Limbal Stem Cell Deficiency in Inflammatory Disorders

Stem cells can be defined as cells that have the capacity to self-renew and the ability to generate differentiated progeny or multiple cell lineages. True stem cells can turn into any type of cells, while progenitor cells are more or less committed to becoming cell types of a particular tissue. Human corneal epithelial stem cells (CESCs) represent a great example and model of adult stem or progenitor cells. Human CESCs have been identified to locate in the basal epithelial layer of the limbus, and thus also referred as to limbal stem cells. We would like to use the both terms, stem and progenitor cells in this chapter based on previous use in the literature for more than two decades. Although the CESCs have been identified to reside at the limbus and many stem cell markers have been proposed, there is no consensus to date regarding the definitive markers for CESCs, and identification and isolation of these cells are still challenging. Based on evaluation of a variety of proposed markers, we have characterized that the CESCs located in the basal layer of human limbal epithelium are small primitive cells expressing three patterns of molecular markers, which represent a unique phenotype of putative corneal epithelial stem or progenitor cells. Based on adult stem cell criteria and the putative limbal stem cell phenotype, our group has attempted to enrich for human CESCs through novel approaches including cell-sizing, adhering to extracellular matrix collagen type IV, and cell sorting for side population or for expression of ABCG2 or connexin 43 cell surface markers. The 5 clonogenic populations isolated from limbal epithelium and its cultures by different methods show the properties that are characteristics of adult stem/progenitor cells: 1) relatively undifferentiated, 2) high proliferative potential, 3) self-renewal. Expansion and cultivation of corneal epithelial progenitor cells have been achieved using different methods, such as limbal tissue explant culture, and limbal epithelial cell suspension co-culture with mouse 3T3 fibroblast feed layer. To avoid the use of xeno-components, two cell lines of commercial human fibroblasts have been identified that support human corneal epithelial regeneration, and have potential use in replacing mouse 3T3 cells for corneal tissue bioengineering. The concept of CESCs has formed the basis for identifying a class of blinding diseases that display features of corneal epithelial stem cell deficiency or limbal stem cell deficiency (LSCD), where the limbal epithelium is damaged. LSCD is characterized by persistent or recurrent epithelial defects, ulceration, corneal vascularization, chronic inflammation, scarring, and conjunctivalization (conjunctival epithelial ingrowth). Only transplantation of CESCs can restore vision. Due to an increasing shortage of corneal donors, corneal tissue engineering is becoming an important discipline that holds great promise for corneal reconstruction. CESCs and optical substrates are known to be the most important factors for corneal tissue bioengineering in regenerative medicine. Our team has recently explored the utilization of natural donor corneal stroma in corneal tissue engineering. In combination with fresh limbal epithelium containing stem cells, and the donor corneal stroma, a great source of natural optical substrate, we developed a native-like corneal equivalent construct with proliferative potential. This corneal construct provides a new clinical cell therapy for corneal reconstruction.

Objective To observe the clinical effect of amniotic membrane transplantation and autologous corneal limbal stem cell transplantation in the treatment of primary pterygium. Methods Seventy-five eyes of 68 cases of pterygium were randomly divided into two groups: the stem cell transplantation groups, 38 eyes of 35 cases, underwent pterygium excision combined with autologous limbal stem cell transplantation; amniotic membrane transplantation group, 37 eyes of 33 cases, underwent pterygium excision combined with amniotic membrane transplantation. The postoperative recurrence, corneal wound healing time and complications of two groups were observed. Results The recurrence rate of stem cell transplantation group was 2.6%, the recurrence rate of amniotic membrane transplantation group was 18.9%, and the recurrence rate of stem cell transplantation group was lower than that of amniotic membrane transplantation group. In the stem cell transplantation group, corneal wound healing time was average (3.49±0.50) days, the average time of corneal wound healing time was (5.28±1.37) days. The corneal wound healing time of the stem cell transplantation group was shorter than that of the amniotic membrane transplantation group, the difference was statistically significant (t=7.550, P=0.000). Conclusion The effect of autologous corneal limbal stem cell transplantation for primary pterygium, with shorter healing time, lower recurrence rate and fewer complications, is better than that of amniotic membrane transplantation. Key words: Pterygium; Transplantation; Stem cell; Amniotic membrane

