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Year : 2004  |  Volume : 52  |  Issue : 1  |  Page : 5-22

Limbal stem cell transplantation

Cornea and Anterior Segment Service, L V Prasad Eye Institute, Hyderabad, India

Correspondence Address:
M Fernandes
Cornea and Anterior Segment Service, L V Prasad Eye Institute, Hyderabad
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Source of Support: None, Conflict of Interest: None

PMID: 15132374

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The past two decades have witnessed remarkable progress in limbal stem cell transplantation. In addition to harvesting stem cells from a cadaver or a live related donor, it is now possible to cultivate limbal stem cells in vitro and then transplant them onto the recipient bed. A clear understanding of the basic disease pathology and a correct assessment of the extent of stem cell deficiency are essential. A holistic approach towards management of limbal stem cell deficiency is needed. This also includes management of the underlying systemic disease, ocular adnexal pathology and dry eye. Conjunctival limbal autografts from the healthy contralateral eye are performed for unilateral cases. In bilateral cases, tissue may be harvested from a cadaver or a living related donor; prolonged immunosuppression is needed to avoid allograft rejection in such cases. This review describes the surgical techniques, postoperative treatment regimes (including immunosuppression for allografts), the complications and their management. The short and long-term outcomes of the various modalities reported in the literature are also described.

How to cite this article:
Fernandes M, Sangwan VS, Rao SK, Basti S, Sridhar MS, Bansal AK, Dua HS. Limbal stem cell transplantation. Indian J Ophthalmol 2004;52:5-22

How to cite this URL:
Fernandes M, Sangwan VS, Rao SK, Basti S, Sridhar MS, Bansal AK, Dua HS. Limbal stem cell transplantation. Indian J Ophthalmol [serial online] 2004 [cited 2024 Feb 25];52:5-22. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?2004/52/1/5/14637

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More than two decades ago, penetrating keratoplasty done for vascularised corneal scars with conjunctivali-sation invariably failed. This was because the transplanted donor epithelium consisted of transient amplifying cells (with limited proliferative potential) and lacked corneal epithelial stem cells. Our understanding of the existence of limbal stem cells and their functions has evolved over the years. Restoration of ocular surface integrity and the limbal barrier by limbal stem cell transplantation is essential in patients with limbal stem cell deficiency. This review provides an overview of the basic concepts of limbal stem cells, the aetiology and clinical features of limbal stem cell deficiency, and describes the management of limbal stem cell deficiency by limbal transplantation.

The corneal epithelial cells undergo constant renewal and regeneration. Cells from the surface are desquamated and replaced by proliferating basal epithelial cells from the periphery. The cells undergo vertical and horizontal movements. [1],[2],[3],[4],[5],[6],[7],[8],[9],[10]This state of dynamic equilibrium is maintained by a sub-population of cells, the stem cells , residing within the palisades of Vogt at the limbus.[11] There are no direct markers for the stem cells. Their existence at the limbus is suggested by indirect clinical[12],[13] and experimental evidence.[10], [14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25]These cells are poorly differentiated with a slow cell cycle, long life span and a high capacity for error-free self renewal. The characteristics of stem cells and the unique features of the limbal basal epithelium have been described in recent reviews.[26],[27] The limbal stem cells are responsible for corneal epithelial renewal and regeneration, and function as a barrier, preventing conjunctival epithelium from growing onto the cornea. [15],[16],[17],[18]

In addition, corneal epithelial stem cells like other epithelial stem cells are developmentally committed to give rise only to cells of their own particular tissue type.[28] They lack the plasticity exhibited by haematopoietic stem cells which may transdifferentiate into skeletal muscle, endothelial cells, liver and brain.[29]

  Limbal Stem Cell Deficiency Top

Aetiology and classification

Limbal stem cell deficiency (LSCD) may be primary or secondary.[11] Primary LSCD is characterised by the absence of identifiable external factors and an insufficient microenvironment to support the limbal stem cells. This includes aniridia and erythrokerato-derma. Multiple endocrine deficiency, neurotrophic keratopathy and peripheral inflammation were added to this group later.[21] Secondary LSCD occurs due to the destruction of limbal stem cells by external factors. These have been compiled in a recent review[26] and include chemical or thermal injuries, ultraviolet and ionising radiation, Stevens-Johnson Syndrome (SJS), ocular cicatricial pemphigoid (OCP), contact lens wear, severe microbial infection and multiple ocular surgeries.

