Home Print this page Email this page Users Online: 443
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2016  |  Volume : 4  |  Issue : 1  |  Page : 3-12

Limbal stem cell deficiency: Current management

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 Submission04-Nov-2014
Date of Acceptance14-Apr-2015
Date of Web Publication19-Jan-2016

Correspondence Address:
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
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2320-3897.174344

Rights and Permissions

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 2023 Mar 25];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) Top

SCs are undifferentiated biological cells that are capable of proliferation, self-maintenance, and producing a large number of differentiated progeny. [1] They are present in all self-renewing tissues like blood, testis, and stratified squamous epithelia. [2] 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. [3] 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. [4],[5]

  SCs at the Limbus Top

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. [6] 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. [7],[8] 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. [9]

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. [10] 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.)

Click here to view

  Etiology of LSCD Top

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].
Table 1: Summary of the causes of limbal stem cell disorder

Click here to view

  Clinical Presentation Top

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

Click here to view

  Diagnosis of LSCD Top

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:

  1. 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.
  2. 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. [11]
  3. 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. [12]
  4. Superficial and deep vascularization: During initial stages of LSCD, localized vascularization may gradually spread to cover the entire cornea.
  5. 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.
  6. Fibrovascular pannus: The epithelial cover of the denuded cornea is associated with the encroachment of fibrovascular tissue of varying thickness.
  7. 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 Top

Most of the times, clinical diagnosis using slit-lamp biomicroscopy is sufficient. For research and documentation, laboratory diagnosis is useful.

  1. 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. [13]
  2. 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. [14]
  3. 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. [15]
  4. Histopathology of the resected fibrovascular pannus and demonstration of the conjunctival epithelial phenotype is confirmatory.

  Management Top

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. [13] 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., [16] 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. [17],[18],[19] 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. [20],[21] 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. [22]

  Unilateral Partial LSCD Top

Conservative management

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. [23] Gas permeable scleral contact lenses may have therapeutic and visual benefits. [24],[25] 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., [26] 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.

Total LSCD

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. [27] Limbal SCs may only be transplanted with a carrier tissue, that may be either conjunctival (conjunctival limbal graft) or cornea (keratolimbal graft). [27] 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. [28] 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

Click here to view
Table 3: Studies of surgical procedures for limbal stem cell deficiency

Click here to view

  Conjunctival Limbal Autograft (CLAU) Top

CLAU was described by Kenyon and Tseng in 1989. [29] The procedure is most beneficial for patients with unilateral partial or total LSCD due to chemical injury, multiple surgeries, or contact lens use.

Surgical technique

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 Top

Tseng et al., [29] 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., [30] 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. [31],[32]

  Keratolimbal Allograft (KLAL) Top

KLAL,a surgical procedure performed to treat severe bilateral ocular surface disorders and systemic immunosuppression, is mandatory for the success of these cases. [33] 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.

Surgical technique

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 Top

The technique was first described by Tsai and Tseng [33] 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. [34],[35] 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.). [36] 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). [37]

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. [38] 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.

Postoperative management

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

Click here to view


Pellegrini and De Luca [39] first described the results of cultured autologous corneal epithelial cell transplantation. In large series by Rama et al., (107 eyes), [40] Sangwan et al., (200 eyes), [41] and Di Iorio et al., (166 eyes); [42] 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. [41] Repeat procedure for failed cases also have shown promising results with success rates being 66% [43] and 82% [40] .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. [41]

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. [43] 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. [44] 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. [45]

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). [46] 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) Top

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. [47] 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.

Surgical technique

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

Click here to view


Sangwan et al., [47] 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. [48]

  Conclusion Top

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.