Limbal stem cell deficiency (LSCD) can cause significant corneal vascularization and scarring and often results in serious visual morbidity. An early and accurate diagnosis can help prevent the same with a timely and appropriate intervention. This review aims to provide an understanding of the different diagnostic tools and presents an algorithmic approach to the management based on a comprehensive clinical examination. Although the diagnosis of LSCD usually relies on the clinical findings, they can be subjective and non-specific. In such cases, using an investigative modality offers an objective method of confirming the diagnosis. Several diagnostic tools have been described in literature, each having its own advantages and limitations. Impression cytology and in vivo confocal microscopy (IVCM) aid in the diagnosis of LSCD by detecting the presence of goblet cells. With immunohistochemistry, impression cytology can help in confirming the corneal or conjunctival source of epithelium. Both IVCM and anterior segment optical coherence tomography can help supplement the diagnosis of LSCD by characterizing the corneal and limbal epithelial changes. Once the diagnosis is established, one of various surgical techniques can be adopted for the treatment of LSCD. These surgeries aim to provide a new source of corneal epithelial stem cells and help in restoring the stability of the ocular surface. The choice of procedure depends on several factors including the involvement of the ocular adnexa, presence of systemic co-morbidities, status of the fellow eye and the comfort level of the surgeon. In LSCD with wet ocular surfaces, autologous and allogeneic limbal stem cell transplantation is preferred in unilateral and bilateral cases, respectively. Another approach in bilateral LSCD with wet ocular surfaces is the use of an autologous stem cell source of a different epithelial lineage, like oral or nasal mucosa. In eyes with bilateral LSCD with significant adnexal issues, a keratoprosthesis is the only viable option. This review provides an overview on the diagnosis and treatment of LSCD, which will help the clinician choose the best option amongst all the therapeutic modalities currently available and gives a clinical perspective on customizing the treatment for each individual case.

To report the outcomes of Boston type I keratoprosthesis (BKPro) in the management of ocular burn injuries. This was a prospective study including all cases of BKPro implantation for ocular burns at the External Diseases and Cornea Service of the Federal University of São Paulo, between February 2008 and February 2010. Ten patients (10 eyes) were enrolled. Procedures performed to manage ocular injury were identified, and data were collected regarding patients' ocular history, surgical procedure(s) performed, and postoperative outcomes, including visual acuity, retention, complications and required surgical procedures. A total of 11 Type 1 BKPro were implanted in 10 eyes of 10 patients. The mean follow-up period was 25.7 ± 10.8 months. Preoperative best-corrected visual acuity (BCVA) ranged from count fingers to light perception. Postoperative BCVA was better than 20/200 in 90% of the patients and better than 20/60 in 60% of the patients. The overall BKPro retention rate was 90%. The most common complications were retroprosthetic membrane formation (50%) and persistent corneal epithelial defect evolving to corneal melting (40%). Patients who underwent ocular surface procedures such as limbal transplantation prior to BKPRo implantation had a lower incidence of corneal melting/thinning (p = 0.07), although this was not statistically significant. The anatomical and functional results identified in this study support the use of BKPro in managing bilateral limbal stem cell deficiency secondary to ocular burns.

To report the midterm outcomes of autologous limbal stem cell transplantation cultivated on amniotic membrane (AM) with or without subsequent penetrating keratoplasty (PKP) in patients with total unilateral limbal stem cell deficiency (LSCD). Eight eyes of 8 consecutive patients with unilateral total LSCD underwent autologous limbal stem cell transplantation cultivated on AM. Four eyes underwent subsequent optical PKP. Main outcome measures were corneal vascularization and transparency. The patients were followed for 34.0 +/- 13.5 months (6-48 months). Seven cases had a stable corneal epithelium with marked decrease in opacification and vascularization. Progressive sectorial conjunctivalization was evident in all cases with subsequent PKP at the last follow-up. Primary failure was observed in one case because of exposure. Transplantation of autologous stem cells cultivated on AM with or without subsequent PKP seems to be an effective way for visual rehabilitation in total LSCD. More work with more cases and longer follow-up are needed to optimize this procedure to provide and maintain an adequate supply of limbal stem cells in these patients.

Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Objective To analyze the effect of pterygium excision combined with autologous limbal stem cell transplantation for patients with pterygium. Methods 42 pterygium patients (66 eyes) treated at our hospital from March, 2015 to May, 2016 were included into the study; Using random number table method, they were divided into a control group and an observation group, 33 eyes for each group. The control group were treated with pterygium excision and the observation group with pterygium excision and autolgous corneal limbal stem cell transplantation. Both groups were followed by 6 months. Results After the surgery, the uncorrected visual acuity was higher in the observation group than in the control group [(0.83±0.15) vs. (0.73±0.15)]. The corneal epithelial repair time and stitches-off time were shorter in the observation group than in the control group [(2.12±0.68) d vs. (2.67±0.96) d and (4.97±0.81) d vs. (5.70±0.92) d], with statistical differences (both P<0.05). After the treatment, the cured rate was higher in the observation group than in the control group (93.94% vs. 75.76%) , with a statistical differences (P<0.01). Conclusion Pterygium excision combined with limbal stem cell translations has good overall effect and can improve the pterygium corneal astigmatism and vision. Key words: Pterygium excision; Limbal stem cell transplantation; Pterygium; Clinical effect

ABSTRACTThough the corneoscleral limbus has been studied for more than 150 years, our ability to successfully treat those with limbal stem cell deficiency (LSCD) is still a work in progress. Today, the only definitive cure for LSCD is stem cell transplantation. This review briefly discusses the evolution of our understanding of limbal stem cell function, provides an outline of the history of autologous limbal stem cell (LSC) transplantation, and discusses current techniques and future strategies for LSCD treatment.

The transplantation of limbal stem cells is one of the most challenging surgical approaches in ocular surface reconstruction. Partial and unilateral limbal stem cell insufficiency (LSCI) can be treated by fractionated abrasion or autologous limbal stem cell transplantation from the fellow eye. In cases of advanced bilateral and partial LSCI, ex vivo expansion of limbal stem cells on amniotic membranes or fibrin can be performed but all patients with complete bilateral LSCI must rely on allogenic limbal stem cell transplantation with high immunological risks. Attempts to combine allogenic limbal stem cell transplantation with mitomycin C and amniotic membrane transplantation are promising. In the laboratory, attempts to transdifferentiate bone marrow stem cells into corneal epithelial cells have been without success. Nonetheless, transdifferentiation of hair follicle stem cells into corneal epithelial cells looks promising. In parallel, research on the limbal stem cell niche is ongoing to elucidate the natural environment of limbal stem cells in order to improve ex vivo culture.

Ocular surface defects represent one of the most common causes of impaired vision or even blindness. For treatment, keratoplasty represents the first choice. However, if corneal defects are more extensive and associated with a limbal stem cell (LSC) deficiency, corneal transplantation is not a sufficient therapeutic procedure and only viable approach to treatment is the transplantation of LSCs. When the LSC deficiency is a bilateral disorder, autologous LSCs are not available. The use of allogeneic LSCs requires strong immunosuppression, which leads to side-effects, and the treatment is not always effective. The alternative and perspective approach to the treatment of severe ocular surface injuries and LSC deficiency is offered by the transplantation of autologous mesenchymal stem cells (MSCs). These cells can be obtained from the bone marrow or adipose tissue of the particular patient, grow well in vitro and can be transferred, using an appropriate scaffold, onto the damaged ocular surface. Here they exert beneficial effects by possible direct differentiation into corneal epithelial cells, by immunomodulatory effects and by the production of numerous trophic and growth factors. Recent experiments utilizing the therapeutic properties of MSCs in animal models with a mechanically or chemically injured ocular surface have yielded promising results and demonstrated significant corneal regeneration, improved corneal transparency and a rapid healing process associated with the restoration of vision. The use of autologous MSCs thus represents a promising therapeutic approach and offers hope for patients with severe ocular surface injuries and LSC deficiency.