Clinical features

The symptoms of LSCD are decreased vision, redness, watering, photophobia and recurrent attacks of pain.[26] The hallmark of LSCD is a triad of signs: conjuncti-valisation, neovascularisation and chronic inflam-mation.[20], [23],[24],[25]The fluorescein-stained conjunctivalised cornea has a stippled appearance,[6],[30] and there may be loss of palisades of Vogt in an area known to have palisades prior to the insult.[31],[32] In unilateral cases, it is useful to compare the limbus in the affected quadrants with the corresponding areas of the unaffected fellow eye. Other features include recurrent and persistent epithelial defects, superficial vascularisation, scarring, thick fibrovascular pannus, ulceration, melting and perforation.[26]

The diagnosis of LSCD is based mainly on clinical signs and may be confirmed by laboratory tests. These include impression cytology, histopathology and fluorophotometry. Impression cytology of the perilimbal area with nitrocellulose acetate paper can detect the presence of goblet cells on the cornea.[21],[33] But the goblet cells may not be detected in the presence of severe ocular inflammation.[34] Histopathology of the resected pannus in selected cases such as vernal keratoconjunctivitis and xeroderma pigmentosum is confirmatory. Fluorophotometry quantifies the corneal barrier dysfunction at the subclinical level.[35] It may also be useful in confirming the diagnosis of LSCD and in the restoration of the barrier function after limbal stem cell transplantation.

  Management of Limbal Stem Cell Deficiency Top

Limbal stem cell transplantation is the only treatment for limbal stem cell deficiency. The success of limbal transplantation depends on a variety of factors and may be adversely affected by concomitant lid pathology, dry eye and uncontrolled systemic disorders. Hence, the management of LSCD also involves the treatment of associated adnexal conditions, management of dry eye and management of systemic diseases. A detailed description of these conditions is outside the scope of this review.

Theoretically it is possible to restore stem cell function by expanding the stem cell population through modulations in the microenvironment, or inducing transient amplifying cell mitosis with the use of appropriate factors.[36] Currently, surgical intervention is the only means of restoring the ocular surface in cases with limbal stem cell dysfunction.

The algorithm for management of LSCD depends on whether the condition is unilateral or bilateral and involves some or all of the limbal stem cells [Figure - 1].

In unilateral cases with partial LSCD, limbal stem cell transplantation is not required. The patient may be kept under close observation,[37] subjected to repeated mechanical debridement also known as sequential sector conjunctival epitheliectomy (SSCE)[37] or amniotic membrane transplantation. [38],[39],[40] Ipsilateral translocation to an area of partial LSCD is shown to have a good outcome in a small series of patients.[41] Unilateral cases with total LSCD may benefit from either a conjunctival limbal autograft, or transplantation of cultured limbal stem cells from the fellow eye or from a living-related donor in a one-eyed patient. In bilateral conditions with total LSCD, an allograft limbal transplant utilising donor tissue from a cadaver or a living relative is the only option. In partial stem cell deficiency, healthy limbal tissue cultured from an unaffected site may be considered.

A classification scheme for epithelial transplantation procedures has been described[42] in order to do away with the confusion regarding the transplanted tissue type and the surgical technique. With the evolution of autologous or homologous cultured stem cells, it is necessary to modify this classification further. [Table - 1] describes the terminology for limbal transplantation procedures. The limbal stem cells may be drawn either from the fellow eye (autograft), a cadaver (allograft) or a living relative (allograft). The limbal stem cells are harvested with a carrier which may be either the conjunctiva (conjunctival-limbal auto- or allograft) or cornea (kerato-limbal allograft).

Surgical techniques

In all limbal transplantation procedures the preparation of the recipient bed remains the same and hence it is described at the beginning of this section. The various limbal transplantation procedures include conjunctival limbal autograft, keratolimbal allograft, conjunctival limbal allograft and cultured corneal epithelial or limbal stem cell transplantation. In addition to the surgical technique, a review of literature of the outcomes of each of these techniques has been included. The outcome of simultaneous or sequential corneal transplantation has been described.

Preparation of recipient bed

Under peribulbar anaesthesia a conjunctival peritomy is done 4-5mm from the limbus. The pannus is dissected off the cornea. Deeper layers of corneal stroma are avoided by using a bevel-up Beaver blade. Hemostasis is achieved with wet-field cautery or dilute adrenaline (1:10,000). Symblephara are released from the limbus, allowing the conjunctiva to fall back. Care is taken to avoid damage to the rectus muscles. When a kerato-limbal allograft is planned, a lamellar dissection of the sclera is carried out to enable a flush fit when the donor corneo-scleral rim is sutured in place.

