Targeting limbal epithelial stem cells: master conductors of corneal epithelial regeneration from the bench to multilevel theranostics.

The cornea is the outermost layer of the eye and plays an essential role in our visual system. Limbal epithelial stem cells (LESCs), which are localized to a highly regulated limbal niche, are the master conductors of corneal epithelial regeneration. Damage to LESCs and their niche may result in limbal stem cell deficiency (LSCD), a disease confused ophthalmologists so many years and can lead to corneal conjunctivalization, neovascularization, and even blindness. How to restore the LESCs function is the hot topic for ocular scientists and clinicians around the world. This review introduced LESCs and the niche microenvironment, outlined various techniques for isolating and culturing LESCs used in LSCD research, presented common diseases that cause LSCD, and provided a comprehensive overview of both the diagnosis and multiple treatments for LSCD from basic research to clinical therapies, especially the emerging cell therapies based on various stem cell sources. In addition, we also innovatively concluded the latest strategies in recent years, including exogenous drugs, tissue engineering, nanotechnology, exosome and gene therapy, as well as the ongoing clinical trials for treating LSCD in recent five years. Finally, we highlighted challenges from bench to bedside in LSCD and discussed cutting-edge areas in LSCD therapeutic research. We hope that this review could pave the way for future research and translation on treating LSCD, a crucial step in the field of ocular health.

Outcomes and Complications of Limbal Stem Cell Allograft Transplantation: A Report by the American Academy of Ophthalmology.

PURPOSE: To review the published literature on the safety and outcomes of keratolimbal allograft (KLAL) transplantation and living-related conjunctival limbal allograft (lr-CLAL) transplantation for bilateral severe/total limbal stem cell deficiency (LSCD). METHODS: Literature searches were last conducted in the PubMed database in February 2023 and were limited to the English language. They yielded 523 citations; 76 were reviewed in full text, and 21 met the inclusion criteria. Two studies were rated level II, and the remaining 19 studies were rated level III. There were no level I studies. RESULTS: After KLAL surgery, best-corrected visual acuity (BCVA) improved in 42% to 92% of eyes at final follow-up (range, 12-95 months). The BCVA was unchanged in 17% to 39% of eyes and decreased in 8% to 29% of eyes. Two of 14 studies that evaluated the results of KLAL reported a notable decline in visual acuity over time postoperatively. Survival of KLAL was variable, ranging from 21% to 90% at last follow-up (range, 12-95 months) and decreased over time. For patients undergoing lr-CLAL surgery, BCVA improved in 31% to 100% of eyes at final follow-up (range, 16-49 months). Of the 9 studies evaluating lr-CLAL, 4 reported BCVA unchanged in 30% to 39% of patients, and 3 reported a decline in BCVA in 8% to 10% of patients. The survival rate of lr-CLAL ranged from 50% to 100% at final follow-up (range, 16-49 months). The most common complications were postoperative elevation of intraocular pressure, persistent epithelial defects, and acute allograft immune rejections. CONCLUSIONS: Given limited options for patients with bilateral LSCD, both KLAL and lr-CLAL are viable choices that may provide improvement of vision and ocular surface findings. The studies trend toward a lower rejection rate and graft failure with lr-CLAL. However, the level and duration of immunosuppression vary widely between the studies and may impact allograft rejections and long-term graft survival. Complications related to immunosuppression are minimal. Repeat surgery may be needed to maintain a viable ocular surface. Reasonable long-term success can be achieved with both KLAL and lr-CLAL with appropriate systemic immunosuppression. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Long-term ocular surface stability in conjunctivolimbal autograft and ocular surface safety in the donor eyes.

