Production and Limbal Lineage Commitment of Aniridia Patient-Derived Induced Pluripotent Stem Cells.

Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development, and aniridia-associated keratopathy (AAK). This progressive corneal opacification resembles late-onset limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. The factors leading to AAK are not known and defects in native LSC differentiation and/or features leading to ocular surface dysfunction like inflammation and loss of innervation could contribute to development of AAK. Here, we produced induced pluripotent stem cells (hiPSC) from 3 AAK patients and examined whether PAX6 haploinsufficiency affects LSC lineage commitment. During LSC differentiation, characterization of the AAK lines showed lowered PAX6 expression as compared to wild type (WT) controls and expression peak of PAX6 during early phase of differentiation was detected only in the WT hiPSC lines. Whether it reflects developmental regulation remains to be studied further. Nevertheless, the AAK-hiPSCs successfully differentiated toward LSC lineage, in line with the presence of LSCs in young patients before cell loss later in life. In addition, patient-specific LSCs showed similar wound healing capacity as WT cells. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected without clear correlation to AAK. As development and maintenance of corneal epithelium involves an interplay between LSCs and their environment, the AAK-hiPSCs generated here can be further used to study the crosstalk between LSCs and limbal niche including, eg, corneal immune cells, stroma cells, and neurons.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Phenotypic and functional characterization of aqueous humor derived extracellular vesicles.

Extracellular vesicles (EVs) are double membrane vesicles, abundant in all biological fluids. However, the characterization of EVs in aqueous humor (AH) is still limited. The aim of the present work was to characterize EVs isolated from AH (AH-EVs) in terms of surface markers of cellular origin and functional properties. We obtained AHs from patients with cataract undergoing surgical phacoemulsification and insertion of intraocular lenses (n = 10). Nanoparticle tracking analysis, electron microscopy, super resolution microscopy and bead-based cytofluorimetry were used to characterize EVs from AH. Subsequently, we investigated the effects of AH-EVs on viability, proliferation and wound healing of human immortalized keratinocyte (HaCaT) cells in vitro in comparison with the effect of mesenchymal stromal cell-EVs (MSC-EVs). AH-EVs had a mean size of around 100 nm and expressed the classical tetraspanins (CD9, CD63 and CD81). Super resolution microscopy revealed co-expression of CD9, CD63 and CD81. Moreover, cytofluorimetric analysis highlighted the expression of mesenchymal, stem, epithelial and endothelial markers. In the in vitro wound healing assay on HaCaT cells, AH-EVs induced a significantly faster wound repair, comparable to the effects of MSC-EVs, and promoted HaCaT cell viability and proliferation. We provide evidence, herein, of the possible AH-EV origin from stromal cells, limbal epithelial/stem cells, ciliary epithelium and corneal endothelium. In addition, we showed their in vitro proliferative and regenerative capacities.

Long-term PM2.5 exposure disrupts corneal epithelial homeostasis by impairing limbal stem/progenitor cells in humans and rat models.

BACKGROUND: Limbal stem/progenitor cells (LSPCs) play a crucial role in maintaining corneal health by regulating epithelial homeostasis. Although PM2.5 is associated with the occurrence of several corneal diseases, its effects on LSPCs are not clearly understood. METHODS: In this study, we explored the correlation between PM2.5 exposure and human limbal epithelial thickness measured by Fourier-domain Optical Coherence Tomography in the ophthalmologic clinic. Long- and short-term PM2.5 exposed-rat models were established to investigate the changes in LSPCs and the associated mechanisms. RESULTS: We found that people living in regions with higher PM2.5 concentrations had thinner limbal epithelium, indicating the loss of LSPCs. In rat models, long-term PM2.5 exposure impairs LSPCs renewal and differentiation, manifesting as corneal epithelial defects and thinner epithelium in the cornea and limbus. However, LSPCs were activated in short-term PM2.5-exposed rat models. RNA sequencing implied that the circadian rhythm in LSPCs was perturbed during PM2.5 exposure. The mRNA level of circadian genes including Per1, Per2, Per3, and Rev-erbα was upregulated in both short- and long-term models, suggesting circadian rhythm was involved in the activation and dysregulation of LSPCs at different stages. PM2.5 also disturbed the limbal microenvironment as evidenced by changes in corneal subbasal nerve fiber density, vascular density and permeability, and immune cell infiltration, which further resulted in the circadian mismatches and dysfunction of LSPCs. CONCLUSION: This study systematically demonstrates that PM2.5 impairs LSPCs and their microenvironment. Moreover, we show that circadian misalignment of LSPCs may be a new mechanism by which PM2.5 induces corneal diseases. Therapeutic options that target circadian rhythm may be viable options for improving LSPC functions and alleviating various PM2.5-associated corneal diseases.

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering.

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.

The Role of Sensory Innervation in Homeostatic and Injury-Induced Corneal Epithelial Renewal.

The cornea is the window through which we see the world. Corneal clarity is required for vision, and blindness occurs when the cornea becomes opaque. The cornea is covered by unique transparent epithelial cells that serve as an outermost cellular barrier bordering between the cornea and the external environment. Corneal sensory nerves protect the cornea from injury by triggering tearing and blink reflexes, and are also thought to regulate corneal epithelial renewal via unknown mechanism(s). When protective corneal sensory innervation is absent due to infection, trauma, intracranial tumors, surgery, or congenital causes, permanent blindness results from repetitive epithelial microtraumas and failure to heal. The condition is termed neurotrophic keratopathy (NK), with an incidence of 5:10,000 people worldwide. In this report, we review the currently available therapeutic solutions for NK and discuss the progress in our understanding of how the sensory nerves induce corneal epithelial renewal.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (ß-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study.