Conjunctival limbal autograft

The candidates most likely to benefit from a conjunctival limbal autograft are those with unilateral partial or total LSCD, either due to chemical / thermal injuries, multiple surgeries, following excision of large tumours, pterygium surgery or contact lens induced keratopathy. The fellow eye provides the donor tissue. It is imperative to exclude LSCD in the donor especially since some injuries may be bilateral and involvement of the other eye may not be clinically apparent. Careful evaluation will help determine subtle damage such as the alteration or absence of the palisades of Vogt that indicates damage to the stem cells.[31],[32] Basti et al[43] noted progressive conjunctivalization in 3 cases following conjunctival limbal autograft, probably due to attenuation of limbal stem cell function with time. They emphasised the need for careful case selection. In their series, pseudopterygium formation was seen in one donor eye of a patient with history of bilateral chemical injury but without clinical evidence of LSCD. The optimum amount of limbal tissue that may be harvested from the donor eye has not been determined though excision up to eight clock hours has been recommended. In our opinion, excision and transplantation of more than six clock hours is best avoided for fear of precipitating LSCD in the donor. It is yet unknown how these donor eyes will respond to an epithelial insult in future and hence a conservative removal may be prudent. Experimental evidence shows the development of LSCD in a rabbit model.[20],[23] Longterm results are awaited as failure due to stem cell attrition ascribed to inflammation and subclinical donor cell disease has been reported.[44],[45]

Careful donor selection[45] and use of topical corticosteroids preoperatively[36] are useful. The use of cultured limbal stem cells[46] however, may also minimise the risk of iatrogenic LSCD in the donor eyes.

Surgical technique

The donor eye is operated under local or general anaesthesia under aseptic conditions. Three to four mm from the limbus, the conjunctiva is tented with Hoskins forceps and a small incision is made with the conjunctival spring scissors. A peritomy of 6 clock hours is done parallel to the limbus. The conjunctival flap without the underlying Tenon's capsule is reflected forwards onto the cornea and progressively undermined to reach the vascular arcades. The undermined limbus and conjunctiva are excised with Vannas' scissors. Minimal corneal stroma should be dissected. Care is taken to avoid damage to the limbus during excision. One must avoid buttonholing and dessication of the donor tissue.

The donor tissue is sutured in the correct anatomical position with interrupted 10-0 monofilament nylon sutures [Figure - 2]. A bandage contact lens is usually applied to prevent dislodging of the donor tissue by the shearing action of the lids, and to avoid injury to the growing epithelial edge.

Theoretically, this technique could result in partial removal of limbal tissue without the support of the stromal microenvironment.[47] Experimentally however, the yield of limbal stem cells cultured from a live donor is much higher than that from cadaveric tissue (Vemuganti GK, Kashyap S, Sangwan VS, Singh S. The proliferative potential of stem cells from fresh versus cadaveric limbal tissues. Invest Ophthalmol Vis Sci 2002;43:S 1623).

It is imperative to ensure that the donor eye is healthy. Harvesting of donor tissue from the apparently unaffected fellow eye in bilateral conditions like contact lens induced epitheliopathy will result in failure.[44],[48] A similar observation was made by one of the authors (SB)[43] in a patient with bilateral chemical injury with subclinical donor eye affliction.


There are several reports in the literature of conjunctival limbal auto transplantation.[43],[44], [48],[49],[50],[51],[52],[53],[54]Most are small case series and hence it is difficult to derive any conclusion. Few reports, however, deserve special mention. Kenyon and Tseng[49] first described the technique of limbal autograft transplantation in a series of 26 patients. The indications were chemical and thermal injuries in 22 (84.6%), contact lens induced keratopathy in 3 (11.5%) and stem cell deficiency following multiple surgeries in 1(3.8%). They fashioned two free limbal grafts that encompassed 8 clock hours of the recipient limbus from the apparently healthy contralateral eye. The mean follow-up was 18 months (2-45 months). In 10 (38.5%) patients with persistent epithelial defects for 3 weeks to 4 years, there was rapid re-epithelialisation (1-4 weeks). Failure of epithelialisation was seen in 3 (11.5%) eyes. Substantial improvement was noted in vision and reduction in stromal vascularisation.

