|Year : 2016 | Volume
| Issue : 1 | Page : 3-12
Limbal stem cell deficiency: Current management
Ikeda Lal1, Nidhi Gupta2, Jyoti Purushotham3, Virender S Sangwan4
1 Cornea and Anterior Segment Services, Lakshmi Vara Prasad Eye Institute, Kallam Anji Reddy Campus, L. V. Prasad Marg, Banjara Hills, Hyderabad, India
2 Cornea and Anterior Segment Services, Dr. Shroff's Charity Eye Hospital, Daryaganj, New Delhi, India
3 Nehru Fullbright Nehru Research Scholar, University of Rochester, Brien Holden Research Centre, New York, USA
4 Dr. Paul Dubord Chair in Cornea Director, Center for Ocular Regeneration (CORE) and Director, Srujana-Center for Innovation (LVP_MITRA Program), Lakshmi Vara Prasad Eye Institute, Kallam Anji Reddy Campus, L. V. Prasad Marg, Banjara Hills, Hyderabad, India
|Date of Submission||04-Nov-2014|
|Date of Acceptance||14-Apr-2015|
|Date of Web Publication||19-Jan-2016|
Virender S Sangwan
Director, Center for Ocular Regeneration (CORE) and LVP-MITIZA Center for Innovation, Dr. Paul Dubord Chair in Cornea, Lakshmi Vara Prasad Eye Institute, Kallam Anji Reddy Campus, Lakshmi Vara Prasad Marg, Banjara Hills, Hyderabad - 500 034, Telangana
Source of Support: None, Conflict of Interest: None
Limbal stem cells (SCs) represent a key component of the corneal-conjunctival barrier and play a vital role in the regeneration and replacement of the corneal epithelium. These SCs are located in the basal region of the limbus, in the palisades of Vogt.Limbal SCs may be lost in a wide variety of conditions, the most common being chemical injury, thermal burns, Stevens-Johnson syndrome (SJS), and vernal keratoconjunctivitis. Limbal SC deficiency (LSCD) may result in a painful and blinding ocular surface disorder leading to epithelial defects and conjunctivilization of the cornea. The management of LSCD depends primarily on the laterality, extent of involvement of the eye, and the condition of the other eye. Amniotic membrane transplantation is an established technique used in acute stage to minimize the primary insult and prevent the development of LSCD. In cases of established LSCD, transplanted tissue may be derived from autologous or allogenic sources. Long-term results of cultured limbal epithelial stem cell transplant have demonstrated greater therapeutic success when compared to previous forms of treatment. Moreover, with the help of advances in microsurgical techniques and immunosuppressant drugs, clinical assessment of the outcome of ocular surface rehabilitative procedures grows increasingly optimistic. This article describes the role of SCs in corneal epithelial regeneration, as well as the etiology and clinical features of LSCD.
Keywords: Allolimbal transplant, amniotic membrane transplant, autologous limbal graft, limbal transplant, ocular surface reconstruction, keratolimbal transplant
|How to cite this article:|
Lal I, Gupta N, Purushotham J, Sangwan VS. Limbal stem cell deficiency: Current management. J Clin Ophthalmol Res 2016;4:3-12
|How to cite this URL:|
Lal I, Gupta N, Purushotham J, Sangwan VS. Limbal stem cell deficiency: Current management. J Clin Ophthalmol Res [serial online] 2016 [cited 2022 Jun 29];4:3-12. Available from: https://www.jcor.in/text.asp?2016/4/1/3/174344
The ocular surface is the first line of defense for the eye. It is defined by the epithelium covering the sclera, conjunctiva, and cornea. The presence of smooth corneal epithelium with stable tear film is essential for the maintenance of corneal clarity. The pleuripotent SCs at the basal layer of the limbus essentially has two functions: Renewal and regeneration of corneal epithelial cells and to serve as a barrier between the corneal and conjunctival epithelium. Any condition that either damages the limbal SCs, or causes loss of homeostasis between epithelial cell regeneration and desquamation results in the state called limbal stem cell deficiency (LSCD). This state of LSCD is characterized by the invasion of irregular and thickened conjunctival epithelium onto the corneal surface resulting in the loss of corneal clarity. However, with the advent of novel microsurgical techniques and immunosuppressive drugs, the treatment options have grown significantly. This article provides an overview of the etiology, clinical features, and therapeutic options for management of LSCD.