PURPOSE: The current study is aimed to present the long-term results of the patients who underwent conjunctivolimbal autograft (CLAU) as the primary operation in unilateral limbal stem cell deficiency and the ocular surface safety of the donor eyes. The patients were followed up for five years or longer. METHODS: The records of all patients who underwent CLAU as the primary operation were retrospectively analyzed. Additional ocular surface operations, ocular surface stability, best-corrected visual acuity (BCVA), and ocular surface status of the donor eyes were investigated. RESULTS: The mean age of the patients at the time of transplantation was 35.07 ± 12.9 (12-60). Twenty-nine eyes of 29 patients were followed up for an average of 97.82 ± 34.45 (60-186) months. Additional ocular surface operation was required in 27.58% (8/29) of the eyes in order to achieve a stable ocular surface. Ocular surface stability was achieved in 82.75% (24/29) of the eyes at the end of the follow-up period. BCVA increased from 1.78 ± 0.82 to 0.91 ± 0.92 logMAR at the last visit (p < 0.001). Corneal ectasia and vascularization developed in one donor eye in the fifth postoperative year. CONCLUSIONS: CLAU tissues provide ocular surface stability with a successful vision result in the long term. CLAU theoretically carries risks including limbal stem cell deficiency in the donor eye. In the long-term follow-up of donor eyes after CLAU, ectasia and limbal stem cell deficiency were observed in one eye.

Limbal graft transplantation: a rare implementation in pediatric limbal stem cell deficiency.

PURPOSE: To evaluate limbal graft transplantation success in pediatric patients with chemical injury-induced limbal stem cell deficiency (LSCD) using the 'LSCD Working Group' staging system. METHODS: Medical records of 11 eyes of 11 children who underwent limbal graft transplantation (limbal autograft/limbal allograft) were included. Surgical success was defined as improvement in the post-operative 1st year LSCD stage. RESULTS: The mean age was 12 ± 5 (4-17) years. Causative agent was alkaline in 4(36.4%) and acid in 3(27.2%) patients. Limbal autograft was performed in 9 (81.8%) eyes with unilateral LSCD, and allograft transplantation was performed in 2 (18.2%) eyes with bilateral LSCD. The mean follow-up time was 33.89 ± 30.73 (12-102.33) months. The overall limbal graft transplantation success rate was 72.7%. Among 9 patients who receive limbal autograft, 8 had improvement in post-operative LSCD stage, 1 had stable LSCD stage. Of the 2 patients who receive limbal allograft, post-operative LSCD stage remained the same in 1 and worsened in 1 patient. The mean time between injury and the surgery was 30.47 ± 30.08 (7-108.47) months. Penetrating keratoplasty was performed in 3 (27.2%) of 11 patients following limbal graft transplantation. CONCLUSION: Management of LSCD in children is challenging and appears to be somewhat different from that of adults. Limited data in the literature indicate that cultivated or simple limbal epithelial transplantations (CLET/SLET) are primarily preferred in children. Although the tendency to take small tissue from the healthy eye is noteworthy, conventional limbal allograft and autograft transplantations also show promising results without any further complications in at least 1 year follow-up period.

[Experimental evaluation of the efficacy of tissue-engineered constructs in the treatment of limbal stem cell deficiency]. / Eksperimental'naya otsenka effektivnosti primeneniya tkaneinzhenernoi konstruktsii v lechenii limbal'noi nedostatochnosti.

Limbal stem cell deficiency (LSCD) is one of the leading factors negatively affecting the success of keratoplasty, and its treatment remains an urgent problem in ophthalmology. With the development of regenerative medicine, one of the promising approaches is the transplantation of tissue-engineered constructs from cultured limbal stem cells (LSCs) in biopolymer carriers. PURPOSE: This study was conducted to develop an experimental model of LSCD and evaluate the effectiveness of transplantation of a tissue-engineered construct consisting of cultured cells containing a population of LSCs and a collagen carrier. MATERIAL AND METHODS: The study was performed on 12 rabbits and included several stages. At the first stage, the physiological effects of collagen matrix implantation into the limbal zone were studied. At the second stage, tissue-engineered constructs consisting of LSCs on a collagen matrix were formed and their effect on the regeneration processes in the experimental LSCD model was analyzed. The animals were divided into 2 groups: surgical treatment (transplantation of the tissue-engineered construct) was used in the experimental group, and conservative treatment was used in the control group. Slit-lamp biomicroscopy with photo-registration, fluorescein corneal staining, optical coherence tomography of the anterior segment of the eye, and impression cytology were used to assess the results. RESULTS: No side reactions were observed after implantation of the collagen matrix into the limbal zone. One month after surgical treatment of the LSCD model in the experimental group, complete epithelization with minor manifestations of epitheliopathy was observed. In the control group, erosion of the corneal epithelium was noted. The time of corneal epithelization in the experimental and control groups was 9.2±2.95 and 46.20±12.07 days, respectively (p=0.139). According to the data of impression cytology, in the experimental group there were no goblet cells in the central part of the cornea, which indicates the restoration of corneal type epithelial cells, in contrast to the control group. CONCLUSION: Transplantation of a tissue-engineered construct from cultured limbal cells on a collagen membrane should be considered as a promising method for the treatment of limbal stem cell deficiency.

Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency.

PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Insights into mustard gas keratopathy- characterizing corneal layer-specific changes in mice exposed to nitrogen mustard.

Exposure to mustard agents, such as sulfur mustard (SM) and nitrogen mustard (NM), often results in ocular surface damage. This can lead to the emergence of various corneal disorders that are collectively referred to as mustard gas keratopathy (MGK). In this study, we aimed to develop a mouse model of MGK by using ocular NM exposure, and describe the subsequent structural changes analyzed across the different layers of the cornea. A 3 µL solution of 0.25 mg/mL or 5 mg/mL NM was applied to the center of the cornea via a 2-mm filter paper for 5 min. Mice were evaluated prior to and after exposure on days 1, 3, 7, 14, and 28 for 4 weeks using slit lamp examination with fluorescein staining. Anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) tracked changes in the epithelium, stroma, and endothelium of the cornea. Histologic evaluation was used to examine corneal cross-sections collected at the completion of follow-up. Following exposure, mice experienced central corneal epithelial erosion and thinning, accompanied by a decreased number of nerve branches in the subbasal plexus and increased activated keratocytes in the stroma in both dosages. The epithelium was recovered by day 3 in the low dose group, followed by exacerbated punctuate erosions alongside persistent corneal edema that arose and continued onward to four weeks post-exposure. The high dose group showed persistent epitheliopathy throughout the study. The endothelial cell density was reduced, more prominent in the high dose group, early after NM exposure, which persisted until the end of follow-up, along with increased polymegethism and pleomorphism. Microstructural changes in the central cornea at 4 weeks post-exposure included dysmorphic basal epithelial cells and reduced epithelial thickness, and in the limbal cornea included decreased cellular layers. We present a mouse model of MGK using NM that successfully replicates ocular injury caused by SM in humans who have been exposed to mustard gas.

Development and validation of a reliable rabbit model of limbal stem cell deficiency by mechanical debridement using an ophthalmic burr.

A simple and reproducible method is necessary to generate reliable animal models of limbal stem cell deficiency (LSCD) for assessing the safety and efficacy of new therapeutic modalities. This study aimed to develop and validate a rabbit model of LSCD through mechanical injury. The corneal and limbal epithelium of New Zealand White rabbits (n = 18) were mechanically debrided using an ophthalmic burr (Algerbrush II) with a 1.0-mm rotating head after 360° conjunctival peritomy. The debrided eyes were serially evaluated for changes in corneal opacity, neo-vascularization, epithelial defect and corneal thickness using clinical photography, slit lamp imaging, fluorescein staining, and anterior segment optical coherence tomography scanning (AS-OCT). Following this, an assessment of histopathology and phenotypic marker expression of the excised corneas was conducted. The experimental eyes were grouped as mild (n = 4), moderate (n = 10), and severe (n = 4) based on the grade of LSCD. The moderate group exhibited abnormal epithelium, cellular infiltration in the stroma, and vascularization in the central, peripheral, and limbal regions of the cornea. The severe group demonstrated central epithelial edema, peripheral epithelial thinning with sparse goblet cell population, extensive cellular infiltration in the stroma, and dense vascularization in the limbal region of the cornea. A significant decrease in the expression of K12 and p63 (p < 0.0001) was observed, indicating the loss of corneal epithelium and limbal epithelial stem cells in the LSCD cornea. This study demonstrates that the Alger brush-induced mechanical debridement model provides a reliable model of LSCD with comprehensive clinic-pathological features and that is well suited for evaluating novel therapeutic and regenerative approaches.