One of the authors (SKR) carried out conjunctival limbal autograft in 16 eyes with LSCD following chemical injury.[53] Nine of these underwent late surgery (more than 4 months after the injury). In this subset of patients, re-epithelialisation was faster (156.1 vs 8.36.7 days) and the visual outcome was better. The authors advise against this technique when the limbal bed is still avascular. They recommend procedures like tenonplasty to facilitate vascularisation of the limbal bed. Coexisting stromal inflammation is an unhealthy microenviron-ment for survival of the transplanted limbal stem cells (Tsai RJF, Tseng SCG. Effects of disease chronicity and keratectomy on the outcome of limbal transplantation. Invest Ophthalmol Vis Sci 1990;31:S 469). A modification in the technique has been described.[54] In the immediate postoperative period following conjunctival limbal autograft, the conjunctival epithelium is closely monitored. If the conjunctival epithelium encroaches onto the cornea, mechanical debridement is done under topical anaesthesia.

Keratolimbal allograft

When using cadaveric tissue, the stem cell carrier may be either conjunctiva or cornea. Only 2 cases of cadaveric conjunctival limbal autograft have been described in the literature.[55] This technique is not routinely used.

Surgical Technique

1. Donor Tissue Selection . Tissue from the youngest possible donor with an upper limit of 50 years is recommended. Surgery should be performed within 72 hours as the cells are expected to be more active and vital.[56]

2. Donor Tissue Preparation. While harvesting the tissue, damage to the epithelium should be avoided, and a peripheral skirt of conjunctiva about 3-4mm wide should be included, if possible. This minimises trauma to the limbal stem cells. In addition, the scleral rim should be 4-5mm wide.

Preparation of the donor lenticule may be as follows:

a) The central cornea is trephined from the corneo-scleral rim. Lamellar dissection of the peripheral rim of tissue to 1/3-1/2 depth[57] or involving minimal stroma[34] is carried out. One of the authors (SKR) prefers to place the rim upside down on a bed of viscoelastic and dissect away the posterior layers in a spiral fashion until a very thin lenticule is obtained.

b) If the whole globe is used, a vacuum trephine may be used centrally, set to a depth of 150 and the dissection of the peripheral cornea and limbus may be carried out with a diamond knife.[58]

c) Since the freehand dissection of either the whole globe or the corneo-scleral rim is tedious, time consuming and technically demanding, with the risk of damage to the limbal stem cell in the process, an alternate technique is to use a microkeratome-based limbal harvester with a 16mm suction head.[59] The viability of harvested limbal stem cells by this technique is not yet determined though their morphology appears unaltered.

The advantages of using a fresh whole globe are rapid transportation and better stabilisation of the cornea during dissection. The lenticules should not be too thick otherwise they may dislodge due to blinking or may result in dellen formation.

3. Placement of Donor Tissue. The lenticules should be oriented in the proper anatomical position with the anterior corneal edge overlying the recipient limbus. This edge should lie flush with the cornea. Using 2 corneo-scleral rims or whole globes, 360 coverage may be ensured by placing the lenticules end-to-end. Sodium hyaluronate may be used to protect the epithelium. [Figure - 3] depicts the surgical technique of excision of donor lenticules from a corneo-scleral rim and anchoring of these lenticules to the recipient bed along with penetrating keratoplasty. Amniotic membrane has been used as an adjunct to limbal transplantation[38] as it helps reconstruct the perilimbal stroma and reduces inflammation and vascularisation. [Figure - 4] shows successful ocular surface reconstruction in a patient with total LSCD resulting from chemical injury.

The advantages and disadvantages of keratolimbal allograft are outlined in Table 2. The current recommendations for cadaveric limbal allograft may limit the amount of tissue available for transplantation since the viability of the limbal stem cells in storage is not known. The potential of culturing limbal stem cells from organ-cultured limbal rings and fresh cadaveric globes has been examined.[60] The yield from the latter was more than 60% successful


The outcome of keratolimbal allograft by several groups has been summarised in [Table - 3]. In 1984, Richard Thoft[61] described a technique of transplanting cadaveric corneal epithelial lenticules (keratoepithelioplasty) in patients with persistent epithelial defects. The ocular surface and visual acuity improved in 3 of 4 patients despite the fact that limbal stem cells were not included in the lenticules and the patients were not immunosuppressed. Subsequently the same group[62] published similar results with a modified technique that included the limbal stem cells in the donor lenticule.