| Basic Properties of Stem Cells(SCs)|| |
SCs are undifferentiated biological cells that are capable of proliferation, self-maintenance, and producing a large number of differentiated progeny.  They are present in all self-renewing tissues like blood, testis, and stratified squamous epithelia.  However, they are a small subpopulation of the total tissue and have been estimated to make up only 0.5-10% of the total cell population.  There are unique inherent properties of SCsthat help them accomplish the task of cellular replacement and tissue regeneration. These features include poor differentiation, error-free proliferation, slow cycling, asymmetric cell division, anatomical protection, long life span, and specialized regulation. ,
| SCs at the Limbus|| |
The corneal epithelium is composed of five to six layers of nonkeratinized, stratified squamous epithelial cells. The conjunctival epithelium consists of nonkeratinized, stratified columnar, vascularized epithelium with mucin secreting goblet cells and is one to two cell layers thick.  At the limbus, there is a gradual transition from corneal to conjunctival epithelium. The unique architecture of the limbus consists of seven to 10 layers of cells and the limbal basement membrane is undulated, which clinically appears as palisades of Vogt. , This specialized microenvironment gives protection and nourishment to the essential SCsand is called the "niche". The fate of a SC, whether it is quiescence, apoptosis, division, or differentiation is governed by the interaction between the SCand the niche. 
Whenever there is a demand for tissue regeneration, the SCs divide. The asymmetric division of SCs leads to one transient amplifying cell (TAC) and the other daughter cell remains as the SC. Both SCs and TACs are progenitor cells located in the basal epithelium and give rise to postmitotic cells (PMCs) of the suprabasal layers and further to terminally differentiated cells (TDCs) of the superficial layer. Ultimately, TDCs are responsible for the expression of functional attributes of a particular tissue. The resulting cellular hierarchy contains heterogeneous populations ordered as follows according to increasing cell maturity: SC-TAC-PMC-TDC [Figure 1]. To maintain the health of the tissue, cellular proliferation, and differentiation in a coordinate manner across different levels of the hierarchy is indispensable. The uniform architecture of corneal epithelium can only be maintained by the rapid turnover of these cells.The 'X,Y,Z hypothesis' of corneal epithelial maintenance was proposed in 1983 by Thoft and Friend.  This hypothesis essentially states that when corneal epithelial cells are shed from the surface of the corneal epithelium (component Z), the lost cells are replaced by the migration of cells from the periphery of the corneal epithelium to the center (component X) and by the migration of cells from the basal layers of the corneal epithelium to the surface (component Y). Therefore, corneal epithelial homeostasis results when X+Y = Z. Conversely, when either X or Y fail to occur, the corneal epithelium cannot be maintained.
|Figure 1: Ocular surface epithelia covering the cornea, limbus, and conjunctiva. The conjunctival epithelium differs from the limbal-corneal epithelium in that it has mucin-expressing goblet cells. Some of the limbal basal epithelial cells are considered to be stem cells (SCs) for the corneal epithelium. By means of centripetal movement (open arrow), SCs generate corneal transient amplifying cells (TACs) located in the corneal epithelial basal layer. Both SCs and TACs are regarded as progenitor cells in the proliferative compartment, and they give rise to post-mitotic cells of the suprabasal layers, as well as terminally differentiated cells of the superficial layers. The latter two cell types belong to the differentiative compartment. Suprabasal cell movement at the limbus creates a barrier to separate the conjunctival epithelium from the corneal epithelium (solid arrow). (Adapted from Brightbill FS ed, Corneal Surgery: Theory, Technique, and Tissue. Edition3. St. Louis, Missouri. Mosby-Year Book, Inc. 1999.)|
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| Etiology of LSCD|| |
The LSCD may result from the destruction of the supporting SC niche, as occurs in hereditary diseases like aniridia or acquired disorders which lead to chronic inflammation of the limbus. However, the more common etiology is related to external factors that destroy the limbal SCssuch as Stevens-Johnson syndrome (SJS) and chemical or thermal burns [Table 1].