Future directions

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? [73]

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Potten CS, Loeffler M. Epidermal cell proliferation. Changes with time in the proportion of isolated, paired and clustered labeled cells in sheets of murine epidermis. Virchows Arch B Cell Pathol Incl Mol Pathol 1987;53:279-85.  Back to cited text no. 1
Cairnie AB. Homoeostasis in the small intestine. In Stem cells of renewing cell populations. Cairnie AB, Lala PK, Osmond DG. New York: Academic Press; 1976:67-77.  Back to cited text no. 2
Potten CS, Morris RJ. Epithelial stem cells in vivo. J Cell Sci Suppl 1988;10:45-62.  Back to cited text no. 3
Tseng SC. Concept and application of limbal stem cells. Eye (Lond) 1989;3:141-57.  Back to cited text no. 4
Boulton M, Albon J. Stem cells in the eye. Int J Biochem Cell Biol 2004;36:643-57.  Back to cited text no. 5
Kenyon KR. Anatomy and pathology of the ocular surface. Int Ophthalmol Clin. 1979; 19:3-35.  Back to cited text no. 6
Goldberg MF, Bron AJ. Limbal palisades of Vogt. Trans Am Ophthalmol Soc 1982;80:155-71.  Back to cited text no. 7
Gipson IK. The epithelial basement membrane zone of the limbus. Eye (Lond) 1989; 3:132-40.  Back to cited text no. 8
Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell 1997; 88:287-98.  Back to cited text no. 9
Thoft RA, Friend J. The X,Y,Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 1983; 24:1442-3.  Back to cited text no. 10
Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol 1998; 82:1407-11.  Back to cited text no. 11
Dua HS, Forrester JV. The corneoscleral limbus in human corneal epithelial wound healing. Am J Ophthalmol 1990;110:646-56.  Back to cited text no. 12
Puangsricharen V, TsengSC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995; 102:1476-85.  Back to cited text no. 13
Sacchetti M, Lambiase A, Cortes M, Sgrulletta R, Bonini S, Merlo D, et al. Clinical and cytological findings in limbal stem cell defiency. Graefes Arch Clin Exp Ophthalmol 2005; 243:870-6.  Back to cited text no. 14
Dua HS, Miri A, Alomar T, Yeung AM, Said DG. The role of limbal stem cells in corneal epithelial maintenance: Testing the dogma. Ophthalmology 2009; 116:856-63.  Back to cited text no. 15
Prabhasawat P, Tesavibul N, Prakairungthong N, Booranapong W. Efficacy of amniotic membrane patching for acute chemical and thermal ocular burns. J Med Assoc Thai 2007; 90:319-26.  Back to cited text no. 16
Ciralsky JB, Sippel KC, Gregory DG. Current ophthalmologic treatment strategies foracuteand chronicStevens-Johnson syndrome and toxic epidermal necrolysis. Curr Opin Ophthalmol 2013; 24:321-8.  Back to cited text no. 17
Hsu M, Jayaram A, Verner R, Lin A, Bouchard C. Indications and outcomes of amniotic membrane transplantation in the management of acute Stevens-Johnson syndrome and toxic epidermal necrolysis: Acase-control study. Cornea 2012; 31:1394-402.  Back to cited text no. 18
Gregory DG. Treatment of acute Stevens-Johnson syndrome and toxic epidermal necrolysis using amniotic membrane: A review of 10 consecutive cases. Ophthalmology 2011; 118:908-14.  Back to cited text no. 19
Kheirkhah A, Johnson DA, Paranjpe DR, Raju VK, Casas V, Tseng SC. Temporary sutureless amniotic membrane patch for acute alkaline burns. Arch Ophthalmol 2008; 126:1059-66.  Back to cited text no. 20
Tomlins PJ, Parulekar MV, Rauz S. "Triple-TEN" in the treatment ofacuteocular complications from toxic epidermal necrolysis. Cornea 2013; 32:365-9.  Back to cited text no. 21
De Sousa JL, Daya S, Malhotra R. Adnexal surgery in patients undergoing ocular surface stem cell transplantation. Ophthalmology 2009; 116:235-42.  Back to cited text no. 22
Shortt AJ, Secker GA, Notara MD, Limb GA, Khaw PT, Tuft SJ, et al. Transplantation of ex vivo cultured limbal epithelial stem cells: A review of techniques and clinical results. Surv Ophthalmol 2007; 52:483-502.  Back to cited text no. 23
Romero-Rangel T, Stavrou P, Cotter J, Rosenthal P, Baltatzis S, Foster CS. Gas-permeable scleral contact lens therapy in ocular surface disease. Am J Ophthalmol 2000; 130:25-32.  Back to cited text no. 24
Rosenthal P, Cotter JM, Baum J. Treatment of persistent corneal epithelial defect with extended wear of a fluid-ventilated gas-permeable scleral contact lens. Am J Ophthalmol 2000; 130:33-41.  Back to cited text no. 25
Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J Ophthalmol 1994; 78:401-8.  Back to cited text no. 26