Several authors have described their results of keratolimbal allograft with improvement in the ocular surface varying from 57 to 83.3%.[34],[38],[42],[57],[58],[63] There is clearly an improvement in the intermediate term outcome with the use of immunosuppression.

Two recent reports[64],[65] have described the longterm outcome of keratolimbal allograft with an average follow-up of 3 and 5 years each. Both groups noted a progressive decline in the visual acuity and graft survival with time. The overall change in ambulatory vision (best-corrected visual acuity, 6/60) was 76.6% 6.8% after one year. This decreased to 53.6% 9.9% and 44.6% 11.6% at 3 and 5 years respectively.[64] SJS patients fared worse than the others. The survival of the allograft also decreased with time from 76.9%6.7% at one year to 47.4%11.7% at 3 years and 23.7%17.5% at 5 years. Simultaneous penetrating keratoplasty and limbal transplant resulted in a less favourable outcome after one year though no difference was noted at 3 years. The survival of a second limbal transplant was better than the first.[64]

Ilari and Daya[65] reported that use of systemic Cyclosporine (CsA) did not prevent allograft rejection but resulted in prolonged graft survival, probably by decreasing chronic rejection.

Conjunctival-limbal allograft

In one-eyed patients with total LSCD, or those with bilateral LSCD, living related conjunctival limbal allograft is a good option. However, similar to the conjunctival limbal autograft, the major concern in conjunctival limbal allograft is harvesting of tissue from a healthy eye and its possible consequences. In addition, the need for immunosuppression is inevitable and the potential risk of adverse effects is unavoidable. Conjunctival limbal allograft is thus a viable option if HLA matched tissue is procured from a living related donor. But despite HLA matching, the recipient must be immunosuppressed indefinitely. [Figure - 5] shows a patient with successful ocular surface reconstruction following living related conjunctival limbal allograft.

Surgical Technique

1. Donor Tissue Preparation . Peribulbar or topical anaesthesia is used, and surgery performed either simultaneously in an adjacent operating room[66] or the donor is operated first, followed immediately by the recipient.[67]

The surgical procedure is similar to that for conjunctival limbal autograft and involves dissecting 2 clock hours each of limbal tissue superiorly and inferiorly. The tissue may be stored either in balanced salt solution[67] or in Ringer's lactate solution.[66] The donor conjunctiva is advanced to the limbus and sutured with 10-0 vicryl or just left alone.

2. Placement of Donor Tissue . The surgical technique is the same as that described for conjunctival limbal autograft in an earlier section.

With the advent of cultured corneal epithelial stem cells, patients with bilateral ocular surface disease and their relatives may benefit by total coverage of the limbus in the former and minimal tissue requirement from the latter. [Table - 4] highlights the advantages and disadvantages of conjunctival limbal allo-transplantation.


Four groups of authors[66],[67],[68],[69]have reported results of their work in this field. Kwitko and co-authors[68] pioneered conjunctival transplantation from a live donor. A success rate of 91.6% (11/12) with an improvement in visual acuity of 5/11 (45.5%) was seen in patients with bilateral ocular surface disease. This was despite the fact that limbal tissue was not included in the lenticules, 360 coverage of limbus was not attempted, and none of their patients were on immunosuppression.

A subsequent study[66] wherein none of the patients were on immunosuppression following living related conjunctival limbal allograft from histocompatible donors, prompted the authors to recommend the use of histocompatible keratolimbal allograft with total limbal coverage along with immunosuppression since none of the eyes had an improvement in vision or the ocular surface.

It has been suggested that limbal transplantation may be carried out in two phases: structural reconstruction by keratolimbal allograft and associated lid surgeries, followed by surface restoration preferably with living related conjunctival limbal allograft to re-establish the limbal barrier.[67] Success rates of 80% each in surface restoration and visual rehabilitation were achieved.

Most recently, a single case report has highlighted effective ocular surface reconstruction in a patient with Stevens Johnson syndrome.[69] The keratinised skin-like epithelium was removed from the ocular surface. Limbal and bulbar tissue were transplanted from a living related donor. One year after surgery, the mucosal epithelium was maintained though some keratinisation was noted.