| Clinical Presentation|| |
Patients with LSCD may present with decreased vision, photophobia, tearing, blepharospasm, ocular pain, and redness. Depending on the extent of limbal involvement, LSCD can be partial or total and unilateral or bilateral. An abnormal epithelial sheet covering a variable area of the cornea may be reflective of mild to severe LSCD. By contrast, the condition is decidedly severe when a part of the cornea, usually including the pupillary area, is covered by a thick fibrovascular pannus. [Figure 2] illustrates the spectrum of manifestations of LSCD.
|Figure 2: Spectrum of manifestations of LSCD. (a) Total LSCD with granuloma formation, (b) Symblepharon, (c) LSCD secondary to vernal keratoconjunctivitis, (d) Partial LSCD, (e) Persistent epithelial defect, (f) Stippled staining on fluorescein stain|
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| Diagnosis of LSCD|| |
The diagnosis of LSCD is based primarily on clinical signs and may be confirmed by laboratory tests. Such tests include impression cytology, histopathology, fluorophotometry, and confocal microscopy. The primary clinical signs of the disease are:
- Loss of limbal anatomy: The normal limbal architecture with rows of palisades and perilimbal vascular arcades are well-defined at the superior and the inferior limbus. There may be loss of palisades of Vogt in an area known to have palisades prior to the insult. Other changes such as scarring, superficial vascularization, or limbal hyperemia indicating chronic inflammation can also be seen.
- Irregular, thin epithelium: During the initial stages of the disease process, progressive loss of a superficial segment of limbal epithelium may occur. Consequently, the sheet of conjunctival/metaplastic epithelium covers the cornea.This epithelium is usually thin and irregular as can be seen by the pooling of fluorescein dye at the junction of the abnormal and remaining normal epithelium. 
- Stippled fluorescein staining of the area covered by abnormal epithelium: The abnormal metaplastic epitheliumconjunctivalization is stained by fluorescein. This stippled fluorescein staining can occur in variable patterns like columns, whorls, or wedges that are easily identified. 
- Superficial and deep vascularization: During initial stages of LSCD, localized vascularization may gradually spread to cover the entire cornea.
- Persistent epithelial defects leading to ulceration, melting, and perforation: Repeated cycles of epithelial breakdown and healing are an important sign of LSCD.The edges of a persistent epithelial defect have a distinct, rolled-up or heaped appearance. Over the time, progressive melting of the corneal stroma with perforation can occur.
- Fibrovascular pannus: The epithelial cover of the denuded cornea is associated with the encroachment of fibrovascular tissue of varying thickness.
- Scarring, keratinization, and calcification: Eventually, by the end stage of the disease, the eye becomes quiet and there may be calcification of the affected tissue. In eyes with associated dryness, the epithelial cover becomes totally or partially keratinized.
| Laboratory Diagnosis of LSCD|| |
Most of the times, clinical diagnosis using slit-lamp biomicroscopy is sufficient. For research and documentation, laboratory diagnosis is useful.
- Impression cytology provides cytological evidence of LSCD. The procedure is simple and non invasive; it involves the application of cellulose acetate filter paper onto the ocular surface in order to remove a small quantity of surface cells. This sample is then examined for the presence of goblet cells as evidence of conjunctivalization. 