Daya SM, Chan CC, Holland EJ, Members of The Cornea Society Ocular Surface Procedures Nomenclature Committee. Cornea society nomenclature for ocular surface rehabilitative procedures. Cornea 2011; 30:1115-9.  Back to cited text no. 27
Jenkins C, Tuft S, Liu C, Buckley R. Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy. Eye (Lond) 1993; 7:629-33.  Back to cited text no. 28
Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 1989; 96:709-22.  Back to cited text no. 29
Rao SK, Rajagopal R, Sitalakshmi G, Padmanabhan P. Limbal autografting: Comparison of results in the acute and chronic phases of ocular surface burns. Cornea 1999; 18:164-71.  Back to cited text no. 30
Miri A, Said DG, Dua HS. Donor site complications in autolimbal and living-related allolimbal transplantation. Ophthalmology 2011; 118:1265-71.  Back to cited text no. 31
Dua HS, Azuara-Blanco A. Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency. Br J Ophthalmol 2000; 84:273-8.  Back to cited text no. 32
Tsai RJ, Tseng SC. Human allograft limbal transplantation for corneal surface reconstruction. Cornea 1994; 13:389-400.  Back to cited text no. 33
Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H, et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med 1999; 340:1697-703.  Back to cited text no. 34
Solomon A, Ellies P, Anderson DF, Touhami A, Grueterich M, Espana EM, et al. Long-term outcome of keratolimbal allograft with or without penetrating keratoplasty for total limbal stem cell deficiency. Ophthalmology2002; 109:1159-66.  Back to cited text no. 35
Shimazaki J, Maruyama F, Shimmura S, Fujishima H, Tsuboto K. Immunologic rejection of the central graft after limbal allograft transplantation combined with penetrating keratoplasty. Cornea 2001; 20:149-52.  Back to cited text no. 36
Ilari L, Daya SM. Long-term outcomes of keratolimbal allograft for the treatment of severe ocular surface disorders. Ophthalmology 2002; 109:1278-84.  Back to cited text no. 37
Tsuboto K, Toda I, Saito H, Shinozaki N, Shimazaki J. Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders. Ophthalmology 1995; 102:1486-96.  Back to cited text no. 38
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997; 349:990-3.  Back to cited text no. 39
Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010; 363:147-55.  Back to cited text no. 40
Sangwan VS, Basu S, Vemuganti GK, Sejpal K, Subramaniam SV, Bandyopadhyay S, et al. Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: A10-year study. Br J Ophthalmol 2011; 95: 1525-9.  Back to cited text no. 41
Di Iorio E, Ferrari S, Fasolo A, Bohm E, Ponzin D, Barbaro V. Techniques for culture and assessment of limbal stem cell grafts. Ocul Surf 2010; 8:146-53.  Back to cited text no. 42
Basu S, Ali H, Sangwan VS. Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol 2012; 153:643-50, 650.  Back to cited text no. 43
VaziraniJ, Basu S, Kenia H, Ali MH, Kacham S, Mariappan I, et al. Unilateral partial limbal stem cell deficiency: Contralateral versus ipsilateral autologous cultivated limbal epithelia ltransplantation. Am J Ophthalmol2014; 157:584-90.  Back to cited text no. 44
Sejpal K, Ali MH, Maddileti S, Basu S, Ramappa M, Kekunnaya R, et al. Cultivated limbal epithelial transplantation in children withocular surface burns. JAMA Ophthalmol 2013; 131:731-6.  Back to cited text no. 45
Basu S, Mohamed A, Chaurasia S, Sejpal K, Vemuganti GK, Sangwan VS. Clinical outcomes of penetrating keratoplasty after autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol 2011; 152:917-24.  Back to cited text no. 46
Sangwan VS, Basu S, MacNeil S, Balasubramanian D. Simple limbal epithelial transplantation (SLET): A novel surgical technique for the treatment of unilateral limbal stem cell deficiency. Br J Ophthalmol 2012; 96:931-4.  Back to cited text no. 47
Amescua G, Atallah M, Nikpoor N, Galor A, Perez VL. Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency. Am J Ophthalmol 2014; 158:469-75.  Back to cited text no. 48
Holland EJ. Epithelial transplantation for the management of severe ocular surface disease. Trans Am Ophthalmol Soc 1996; 94:677-743.  Back to cited text no. 49
Tan DT, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology 1996; 103:29-36.  Back to cited text no. 50
Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol 1998; 116:431-41.  Back to cited text no. 51
Reinhard T, Sundmacher R, Spelsberg H, Althaus C. Homologous penetrating central limbo-keratoplasty (HPCLK) in bilateral limbal stem cell insufficiency. Acta Ophthalmol Scand 1999;77:663-7.  Back to cited text no. 52