Cultured corneal epithelial or limbal stem cell transplantation

Currently, culturing corneal epithelial stem cells is the most exciting and promising technique in limbal transplantation. It is possible to culture stem cells using a small amount of tissue thereby minimising the damage to the donor surface and the potential limbal epithelial exhaustion. Since only epithelial cells without Langerhan's cells and blood vessels are transplanted, theoretically it is possible that the incidence of rejection may also be reduced in homologous cultured limbal stem cell transplantation in bilateral ocular surface disease. The concept of culturing stem cells was derived from the use of cultured human epidermal cells as autologous grafts in burns patients and in plastic and reconstructive surgery.[70],[71] Rheinwald and Green[72] established a human epidermal keratinocyte-culturing method involving the use of 3T3 feeder layers. This was subsequently applied to limbal stem cells.

Surgical technique

Briefly, the technique entails a limbal biopsy from the donor (approximately 1mm[2]). The tissue is either trypsinised to disaggregate the cells[46] or cultured directly.[73] The corneal limbal epithelial cells are co-cultured with 3T3 fibroblasts inactivated by either g irradiation[72] or mitomycin-C,[73] in Dulbecco's minimum essential medium (DMEM) and Ham's F12 medium with addition of several supplements. The medium is changed every 3 days and confluent secondary cultures are released from the plastic dish enzymatically. These grafts may be mounted on petrolatum gauze or a bandage soft contact lens.[46] More recently, de-epithelialized amniotic membrane has been used as a carrier experimentally[73],[74],[75]and clinically. [76],[77],[78],[79]Besides its known properties of promoting epithelialisation,[80],[81] reducing inflammation and scarring,[82] preserving and maintaining existing limbal stem cells,[40] amniotic membrane serves as a natural substrate on which limbal stem cells can grow and proliferate.[74],[75] It also enables easier handling of the cultured limbal stem cells. It is believed that the amniotic membrane also acts as a barrier to immune cells,[82],[83] decreases the immune response by inhibiting IL-1β and IL8 expression[84] and produces antiangiogenic proteins.[85] All these properties are beneficial to limbal stem cell transplantation; whether the concomitant use of amniotic membrane may help reduce the risk of rejection in allo-transplantation is yet undetermined. Fibrin substrate has also been used to culture limbal stem cells.[86] The culture system may be maintained for 14-28 days[76],[77],[78],[79]and either transferred to the recipient bed or subjected to air-lifting to promote epithelial tight junction formation and stratification. [76],[78] The cultured stem cells and their carrier are transferred onto the recipient bed, anchored to the limbus with 10-0 nylon sutures and to the surrounding conjunctiva with 8-0 vicryl sutures. Care should be taken to protect these cells with sodium hyaluronate. A bandage contact lens may be used postoperatively.


Pellegrini et al[46] first described the results of cultured autologous corneal epithelial cells in two patients followed up for more than two years. Penetrating keratoplasty was done in one patient after 4 months and vision improved to 6/24. In the other it was counting fingers at one metre without additional surgery. There was no recurrence of conjunctivalisation and the surface remained stable. Corneal biopsies in both eyes approximately 1Z\x years after limbal transplantation, were positive for cornea specific keratin K3. Similar encouraging results are reported by others.[76],[77]

In a series of 13 cases [SJS (8), OCP (3), burns (2)] with total LSCD, the results of cultured corneal epithelial transplantation were not as encouraging.[79] Corneal epithelialisation was achieved in only 6 of 13 eyes (46.2%). Four of 13 eyes (30.8%) had corneal perforations. The poor outcome was attributed to severe ocular surface disease with dry eye and cicatrisation along with the use of cultivated allotransplants.

One of the authors has described (Sangwan VS, Vemuganti GK, Singh S, Kashyap S, Ifthekar G, Rao GN. Early results of ocular surface reconstruction in unilateral severe limbal stem cell deficiency using autologous cultured limbal and conjunctival stem cells. Invest Ophthalmol Vis Sci 2002;43:S 2992) successful ocular surface reconstruction 1-8 months following cultured limbal autograft in 18/24(75%) eyes with unilateral stem cell deficiency due to chemical burns. Cultured conjunctiva was also transplanted in 18 eyes with severe conjunctival damage. The surface was restored in 17(94.4%) of these eyes. Longterm results with this novel method of co-culturing limbal and conjunctival epithelium are awaited. [Figure - 6] shows a patient with unilateral total LSCD managed by cultured limbal stem cells and cultured limbal autograft.

While the results are very promising in selected cases, there are several unanswered questions regarding the nature of cells on the surface and the strength of their adherence to the stroma, whether they return to their niche at the limbus, and their longterm survival.