Insights into mustard gas keratopathy: Characterizing corneal layer-specific changes in mice exposed to nitrogen mustard.

Exposure to mustard agents, such as sulfur mustard (SM) and nitrogen mustard (NM), often results in ocular surface damage. This can lead to the emergence of various corneal disorders that are collectively referred to as mustard gas keratopathy (MGK). In this study, we aimed to develop a mouse model of MGK by using ocular NM exposure, and describe the subsequent structural changes analyzed across the different layers of the cornea. A 3 µL solution of 0.25 mg/mL NM was applied to the center of the cornea via a 2-mm filter paper for 5 min. Mice were evaluated prior to and after exposure on days 1 and 3, and weekly for 4 weeks using slit lamp examination with fluorescein staining. Anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) tracked changes in the epithelium, stroma, and endothelium of the cornea. Histologic evaluation and immunostaining were used to examine corneal cross-sections collected at the completion of follow-up. A biphasic ocular injury was observed in mice exposed to NM, most prominent in the corneal epithelium and anterior stroma. Following exposure, mice experienced central corneal epithelial erosions and thinning, accompanied by a decreased number of nerve branches in the subbasal plexus and increased activated keratocytes in the stroma. The epithelium was recovered by day 3, followed by exacerbated punctuate erosions alongside persistent stromal edema that arose and continued onward to four weeks post-exposure. The endothelial cell density was reduced on the first day after NM exposure, which persisted until the end of follow-up, along with increased polymegethism and pleomorphism. Microstructural changes in the central cornea at this time included dysmorphic basal epithelial cells, and in the limbal cornea included decreased cellular layers and p63+ area, along with increased DNA oxidization. We present a mouse model of MGK using NM that successfully replicates ocular injury caused by SM in humans who have been exposed to mustard gas. Our research suggests DNA oxidation contributes to the long-term effects of nitrogen mustard on limbal stem cells.

FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement.

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/- mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6 +/- mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6 +/- mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Clinical efficacy of a new surgical technique of oral mucosal epithelial transplantation for severe ocular surface disorders.

BACKGROUND: Severe ocular surface disorders are one of the major blinding diseases, and a paucity of original tissue obscures successful reconstruction. We developed a new surgical technique of direct oral mucosal epithelial transplantation (OMET) to reconstruct severely damaged ocular surfaces in 2011. This study elaborates on the clinical efficacy of OMET. METHODS: A retrospective review of patients with severe ocular surface disorders who underwent OMET from 2011 to 2021 at the Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine was conducted. Patients who were followed up for at least 3 months postoperatively and had sufficient pre or postoperative records were included. Surgical efficacy was evaluated by comparing the best-corrected visual acuity (BCVA), corneal transparency, neovascularization grade, and symblepharon grade. Additionally, postoperative ocular surface impression cytology was used to study the morphology of the newborn epithelial cells. RESULTS: Forty-eight patients (49 eyes; mean age: 42.55 ± 12.40 years, range:12-66 years) were enrolled in the study. The etiology included chemical burns (30 eyes), thermal burns (16 eyes), explosive injuries (1 eye), Stevens-Johnson syndrome (1 eye), and multiple pterygiums (1 eye). The mean follow-up period was 25.97 ± 22.99 months. Postoperatively, 29 eyes (59.18%) showed improved corneal transparency, 26 eyes (53.06%) had improved BCVA, 47 eyes (95.92%) had a stable epithelium until the final follow-up, 44 eyes (89.80%) had a reduced neovascularization grade. Of the 20 eyes with preoperative symblepharon, 15 (75%) were completely resolved, and five (25%) were partially resolved. Impression cytological studies showed no postoperative conjunctival invasion onto the corneal surface. CONCLUSIONS: OMET is a safe and effective surgical technique for reconstruction in severe ocular surface disorder by maintaining a stable epithelium and reducing the neovascularization and symblepharon grade.

