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.

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.

Durvalumab-associated limbal stem cell deficiency and secondary corneal perforation.

Purpose: We report a patient with bilateral limbal stem cell deficiency (LSCD) like clinical manifestations and secondary corneal perforation presumably induced by durvalumab following its use for the treatment of non-small cell lung carcinoma. Observations: A 65-year-old male diagnosed with non-small cell lung carcinoma was treated with monthly durvalumab infusions. Two months after starting durvalumab, the patient was found to have bilateral severe keratoconjunctivitis and LSCD-like clinical findings. Despite topical management and oral prednisone for presumed ocular cicatricial pemphigoid, the patient continued to worsen clinically. The patient was transferred to our institution about one year later with persistent inflammation. The patient eventually developed a corneal perforation of the left eye, which required the application of cyanoacrylic tissue adhesive. Due to the lack of response to oral prednisone, durvalumab was discontinued with the approval of the patient's oncologist. Several months following the discontinuation of durvalumab, the conjunctival inflammation subsided, and corneal epithelial breakdown and ulceration resolved. Conclusions: We report an association between durvalumab and the development of bilateral LSCD-like clinical findings with subsequent corneal perforation. We hope this case reinforces the importance of routine ophthalmologic follow-up after starting any cancer treatment, especially in patients with symptoms and signs suggesting ocular surface disease or inflammation.

The effect of cultured autologous oral mucosal epithelial cells on ocular surface reconstruction.

Introduction: Oral epithelial cells were recently shown to be able to differentiate into corneal epithelium, and the efficacy of cultured autologous oral mucosal epithelial cells (CAOMEC) has been suggested by the presence of epithelium replacement. Therefore, the aim of this study was to evaluate the treatment outcome in limbal stem cell deficiency (LSCD) by adding CAOMEC to regular amniotic membrane (AM) treatment. Material and methods: Eyes with LSCD were randomized to two groups to undergo either autologous oral mucosal epithelial cell sheet (CAOMECS) combined with AM transplantation (A group) or AM transplantation alone (B group). Clinical outcome measures were corneal epithelium healing, best corrected visual acuity, symblepharon, corneal transparency, corneal neovascularization and ocular surface inflammation. Results: The normal corneal epithelialization rate in group A (73.33%) was higher than that in group B (35.48%), and the average healing time was shorter (3.45 ±2.12 weeks vs. 4.64 ±1.63 weeks). The symblepharon in the above two groups was improved in the first 3 months after surgery, but after 6 months, part of the B group had recurrence. In improving corneal transparency, group A has obvious advantages. Corneal neovascularization (CNV) was improved to some extent in the first 3 months after surgery, but group A (1.47 ±0.64) was better than group B (1.94 ±0.85) after 6 months. Both groups can improve the inflammatory state to some extent. Conclusions: The transplantation of CAOMECS offers a viable and safe alternative in the reconstruction of a stable ocular surface. The effect is better than that of traditional AM transplantation, mainly in promoting corneal epithelialization, improving ocular surface structure, and reducing fiber and vascular infiltration.

Prevalence of limbal stem cell deficiency at an academic referral center over a two-year period.

Aim: To evaluate the prevalence and clinical characteristics of limbal stem cell deficiency (LSCD) in the setting of a tertiary referral cornea practice at an academic center. Patient and methods: A retrospective chart review was performed to identify all unique medical record numbers (MRNs) presenting to a single cornea specialist (JHH) at the University of Minnesota during calendar years 2019 and 2020. Records were queried and confirmed for a diagnosis of LSCD. Clinical characteristics of identified patients, including demographics, etiology of LSCD, severity of LSCD, treatment, and best corrected visual acuity (BCVA) at final follow-up, were documented. Results: In total 1436 unique MRNs were identified over the study period. There were 61 individuals (91 eyes) diagnosed with LSCD, resulting in a prevalence of 4.25% (95% CI, 3.33-5.42). Of 91 eyes, 60 eyes were bilateral (65.9%). Among all eyes, ocular surface burns were the most common etiology (18.7%) followed by iatrogenic or medicamentosa (15.4%). There were 51 eyes (56.0%) that underwent some form of transplantation. The median BCVA at final follow-up was Snellen 20/80 (range 20/20 to no light perception). Conclusions: The prevalence of LSCD found at a cornea subspecialty tertiary referral center in our study was much higher than previously reported prevalence rates. This may reflect referral bias and potential underdiagnosis of LSCD in practices outside of subspecialty referral centers. The high prevalence rate in our study also suggests that LSCD patients are concentrated in subspecialty referral practices, with many having high morbidity disease. This constitutes a major health burden for these practices.