Corneal transplantation

The ultimate aim of any transplant surgery is to replace the diseased tissue with normal functioning tissue. After restoring ocular surface stability by limbal transplantation, visual rehabilitation may be carried out by penetrating keratoplasty. The visual acuity of a patient with ocular surface disease who has undergone limbal stem cell transplantation may improve without any further surgical intervention. However, in cases where the corneal stromal opacification hampers visual recovery, a penetrating keratoplasty may be necessary. [Figure - 7] shows a patient who had recurrence of gelatinous droplet dystrophy following penetrating keratoplasty successfully managed by simultaneous keratolimbal allograft and penetrating keratoplasty.

Some authors[66],[87],[88] suggest that penetrating keratoplasty and limbal stem cell transplant should be done at the same sitting. Since the same donor tissue is utilised to deliver a lower antigenic load to the recipient, theoretically the risk of allograft rejection is minimised. Additionally, the transient amplifying cells are preserved, thereby lessening the chance of ocular surface breakdown in the peri-operative period.

Croasdale[56] recommends penetrating keratoplasty after an interval of 3 months, after the ocular surface has stabilised. Others recommend waiting for up to a year since a successful limbal transplant may obviate the need for a penetrating keratoplasty;[65], [67] they instead advocate a deep lamellar keratoplasty if the endothelium is healthy.[65] It is also known that the risk of corneal allograft rejection increases if a transplant is carried out in an inflamed and vascularised recipient bed.[88] The surgery is technically easier. The corneal tissue should have intact epithelium and this should be protected by sodium hyaluronate to avoid epithelial damage while punching the donor cornea and during surgery.


There are several studies wherein simultaneous limbal transplantation and penetrating keratoplasty have been done. These have been summarised in [Table - 3]. Recent evidence[64], [65] however indicates that the combined procedure has a poorer long-term outcome. [Table - 5] highlights isolated cases from amongst series of patients who have undergone limbal transplantation wherein penetrating keratoplasty has been done sequentially. With such a small number of patients, it is difficult to derive a conclusion as to which technique is better. There is a need for prospective studies with a larger sample size and longer follow up.

  Postoperative Care Top


The topical regimen common to both auto- and allografts includes topical antibiotics until the conjunctival and corneal epithelial defects heal, and topical corticosteroids, preferably preservative-free. Topical corticosteroids are tapered based on the existing inflammation and continued long-term on a low maintenance dose. Excessive treatment in the early postoperative period is avoided to avert the retardation of epithelialisation.[47] Preservative-free tear substitutes and autologous serum have been used.[56],[63],[64],[79] Topical cyclosporine A has been used in allolimbal transplantation. [34,[63],[64],[65],[73],[79] The role of immunosuppre-ssants and dosage schedules is described in a later section.


In the immediate postoperative period, very close follow-up is necessary until the epithelium heals. Thereafter, monthly follow-up is needed to monitor recurrence of conjunctivalisation, ocular surface breakdown, rejection and adverse effects of immunosuppression. The bandage soft contact lens and conformer may be removed 6-8 weeks postoperatively.


The mechanism and onset of action, dosage and route of administration, adverse effects of immunosuppres-sants used in limbal transplantation have been briefly described in [Table - 6]. The drugs used include cyclosporin A, cyclophosphamide, azathioprine, tacrolimus, methotrexate and prednisolone.


Intraoperative complications of the recipient include damage to the muscle during symblepharon release, bleeding during superficial keratectomy, and corneal perforation.

Thick donor lenticules result in improper surfacing of tear fluid with subsequent dellen formation. Friction due to lid movement may dislodge the lenticules.

Postoperative complications include epithelial breakdown, limbal allograft rejection, infection and secondary glaucoma.

The predisposing factors of epithelial breakdown include persistent or severe inflammation, [67],[68] lid abnormality,[58] contact lens induced keratopathy,[44] trichiasis[73] and following applanation tonometry.[66] Healing may be hastened by frequent instillation of preservative-free topical lubricants, autologous serum, and hyaluronic acid.[58],[79] Botulinum induced ptosis has successfully been used for the management of persistent epithelial defect.[38]

The incidence of limbal allograft rejection varies from 14% - 75%.[38],[57],[58],[61],[66],[67],[68],[83],[89] The clinical features are tortuous and engorged limbal blood vessels,[57] development of ischemia in a lenticule after remaining healthy and vascularised for sometime and sudden onset of an epithelial defect accompanied by conjunctival inflammation[73] [Table - 7].