- Immunocytochemistry technique specifically recognizes cytokeratin markers expressed by differentiated epithelial cells. Cytokeratin 3 and 12 are present in normal corneal epithelial cells, whereas cytokeratin 19 is expressed by the conjunctival epithelium. So, it is possible to identify whether the corneal or the conjunctival epithelial phenotype is present over the cornea. The cytokeratin profile is known to have a strong clinical correlation and can confirm or refute diagnosis of LSCD. 
- In vivo confocal microscopy may also aid in establishing clinical diagnosis of LSCD and can be used to assess outcomes after surgery. The corneal epithelial cells viewed under a confocal microscope appear well-defined and regular, with bright borders and dark cytoplasm. In superficial layers, they appear flatter and have bright nuclei. Corneal epithelial cells can be clearly differentiated from conjunctival epithelial cells, which are hyperreflective and ill-defined. In addition, goblet cells and blood vessels can be seen using this technique. 
- Histopathology of the resected fibrovascular pannus and demonstration of the conjunctival epithelial phenotype is confirmatory.
| Management|| |
Principles of management
It is imperative to control the florid ocular surface inflammation in the acute stage of the disease either by pharmacological or surgical means. The definitive treatment of LSCDin chronic phase is surgical restoration of the corneal epithelialarchitecture. Corneal transplantation alone does not work in cases of LSCD as the transplanted cornea does not contain limbal SCs and these eyes eventually develop epithelial healing problems and recurrence of LSCD. The SC transplantation procedures are recommended for wet eyes with LSCD, as the SCs do not survive in cases with dry ocular surface like SJS. The eye lid abnormalities must be corrected prior to limbal transplantation procedures. If LSCD is due to underlying immune-mediated disease (MMP, SJS, VKC, etc.), then it is critical to control inflammation using local and/ or systemic immunomodulation.
Acute stage management
It has been recognized that in the acute stage of chemical injury and SJS, inflammation causes greater SC damage than the primary injury itself.  Even if fluorescein staining depicts an ocular surface defect involving the limbus, it does not ascertain that limbal SCsare completely destroyed at basal level. Therefore, the ophthalmologist who first treats the patient is of prime importance in taking effective measures to suppress inflammation and preserve the limbal SCs.
Application of amniotic membrane in the acute stage of insult serves as a biological bandage.
Prabhasawat et al.,  reported that amniotic membrane transplant (AMT) within 5 days of grade II-III chemical burns resulted in faster epithelial healing as well as decreased corneal haze and LSCD when compared with the results of AMT after 5 days of injury. It is well recognized that when performed within 2 weeks of the onset of the ocular effects of SJS, AMT facilitates rapid epithelial healing and reduces inflammation and scarring of the ocular surface. ,, A newer approach involves the implantation of ProKera-an FDA-approved class II medical device consisting of a sheet of cryopreserved amniotic membrane clipped into a dual symblepharon ring system - during the acute stage of chemical injury or SJS. It allows for an early intervention, at bedside, promoting the beneficial effect of reduction of inflammation and early healing of epithelial defect of the cornea and conjunctiva. , Medical management of acute stage disease is equally important, especially use of topical corticosteroid in acute ocular surface burns is of paramount importance.
Chronic stage management
Chronic stage management of LSCD is dependent on a multitude of factors, which needs to be considered before planning an intervention. Factors to be considered are the degree of LSCD, laterality of the disease, extent of conjunctival disease, presence of conjunctival inflammation, nature of the ocular surface, and the age and general health of the patient. Restoration of normal corneal phenotype and, in turn, corneal clarity is highly dependent on the health of the surrounding tissues. As such, rehabilitation of the ocular surface includes efforts to improve the ocular surface environment. Health of the ocular surface environment is maintained by control of inflammation, proper lubrication, adequate eyelid closure, and the elimination of keratinization and symblephara. Any associated eyelid abnormalities such as poor eyelid closure or eyelid malposition must be surgically corrected as well. Furthermore, instabilities in the tear film and dry eye-common accompanying features of LSCD-must be resolved before considering SC transplant. 