[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.

Biologicals and Biomaterials for Corneal Regeneration and Vision Restoration in Limbal Stem Cell Deficiency.

The mammalian cornea is decorated with stem cells bestowed with the life-long task of renewing the epithelium, provided they remain healthy, functional, and in sufficient numbers. If not, a debilitating disease known as limbal stem cell deficiency (LSCD) can develop causing blindness. Decades after the first stem cell (SC) therapy is devised to treat this condition, patients continue to suffer unacceptable failures. During this time, improvements to therapeutics have included identifying better markers to isolate robust SC populations and nurturing them on crudely modified biological or biomaterial scaffolds including human amniotic membrane, fibrin, and contact lenses, prior to their delivery. Researchers are now gathering information about the biomolecular and biomechanical properties of the corneal SC niche to decipher what biological and/or synthetic materials can be incorporated into these carriers. Advances in biomedical engineering including electrospinning and 3D bioprinting with surface functionalization and micropatterning, and self-assembly models, have generated a wealth of biocompatible, biodegradable, integrating scaffolds to choose from, some of which are being tested for their SC delivery capacity in the hope of improving clinical outcomes for patients with LSCD.

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.

Comparative analysis of long-term results of three epithelial cell transplantation procedures for treating limbal stem cell deficiency.

This study compared the long-term outcome of different epithelial transplantation techniques to treat limbal stem cell deficiency (LSCD). We conducted a retrospective 15-year comparative systematic cohort study of patients with LSCD who underwent either cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET), or cultivated oral mucosal epithelial transplantation (COMET). We reviewed the demographic data, etiology, LSCD severity, best-corrected visual acuity, surgical outcomes, and complications. A total of 103 eyes of 94 patients (mean age, 45.0 ± 16.4 years) with LSCD were enrolled. The most common cause of LSCD was chemical injury (42.7 %). The median follow-up time was 75 months. The success rates of CLET, SLET, and COMET were 45.5 %, 77.8 %, and 57.8 %, respectively. The 7-year survival rates after CLET, SLET, and COMET were 50.0 %, 72.2 %, and 53.2 %, respectively. Steven-Johnson syndrome (SJS) had a significantly lower survival rate than other causes (p < 0.001), but SLET had a significantly higher survival rate than CLET (p = 0.018) and COMET (p = 0.047). Visual improvement of more than four Snellen lines was achieved in 53.1 % of successful cases and 28.2 % of failed cases. SJS, Schirmer I test <5 mm, and the presence of postoperative recurrent epithelial defects were significant risk factors for a failed surgery. All epithelial transplantation techniques had favorable long-term surgical outcomes. More than half of the patients achieved a stable ocular surface and visual acuity improvement up to 7 years postoperatively. SLET tends to have a better surgical outcome than CLET and COMET, especially in patients with SJS.

Matrix-Assisted Cell Transplantation for the Treatment of Limbal Stem Cell Deficiency in a Rabbit Model.

With the development of regenerative medicine in ophthalmology, the identification of cells with high proliferative potential in the limbal area has attracted the attention of ophthalmologists and offered a new option for treatment in clinical practice. Limbal stem cell deficiency (LSCD) is an identified eye disease with a difficult and negative outcome, for which the traditional treatment is keratoplasty. This study sought to evaluate the efficacy of matrix-assisted cell transplantation consisting of in vitro-cultured autologous limbal stem cells (LSCs) and type I collagen for the treatment of LSCD in rabbits. LSCD was induced in 10 rabbits by a combination of mechanical limbectomy and alkali burns. Cells were cultured on a plate for 14 days before being transferred to a collagen-based matrix for another 7 days. Rabbits were divided into two groups as follows: the experimental group (five rabbits) received matrix-assisted cell transplantation, while the control group (five rabbits) received only conservative therapy with anti-inflammatory eye drops. During the postoperative period, all rabbits were examined using slit-lamp biomicroscopy with photo-registration and fluorescent staining, impression cytology and anterior segment optical coherence tomography (AS-OCT). Rabbits were euthanized at 30 and 120 days, and their corneas were processed for histology and immunohistochemistry. As a consequence, rabbits in the experimental group demonstrated the restoration of the corneal epithelium and transparency without epithelial defects. Moreover, goblet cells were absent in the central zone of the corneal epithelium. In conclusion, our new method of treatment enhanced the corneal surface and is an effective method of treatment for LSCD in rabbits.