PURPOSE: Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. METHODS: LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30 s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15 s, 30 s, 60 s, 90 s, and 120 s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. RESULTS: In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). CONCLUSIONS: Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5 days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells.

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down). Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot. DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone. Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus.

The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117-CD90+), LM (CD117+CD90-), and LEPC (CD117-CD90-). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4-5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63+ and Ki67+ cells and decreased CK12+ cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.

Influence of graft vascularization on graft survival following homologous limbo-keratoplasty.

PURPOSE: Limbo-keratoplasty enables visual improvement and limbal stem cell transplantation at the same. During follow-up, most grafts show vascularization of the limbus. However, it is unclear whether vascularization is harmful due to immunologic effects or helpful to nourish the limbal stem cells and is therefore necessary for a clear graft. The aim of our study is to analyze the influence of graft vascularization on graft survival following homologous limbo-keratoplasty. METHODS: In this retrospective study, we assessed all consecutive limbo-keratoplasties performed in our hospital. All eyes with suitable photo-documentation were included and divided into two groups (limbal stem cell deficiency and corneal dystrophy). We categorized the grade of vascularization (0, 1, 2, 3, 3b) and analyzed clear graft survival, recurrence of the underlying disease and the endothelial cell density (ECD) with regard to the reason for the graft. Event rates were estimated with the Kaplan-Meier method. RESULTS: A total of 79 eyes with limbal stem cell deficiency and 15 with corneal dystrophies were analyzed. A high degree of graft vascularization had a tendency for better graft survival in limbal stem cell deficiency, whereas in corneal dystrophies, grafts with no vascularization had preferable outcomes. Recurrence-free graft survival was only seen in grade 1 and 3 vascularization in corneal dystrophies. CONCLUSION: Vascularization of the limbus seems to have an impact on the long-term outcome of limbo-keratoplasty. The effect seems to be favorable in limbal stem cell deficiency and on recurrence rates in corneal dystrophies. However, the latter might be overshadowed by an unfavorable immunologic effect in corneal dystrophies where the baseline immunologic risk profile is commonly more favorable than in limbal stem cell deficiency.

ALT (allogeneic limbal transplantation): a new surgical technique for limbal stem cell deficiency.

PURPOSE: Limbal stem cell deficiency (LSCD) is a rare but extremely relevant disease of the eye. LSCD patients often require a variety of surgical procedures, including keratoplasty in some cases. However, the outcome of these surgeries, including opacification and revascularization, is often frustrating due to LSCD relapse. METHODS: We developed a new surgical technique for the treatment of LSCD in which partial allogenic limbal transplantation (ALT) is carried out as part of penetrating keratoplasty (PK). After the PK, 1-8 slices from the limbal tissue of the donor graft are prepared and placed under the double running sutures attaching the corneal graft. This procedure was performed on 14 patients with LSCD, caused by severe ocular burn in 5 cases and by infection in 9. Between one and eight limbal transplants were used depending on the extension of the LSCD. RESULTS: All 14 patients showed stable or increased visual acuity after the ALT surgery compared to their preoperative visual acuity. All of the grafts were integrated into the superficial corneal layers without progression of corneal vascularization beyond the limbal grafts. The median follow-up period was 12 months on average. CONCLUSION: The ALT method seems to be a promising surgical procedure for the treatment of patients with LSCD. It can be properly carried out in the context of keratoplasty and does not require a separate donor tissue. The ALT grafts may offer the possibility of constructing a new limbal region, resulting in stable or even increased visual acuity and the absence of corneal vascularization.

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD.

Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Comparison of different methods to isolate mouse limbal epithelial cells.

Limbal stem cells (LSCs) are the stem cell reservoir for corneal epithelium. The protocol to isolate LSCs from human cornea has been examined and optimized. However, the isolation protocol has not been optimized for mouse cornea, which is crucial for the downstream cell analysis. Here we compared four different isolation methods evolved from the previous reports to obtain mouse limbal epithelial cells which are heterogeneous and contain LSCs in a single-cell suspension: (1) the dissected limbal rim was cut into pieces and digested by 10-cycle incubation in trypsin; (2) after the removal of corneal epithelium by a rotating bur, the remaining eyeball was incubated in dispase at 4 °C for overnight to obtain limbal epithelial sheet, followed by trypsin digestion into a single-cell suspension; (3) same as method 2 except that the incubation was in dispase at 37 °C for 2h and an additional collagenase incubation at 37 °C for 20 min; (4) same as method 3 except that the corneal epithelium was punctured by a 1.5 mm trephine instead of being removed by a rotating bur. Method 1 showed the lowest cell yield, the lowest percentage of single cells, and the lowest number of limbal epithelial stem/progenitor cells in the harvested cells among the four methods, thus not a recommended protocol. Method 2, 3, and 4 isolated a comparable number of K14+ and p63α-bright stem/progenitor cells per eye. The remaining eye globe after cell collection in the three methods showed a complete removal of limbal epithelium albeit different extent of corneal and limbal stromal digestion. Among the three methods, method 2 showed a higher cell viability than method 4; method 3 yielded the lowest cell number; method 4 led to the highest percentage of single cells in cell suspension. Results suggest that method 2, 3, and 4 are preferred methods to isolate heterogeneous-LSCs from mouse corneas.

ß-blocker eye drops affect ocular surface through ß2 adrenoceptor of corneal limbal stem cells.

BACKGROUND: Topical application of ß-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of ß-blocker eye drops on corneal epithelial wound healing. METHODS: Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL ß-blocker levobunolol, ß1-adrenoceptor (ß1AR)-specific antagonist atenolol or ß2-adrenoceptor (ß2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining. RESULTS: Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. CONCLUSION: ß-blocker eye drops impaired corneal wound healing by inhibiting the ß2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective ß1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.

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. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.