Rama P, Bonini S, Lambiase A, Golisano O, Paterna P, De Luca M, et al. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 2001; 72:1478-85.  Back to cited text no. 53
Anderson DF, Ellies P, Pires RT, Tseng SC. Amniotic membrane transplantation for partial limbal stem cell deficiency.Br J Ophthalmol 2001; 85:567-75.  Back to cited text no. 54
Prabhasawat P, Ekpo P, Uiprasertkul M, Chotikavanich S, Tesavibul N. Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction. Clin Ophthalmol 2012; 6:1483-92.  Back to cited text no. 55
Yao Y, Zhang B, Zhou P, Jiang JK. Autologous limbal grafting combined with deep lamellar keratoplasty in unilateral eye with severe chemical or thermal burn at late stage. Ophthalmology 2002; 109:2011-7.  Back to cited text no. 56
Holland EJ, Djalilian AR, Schwartz GS. Management of aniridic keratopathy with keratolimbal allograft: A limbal stem cell transplantation technique. Ophthalmology 2003; 110:125-30.  Back to cited text no. 57
Gomes JA, dos Santos MS, Cunha MC, Mascaro VL et al. Amniotic membrane transplantation for partial and total limbal stem cell deficiency secondary to chemical burn. Ophthalmology 2003; 110:466-73.  Back to cited text no. 58
Shimazaki J, Shimmura S, Tsubota K. Donor source affects the outcome of ocular surface reconstruction in chemical or thermal burns of the cornea. Ophthalmology 2004; 111:38-44.  Back to cited text no. 59
Reinhard T, Spelsberg H, Henke L, Kontopoulos T, Enczmann J, Wernet P, et al. Long-term results of allogenic penetrating limbo-keratoplasty in total limbal stem cell deficiency. Ophthalmology 2004; 111:775-82.  Back to cited text no. 60
Ozdemir O, Tekeli O, Ornek K, Arslanpence A, Yalcindag NF. Limbal autograft and allograft transplantations in patients with corneal burns. Eye (Lond) 2004; 18:241-8.  Back to cited text no. 61
Santos MS, Gomes JA, Hofling-Lima AL, Rizzo LV, RomanoA, Belfort R Jr. Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency. Am J Ophthalmol 2005; 140:223-30.  Back to cited text no. 62
Ivekovic R, Tedeschi-Reiner E, Novak-Laus K, Andrijevic-Derk B, Cima I, Mandic Z. Limbal graft and/or amniotic membrane transplantation in the treatment of ocular burns. Ophthalmologica 2005; 219:297-302.  Back to cited text no. 63
Chandra A, Maurya OP, Reddy B, Kumar G, Pandey K, Bhaduri G. Amniotic membrane transplantation in ocular surface disorders. J Indian Med Assoc 2005; 103:364-6.  Back to cited text no. 64
Sangwan VS, Matalia HP, Vemuganti GK, Fatima A, Ifthekar G, Singh S, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol 2006; 54:29-34.  Back to cited text no. 65
[PUBMED]  Medknow Journal  
Inatomi T, Nakamura T, Koizumi N, Sotozono C, Yokoi N, Kinoshita S. Midterm results on ocular surfacereconstruction using cultivated autologous oral mucosal epithelial transplantation. Am J Ophthalmol 2006; 141:267-75.  Back to cited text no. 66
Sangwan VS, Matalia HP, Vemuganti GK, Fatima A, Ifthekar G, Singh S, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol 2006; 54:29-34.  Back to cited text no. 67
[PUBMED]  Medknow Journal  
Pauklin M,Fuchsluger TA,Westekemper H,Steuhl KP,Meller D. Midterm results of cultivated autologous and allogeneic limbal epithelialtransplantationin limbal stem cell deficiency. Dev Ophthalmol 2010; 45:57-70.  Back to cited text no. 68
Biber JM,Skeens HM,Neff KD,Holland EJ. The cincinnati procedure: Technique and outcomes of combined living-relatedconjunctivallimbalallografts and keratolimbal allografts in severe ocular surface failure. Cornea 2011; 30:765-71.  Back to cited text no. 69
Nakamura T, Takeda K, Inatomi T, Sotozono C, Kinoshita S. Long-term results of autologous cultivated oral mucosal epithelialtransplantationin the scar phase of severe ocular surface disorders. Br J Ophthalmol 2011; 95:942-6.  Back to cited text no. 70
Burillon C, Huot L, Justin V, Nataf S, Chapuis F, Decullier E, et al. Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Invest Ophthalmol Vis Sci 2012; 53:1325-31.  Back to cited text no. 71
Marchini G, Pedrotti E, Pedrotti M, Barbaro V, Di Iorio E, Ferrari S, et al. Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns. Clin Experiment Ophthalmol2012; 40:255-67.  Back to cited text no. 72
Konomi K, Satake Y, Shimmura S, Tsubota K, Shimazaki J. Long-term results ofamniotic membrane transplantation for partial limbal deficiency. Cornea 2013; 32:1110-5.  Back to cited text no. 73