| Unilateral Partial LSCD|| |
Partial LSCD causing peripheral conjunctivalization of the corneal surface rather than damage to the visual axis can be managed conservatively. The conservative measures for treatment of partial LSCD include copious topical lubricants and anti-inflammatory agents, and the use of bandage contact lenses.  Gas permeable scleral contact lenses may have therapeutic and visual benefits. , The fluid interphase in scleral contact lenses appears to aid re-epithelialization through oxygenation, moisture, and protection. However, definitive treatment of any LSCD is surgical. Medical or conservative treatment would not rectify the LSCD.
Surgical treatment is the only option in cases of LSCD where the visual axis is involved and most of the corneal surface is covered with conjunctiva-like epithelium. Dua et al.,  have described the technique of sequential sector conjunctival epitheliectomy (SSCE) for the management of such cases of LSCD. The procedure requires removal of conjunctival epithelium that is covering a sector of the cornea and limbus or adjacent bulbar conjunctiva, so that corneal epithelial cells may regrow over the denuded surface.AMT is useful in such cases and helps in restoration of ocular surface. In many cases of partial LSCD, the debridement of the conjunctival epithelium, amniotic membrane coupled by follow up of the patient may suffice.
The management of diffuse LSCD involves surgical transplantation of limbal SCs. A number of transplantation techniques have been used over the years, and many have been described with variable terminologies. The nomenclature for the ocular surface rehabilitative procedure, given by the Cornea Society, is based on the anatomic source of the donor (conjunctiva, limbus, and oral mucosa), genetic source of the donor (autograft or allograft-cadaveric/living relative/living nonrelative), and cell culture techniques used for donor tissue multiplication.  Limbal SCs may only be transplanted with a carrier tissue, that may be either conjunctival (conjunctival limbal graft) or cornea (keratolimbal graft).  These procedures may be combined with or followed by keratoplasty.
Preparation of the recipient bed
In all transplantation procedures, the preparation of the recipient eye is similar. Briefly, symblephara are released before inserting the speculum and are allowed to fall back. Conjunctival peritomy is done 4-5 mm from the limbus. The conjunctivalized pannus is removed from the corneal surface with blunt dissection. The hemostasis is achieved with wet-field cautery or dilute adrenaline (1:10,000).
The choice of ocular surface rehabilitative procedure depends on whether the condition is unilateral or bilateral and the extent of limbal involvement. Limbal autograft, either conjunctival limbal autograft or transplantation of cultured limbal SCs from the fellow eye, is the procedure of choice in cases of unilateral LSCD. However, it is vital to assess the donor eye and ensure that it is free from any condition that may predispose to development of LSCD prior to conducting the autograft.  In cases of bilateral total LSCD, an allograft limbal transplant utilizing donor tissue from a live relative, a live nonrelated donor, or a cadaveric tissue is required. Alternatively, an exvivo cultivated oral mucosal or conjunctival transplant can be performed. Autologous transplantation is preferred over allogenic donor transplantation as there is no risk of rejection and therefore no need for immunosuppression. These advantages improve the prognosis for a favorable outcome following autograft compared to after allograft procedures.
The algorithm for management of LSCD is given in [Table 2]. The studies conducted on different surgical procedures and their outcomes have been enumerated in [Table 3]. The indication and surgical technique for each has been described along with a review of the outcome. Findings from small case series have been excluded from the discussion, as they are inconclusive.
|Table 2: Algorithm for management of limbal stem cell defi ciency at acute and chronic stage|
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| Conjunctival Limbal Autograft (CLAU)|| |
CLAU was described by Kenyon and Tseng in 1989.  The procedure is most beneficial for patients with unilateral partial or total LSCD due to chemical injury, multiple surgeries, or contact lens use.
