Cells/cytology , Time Factors , Transplantation, Autologous , Visual Acuity , Young AdultABSTRACT
Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.
Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Epithelium, Corneal/cytology , Epithelium, Corneal/metabolism , Gene Expression Regulation, Developmental/drug effects , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Biomarkers , Cell Line , Cell Lineage/genetics , Cells, Cultured , HumansABSTRACT
A recombinant formulation of silk fibroin containing the arginine-glycine-aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal-epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.
Subject(s)
Cornea/cytology , Fibroins/chemistry , Tissue Scaffolds/chemistry , Animals , Biomechanical Phenomena , Bombyx/chemistry , Bombyx/genetics , Cell Adhesion , Cell Proliferation , Cells, Cultured , Coculture Techniques , Corneal Stroma/cytology , Epithelium, Corneal/cytology , Fibroins/genetics , Humans , Limbus Corneae/cytology , Membranes, Artificial , Microscopy, Confocal , Oligopeptides/chemistry , Oligopeptides/genetics , Permeability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Tissue EngineeringABSTRACT
The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.
Subject(s)
Corneal Stroma/metabolism , Limbus Corneae/metabolism , Transcriptome , Aged , Biomarkers/metabolism , Endothelium, Corneal/cytology , Epithelium, Corneal/cytology , Female , Humans , Limbus Corneae/cytology , Male , Middle Aged , Single-Cell Analysis , Stem Cell Niche , Young AdultABSTRACT
Amniotic membrane (AM) transplantation is often used as a treatment for corneal repair, but AM is prone to dissolving and shedding after surgery; multiple transplants will cause pain and financial burden. In this work, human amniotic membrane was firstly decellularized to obtain an AM extracellular matrix (dAM). This dAM was homogenized and extracted to obtain the dAM extract (simplified as dAME). Different forms of administration for corneal injury were performed as liquid drops (diluted dAME), in situ gels (using temperature-dependent Poloxamer 407 as the matrix), and tablets (poly(vinyl alcohol) as the matrix). The cytocompatibility of dAME was evaluated using corneal epithelial cells, corneal stromal cells and fibroblasts as cell models. The results showed that dAME is biocompatible to all these cells. Cells exhibited normal morphology and growth state at a dAME concentration of up to 160 µg mL-1. In vivo, dAME exhibited increased wound healing efficiency in severe corneal injury, being characterized with a shorter healing time for epithelium and a faster recovery for stromal opacity and thickness, compared with those of the control eyes. Different forms of administration have different effects on corneal repair; among them, in situ gels achieved the best therapeutic efficiency. Their biological mechanism was detected via quantitative real-time polymerase chain reaction (qRT-PCR) technology. It was confirmed that dAME plays important roles in promoting the mRNA expression of leucine-rich and immunoglobulin-like domains 1 (LRIG1) and in inhibiting the mRNA of transforming growth factor-ß1 (TGF-ß1).
Subject(s)
Amnion , Corneal Injuries/therapy , Epithelium, Corneal/cytology , Tissue Extracts/therapeutic use , Animals , Cell Survival , Cells, Cultured , Drug Administration Schedule , Fibroblasts , Humans , Rabbits , Stromal CellsABSTRACT
Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFß. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.
Subject(s)
Epithelium, Corneal/metabolism , Eye/physiopathology , Limbus Corneae/metabolism , Stem Cell Niche/physiology , Stem Cells/metabolism , Animals , Epithelium, Corneal/cytology , Eye/metabolism , Humans , Limbus Corneae/cytology , Mechanotransduction, Cellular/physiology , Stem Cells/cytologyABSTRACT
In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.
Subject(s)
Clusterin/genetics , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Gene Expression , Signal Transduction/genetics , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , Tissue Engineering/methods , Transcription Factor AP-1/metabolism , Wound Healing/genetics , Adult , Aged , Cells, Cultured , Child , Clusterin/metabolism , Epithelium, Corneal/metabolism , Fibroblasts/metabolism , Humans , Middle Aged , Promoter Regions, Genetic , Tissue Donors , TransfectionABSTRACT
BACKGROUND: Age-related changes affecting the ocular surface cause vision loss in the elderly. Cisd2 deficiency drives premature aging in mice as well as resulting in various ocular surface abnormalities. Here we investigate the role of CISD2 in corneal health and disease. METHODS: We studied the molecular mechanism underlying the ocular phenotypes brought about by Cisd2 deficiency using both Cisd2 knockout (KO) mice and a human corneal epithelial cell (HCEC) cell line carrying a CRISPR-mediated CISD2KO background. We also develop a potential therapeutic strategy that targets the Ca2+ signaling pathway, which has been found to be dysregulated in the corneal epithelium of subjects with ocular surface disease in order to extend the mechanistic findings into a translational application. FINDINGS: Firstly, in patients with corneal epithelial disease, CISD2 is down-regulated in their corneal epithelial cells. Secondly, using mouse cornea, Cisd2 deficiency causes a cycle of chronic injury and persistent repair resulting in exhaustion of the limbal progenitor cells. Thirdly, in human corneal epithelial cells, CISD2 deficiency disrupts intracellular Ca2+ homeostasis, impairing mitochondrial function, thereby retarding corneal repair. Fourthly, cyclosporine A and EDTA facilitate corneal epithelial wound healing in Cisd2 knockout mice. Finally, cyclosporine A treatment restores corneal epithelial erosion in patients with dry eye disease, which affects the ocular surface. INTERPRETATION: These findings reveal that Cisd2 plays an essential role in the cornea and that Ca2+ signaling pathways are potential targets for developing therapeutics of corneal epithelial diseases. FUNDING: This study was supported by the Ministry of Science and Technology (MOST) and Chang Gung Medical Research Foundation, Taiwan.