Limbal dermoids are congenital, benign, choristomatous growths affecting the corneal-limbal junction. Conventional excision techniques often result in persistent epithelial defects, corneal thinning, and vascularization due to sectoral limbal stem cell deficiency. This study investigated a novel surgical approach for limbal dermoid excision, utilizing Bowman’s membrane lenticule and autologous limbal stem cell transplantation, aimed at improving epithelial healing and visual outcomes. Thirty-four subjects (24 females, 10 males; mean age 8.33 ± 6.47 years) with limbal dermoids underwent the procedure. After dermoid excision, a Bowman’s membrane lenticule was placed over the defect and tucked 1 mm beneath the surrounding tissue. Sectoral limbal reconstruction was then performed using the AutoSLET technique. Pre- and postoperative assessments included visual acuity, corneal thickness, and epithelialization time. Statistical analysis employed paired t-tests. The mean epithelialization time was 3.36 ± 0.74 weeks, indicating rapid healing. Best-corrected visual acuity (BCVA) significantly improved from a preoperative mean of 0.136 ± 0.121 decimal units to a postoperative mean of 0.336 ± 0.214 decimal units (p < 0.001). Corneal thickness also demonstrated a significant increase, rising from a preoperative mean of 294 ± 49.68 microns to a postoperative mean of 484 ± 5.037 microns (p < 0.001). There is a transient edema below the Bowman lenticule observed in many cases, which resolves with deposition of granulation tissue. The findings suggest that the combined use of Bowman’s membrane lenticule and autologous limbal stem cell transplantation offers a promising surgical strategy for limbal dermoid excision. This technique promotes rapid epithelialization and leads to significant improvements in visual acuity and corneal thickness compared to conventional methods. The utilization of Bowman’s membrane as a natural basement membrane and the direct application of limbal stem cells facilitate enhanced epithelial healing and visual rehabilitation. While the study is limited by its small sample size, the results demonstrate the potential of this novel approach in managing limbal dermoids effectively.

Objective: To study the effect of bandage corneal contact lenses after pterygium surgery. Methods: This was a case-control study. Patients with an excised pterygium combined with autologous corneal limbal stem cell transplantation were selected at Tiantai People's Hospital of Zhejiang Province between February and June 2017. Patients were divided into two groups based on postoperative treatment: Experimental group was treated with autologous corneal limbal stem cell transplants combined with bandage corneal contact lenses, control group had routine treatment by covering the eyes after autologous limbal stem cell transplantation. There were 20 patients (20 eyes) in each group. After the operation, corneal irritation symptoms and healing time for the corneal epithelium were observed in the two groups. The data were compared using a t test, and a rank sum test. Results: The pain scores of the patients in the experimental group were significantly lower than the pain scores in the control group, and the difference between the two groups was statistically significant (2 hours postoperative: Z=-3.986, P<0.001; 1 day postoperative: Z=-3.153, P=0.002; 2 days postoperative: Z=-3.216, P=0.001; 3 days postoperative: Z=-2.377, P=0.017; 5 days postoperative: Z=-2.333, P=0.020). The recovery time for the corneal epithelium stained with fluorescein after surgery was clearly shortened in the experimental group, and the difference was statistically significant (Z=-3.714, P<0.001). No infection, recurrence, poor implant healing, subconjunctival cyst or other complications occurred after 2 to 6 months of follow-up. Conclusions: The application of bandage corneal contact lenses after pterygium surgery can relieve pain and irritation symptoms in patients, promote postoperative recovery, reduce the postoperative recurrence rate and improve postoperative quality of life. Key words: pterygium; bandage corneal contact lenses; comforting level

The limbal stem cells niche (LSCN) is an optimal microenvironment that provides the limbal epithelial stem cells (LESCs) and strictly regulates their proliferation and differentiation. Disturbing the LSCN homeostasis can lead to limbal stem cell dysfunction (LSCD) and subsequent ocular surface aberrations, such as corneal stromal inflammation, persistent epithelial defects, corneal neovascularisation, lymphangiogenesis, corneal opacification, and conjunctivalization. As ocular surface disorders are considered the second main cause of blindness, it becomes crucial to explore different therapeutic strategies for restoring the functions of the LSCN. A major limitation of corneal transplantation is the current shortage of donor tissue to meet the requirements worldwide. In this context, it becomes mandatory to find an alternative regenerative medicine, such as using cultured limbal epithelial/stromal stem cells, inducing the production of corneal like cells by using other sources of stem cells, and using tissue engineering methods aiming to produce the three-dimensional (3D) printed cornea. Limbal epithelial stem cells have been considered the magic potion for eye treatment. Epithelial and stromal stem cells in the limbal niche hold the responsibility of replenishing the corneal epithelium. These stem cells are being used for transplantation to maintain corneal epithelial integrity and ultimately sustain optimal vision. In this review, we summarised the characteristics of the LSCN and their current and future roles in restoring corneal homeostasis in eyes with LSCD.

Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.