Animal Models for Limbal Stem Cell Deficiency: A Critical Narrative Literature Review.

This literature review will provide a critical narrative overview of the highlights and potential pitfalls of the reported animal models for limbal stem cell deficiency (LSCD) and will identify the neglected aspects of this research area. There exists significant heterogeneity in the literature regarding the methodology used to create the model and the predefined duration after the insult when the model is supposedly fully fit for evaluations and/or for testing various therapeutic interventions. The literature is also replete with examples wherein the implementation of a specific model varies significantly across different studies. For example, the concentration of the chemical, as well as its duration and technique of exposure in a chemically induced LSCD model, has a great impact not only on the validity of the model but also on the severity of the complications. Furthermore, while some models induce a full-blown clinical picture of total LSCD, some are hindered by their ability to yield only partial LSCD. Another aspect to consider is the nature of the damage induced by a specific method. As thermal methods cause more stromal scarring, they may be better suited for assessing the anti-fibrotic properties of a particular treatment. On the other hand, since chemical burns cause more neovascularisation, they provide the opportunity to tap into the potential treatments for anti-neovascularisation. The animal species (i.e., rats, mice, rabbits, etc.) is also a crucial factor in the validity of the model and its potential for clinical translation, with each animal having its unique set of advantages and disadvantages. This review will also elaborate on other overlooked aspects, such as the anaesthetic(s) used during experiments, the gender of the animals, care after LSCD induction, and model validation. The review will conclude by providing future perspectives and suggestions for further developments in this rather important area of research.

Neovascularization against autologous SLET donor grafts despite successful corneal epithelialization and graft adherence.

PURPOSE: To report a case of neovascularization against autologous grafts after simple limbal epithelial transplantation (SLET) despite successful corneal epithelialization, as well as its subsequent regression without intervention. METHODS: A case report and review of the literature. RESULTS: A 52-year-old woman underwent uncomplicated autologous SLET for asymmetric limbal stem cell deficiency (LSCD) in the left eye. One month after the surgery, the patient had successful adherence of the graft and corneal epithelialization; however, new neovascularization developed in the left eye towards the graft sites. With only a slow taper of topical prednisolone acetate and polymyxin b/trimethoprim, the neovascularization regressed to ghost vessels over the following three months with improvement of her LSCD symptoms and increased clarity of her cornea. CONCLUSION: The limbus does not enjoy relative immune privilege like other parts of the eye; therefore, autologous limbal stem cell transplantation (along with the minimal immune response generated) is valuable for restoration of the ocular surface. Here, we describe neovascularization against autologous donor grafts despite an otherwise uncomplicated surgery and expected epithelialization course. Inflammation-mediated angiogenesis likely initiated the neovascularization, suggesting that immune mediators of inflammation may be inadvertently part of the graft tissue in bilateral LSCD.

Corneal Epithelial Development and the Role of Induced Pluripotent Stem Cells for Regeneration.

Severe corneal disorders due to infective aetiologies, trauma, chemical injuries, and chronic cicatricial inflammations, are among vision-threatening pathologies leading to permanent corneal scarring. The whole cornea or lamellar corneal transplantation is often used as a last resort to restore vision. However, limited autologous tissue sources and potential adverse post-allotransplantation sequalae urge the need for more robust and strategic alternatives. Contemporary management using cultivated corneal epithelial transplantation has paved the way for utilizing stem cells as a regenerative potential. Humaninduced pluripotent stem cells (hiPSCs) can generate ectodermal progenitors and potentially be used for ocular surface regeneration. This review summarizes the process of corneal morphogenesis and the signaling pathways underlying the development of corneal epithelium, which is key to translating the maturation and differentiation process of hiPSCs in vitro. The current state of knowledge and methodology for driving efficient corneal epithelial cell differentiation from pluripotent stem cells are highlighted.

Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency.

PURPOSE: To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). METHODS: A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. RESULTS: No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). CONCLUSION: ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

Ocular Manifestations in Patients Affected by p63-Associated Disorders: Ectrodactyly-Ectodermal Dysplasia-Clefting (EEC) and Ankyloblepharon-Ectodermal Defects-Cleft Lip Palate (AEC) Syndromes.