Two strips of limbal conjunctival grafts, each spanning 6-7 mm limbal arc length, are removed by superficial lamellar keratectomy including 1mm within the limbusfrom superior and inferior limbal quadrants. Depending on the outcome of symblepharonlysis, 5mm or more of the adjacent conjunctiva may also be taken. These two free grafts are transferred and secured to the recipient eye, at the corresponding anatomical sites, by interrupted 10-0 nylon sutures at the limbus and 8-0 vicryl sutures at the sclera. An amniotic membrane patch may be used to cover the lesion site on the donor eye, thereby preventing any possible complications due to surgery. Although there is no defined optimal size for tissue harvested from the donor eye, excision upto 8 clock hours has been transplanted. In our opinion, it is best to avoid excision and transplantation of more than 6 clock hours for the fear of precipitating iatrogenic LSCD in the donor eye. To prevent donor site LSCD, the limbal graft should be partial thickness (40-60 μm).
| Results|| |
Tseng et al.,  observed clinical outcomes of 21 patients with follow-up at 6 months or more and found improved visual acuity (17 cases), rapid surface healing (19 cases), stable epithelial adhesion without recurrent erosions or persistent epithelial defect (20 cases), arrest or regression of corneal neovascularization (15 cases), and probable increased success for lamellar or penetrating keratoplasty (eight cases). No intraoperative complications were encountered and no adverse reactions developed in donor eyes. Impression cytology in selected cases showed restoration of the corneal epithelial phenotype and regression of goblet cells from the recipient cornea. In a similar series by Rao et al.,  the authors recommended procedures like tenonplasty to facilitate vascularization of the limbal bed. However, donor site complications were reported by several groups, raising concerns regarding the safety of this procedure. ,
| Keratolimbal Allograft (KLAL)|| |
KLAL,a surgical procedure performed to treat severe bilateral ocular surface disorders and systemic immunosuppression, is mandatory for the success of these cases.  It is also a surgical option for patients with unilateral SC deficiency who fear damage to the fellow eye during tissue extraction. It involves the transplantation of allogenic cadaveric limbal SCs onto the affected eye. KLAL may be useful if there is no available living relative. It is ideally suited for disease entities that primarily affect the limbus with minimal or no involvement of the conjunctiva, such as aniridia. The procedure may be performed as a 360-degree KLAL for treatment of total LSCD, or as a sectoral KLAL for treatment of partial LSCD.
The source of tissue for KLALis a donor corneoscleral rim preserved in corneal storage medium at 4°C. The age of the donor preferably should be under 50 years for optimal SC yield. The central cornea of the corneoscleral rim is excised with a 7.5mm trephine. Excess peripheral rim is excised leaving approximately 1mm of sclera peripheral to the limbus. The posterior one-half to two-thirds of the ring is then removed by lamellar dissection using a sharp, rounded, steel, crescent blade. The ring-shaped limbal tissue is sutured to the recipient limbal area with interrupted sutures. At the conclusion of this procedure, a penetrating or lamellar keratoplasty may be performed using tissue from the same corneoscleral button.
| Results|| |
The technique was first described by Tsai and Tseng  in 1994 and since then has been reported by others. The long-term success of KLAL with immunosuppressant drugs has been 40-50% at3-5 years after surgery. , In diseases with chronic inflammation, aqueous tear deficiency, and uncorrected lid abnormalities such as SJS, the success of KLAL has been reported to be low (Shimazaki et al.).  This low success rate is attributed to sensitization, which leads to early allograft rejection. Systemic cyclosporine has been reported to prolong graft survival by decreasing chronic rejection in KLAL (Ilari L, Daya SM). 
Conjunctival Limbal Allograft (CLAL)
CLAL is utilized for similar group of patients as KLAL, but it possesses the advantage of transplanting more viable SCs. The surgical procedure of CLAL is identical to CLAU. The source of donor tissue can be cadaveric (c-CLAL), from a live relative (lr-CLAL) or a live non-relative (lnr-CLAL). Immunosuppression is also mandatory for this procedure.