Derivation of Limbal Stem Cells from Human Adult Mesenchymal Stem Cells for the Treatment of Limbal Stem Cell Deficiency.

Approximately 10 million individuals have blindness due to limbal stem cell (LSCs) deficiency, one of the most challenging problems in ophthalmology. To replenish the LSC pool, an autologous extraocular cell source is appropriate, thereby avoiding the risk of immune rejection, the need for immunosuppression and the risk of damaging the contralateral eye. In recent years, adipose-derived mesenchymal stem cells (ADSCs) have been a key element in ocular regenerative medicine. In this study, we developed a protocol for deriving human LSCs from ADSCs compatible with the standard carrier human amniotic membrane, helping provide a stem cell pool capable of maintaining proper corneal epithelial homeostasis. The best protocol included an ectodermal induction step by culturing ADSCs with media containing fetal bovine serum, transforming growth factor-ß inhibitor SB-505124, Wnt inhibitor IWP-2 and FGF2 for 7 days, followed by an LSC induction step of culture in modified supplemental hormonal epithelial medium supplemented with pigment epithelium-derived factor and keratinocyte growth factor for 10 additional days. The optimal differentiation efficiency was achieved when cells were cultured in this manner over vitronectin coating, resulting in up to 50% double-positive αp63/BMI-1 cells. The results of this project will benefit patients with LSC deficiency, aiding the restoration of vision.

Early Clinical Outcomes of the First Commercialized Human Autologous Ex Vivo Cultivated Oral Mucosal Epithelial Cell Transplantation for Limbal Stem Cell Deficiency: Two Case Reports and Literature Review.

The first product in the world for ex vivo cultivated oral mucosal epithelial cell transplantation (COMET) to treat limbal stem cell deficiency (LSCD), named Ocural®, was launched in June 2021 in Japan. COMET was performed on two patients, including the first case in the post-marketing phase of Ocural®. Pathological and immunohistochemical examinations were also carried out using specimens obtained before and after COMET and the spare cell sheet. In case 1, the ocular surface remained free from epithelial defects for approximately six months. In case 2, although defect of the cornea-like epithelia was observed after COMET for one month, it was resolved after the insertion of lacrimal punctal plugs. In case 1, adjuvant treatment was interrupted due to an accident during the second month after COMET, resulting in conjunctival ingrowth and corneal opacity. Eventually, a lamellar keratoplasty was required at six months after COMET. Immunohistochemistry revealed the presence of markers for stem cells (p63, p75), proliferation (Ki-67), and differentiation (Keratin-3, -4, and -13) in both the cornea-like tissue after COMET and a cultivated oral mucosal epithelial cell sheet. In conclusion, Ocural® can be accomplished without major complications, and the stem cells derived from oral mucosa might be successfully engrafted.

Evaluation of the cytotoxic and genotoxic/antigenotoxic effects of resveratrol in human limbal explant cultures.

PURPOSE: Resveratrol (RSV) is a natural polyphenol phytoalexin compound and has long been considered to possess antioxidant and anti-inflammatory effects. In order to exploit the protective potential of RSV in anterior segment diseases, we investigated the possible cytotoxic, genotoxic/antigenotoxic effects of human limbal explant cultures to RSV and MMC or H2O2 alone and in combination. METHODS: A total of 18 limbal explant tissues obtained from three corneal donors were placed on the 12 well tissue culture polystyrene plates and cultured for 14 days. Cell growth from limbal explants was observed by inverted phase contrast microscopy. The cytotoxic effects of RSV was studied by neutral red uptake assay. For the assessment of the genotoxic and antigenotoxic effects, basic alkaline technique of comet assay was performed. RESULTS: It was found that the concentrations of RSV up to 100 µM did not significantly affect the viability of outgrowth cells of limbal explant during 24 h exposure. When compared to negative control, all concentrations of RSV alone caused an increase in DNA strand breakage. Interestingly, 10 µg/mL MMC alone caused similar tail intensity and tail moment values with RSV alone. On the other hand, RSV treatment in all doses seemed to decrease the DNA damage induced by either H2O2 or MMC. CONCLUSION: RSV is an attractive natural compound for the purpose of oxidative stress reduction in ocular surface and can be utilized as a supplement to promote ocular surface regeneration in vivo.