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]

This article has been cited by
1 Evaluation of the factors that influence surgical outcome in conjunctival-limbal allograft transplantation
Murat Kasikci, Ilayda Korkmaz, Melis Palamar, Sait Egrilmez, Ayse Yagci, Ozlem Barut Selver
Eye. 2022;
[Pubmed] | [DOI]
2 Evaluation of Limbal Stem Cell Transplant Success in Ocular Chemical Injury
Ilayda Korkmaz, Melis Palamar, Sait Egrilmez, Mehmet Gurdal, Ayse Yagci, Ozlem Barut Selver
Experimental and Clinical Transplantation. 2022;
[Pubmed] | [DOI]
3 Therapeutic measures for sulfur mustard-induced ocular injury
Ariel Gore,Tamar Kadar,Shlomit Dachir,Vered Horwitz
Toxicology Letters. 2021; 340: 58
[Pubmed] | [DOI]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Basic Properties...
SCs at the Limbus
Etiology of LSCD
Clinical Present...
Diagnosis of LSCD
Laboratory Diagn...
Unilateral Parti...
Conjunctival Lim...
Keratolimbal All...
Simple Limbal Ep...
Article Figures
Article Tables

 Article Access Statistics
    PDF Downloaded982    
    Comments [Add]    
    Cited by others 3    

Recommend this journal