BACKGROUND/AIMS: The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) and Ankyloblepharon-ectodermal defect-cleft lip/palate (AEC) syndromes are rare autosomal dominant diseases caused by heterozygous mutations in the p63 gene. Patients are characterized by abnormalities of the skin, teeth, and hair and have limb defects, orofacial clefting and ectodermal dysplasia. In addition, they often show ocular surface alterations, leading to progressive corneal clouding and eventually blindness. Here, we present 8 cases describing patients affected by EEC (n = 6, with 5 sporadic and 1 familial cases) and AEC (n = 2, both sporadic cases) syndromes. We attempt to provide a description of the ocular disease progression over the years. METHODS: Clinical examinations and monitoring of ocular parameters for the assessment of limbal stem cell deficiency were constantly performed on patients between 2009 and 2023. Quantitative data and comparison with existing cases described in the literature are reported. RESULTS: The therapies supplied to patients were essential for the management of the symptoms, but unfortunately did not halt the progression of the pathology. CONCLUSIONS: A constant monitoring of the patients would help avoid the sudden worsening of symptoms. If the progression of the disease slows down, it would allow for the development of newer therapeutic strategies aimed at correcting the genetic defect.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.

BACKGROUND: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. METHODS: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. RESULTS: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. CONCLUSION: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

A Review of Contact Lens-Induced Limbal Stem Cell Deficiency.

Limbal stem cell deficiency (LSCD) is a pathologic condition caused by the dysfunction and destruction of stem cells, stem cell precursors and limbal cell niche in the corneal epithelium, leading to severe conjunctivalization of the cornea. Etiologies for LSCD span from congenital (aniridia), traumatic (chemical or thermal injuries), autoimmune (Stevens-Johnson syndrome) and iatrogenic disease to contact lens (CL) wear. Of these, CL wear is the least understood and is often a subclinical cause of LSCD. Even with recent advances in LSCD research, limitations persist in establishing the pathogenesis and treatment guidelines for CL-induced LSCD. A literature search was conducted to include original articles containing patients with CL-induced LSCD. This review will critically discuss the complex pathophysiology behind CL-induced LSCD, the underlying risk factors and epidemiology of the disease as well as methods to obtain a diagnosis. Various treatment options will be reviewed based on proposed treatment strategies.

Axenfeld-Reiger syndrome: A search for the missing links.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is a rare cause of congenital glaucoma and may result in loss of vision. ARS is mostly autosomal dominant in nature characterized by developmental abnormalities in the angle of anterior chamber and iris of the eye, also associated with structural abnormalities in the body. AIM: To study and observe the demographics and clinical findings in a very rare ocular disease known as ARS. METHODS: Case records of symptomatic patients attending Ophthalmology outpatient department and diagnosed to have ocular hypertension or glaucoma in 3 years from March 2017 to March 2020 were evaluated to search for cases diagnosed with ARS. Records of all patients diagnosed with ARS were then analysed for demographic and clinical characterization as well as management and success of therapy. RESULTS: Eight out of ten patients with positive clinical signs were symptomatic and had glaucoma. One of these patients had limbal stem cell deficiency and another had vernal keratoconjunctivitis. CONCLUSION: Clinical characterization of ARS is important for making a definitive diagnosis and determining prognosis.

Successful use of conjunctival flaps to prolong survival of type I keratoprosthesis in severe bilateral chemical burns: two case reports.

INTRODUCTION: This report describes the use of conjunctival flaps to enable the survival of type I keratoprosthesis (KPro) in two cases of bilateral severe total limbal stem cell deficiency (LSCD) following chemical burns. PRESENTATION OF CASE: Two patients had a history of bilateral chemical injury with lime. On examination, the presenting vision was light perception to hand motions and both cases had conjunctivalized ocular surfaces with symblepharon. A modified technique of type I keratoprosthesis was used, where the conjunctivalized corneal pannus was dissected and lifted off as an inferior fornix-based conjunctival flap. This was followed by a standard surgical technique of type I KPro. The flap was then secured over the device and optical opening was made two weeks later. Both the patients had stable ocular surfaces with visual acuity of 20/20 at 2-7 years of follow-up. DISCUSSION: In patients with total LSCD with adnexal involvement, type I KPro has unsatisfactory long-term survival because of the risk of repeated epithelial breakdowns and stromal ulceration. With the innovative approach described in this report, type I KPro can be successfully used for sustainable visual improvement in the presence of severe ocular surface disease and symblepharon. CONCLUSION: Conjunctival flaps can be used along with type I KPros to improve long-term survival and give sustainable visual outcomes in cases of bilateral corneal blindness due to advanced ocular surface damage.