Tsuboto et al., observed the outcome of limbal allograft in a case series with nine patients.  The group reported complete epithelialization in five cases at a mean follow-up of 12.3 months after surgery. Two patients underwent episodes of rejection, which were successfully controlled by systemic immunosuppression.
Cultured limbal epithelial transplantation
This is a novel method of transplanting limbal SCs following ex vivo expansion of limbal SCs. The limbal biopsy is performed either from the contralateral eye or the healthy limbus in cases of partial LSCD. In cases of bilateral LSCD, the limbal biopsy is obtained from the eye of a relative or from a cadaveric eye. The extracted tissue contains a population of limbal SCs, which are isolated and expanded in a laboratory to produce a sheet of cultured limbal SCs over the amniotic membrane suitable for transplantation onto the cornea. The advantage of the procedure is that the required limbal biopsy is substantially small and minimizes the risk of precipitating SC failure in the donor eye, and it also provides an option for repeated biopsy. Another theoretical advantage of this new technique over KLAL and lr-CLAL is the reduced risk of allograft rejection due to the absence of antigen-presenting Langerhans cells in the ex vivo cultured SCs.
For patients with visually significant stromal scarring, penetrating keratoplasty is recommended in addition to limbal SC transplant, with at least 3 months interval between the two procedures. For surgeries utilizing allogenic transplant, systemic immunosuppression has been shown to improve transplant success rate. A multidrug regimen administered over the course of 12-18 months is recommended, after which the dosage can be gradually tapered. Specific immunosuppressive drugs with dosage are summarized in [Table 4].
|Table 4: Immunosuppresive drugs post ocular surface rehabilitative procedures|
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Pellegrini and De Luca  first described the results of cultured autologous corneal epithelial cell transplantation. In large series by Rama et al., (107 eyes),  Sangwan et al., (200 eyes),  and Di Iorio et al., (166 eyes);  clinical success was reported in 68, 71, and 80% of eyes, respectively. In the study by Sangwan et al., it was shown that eyes with previous AMG or Penetrating keratoplasty (PK) were more prone to failure of limbal SC transplant, while the timing of limbal transplantation did not affect the eventual outcome as long as the surface inflammation was adequately controlled.  Repeat procedure for failed cases also have shown promising results with success rates being 66%  and 82%  .However, this procedure has the disadvantage of being expensive due to laboratory costs and the risks involved in using xenobiotic materials for culture. Xeno-free culture techniques have been devised, but the cost is a major limiting factor in its widespread application. 
The results of repeat CLET are encouraging in terms of stabilization of ocular surface as well as improvement in visual acuity. Out of 50 eyes, 33 (66%) maintained a completely epithelialized and clinically stable corneal surface. A two-line improvement in visual acuity was seen in 38 of the 50 recipient eyes (76%). None of the donor eyes developed LSCD. The minimum follow-up in this study was 1 year.  For cases with partial LSCD, outcomes are similar and are not dependent on whether the limbal biopsy is taken from the healthy or the affected eye.  In case of children less than 15 years of age, the results of CLET are not as good as adult cases and nearly half of children achieve a stable ocular surface along with improvement in visual acuity. 