Early recognition and treatment of limbal allograft rejection is important to avoid stem cell death and ocular surface breakdown. The protocol recommended by Daya at et[90] al comprises of hourly application of preservative-free topical corticosteroids and subconjunctival or subTenon's depot corticosteroids in the section of rejection. Intravenous methylprednisolone is given in three bolus doses over 3 days followed by oral prednisolone. Parenteral cyclosporine A is stepped up. This regimen is likely to reverse the rejection, but the survival of the stem cells thereafter is questionable.

Though infective keratitis is quite rare following limbal stem cell transplant, should it occur aggressive antimicrobial therapy is necessary.

Glaucoma management in patients with LSCD is difficult. Preoperatively the intraocular pressure should be controlled. The use of Molteno drainage devices preoperatively has been suggested.[38] In the eventuality of raised pressure postoperatively, a shunt may be needed since conjunctival scarring precludes performing trabeculectomy. As a last resort, cyclo-destructive procedures may be carried out in an eye with poor visual potential.

Longterm outcome

While the short and intermediate term results of limbal stem cell transplantation appear promising, the question of their long-term survival is not yet answered. Molecular diagnostic techniques are increasingly used to search for the surviving cells in limbal transplantation patients. [91],[92],[93],[94]

  Conclusion Top

Limbal stem cell transplantation is an effective modality in patients with limbal stem cell deficiency. Success or failure of limbal transplantation may be determined by visual acuity, ocular surface stability, recurrence of conjunctivalisation or symblepharon, complications like limbal allograft rejection that may result in stem cell death, and duration of follow-up.

The outcome is governed by the following:

i) The underlying ocular pathology and its chronicity: In primary limbal stem cell deficiency, the stromal microenvironment may be insufficient to support the growth of limbal stem cells and thus there may be no benefit from limbal transplantation.[95] Similarly in the presence of ocular inflammation, the viability of the stem cells may be adversely affected.

ii) In patients with subnormal tear function, the outcome of limbal stem cell transplantation is poorer.

iii) Immunosuppression is essential in patients receiving limbal allografts, as in liver or kidney transplant patients.

The surgical procedure depends on the amount of limbal stem cell deficiency. There are some patients in whom limbal transplantation will definitely fail. In those with unilateral limbal stem cell deficiency, cultured autologous stem cells may minimise the risk of iatrogenic deficiency in the contralateral eye. For bilateral limbal stem cell deficiency, limbal allografts with aggressive immunosuppression are the only option. Culturing limbal stem cells from living related HLA compatible donors may prove to be superior to keratolimbal allograft in terms of cell viability , longevity, and reduced risk of rejection. Though the results may not be very encouraging, most of these patients are bilaterally blind and this procedure may be the only available option. A single procedure may not suffice and repeat surgeries may be needed to maintain ocular surface stability. Limbal transplantation followed by penetrating or deep lamellar keratoplasty may be more successful than the combined procedure.

  The Future Top

Although the results of recent techniques of limbal transplantation are encouraging, significant problems exist. For instance, difficulty in identifying stem cells due to the lack of specific markers, immunological rejection, difficult culturing techniques and lack of adequate treatment for the tear film dysfunction are but a few predicaments that require further research.

Recently, p63 transcription factor has been identified as a keratinocyte stem cell marker[96] and the development of in vivo techniques of studying the epithelial cell morphology would be of tremendous help in studying transplanted limbal epithelial cells.

Fluorescein-activated cell sorting may be used to isolate pluripotent stem cells from Langerhan's cells in allogeneic transplants thereby reducing the risk of rejection and need for immunosuppression.[97]

Mutations in β IGH3 gene lead to abnormal processing and turnover of keratoepithelin, which has been demonstrated in the deposits of corneas with granular, Avellino, Reis-Buckler's, lattice and Thiel-Behnke dystrophies.[98] These dystrophies are known to recur[99]and it is presumed that the host-derived epithelium continues to produce defective keratoepithelin, suggesting a role for limbal stem cell transplantation and/or gene therapy in the management of these disorders.

Transdifferentiation of adult stem cells into different phenotypes is a burning issue amongst molecular biologists. The ability to transdifferentiate limbal stem cells from other stem cells would enable limbal transplantation without the risk of rejection.

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  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]

  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7]

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