Limbal stem cell deficiency (LSCD) in rats and mice following whole body exposure to sulfur mustard (SM) vapor.

Ocular injuries following sulfur mustard (SM) exposure are characterized by an acute phase expressed by corneal erosions and inflammation of the anterior segment that after a clinically silent period may be followed by irreversible corneal injuries. The latter includes epithelial defects, chronic inflammation and neovascularization (NV), and were defined in rabbits and in humans as Limbal Stem Cell Deficiency (LSCD), that derived from a delayed loss of corneal epithelial stem cells (ESC), due to secondary processes most likely in the epithelial stem cell (SC) niche. The present study expands our research on SM-induced ocular injury to rodents (rats and mice) following whole body vapor exposure, aiming to define whether the delayed development of LSCD is a general characteristic of SM ocular toxicity. Freely moving rats and mice were exposed to SM vapor (155 µg/l, 10 min). Clinical examination was carried out in rats and included a slit-lamp bio-microscopy, up to 6 months. Eyes were taken for histology at different time points following exposure and evaluation included hematoxylin and eosin (H&E) staining for general morphology, PAS for identification of goblet cells and p63 immunohistochemistry for progenitor epithelial cells. Whole body exposure to SM vapor in rats and mice resulted in acute ocular injury characterized by corneal erosions and ocular inflammation. Following a brief recovery period, 80-90% of the exposed eyes developed corneal NV associated with abnormal corneal epithelium, stromal inflammation and endothelial damage. The late injury was accompanied by migration of conjunctival goblet cells to the cornea and a loss of limbal epithelial progenitor cells, indicating LSCD. The long-term ocular injury shown hereby in rats and mice was consistent with the lesions described in rabbits and in human casualties and demonstrated the general phenomenon of limbal epithelial stem cells deficiency in SM ocular toxicity. The delayed manifestation of this pathology points towards a therapeutic window for the development of medical countermeasures in small animals following exposure in a real life scenario.

Labial mucosal epithelium grafting in an ex vivo human donor cornea model.

The purpose of the study was to establish a simple ex vivo corneal re-epithelization model and study the labial mucosal epithelium grafting as a potential approach for ocular surface reconstruction. Four human donor corneal buttons were overstored in a corneal cold storage solution at 4 °C for 32-52 days. Four labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed informed consent. The substantia propria was trimmed off, and the resulting graft was sutured near the corneal limbus with running sutures (thus forming the tissue construct). Constructs were cultured under the standard conditions with the anterior corneal side outwards. After 3 weeks of culture, constructs were removed, washed, and fixed. Sections were stained with hematoxylin and eosin (HE), anti-keratins 4, 13, 19, and p63. Nuclei were counterstained with Hoechst. After the cultivation, all constructs were integral with the attached graft and non-loosened sutures. The native cells were absent in all donor corneas. Histological evaluation demonstrated that the labial mucosal grafts were attached to the Bowman's membrane (BM), and its cellular outgrowths were found to be transit from the graft to the BM over the anterior surface in all constructs. Cells expressed mucosal epithelial keratins 4, 13, and 19, and several were p63-positive in nuclei. In the study, a simple ex vivo corneal re-epithelization model was successfully established. The model was potent in studying the labial mucosal epithelium grafting as an option for autologous ocular surface reconstruction in patients with bilateral 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.

Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems.

Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.

Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells.

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.