In a study by Basu et al., patients with unilateral LSCD underwent PK either as a single stage (along with CLET, n=12) or two-stage (at least 6 weeks after CLET, n = 35) procedure. The mean follow-up was 4.2 ± 1.9 years. Corneal allograft survival rate at 1 year was significantly greater in eyes undergoing two-stage (80 ± 6%; median survival, 4 years) compared with single-stage (25 ± 13%; median survival, 6 months; P = 0.0003) procedure. Visual acuity of 20/40 or better was attained by 71.4% of eyes with clear corneal grafts. Recurrence of LSCD was more common after single-stage (58.3%) than two-stage (14.3%) surgery (P = 0.008).  Thus, it can be concluded that the two-stage approach of cultivated limbal epithelial transplantation followed by PK successfully restores ocular surface stability and vision in eyes with chronic ocular burns. The single-stage approach is associated with poorer clinical outcomes.
| Simple Limbal Epithelial Transplantation (SLET)|| |
SLET is a simplified technique of limbal transplantation, which combines the benefits of both CLAU and cultivated limbal epithelial transplant described by Sangwan et al.  It is a single-stage procedure, does not require an expensive laboratory set up unlike CLET and there is no danger of inducing iatrogenic LSCD in the donor eye. it is convenient for the patients and it is cost-effective because of single stage and no requirement of laboratory expanded cells. Here, ocular surface, after excision of pannus, serves as a natural incubator for cell growth and multiplication.
This technique requires less donor tissue than previously used for conventional autografting. A 2 × 2 mm area of limbal tissue is excised and secured. The donor tissue is divided into 10-15 small pieces and placed onto the amniotic membrane over the recipient bed, avoiding the visual axis. These transplants are secured in place with fibrin glue and a bandage contact lens is applied [Figure 3].
|Figure 3: Surgical steps of SLET. (a) Limbal biopsy from the healthy donor eye, (b) 360° conjunctivalperitomy, (c) pannus dissection, (d) cauterization of scleral bed, (e) amniotic membrane graft placement over the ocular surface, (f) chopping of limbal biopsy into small pieces on Teflon block, (g) placement of biopsy transplants on the cornea, (h) application of fibrin glue to secure the transplants, and (i) bandage contact lens. SLET = Simple limbal epithelial transplant|
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Sangwan et al.,  reported the results of SLET in six patients with unilateral and total LSCDfollowing ocular surface burns. At a mean follow-up of 6 weeks, a completely stable ocular surface was seen in all recipient eyes, and this was maintained long-term at a mean follow-up of 9.2 months. Visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/60 or better in four (66.6%) eyes. Meanwhile, none of the donor eyes developed any complications. Amescua et al., have recently described a sandwich technique of SLET using cryopreserved amniotic membrane where in the transplants are placed between the two layers of AMG. Their outcomes were comparable with the outcomes of the original technique in a case series of four cases. 
| Conclusion|| |
The management of LSCD is a challenge. Currently available therapeutic options have given successful results in reasonably large proportion of treated eyes. The success of a given therapy is dependent on the underlying pathology, the tearand eyelid function, and presence of ocular surface inflammation. However, the role of primary treatment received at the time of insult is crucial, especially in conditions of ocular burn and SJS. The placement of amniotic membrane at an early stage has been shown to prevent or limit damage to the limbal SCs.
Invivo expansion of SCs is an effective technique for unilateral LSCD ineyes with a wet ocular surface, which eliminates the need for an expensive SClaboratory. Currently research is going on to develop alternatives to human amniotic membrane to improve safety and further reduce the cost of the surgery. The challenges include developing a product capable of mass production, nontoxic, safe for human use, and can support the SCs. Other sources of cells are being tested for their potential to regenerate the ocular surface. These include human embryonic SCs, skin epidermal SCs, hair follicle SCs, bone marrow-derived mesenchymal SCs, and immature dental pulp SCs. Although the results of SLET are encouraging, there are still a few questions that remained unanswered like how long does the "stemness" of these SCs last and how many SCs should be transplanted. Moreover, this technique is only effective for patients with a good tear secretion (wet eyes). The ultimate challenge lies in converting dry eyes to wet eyes so that they can benefit from SC transplantation procedures. Could we regrow glandular structures for dry eyes? Can we further refine surgical technique of SLET like quantifying amount of tissue (limbal biopsy, size, and thickness) required for regenerating entire corneal epithelium? 
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]